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Gap junctions in skeletal development and function
Abstract
Gap junctions play a critical role in the coordinated function and activity of nearly all of the skeletal cells. This is not surprising, given the elaborate orchestration of skeletal patterning, bone modeling and subsequent remodeling, as well as the mechanical stresses, strains and adaptive responses that the skeleton must accommodate. Much remains to be learned regarding the role of gap junctions and hemichannels in these processes. A common theme is that without connexins none of the cells of bone function properly. Thus, connexins play an important role in skeletal form and function.
... Bone remodeling, a well-regulated process, represents the relationship between bone formation and resorption and reflects the dynamics of bone tissue under physiological and pathophysiological conditions [1][2][3]. Osteoporosis is a multifactorial disease characterized by a reduction in bone mineral density, leading to fragility and fractures [4,5], and it has a negative impact on public health and quality of life [6]. Osteoporosis is a disease that affects bone remodeling and is one of the most common bone diseases with a high clinical and economic burden. ...
... In humans, 21 members of the connexin family are identified, and they are involved in cell-cell communication and intracellular-extracellular exchange via gap junctions and hemichannels, respectively [8,9]. Connexin 43 (Cx43) is the most abundant gap junctional protein in bone cells, including osteoblasts and osteoclasts, and, therefore, a key player in the function as well as the development of these cells [2,8,[10][11][12]. For physiological bone remodeling and skeletal homeostasis, Cx43 plays an important role in the proliferation, differentiation, and survival of bone cells [2,[8][9][10]. ...
... Connexin 43 (Cx43) is the most abundant gap junctional protein in bone cells, including osteoblasts and osteoclasts, and, therefore, a key player in the function as well as the development of these cells [2,8,[10][11][12]. For physiological bone remodeling and skeletal homeostasis, Cx43 plays an important role in the proliferation, differentiation, and survival of bone cells [2,[8][9][10]. During osteogenic differentiation of human mesenchymal stem cells (hMSCs), gene expression of Cx43 was increased in comparison to undifferentiated cells [13]. ...
Bone remodeling is essential for maintaining bone health. The imbalance between bone formation and bone resorption leads to bone diseases such as osteoporosis. Connexin43 (Cx43) is a gap junction molecule that plays an important role in bone homeostasis. The present study investigates the morphological characteristics of bone trabeculae and the distribution of Cx43 in bone cells using osteoporotic rat models to explore the relationship between osteoporosis and bone remodeling. Female Sprague–Dawley rats were divided into three groups: sham, ovarectomy with food deprivation (OVX+diet), and ovarectomy with steroid administration (OVX+steroid) for 3 and 12 months to induce osteoporosis. The lumbar vertebrae were processed for histomorphometric and immunohistochemical evaluation of the trabeculae and the distribution of Cx43 in bone cells. The data showed a significant reduction in trabecular bone in both osteoporotic groups. After 12 months, the OVX+diet treatment resulted in reduced mineralization and an increase in unmineralized bone. The percentage of alkaline phosphatase-positive areas in the OVX+diet vertebrae was lower at 12 months compared to the sham group. A significant increase in tartrate-resistant acid phosphatase (TRAP) positive osteoclasts was observed in the OVX+diet group. Both osteoporotic groups showed a decrease in Cx43-positive osteoblasts areas. An increase in the number of osteoclasts positive for Cx43 was detected in the OVX+diet group. The changes in Cx43 distribution in bone cells, together with trabecular mineralization, suggest that Cx43 may play a role in the progression of osteoporosis and could be a valuable target to improve bone remodeling.
... To date, five connexin subfamilies have been identified (α, β, γ, δ and ε, or GJA, GJB, GJC, GJD and GJE), twenty-one Cx genes have been identified in the human genome, and twenty Cx genes have been identified in the mouse genome [2,11,12]. Depending on the Cx profile present in a particular cell type, GJs will determine different properties in terms of propagations of electrical signals and permeability to second messengers and metabolites, not only between interconnected cells but also between the intracellular milieu and the surrounding microenvironment [13]. This obviously plays a crucial role in cell physiological properties. ...
... It is permeable to relatively large molecules, with a weak preference for negatively charged particles [15] Osteoblast differentiation and extracellular matrix mineralization [23][24][25][26][27][28] Interconnections between osteocytes and between osteocytes with osteoblasts and osteoclasts, arranging a "functional syncytium" within bone tissue [11,29] Transduction of mechanical into biochemical signals that are propagated through bone tissue by the syncytial network [30][31][32][33] Cell response to biochemical signals from the external medium [28,[34][35][36] Osteoclastogenesis. Osteoclast reabsorption activity [16,[37][38][39][40] Anti-apoptotic effects [13,14,41,42] Interactions between articular cartilage and subchondral bone [43] Chondrocyte differentiation [42,44,45] Arrangement of the articular chondrocyte network [1,46,47] Intercellular propagation of Ca2+ waves following mechanical stimulation of articular chondrocytes [48,49] Implicated in the etiology of osteoarthritis [13,[50][51][52][53] Bone marrow stromal cells Osteogenic differentiation [54][55][56][57][58] Cx45 GJC1 Gjc1 ...
... It is permeable to relatively large molecules, with a weak preference for negatively charged particles [15] Osteoblast differentiation and extracellular matrix mineralization [23][24][25][26][27][28] Interconnections between osteocytes and between osteocytes with osteoblasts and osteoclasts, arranging a "functional syncytium" within bone tissue [11,29] Transduction of mechanical into biochemical signals that are propagated through bone tissue by the syncytial network [30][31][32][33] Cell response to biochemical signals from the external medium [28,[34][35][36] Osteoclastogenesis. Osteoclast reabsorption activity [16,[37][38][39][40] Anti-apoptotic effects [13,14,41,42] Interactions between articular cartilage and subchondral bone [43] Chondrocyte differentiation [42,44,45] Arrangement of the articular chondrocyte network [1,46,47] Intercellular propagation of Ca2+ waves following mechanical stimulation of articular chondrocytes [48,49] Implicated in the etiology of osteoarthritis [13,[50][51][52][53] Bone marrow stromal cells Osteogenic differentiation [54][55][56][57][58] Cx45 GJC1 Gjc1 ...
Gap junctions (GJs) formed by connexins (Cxs) play an important role in the intercellular communication within most body tissues. In this paper, we focus on GJs and Cxs present in skeletal tissues. Cx43 is the most expressed connexin, participating in the formation of both GJs for intercellular communication and hemichannels (HCs) for communication with the external environment. Through GJs in long dendritic-like cytoplasmic processes, osteocytes embedded in deep lacunae are able to form a functional syncytium not only with neighboring osteocytes but also with bone cells located at the bone surface, despite the surrounding mineralized matrix. The functional syncytium allows a coordinated cell activity through the wide propagation of calcium waves, nutrients and anabolic and/or catabolic factors. Acting as mechanosensors, osteocytes are able to transduce mechanical stimuli into biological signals that spread through the syncytium to orchestrate bone remodeling. The fundamental role of Cxs and GJs is confirmed by a plethora of investigations that have highlighted how up- and downregulation of Cxs and GJs critically influence skeletal development and cartilage functions. A better knowledge of GJ and Cx mechanisms in physiological and pathological conditions might help in developing therapeutic approaches aimed at the treatment of human skeletal system disorders.
... For example, osteocytes can respond to fluid shear by opening Cx43-HCs to release PGE2 and ultimately promote new bone synthesis [30,31]. Other less distributed Cx members are also important to musculoskeletal activities, such as Cx37 and Cx45 in the osteoblast plasma membrane and Cx46, which was identified as a component of monomeric structures in the Golgi of osteoblasts [32][33][34]. Studies have shown that the appearance of Cx37 in osteocytes is necessary for the differentiation of osteoclasts [35,36]. ...
... For GJs in chondrocytes, studies have suggested that intercellular communication among human chondrocytes may occur via GJ channels [119]. Some experiments have shown that Cx43 is expressed in animal chondrocytes; more importantly, Cx43 can form normal GJ channels but is only detected in dense monolayer and micro mass cultures [17,33,106,[122][123][124][125][126]. However, Cx43-GJs are not only located in clusters of chondrocytes as previously thought [11], which means that distant chondrocytes in mature articular cartilage can connect with one another via special physical connections, and it was finally demonstrated that cell-to-cell communication between highly specialized and distant chondrocytes in cartilage occurs through GJ channels that are predominantly composed of Cx43, which allows for the exchange of various molecules, including ions, second messengers, miRNAs and other compounds, such as glucose and amino acids [126][127][128]. ...
Connexin43, which is the most highly expressed connexin subtype in the musculoskeletal system, exists in a variety of bone cells, synovial tissue, and cartilage tissue. Connexin43 has been suggested to be a key regulator of bone homeostasis. Studies have shown aberrant Connexin43 expression in musculoskeletal disorders, such as osteoporosis, osteoarthritis, and rheumatoid arthritis. During cellular activities, Connexin43 can participate in the formation of functionally specific gap junctions and hemichannels and can exert independent cellular regulatory and signaling functions through special C-termini. The critical role of Connexin43 in physiological development and disease progression has been gradually revealed. In this article, the function of Connexin43 in musculoskeletal tissues is summarized, revealing the potential role of Connexin43 as a key target in the treatment of related bone and muscle disorders and the need for further discovery.
... 16 Gap junctions are indispensable for the ossification process, presumably allowing diffusion of ions or small signaling molecules between condensing cells to induce osteoblast differentiation for the skull roof. [17][18][19][20] The cranial dermal bones ossify exclusively with osteoblasts, thus lacking the chondrocytes present in endochondral ossification, and undergo what is referred to as intramembranous ossification. [21][22][23] The molecular mechanisms that induce mesenchymal condensations for cranial dermal bone ossification remain enigmatic. ...
... In the condensations, cellular membranes of adjacent cells closely contacted each other ( Figure 3H,I), implying that the cells were interlinked by microscale architectures, such as gap junctions, which are indispensable for osteoblast differentiation. 17,48 In regions where mesenchymal cells had condensed, a small space was created inside the cell condensations ( Figure 3I, arrows). Bone matrix would be deposited presumably into this space at later stages ( Figure 3I). ...
Background
The cellular and molecular mechanisms initiating vertebrate cranial dermal bone formation is a conundrum in evolutionary and developmental biology. Decades of studies have determined the developmental processes of cranial dermal bones in various vertebrates and identified possible inducers of dermal bone. However, evolutionarily derived characters of current experimental model organisms, such as non‐homologous frontal bones between teleosts and sarcopterygians, hinder investigations of ancestral and conserved mechanisms of vertebrate cranial dermal bone induction. Thus, investigating such mechanisms with animals diverging at evolutionarily informative phylogenetic nodes is imperative.
Results
We investigated the cellular foundations of skull frontal bone formation in the spotted gar Lepisosteus oculatus, a basally branching non‐teleost actinopterygian. Whole‐mount bone and cartilage staining and hematoxylin‐eosin section staining revealed that mesenchymal cell condensations in the frontal bone of spotted gar develop in close association with the underlying cartilage. We also identified novel aspects of frontal bone formation: enrichment of F‐actin, cellular membranes, and E‐cadherin in condensing cells, and extension of podia‐like structures from osteoblasts to the frontal bone, which may be responsible for bone mineral transport.
Conclusion
This study highlights the process of frontal bone formation with dynamic architectural changes of mesenchymal cells in spotted gar, an emerging non‐teleost fish model system, illuminating supposedly ancestral and likely conserved developmental mechanisms of skull bone formation among vertebrates.
... us, isoflavones can bind to ERs in adipose tissue to regulate estrogen biosynthesis [65]. e protein expressed by the Cx43 acts on cell-to-cell communication allowing the signaling of important molecules for cellular development [66,67]. In the included studies, the expression of Cx43 gene was higher in LC, seminiferous epithelium, spermatogonia, spermatocytes, and spermatozoa of animals treated with isoflavone aglycone (genistein). ...
... Endocrine dysregulation during spermatogenesis leads to the production of defective sperm, with problems of motility, capacity, and viability [75]. e decrease in FSH observed in our findings may cause a reduction in cyclic adenosine monophosphate (cAMP) and androgen-binding protein (ABP) levels compromising spermatogenesis [67]. Studies have also indicated that estrogen intake causes a decrease in LH and FSH serum levels associated with an increase in prolactin serum levels [63]. ...
