Nephronectin promotes osteoblast differentiation via the epidermal growth factor-like repeats

We found that nephronectin was significantly down-regulated by TGF-β1. To determine the function of nephronectin in osteogenesis, we generated various constructs to produce stable MC3T3-E1 cell lines, expressing and secreting nephronectin protein, including full-length (Npnt), lacking EGF-like repeats (Np-MAM), and lacking RGD and MAM domains (Np-EGF). We demonstrated that nephronectin promotes differentiation during osteoblast differentiation and the EGF-like repeats were essential. Lack of these repeats resulted in inhibiting the change in morphology. Over-expression of nephronectin results in earlier formation of bone nodules than the vector control. ERK activation is essential for nephronectin-induced osteoblast differentiation.     

Transforming growth factor-β inhibits nephronectin-induced osteoblast differentiation

We used cDNA microarray to identify transforming growth factor beta (TGF-β) responsive target genes during osteoblast development and found that nephronectin (Npnt) is one such gene that is significantly down-regulated. Here we report the role of TGF-β in regulating Npnt-mediated osteoblast differentiation. We found that the effect of TGF-β on Npnt expression is associated with a change in cell morphology in a dose-dependent manner. Npnt-induced osteoblast differentiation was also inhibited by TGF-β, which changed cell morphology from cuboidal to fibroblastic, an indication that osteoblast differentiation was disrupted. Furthermore, TGF-β inhibited differentiation of osteoblasts transfected with various truncated Npnt constructs, suggesting that TGF-β can exert a down-stream effect on Npnt function. Our results suggest that TGF-β can inhibit osteoblast differentiation through various mechanisms.