Project description:This study sought to understand the regulation of an osteoclastic protein-tyrosine phosphatase (PTP-oc), a positive regulator of osteoclast activaty. Our past studies suggested that PTP-oc is regulated post-transcriptionally. The 3'-UTR of PTP-oc mRNA contains a target site for <i>miR17</i>. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of <i>miR17</i> (expressed as one of the six cluster genes of <i>miR17~92</i>) and <i>PTP-oc</i> mRNA. Overexpression of pre-<i>miR17~92</i> in mouse osteoclast precursors reduced <i>PTP-oc</i> mRNA level and the size of the derived osteoclasts; whereas deletion of <i>miR17~92</i> or inhibition of <i>miR17</i> resulted in the formation of larger osteoclasts containing more nuclei that expressed higher <i>PTP-oc</i> mRNA levels and created larger resorption pits. Thus, PTP-oc-mediated osteoclast activation is modulated in part by <i>miR17~92</i>, particularly <i>miR17</i>. The <i>miR17~92</i> osteoclast conditional knockout (cKO) mutants, generated by breeding <i>miR17~92<sup>loxp/loxp</sup></i> mice with <i>Ctsk-Cre</i> mice, had lower Tb.BV/TV, Tb.BMD, Tb.Conn-Dens, Tb.N, and Tb.Th, but larger Tb.Sp, and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow-derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of <i>miR17~92</i> in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular <i>miR17</i> level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, <i>miR17-92</i> negatively regulates the osteoclast activity, in part via the <i>miR17</i>-mediated suppression of PTP-oc in osteoclasts.
Project description:Bone homeostasis is a metabolic balance between the new bone formation by osteoblasts and old bone resorption by osteoclasts. Excessive osteoclastic bone resorption results in low bone mass, which is the major cause of bone diseases such as rheumatoid arthritis. Small GTPases Rac1 is a key regulator of osteoclast differentiation, but its exact mechanism is not fully understood. ELMO and DOCK proteins form complexes that function as guanine nucleotide exchange factors for Rac activation. Here, we report that ELMO1 plays an important role in differentiation and bone resorption of osteoclasts. Osteoclast precursors derived from bone marrow monocytes (BMMs) of Elmo1<sup>-/-</sup> mice display defective adhesion and migration during differentiation. The cells also have a reduced activation of Rac1, p38, JNK, and AKT in response to RANKL stimulation. Importantly, we show that bone erosion is alleviated in Elmo1<sup>-/-</sup> mice in a rheumatoid arthritis mouse model. Taken together, our results suggest that ELMO1, as a regulator of Rac1, regulates osteoclast differentiation and bone resorption both <i>in vitro</i> and <i>in vivo</i>.
Project description:Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating ?-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.
Project description:Activation of osteoclasts during orthodontic tooth treatment is a prerequisite for alveolar bone resorption and tooth movement. However, the key regulatory molecules involved in osteoclastogenesis during this process remain unclear. Long noncoding RNAs (lncRNAs) are a newly identified class of functional RNAs that regulate cellular processes, such as gene expression and translation regulation. Recently, lncRNAs have been reported to be involved in osteogenesis and bone formation. However, as the most abundant noncoding RNAs in vivo, the potential regulatory role of lncRNAs in osteoclast formation and bone resorption urgently needs to be clarified. We recently found that the lncRNA Nron (long noncoding RNA repressor of the nuclear factor of activated T cells) is highly expressed in osteoclast precursors. Nron is downregulated during osteoclastogenesis and bone ageing. To further determine whether Nron regulates osteoclast activity during orthodontic treatment, osteoclastic Nron transgenic (Nron cTG) and osteoclastic knockout (Nron CKO) mouse models were generated. When Nron was overexpressed, the orthodontic tooth movement rate was reduced. In addition, the number of osteoclasts decreased, and the activity of osteoclasts was inhibited. Mechanistically, Nron controlled the maturation of osteoclasts by regulating NFATc1 nuclear translocation. In contrast, by deleting Nron specifically in osteoclasts, tooth movement speed increased in Nron CKO mice. These results indicate that lncRNAs could be potential targets to regulate osteoclastogenesis and orthodontic tooth movement speed in the clinic in the future.
