Research Scientist Columbia University Medical Center New York, New York, United States
Introduction: Multiple myeloma cells activate osteoclasts by producing osteoclastogenic factors. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is one of the critical osteoclastogenic factors highly secreted by MM cells (JCI 2016). Further studies indicated that checkpoint inhibitor PD-1H/VISTA functions as the MMP-13 receptor on osteoclasts and mediates the MMP-13-induced osteoclast activation. Interestingly, PD-1H pulldown mass spectrum assay suggests that PD-1H associates with cytoskeleton proteins. Further, we found PD-1H regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Since Rho GTPases substrates Rac1/2 are the key regulators of the dynamic actin cytoskeleton rearrangements, we investigated its role in PD-1H mediated OCL formation/activation.
Methods: Bone marrow mononuclear cells from WT or Pd-1h-/- mice were cultured in osteoclast differentiation medium without or with MMP-13. Activated Rac1 was detected in Rac1 pull-down complex from whole cell lysates by WB. Localization of c-Src, Rac1, PD-1H and F-actin ring in osteoclast were detected by confocal immunofluorescence (IF) microscope and activation of c-Src was detected by western blotting. The binding of c-Src with PD-1H and 1-215 mutant was checked by co-IP assay after co-expressed in HEK 293 cells.
Results: Our results showed that MMP-13 activated Rac1 in WT, but not Pd-1h-/- osteoclasts. IF staining indicated that PD-1H and Rac1 co-localized, especially at the F-actin belt in WT osteoclasts. Consistent with the decreased Rac1 activation and F-actin belt formation observed in Pd-1h-/- osteoclasts, Rac1 failed to localize at F-actin-rich areas in Pd-1h knockout cells. Non-receptor tyrosine kinase c-Src associates with RANK and mediates RANKL-induced Rac1 activation and cytoskeleton reorganization. Confocal IF staining of the WT osteoclasts indicated that c-Src and PD-1H almost completely co-localized on the F-actin sealing belt and perinuclear area. Co-immunoprecipitation assays confirmed the direct binding of c-Src to the C-terminal intracellular domain of PD-1H as a PD-1H 1-215 mutant lacking its intracellular domain failed to bind c-Src. IF staining of c-Src, PD-1H and F-actin indicated that in contrast to WT osteoclasts, c-Src in Pd-1h-/- osteoclasts mainly accumulated in perinuclear areas, but not in the F-actin belt hereby not allowing appropriate cytoskeleton reorganization necessary for OCL formation. Finally, MMP-13 induced c-Src phosphorylation/activation in WT osteoclasts, which was impaired in Pd-1h-/- cells.
Conclusions: This study reveals the novel role of MMP-13/PD-1H signaling in the regulation of osteoclasts cytoskeleton reorganization. MMP-13 binds to PD-1H and promotes c-Src/Rac1 signaling activation and subsequently promotes osteoclast bone resorption activities. This study hence revealed a novel role of the checkpoint inhibitor PD-1H/VISTA in osteoclast cytoskeleton regulation and subsequently multiple myeloma bone disease.
