Collagenase-1 and -3 Differentially Regulate MG63 Cell Osteoblastic Differentiation

Objective: We have previously shown that collagenase-1 (MMP-1) and -3 (MMP-13) differentially enhance expression of osteoblastic markers in a heterogenous population of primary human periodontal ligament cells. In these studies we examined the effects of MMP-1 and -13 on osteoblastic differentiation of an osteoblastic cell line and delineated the hierarchy of osteoblast-specific transcription factors and markers that are regulated by these two MMPs. Methods: The expression of collagenase-1 and/or -3 was enhanced or repressed in MG63 (plating density 20,000 cells/cm²) by transfection with cDNA or siRNA to these MMPs, respectively. After 18, 36 and 72 hours of culture, the levels and activity of collagenase-1 and -3 in cell-conditioned medium were determined by Western-blotting and MMP-1 or -13 activity assays. Cell extracts were used for measuring alkaline phosphatase (AP) activity, and qRT-PCR was used to analyze BMP-2, TGF-ß1, DLX5, MSX2, Runx2, osterix (Osx), osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC). Results: The overexpression of MMP-1 resulted in significant decreases in BMP-2, AP, OP and BSP and increases in TGF-ß1, DLX5 and MSX2. MMP-13 overexpression resulted in significant decreases in BMP-2, DLX5, Runx2, OP and BSP, and increases in TGF-ß1, MSX2 and OC. The knockdown of MMP-1 caused significant increases in all osteoblastic markers. MMP-13 knockdown produced significant increases in TGF-ß1, MSX2 and Osx but decreases in DLX5, Runx2 and OC. Suppression of both MMPs resulted in significant increases of all osteoblastic markers except DLX5 and Runx2. Conclusion: These data show that collagenase-1 and -3 differentially regulate the expression of osteoblastic markers in MG63 cells. The selective modulation of osteoblastic phenotype by the two collagenases may result from the divergence of specific pathways regulating osteoblastic differentiation. (Studies supported by NIH R01 DE16671).