Moursi A M, Globus R K, Damsky C H
Department of Stomatology, University of California San Francisco, San Francisco, CA 94143, USA.
J Cell Sci. 1997 Sep;110 ( Pt 18):2187-96. doi: 10.1242/jcs.110.18.2187.
We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding 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 alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, 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 alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 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 anti-integrin 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-alpha5 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 alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 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 alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.
我们先前的研究表明，抗纤连蛋白抗体或含有中央细胞结合结构域的可溶性纤连蛋白片段可在体外抑制胎儿颅骨成骨细胞形成矿化结节。这些发现表明纤连蛋白在成骨细胞分化和形态发生中起关键作用。在本研究中，我们验证了以下假设：纤连蛋白对骨生成的影响是通过与成骨细胞上纤连蛋白的整合素受体直接相互作用介导的。免疫细胞化学分析在胎儿大鼠颅骨组织以及处于分化各阶段的培养成骨细胞中鉴定出整合素纤连蛋白受体α5β1。在组织和细胞培养的各个阶段还鉴定出了其他三种可结合纤连蛋白以及其他基质成分的整合素，即α3β1、α8β1和αvβ3。免疫沉淀数据显示，在成骨细胞分化过程中α5β1水平保持恒定，而在成熟矿化培养物中α3β1和α8β1水平下降。为了确定整合素纤连蛋白受体是否是成骨细胞形成矿化结节所必需的，我们检测了在存在和不存在功能干扰性抗整合素抗体的情况下结节形成的程度。从汇合时（第3天）开始，抗体持续存在于培养物中，并在第10天和第20天测量结节形成情况。一种抗α5整合素亚基抗体在两个时间点均将结节形成减少至对照值的5%以下。结节形成的抑制是可逆的，且不影响细胞附着和活力。针对α3β1和α8β1的功能干扰性抗体也减少了结节形成，降至对照值的20%以下。相比之下，针对αvβ3和αvβ5的功能干扰性抗体不影响结节形成，表明所观察到的抑制确实具有特异性。为了确定抗体处理对基因表达的影响，检测了稳态mRNA表达，发现成骨细胞标志物碱性磷酸酶和骨钙素的表达受到抑制。总之，这些结果表明成骨细胞与细胞外基质的直接相互作用是由一组特定的整合素受体介导的，包括α5β1、α3β1和α8β1。我们进一步得出结论，特定的α5β1纤连蛋白受体介导了成骨细胞与纤连蛋白之间对于骨形态发生和成骨细胞分化都至关重要的相互作用。