Isoflavone is a phytoestrogen found in different types of food that can act as endocrine disrupters leading to testicular dysfunction. Currently, fragmented data on the action of this compound in the testicles make it difficult to assess its effects to define a safe dose. Thus, we systematically reviewed the preclinical evidence of the impact of isoflavone on testicular function. We also determined which form (aglycones or glycosylated) was the most used, which allowed us to understand the main biological processes involved in testicular function after isoflavone exposure. This systematic review was carried out according to the PRISMA guidelines using a structured search on the biomedical databases MEDLINE (PubMed), Scopus, and Web of Science, recovering and analyzing 22 original studies. The bias analysis and the quality of the studies were assessed by the criteria described in the risk of bias tool developed by SYRCLE (Systematic Review Centre for Laboratory Animal Experimentation). The aglycones and glycosylated isoflavones proved to be harmful to the reproductive health, and the glycosylates at doses of 50, 100, 146, 200, 300, 500, and 600 mg/kg, in addition to 190 and 1000 mg/L, appear to be even more harmful. The main testicular pathologies resulting from the use of isoflavones are associated with Leydig cells resulting from changes in molecular functions and cellular components. The most used isoflavone to evaluate testicular changes was the genistein/daidzein conjugate. The consumption of high doses of isoflavones promotes changes in the functioning of Leydig cells, inducing testicular changes and leading to infertility in murine models.
... Connexins are found within all types of bone cells, and Cx43 is the most expressed one both in vivo and in vitro [140,141]. Expression of other types of Cxs, such as Cx45, Cx46 and more recently Cx37 has also been reported [142,143]. Bone cells (mostly osteoblasts, osteocytes and osteoprogenitors) are interconnected via functional Cx43 containing-gap junctions allowing them to orchestrate bone development and turnover. ...
... These studies highlighted a function of Cx43 for osteoblasts and osteocytes differentiation, function and survival. Nevertheless, whether this function occurred through a gap-junction dependent-or independent (as hemichannel or as scaffolding partner) mechanism remains unclear [140,141]. ...
Over the past 50 years, increasing experimental evidences have established that connexins (Cxs) and gap junctional intercellular communication (GJIC) ensure an important role in both the onset and development of cancerous processes. In the present review, we focus on the impact of Cxs and GJIC during the development of prostate cancer (PCa), from the primary growth mainly localized in acinar glands and ducts to the distant metastasis mainly concentrated in bone. As observed in several other types of solid tumours, Cxs and especially Cx43 exhibit an ambivalent role with a tumour suppressor effect in the early stages and, conversely, a rather pro-tumoral profile for most of invasion and dissemination steps to secondary sites. We report here the current knowledge on the function of Cxs during PCa cells migration, cytoskeletal dynamics, proteinases activities and the cross talk with the surrounding stromal cells in the microenvironment of the tumour and the bones. In addition, we discuss the role of Cxs in the bone tropism even if the prostate model is rarely used to study the complete sequence of cancer dissemination compared to breast cancer or melanoma. Even if not yet fully understood, these recent findings on Cxs provide new insights into their molecular mechanisms associated with progression and bone targeted behaviour of PCa.
... Connexins are found within all types of bone cells, and Cx43 is the most expressed one both in vivo and in vitro [140,141]. Expression of other types of Cxs, such as Cx45, Cx46 and more recently Cx37 has also been reported [142,143]. Bone cells (mostly osteoblasts, osteocytes and osteoprogenitors) are interconnected via functional Cx43 containing-gap junctions allowing them to orchestrate bone development and turnover. ...
... These studies highlighted a function of Cx43 for osteoblasts and osteocytes differentiation, function and survival. Nevertheless, whether this function occurred through a gap-junction dependent-or independent (as hemichannel or as scaffolding partner) mechanism remains unclear [140,141]. ...
This article is a report of the “International Colloquium on Gap junctions: 50 Years of Impact on Cancer” that was held 8–9 September 2016, at the Amphitheater “Pôle Biologie Santé” of the University of Poitiers (Poitiers, France). The colloquium was organized by M. Mesnil (Université de Poitiers, Poitiers, France) and C. Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248–1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized here below. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
... 17 18 CX43 is the predominant gap junction detected in osteoblasts, osteoclasts, osteocytes, and chondrocytes, although CX45, CX46, CX26, and CX37 are also expressed in bone cells. [19][20][21][22][23][24][25] Mutations in connexin genes are associated with a large variety of disorders affecting many organs. 26 Specific to bone, mutations in CX43 are responsible for AR CMD and oculodentodigital dysplasia (ODDD), characterized by syndactyly, microphthalmia, craniofacial and dental abnormalities. ...
Craniometaphyseal dysplasia (CMD), a rare craniotubular disorder, occurs in an autosomal dominant (AD) or autosomal recessive (AR) form. CMD is characterized by hyperostosis of craniofacial bones and flaring metaphyses of long bones. Many patients with CMD suffer from neurological symptoms. To date, the pathogenesis of CMD is not fully understood. Treatment is limited to decompression surgery. Here, we report a knock in (KI) mouse model for AR CMD carrying a R239Q mutation in CX43. Cx43KI/KI mice replicate many features of AR CMD in craniofacial and long bones. In contrast to Cx43+/+ littermates, Cx43KI/KI mice exhibit periosteal bone deposition and increased osteoclast (OC) numbers in the endosteum of long bones, leading to an expanded bone marrow cavity and increased cortical bone thickness. Although formation of Cx43+/+ and Cx43KI/KI resting OCs are comparable, on bone chips the actively resorbing Cx43KI/KI OCs resorb less bone. Cortical bones of Cx43KI/KI mice have an increase in degenerating osteocytes and empty lacunae. Osteocyte dendrite formation is decreased with reduced expression levels of Fgf23, Sost, Tnf-α, IL-1β, Esr1, Esr2, and a lower Rankl/Opg ratio. Female Cx43KI/KI mice display a more severe phenotype. Sexual dimorphism in bone becomes more evident as mice age. Our data show that the CX43R239Q mutation results in mislocalization of CX43 protein and impairment of gap junction and hemichannel activity. Different from CX43 ablation mouse models, the CX43R239Q mutation leads to the AR CMD-like phenotype in Cx43KI/KI mice not only by loss-of-function but also via a not yet revealed dominant function.
... Recently, Cx43 was found to be the most abundantly expressed Cx in human bone and cartilage and plays an important role in maintaining the functional homeostasis of cartilage. In addition to the classic GJ intercellular communication (GJIC), there are also barrier-free interstitial junctions in the membrane that act as direct conduits between the cytoplasm and the extracellular environment (13,14). The ability of GJIC to transmit biophysics to the entire network of bone cells and osteoblasts suggests that GJIC may be an important mechanism by which the mechanical forces are transmitted through bone and subsequently lead to bone formation. ...
Background and Objective
Connexin 43 (Cx43) is the main gap junction (GJ) protein and hemichannel protein in bone tissue. It is involved in the formation of hemichannels and GJs and establishes channels that can communicate directly to exchange substances and signals, affecting the structure and function of osteocytes. CX43 is very important for the normal development of bone tissue and the establishment and balance of bone reconstruction. However, the molecular mechanisms by which CX43 regulates osteoblast function and homeostasis have been less well studied, and this article provides a review of research in this area.
Methods
We searched the PubMed, EMBASE, Cochrane Library, and Web of Science databases for studies published up to June 2023 using the keywords Connexin 43/Cx43 and Osteocytes. Screening of literatures according to inclusion and exclusion guidelines and summarized the results.
Key Content and Findings
Osteocytes, osteoblasts, and osteoclasts all express Cx43 and form an overall network through the interaction between GJs. Cx43 is not only involved in the mechanical response of bone tissue but also in the regulation of signal transduction, which could provide new molecular markers and novel targets for the treatment of certain bone diseases.
Conclusions
Cx43 is expressed in osteoblasts, osteoclasts, and osteoclasts and plays an important role in regulating the function, signal transduction, and mechanotransduction of osteocytes. This review offers a new contribution to the literature by summarizing the relationship between Cx43, a key protein of bone tissue, and osteoblasts.
... Recently, connexin 43 (Cx43) was found to be the most abundantly expressed Cx in human bone and cartilage and plays an important role in maintaining the functional homeostasis of cartilage. In addition to the classic GJ intercellular communication (GJIC), there are also barrier-free interstitial junctions in the membrane that act as direct conduits between the cytoplasm and the extracellular environment (13,14). The ability of GJIC to transmit biophysics to the entire network of bone cells and osteoblasts suggests that GJIC may be an important mechanism by which the mechanical forces are transmitted through bone and subsequently lead to bone formation. ...
... 13 Each apposed cell contributes a hemichannel to the formation of a complete channel, the gap junction. 14 So far, at least 20 connexin isoforms in humans and mice have been reported. 15 Among them, connexin43 (Cx43) shows the most abundant expression in osteocytes. ...
Gap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes' GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes' GJIC in aged male mice and its mechanism. Methods Changes in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks. Results The AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes' GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo. Conclusion Our study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing. Cite this article: Bone Joint Res 2022;11(7):413-425.
... 13 Each apposed cell contributes a hemichannel to the formation of a complete channel, the gap junction. 14 So far, at least 20 connexin isoforms in humans and mice have been reported. 15 Among them, connexin43 (Cx43) shows the most abundant expression in osteocytes. ...
Aims
Gap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism.
Methods
Changes in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks.
Results
The AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes’ GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo.
Conclusion
Our study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing. Cite this article: Bone Joint Res 2022;11(7):413–425.
... You're reading In silico analysis to reveal underlying trans differentiation mechanism of Mesenchymal Stem Cells into Osteocytes Introduction Bone gives shape to the body and also protects internal delicate organs against external insult [1]. Bone is affected by different types of stimuli and factors [2,3] such as cell-cell communication via gap junction, paracrine, endocrine, and autocrine factors [4]. The remodeling process is controlled by organized and coordinated interactions between different bone cells [5]. ...
ackground: Bone is a mineralized dynamic tissue, helps to protect and support the body. Osteoarthritis damages the cartilage and is responsible for the degeneration of the bone. Many cell-based therapies are available to repair the damage however, the non-availability of autologous cells and slows healing during regeneration of the damaged bone present major constraints. Hence, there is a need to search for a convenient and easily available cell source that can not only be used to repair the bone but can also enhance its regenerative potential. β-glycerophosphate, dexamethasone, and L-ascorbic-2-phosphate can differentiate mesenchymal stem cells (MSCs) into osteocytes. So far, the interaction of these compounds with osteocytes-specific proteins has not been studied. In this study, in silico analysis was performed to investigate the interaction of proteins with osteocytes specific compounds at the amino acids level. Methods: 3D structures of Dexamethasone and L-ascorbic-2-phosphate (ascorbic acid) were drawn using Molecular Operating Environment (MOE). Then absorption, distribution, metabolism, and excretion (ADME) analysis was achieved using an online tool of "Swiss Package". By Ramachandran plot, the predicted model of ALPL, MMP13, Osteonectin, and RunX2 proteins were evaluated. Then docking of these proteins with Dexamethasone and L-ascorbic-2-phosphate was performed. Results: L-ascorbic-2-phosphate and Dexamethasone docked within the binding pockets of ALPL, RunX2, MMP13, and Osteonectin proteins, expressed in the bone cells. These compounds also showed good drug-likeness and pharmacokinetics properties. Conclusion: It is concluded that β-glycerophosphate, dexamethasone, and L-ascorbic-2-phosphate are novel substrates for osteogenic differentiation. These compounds could increase the healing and regenerative potential of bone cells by enhancing the expression of osteocytes specific proteins. B Abstract www.als-journal.com/ ISSN 2310-5380/ December 2021
... Unlike gap junctions, which mediate intercellular communication between adjacent cells, hemichannels facilitate the exchange of small molecules (<1.2 kDa) between the cell and the extracellular microenvironment [10]. Connexin 43 (Cx43), a gap junction/hemichannelforming protein, is a major connexin subtype expressed in bone cells and plays a crucial role in bone function, including osteoblast proliferation and differentiation, osteocyte mechanotransduction and survival, as well as bone development and bone mass after birth [11,12]. ...
Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes’ resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.
