Article

Menopause transition promotes distinct modulation of mRNAs and miRNAs expression in calvaria and bone marrow osteoblastic cells

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Abstract

Investigation on functional genome research may contribute to the knowledge of functional roles of different mRNAs and miRNAs in bone cells of osteoporotic animals. Currently, few studies indicate the changes in gene modulation that osteoporosis causes in osteoblastic cells from different sites. Thus, the purpose of this investigation was to evaluate cell viability, alkaline phosphatase activity and modulation of mRNAs/miRNAs in osteoblastic cells from calvaria and bone marrow by means of microarray technology. Wistar female rats were divided in sham operated and ovariectomized groups. After 150 days of ovariectomy, cells were isolated from both sites to perform cell culture. Results showed that calvaria cells from ovariectomized rats had a decrease in viability when compared to control groups and to bone marrow cells from osteoporotic rats after 3 days. Alkaline phosphatase activity decreased in calvaria cells from ovariectomized rats whereas it was increased in bone marrow osteoblastic cells in the same group. Microarray data analysis showed 5447 differentially expressed mRNAs and 82 differentially expressed miRNAs in calvaria cells. The same way, 4399 mRNAs and 54 miRNAs were expressed in bone marrow cells. mRNAs associated to bone metabolism such as Anxa5, Sp7, Spp1, Notch1 were distinctively modulated in both sites, as well as miRNAs such as miR-350, miR-542-3p, miR-204-5p and miR-30e- 3p. The RNA species identified in this study could be further used as targets for treatment or prevention of osteoporosis.

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... We read with great interest the recent report by Semeghini et al. (2017), Cell Biol Int, "Menopause transition promotes distinct modulation of mRNAs and miRNAs expression in calvaria and bone marrow osteoblastic cells," which appeared on 24 May 2017 in Cell Biology International. The results of the report are very helpful for us; however, from our perspective, the author's method in bioinformatics analysis is inappropriate: Student's t-test is an inappropriate statistical method for detecting differentially expressed mRNA or miRNA in osteoblastic cells from calvaria of ovariectomized rats compared to control or in osteoblastic cells from bone marrow of ovariectomized rats compared to control. ...
... We read with great interest the recent report by Semeghini et al. (2017), "Menopause transition promotes distinct modulation of mRNAs and miRNAs expression in calvaria and bone marrow osteoblastic cells," which appeared on 24 May 2017 in Cell Biology International. The results of the report are very helpful for us; however, from our perspective, the author's method in bioinformatics analysis is inappropriate. ...
Article
We read with great interest the recent report by Semeghini et al., "Menopause transition promotes distinct modulation of mRNAs and miRNAs expression in calvaria and bone marrow osteoblastic cells," which appeared on 24 May 2017 in Cell Biology International. The results of the report are very helpful for us, however, from our perspective, the author's methods in bioinformatics analysis is inappropriate: Student's t-tests is an inappropriate statistical method for detecting differentially expressed mRNA or miRNA in osteoblastic cells from calvaria of ovariectomized rats compared to control or in osteoblastic cells from bone marrow of ovariectomized rats compared to control.
... Furthermore, a notable inverse association was observed between the expression of miR-542-3p messenger RNA in rats that underwent ovariectomy . The analysis of microarray data also indicated that miR-542-3p exhibited differential expression in the bone marrow cells of rats that underwent ovariectomy (Semeghini et al., 2018). The findings of our study indicated a decrease in the expression of miR-542-3p in individuals with osteoporosis. ...
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... These results differ from other investigations that used cells from different sites such as bone marrow, when osteoblastic cells showed higher or similar cell proliferation in OVX groups when compared to sham group (14). Nevertheless, Semeghini et al. (15) also showed lower cell proliferation of calvaria osteoblastic cells from female ovariectomized whereas bone marrow cells from sham and OVX groups had similar proliferation. ...