Project description:The axon guidance cue SLIT3 was identified as an osteoanabolic agent in two recent reports. However, these reports conflict in their nomination of osteoblasts versus osteoclasts as the key producers of skeletal SLIT3 and additionally offer conflicting data on the effects of SLIT3 on osteoclastogenesis. Here, aiming to address this discrepancy, we found no observable SLIT3 expression during human or mouse osteoclastogenesis and the only modest SLIT3-mediated effects on osteoclast differentiation. Conditional deletion of SLIT3 in cathepsin K (CTSK)-positive cells, including osteoclasts, had no effect on the number of osteoclast progenitors, in vitro osteoclast differentiation, overall bone mass, or bone resorption/formation parameters. Similar results were observed with the deletion of SLIT3 in LysM-positive cells, including osteoclast lineage cells. Consistent with this finding, bone marrow chimeras made from Slit3 -/- donors that lacked SLIT3 expression at all stages of osteoclast development displayed normal bone mass relative to controls. Taken in context, multiple lines of evidence were unable to identify the physiologic function of osteoclast-derived SLIT3, indicating that osteoblasts are the major source of skeletal SLIT3.
Project description:OBJECTIVE:To investigate whether ?-catenin signaling in chondrocytes regulates osteoclastogenesis, thereby contributing to postnatal bone growth and bone remodeling. METHODS:Mice with conditional knockout (cKO) or conditional activation (cAct) of chondrocyte-specific ?-catenin were generated. Changes in bone mass, osteoclast numbers, and osteoblast activity were examined. The mechanisms by which ?-catenin signaling in chondrocytes regulates osteoclast formation were determined. RESULTS:The ?-catenin cKO mice developed localized bone loss, whereas cAct mice developed a high bone mass phenotype. Histologic findings suggested that these phenotypes were caused primarily by impaired osteoclast formation, rather than impaired bone formation. Further molecular signaling analyses revealed that ?-catenin signaling controlled this process by regulating the expression of the RANKL and osteoprotegerin (OPG) genes in chondrocytes. Activation of ?-catenin signaling in chondrocytes suppressed Rankl gene transcription through a glucocorticoid receptor-dependent mechanism. The severe bone loss phenotype observed in ?-catenin cKO mice was largely restored by treatment with human recombinant OPG or transgenic overexpression of Opg in chondrocytes. CONCLUSION:?-catenin signaling in chondrocytes plays a key role in postnatal bone growth and bone remodeling through its regulation of osteoclast formation.
Project description:The cytokines RANKL and TNF activate NF-?B signaling in osteoclast precursors (OCPs) to induce osteoclast (OC) formation. Conversely, TNF can limit OC formation through NF-?B p100, which acts as an inhibitor, and TNF receptor-associated receptor 3 (TRAF3); however, a role for TRAF3 in RANKL-mediated OC formation is unknown. We found that TRAF3 limits RANKL-induced osteoclastogenesis by suppressing canonical and noncanonical NF-?B signaling. Conditional OC-specific Traf3-KO (cKO) mice had mild osteoporosis and increased OC formation. RANKL induced TRAF3 degradation via the lysosome/autophagy system. The autophagy/lysosome inhibitor chloroquine reduced RANKL-induced OC formation and function by increasing TRAF3 expression in OCPs in vitro and in vivo. Although chloroquine had no effect on basal bone resorption, it inhibited parathyroid hormone- and ovariectomy-induced OC activation in WT, but not cKO, mice. Deletion of the transcription factor gene Relb resulted in increased TRAF3 expression in OCPs, which was associated with decreased RANKL-induced TRAF3 degradation. RelB directly increased expression of BECN1, a key autophagy regulator, by binding to its promoter. These data indicate that autophagic/lysosomal degradation of TRAF3 is an important step in RANKL-induced NF-?B activation in OCPs. Furthermore, treatments that increase TRAF3 levels in OCPs, including pharmacological inhibition of its degradation with compounds such as chloroquine, may limit bone destruction in common bone diseases.
