TACE-Mediated Shedding Supports a Role for Semaphorin 4D Expressed on Osteoclasts in Up-Regulation of RANKL-Induced Osteoclastogenesis
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CitationKim, Jaeyoung. 2017. TACE-Mediated Shedding Supports a Role for Semaphorin 4D Expressed on Osteoclasts in Up-Regulation of RANKL-Induced Osteoclastogenesis. Doctoral dissertation, Harvard School of Dental Medicine.
AbstractBackground: This proposal targets a recently identified bone anabolic mechanism (Semaphorin 4D as an anti-coupling factor) that is elicited by bone cell-cell communication. During bone remodeling, osteoblast-activating molecules are released from demineralized bone matrix and osteoclasts to replace the same amount of resorbed bone by osteoclasts. According to Howard et al, those anabolic molecules released by activated osteoclast are termed as “coupling factors”. Important to this study, osteoclast was also found to produce an anti-coupling factor, Semaphorin 4D (Sema4D) to suppress osteoblastogenesis until bone resorption is accomplished. However, possible effects of Sema4D on RANKL-mediated osteoclastogenesis is still unknown. Besides, according to Li Zhu et al, extracellular domain of Sema4D is shed from the platelet surface by the metalloprotease TACE (TNF-converting enzyme), also known as ADAM17, is required for this to occur. However, expression and function of TACE on osteoclasts is also unknown. Based on these lines of evidence, it is hypothesized that TACE expressed on osteoclast cells would shed the membrane bound Sema4D into soluble Sema4D which in turn, promote RANKL-induced osteoclastogenesis and suppress osteoblastogenesis. To test above noted hypothesis, the aim of this study is to evaluate the possible impact of Sema4D on osteoclastogenesis by investigating the effect of anti-Sema4D monoclonal antibody (anti-Sema4D-mAb) on osteoclastogenesis and to evaluate the expression and function of TACE on osteoclast cells by examining the effects of silencing TACE mRNA and anti-TACE-mAb on osteoclastogenesis and osteoblastogenesis.
Materials and Methods: in vitro study, detecting the expression of Sema4D on osteoclast cells and effects of Sema4D were examined by western-blotting analysis and TRAP staining/Pit formation assay, respectively. ALP assay also performed to examine the effects of Sema4D on osteoblastogenesis by culturing osteoblast cells in the osteoclast cultured supernatant. Also, TACE and MT1-MMP expressed on osteoclast cells were detected via western blot assay, RT-PCR, and immune staining and fluorescence microscopy. Differentiation of osteoclast cells was evaluated through TRAP staining and pit formation assay under RNA mediated silencing of TACE and MT1-MMP in a loss of function confirmed by western blot and RT-PCR. Osteoblast cells are cultured with and without siTACE and siMT1-MMP treated osteoclast-cultured medium to examine the function of TACE and MT1-MMP on osteoblastogenesis through ALP assay. As for a proof of concept, the expression of membrane bound- and soluble Sema4D was detected by western blot, PCR and immunostaining and fluorescence microscope under treatment of anti TACE- and MT1-MMP mAb, followed by TRAP/pit formation assay and ALP was conducted on osteoclastogenesis and osteoblastogenesis, respectively.
Results: Sema4D was also expressed on osteoclast cells as membrane bound forms and soluble forms. The level of osteoclastogenesis was significantly suppressed in the group with anti-Sema4D-mAb compared to non-treated control group while recombinant Sema4D added group showed significant higher expression of osteoclastogenesis. Mature osteoclasts with more than 10 nuclei per cell have higher activity in pit formation (p<0.01). Furthermore, recombinant Sema4D and culture supernatant from RANKL-stimulated osteoclasts suppressed the in vitro osteoblastogenesis, as determined by ALP assay. TACE and MT1-MMP was also expressed on osteoclast cells. When they lost their function by silencing RNA, osteoclastogenesis was inhibited while osteoblastogenesis was enhanced. As proof of concept with monoclonal antibody, osteoclastogenesis was suppressed while osteoblastogenesis was enhanced via anti-TACE-mAb and anti-MT1-MMP-mAb as determined by TRAP/pit formation and ALP assay, respectively.
Conclusion: Sema4D can elicit anti-coupling effect on bone remodeling processes via two different ways. Sema4D initially expressed on the surface of preosteoclast can serve as a ligand to inhibit osteoblastogenesis via preosteoclast-preosteoblast interactions (Negishi-Koga et al. 2011) and then, after being shed from the surface, serve as a bioactive soluble molecule capable of interacting with receptors expressed on osteoclast precursor cells as well as on remote cells. In order for this to occur, TACE and MT1-MMP plays a crucial role in shedding the extracellular domain of Sema4D to function them as autocrine. Besides, among the known Sema4D receptors, CD72, but not Plexin B1/B2, expressed on the cell surface of osteoclast functions as receptor for solubilized Sema4D. These results suggested that TACE and MT1-MMP expressed on osteoclast cells would shed the membrane bound Sema4D into soluble Sema4D which in turn, promote RANKL-induced osteoclastogenesis and suppress osteoblastogenesis. Contrast to osteoblasts of which Plexin B2 function as receptor for Sema4D, Sema4D binds with CD72 expression on osteoclasts for the promotion of RANKL-mediated osteoclastogenesis.
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