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Dietary compound gossypetin inhibits bone resorption through down-regulating lysosomal cathepsin K activity and autophagy-related protein induction in actin ring-bearing osteoclasts
Abstract
Gossypetin, usually isolated from the flowers and the calyx of Hibiscus sabdariffa, possesses anti-microbial and anti-atherosclerotic effects. However, anti-osteoporotic effects of gossypetin have not been elucidated. Gossypetin attenuated RANKL-induced multinucleated osteoclast formation with enhanced TRAP activity and blunted bone resorption active in osteoclasts. Gossypetin inhibited the actin ring formation and αvβ3 integrin induction for sealing zones. This compound suppressed the induction of CAII, V-ATPase, ClC-7 and Ae2, all required for secretion of proton and chloride ions into resorption lacunae. Furthermore, gossypetin reduced lysosomal cathepsin K transcription and MMP-9 activity, blunting accumulation of lysosomes in osteoclasts displaying an actin ring. The presence of gossypetin deterred the induction of Rab7, and Atg12–Atg5 conjugate and Atg7 involved in LC3 lipidation, all prerequisites to osteoclast ruffled border formation. These observations demonstrate for the first time that gossypetin was effective in retarding ruffled border formation and acidification in a sealed microenvironment of osteoclast resorption lacunae.
... However, the mechanistic efficacy of these compounds in counteracting bone loss remains elusive. Gossypetin, a bioactive compound of Hibiscus sabdariffa, antagonized bone resorption through inhibiting the ruffled border formation [27]. On the basis of the evidence that bone resorption in osteoclasts relies on a molecular apparatus linking lysosomes to microtubules, the present study examined whether aesculetin ( Figure 1A) and its glucoside aesculin (Figure 2A) inhibited bone resorption through disturbing the lysosomal intracellular trafficking to the ruffled border in RANKL-exposed murine Raw 264.7 macrophages. ...
... This study found that aesculetin inhibited the induction of LC3, Atg5, and Atg7 involved in LC3 lipidation of osteoclasts, indicating that this compound inhibited fusion of LC3II-decorated membrane with secretory lysosomes. Similarly, gossypetin diminished the ATG induction and lysosomal cathepsin K activity in actin ring-bearing osteoclasts [27]. Furthermore, it was shown that LC3 associates with microtubules in proximity of the actin ring and regulates Cdc42 [23]. ...
For the optimal resorption of mineralized bone matrix, osteoclasts require the generation of the ruffled border and acidic resorption lacuna through lysosomal trafficking and exocytosis. Coumarin-type aesculetin is a naturally occurring compound with anti-inflammatory and antibacterial effects. However, the direct effects of aesculetin on osteoclastogenesis remain to be elucidated. This study found that aesculetin inhibited osteoclast activation and bone resorption through blocking formation and exocytosis of lysosomes. Raw 264.7 cells were differentiated in the presence of 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and treated with 1–10 μM aesculetin. Differentiation, bone resorption, and lysosome biogenesis of osteoclasts were determined by tartrate-resistance acid phosphatase (TRAP) staining, bone resorption assay, Western blotting, immunocytochemical analysis, and LysoTracker staining. Aesculetin inhibited RANKL-induced formation of multinucleated osteoclasts with a reduction of TRAP activity. Micromolar aesculetin deterred the actin ring formation through inhibition of induction of αvβ3 integrin and Cdc42 but not cluster of differentiation 44 (CD44) in RANKL-exposed osteoclasts. Administering aesculetin to RANKL-exposed osteoclasts attenuated the induction of autophagy-related proteins, microtubule-associated protein light chain 3, and small GTPase Rab7, hampering the lysosomal trafficking onto ruffled border crucial for bone resorption. In addition, aesculetin curtailed cellular induction of Pleckstrin homology domain-containing protein family member 1 and lissencephaly-1 involved in lysosome positioning to microtubules involved in the lysosomal transport within mature osteoclasts. These results demonstrate that aesculetin retarded osteoclast differentiation and impaired lysosomal trafficking and exocytosis for the formation of the putative ruffled border. Therefore, aesculetin may be a potential osteoprotective agent targeting RANKL-induced osteoclastic born resorption for medicinal use.
... Accelerated bone resorption via osteoclastic cells in osteoporotic rats in the present work was illuminated by the increased serum level of TRAP and CTSK; lysosomal proteases enzymes that are expressed in osteoclast. The ruffled borders of osteoclast start degrading bone matrix through the fusion of lysosomes with TRAP and CTSK [53]. It has been reported that, deletion of in-vivo CTSK in osteoclastic cells resulted in the activation of osteoblastic cells which in turn enhanced bone formation [54]. ...
Chloroquine (CQ); a lysosomotropic agent used for decade ago as anti-malarial, was tested against aging induced osteoporosis. Osteoporosis in male rats was induced using D-galactose (D-gal) as a reducing sugar at a dose of 200 mg/kg/day; i.p. Osteoporotic rats were orally treated with CQ (10 mg/kg/day) for four successive weeks. Bone densitometry of tibia and femur were evaluated. Bone formation biomarkers; osteoprotegrin (OPG), bone specific alkaline phosphatse (BALP), and osteocalcin (OCN), and bone resorption biomarker; receptor activator of nuclear factor kappa-B ligand (RANKL), cathepsin-k (CTSK), tartrate-resistant acid phosphatase (TRAP) were estimated. Moreover, the expression of extracellular regulated kinase (ERK) in bone was determined. CQ ameliorated the bone detrimental changes induced by D-galactose. It enhanced bone health as revealed by measurement of bone densitometry, halted the activation of receptor activator of nuclear factor kappa-B ligand (RANKL) and reduced bone manifestation of ERK. Furthermore, CQ treatment abated serum cathepsin-k (CTSK) and serum tartrate-resistant acid phosphatase (TRAP) thus inhibited osteoclastogenesis and consequently restored the RANKL/OPG ratio. CQ demonstrated an antioxidant effect in bone where it increased both Catalase (CAT) and Superoxide dismutase (SOD). These CQ preserving effect in rats treated with D-galactose were confirmed by the histopathological examination. The present study points to the potential therapeutic effect of CQ as anti-osteoporotic agent possibly through its antioxidant effects and suppression of ERK associated osteoclastogenesis.
... anti-lipidemic, antimicrobial, anti-osteoporotic, and hepatoprotective(Dwi Antika et al., 2016;Salvamani et al., 2014).Allicin (diallyl thiosulfate or S-allyl cysteine sulfoxide) is a natural volatile compound containing sulfur which is predominantly present in garlic, Allium sativum (family Alliaceae). Some other Allium species containing allicin are A. ampeloprasum (elephant garlic), A. ursinum (wild garlic), A. victorialis (alpine leek), and A. vineale (field garlic)(Moss & Ramji, 2016;Salehi et al., 2019). ...
Under physiological conditions, endothelial cells act as protective barrier which prevents direct contact of blood with circulating factors via production of tissue plasminogen activator. Risk factors of metabolic disorders are responsible to induce endothelial dysfunction and may consequently lead to prognosis of atherosclerosis. This article summarizes the process of atherosclerosis which involves number of sequences including formation and interaction of AGE‐RAGE, activation of polyol pathway, protein kinase C, and hexosamine‐mediated pathway. All these mechanisms can lead to the development of oxidative stress which may further aggravate condition. Different pharmacological interventions are being used to treat atherosclerosis, however, these might be associated with mild to severe side effects. Therefore, plant‐based bioactive compounds having potential to combat and prevent atherosclerosis in diabetic patients are attaining recent focus. By understanding process of development and mechanisms involved in atherosclerotic plaque formation, these bioactive compounds can be better option for future therapeutic interventions for atherosclerosis treatment.