... JID: ACTBIO [m5G; April 15, 2021;18:34 ] ditions in the cytoplasmic processes within the lacuna-canalicular network (LCN). While gap junctions between osteoblastic and osteocyte processes are found in this region [55] , the main bulk of the osteoblast cell body is located outside of the LCN, such as on the Haversian canal surface. Due to the Haversian canal being 10 0 0 times larger than the LCN [56] , shear stress and fluid velocity on the Haversian canal surface are expected to be notably lower than those in the LCN region [14] . ...
The reliability of conventional cell culture studies to evaluate biomaterials is often questioned, as in vitro outcomes may contradict results obtained through in vivo assays. Microfluidics technology has the potential to reproduce complex physiological conditions by allowing for fine control of microscale features such as cell confinement and flow rate. Having a continuous flow during cell culture is especially advantageous for bioactive biomaterials such as calcium-deficient hydroxyapatite (HA), which may otherwise alter medium composition and jeopardize cell viability, potentially producing false negative results in vitro. In this work, HA was integrated into a microfluidics-based platform (HA-on-chip) and the effect of varied flow rates (2, 8 and 14 µl/min, corresponding to 0.002, 0.008 and 0.014 dyn/cm², respectively) was evaluated. A HA sample placed in a well plate (HA-static) was included as a control. While substantial calcium depletion and phosphate release occurred in static conditions, the concentration of ions in HA-on-chip samples remained similar to those of fresh medium, particularly at higher flow rates. Pre-osteoblast-like cells (MC3T3-E1) exhibited a significantly higher degree of proliferation on HA-on-chip (8 μl/min flow rate) as compared to HA-static. However, cell differentiation, analysed by alkaline phosphatase (ALP) activity, showed low values in both conditions. This study indicates that cells respond differently when cultured on HA under flow compared to static conditions, which indicates the need for more physiologically relevant methods to increase the predictive value of in vitro studies used to evaluate biomaterials.
Statement of Significance
There is a lack of correlation between the results obtained when testing some biomaterials under cell culture as opposed to animal models. To address this issue, a cell culture method with slightly enhanced physiological relevance was developed by incorporating a biomaterial, known to regenerate bone, inside of a microfluidic platform that enabled a continuous supply of cell culture medium. Since the utilized biomaterial interacts with surrounding ions, the perfusion of medium allowed for shielding of these changes similarly as would happen in the body. The experimental outcomes observed in the dynamic platform were different than those obtained with standard static cell culture systems, proving the key role of the platform in the assessment of biomaterials.
... The principal structural component, the membrane protein Connexin, is organized into a hexameric structure called Connexon which associates to form the functional double membrane gap junction [3][4][5][6]. Connexins are widely expressed and have also been demonstrated to be present in skeletal tissue [7,8], corneal epithelium, cardiac tissue [9][10][11][12] and are absent in erythrocytes [13,14]. ...
Aim
Drug delivery is crucial for therapeutic efficacy and gap junction communication channels (GJIC) facilitate movement within the tumour. Pro-drug activation, a modality of cancer therapy leads to Ganciclovir triphosphate (GCV-TP) incorporation into newly synthesized DNA resulting in cell death. The objective was to enhance, with Histone deacetylase inhibitors (HDACi) and All Trans Retinoic Acid (ATRA), GJIC, crucial for drug delivery, and with combination, abrogate the observed detrimental effect of Dexamethasone (DXM).
Methods
Cell lines (NT8E, and HeLa) were pre-treated with Valproic Acid (VPA) (1 mM), 4 Phenyl Butyrate (4PB) (2 mM), ATRA (10 μM) and Dexamethasone (1 μM). Protein quantitated with the Bicinchoninic (BCA) assay for cell lysates, membrane and soluble fractions was assessed with Western blotting for Connexins (43, 26 and 32) and E-Cadherin. A qRT-PCR was done for CX 43-GJA1, CX 26-GJB2, CX 32-GJB1 and E-Cadherin, and normalized with Glyceraldehyde Phosphate dehydrogenase (GAPDH). Further, localization of Connexins (CX) and E-Cadherin, GJIC competence, pre-clinical in-vitro studies and the mechanism of cell death were evaluated.
Results
There was no toxicity or change in growth patterns observed with the drugs. In both the cell lines CX 43 localized to the membrane whereas CX 32 and CX 26 were present but not membrane bound. E-Cadherin was present on the membrane in NT8E and completely absent in HeLa cells. Effects of HDACi, DXM and ATRA were seen on the expression of Connexins and E-Cadherin in both the cell lines. NT8E and HeLa cell lines showed enhanced GJIC with 4PB [30 %], VPA [36 %] and ATRA [54 %] with a 60 % increase in cytotoxicity and an abrogation of Dexamethasone inhibition on combination with VPA or ATRA.
Conclusion
An enhancement of GJIC function by HDACi and ATRA increased cytotoxicity and could be effective in the presence of Dexamethasone, when combined with ATRA or VPA.
... (2) Applied current could change the cell gap junctions, which affect the exchange of certain signaling molecules such as calcium, cyclic nucleotides, and inositol phosphates [64]. Much evidence indicates that gap junction communication is necessary for the development and maintenance of a differentiated osteoblast phenotype, including the production of ALP, OCN, bone sialoprotein, and collagen type I (COL I) [65]. ...
Background: Reconstruction of bone fractures and defects remains a big challenge in orthopedic surgery. While regenerative engineering has advanced the field greatly using a combination of biomaterial scaffolds and stem cells, one matter of difficulty is inducing osteogenesis in these cells. Recent works have shown electricity’s ability to promote osteogenesis in stem cell lines when seeded in bone scaffolds; however, typical electrical stimulators are either (a) externally housed and require overcomplex percutaneous wires be connected to the implanted scaffold or (b) implanted non-degradable devices which contain toxic batteries and require invasive removal surgeries. Objective: Here, we establish a biodegradable, piezoelectric Poly-L-Lactic Acid (PLLA) scaffold that uses external, non-invasive ultrasound to generate an electric charge that promotes stem cell osteogenesis. Methods: Demonstration of this system included (1) development of a piezoelectric PLLA mesh, (2) verification of its piezoelectric efficacy and degradation, (3) manufacturing of a PLLA scaffold, (4) in vitro testing of the system’s ability to enhance bone regeneration compared to a control, and (5) using assessments of cell proliferation and differentiation through protein, mineral, and gene assays. Results: Ultimately a 3000rpm electrospun PLLA nanofiber film that could output 40mV when stimulated with 40kHz 0.4W/cm2 ultrasound was assembled into a bone scaffold and seeded with adipose-derived stem cells (ADSCs). In vitro testing showed that relative to a control, in cells subjected to the experimental conditions alkaline phosphatase production increased 5-fold, mineral production increased 18-fold, osteocalcin gene 40-fold, and osterix gene 100-fold. Conclusion: The production of surface-level charge from ultrasonic stimulation of PLLA and the use of that charge to promote osteogenic differentiation in ADSCs was successfully demonstrated. The fact that PLLA was successfully used in combination with externally applied ultrasound to produce electrical charge opens up new frontiers for the field of tissue regeneration. This advancement helps make tissue engineering a tool that can tackle problems of even greater magnitude.
... The cell-cell communication via gap junction in hBMSCs plays a crucial role in osteogenesis. Cx43 is a major connexin in gap junction communication in order to coordinate the synthesis of new bone [43]. Fig. 8A shows the immunostaining of Cx43 in hBMSCs, in which the cells appeared to have a close connection when cultured on S0-S3 for 10 days. ...
The micro/nano hybrid structure is considered to be a biomaterial characteristic to stimulate osteogenesis by mimicking the three-dimensional structure of the bone matrix. However, the mechanism of the hybrid structure induced osteogenic differentiation of stem cells is still unknown. For elucidating the mechanisms, one of the challenge is to directly fabricate micro/nano hybrid structure on bioceramics because of its brittleness. In this study, hydroxyapatite (HA) bioceramics with the micro/nano hybrid structure were firstly fabricated via a hydrothermal treatment and template method, and the effect of the different surface structures on the expression of integrins, BMP2 signaling pathways and cell-cell communication was investigated. Interestingly, the results suggested that the osteogenic differentiation induced by micro/nano structures was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, while activated BMP2 could in turn activate integrins and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro-and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. Statement of Significance The micro/nano hybrid structure has been found to have synergistic bioactivity on osteogenesis. However, it is still a challenge to fabricate the hybrid structure directly on the bioceramics, and the role of micro-and nano-structure, in particular the mechanism of the micro/nano-hybrid structure induced stem cell differentiation is still unknown. In this study, we firstly fabricated hydroxyapatite bioceramics with the micro/nano hybrid structure, and then investigated the effect of different surface structure on expression of integrins, BMP2 signaling pathways and cell-cell communication. Interestingly, we found that the osteogenic differentiation induced by structure was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, and activated BMP2 could in turn activate some integrin subunits and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro-and nano-structures led to the syner-gistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect.
... Connexin 43 (Cx43) forms gap junctions that mediate osteocyte coupling and are critical for maintaining proper bone physiology, including proliferation, survival and differentiation of osteoblasts, and skeletal development. [11][12][13] Hemichannels (HCs), unpaired gap junction channels, are extensively involved in the communication between osteocytes and their extracellular environment, and play important roles in autocrine/paracrine signaling, cell survival, and mechanotransduction. [14][15][16][17] Connexin-based gap junctions and HCs allow the passage of small molecules (MW < 1 000 Da), such as ions, essential metabolites, and second messengers, such as Ca 2+ , IP3, NAD + , prostaglandin E 2 (PGE 2 ), cAMP, cGMP, ADP, and ATP. ...
Channels that form between cells and their extracellular environment help protect bone tissue from the damage wrought by low estrogen levels, a major cause of bone loss in post-menopausal women. Jean Jiang from the UT Health San Antonio and colleagues showed that depleting the estrogen hormone in mouse bone cells reduced levels of connexin 43 and impaired the protein’s ability to forms pores known as ‘hemichannels’. The researchers surgically removed the ovaries of various mouse strains to induce estrogen deficiencies. They found that transgenic mice without working hemichannels had reduced bone mass compared to normal mice or mice that could make hemichannels but lacked the ability for those channels to come together to form complete passageways. The findings highlight the importance of connexin 43 hemichannels in protecting bone tissue against osteoporosis.
... 7,21 However, cell-to-cell crosstalk through gap junction pathways of the articular cartilage is important in maintaining homeostasis. 22 Up to now, pathways mediated by GJIC in chondrocytes have been less studied. Cx43 determines the fate of gap junctions for Cx43 is reported to be the predominant structure of connexin family in chondrocytes, and the expression level of Cx43 determines the structure and number of GJIC channels. ...
Purposes: Gap junction intercellular communication (GJIC) exhibits a key role in maintaining the homeostasis of articular cartilage. Connexin43 (Cx43) protein is predominant in the structures that form gap junctions. We aim to determine the potential underlying mechanisms of TGF-β1 (Transforming growth factor-β1)—regulated cell communication in chondrocytes.
Materials and methods: After exposure of chondrocytes to recombinant TGF-β1, quantitative real-time PCR was used to detect expression levels of Cx43 mRNA. Western blot analysis was used to check Cx43 and mitogen-activated protein kinase (MAPK) family components. Immunofluorescence staining was performed to confirm ERK-MAPK pathway activation and Cx43 protein distribution. MAPK inhibitors (ERK inhibitor U0126, JNK inhibitor SP 600125 and P38 inhibitor SP 203580) were applied to verify the specificity effects of ERK-MAPK pathway. GJIC between chondrocytes were evaluated using Scrape loading/dye transfer (SLDT) assay.
Results: It was first found that TGF-β1modulatedthe Cx43protein expressions and its sub-cellular distribution. TGF-β1 promoted gap junction intercellular communication (GJIC) formations in chondrocytes, especially in a higher cell intensity. ERK-MAPK signaling pathway was activated in TGF-β1-mediated gap junctions among chondrocytes. Furthermore, the inhibitor of ERK attenuated the increases of Cx43 expressions and functional gap junction formations induced by TGF-β1, while cross-talk between ERK-MAPK and Smad signal pathways exists shown in the process.
Conclusions: This study provides evidence to show the importance of the ERK-MAPK pathway in TGF-β1—mediated Cx43 expression and functional gap junction formation.
... SCientifiC RepoRts | (2018) 8:14792 | DOI: 10.1038/s41598-018-33247-8 kinases, and furthermore, they are shared by the same core sequence conservation and structure from the point of structural biology 21 , we are interested in whether the profound morphological shift of osteocytes will occur by the induction of FGF7. ...
