Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

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Abstract
We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cellbinding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin’s effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor α5β1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, α3β1, α8β1 and αvβ3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that α5β1 levels are constant throughout osteoblast differentiation whereas levels of α3β1 and α8β1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing antiintegrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-α5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against α3β1 and α8β1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to αvβ3 and αvβ5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes α5β1, α3β1 and α8β1. We further conclude that the specific α5β1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

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