Browsing School, Graduate by Subject "RANKL"
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Cyclic AMP (cAMP) as a Putative Second Messenger Communicated by Gap Junctions in BoneSkeletal homeostasis is crucial to maintaining bone quality. In the absence of balanced bone formation and bone resorption, skeletal disease develops. Numerous studies show that the gap junction protein connexin43 (Cx43) plays a role in the function of bone forming osteoblasts and osteocytes, as well as the coordination of bone resorption by these cells. Little is known about how Cx43 influence skeletal homeostasis. In particular, the identities of the second messenger signals that are communicated by bone cells to regulate skeletal homeostasis are unclear. Here, we assessed the role of cAMP as a biologically relevant second messenger communicated by Cx43 containing gap junctions among bone cells and its regulation of genes involved in skeletal homeostasis, including sclerostin and RANKL in UMR106- cells. Methods: UMR106-cells were cultured and transfected with different plasmid constructs to manipulate cAMP levels (constitutively active GalphaS), as well as Cx43 expression. Luciferase reporter assays and western blots were used to assess cAMP-dependent signaling. Regulation of the SOST and RANKL were assessed by quantitative RT-PCR. Results: The in vitro studies conducted have shown the synergism between Cx43 and G-alphaS, which generates cAMP, confirming the amplified signal when these two were expressed together. Further, a modified parachute assay revealed that cell-to-cell contact was required for the sharing of cAMP-dependent signals by Cx43 expressing bone cells. Cx43 and cAMP combine to potently increase RANKL (a stimulator of bone resorption) expression and suppress SOST expression (An inhibitor of bone formation). Conclusion: There was up regulated signals when Cx43 and G-alphaS were co-expressed implying the role of the second messenger cAMP in cell-to-cell communication by bone cells. In addition, these signals converged on the expression of RANKL and sclerostin, two genes that play a major role in bone turnover. Accordingly, this pathway may represent one of the ways that gap junctions regulate skeletal homeostasis and could be a target of therapeutic intervention for skeletal disease.
Interleukin-4: a molecular switch from osteoclasts to multinucleated giant cellsInterleukin 4 (IL-4) inhibited Receptor Activator of NF-κB Ligand (RANKL)-induced osteoclast (OC) differentiation, while at the same time promoted macrophage fusion to form multinucleated giant cells (MNG). This was dependent on the expression of signal transducer and activator of transcription-6 (STAT6). The precise mechanism of this is not understood. The regulation of one typical osteoclastogenic gene, tartrate-resistant acid phosphatase (TRAP), by IL-4 was studied in detail. RANKL significantly upregulated TRAP expression and its expression was inhibited in the presence of IL-4. We found that IL-4 activated-STAT6 directly bound to the TRAP promoter. However, the effect of STAT6 binding was to modestly enhance TRAP transcription. The ability of IL-4 to inhibit RANKL- induced TRAP was through an indirect mechanism: IL-4 significantly inhibited the expression of the transcription factor NFATc1. The IL-4-induced downregulation of NFATc1 was responsible for the downregulation of TRAP. Several groups have proposed that IL-4 inhibits osteoclastogenesis by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not inhibit the ability of RANKL to activate the canonical NF-κB pathway. Rather, IL-4 inhibited activation of the alternative pathway. IL-4 also upregulated p105/50 expression in the presence of RANKL. IL-4 did not inhibit osteoclastogenesis in p105/50 deficient bone marrow-derived macrophages (BMM). However, in p105/50 deficient BMM, the formation of both multinucleated OC and MNG induced by RANKL or IL-4 respectively was impaired. This suggests that NF-κB also played an important role in IL-4-induced macrophage fusion. In addition, an inhibitor of NF-κB activation effectively blocked both OC and MNG formation. Furthermore, transduction of BMM with p50, p65, p52 or RelB enhanced both OC and MNG formation. These results suggest that NF-κB molecules participates in the IL-4-induced MNG formation and that alteration of the NF-κB pathway may induce a switch in cell fate from OC to MNG.
Role of Lipopolysaccharide and RANKL in Osteoclastogenesis: Potential Inhibitory Effects of C-Phycocyanin on the Respective Molecular Pathways of OsteoclastogenesisMany skeletal diseases are characterized by excessive bone loss. Bone loss is mediated by osteoclasts, which are differentiated from cells of the monocyte/macrophage lineage upon stimulation of two indispensable factors, the RANKL and M-CSF. Lipopolysaccharide (LPS), a bacterial pathogenic factor, has also been shown to engage in osteoclastogenesis during inflammatory events actively. C-phycocyanin (C-PC) is a phycobiliprotein found in the blue-green algae that showed many therapeutic effects, including anti-arthritic and anti-inflammatory properties. However, the exact mechanism by which LPS regulates osteoclastogenesis and also the impact of C-PC on osteoclastogenesis needs further elucidations. We studied the osteoclast differentiation process in vitro using RAW 264.7 macrophage cell line. First, we showed that LPS induced osteoclastogenesis in RANKL-primed cells in vitro. LPS elicited osteoclastogenic mechanism by signaling through the TLR4 receptor, which is expressed in osteoclast precursors. Here we also found that TNF-α secreted by osteoclast precursors in response to TLR4 stimulation regulated the processes of osteoclastogenesis via TNF-R signaling. Second, we tested the inhibitory effect of C-PC on osteoclastogenesis. We showed here that C-PC strongly inhibited the early stage of osteoclast differentiation, thus significantly suppressing RANKL- and LPS- mediated osteoclastogenesis. Nonetheless, osteoblast differentiation and activity were not affected by C-PC. Reactive oxygen species (ROS) are generated during RANKL-mediated osteoclast differentiation. While studying the possible mechanisms of osteoclast differentiation, we found that C-PC a) attenuated RANKL-induced ROS; and b) interfered with RANKL-stimulated NF-κB signaling by preventing the degradation of cytosolic IκB-α; subsequently, these resulted in the loss of sequential activation of the osteoclastogenic downstream markers such as c-Fos and NFATc1. We propose that a unique mechanism of osteoclastogenesis is mediated by bacterial LPS that can be targeted during inflammatory-mediated bone loss. Also, C-PC could potentially be used as a therapeutic compound in osteolytic diseases caused by osteoclast activation without affecting osteoblast function.