It is well recognized that osteocytes communicate with each other via gap junctions and that connxin43 (Cx43) shows its great potential in gap junction for the contribution enabling transmission of small molecules and operating in an autocrine/a paracrine manner. Fibroblast growth factors (FGFs) play significant roles in new bone formation and adult bone remodeling, and FGF signaling is regulated by the precise spatiotemporal approaches. However, the influence of FGF7 on osteocyte cell processes is not well elucidated. In this study, we aimed to examine the impact of FGF7 on osteocyte cell processes by characterizing the expression of Cx43 and to reveal the underlying mechanism regulating this cell process. We first found that the mRNA level of FGF7 was higher relative to other FGF family members both in osteocytes cell line (MLO-Y4) and bone tissue. We then demonstrated that FGF7 could increase the expression of Cx43 in osteocytes and promote the cell processes in the form of gap junctions between osteocytes. This modulation was due to the FGF7-induced cytoplasmic accumulation and resultant nuclear translocation of β-catenin. Our results could help us to further understand the importance of FGF7 on bone cell behavior and bone physiology and even pathology.
... The cell-cell communication via gap junction in hBMSCs plays a crucial role in osteogenesis. Cx43 is a major connexin in gap junction communication in order to coordinate the synthesis of new bone [43]. Fig. 8A shows the immunostaining of Cx43 in hBMSCs, in which the cells appeared to have a close connection when cultured on S0-S3 for 10 days. ...
Statement of significance:
The micro/nano hybrid structure has been found to have synergistic bioactivity on osteogenesis. However, it is still a challenge to fabricate the hybrid structure directly on the bioceramics, and the role of micro- and nano-structure, in particular the mechanism of the micro/nano-hybrid structure induced stem cell differentiation is still unknown. In this study, we firstly fabricated hydroxyapatite bioceramics with the micro/nano hybrid structure, and then investigated the effect of different surface structure on expression of integrins, BMP2 signaling pathways and cell-cell communication. Interestingly, we found that the osteogenic differentiation induced by structure was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, and activated BMP2 could in turn activate some integrin subunits and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect.
... Gap junction plays a critical role in the coordinated function and activity of nearly all skeletal cells. Its function in bone physiology has been widely discussed and summarized elsewhere [60,61]. To summarize, gap junctions play a significant role in signal transduction that affects bone modeling and remodeling processes. ...
An emerging concept in intercellular communication in mammals is that communication can be mediated by exchange of genetic material, mainly in the form of RNAs. In this review, we discuss recent studies that describe the trafficking of genetic material with a focus on bone cell communication. Three major carriers are discussed: gap junctions, protein-binding complexes, and genetic material exchange mediated by extracellular vesicles. While protein-level exchange has been well documented, no review has summarized the novel paradigm of cell-to-cell communication by genetic information exchange in bone tissues or its biological relevance in terms of bone homeostasis and bone-related diseases. The purpose of this review is to promote further understanding of this novel discovery regarding bone cell communication and provide references for further investigations.
... Osteoblasts and osteocytes also express Cx45 and Cx46, although the function of these connexins in bone cells is not yet known [23][24][25]. More recently, the expression of Cx37 was demonstrated in osteoclasts, osteoblasts, and osteocytes [26,27], and its ubiquitous deletion leads to decreased bone mass due to defective osteoclast differentiation and to altered bone matrix composition [27,28]. ...
Purpose of review:
To discuss current knowledge on the role of connexins and pannexins in the musculoskeletal system.
Recent findings:
Connexins and pannexins are crucial for the development and maintenance of both bone and skeletal muscle. In bone, the presence of connexin and more recently of pannexin channels in osteoblasts, osteoclasts, and osteocytes has been described and shown to be essential for normal skeletal development and bone adaptation. In skeletal muscles, connexins and pannexins play important roles during development and regeneration through coordinated regulation of metabolic functions via cell-to-cell communication. Further, under pathological conditions, altered expression of these proteins can promote muscle atrophy and degeneration by stimulating inflammasome activity. In this review, we highlight the important roles of connexins and pannexins in the development, maintenance, and regeneration of musculoskeletal tissues, with emphasis on the mechanisms by which these molecules mediate chemical (e.g., ATP and prostaglandin E2) and physical (e.g., mechanical stimulation) stimuli that target the musculoskeletal system and their involvement in the pathophysiological changes in both genetic and acquired diseases.
... 1A, 2A, 4A). It is possible that disruption of the plasma membrane by the transfection reagents disrupts the cell-to-cell communication needed for mineralization (Cheng et al. 2000;Furlan et al. 2001;Stains and Civitelli 2005). Normally decreases in SOST and DKK1 secretion would increase mineralization. ...
Previously, our laboratory identified ECE-1, encoding endothelin-converting enzyme-1 (ECE-1), as a positional candidate for a pleiotropic quantitative trait locus affecting femoral size, shape, and biomechanical performance. We hypothesized that endothelin-1 (ET-1) signaling promotes osteogenesis. Exposure of immortalized mouse osteoblast (TMOb) cells to big ET-1 increased mineralization. Following big ET-1 treatment, we measured the secretion of insulin-like-growth factor-1 (IGF1), dickkopf-homolog-1 protein 1 (DKK1), and sclerostin (SOST). In each case, big ET-1 signaling changed secretion in a manner that favored increased osteogenic activity. Treatment with ECE-1, endothelin receptor A (EDNRA), or WNT receptor antagonists inhibited the big ET-1-mediated increase in mineralization. In the presence of big ET-1, message levels of Runx2, Igf1, Dkk1, and Sost are uncoupled from protein production, suggesting posttranscriptional regulation. To evaluate the role of big ET-1 in normal bone physiology, we inhibited EDNRA signaling during mineralization in the absence of exogenous ET-1. EDNRA blockade reduced mineralization, decreased IGF1, and increased DKK1 and SOST secretion, responses opposite to those induced by exogenous big ET-1. Pharmacological and siRNA knockdown to inhibit ECE-1 reduced mineralization and IGF1 secretion with decreasing DKK1 and decreasing or stable SOST secretion, suggesting a further, unknown role of ECE-1 in osteoblast maturation. Previously we identified miR 126-3p as a potential ET-1-responsive regulator of SOST in murine cells. Overexpression of miR126-3p increased mineralization in TMOb cells and decreased SOST secretion. Osteoblasts express the ET-1 signaling pathway and ET-1 signaling is necessary for normal osteoblast differentiation and mineralization, acting through regulation of miRs that target osteogenic molecules.
... Proteins and nucleic acids cannot pass through these channels. Connexins are widely expressed and to name a few, have been shown to be present in skeletal tissue, corneal epithelium and cardiac tissue [33][34][35]. They are absent in erythrocytes and lymphocytes [36]. ...
Cancer is a dreadful disease for which multitudinous research efforts are being channelized to discover new drugs and treatment strategies.
... Due to the role of osteocytes as primary mechanosensor cells in bone and their extensive communication network, there has long been proposed an important role of Cxs in the response of these cells to mechanical stimulation(Lloyd et al ., 2014;Plotkin et al ., 2013). Several Cxs, including Cx43, Cx45, Cx46 and Cx37, have been characterized in osteoblasts and osteocytes(Stains et al ., 2005;Pacheco-Costa et al ., 2014); although Cx43 has been the best studied so far in osteocytes(Civitelli et al ., 1993;Schirrmacher et al ., 1992;Yellowley et al ., 2000). Fluid flow-induced stress has been shown to trigger the opening of Cx43 hemichannels associated with the anti- ...
Bone is one of the most adaptable tissues in the body as it is continuously subjected to load bearing. In fact, mechanical loading is an important regulator of bone mass. The skeleton adjusts to load by changing its mass, shape and microarchitecture, depending on the magnitude of the strain. Mechanical stimulation is necessary for the development of the skeleton, whereas in adults physiological levels of strain help maintain bone mass by reducing bone resorption. On the other hand, an excessive level of strain or bone disuse induces bone loss. Osteocytes are long-lived cells comprising more than 90% of bone cellularity, which are embedded in the bone matrix forming a functional syncytium extending to the bone surface. These cells are considered to be the main bone cells responsible for translating mechanical strain into regulatory signals for osteoblasts and osteoclasts, leading to adapting bone responses to environmental changes. In this review, we discuss the complexity and well-orchestrated events that occur in bone mechanotransduction, focusing on osteocyte viability as an important biological response in this respect. Elucidation of the molecular mechanisms of bone mechanotransduction and the key role of osteocytes is opening new avenues for the treatment of bone loss-related diseases.
... There are several factors: ionic concentration, second messengers, transjunctional potential, soluble factors, and gap junctional channels induced by protein phosphorylation [80]. GJIC has been implicated in the control of cell proliferation and differentiation in various normal tissues and bone [81], and GJIC dysfunction or mutations of connexin genes have been linked to several human pathological lesions. ...
Objective: The present review describes endothelins (ETs) and endothelin receptors (ETRs) involved in the remodeling of mesenchymal tissue and bone formation, as processes of wound healing and skeletal tissue remodeling. Methods: We reviewed 136 manuscripts and papers, concerning about ET and ETR, how (1) ET and ETR stimulate osteoblastic activities, (2) P⁴²/P⁴⁴ MAP kinase activation is mediated in ET-1-induced IL-6 synthesis, (3) ET induces Ca²⁺ mobilization and the activation of protein kinase C, (4) ET-1 regulates phosphorylation of connexin-43, and connexin-43 is predominant in the differentiation of osteogenic cells, and (5) ET inhibited the mutual process of osteoblasts. Results: Since the discovery of ETs and ETRs, the vascular physiology and biological mechanism of ETs and ETRs have been highlighted and have indicated that ET-1 mediates the proliferation and differentiation of osteoblastic (progenitor) cells. ET-1 binds to ETAR and osteoblasts, osteoclasts, and chondrocytes reactive to ET-1 mRNA, and ET-1 production by these cells are enhanced by BMP-7. ET exhibits a mitogenic function in connective and bone tissues. ET recognizes angiogenesis with VEGF, and cooperative ET may play a role in osteogenesis via matrix formation. Metastatic tumor cells produce ET-1, which stimulates new bone formation; this finding involves osteoblastic tumor metastases, a process that inhibits ETAR antagonists. Understanding the multiple biophysiological functions of the ET axis is beneficial for application of new treatment employing tissue regeneration. Conclusion: ET is one of the potent mediators of bone regeneration and morphogenesis, including skeletal formation, in craniofacial development.
Osteoarthritis (OA) is a progressive degenerative disease that causes severe pain and functional limitation, especially during locomotion. It is the most common arthritis type that damages the surface of articular cartilage until the underlying bone. In the past decade, the scientific community has made a considerable effort to improve or discover therapeutical products used as a form of conservative treatment capable of restoring the damaged articular tissue, avoiding, as far as possible, the use of surgical practices. The most common and direct nonoperative application available for OA treatment is the viscosupplementation (VS) procedure that demonstrates a safe, effective method and is less painful for the patients. The most recent works dealing with the design, development, and validation of viscosupplement products in preclinical and clinical trials for OA treatment are overviewed herein. In general, despite the development of new products, hyaluronic acid continues to be among the most reported intra‐articular viscosupplement products used in clinical trials, typically used as an isolated product or conjugated with other biologicals or drugs, such as platelet‐rich plasma and corticosteroids (CS). However, this issue is still demanding innovation. Approaches comprising new biomaterials as VS products, with intrinsic bioactivity, economical, and environmental friendliness, are required.
Bones play several roles in the human body, providing structure, protecting organs, anchoring muscles, and storing calcium. Therefore, it is fundamental to keep the bone health as close as possible to the optimum. While it is important to build strong and healthy bones during childhood and adolescence, every effort should be taken to protect bone health, during adulthood, too. Bones are continuously changing — new bone is made, and old bone is broken down. At young ages, the body makes new bone faster than it breaks down the old bones, consequently bone mass increases. Most people reach their peak bone mass around age 30. After that, bone remodeling continues, but bones start to lose slightly more bone mass than what they gain. This paves the way for osteoporosis or what is known as “thinning of the bones,” a condition that causes bones to become weak and brittle. This relies on how much bone mass has been attained during the adulthood and how rapidly the body loses it after that. The higher the peak bone mass, the more bone the person would have “in the bank” and the less likely to develop osteoporosis as he/she gets older. Building healthy bones is the only way to reduce the enormous personal and economic costs of osteoporosis — a major cause of pain, disability, and premature death affecting both women and men.