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Recent studies suggest that osteoporosis, in addition to the damage caused in long bones, may cause deterioration in the jaws, especially in alveolar bone sites, with effects in the progress of periodontal disease as well as in bone healing. The aim of this study was to evaluate the effect of osteoporosis in the metabolism of rat alveolar bone osteoblasts. There were used 10 female rats divided in two experimental groups (Sham and OVX), which were ovariectomized and after 8 weeks euthanized to collect mandibular bone samples in order to isolate osteoblastic cells. The cells were cultured in 24-well plates to perform the in vitro experiments. After 7, 10 and 14 days, there were evaluated cell proliferation by MTT assay, in situ detection of alkaline phosphatase (ALP) as well as mineralized nodules and expression of genes associated to bone remodeling. Results showed that at 7, 10 and 14 days cell proliferation was lower for OVX group. In situ detection of ALP was higher at 7 days and lower at 10 and 14 days in OVX group. At 17 and 21 days, OVX group had a significative decrease of mineralization nodules. There was a downregulation in the expression of Alp, Bglap and Runx2 genes and an upregulation of Opg in OVX group, whereas Opn and Rankl modulation was similar between the evaluated groups. Our results suggest that osteoporosis has a deleterious effect on alveolar bone cells from ovariectomized rats, which might affect the treatment of diseases associated to the jaw bones.
... O steoporosis is characterized by a loss of bone mass and weakening of its microarchitecture, leading to fragility and increased fracture risk. 1 Besides clinical features, investigations showed that osteoporosis may affect functional activity and the expression of mRNAs and miRNAs in osteoblastic cells from healthy and ovariectomized rats, as well as promote distinct gene expression profiles among osteoblasts from different sites, such as femur and calvaria. 2 The habit of drinking caffeinated beverages is very common in America and Europe, and several epidemiological studies have shown that a high dietary caffeine intake is associated with reduced bone mineral density and increased urinary calcium loss. 3 Caffeine is the main component of coffee, chocolate, tea, energy and soft drinks, as well as medicines. ...
... Huang et al. demonstrated that miR-204 inhibited osteogenesis and promoted adipogenesis of bone marrow stem cells through negative regulation of Runx2, an important transcription factor for chondrogenesis and osteogenesis [32]. In a rat model of osteoporosis, miR-204-5p was one of the microRNAs associated with bone metabolism in bone marrow osteoblastic cells [33]. In our current study, we found that miR-204-5p was 4.56-fold up-regulated in aged osteoblasts, which was validated in a GEO array of trabecular bone specimens from osteoporotic hip fractures in elderly women [26]. ...
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With regard to Hedgehog signaling in mammalian development, the majority of research has focused on Gli2 and Gli3 rather than Gli1. This is because Gli1−/− mice do not show any gross abnormalities in adulthood, and no detailed analyses of fetal Gli1−/− mice are available. In this study, we investigated the physiological role of Gli1 in osteogenesis. Histological analyses revealed that bone formation was impaired in Gli1−/− fetuses compared with WT fetuses. Gli1−/− perichondrial cells expressed neither runt-related transcription factor 2 (Runx2) nor osterix, master regulators of osteogenesis, in contrast to WT cells. In vitro analyses showed that overexpression of Gli1 up-regulated early osteogenesis-related genes in both WT and Runx2−/− perichondrial cells, and Gli1 activated transcription of those genes via its association with their 5′-regulatory regions, underlying the function of Gli1 in the perichondrium. Moreover, Gli1−/−;Gli2−/− mice showed more severe phenotypes of impaired bone formation than either Gli1−/− or Gli2−/− mice, and osteoblast differentiation was impaired in Gli1−/−;Gli3−/− perichondrial cells compared with Gli3−/− cells in vitro. These data suggest that Gli1 itself can induce early osteoblast differentiation, at least to some extent, in a Runx2-independent manner. It also plays a redundant role with Gli2 and is involved in the repressor function of Gli3 in osteogenesis. On the basis of these findings, we propose that upon Hedgehog input, Gli1 functions collectively with Gli2 and Gli3 in osteogenesis.
