Matrix GLA protein modulates differentiation induced by done morphogeneticprotein-2 in C3H10T1/2 cells

Matrix GLA protein (MGP) is ubiquitously expressed with high accumulation i n bone and cartilage, where it was found to associate with bone morphogenet ic proteins (BMP) during protein purification. To test whether MGP affects BMP-induced differentiation, three sets of experiments were performed. Firs t, pluripotent C3H10T1/2 cells transfected with human MPG (hMGP) or antisen se to hMGP (AS-hMGP) were treated with BMP-2. In cells overexpressing hMGP, osteogenic and chondrogenic differentiation was inhibited indicating decre ased BMP-2 activity. Conversely, in cells overexpressing AS-hMGP, BMP-2 act ivity was enhanced. Second, cells were prepared from homozygous and heteroz ygous MPG-deficient mice aortas. When treated with BMP-2, these cells under went chondrogenic and osteogenic differentiation, respectively, whereas con trols did not. Third, FLAG-tagged hMGP with the same biological effect as n ative hMGP inhibited BMP-induced differentiation, when exogenously added to culture media. Together, these results suggest that MGP modulates BMP acti vity. To test whether hMGP fragments would retain the effect of full-length hMGP, three subdomains were overexpressed in C3H10T1/2 cells, In cells exp ressing the mid-region, alone (amino acids (aa) 35-54) or in combination wi th the N terminus (aa 1-54) but not the C terminus (aa 35-84), osteogenic d ifferentiation was enhanced and occurred even without added BMP-2. Thus, tw o subdomains had the opposite effect of full-length hMGP, possibly due to d ifferent expression levels or domain characteristics.