Practical applications
Atherosclerosis is one of major underlying disorders of cardiovascular diseases which occur through multiple mechanisms and is associated with metabolic disorders. Conventional therapeutic interventions are not only used to treat atherosclerosis, but are also commonly associated with mild to severe side effects. Therefore, nowadays, bioactive compounds having potential to combat and prevent atherosclerosis in diabetic patients are preferred. By understanding mechanisms involved in atherosclerotic plaque formation, bioactive compounds can be better understood for treatment of atherosclerosis. In this manuscript, we have focused on treatment strategies of atherosclerosis using bioactive compounds notably alkaloids and flavonoids having diverse pharmacological and therapeutic potentials with special focus on the mechanism of action of these bioactive compounds suitable for treatment of atherosclerosis. This manuscript will provide the scientific insights of bioactive compounds to researchers who are working in the area of drug discovery and development to control pathogenesis and development of atherosclerosis and its associated cardiometabolic disorders.
Great attention was recently given to the importance of RAS in controlling inflammatory bone diseases, following the discovery of its local existence in skeletal tissues. Local RAS was found to be expressed on osteoblastic and osteoclastic cells and to exert its action via angiotensin II (AngII) receptors, including angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptors. Telmisartan (TLM), an AT1R blocker (ARBs), was investigated in the present study for its therapeutic effect on bone health in osteoporotic rats. D‐galactose, a reducing sugar at a dose of 200 mg/kg/day/i.p. was used to induce osteoporosis in male rats. TLM, at a dose of 5 mg/kg/day, was orally introduced in the osteoporotic rats for four consecutive weeks. Tibia and femur bone densitometry were estimated, bone formation and bone resorption biomarkers serum levels were measured, mineral content in blood was also valued, and finally the extracellular regulated kinase (ERK) expression in bone was determined. TLM considerably improved the deleterious effect of D‐galactose on bone mineral density. It blunted serum bone specific alkaline phosphatase and osteocalcin while elevating serum osteoprotegrin (OPG). On the other hand, TLM turned off the pronounced elevation in serum receptor activator of nuclear factor‐κβ ligand (RANKL), tartrate‐resistant acid phosphatase and cathepsin‐k. Furthermore, it significantly hindered the bone expression of ERK which hampered osteoclastogenesis. AT1R inhibition abolished the rise in serum calcium and phosphorus and normalized serum superoxide dismutase and catalase. These TLM protective effects in D‐galactose‐treated rats were confirmed by the histopathological examination. The results all together denote the potential therapeutic value of ARBs therapy in osteoporosis.
Osteoclasts are cells of haematopoietic origin that are uniquely specialized to degrade bone. Under physiological conditions, the osteoclastogenesis pathway depends on macrophage colony-stimulating factor 1 (CSF-1, also known as M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). However, an emerging hypothesis is that alternative pathways of osteoclast generation might be active during inflammatory arthritis. In this Perspectives article, we summarize the physiological pathway of osteoclastogenesis and then focus on experimental findings that support the hypothesis that infiltrating inflammatory cells and the cytokine milieu provide multiple routes to bone destruction. The precise identity of osteoclast precursor(s) is not yet known. We propose that myeloid cell differentiation during inflammation could be an important contributor to the differentiation of osteoclast populations and their associated pathologies. Understanding the dynamics of osteoclast differentiation in inflammatory arthritis is crucial for the development of therapeutic strategies for inflammatory joint disease in children and adults.
Hibiscus sabdariffa L. (Hs, roselle; Malvaceae) has been used traditionally as a food, in herbal drinks, in hot and cold beverages, as a flavouring agent in the food industry and as a herbal medicine. In vitro and in vivo studies as well as some clinical trials provide some evidence mostly for phytochemically poorly characterised Hs extracts. Extracts showed antibacterial, anti-oxidant, nephro- and hepato-protective, renal/diuretic effect, effects on lipid metabolism (anti-cholesterol), anti-diabetic and anti-hypertensive effects among others. This might be linked to strong antioxidant activities, inhibition of α-glucosidase and α-amylase, inhibition of angiotensin-converting enzymes (ACE), and direct vaso-relaxant effect or calcium channel modulation. Phenolic acids (esp. protocatechuic acid), organic acid (hydroxycitric acid and hibiscus acid) and anthocyanins (delphinidin-3-sambubioside and cyanidin-3-sambubioside) are likely to contribute to the reported effects.
More well designed controlled clinical trials are needed which use phytochemically characterised preparations. Hs has an excellent safety and tolerability record.
We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytotic route from the ruffled border to the functional secretory domain (FSD) in bone-penetrating osteoclasts. Cells of rat or human origin were cultured on bovine bone slices and analyzed via confocal microscopy. Helix pomatia lectin binding indicated that transcytotic vesicles expose aberrant N-acetylgalactosamine glycoconjugates, which is associated with a poor prognosis for a range of metastasizing human adenocarcinomas. Transcytotic vesicles fuse with the autophagosomal compartments and represent raft concentrates. Furthermore, the results of a vertical vesicle analysis suggest that multiple vesicle populations arise from the ruffled border and that some of these vesicles undergo a maturation process along the transcytotic route. Finally, our data suggest that the targeting of these membrane pathways may be determined by a novel F-actin-containing and FSD-circumscribing molecular barrier.
Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl− channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H+-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H+-ATPase of the osteoclast ruffled membrane.
Autophagy describes the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. Autophagy is essentially required to prevent accumulation of cellular damage and to ensure cellular homeostasis. Indeed, impaired autophagy has been implicated in a variety of different diseases. We examined the role of autophagy in inflammatory bone loss. We demonstrated that autophagy is activated by the pro-inflammatory cytokine tumor necrosis factor (TNF/TNFα) in osteoclasts of patients with rheumatoid arthritis (RA). Autophagy induces osteoclast differentiation and stimulates osteoclast-mediated bone resorption in vitro and in vivo, thereby highlighting autophagy as a novel mediator of TNF-induced bone resorption.
Purpose:
To evaluate the protective effect of gossypetin (GTIN) against gamma (γ)-radiation-mediated DNA damage.
Materials and methods:
Increasing concentrations (10-150 μM) of GTIN were incubated with supercoiled DNA 1 h prior exposure to γ-radiation in the range of 5-Gy absorbed dose from Co(60) γ source. To establish the effective protective concentration of GTIN, supercoiled DNA was pre-incubated with 50 μM of GTIN for 1 h followed by exposure of 5, 10 and 20 Gy doses of γ-radiation. Moreover, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical, nitric oxide (NO) scavenging, metal chelating activity and ferric reducing antioxidant power (FRAP) of GTIN were measured and compared with standards. The flowcytometric analysis and radiation-induced genomic DNA damage by comet assay were employed to estimate the level of intracellular reactive oxygen species (ROS) using isolated murine hepatocytes.
Results:
GTIN was able to effectively scavenge different free radicals in in vitro situations. It could significantly prevent radiation induced supercoiled and genomic DNA damage with reduced comet parameters. It also acted as a potent scavenger of the radiation induced ROS.
Conclusions:
GTIN ameliorated radiation-induced oxidative stress and DNA damage by its free-radical scavenging activity.