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Osteoporosis is a major health problem worldwide, and is projected to increase exponentially due to the aging of the population. The absolute fracture risk in individual subjects is calculated by the use of algorithms which include bone mineral density (BMD), age, gender, history of prior fracture and other risk factors. This review describes the laboratory investigations into osteoporosis which include serum calcium, phosphate, creatinine, alkaline phosphatase and 25-hydroxyvitamin D and, additionally in men, testosterone. Parathyroid hormone (PTH) is measured in patients with abnormal serum calcium to determine its cause. Other laboratory investigations such as thyroid function testing, screening for multiple myeloma, and screening for Cushing's syndrome, are performed if indicated. Measurement of bone turnover markers (BTMs) is currently not included in algorithms for fracture risk calculations due to the lack of data. However, BTMs may be useful for monitoring osteoporosis treatment. Further studies of the reference BTMs serum carboxy terminal telopeptide of collagen type I (s-CTX) and serum procollagen type I N-terminal propeptide (s-PINP) in fracture risk prediction and in monitoring various treatments for osteoporosis may help expedite their inclusion in routine clinical practice.
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MicroRNAs (miRNAs) repress cellular protein levels to provide a sophisticated parameter of gene regulation that coordinates a broad spectrum of biological processes. Bone organogenesis is a complex process involving the differentiation and crosstalk of multiple cell types for formation and remodeling of the skeleton. Inhibition of mRNA translation by miRNAs has emerged as an important regulator of developmental osteogenic signaling pathways, osteoblast growth and differentiation, osteoclast-mediated bone resorption activity and bone homeostasis in the adult skeleton. miRNAs control multiple layers of gene regulation for bone development and postnatal functions, from the initial response of stem/progenitor cells to the structural and metabolic activity of the mature tissue. This Review brings into focus an emerging concept of bone-regulating miRNAs, the evidence for which has been gathered largely from in vivo mouse models and in vitro studies in human and mouse skeletal cell populations. Characterization of miRNAs that operate through tissue-specific transcription factors in osteoblast and osteoclast lineage cells, as well as intricate feedforward and reverse loops, has provided novel insights into the supervision of signaling pathways and regulatory networks controlling normal bone formation and turnover. The current knowledge of miRNAs characteristic of human pathologic disorders of the skeleton is presented with a future goal towards translational studies.
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This study aimed at evaluating the bone repair process in ovariectomized rats submitted to an irradiation procedure. For this purpose, one hundred rats were randomly divided in four experimental groups: control, ovariectomized, irradiated and irradiated/ovariectomized. A bone defect was made on all animals' tibias. Three days after surgery, only irradiated and irradiated/ovariectomized rats received 8 Gy of gamma rays on the lower limbs region. The animals were sacrificed 7, 14, 21 and 28 days after surgery in order to assess the repair process. It was possible to observe a delay in the bone repair process in the irradiated/ovariectomized group, in which there was a remarkable association between estrogen deficiency and ionizing radiation resulting in the reduction of newly formed bone production, thus accelerating the resorption process.
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As the world population rises, osteoporotic fracture is an emerging global threat to the well-being of elderly patients. The process of fracture healing by intramembranous ossification or/and endochondral ossification involve many well-orchestrated events including the signaling, recruitment and differentiation of mesenchymal stem cells (MSCs) during the early phase; formation of a hard callus and extracellular matrix, angiogenesis and revascularization during the mid-phase; and finally callus remodeling at the late phase of fracture healing.
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Background A number of genes have been identified to be related with primary osteoporosis while less is known about the comprehensive interactions between regulating genes and proteins. We aimed to identify the differentially expressed genes (DEGs) and regulatory effects of transcription factors (TFs) involved in primary osteoporosis.