Purposes:
Cell-cell communication is an essential process to respond to biological stimuli and sustain the microenvironmental homeostasis of human periodontal ligament stem cells (hPDLSCs). Connective tissue growth factor (CTGF), a critical secreted matrix protein, exhibits significant tasks in regulating the cell-cell and cell-matrix interactions. This study aimed to explore the relationship between CTGF and cell communication and the underlying mechanism.
Methods:
qRT-PCR was used to detect CCN family, connexin and pannexin family expression in hPDLSCs. Stimulation with CTGF, cell migration assay was performed to examine the wound repair. The scrape loading/dye transfer assay was employed to access lucifer Yellow molecules transfer efficiency mediated by cell-cell communication. Connexin43 (Cx43), Pannexin1 (Panx1), MAPK, and the PI3K/Akt signaling pathway proteins were examined via Western blotting. Immunofluorescence was applied to visualize the localization of specific proteins within cells. Corresponding pathway inhibitors were applied to hPDLSCs to detect Cx43, Panx1 expression and intercellular communication induced by CTGF.
Results:
Our result showed that CTGF was the second most expressed CCN family member in hPDLSCs. Cx43 and Panx1 were the most widely expressed gap junction hemichannels in hPDLSCs. CTGF enhanced hPDLSCs migration in a dose-dependent manner. CTGF promoted cell-cell communication by up-regulating Cx43 and Panx1. CTGF induced Akt, JNK, and p38 phosphorylation and subcellular relocation. Inhibiting corresponding pathways reduced Cx43 expression, thereby weakening CTGF-induced cell-cell communication. However, the Panx1 expression in CTGF-treated hPDLSCs mainly depended on PI3K/Akt signaling.
Conclusion:
We provided novel evidence that CTGF promoted cell-cell communication in hPDLSCs through MAPK and PI3K pathway. This article is protected by copyright. All rights reserved.
Communication processes among the cells of bone are essential for the structure and function of the organ. After it was proposed that communication from the osteoblast lineage to hemopoietic cells initiated osteoclastogenesis, the molecular controls were identified to be the tumour necrosis factor ligand and receptor families. This was followed by revelation of very many signalling processes among the cells of bone that regulate the three phases of bone remodelling, the resorption, reversal and formation phases. In many instances the ways in which these mechanisms operate can determine how drugs act on bone, whether they be inhibitors of resorption or promoters of formation.
Osteoblasts are the only cells that can give rise to bones in vertebrates. Thus, one of the most important functions of these metabolically active cells is mineralized matrix production. As osteoblasts have a limited lifespan, they must be constantly replenished by pre-osteoblasts, their immediate precursors. As disruption of the regulation of bone-forming osteoblasts results in a variety of bone diseases, a better understanding of the origin of these cells by defining the mechanisms of bone development, remodeling, regeneration is central to the development of novel therapeutic approaches. In recent years, substantial new insights into the origin of osteoblasts - largely owing to rapid technological advances in murine lineage-tracing approaches and other single-cell technologies - have been obtained. Collectively, these findings indicate that osteoblasts involved in bone formation under various physiological, pathological, and therapeutic conditions can be obtained from numerous sources. The origins of osteoblasts include - but are not limited to - chondrocytes in the growth plate, stromal cells in the bone marrow, quiescent bone-lining cells on the bone surface, and specialized fibroblasts in the craniofacial structures, such as sutures and periodontal ligaments. As osteoblasts can be generated from local cellular sources, bones can flexibly respond to regenerative and anabolic cues. However, whether osteoblasts derived from different cellular sources have distinct functions remains to be investigated. Currently, we are at the initial stage to aptly unravel the incredible diversity of the origins of bone-forming osteoblasts.
During natural tissue regeneration, tissue microenvironment and stem cell niche including cell–cell interaction, soluble factors, and extracellular matrix (ECM) provide a train of biochemical and biophysical cues for modulation of cell behaviors and tissue functions. Design of functional biomaterials to mimic the tissue/cell microenvironment have great potentials for tissue regeneration applications. Recently, electroactive biomaterials have drawn increasing attentions not only as scaffolds for cell adhesion and structural support, but also as modulators to regulate cell/tissue behaviors and function, especially for electrically excitable cells and tissues. More importantly, electrostimulation can further modulate a myriad of biological processes, from cell cycle, migration, proliferation and differentiation to neural conduction, muscle contraction, embryogenesis, and tissue regeneration. In this review, endogenous bioelectricity and piezoelectricity are introduced. Then, design rationale of electroactive biomaterials is discussed for imitating dynamic cell microenvironment, as well as their mediated electrostimulation and the applying pathways. Recent advances in electroactive biomaterials are systematically overviewed for modulation of stem cell fate and tissue regeneration, mainly including nerve regeneration, bone tissue engineering, and cardiac tissue engineering. Finally, the significance for simulating the native tissue microenvironment is emphasized and the open challenges and future perspectives of electroactive biomaterials are concluded. This review provides a comprehensive understanding of the endogenous bioelectricity and piezoelectricity, as well as recent progress in the study of electroactive biomaterials and systems, and their mediated electrostimulation in tissue regeneration.
The facts that most tissues or organs consist of a variety of cells suggest that interactions between different types of cells play critical roles in tissue or organ development. In tissue engineering, the effects of biomaterials on cell-cell interactions have recently attracted increasing attention for better elucidating the mechanisms through which biomaterials promote tissue regeneration. Numerous studies have focused on these effects of biomaterials on cell-cell interactions. In this review, comprehensive information was provided about the existing cell co-culture technologies and the main behavioral modes of cell-cell interactions. The effects of biomaterials on the cell-cell interactions in various types of tissue regeneration have been summarized and discussed. In the end, the existing problems and future perspectives that would help promote the research of biomaterials in tissue engineering have been proposed. This article can help researchers to understand the progress and importance of studying the effects of biomaterials on cell-cell interactions in tissue engineering and to choose the optimal cell-cell co-culture models for designing experiments.
Gap junctions mediate cellular communication and homeostasis by controlling the intercellular exchange of small and hydrophilic molecules and ions. Gap junction channels are formed by the docking of 2 hemichannels of adjacent cells, which in turn are composed of 6 connexin subunits. Connexin proteins as such can also control the cellular life cycle independent of their channel activities. This has been most demonstrated in the context of cell growth and cell death. Different mechanisms are involved mainly related to direct interaction with cell growth or cell death regulators, but also implying effects on the expression of cell growth and cell death regulators. The present paper focuses on these atypical roles of connexin proteins.
Many cancers commonly metastasize to bone. After entering the bone, cancer cells can interact with surrounding stromal cells, which ultimately influences metastasis progression. Extracellular vesicles, direct cell contact and gap junctions, and cytokines are all mechanisms of intercellular communication that have been observed to occur in the bone microenvironment. These methods of cellular crosstalk can occur between cancer cells and a variety of stromal cells, with each interaction having a different impact on cancer progression. Communication between cancer cells and bone-resident cells has previously been implicated in processes such as cancer cell trafficking and arrest in bone, cancer cell dormancy, cancer cell reactivation, and proliferation. In this chapter we review innovative techniques and model systems that can be used to study bidirectional crosstalk between cancer cells and stromal cells in the bone, with an emphasis specifically on bone-metastatic breast cancer. Investigating how metastatic cancer cells interact with, and are influenced by, the bone microenvironment is crucial to better understanding of the progression of bone metastasis.
Objective:
To explore the effect of connexin 43 (Cx43) silence on the apoptosis in mouse chondrocyte under mechanical stress.
Methods: Mouse chondrocyte ATDC5 cells were divided into a control group, a mechanical stress group, a Cx43 siRNA transfection group, a scramble siRNA transfection group, a mechanical stress+scramble group, and a mechanical stress+siCx43 group. Flexcell FX-5000 system was used to produce mechanical stress on ATDC5 cells cultured in vitro. The mRNA and protein level of Cx43 was detected by quantitative RT-PCR (RT-qPCR) and Western blot. The cell activity and cell apoptosis was detected by cell counting kit-8 (CCK-8) method and flow cytometry, respectively. Caspase-3 activity was detected by colorimetric assay. The protein expression of Bcl-2, Bax, p-JNK and JNK was detected by Western blot.
Results: Mechanical stress upregulated the mRNA and protein expression of Cx43 (both P<0.05). Transfection of Cx43 siRNA significantly decreased Cx43 mRNA and protein level (both P<0.05). After stimulation with mechanical stress, chondrocyte viability was significantly decreased, whereas cell apoptosis and caspase-3 activity were increased (both P<0.05). Mechanical stress obviously upregulated Bax protein level, and downregulated Bcl-2 protein expression and Bcl-2/Bax (both P<0.05). Cx43 siRNA transfection significantly increased cell viability, inhibited cell apoptosis and caspase-3 activity (both P<0.05). Cx43 siRNA also inhibited Bax expression, and increased the Bcl-2 protein expression and Bcl-2/Bax (both P<0.05). Furthermore, Cx43 siRNA significantly suppressed the p-JNK expression induced by mechanical stress (P<0.05).
Conclusion: Cx43 silence inhibits mechanical stress-induced apoptosis in chondrocyte, which might be mediated by JNK signaling pathway.
The circulatory system in long bones is incompletely understood. A new study published in Nature Metabolism unveils the presence of dense vascular networks in long bones that facilitate the egress of bone marrow cells and potentially the exchange of nutrients between the bone marrow and the systemic circulation.
The connexin family of channel-forming proteins is present in every tissue type in the human anatomy. Connexins are best known for forming clustered intercellular channels, structurally known as gap junctions, where they serve to exchange members of the metabolome between adjacent cells. In their single-membrane hemichannel form, connexins can act as conduits for the passage of small molecules in autocrine and paracrine signalling. Here, we review the roles of connexins in health and disease, focusing on the potential of connexins as therapeutic targets in acquired and inherited diseases as well as wound repair, while highlighting the associated clinical challenges.
A wide range of techniques and methods are actively invented by clinicians and scientists who are dedicated to the field of musculoskeletal tissue regeneration. Biological, chemical, and physiological factors, which play key roles in musculoskeletal tissue development, have been extensively explored. However, physical stimulation is increasingly showing extreme importance in the processes of osteogenic and chondrogenic differentiation, proliferation and maturation through defined dose parameters including mode, frequency, magnitude, and duration of stimuli. Studies have shown manipulation of physical microenvironment is an indispensable strategy for the repair and regeneration of bone and cartilage, and biophysical cues could profoundly promote their regeneration. In this article, we review recent literature on utilization of physical stimulation, such as mechanical forces (cyclic strain, fluid shear stress, etc.), electrical and magnetic fields, ultrasound, shock waves, substrate stimuli, etc., to promote the repair and regeneration of bone and cartilage tissue. Emphasis is placed on the mechanism of cellular response and the potential clinical usage of these stimulations for bone and cartilage regeneration.
Connexons form the basis of hemichannels and gap junctions. They are composed of six tetraspan proteins called connexins. Connexons can function as individual hemichannels, releasing cytosolic factors (such as ATP) into the pericellular environment. Alternatively, two hemichannel connexons from neighbouring cells can come together to form gap junctions, membrane-spanning channels that facilitate cell-cell communication by enabling signalling molecules of approximately 1 kDa to pass from one cell to an adjacent cell. Connexins are expressed in joint tissues including bone, cartilage, skeletal muscle and the synovium. Indicative of their importance as gap junction components, connexins are also known as gap junction proteins, but individual connexin proteins are gaining recognition for their channel-independent roles, which include scaffolding and signalling functions. Considerable evidence indicates that connexons contribute to the function of bone and muscle, but less is known about the function of connexons in other joint tissues. However, the implication that connexins and gap junctional channels might be involved in joint disease, including age-related bone loss, osteoarthritis and rheumatoid arthritis, emphasizes the need for further research into these areas and highlights the therapeutic potential of connexins.