Project description:Although leukotriene B(4) (LTB(4)) is produced in various inflammatory diseases, its functions in bone metabolism remain unknown. Using mice deficient in the high-affinity LTB(4) receptor BLT1, we evaluated the roles of BLT1 in the development of two bone resorption models, namely bone loss induced by ovariectomy and lipopolysaccharide. Through observations of bone mineral contents and bone morphometric parameters, we found that bone resorption in both models was significantly attenuated in BLT1-deficient mice. Furthermore, osteoclasts from BLT1-deficient mice showed reduced calcium resorption activities compared with wild-type osteoclasts. Osteoclasts expressed BLT1, but not the low-affinity LTB(4) receptor BLT2, and produced LTB(4). LTB(4) changed the cell morphology of osteoclasts through the BLT1-Gi protein-Rac1 signaling pathway. Given the causal relationship between osteoclast morphology and osteoclastic activity, these findings suggest that autocrine/paracrine LTB(4) increases the osteoclastic activity through the BLT1-Gi protein-Rac1 signaling pathway. Inhibition of BLT1 functions may represent a strategy for preventing bone resorption diseases.
Project description:Cdc42 mediates bone resorption principally by stimulating osteoclastogenesis. Whether its sister GTPase, Rac, meaningfully impacts upon the osteoclast and, if so, by what means, is unclear. We find that whereas deletion of Rac1 or Rac2 alone has no effect, variable reduction of Rac1 in osteoclastic cells of Rac2(-/-) mice causes severe osteopetrosis. Osteoclasts lacking Rac1 and Rac2 in combination (Rac double-knockout, RacDKO), fail to effectively resorb bone. By contrast, osteoclasts are abundant in RacDKO osteopetrotic mice and, unlike those deficient in Cdc42, express the maturation markers of the cells normally. Hence, the osteopetrotic lesion of RacDKO mice largely reflects impaired function, and not arrested differentiation, of the resorptive polykaryon. The dysfunction of RacDKO osteoclasts represents failed cytoskeleton organization as evidenced by reduced motility of the cells and their inability to spread or generate the key resorptive organelles (i.e. actin rings and ruffled borders), which is accompanied by abnormal Arp3 distribution. The cytoskeleton-organizing capacity of Rac1 is mediated through its 20-amino-acid effector domain. Thus, Rac1 and Rac2 are mutually compensatory. Unlike Cdc42 deficiency, their combined absence does not impact upon differentiation but promotes severe osteopetrosis by dysregulating the osteoclast cytoskeleton.
Project description:Signal Transducer and Activator of Transcription 3 (STAT3) has recently been shown to be involved in bone development and has been implicated in bone diseases, such as Job's Syndrome. Bone growth and changes have been known for many years to differ between sexes with male bones tending to have higher bone mass than female bones and older females tending to lose bone mass at faster rates than older males. Previous studies using conditional knock mice with Stat3 specifically deleted from the osteoblasts showed both sexes exhibited decreased bone mineral density (BMD) and strength. Using the Cre-Lox system with Cathepsin K promotor driving Cre to target the deletion of the Stat3 gene in mature osteoclasts (STAT3-cKO mice), we observed that 8-week old STAT3-cKO female femurs exhibited significantly lower BMD and bone mineral content (BMC) compared to littermate control (CN) females. There were no differences in BMD and BMC observed between male knock-out and male CN femurs. However, micro-computed tomography (?CT) analysis showed that both male and female STAT3-cKO mice had significant decreases in bone volume/tissue volume (BV/TV). Bone histomorphometry analysis of the distal femur, further revealed a decrease in bone formation rate and mineralizing surface/bone surface (MS/BS) with a significant decrease in osteoclast surface in female, but not male, STAT3-cKO mice. Profiling gene expression in an osteoclastic cell line with a knockdown of STAT3 showed an upregulation of a number of genes that are directly regulated by estrogen receptors. These data collectively suggest that regulation of STAT3 differs in male and female osteoclasts and that inactivation of STAT3 in osteoclasts affects bone turnover more in females than males, demonstrating the complicated nature of STAT3 signaling pathways in osteoclastogenesis. Drugs targeting the STAT3 pathway may be used for treatment of diseases such as Job's Syndrome and osteoporosis.