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Objective: Expression of a large number of genes during differentiation of undifferentiated pulp cells into odontoblastic cells is still unknown, hence the aim of this investigation was to compare undifferentiated pulp cells (OD-21) and odontoblast-like cells (MDPC-23) through the assessment of cell stimulation and gene expression profiling. Design: The cells were cultured and after the experimental periods, there were evaluated cell proliferation and viability as well as alkaline phosphatase activity (ALP) and mineralization nodules. To evaluate gene expression it was used fluorescence cDNA microarray technology in addition to bioinformatics programmes such as SAM (significance analysis of microarrays). Gene expression was validated by Real Time PCR (qPCR). Results: The results showed that viability was above 80% in both cells, cell proliferation and ALP activity was higher in MDPC-23 cells and mineralization nodules were present only in the cultures of odontoblast-like cells. There were observed genes associated to odontogenesis with similar behaviour in both cell types, such as Il10, Traf6, Lef1 and Hspa8. Regions of the heatmap showed differences in induction and repression of genes such as Jak2 and Fas. Conclusion: OD-21 cells share many genes with similar behaviour to MDPC-23 cells, suggesting their potential to differentiate into odontoblasts.
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Osteoporosis is a degenerative disease that primarily affects postmenopausal women. Based on panoramic radiographs, several assessment methods have been proposed for the diagnosis and evaluation of bone changes and as a predictor of osteoporosis for example the mandibular index. The purpose of this study is to compare the assessment of mandibular indices on panoramic and cross-sectional images. Forty-four cone beam computed tomography (CBCT) images from postmenopausal female subjects aged more than 45 years without systemic changes were selected for this study. From those images, cross-sectional and panoramic reconstruction images were assembled into a template for evaluation. The evaluation was conducted by observing the panoramic images and parasagittal sections. The appearance of the inferior cortex of the mandible was classified according to the mandibular index: C1, the endosteal margin of the cortex was even and sharp; C2, the endosteal margin presented semilunar defects or appeared to form endosteal cortical residues; or C3, the cortical layer formed heavy endosteal cortical residues and was clearly porous. Based on Wilcoxon statistical test (p > 0.01), the data showed no statistically significant difference between the exams. The mandibular index assigned in tomographic images is comparable to that obtained in panoramic images, indicating a valid use of the index in CBCT images, which can lead to the identification of patients with bone mass loss and a premature referral to further exams and treatment.
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Clinically, bone marrow stromal cells (BMCs) are the most common source of osteoprogenitor cells. Its harvest process, however, is invasive to patients. Previous reports have shown the potential advantages of using periosteum-derived cells (PDCs) as a source of cell-based transplant therapy. The objective of our study was to characterize the osteoblastic differentiation and mineralization ability of PDCs versus BMCs and osteoblasts (OBs). BMCs, OBs, and PDCs were isolated from 4-week-old male Wistar rats. To characterize the differentiation ability of the cells, MTS assay, alkaline phosphatase (ALP) activity staining, picrosirius red staining, and alizarin red staining were performed. Immunohistochemistry was performed on paraffin sections of calvarial periosteum to determine the presence of mesenchymal stem cells. PDCs showed the greatest proliferation rate compared with BMCs and OBs. Matured collagenous matrix formation was observed in PDCs and BMCs. ALP-positive cells and in vitro mineralization were evident in all cell types analyzed; however, that of PDCs was not comparable to that of the OBs and BMCs. Immunohistochemistry revealed the presence of STRO-1-and CD105-positive cells in the cambium layer of the periosteum. PDCs have remarkable proliferative ability, but contain only a small population of osteogenic cells compared with BMCs and OBs. Although cell activity can be affected by various factors, such as age, culture condition, additives, and so forth, PDCs are likely not the source of OBs, although they might provide matrices that indirectly aid in bone formation.
