MMP-2 regulation of osteoclast formation

The association between extracellular domains of integrin associated protein (IAP) and the transmembrane protein SHPS-1 has been reported to regulate osteoclast formation. We have demonstrated that the IAP-SHPS-1 association in smooth muscle and endothelial cells is regulated by MMP-2 cleavage of IAP. Patients with an MMP-2 deficiency exhibit higher levels of osteoclast formation and subsequent bone resorption. Objectives: To determine whether regulation of MMP-2 cleavage of IAP regulates osteoclast formation. Methods: Bone marrow cells (BMC) were isolated from 12-week old male C57/B6 mice. Osteoclast (OC) formation was stimulated by the addition of sRANK-L (50ng/ml) and mCSF (25ng/ml). BMC were incubated +/- modulators of IAP cleavage (a highly specific MMP-2 inhibitor, SB-3CT, which protects IAP from cleavage or an anti-IAP antibody that accelerates IAP cleavage) for 7 days. TRAP positive cells with three or more nuclei were counted as OC. Parallel cultures of cells were lysed and IAP was visualized by immunoblotting with an anti-IAP antibody. Results: The addition of sRANK-L and mCSF stimulated marked OC formation. In the presence of the MMP-2 inhibitor OC formation was further increased 1.7 fold (p‹0.05). However, addition of anti-IAP antibody resulted in a 1.3 fold decrease in OC formation compared with sRANKL and mCSF alone (p‹0.05). A significant increase in the amount of intact IAP could be detected from the lysates treated with RANK-L (osteoclast positive) compared with BMC lysates not treated (osteoclast negative). The increased formation of OC in the presence of the MMP-2 inhibitor was associated with a significant increased in intact IAP whereas the decrease in osteoclast formation in the presence of the anti IAP antibody was associated with a significant decrease in the amount of intact IAP. Conclusion: These data support the hypothesis that MMP-2 mediated cleavage of IAP regulates OC formation.