We investigated the role of Akt, a downstream effector of phosphatidylinositol 3-kinase, in bone-resorbing activity of mature osteoclasts. Treatment with a specific Akt inhibitor disrupted sealing zone formation and decreased the bone-resorbing activity of osteoclasts. The normal microtubule structures were lost and the Akt inhibitor reduced the amount of acetylated tubulin, which reflects stabilized microtubules, whereas forced Akt activation by adenovirus vectors resulted in the opposite effect. Forced Akt activation increased the binding of the microtubule-associated protein APC, the APC-binding protein end-binding protein 1 (EB1) and dynactin, a dynein activator complex, with microtubules. Depletion of Akt1 and Akt2 resulted in a disconnection of APC/EB1 and a decrease in bone-resorbing activity along with reduced sealing zone formation, both of which were recovered upon the addition of LiCl, a GSK-3β inhibitor. The Akt1 and Akt2 double knock-out mice exhibited osteosclerosis due to reduced bone resorption. These findings indicate that Akt controls the bone-resorbing activity of osteoclasts by stabilizing microtubules via a regulation of the binding of microtubule associated proteins. © 2012 American Society for Bone and Mineral Research.
It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
Osteoclasts are multinucleated cells that are responsible for resorption of bone, and increased activity of these cells is associated with several common bone diseases, including postmenopausal osteoporosis. Upon adhesion to bone, osteoclasts become polarized and reorganise their cytoskeleton and membrane to form unique domains including the sealing zone (SZ), which is a dense ring of F-actin-rich podosomes delimiting the ruffled border (RB), where protons and proteases are secreted to demineralise and degrade the bone matrix, respectively. These processes are dependent on the activity of small GTPases. Rho GTPases are well known to control the organization of F-actin and adhesion structures of different cell types, affecting subsequently their migration. In osteoclasts, RhoA, Rac, Cdc42, RhoU and also Arf6 regulate podosome assembly and their organization into the SZ. By contrast, the formation of the RB involves vesicular trafficking pathways that are regulated by the Rab family of GTPases, in particular lysosomal Rab7. Finally, osteoclast survival is dependent on the activity of Ras GTPases. The correct function of almost all these GTPases is absolutely dependent on post-translational prenylation, which enables them to localize to specific target membranes. Bisphosphonate drugs, which are widely used in the treatment of bone diseases such as osteoporosis, act by preventing the prenylation of small GTPases, resulting in the loss of the SZ and RB and therefore inhibition of osteoclast activity, as well as inducing osteoclast apoptosis. In this review we summarize current understanding of the role of specific prenylated small GTPases in osteoclast polarization, function and survival.
Vacuolar H+-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role
in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait
construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts
(B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between
NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts.
Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC50 of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or
receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors
may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.
Osteoclasts are multinucleated bone-resorbing cells responsible for constant remodeling of bone tissue and for maintaining calcium homeostasis. The osteoclast creates an enclosed space, a lacuna, between their ruffled border membrane and the mineralized bone. They extrude H(+) and Cl(-) into these lacunae by the combined action of vesicular H(+)-ATPases and ClC-7 exchangers to dissolve the hydroxyapatite of bone matrix. Along with intracellular production of H(+) and HCO(3)(-) by carbonic anhydrase II, the H(+)-ATPases and ClC-7 exchangers seems prerequisite for bone resorption, because genetic disruption of either of these proteins leads to osteopetrosis. We aimed to complete the molecular model for lacunar acidification, hypothesizing that a HCO(3)(-) extruding and Cl(-) loading anion exchange protein (Ae) would be necessary to sustain bone resorption. The Ae proteins can provide both intracellular pH neutrality and serve as cellular entry mechanism for Cl(-) during bone resorption. Immunohistochemistry revealed that Ae2 is exclusively expressed at the contra-lacunar plasma membrane domain of mouse osteoclast. Severe osteopetrosis was encountered in Ae2 knockout (Ae2-/-) mice where the skeletal development was impaired with a higher diffuse radio-density on x-ray examination and the bone marrow cavity was occupied by irregular bone speculae. Furthermore, osteoclasts in Ae2-/- mice were dramatically enlarged and fail to form the normal ruffled border facing the lacunae. Thus, Ae2 is likely to be an essential component of the bone resorption mechanism in osteoclasts.
Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruffled borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts in vitro revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.
The alpha(v)beta(3) integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to alpha(v)beta(3), inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of alpha(v)beta(3) integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC(50 )= 0.1 nM) than those required to detach osteoclasts from bone (IC(50 ) approximately 1 microM); (2) echistatin (IC(50 )= 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) alpha(v)beta(3) integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), alpha(v)beta(3) disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC(50 )= 13 nM), and alpha(v)beta(3) visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that alpha(v)beta(3) integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC(50 )= 0.1 nM) and maintenance of the sealing zone (IC(50) approximately 10 nM).
Osteoclasts are multinucleated cells responsible for bone resorption. They have developed an efficient machinery for dissolving crystalline hydroxyapatite and degrading organic bone matrix rich in collagen fibers. When initiating bone resorption, osteoclasts become polarized, and three distinct membrane domains appear: a ruffled border, a sealing zone and a functional secretory domain. Simultaneously, the cytoskeleton undergoes extensive re-organisation. During this process, the actin cytoskeleton forms an attachment ring at the sealing zone, the membrane domain that anchors the resorbing cell to bone matrix. The ruffled border appears inside the sealing zone, and has several characteristics of late endosomal membrane. Extensive vesicle transport to the ruffled border delivers hydrochloric acid and proteases to an area between the ruffled border and the bone surface called the resorption lacuna. In this extracellular compartment, crystalline hydroxyapatite is dissolved by acid, and a mixture of proteases degrades the organic matrix. The degradation products of collagen and other matrix components are endocytosed, transported through the cell and exocytosed through a functional secretory domain. This transcytotic route allows osteoclasts to remove large amounts of matrix-degradation products without losing their tight attachment to underlying bone. It also facilitates further processing of the degradation products intracellularly during the passage through the cell.
Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion-activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion-targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2-gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.
Collectively, plants contain several different families of natural products among which are compounds with weak estrogenic or antiestrogenic activity toward mammals. These compounds, termed phytoestrogens, include certain isoflavonoids, flavonoids, stilbenes, and lignans. The best-studied dietary phytoestrogens are the soy isoflavones and the flaxseed lignans. Their perceived health beneficial properties extend beyond hormone-dependent breast and prostate cancers and osteoporosis to include cognitive function, cardiovascular disease, immunity and inflammation, and reproduction and fertility. In the future, metabolic engineering of plants could generate novel and exquisitely controlled dietary sources with which to better assess the potential health beneficial effects of phytoestrogens.
The anti-obesity potential of dietary fibre (DF) from Hibiscus sabdariffa (ground calyces) and Agave tequilana (agave-fructans) as an additive in the feed of animals subjected to a high fat diet was examined. Over a 5-week period, the effects of each DF alone, and in combination, were examined with respect to food intake, weight gain, adiposity, plasma metabolic profile, appetite regulation and inflammatory biomarkers in white and brown adipose tissue. Supplementation with the ground H. sabdariffa calyces was associated with a reduction in body weight, adiposity, plasma total cholesterol and glucose. As novel findings, the addition of H. sabdariffa increased plasma GLP1, reduced proinflammatory cytokines in white adipose tissue and increased IL6 in brown adipose tissue; it therefore appears to be a promising source of functional DF. These effects were partially maintained when H. sabdariffa was supplied in combination with A. tequilana DF and were independent of appetite reduction.
Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury. Gossypetin, a naturally occurring hexahydroxy flavone, has been shown to possess antimutagenic, antioxidant, antimicrobial, and antiatherosclerotic effects. In this study, the atheroprotective role of gossypetin was examined in endothelial cells. The protective effect of gossypetin against ox-LDL-induced injury in human umbilical vein endothelial cells (HUVECs) was first noted at 0.1-0.5 μM. Gossypetin showed potential in reducing the ox-LDL-dependent apoptosis as demonstrated by morphological and biochemical features, including apoptotic bodies formation, distribution of hypodiploid phase and caspase-3 activation. Next, the ox-LDL-induced formation of acidic vesicular organelles and the upregulation of the autophagy-related genes (LC3 and Beclin-1) were enhanced by gossypetin. The gossypetin-triggered autophagic flux was further confirmed by an increase of the LC3-II level under the pretreatment of an autophagy inhibitor chloroquine (CQ). In addition, silencing of Beclin-1 inhibited both the gossypetin mediated protective affects and the autophagic process. Molecular data indicated that autophagic effect of gossypetin might be mediated via class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt cascade signalings, as demonstrated by the usage of a class III PI3K inhibitor 3-methyladenine (3-MA), and a PTEN inhibitor SF1670. Finally, gossypetin improved atherosclerotic lesions and endothelial injury in vivo. Our data imply that gossypetin upregulates the autophagic pathway, which led to subsequent reduction of ox-LDL-induced atherogenic endothelial cell injury and apoptosis, and provide a new mechanism for the antiatherosclerotic activity of gossypetin.
Postmenopausal osteoporosis is a silent systemic progressive disease characterised by a decrease in bone mass per unit volume. This condition compromises the physical strength of the skeleton and increases the susceptibility to fractures on minor trauma. The imbalance between bone formation and bone resorption is known to be responsible for postmenopausal bone loss. Estrogen deficiency contributes to bone loss by increasing the production of pro-inflammatory cytokines by bone marrow and bone cells. Clinical and molecular evidence indicates that estrogen-regulated cytokines exert regulatory effects on bone turnover implicating their role as being the primary mediators of the accelerated bone loss at menopause. The current perspective on the role and interaction of cytokines such as IL-1, IL-4, IL-6, IL-17, TNF, IFN-γ and TGF-β in bone loss linked with estrogen deficiency is reviewed. Current treatment options and emerging drug therapies in the management of postmenopausal osteoporosis are also evaluated.
Background:
Bone remodelling maintains skeletal integrity by osteoclasts removing foci of damaged bone and osteoblasts replacing them with new bone. Diseases associated with increased bone resorption have increased remodelling often with inadequate bone formation and increased risk of fracture. New therapies are needed for these diseases to reduce resorption and increase formation.
Design:
The molecular mechanisms regulating osteoclast and osteoblast functions have become better understood in the past 20 years and have led to questioning of the long-held notion that osteoblastic cells have the dominant regulatory role over osteoclastic cells in bone remodelling. Here, we review current knowledge of how osteoclast formation and functions are regulated and describe how enhanced understanding of these has led to development of new drugs for the management of common bone diseases characterized by increased bone resorption.
Results:
Osteoclast formation and functions are regulated by cytokines, especially receptor activator of NF-κB ligand (RANKL) and macrophage-colony-stimulating factor (M-CSF). The differentiation, activity and lifecycle of osteoclasts are regulated in part by other cells that reside within the bone. These include osteoblasts, osteocytes and immune cells, which express these cytokines in response to most factors that promote bone resorption. RANKL and M-CSF activate numerous signalling pathways, which are potential targets for therapeutic intervention. Importantly, osteoclastic cells also function as positive and negative regulators of osteoblastic bone formation.
Conclusions:
There are multiple targets within osteoclasts for pharmacologic intervention to prevent bone loss in osteoporosis and other resorptive bone diseases. However, novel therapies could also affect osteoblastic cell functions.
Bone-remodeling imbalance induced by increased osteoclast formation and bone resorption is known to cause skeletal diseases such as osteoporosis. The reduction of estrogen levels at menopause is one of the strongest risk factors developing postmenopausal osteoporosis. This study investigated osteoprotective effects of the dihydrochalcone phloretin found in apple tree leaves on bone loss in ovariectomized (OVX) C57BL/6 female mice as a model for postmenopausal osteoporosis. OVX demoted bone mineral density (BMD) of mouse femurs, reduced serum 17β-estradiol level and enhanced serum receptor activator of NF-κB ligand (RANKL)/osteoprotegerin ratio with uterine atrophy. Oral administration of 10mg/kg phloretin to OVX mice for 8 weeks improved such effects, compared to sham-operated mice. Phloretin attenuated TRAP activity and cellular expression of β3 integrin and carbonic anhydrase II augmented in femoral bone tissues of OVX mice. This study further examined that osteogenic activity of phloretin in RANKL-differentiated Raw 264.7 macrophages into mature osteoclasts. Phloretin at 1-20μM stimulated Smac expression and capase-3 activation concurrently with nuclear fragmentation of multi-nucleated osteoclasts, indicating that this compound promoted osteoclast apoptosis. Consistently, phloretin enhanced bcl-2 induction but diminished bax expression. Furthermore, phloretin activated ASK-1-diverged JNK and p38 MAPK signaling pathways in mature osteoclasts, whereas it dose-dependently inhibited the RANKL-stimulated activation of ERK. Therefore, phloretin manipulated ASK-1-MAPK signal transduction leading to transcription of apoptotic genes. Phloretin was effective in preventing estrogen deficiency-induced osteoclastogenic resorption.
Kaempferol is one of the most common flavonoid that is present in a variety of vegetables and fruits and has effects on bone metabolism. The present study was performed to define the effects of kaempferol on interleukin (IL)-1β-stimulated receptor activator of NF-κB ligand (RANKL)-mediated osteoclast differentiation. Bone marrow cells were harvested from 6-week-old male imprinting control region mice, and the differentiation of osteoclasts from these cells was evaluated by tartrate-resistant acid phosphatase staining and resorption pit formation assay. Phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated p38, phosphorylated c-Jun amino-terminal kinase, NF-κB (p65), IκBα, c-Fos, and nuclear factor of activated T cells c1 (NFATc1) expressions were examined by Western blotting and quantitative RT-PCR. Kaempferol inhibits IL-1β-stimulated, RANKL-mediated osteoclast differentiation and also inhibits IL-1β-stimulated, RANKL-mediated phosphorylation of ERK 1/2, p38 and JNK MAP kinases, and expressions of c-Fos and NFATc1. These results indicate that kaempferol has an inhibitory role in the bone loss by preventing osteoclast formation and suggest that it might be a novel therapeutic agent for the treatment of inflammatory arthritis by managing bone destruction.