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Growing evidence shows the possibility of role of microRNAs (miRNA) in regulating bone mass. We investigated the change of miRNAs and mRNA expression profiles in bone tissue in ovariectomized mice model and evaluated regulatory mechanism of bone mass mediated by miRNAs in estrogen deficiency state. Eight-week old female C3H/HeJ mice underwent ovariectomy (OVX) or sham operation (Sham-op) and their femur and tibia were harvested to extract total bone RNAs after 4 weeks for microarray analysis. Eight miRNAs (miR-127, -133a, -133a*, -133b, -136 -206, -378, -378*) were identified to be upregulated after OVX while one miRNA (miR-204) was downregulated. Concomitant analysis of mRNA microarray revealed that 658 genes were differentially expressed between OVX and Sham-op mice. Target prediction of differentially expressed miRNAs identified potential targets and integrative analysis using the mRNA microarray results showed that PPARγ and CREB pathways are activated in skeletal tissues after ovariectomy. Among the potential candidates of miRNA, we further studied the role of miR-127 in vitro, which exhibited the greatest changes after OVX. We also studied the effects of miR-136, which has not been studied in the context of bone mass regulation. Transfection of miR-127 inhibitor has enhanced osteoblastic differentiation in UAMS-32 cells as measured by alkaline phosphatase activities and mRNA expression of osteoblast-specific genes while miR-136 precursor has inhibited osteoblastic differentiation. Furthermore, transfection of both miR-127 and miR-136 inhibitors enhanced the osteocyte-like morphological changes and survival in MLO-Y4 cells while precursors of miR-127 and -136 has aggravated dexamethasone-induced cell death. Both of the precursors enhanced osteoclastic differentiation in bone marrow macrophages, indicating that both miR-127 and -136 are negatively regulating bone mass. Taken together, these results suggest a novel insight into the association between distinct miRNAs expression and their possible role through regulatory network with mRNAs in the pathogenesis of estrogen deficiency induced osteoporosis.
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
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Currently, osteoporosis affects over half of our population beyond the age of 50, and hip fractures related to osteoporosis accounted for direct costs of $18 billion in 2002 ("About Osteoporosis: Fast Facts", 2006). The average length of a hospital stay for a primary fracture diagnosis is nearly one week, and approximately 25% of previously independent older patients who sustain hip fractures remain in long-term care for over a year. In response to the necessity for improved fracture care and shortened healing time, the field of orthopaedic surgery has begun to turn toward cellular and molecular biology research for the next answer. The goal of the proposed research is to determine if current treatment and potentially new therapeutic compounds are capable of regulating bone cell function. Osteoblast and osteoclast cells were treated for periods of 24, 48 and 72 hours in the presence of estrogen, demineralized bone matrix proteins, or an antagonist to neuropeptide Y. Following the incubation, cell viability, cell function, and morphology were determined. The results indicated a significant increase in osteoblast proliferation and alkaline phosphate production in cells treated with estrogen and DBM without evidence of cellular damage. DBM and estrogen did not affect osteoclast cell numbers, while neuropeptide Y antagonist reduced osteoclast numbers. The data shows Y antagonist may be a useful and safe compound that could be used in the treatment of osteoporotic fractures.
Article
Kawai M, de Paula FJA, Rosen CJ (Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka, Japan; University of São Paulo, Ribeirão Preto, SP, Brazil; and Maine Medical Center Research Institute, Scarborough, ME, USA). New insights into osteoporosis: the bone–fat connection (Review). J Intern Med 2012; 272: 317–329. Abstract. Osteoporosis and obesity are chronic disorders that are both increasing in prevalence. The pathophysiology of these conditions is multifactorial and includes genetic, environmental and hormonal determinants. Although it has long been considered that these are distinct disorders rarely found in the same individual, emerging evidence from basic and clinical studies support an important interaction between adipose tissue and the skeleton. It is proposed that adiposity may influence bone remodelling through three mechanisms: (i) secretion of cytokines that directly target bone, (ii) production of adipokines that influence the central nervous system thereby changing sympathetic impulses to bone and (iii) paracrine influences on adjacent skeletal cells. Here we focus on the current understanding of bone–fat interactions and the clinical implications of recent studies linking obesity to osteoporosis.