In diabetic retinopathy, high glucose (HG)-mediated breakdown in cell-cell communication promotes disruption of retinal homeostasis. Several studies indicate that HG condition alters expression of connexin genes and subsequent gap junction intercellular communication (GJIC) in retinal vascular cells and non-vascular cells. A serious consequence of disrupted cell-cell communication is apoptosis and breakdown of the blood-retinal barrier (BRB). More recently, studies suggest adverse effects from HG on retinal Müller cells. This article focuses on HG-mediated changes in connexin expression and GJIC and their subsequent effects on the breakdown of retinal homeostasis, cell death, compromised vascular permeability, and interactions between endothelial cells, pericytes and retinal Müller cells in the pathogenesis of diabetic retinopathy. Additionally, options for rectifying disrupted homeostasis under HG condition associated with diabetic retinopathy are reviewed.
Statement of significance:
Tissue engineering can regenerate or replace tissue or organs through combining cells, biomaterials and growth factors. Normally, for repairing a specific tissue, only one type of cells, one kind of biomaterials, and specific growth factors are used to support cell growth. In this study, we proposed a novel tissue engineering approach by simply using co-cultured cells and combined biomaterial signals. Using a skin tissue engineering model, we successfully proved that the combined biomaterial signals such as surface nanostructures and bioactive ions could synergistically stimulate the cell-cell communication in co-culture system through paracrine effects and gap junction activation, and regulated expression of growth factors and extracellular matrix proteins, resulting in an activated tissue engineering constructs that significantly enhanced skin regeneration.
It has been widely reported that chemical, structural or mechanical signals of biomaterials can impact cell behaviors and tissue regeneration, but few studies have investigated the effects of biomaterial signals on cell–cell interactions although communications between cells are critical for tissue regeneration. Our recent studies have shown that chemical signals of bioglass (BG) can stimulate communications in bone marrow stromal cells and endothelial cells, which results in enhanced angiogenesis and osteogenesis. Considering the facts that, in vivo, different biomaterial signals may simultaneously affect cell–cell interactions, in this study, we proposed that combining chemical and structural signals of biomaterials may further improve cell–cell interactions. Results proved that combined structural signals of aligned electrospun nanofibers and chemical signals of BG ionic products could significantly stimulate interactions between co-cultured bone marrow stromal cells and endothelial cells through both of paracrine effects and junctional communications as compared to single type of biomaterial signals. Further study indicated that both chemical signals of BG and structural signals of electrospun nanofibers played important role in stimulating paracrine effects while for improving junctional communication, structural signals of electrospun nanofibers played a more important role than chemical signals of BG, which resulted in enhanced vascularization and osteogenic differentiation in co-cultures. Therefore, applying combined biomaterial signals to activate cell–cell interactions is a promising strategy for enhancing tissue regeneration.
The effects of extracellular calcium on osteogenic differentiation capacity of human bone-derived mesenchymal stromal cells with special regard to connexin 43 (cx43) have been investigated by means of cell culture experiments. Mesenchymal stromal cells isolated from human cancellous bone were cultured on tissue culture plates at different calcium ion (Ca²⁺) concentrations (1.8 mmol l⁻¹, 10 mmol l⁻¹, 20 mmol l⁻¹). Cell responses were evaluated by quantitative RT-PCR, immunofluorescence staining, and Lucifer Yellow fluorescence uptake experiments. It could be shown that increasing Ca²⁺ concentrations correlate with increasing cx43 and bone sialoprotein mRNA levels as well as with enhanced cx43 fluorescence signaling and matrix mineralization of the cultures as shown by von Kossa staining. Hemichannel gating − assessed by Lucifer Yellow uptake − increases with increasing extracellular Ca²⁺ concentrations suggesting that regulatory effects at the hemichannel level are calcium-dependent.
Background
Bone remodelling is dependent on the balance between bone resorbing osteoclasts and bone forming osteoblasts. We have shown previously that osteoclasts contain gap-junctional protein connexin-43 and that a commonly used gap-junctional inhibitor, heptanol, can inhibit osteoclastic bone resorption. Since heptanol may also have some unspecific effect unrelated to gap-junctional inhibition we wanted to test the importance of gap-junctional communication to osteoclasts using a more specific inhibitor.
Methods
A synthetic connexin-mimetic peptide, Gap 27, was used to evaluate the contribution of gap-junctional communication to osteoclastic bone resorption. We utilised the well-characterised pit-formation assay to study the effects of the specific gap-junctional inhibitor to the survival and activity of osteoclasts.
Results
Gap 27 caused a remarked decrease in the number of both TRAP-positive mononuclear and multinucleated rat osteoclasts cultured on bovine bone slices. The decrease in the cell survival seemed to be restricted to TRAP-positive cells, whereas the other cells of the culture model seemed unaffected. The activity of the remaining osteoclasts was found to be diminished by measuring the percentage of osteoclasts with actin rings of all TRAP-positive cells. In addition, the resorbed area in the treated cultures was greatly diminished.
Conclusions
On the basis of these results we conclude that gap-junctional communication is essential for the action of bone resorbing osteoclasts and for proper remodelling for bone.
The distribution, expression, and functionality of gap junctions was examined in bovine chondrocytes (BCs) isolated from mature articular cartilage. BC cells displayed immunoreactivity for connexin 43 (Cx43), a specific gap junction protein. Cx43 protein expression was confirmed by Western blot analysis, and Cx43 mRNA was detected by nuclease protection assay. Additionally, BCs were shown to be functionally coupled, as revealed by dye transfer studies, and octanol, a gap junction uncoupler, greatly attenuated coupling. Furthermore, confocal microscopy of fluo-3 loaded BC cells revealed that deformation-induced cytosolic Ca2+ ion (Ca2+) signals propagated from cell-to-cell via gap junctions. To our knowledge, this is the first evidence suggesting that chondrocytes isolated from adult articular cartilage express functional gap junctions.
Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.
Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43
(Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos
to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result
of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart.
This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among
connexins in other parts of the developing fetus.
Gap junctions which comprise a family of proteins called connexins have been implicated in the morphogenesis of the chick limb bud. We have examined the expression patterns of two members of the connexin family, connexin43 (Cx43) and connexin42 (Cx42), during the early development of the chick limb bud and embryo by in situ hybridization. Cx43 mRNA is expressed in high amounts in the apical ectodermal ridge (AER), which promotes the outgrowth of the mesodermal cells of the limb bud, and in the ectopic AER of the limb buds of polydactylous diplopodia-5 mutant embryos. In contrast, little Cx43 expression is detectable in nonridge limb ectoderm at early stages of limb development. These results suggest that Cx43 gap junctions may integrate the activity of the cells comprising the AER and compartmentalize them into a functionally distinct entity capable of directing limb outgrowth. In addition, Cx43 exhibits high expression in the posterior subridge mesoderm of the early limb bud that is growing out in response to the AER, but little expression in the anterior mesoderm. This graded distribution of Cx43 transcripts correlates with a functional gradient of gap junctional communication along the anteroposterior (AP) axis, and suggests that Cx43 gap junctions may be involved in pattern formation across the AP axis. At later stages of development, Cx43 is transiently expressed in high amounts in the precartilage condensations of the carpals and metacarpals, at a time when critical cell-cell interactions are occurring that trigger cartilage differentiation. In contrast, in the developing limb, Cx42 is expressed exclusively by the central artery. In the remainder of the chick embryo, Cx42 is expressed in high amounts by the vessels comprising the arterial vasculature, but is not expressed by the venous vasculature. Thus, Cx42 gap junctions may be involved in specification of the arterial vasculature of the limb and embryo. Cx42, but not Cx43, is expressed in the ventricle of the heart, and by cells along the intrasclerotomal fissure that separates the rostral and caudal halves of the sclerotome of somites into distinct communication compartments.
We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.
Polyclonal antisera to synthetic peptides matching sequences on the cytoplasmic regions of connexin-43, a gap junction protein first identified in rat heart, have been used to immunolabel gap junctions in the calvarial bone, maintained intact as in vivo, of 1- to 2-week-old rats. The specimens were examined in reflection and fluorescence modes by scanning laser confocal microscopy, and the numbers of gap junctions and their sizes estimated. The mean number of connexin-43 immunolabelled junctions per osteoblast (n = 65) was 15.3 (SD +/- 4.5). The mean length of 227 junctions, selected for the sharpness of the image of the fluorescent spot, was 0.67 micron (SD +/- 0.18; range 0.37-1.29 microns) and their mean area 0.26 micron2 (SD +/- 0.145; range 0.075-0.93 micron2); these probably fell within the upper half of the total size range. Gap junctions were detected between preosteoblasts, osteoblasts, osteocytes and chondrocytes, and between these juxtaposed cell types. In addition, connexin-43 immunolabelled junctions were found between some osteoclasts and overlying mononuclear cells at active sites of resorption.
To assess whether connexin (Cx) expression contributes to insulin secretion, we have investigated normal and tumoral insulin-producing cells for connexins, gap junctions, and coupling. We have found that the glucose-sensitive cells of pancreatic islets and of a rat insulinoma are functionally coupled by gap junctions made of Cx43. In contrast, cells of several lines secreting insulin abnormally do not express Cx43, gap junctions, and coupling. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta-cells, showed an expression of the insulin gene and an insulin content that were markedly elevated, compared with those observed in both wild-type (uncoupled) cells and in transfected cells overexpressing Cx43. These findings indicate that adequate levels of Cx-mediated coupling are required for proper insulin production and storage.
Gap junction channels are formed by paired oligomeric membrane hemichannels called connexons, which are composed of proteins of the connexin family. Experiments with transfected cell lines and paired Xenopus oocytes have demonstrated that heterotypic intercellular channels which are formed by two connexons, each composed of a different connexin, can selectively occur. Studies by Stauffer [Stauffer, K. A. (1995) J. Biol. Chem. 270, 6768-6772] have shown that recombinant Cx26 and Cx32 coinfected into insect cells may form heteromeric connexons. By solubilizing and subfractionating individual connexons from ovine lenses, we show by immunoprecipitation that connexons can contain two different connexins forming heteromeric assemblies in vivo.
To evaluate whether intercellular coupling via connexin43 gap junction channels modulates hormonal responsiveness of cells in contact, we have created osteoblastic cell lines deficient in connexin43. Osteoblastic ROS 17/2.8 cells were transfected with a plasmid containing an antisense cDNA construct to rat connexin43. Control transfection did not alter cell-to-cell coupling nor connexin43 mRNA or protein expression relative to nontransfected ROS 17/2.8 cells. In contrast, stable transfection with an antisense connexin43 cDNA resulted in two clones, RCx4 and RCx16, which displayed significant decreases in connexin43 mRNA and protein expression and were dramatically deficient in cell-to-cell coupling. Phenotypically, all transfectants retained osteoblastic characteristics. However, cells rendered connexin43-deficient through antisense transfection displayed a dramatic attenuation in the cAMP response to parathyroid hormone. Alterations in hormonal responses were not due to changes in parathyroid hormone receptor number or binding kinetics nor to alterations in adenylyl cyclase activity. These results indicate that gap junctions may be required for mediating hormonal signals. Furthermore, these experiments support a regulatory role for connexin43-mediated intercellular communication in the modulation of hormonal responses within elaborately networked bone cells.
Holt-Oram syndrome is characterized by upper limb malformations and cardiac septation defects. Here, we demonstrate that mutations in the human TBX5 gene underlie this disorder. TBX5 was cloned from the disease locus on human chromosome 12q24.1 and identified as a member of the T-box transcription factor family. A nonsense mutation in TBX5 causes Holt-Oram syndrome in affected members of one family; a TBX5 missense mutation was identified in affected members of another. We conclude that TBX5 is critical for limb and heart development and suggest that haploinsufficiency of TBX5 causes Holt-Oram syndrome.
Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.
Reduction of gap junctional communication in v-src transformed cells is accompanied by tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). Cx43 is phosphorylated
on tyrosine by v-Src. The Src homology 3 (SH3) and Src homology 2 (SH2) domains of v-Src mediate interactions with substrate
proteins. SH3 domains interact with proline-rich peptide motifs. SH2 domains associate with short amino acid sequences containing
phosphotyrosine. We present evidence that the SH3 and SH2 domains of v-Src bind to proline-rich motifs and a phosphorylated
tyrosine residue in the C-terminal tail of Cx43. Cx43 bound to the SH3 domain of v-Src, but not c-Src, in vitro. Tyrosine-phosphorylated Cx43 bound to the SH2 domain of v-Src in vitro. v-Src coprecipitated with Cx43 from v-src-transformed Rat-1 fibroblasts. Mutations in the SH3 and SH2 domains of v-Src, and in the proline-rich region or tyrosine
265 of Cx43, reduced interactions between v-Src and Cx43 in vivo. Tyrosine phosphorylation of Cx43 was dependent on the association of v-Src and Cx43. These results provide further evidence
for the direct involvement of v-Src in tyrosine phosphorylation of Cx43 and inhibition of gap junctional communication in
v-src-transformed cells.
Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected.
Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.
Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of negatively charged dyes between UMR 106-01 cells. This stimulatory effect lasted for at least 4 h. Both PGE2 and PTH increased steady-state levels of Cx43 mRNA, but only after 2-4 h of incubation. Transfection with a Cx43 gene construct linked to luciferase showed that this effect of PTH was the result of transcriptional upregulation of Cx43 promoter. Stimulation of dye coupling and Cx43 gene transcription were reproduced by forskolin and 8Br-cAMP. Exposure to PGE2 for 30 min increased Cx43 abundance at appositional membranes in UMR 106-01, whereas total Cx43 protein levels increased only after 4-6 h of incubation with either PGE2 or PTH. Inhibition of protein synthesis by cycloheximide did not affect this early stimulation of dye coupling, but it significantly inhibited the sustained effect of PTH and forskolin on cell coupling. In summary, both PTH and PGE2, presumably through cAMP production, enhance gap junctional communication in osteoblastic cell cultures via two mechanisms: initial rapid redistribution of Cx43 to the cell membrane, and later stimulation of Cx43 gene expression. Modulation of intercellular communication represents a novel mechanism by which osteotropic factors regulate the activity of bone forming cells.
To further characterize how gap junction-dependent Ca2+ waves propagate between sheep lens cells, we examined the possible roles of inositol 1,4,5-trisphosphate (IP3), Ca2+ and cyclic ADP-ribose (cADPR) in mediating intercellular Ca2+ waves. Second messengers were microinjected into a single cell in a monolayer of sheep lens cells while monitoring cytosolic Ca2+ with fura-2 and fluorescence microscopy. All three compounds initiated intercellular Ca2+ waves, but more cells responded following the injection of either IP3 or cADPR than responded following the injection of Ca2+. When either IP3 or cADPR was co-injected with the Ca2+ chelator EGTA, cytosolic Ca2+ in the injected cell decreased but cytosolic Ca2+ in the adjacent cells increased, indicating that the intercellular messenger was IP3 or cADPR, rather than Ca2+. The phospholipase C inhibitor U73122 eliminated mechanically initiated intercellular Ca2+ waves, indicating that mechanical initiation probably requires IP3 production. In U73122-treated cells, injected IP3 initiated an intercellular Ca2+ wave in which the number of cells responding increased as the amount of IP3 injected increased, indicating that the distance traveled by the Ca2+ wave was dependent on cell-to-cell diffusion of IP3. In contrast, the ability of cADPR both to increase cytosolic Ca2+ in the injected cell and to initiate intercellular Ca2+ waves was greatly attenuated by U73122. In conclusion, Ca2+, IP3 and cADPR can all mediate intercellular Ca2+ waves by passing through gap junction channels, but both IP3 and cADPR are more effective intercellular messengers than Ca2+.
Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.
Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in alpha1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for alpha1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing alpha1 connexins, and from alpha1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative alpha1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and alpha1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous alpha1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and alpha1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the alpha1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium- an elevation in the CMV43 mice vs. a reduction in the alpha1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non-neural crest- derived tissues. Overall, these findings suggest that gap junction communication mediated by alpha1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of alpha1 connexin function.
Nature Genetics 15, 30–35 (1997). The name of the gene TBX5 was unintentionally left out of the title of this report. We regret any difficulty this caused.
To assess whether connexin (Cx) expression contributes to insulin secretion, we have investigated normal and tumoral insulin-producing cells for connexins, gap junctions, and coupling. We have found that the glucose-sensitive cells of pancreatic islets and of a rat insulinoma are functionally coupled by gap junctions made of Cx43. In contrast, cells of several lines secreting insulin abnormally do not express Cx43, gap junctions, and coupling. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta-cells, showed an expression of the insulin gene and an insulin content that were markedly elevated, compared with those observed in both wild-type (uncoupled) cells and in transfected cells overexpressing Cx43. These findings indicate that adequate levels of Cx-mediated coupling are required for proper insulin production and storage.
Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of negatively charged dyes between UMR 106-01 cells. This stimulatory effect lasted for at least 4 h. Both PGE2 and PTH increased steady-state levels of Cx43 mRNA, but only after 2–4 h of incubation. Transfection with a Cx43 gene construct linked to luciferase showed that this effect of PTH was the result of transcriptional upregulation of Cx43 promoter. Stimulation of dye coupling and Cx43 gene transcription were reproduced by forskolin and 8Br-cAMP. Exposure to PGE2 for 30 min increased Cx43 abundance at appositional membranes in UMR 106-01, whereas total Cx43 protein levels increased only after 4–6 h of incubation with either PGE2 or PTH. Inhibition of protein synthesis by cycloheximide did not affect this early stimulation of dye coupling, but it significantly inhibited the sustained effect of PTH and forskolin on cell coupling. In summary, both PTH and PGE2, presumably through cAMP production, enhance gap junctional communication in osteoblastic cell cultures via two mechanisms: initial rapid redistribution of Cx43 to the cell membrane, and later stimulation of Cx43 gene expression. Modulation of intercellular communication represents a novel mechanism by which osteotropic factors regulate the activity of bone forming cells. J. Cell. Biochem. 68:8–21, 1998. © 1998 Wiley-Liss, Inc.
Effective bone remodeling requires the coordination of bone matrix deposition by osteoblastic cells, which may occur via soluble mediators or via direct intercellular communication. We have previously identified two mechanisms by which rat osteoblastic cell lines coordinate calcium signaling among cells: autocrine activation of P2 (purinergic) receptors leading to release of intracellular calcium stores, and gap junction-mediated communication resulting in influx of extracellular calcium. In the current work we asked whether human osteoblastic cells (HOB) were capable of mechanically induced intercellular calcium signaling, and if so, by which mechanisms. Upon mechanical stimulation, human osteoblasts propagated fast intercellular calcium waves, which required activation of P2 receptors and release of intracellular calcium stores but did not require calcium influx or gap junctional communication. After the fast intercellular calcium waves were blocked, we observed slower calcium waves that were dependent on gap junctional communication and influx of extracellular calcium. These results show that human osteoblastic cells can propagate calcium signals from cell to cell by two markedly different mechanisms and suggest that these two pathways may serve different purposes in coordinating osteoblast functions.
Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co-ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx-1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face. © 2001 Wiley-Liss, Inc.
Gap junctional communication has been implicated in numerous cellular processes. However, the repertoire of specific transjunctional substances which mediate these processes remains relatively unexplored. A few selected secondary messengers have been identified, at least indirectly (e.g., cAMP and IP3) and phenotypic complementation experiments have indicated that gap junctions enable communicating cells to distribute nucleotide pools as a shared resource. The latter would include high energy compounds such as ADP and ATP, allowing cells to share energy resources. We have utilized a nonbiased process to directly capture, identify, and quantify transjunctional compounds from C6 glioma cells, the transformed phenotype of which has been ameliorated by transfection with connexin43 (Cx43). This technique involves the direct isolation, identification, and quantitation of radioactive transjunctional molecules that travel from metabolically labeled "donor" cells to "receiver" cells. This report demonstrates that ADP and/or ATP represents over 6% of the transjunctional material derived from glucose in Cx43-transfected C6 glioma cells. Furthermore, equilibration of these high energy metabolites among first order neighbors is shown to occur in less than 20 min of communication.
Osteoblasts were investigated by two methods, electrical conductance and dye injection. Current injection into one cell caused a change in the recorded transmembrane potential of a second cell, indicating high conductance pathways between the two cells. Dyes injected into a single osteoblast were transmitted to numerous surrounding cells.
Osteoblasts in the diaphysis of the tibia during endochondral ossification in young rats are attached to one another by nexus, by "adhaerens" junctions, and by simple appositions. "Adhaerens" junctions and nexus also occur between preosteoblasts and osteoblasts. Furthermore, to osteoblasts exhibit a network of microfilament bundles in the cell periphery overlying the osteoid. From this network bundles extend into the cell processes which protrude into the unmineralized matrix. The mean diameter of individual microfilaments is 5.9 +/- 0.06 nm. A possible role of nexus and microfilaments in controlled bone growth and differentiation is discussed.
The presence of gap junctions between osteoblastic cells has been previously reported. For this study we used the rat osteosarcoma cell line UMR 106, which expresses the osteoblastic phenotype, as a model to characterize further the nature, physiology, and regulation of gap junctions. Northern blot analysis identified a 3.0-kilobase RNA species corresponding to the gap junction protein connexin 43. The presence of two other connexin RNA species (26 and 32) could not be detected by this method in these cells. The identified connexin RNA was amplified by reverse transcription coupled to polymerase chain reaction; the sequence of the amplified product appears identical to the sequence of a cloned rat heart connexin 43 gene. After treatment with PTH, forskolin, and 8-Br-cAMP (a cAMP analog), the levels of connexin 43 RNA in UMR 106 cells increased. Further evidence for the role of PTH and cAMP in the physiology of gap junctions in these cells was obtained with Lucifer yellow dye transfer experiments. Gap-junctional intercellular communication increased in response to PTH and forskolin (an inducer of adenylate cyclase activity). Expression of connexin 43 RNA increased severalfold in response to PTH in a concentration- and time-dependent fashion. Connexin 43 RNA and its PTH-mediated stimulation were also observed in several other osteoblastic cell lines. The roles of PTH and forskolin in regulating the physiological state of gap junctions were confirmed in primary cultures of rat calvaria osteoblasts.
Two types of calcium (Ca2+) signaling-propagating intercellular Ca2+ waves of increasing intracellular Ca2+ concentration ([Ca2+]i) and nonpropagating oscillations in [Ca2+]i-co-exist in a variety of cell types. To investigate this difference in Ca2+ signaling, airway epithelial cells were loaded with heparin, an inositol 1,4,5-triphosphate (IP3) receptor antagonist, by pulsed, high-frequency electroporation. Heparin inhibited propagation of intercellular Ca2+ waves but not oscillations of [Ca2+]i. In heparin-free cells, Ca2+ waves propagated through cells displaying [Ca2+]i oscillations. Depletion of intracellular Ca2+ pools with the Ca2+-pump inhibitor thapsigargin also inhibited the propagation of Ca2+ waves. These studies demonstrate that the release of Ca2+ by IP3 is necessary for the propagation of intercellular Ca2+ waves and suggest that IP3 moves through gap junctions to communicate intercellular Ca2+ waves.
A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20/21) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities.
The ability of articular cartilage to undergo reversible deformation is dependent upon the organization of specific macromolecules within the extracellular matrix. This abundant matrix is elaborated by a small number of chondrocytes which maintain homeostasis via a synchronized balance between anabolism and catabolism. Type II collagen together with smaller amounts of other collagens form the fibrous network of the tissue in which are "entrapped" the aggregating proteoglycans in an underhydrated form. Small amounts of nonaggregating dermatan sulfate containing proteoglycans and matrix proteins are also present in the matrix but their function is less well understood.
Ultrastructural studies were carried out on intercellular relationships during osteocyte differentiation in primary parallel-fibred bone of newborn rabbit. It was found that throughout the whole differentiative process preosteocytes are in close relationship with the neighboring cells (osteoblasts, osteocytes) by means of variously-shaped intercellular contacts (invaginated finger-like, side-to-side, and end-to-end) and two types of specialized junctions: gap and adherens. The pivotal role played by these contacts and junctions in osteocyte differentiation and activity is considered in the context of their particular functional significance.
Intercellular communication of epithelial cells was examined by measuring changes in intracellular calcium concentration ([Ca2+]i). Mechanical stimulation of respiratory tract ciliated cells in culture induced a wave of increasing Ca2+ that spread, cell by cell, from the stimulated cell to neighboring cells. The communication of these Ca2+ waves between cells was restricted or blocked by halothane, an anesthetic known to uncouple cells. In the absence of extracellular Ca2+, the mechanically stimulated cell showed no change or a decrease in [Ca2+]i, whereas [Ca2+]i increased in neighboring cells. Iontophoretic injection of inositol 1,4,5-trisphosphate (IP3) evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. These results support the hypothesis that IP3 acts as a cellular messenger that mediates communication through gap junctions between ciliated epithelial cells.