Chronic inflammatory processes close to bone often lead to loss of bone in diseases such as rheumatoid arthritis, periodontitis, loosened joint prosthesis and tooth implants. This is mainly due to local formation of bone resorbing osteoclasts which degrade bone without any subsequent coupling to new bone formation. Crucial for osteoclastogenesis is stimulation of mononuclear osteoclast progenitors by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) which induces their differentiation along the osteoclastic lineage and the fusion to mature, multinucleated osteoclasts. M-CSF and RANKL are produced by osteoblasts/osteocytes and by synovial and periodontal fibroblasts and the expression is regulated by pro- and anti-inflammatory cytokines. These cytokines also regulate osteoclastic differentiation by direct effects on the progenitor cells. In the present overview, we introduce the basic concepts of osteoclast progenitor cell differentiation and summarize the current knowledge on cytokines stimulating and inhibiting osteoclastogenesis by direct and indirect mechanisms.
Gossypetin, a flavone originally isolated from Hibiscus species, has been shown to possess antioxidant, antimicrobial, and antimutagenic activities. Here, we investigated the mechanism(s) underlying the anti-atherosclerotic potential of gossypetin. 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay showed the addition of > 50 μM of gossypetin could scavenge over 50% of DPPH radicals. The inhibitory effects of gossypetin on the lipid and protein oxidation of LDL were defined by thiobarbituric acid relative substances (TBARS) assay, the relative electrophoretic mobility (REM) of oxidized LDL (ox-LDL), and fragmentation of apoB in the Cu(2+)-induced oxidation of LDL. Gossypetin showed potential in reducing ox-LDL-induced foam cell formation and intracellular lipid accumulation, and uptake ability of macrophages under non-cytotoxic concentrations. Molecular data showed that these influences of gossypetin might be mediated via peroxisome proliferator-activated receptor α (PPARα)/ liver-X receptor α (LXRα)/ ATP-binding cassette transporter A1 (ABCA1) and PPARγ/ scavenger receptor CD36 pathways, as demonstrated by the transfection of PPARα siRNA or PPARγ expression vector. Our data implied that gossypetin regulated the PPARs signals, which in turn led to stimulation of cholesterol removal from macrophages and delay atherosclerosis. These results suggested that gossypetin potentially could be developed as an anti-atherosclerotic agent.
Context
Although postmenopausal hormone replacement therapy (HRT) is widely
used in the United States, new evidence about its benefits and harms requires
reconsideration of its use for the primary prevention of chronic conditions.Objective
To assess the benefits and harms of HRT for the primary prevention of
cardiovascular disease, thromboembolism, osteoporosis, cancer, dementia, and
cholecystitis by reviewing the literature, conducting meta-analyses, and calculating
outcome rates.Data Sources
All relevant English-language studies were identified in MEDLINE (1966-2001),
HealthSTAR (1975-2001), Cochrane Library databases, and reference lists of
key articles. Recent results of the Women's Health Initiative (WHI) and the
Heart and Estrogen/progestin Replacement Study (HERS) are included for reported
outcomes.Study Selection and Data Extraction
We used all published studies of HRT if they contained a comparison
group of HRT nonusers and reported data relating to HRT use and clinical outcomes
of interest. Studies were excluded if the population was selected according
to prior events or presence of conditions associated with higher risks for
targeted outcomes.Data Synthesis
Meta-analyses of observational studies indicated summary relative risks
(RRs) for coronary heart disease (CHD) incidence and mortality that were significantly
reduced among current HRT users only, although risk for incidence was not
reduced when only studies that controlled for socioeconomic status were included.
The WHI reported increased CHD events (hazard ratio [HR], 1.29; 95% confidence
interval [CI], 1.02-1.63). Stroke incidence but not mortality was significantly
increased among HRT users in the meta-analysis and the WHI. The meta-analysis
indicated that risk was significantly elevated for thromboembolic stroke (RR,
1.20; 95% CI, 1.01-1.40) but not subarachnoid or intracerebral stroke. Risk
of venous thromboembolism among current HRT users was increased overall (RR,
2.14; 95% CI, 1.64-2.81) and was highest during the first year of use (RR,
3.49; 95% CI, 2.33-5.59) according to a meta-analysis of 12 studies. Protection
against osteoporotic fractures is supported by a meta-analysis of 22 estrogen
trials, cohort studies, results of the WHI, and trials with bone density outcomes.
Current estrogen users have an increased risk of breast cancer that increases
with duration of use. Endometrial cancer incidence, but not mortality, is
increased with unopposed estrogen use but not with estrogen with progestin.
A meta-analysis of 18 observational studies showed a 20% reduction in colon
cancer incidence among women who had ever used HRT (RR, 0.80; 95% CI, 0.74-0.86),
a finding supported by the WHI. Women symptomatic from menopause had improvement
in certain aspects of cognition. Current studies of estrogen and dementia
are not definitive. In a cohort study, current HRT users had an age-adjusted
RR for cholecystitis of 1.8 (95% CI, 1.6-2.0), increasing to 2.5 (95% CI,
2.0-2.9) after 5 years of use.Conclusions
Benefits of HRT include prevention of osteoporotic fractures and colorectal
cancer, while prevention of dementia is uncertain. Harms include CHD, stroke,
thromboembolic events, breast cancer with 5 or more years of use, and cholecystitis.
Approximately 38% of postmenopausal women in the United States in 1995
used hormone replacement therapy (HRT), estrogen with or without progestin,
to treat symptoms of menopause and prevent chronic conditions such as cardiovascular
disease and osteoporosis.1 Although treatment
of symptoms of menopause, such as hot flashes and urogenital atrophy, among
others, is a common indication for short-term use, potential preventive effects
of HRT on long-term health outcomes have become an increasingly important
consideration.
In 1996, the second US Preventive Services Task Force (USPSTF) determined
that there was insufficient evidence to recommend for or against HRT for all
women but thought that individual decisions should be based on patient risk
factors, an understanding of the probable benefits and harms, and personal
preferences.2 Many studies have been published
since these recommendations were released, including the first report from
the Women's Health Initiative (WHI),3 a large
randomized primary prevention trial, and the Heart and Estrogen/progestin
Replacement Study (HERS),4 a secondary prevention
trial reporting multiple outcomes.4- 6
This review was initiated to aid the current USPSTF in making new recommendations
that will be released this fall. The focus of the USPSTF is to develop recommendations
on screening, counseling, and chemoprophylaxis for asymptomatic populations.
We conducted systematic searches of the literature on postmenopausal
HRT use and its effectiveness for primary prevention of chronic conditions
and its effects on harmful outcomes. Treatment of symptoms of menopause and
use of HRT for treatment of a preexisting condition are outside the scope
of the USPSTF recommendation, and this literature was not reviewed. We focused
on primary outcomes such as myocardial infarction (MI) rather than intermediate
outcomes such as lipid levels. To provide an overview of benefits and harms,
we conducted several meta-analyses and used these results, as well as those
from selected published articles, to calculate numbers of events prevented
or caused by HRT in a hypothetical population of postmenopausal women.
Chronic inflammation affects bone metabolism leading to disequilibrium in the rates of bone resorption and repair and subsequently to local and generalized bone loss. Osteoporosis represents an important co-morbidity of Rheumatoid Arthritis (RA) patients, which exhibit increased fracture risk. Osteoclasts play a pivotal role in the development and progression of bone loss, while resident synovial cells such as T cells, monocytes and synovial fibroblasts have been identified as sources of osteoclast differentiation signals in RA. This process is mainly mediated through the receptor activator of nuclear-κappa B ligand (RANKL) signalling system, which is upregulated by numerous proinflammatory cytokines involved in the pathogenesis of RA. Improved knowledge of the association between cells and cytokines of the immune system and their relationship to bone remodelling has revealed several promising targets for the treatment of inflammatory bone loss in RA. In this respect, initiation of biologic therapies targeting inflammatory cytokines and/or lymphocyte activation have modified RA therapy not only by blocking local and systemic inflammatory cascades but also by providing beneficial effects against bone and joint degradation. In this article we briefly present the modern view of the mechanisms that govern inflammatory bone loss, highlighting the role of cytokine-induced molecular pathways, and discuss in detail the effects of different biologic treatment strategies on bone mass in RA patients.
Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast- and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their sphingosine kinase 1 (Sphk1) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of sphingosine-1-phosphate (S1P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S1P1,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S1P.
Aims:
Quercetin, a flavonoid present in vegetables, has anti-inflammatory properties and potential inhibitory effects on bone resorption. Up to date, the effect of quercetin on lipopolysaccharide (LPS)-induced osteoclastogenesis has not yet been reported. In the current study, we evaluated the effect of quercetin on LPS-induced osteoclast apoptosis and bone resorption.
Methods:
RAW264.7 cells were non-treated, treated with LPS alone, or treated with both LPS and quercetin. After treatment, the number of osteoclasts, cell viability, bone resorption and osteoclast apoptosis were measured. The expressions of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP9) and cathepsin K (CK) were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of receptor activator of nuclear factor-ĸB (RANK), tumor necrosis factor receptor-associated factor 6 (TRAF6), cyclooxygenase-2 (COX-2), Bax, Bcl-2 and mitogenactivated protein kinases (MAPKs) were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with MAPK inhibitors.
Results:
LPS directly promoted osteoclast differentiation of RAW264.7 cells and upregulated the protein expression of RANK, TRAF6 and COX-2; while quercetin significantly decreased the number of LPS-induced osteoclasts in a dose-dependent manner. None of the treatments increased cytotoxicity in RAW264.7 cells. Quercetin inhibited mRNA expressions of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2 in LPS-induced mature osteoclasts. Quercetin also induced apoptosis and inhibited bone resorptive activity in LPS-induced mature osteoclasts. Furthermore, quercetin promoted the apoptotic signaling pathway including increasing the phosphorylation of p38-MAPK, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), and Bax, while inhibited Bcl-2 expression.
Conclusions:
Quercetin could supress LPS-induced osteoclast bone resorption through blocking RANK signaling and inhibiting the expression of osteoclast-related genes. Quercetin also promoted LPS-induced osteoclast apoptosis via activation of the MAPK apoptotic signaling pathway. These findings suggest that quercetin could be of potential use as a therapeutic agent to treat bacteria-induced bone resorption.
Bone-remodeling imbalance resulting in more bone resorption than bone formation is known to cause skeletal diseases such as osteoporosis. Phloretin, a natural dihydrochalcone compound largely present in apple peels, possesses antiphotoaging, and antiinflammatory activity.
Phloretin inhibited receptor activator of NF-κB ligand (RANKL)-induced formation of multinucleated osteoclasts and diminished bone resorption area produced during the osteoclast differentiation process. It was also found that ≥ 10 μM phloretin reduced RANKL-enhanced tartrate-resistance acid phosphatase activity and matrix metalloproteinase-9 secretion in a dose-dependent manner. The phloretin treatment retarded RANKL-induced expression of carbonic anhydrase II, vacuolar-type H(+) -ATPase D2 and β3 integrin, all involved in the bone resorption. Furthermore, submicromolar phloretin diminished the expression and secretion of cathepsin K elevated by RANKL, being concurrent with inhibition of TRAF6 induction and NF-κB activation. RANKL-induced activation of nuclear factor of activated T cells c1 (NFATc1) and microphthalmia-associated transcription factor was also suppressed by phloretin.
These results demonstrate that the inhibition of osteoclast differentiation and bone resorption by phloretin entail a disturbance of TRAF6-NFATc1-NF-κB pathway triggered by RANKL. Therefore, phloretin may be a potential therapeutic agent targeting osteoclast differentiation and bone resorption in skeletal diseases such as osteoporosis.
The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.
Bone remodelling is an active and dynamic process that relies on the correct balance between bone resorption by osteoclasts and bone deposition by osteoblasts. Moreover, these two functions must be tightly coupled not only quantitatively, but also in time and space. When the coupling is lost, the correct bone mass could be compromised, leading to several skeletal pathologies. Indeed, bone loss and osteoporosis are the result of an increased osteoclast function and/or a reduced osteoblast activity. In contrast, other pathologies are related to osteoclast failure to resorbe bone, such as osteopetrosis, a rare genetic disorder characterized by an increased bone mass and also linked to an impairment of bone marrow functions. Starting from these assumptions, it is necessary to more deeply understand the molecular mechanisms regulating bone cell functions. Indeed, recent studies evidenced a complex interplay between the immune and skeletal systems, which share several regulatory molecules including cytokines, receptors and transcription factors. These data allowed to more deeply understand the mechanisms underlying bone mass regulation and could open new avenue to identify target molecules for alterantive therapies more efficacious against bone diseases.
Microtubule-associated protein 1 light chain-3 (LC3) plays a critical role in autophagosome formation during autophagy; however, its potential alternative functions remain largely unexplored. Here we demonstrate a discrete role for LC3 in osteoclast, a specialized bone-resorbing cell that requires a dynamic microtubule network for its activity. We found that an increase in the conversion of soluble LC3-I to lipid-bound LC3-II in mature osteoclast was correlated with osteoclast activity, but not with autophagic activity. Knockdown of LC3 using small interfering RNA did not affect TRAP-positive multinucleated cell formation, but suppressed actin ring formation, cathepsin K release, and the subsequent bone-resorbing capacity of osteoclasts. LC3 mediated this function by associating with microtubules and regulating Cdc42 activity. More importantly, LC3-II protein levels were reduced by the Atg5 knockdown, and this knockdown led to decrease in Cdc42 activity, indicating that LC3-II is critical for Cdc42 activity. Overexpression of a constitutively active form of Cdc42 partially rescued the phenotype induced by LC3 knockdown. Our results demonstrate that LC3 contributes to the regulatory link between the microtubule and Cdc42 involved in bone-resorbing activity, providing evidence for a role for LC3 in mediating diverse cellular functions beyond its role as an autophagy protein.
The osteoclast represents one of the most highly specialized cells within the human body, which operates within a microenvironment of diverse cellular populations and matrix proteins. Moreover, the osteoclast directly effects and is affected by these surroundings in a delicate relationship of cellular differentiation, bone resorption, and controlled apoptosis. The result is the maintenance of adequate bone mass throughout life. Unsurprisingly, disturbances within this environment or the molecular regulation of normal osteoclast biology has profound effects on skeletal homeostasis and crippling physical manifestations. This review will summarize current literature describing normal and pathological osteoclast biology and highlight the benefits of osteoclast-targeted therapy to combat skeletal disorders.
Osteoclasts resorb bone via the ruffled border, whose complex folds are generated by secretory lysosome fusion with bone-apposed plasma membrane. Lysosomal fusion with the plasmalemma results in acidification of the resorptive microenvironment and release of CatK to digest the organic matrix of bone. The means by which secretory lysosomes are directed to fuse with the ruffled border are enigmatic. We show that proteins essential for autophagy, including Atg5, Atg7, Atg4B, and LC3, are important for generating the osteoclast ruffled border, the secretory function of osteoclasts, and bone resorption in vitro and in vivo. Further, Rab7, which is required for osteoclast function, localizes to the ruffled border in an Atg5-dependent manner. Thus, autophagy proteins participate in polarized secretion of lysosomal contents into the extracellular space by directing lysosomes to fuse with the plasma membrane. These findings are in keeping with a putative link between autophagy genes and human skeletal homeostasis.