Article
The objective of this study was to identify the differences between osteoblasts derived from normal adult rat mandibles and osteoporotic adult rats. An osteoporotic animal model was established by performing a bilateral ovariectomy (ovx group). The proliferation and differentiation abilities of osteoblasts were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide), alkaline phosphatase (ALP) and osteocalcin release (OC) assays. Transmission electron microscopy (TEM) was performed to assess differences in the ultrastructure. Proliferating cell nuclear antigen (PCNA) and uncoupling protein 2 (UCP2) protein concentrations were analyzed by Western blot. In addition, UCP2 protein in osteoblasts was assessed by immunohistochemistry staining. ATP and reactive oxygen species (ROS) concentrations were analyzed separately with ATP and ROS quantification kits. At four and 12 weeks after the operation, osteoblasts of the ovx group showed earlier attachment, fewer dead cells and faster growth compared with cells in the sham group. TEM showed that osteoblasts of the ovx group had fewer folds, lysosomes, peroxisomes and less rough endoplasmic reticulum. The results of the MTT, ALP activity and OC assays were all higher in osteoblasts from the ovx group at four or 12 weeks postsurgery than osteoblasts from the sham group. PCNA protein concentrations in the ovx group increased significantly compared with those of the sham group at four or 12 weeks after the operation, but UCP2 concentrations decreased over the same time period. UCP2 immunohistochemical staining of osteoblasts showed that the protein was concentrated in the cytoplasm and that the osteoblasts from the sham group had higher expression than those from the ovx group. The ATP and ROS concentrations of the ovx groups were significantly higher than the sham groups at four or 12 weeks postsurgery. Therefore, we concluded that there are differences in cell ultrastructure, proliferation, differentiation, ATP and ROS concentrations, and PCNA and UCP2 protein expression levels in osteoblasts from the mandibles of rats of the ovx group compared with those from the sham group.
Article
Age-related bone loss is associated with significant changes in bone remodeling characterized by decreased trabecular and periosteal bone formation relative to bone resorption, resulting in bone fragility and increased risk of fractures. Prevention or reversal of age-related decrease in bone mass and increase in bone fragility has been based on inhibition of bone resorption using anticatabolic drugs. The current challenge is to promote osteoblastogenesis and bone formation to prevent age-related bone deterioration. A limited number of approved therapeutic molecules are available to activate bone formation. Therefore, there is a need for anabolic drugs that promote bone matrix apposition at the endosteal, endocortical, and periosteal envelopes by increasing the number of osteoblast precursor cells and/or the function of mature osteoblasts. In this study, we review current therapeutics promoting bone formation and anabolic molecules targeting signaling pathways involved in osteoblastogenesis, based on selected full-text articles searched on Medline search from 1990 to 2010. We present current therapeutic approaches focused on intermittent parathyroid hormone and Wnt signaling agonists/antagonists. We also discuss novel approaches for prevention and treatment of defective bone formation and bone loss associated with aging and osteoporosis. These strategies targeting osteoblastic cell functions may prove to be useful in promoting bone formation and improving bone strength in the aging population.
Article
Parathyroid hormone (PTH) suppresses Dickkopf 1 (Dkk1) expression in osteoblasts. To determine whether this suppression is essential for PTH-mediated Wnt signaling and bone formation, we examined mice that overexpress Dkk1 in osteoblasts (Dkk1 mice). Dkk1 mice were osteopenic due to abnormal osteoblast and osteoclast activity. When fed a low-calcium diet, and in two other models of hyperparathyroidism, these mice failed to develop the peritrabecular stromal cell response ("osteitis fibrosis") and new bone formation seen in wild-type mice. Despite these effects of Dkk1 overexpression, PTH still activated Wnt signaling in Dkk1 mice and in osteoblastic cells cultured from these mice. In cultured MC3T3E1 preosteoblastic cells, PTH dramatically suppressed Dkk1 expression, induced PKA-mediated phosphorylation of beta-catenin, and significantly enhanced Lef1 expression. Our findings indicate that the full actions of PTH require intact Wnt signaling but that PTH can activate the Wnt pathway despite overexpression of Dkk1.
