Extracellular Matrix Modulation of rhGDF-5-Induced Cellular Differentiation

Abstract

Growth and differentiation factor-5 (GDF-5) is a divergent member of the TGF-β/bone morphogenetic protein (BMP) superfamily that is required for proper skeletal patterning and joint development in the vertebrate limb. Based on the homology of GDF-5 to other bone-inducing BMP family members, the inductive activity of a recombinant form of human GDF5 (rhGDF-5), and the influence of the extracellular matrix (ECM) on this inductive activity was evaluated in a series of well-defined in vitro assays. Fetal rat calvarial (FRC) cells were plated on various purified extracellular matrix proteins in the presence of rhGDF-5 (100 ng/mL) for 3 weeks and scored for differentiation at the level of morphology, overall proteoglycan synthesis and deposition, aggrecan and Type II collagen mRNA and protein expressions. Results show that GDF-5 stimulated chondrogenic nodule formation by FRC cells plated on Type I collagen but to a lesser extent on tissue culture plastic or fibronectin. These chondrogenic nodules stained heavily with Alcian blue and expressed chondrogenic markers such as Type II collagen and aggrecan, as judged by immunohistochemical and RT-PCR analyses, respectively. Cells in the monolayer that surrounded the nodules did not express the chondrogenic markers. The molecular signaling mechanism by which GDF-5 induces chondrogenesis in FRC cells in the presence of Type I collagen was investigated using well-characterized modulators of intracellular signaling mediators. Results show that the ligand-dependent chondrogenesis was inhibited by the calcium ionophore A23187, rapamycin but not by dibutyryl-cAMP, Na₃/VO₄, or EGTA. The known effects of A23187 and rapamycin on intracellular signaling pathway suggest that the GDF-5/Type I collagen-induced chondrogenesis is mediated through modulation of intracellular calcium concentration accompanied by activation of the p70 S6 kinase (p70^s6k) signaling pathway. Together, these results indicate that cellular interaction with Type I collagen significantly enhances the differentiating activity of GDF-5. This effect is likely mediated by the convergence of downstream matrix and growth factor receptor signaling pathways.