The unique ability of the osteoclasts to resorb the calcified bone matrix is dependent on secretion of hydrochloric acid. This process is mediated by a vacuolar H+ ATPase (V-ATPase) and a chloride-proton antiporter. The structural subunit of the V-ATPase, a3, is highly specific for osteoclasts, and mutations in a3 lead to infantile malignant osteopetrosis, a phenomenon characterized by increased bone mass, an increased number of non-resorbing osteoclasts, and a complete lack of bone resorption. Importantly, these individuals have normal or even increased osteoblast numbers and bone formation suggesting that the osteoclasts, but not their resorptive capability, relay an anabolic signal, and, hence, that bone formation can be uncoupled from bone resorption when the a3 subunit is eliminated by mutations, or possibly by pharmacological intervention. The pharmacological profile of the a3 subunit as a highly specific target with a mode of action profile augmenting uncoupling and sustained bone formation, as derived from osteopetrotic patients and mice, highlights the relevance of the V-ATPase in future osteoporosis drug development. However, as illustrated by numerous attempts at developing specific inhibitors of the osteoclastic V-ATPase it is a very difficult target to work with, and an inhibitor possessing the desired profile remains elusive, although highly promising approaches recently have been launched.
Bone destruction is a frequent and clinically serious event in patients with rheumatoid arthritis (RA). Local joint destruction can cause joint instability and often necessitates reconstructive or replacement surgery. Moreover, inflammation-induced systemic bone loss is associated with an increased fracture risk. Bone resorption is a well-controlled process that is dependent on the differentiation of monocytes to bone-resorbing osteoclasts. Infiltrating as well as resident synovial cells, such as T cells, monocytes and synovial fibroblasts, have been identified as sources of osteoclast differentiation signals in RA patients. Pro-inflammatory cytokines are amongst the most important mechanisms driving this process. In particular, macrophage colony-stimulating factor, RANKL, TNF, IL-1 and IL-17 may play dominant roles in the pathogenesis of arthritis-associated bone loss. These cytokines activate different intracellular pathways to initiate osteoclast differentiation. Thus, over the past years several promising targets for the treatment of arthritic bone destruction have been defined.
Bone resorption is required for skeletal modelling during bone growth and for mineral homeostasis and bone remodelling throughout life. Osteoclasts are multinucleated cells that are uniquely specialised to carry out this physiological bone resorption. As osteolysis is a feature of most diseases of bone and joint, osteoclasts also play a role in pathological bone resorption, the extent of which is a function of the cellular and molecular mechanisms that govern their formation and function.
To treat systemic bone loss as in osteoporosis and/or focal osteolysis as in rheumatoid arthritis or periodontal disease, most approaches target the osteoclasts, the cells that resorb bone. Bisphosphonates are currently the most widely used antiresorptive therapies. They act by binding the mineral component of bone and interfere with the action of osteoclasts. The nitrogen-containing bisphosphonates, such as alendronate, act as inhibitors of farnesyl-pyrophosphate synthase, which leads to inhibition of the prenylation of many intracellular signaling proteins. The discovery of RANKL and the essential role of RANK signaling in osteoclast differentiation, activity and survival have led to the development of denosumab, a fully human monoclonal antibody. Denosumab acts by binding to and inhibiting RANKL, leading to the loss of osteoclasts from bone surfaces. In phase 3 clinical studies, denosumab was shown to significantly reduce vertebral, nonvertebral and hip fractures compared with placebo and increase areal BMD compared with alendronate. In this review, we suggest that the key pharmacological differences between denosumab and the bisphosphonates reside in the distribution of the drugs within bone and their effects on precursors and mature osteoclasts. This may explain differences in the degree and rapidity of reduction of bone resorption, their potential differential effects on trabecular and cortical bone, and the reversibility of their actions.
Integrins are a large family of heteromeric cell surface receptors composed of non-covalently bound alpha and beta subunits which interact with extracellular matrix molecules, serum constituents and the adhesion molecules of the immunoglobulin family. The extracellular domains of many integrins recognize the RGD (Arg-Gly-Asp) tripeptide found in several extracellular macromolecules such as fibronectin, vitronectin, fibrinogen and osteopontin. The vitronectin receptor, alpha v beta 3 integrin, is highly expressed in osteoclasts, the bone resorbing cells, and binds many of these RGD containing proteins including osteopontin, which is abundant in bone. Antibodies to alpha v beta 3, RGD peptides and RGD containing proteins such as echistatin, and kistrin were shown to inhibit bone resorption in vitro and in vivo. The identity of the alpha v beta 3 natural ligand and its mode of action in bone are so far not known. In addition to the very high levels of alpha v beta 3, mammalian osteoclasts also express alpha 2 beta 1, a collagen/laminin receptor and alpha v beta 1, another vitronectin receptor. Signaling events that follow substrate recognition by osteoclasts are not well understood. RGD containing peptides and proteins modulate [Ca2+] transients in osteoclasts and phosphatidylinositol 3-kinase and pp60c-src are associated with alpha v beta 3 in these cells. alpha v and beta 3 genes were shown to be regulated by the calciotropic hormone 1,25(OH)2D3 and by a number of cytokines known to be modulators of bone metabolism. In summary, elucidation of the interactions of osteoclast integrins with components of bone matrix, may lead to further understanding of the mechanism of bone resorption.
The skeleton is a metabolically active organ that undergoes continuous remodeling throughout life. This remodeling is necessary both to maintain the structural integrity of the skeleton and to subserve its metabolic functions as a storehouse of calcium and phosphorus. These dual functions often come into conflict under conditions of changing mechanical forces or metabolic and nutritional stress. The bone remodeling cycle involves a complex series of sequential steps that are highly regulated. The "activation" phase of remodeling is dependent on the effects of local and systemic factors on mesenchymal cells of the osteoblast lineage. These cells interact with hematopoietic precursors to form osteoclasts in the "resorption" phase. Subsequently, there is a "reversal" phase during which mononuclear cells are present on the bone surface. They may complete the resorption process and produce the signals that initiate formation. Finally, successive waves of mesenchymal cells differentiate into functional osteoblasts, which lay down matrix in the "formation" phase. The effects of calcium-regulating hormones on this remodeling cycle subserve the metabolic functions of the skeleton. Other systemic hormones control overall skeletal growth. The responses to changes in mechanical force and repair of microfractures, as well as the maintenance of the remodeling cycle, are determined locally by cytokines, prostaglandins, and growth factors. Interactions between systemic and local factors are important in the pathogenesis of osteoporosis as well as the skeletal changes in hyperparathyroidism and hyperthyroidism. Local factors are implicated in the pathogenesis of the skeletal changes associated with immobilization, inflammation, and Paget disease of bone.
Osteoporosis, a disease endemic in Western society, typically reflects an imbalance in skeletal turnover so that bone resorption
exceeds bone formation. Bone resorption is the unique function of the osteoclast, and anti-osteoporosis therapy to date has
targeted this cell. The osteoclast is a specialized macrophage polykaryon whose differentiation is principally regulated by
macrophage colony-stimulating factor, RANK ligand, and osteoprotegerin. Reflecting integrin-mediated signals, the osteoclast
develops a specialized cytoskeleton that permits it to establish an isolated microenvironment between itself and bone, wherein
matrix degradation occurs by a process involving proton transport. Osteopetrotic mutants have provided a wealth of information
about the genes that regulate the differentiation of osteoclasts and their capacity to resorb bone.
Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclast and bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases. This study summarizes the characterization and role of each type of ion transport and defines the main biochemical mechanisms involved in the dissolution of bone mineral during bone resorption. The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar H+-adenosine triphosphatase (ATPase) coupled with Cl- conductance localized to the ruffled membrane. Carbonic anhydrase II (CAII) provides the proton source for extracellular acidification by H+-ATPase and the HCO3- source for the HCO3-/Cl- exchanger. Whereas some transporters are responsible for the bone resorption process, others are essential for pH regulation in the osteoclast. The HCO3-/Cl- exchanger, in association with CAII, is the major transporter for maintenance of normal intracellular pH. An Na+/H+ antiporter may also contribute to the recovery of intracellular pH during early osteoclast activation. Once this mechanism has been rendered inoperative, another conductive pathway translocates the protons and modulates cytoplasmic pH. Inward-rectifying K+ channels may also be involved by compensating for the external acidification due to H+ transport. These different effects of transport processes, either on bone resorption or pH homeostasis, increase the number of possible sites for pharmacological intervention in the treatment of metabolic bone diseases.
Patients with pycnodysostosis, a rare skeletal dysplasia, present with bone abnormalities such as short stature, acroosteolysis of distal phalanges, and skull deformities. The disease is caused by a deficiency of the cysteine protease cathepsin K which is responsible for degradation of collagen type I and other bone proteins. Osteoclasts, bone cells of hematopoietic origin responsible for bone mineral as well as protein matrix degradation, are dysfunctional in patients with pycnodysostosis due to mutations in the cathepsin K gene. Cathepsin K deficient osteoclasts can demineralize bone but cannot degrade the protein matrix. Mutations in the cathepsin K gene disrupting wild type cathepsin K activity have been described in patients with pycnodysostosis. Animal models of cathepsin K deficiency have been created and provide a valuable tool to study osteoclast function and treatment for cathepsin K deficiency. Understanding the regulation and role of cathepsin K in osteoclast function is important for designing future therapies for pycnodysostosis. Cathepsin K inhibitors will be useful in pathological processes involving excess osteoclast activation and bone resorption such as osteoporosis, bone metastasis and multiple myeloma. This review will discuss the bone remodeling cycle, the human disease pycnodysostosis caused by cathepsin K deficiency and cathepsin K activity and regulation.
Although postmenopausal hormone replacement therapy (HRT) is widely used in the United States, new evidence about its benefits and harms requires reconsideration of its use for the primary prevention of chronic conditions.
To assess the benefits and harms of HRT for the primary prevention of cardiovascular disease, thromboembolism, osteoporosis, cancer, dementia, and cholecystitis by reviewing the literature, conducting meta-analyses, and calculating outcome rates.
All relevant English-language studies were identified in MEDLINE (1966-2001), HealthSTAR (1975-2001), Cochrane Library databases, and reference lists of key articles. Recent results of the Women's Health Initiative (WHI) and the Heart and Estrogen/progestin Replacement Study (HERS) are included for reported outcomes.
We used all published studies of HRT if they contained a comparison group of HRT nonusers and reported data relating to HRT use and clinical outcomes of interest. Studies were excluded if the population was selected according to prior events or presence of conditions associated with higher risks for targeted outcomes.
Meta-analyses of observational studies indicated summary relative risks (RRs) for coronary heart disease (CHD) incidence and mortality that were significantly reduced among current HRT users only, although risk for incidence was not reduced when only studies that controlled for socioeconomic status were included. The WHI reported increased CHD events (hazard ratio [HR], 1.29; 95% confidence interval [CI], 1.02-1.63). Stroke incidence but not mortality was significantly increased among HRT users in the meta-analysis and the WHI. The meta-analysis indicated that risk was significantly elevated for thromboembolic stroke (RR, 1.20; 95% CI, 1.01-1.40) but not subarachnoid or intracerebral stroke. Risk of venous thromboembolism among current HRT users was increased overall (RR, 2.14; 95% CI, 1.64-2.81) and was highest during the first year of use (RR, 3.49; 95% CI, 2.33-5.59) according to a meta-analysis of 12 studies. Protection against osteoporotic fractures is supported by a meta-analysis of 22 estrogen trials, cohort studies, results of the WHI, and trials with bone density outcomes. Current estrogen users have an increased risk of breast cancer that increases with duration of use. Endometrial cancer incidence, but not mortality, is increased with unopposed estrogen use but not with estrogen with progestin. A meta-analysis of 18 observational studies showed a 20% reduction in colon cancer incidence among women who had ever used HRT (RR, 0.80; 95% CI, 0.74-0.86), a finding supported by the WHI. Women symptomatic from menopause had improvement in certain aspects of cognition. Current studies of estrogen and dementia are not definitive. In a cohort study, current HRT users had an age-adjusted RR for cholecystitis of 1.8 (95% CI, 1.6-2.0), increasing to 2.5 (95% CI, 2.0-2.9) after 5 years of use.
Benefits of HRT include prevention of osteoporotic fractures and colorectal cancer, while prevention of dementia is uncertain. Harms include CHD, stroke, thromboembolic events, breast cancer with 5 or more years of use, and cholecystitis.
Osteoclasts are multinucleated hematopoietic cells specialised for bone resorption. Dissolution of the inorganic fraction of the bone matrix is mediated by acidification of the bone surface in contact with the osteoclast whereas secreted lysosomal enzymes digest organic components. Through massive exocytosis, the plasma membrane in contact with the bone surface enlarges into the ruffled border, which has unusual features more similar to endosomal/lysosomal membranes. Maintenance of the ruffled border during resorption is achieved through a balance between exocytosis and endocytosis. Inactivation of proteins necessary for the extracellular acidification or of the proteases involved in matrix degradation leads to osteopetrosis; a disease characterised by dense bones.
Osteoclasts are specialized cells derived from the monocyte/macrophage haematopoietic lineage that develop and adhere to bone matrix, then secrete acid and lytic enzymes that degrade it in a specialized, extracellular compartment. Discovery of the RANK signalling pathway in the osteoclast has provided insight into the mechanisms of osteoclastogenesis and activation of bone resorption, and how hormonal signals impact bone structure and mass. Further study of this pathway is providing the molecular basis for developing therapeutics to treat osteoporosis and other diseases of bone loss.
Hundreds of millions of people worldwide are affected by bone-related diseases, such as osteoporosis and rheumatoid arthritis. Understanding the molecular mechanisms of bone metabolism is crucial for developing novel drugs for treating such diseases. In particular, genetic experiments showing that the receptor activator of NF-kappaB (RANK), its ligand RANKL, and the decoy receptor OPG are essential, central regulators of osteoclast development and osteoclast function were significant turning points in our understanding of bone diseases. RANKL-RANK signaling activates a variety of downstream signaling pathways required for osteoclast development. Moreover, molecular cross-talk between RANKL-RANK and other ligand-receptor systems fine-tunes bone homeostasis in normal physiology and disease. Designing novel drugs that target RANKL-RANK and their signaling pathways in osteoclasts could potentially revolutionize the treatment of many diseases associated with bone loss such as arthritis, tooth loss, cancer metastases or osteoporosis.