Article
Differentiation of mesenchymal stem cells into a particular lineage is tightly regulated, and malfunction of this regulation could lead to pathological consequences. Patients with osteoporosis have increased adipocyte accumulation, but the mechanisms involved remain to be defined. In this study, we aimed to investigate if microRNAs regulate mesenchymal progenitor cells and bone marrow stromal cell (BMSC) differentiation through modulation of Runx2, a key transcription factor for osteogenesis. We found that miR-204 and its homolog miR-211 were expressed in mesenchymal progenitor cell lines and BMSCs and their expression was induced during adipocyte differentiation, whereas Runx2 protein expression was suppressed. Retroviral overexpression of miR-204 or transfection of miR-204 oligo decreased Runx2 protein levels and miR-204 inhibition significantly elevated Runx2 protein levels, suggesting that miR-204 acts as an endogenous attenuator of Runx2 in mesenchymal progenitor cells and BMSCs. Mutations of putative miR-204 binding sites upregulated the Runx2 3'-UTR reporter activity, suggesting that miR-204/211 bind to Runx2 3'-UTR. Perturbation of miR-204 resulted in altered differentiation fate of mesenchymal progenitor cells and BMSCs: osteoblast differentiation was inhibited and adipocyte differentiation was promoted when miR-204 was overexpressed in these cells, whereasosteogenesis was upregulated and adipocyte formation was impaired when miR-204 was inhibited. Together, our data demonstrated that miR-204/211 act as important endogenous negative regulators of Runx2, which inhibit osteogenesis and promote adipogenesis of mesenchymal progenitor cells and BMSCs.
Article
Osteoporosis is a complex skeletal disorder in which compromised bone strength increases the risk of fragility fractures. Recent scientific advances in bone biology and immunology have greatly expanded our insights into the pathogenesis of osteoporosis. For those with osteoporotic fractures, however, the physical and psychological effects remain severe. Primary care physicians need to understand the basic mechanisms of bone physiology and pathophysiology in order to both prevent and treat this devastating disorder.
Article
Bone remodeling is a complex process regulated by systemic hormones and local factors. The exact role of growth factors has not been defined, but it is apparent that factors such as the FGFs stimulate preosteoblast cell replication, which results in an increased osteoblast population capable of synthesizing bone matrix. Other factors, such as IGF I and TGFβ, stimulate cell replication and differentiated function, whereas β2 m and binding proteins probably affect the availability of a factor and the binding to its receptor. It is suspected that the local factors are important in the coupling of bone formation to bone resorption, but their exact mode of action has not been defined. Lymphokines and monokines stimulate bone resorption and preosteoblast cell replication, suggesting a possible role in coupling, but their effects may be more relevant to pathological than to physiological events. The local growth factors found in bone are not unique, suggesting lack of specificity for skeletal tissue. The specificity for this tissue may depend upon the particular cellular event modulated, the state of cell differentiation, the combination of factors to which cells are exposed, the sequence of factor exposure, and their interactions with hormones. Future work should address these issues and determine if systemic hormones enhance or inhibit the synthesis of local factors or modulate their activities, as it was recently shown for PTH. Additional research is needed to understand the exact processes affected by the various factors, their mechanism of action, and their effects in vivo. Although our knowledge about local regulators of skeletal growth is still limited, it is likely that future investigations will reveal that they have an important role in bone remodeling and in the pathogenesis of metabolic bone disease.