Mounting experimental evidence indicates that osteoblasts may be cellular intermediaries in the local activation of bone remodeling. To elucidate the role of these cells in activation, we examined the effects of prostaglandins (PGs), known resorption stimulators, on cell shape and intercellular junctional relationships in osteoblasts cultured from rat fetal calvaria. Exposure to PGE2 and PGE1, promoters of bone resorption, rapidly (within 20 min) converted the osteoblasts from a flattened to a stellate shape (shape change), and markedly increased the appearance of intercellular (gap) junctions within 10 min. Both effects were directly related to the prostaglandin concentration, as little as 1 nM being effective. PGE1, but not PGB1, PGF1 alpha, PGD2, and PGF2 alpha, mimicked the substantial effect of PGE2 on shape change. Shape change and gap junction formation appear to arise independently. PTH, an inducer of shape change, did not affect the number of gap junctions appreciably. Colchicine, a microtubule polymerization inhibitor, and trifluoperazine, an inhibitor of calmodulin action, blunted PGE2-mediated shape change but not the effect of PGE2 on gap junctions. Shape change and gap junction formation may be important events in local activation, shape changes in surface osteoblasts serving to expose bone surfaces which are chemotactic for osteoclasts and gap junctions propagating locally initiated activation messages.
Cell membrane specializations occur at contact sites between adjacent osteoblasts and osteoblasts and osteocytes. These junctions have been described by other investigators as being important in preventing the extracellular movement of material around bone cells. Previously we described how certain small proteins circumvented the osteoblast population and rapidly penetrated the canalicular-osteocyte system. In the present study we used lanthanum colloid as an extracellular marker, the lanthanum readily penetrated the bone cell junctions and the extracellular space of bone. Morphologically, these junctions were not "tight" or "occluding" structures, but resembled "gap" junctions. These gap junctions contained elements which formed intercellular bridges between adjacent cells but also maintained a 2 nm space between cells that contained extracellular fluid. These gap junctions may have an important function in the control or coordination of bone cell activity throughout a given volume of bone.
The Holt-Oram syndrome is an autosomal dominant condition characterized by skeletal abnormalities that are frequently accompanied by congenital cardiac defects. The cause of these disparate clinical features is unknown. To identify the chromosomal location of the Holt-Oram syndrome gene, we performed clinical and genetic studies.
Two large families with the Holt-Oram syndrome were evaluated by radiography of the hands, electrocardiography, and transthoracic echocardiography. Genetic-linkage analyses were performed with polymorphic DNA loci dispersed throughout the genome to identify a locus that was inherited with the Holt-Oram syndrome in family members.
A total of 19 members of Family A had Holt-Oram syndrome with mild-to-moderate skeletal deformities, including triphalangeal thumbs and carpal-bone dysmorphism. All affected members of Family A had moderate-to-severe congenital cardiac abnormalities, such as ventricular or atrial septal defects or atrioventricular-canal defects. Eighteen members of a second kindred (Family B) had Holt-Oram syndrome with moderate-to-severe skeletal deformities, including phocomelia. Twelve of the affected members had no cardiac defects; six had only atrial septal defects. Genetic analyses demonstrated linkage of the disease in each family to polymorphic loci on the long arm of chromosome 12 (combined multipoint lod score, 16.8). These data suggest odds greater than 10(16):1 that the genetic defect for Holt-Oram syndrome is present on the long arm of chromosome 12 (12q2).
Mutations in a gene on chromosome 12q2 can produce a wide range of disease phenotypes characteristic of the Holt-Oram syndrome. This gene has an important role in both skeletal and cardiac development.
The pattern of connexin43 expression in developing chick limb buds was examined using a site-specific polyclonal antibody and confocal microscopy. Connexin43 is expressed at stages of limb development when epithelial-mesenchymal interactions are occurring that mediate morphogenesis. Extensive labeling was observed in the apical ectodermal ridge and labeling was also found in underlying mesenchyme cells at the tip of the bud. In mouse limb buds, the same gap junction protein is expressed only in the apical ridge. Manipulations of developing chick wing buds show that mesenchymal expression of connexin43 appears to be controlled by the apical ectodermal ridge. When the apical ridge is surgically removed and limb truncations result, mesenchymal labeling is markedly reduced and conversely the grafting of an additional ridge induces connexin43 expression between underlying mesenchymal cells which do not normally show expression at this stage of development. In addition, a treatment with retinoic acid that flattens the apical ridge and inhibits bud outgrowth reduces expression in both mesenchymal and epithelial tissues. The abolition of connexin43 expression in mesenchymal and epithelial domains when bud outgrowth is halted suggests that synthesis of this gap junction protein is related to the epithelial-mesenchymal interactions that mediate morphogenesis of the bud.
We have used the quail-chick chimera technique to study the origin of the bones of the skull in the avian embryo. Although the contribution of the neural crest to the facial and visceral skeleton had been established previously, the origin of the vault of the skull (i.e. frontal and parietal bones) remained uncertain. Moreover formation of the occipito-otic region from either the somitic or the cephalic paraxial mesoderm had not been experimentally investigated. The data obtained in the present and previous works now allow us to assign a precise embryonic origin from either the mesectoderm, the paraxial cephalic mesoderm or the five first somites, to all the bones forming the avian skull. We distinguish a skull located in front of the extreme tip of the notochord which reaches the sella turcica and a skull located caudally to this boundary. The former (‘prechordal skull’) is derived entirely from the neural crest, the latter from the mesoderm (cephalic or somitic) in its ventromedial part (‘chordal skull’) and from the crest for the parietal bone and for part of the otic region. An important point enlighten in this work concerns the double origin of the corpus of the sphenoid in which basipresphenoid is of neural crest origin and the basipostsphenoid is formed by the cephalic mesoderm. Formation of the occipito-otic region of the skeleton is particularly complex and involves the cooperation of the five first somites and the paraxial mesoderm at the hindbrain level. The morphogenetic movements leading to the initial puzzle assembly could be visualized in a reproducible way by means of small grafts of quail mesodermal areas into chick embryos.
The data reported here are discussed in the evolutionary context of the ‘New Head’ hypothesis of Gans and Northcutt (1983, Science, 220, 268-274).
Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues-processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-beta) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-beta and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-beta for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by 3H-thymidine (3H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of 3H-TdR at doses of 50-800 ng/ml. Similarly, TGF-beta inhibited uptake of 3H-TdR at doses of 2-32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 microgram/ml) or higher dose of TGF-beta (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-beta on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-beta (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-beta on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-beta at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC50) calculated for BMP-2 and TGF-beta at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-beta. The observation may suggest that TGF-beta may have effects upon cytoskeletal elements in osseous tissues.
Osteoarthritis is a slow progressive disease characterized by destruction of the articular cartilage. The degradation of extracellular matrix components is mainly mediated by a family of enzymes, the metalloproteinases (MMPs), which are active at neutral pH. Interleukin-1 (IL-1) is a small peptide, active in autocrine and paracrine fashions. In vitro IL-1 increases the production of MMPs and inhibits the synthesis of collagen type II and proteoglycans. Its role in osteoarthritis is based on several findings: IL-1 is detectable in the synovial fluid and in the cartilage matrix of osteoarthritic joints; in vivo its deleterious actions can be reproduced by intra-articular injection of recombinant IL-1; biochemical changes observed in the cartilage matrix from osteoarthritic joints resemble those induced in vitro by IL-1; finally, antagonists of IL-1 are capable in vivo of preventing or at least diminishing the degradation of cartilage matrix components in several models of experimental arthritis. Interleukin-1 appears to be a main factor mediating cartilage matrix destruction. However, its role in human osteoarthritis, although highly probable, remains to be determined.
The Gja1 gene encoding the gap junction connexin 43 (Cx43) is dynamically regulated during limb morphogenesis. Transcript expression is found in many regions of the limb bud known to be important in regulating limb growth and patterning. In the newly emerged limb bud, Gja1 transcripts are first expressed in the ventrodistal margin of the ectoderm, and later transcript expression is localized to the apical ectodermal ridge (AER). Interestingly, transcript expression in the ventrodistal ectoderm is initiated left/right asymmetrically, with some strain backgrounds showing reverse sidedness in the fore vs. hindlimb buds. In legless, a mouse mutant exhibiting both limb and left/right patterning defects, Gja1 transcripts could not be detected in this region. However, in the i.v./i.v. embryo, a mutant with randomization of body situs the same pattern of Gja1 asymmetry was found in the limb ectoderm regardless of body situs. This suggests that Gja1 transcript expression is not directly linked to signaling pathways involved in specification of the left/right axis. In addition to transcript expression in the apical ectodermal ridge, Gja1 transcripts were also found at high levels in the ventral ectoderm. In the limb bud mesenchyme, Gja1 transcripts were distributed in a posterior distal gradient, coincident with tissue known to have polarizing activity. With limb outgrowth and the initiation of limb mesenchyme condensation. Gja1 transcripts were localized in the presumptive progress zone, and in the condensing mesenchyme. In more proximal regions of the limb where mesenchyme differentiation has been initiated, Gja1 transcripts were expressed only in the outer mesenchymal cells comprising the presumptive perichondrium. Further analysis of transgenic mice ectopically expressing Wnt-1 in the limb mesenchyme revealed alterations in the pattern of Gja1 transcript expression in conjunction with the perturbation of limb mesenchyme condensation and differentiation. Together, these findings indicate that Cx43 gap junctions may mediate cell-cell interactions important in cell signaling processes involved in limb growth and patterning.
Intercellular communication allows the co-ordination of cell metabolism between tissues as well as sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks that favour the intercellular exchange of nutrients and second messengers. Heterologous intercellular communication was studied in co-cultures of articular chondrocytes and HIG-82 synovial cells by measuring mechanically induced cytosolic changes in Ca2+ ion levels by digital fluorescence video imaging. In confluent co-cultures, mechanical stimulation induced intercellular Ca2+ waves that propagated to both cell types with similar kinetics. Intercellular wave spreading was inhibited by 18alpha-glycyrrhetinic acid and by treatments inhibiting the activation of purinoreceptors, suggesting that intercellular signalling between these two cell types occurs both through gap junctions and ATP-mediated paracrine stimulation. In rheumatoid arthritis the formation of the synovial pannus induces structural changes at the chondrosynovial junction, where chondrocyte and synovial cells come into close apposition: these results provide the first evidence for direct intercellular communication between these two cell types.
Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development.
Tumour necrosis factor alpha (TNF-alpha) plays an important role in orchestrating inflammatory responses with the vascular endothelium as main target cell type, and was found to promote migration of endothelial cells, as occurs in wound healing processes. Substantial evidence exists that endothelial cell migration in wound healing is related to changes in cell coupling by means of gap junctions. Gap junctions are agglomerates of cell-to-cell channels that allow direct electrical and metabolic communication between cells. The authors have investigated whether TNF-alpha alters the expression of gap junction proteins (connexins, Cx) between human umbilical vein endothelial cells (HUVEC), thereby changing the extent of intercellular communication, as measured by dye coupling. Under control conditions, Cx43, Cx40, and Cx37 protein and mRNA were present in HUVEC. After exposure to 0.5 nM TNF-alpha for 48 h, however, the authors were no longer able to detect Cx37 and Cx40 protein, whereas Cx43 levels seemed unaltered but showed more perinuclear staining. After 24 and 48 h exposure to TNF-alpha, levels of Cx37 and Cx40 mRNA, were reduced, while the level of Cx43 mRNA remained unaltered, suggesting transcriptional regulation. If TNF-alpha was removed from the medium, Cx37 and Cx40 expression was restored within 24 h. The modulation of connexin expression by TNF-alpha resulted in a decrease in dye coupling of 40%.
MEETING REPORTS constellations of behavior. To this end, at least a dozen new population wide twin registers are being developed in addition to the extant Scandinavian registers 7. These registers will also be valuable for QTL analyses using sib-pairs and for analyses that integrate quantitative and molecular genetics. In summary, this year's BGA meeting presented results from a wide variety of methods by which the influence of genes on complex behaviors can be explored. It is predicted that over the next decade the work of members of this group will become more and more closely linked with researchers working in the field of molecular genetics as the search for specific genes progresses.