Article
Both osteoblasts and osteoclasts are derived from progenitors that reside in the bone marrow; osteoblasts belong to the mesenchymal lineage of the marrow stroma, and osteoclasts to the hematopoietic lineage. The development of osteoclasts from their progenitors is dependent on stromal-osteoblastic cells, which are a major source of cytokines that are critical in osteoclastogenesis, such as interleukin-6 and interleukin-11. The production of interleukin-6 by stromal osteoblastic cells, as well as the responsiveness of bone marrow cells to cytokines such as interleukin-6 and interleukin-11, is regulated by sex steroids. When gonadal function is lost, the formation of osteoclasts as well as osteoblasts increases in the marrow, both changes apparently mediated by an increase in the production of interleukin-6 and perhaps by an increase in the responsiveness of bone marrow progenitor cells not only to interleukin-6 but also to other cytokines with osteoclastogenic and osteoblastogenic properties. The cellular activity of the bone marrow is also altered by the process of aging. Specifically, senescence may decrease the ability of the marrow to form osteoblast precursors. The association between the dysregulation of osteoclast or osteoblast development in the marrow and the disruption of the balance between bone resorption and bone formation, resulting in the loss of bone, leads to the following notion. Like homeostasis of other regenerating tissues, homeostasis of bone depends on the orderly replenishment of its cellular constituents. Excessive osteoclastogenesis and inadequate osteoblastogenesis are responsible for the mismatch between the formation and resorption of bone in postmenopausal and age-related osteopenia. The recognition that changes in the numbers of bone cells, rather than changes in the activity of individual cells, form the pathogenetic basis of osteoporosis is a major advance in understanding the mechanism of this disease.
Article
The adult skeleton regenerates by temporary cellular structures that comprise teams of juxtaposed osteoclasts and osteoblasts and replace periodically old bone with new. A considerable body of evidence accumulated during the last decade has shown that the rate of genesis of these two highly specialized cell types, as well as the prevalence of their apoptosis, is essential for the maintenance of bone homeostasis; and that common metabolic bone disorders such as osteoporosis result largely from a derangement in the birth or death of these cells. The purpose of this article is 3-fold: 1) to review the role and the molecular mechanism of action of regulatory molecules, such as cytokines and hormones, in osteoclast and osteoblast birth and apoptosis; 2) to review the evidence for the contribution of changes in bone cell birth or death to the pathogenesis of the most common forms of osteoporosis; and 3) to highlight the implications of bone cell birth and death for a better understanding of the mechanism of action and efficacy of present and future pharmacotherapeutic agents for osteoporosis.
Article
Osteoporosis afflicts 75 million persons in the United States, Europe and Japan and results in more than 1.3 million fractures annually in the United States. Because osteoporosis is usually asymptomatic until a fracture occurs, family physicians must identify the appropriate timing and methods for screening those at risk. Prevention is the most important step, and women of all ages should be encouraged to take 1,000 to 1,500 mg of supplemental calcium daily, participate in regular weight-bearing exercise, avoid medications known to compromise bone density, institute hormone replacement therapy at menopause unless contraindicated and avoid tobacco and excessive alcohol intake. All postmenopausal women who present with fractures as well as younger women who have risk factors should be evaluated for the disease. Physicians should recommend bone mineral density testing to younger women at risk and postmenopausal women younger than 65 years who have risk factors for osteoporosis other than being postmenopausal. Bone mineral density testing should be recommended to all women 65 years and older regardless of additional risk factors. Bone mineral density screening should be used as an adjunct to clinical judgment only if the results would influence the choice of therapy or convince the patient to take appropriate preventive measures.
Article
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Emerging insights into the pathophysiology of osteoporosis
  • S C Manolagas
  • R L Jilka
Manolagas SC, Jilka RL. (1995). Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N Engl J Med 332: 305-11.
Mammalian microRNAs: a small world for fine-tuning gene expression
  • Sevignani