Abstract
Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation.
Highlights
We found an increase in the mean fluorescence intensity (MFI) of ␣4 or 3 after differentiation, a decrease in ␣v, and no change in ␣5, ␣9, and 1 expression (Fig. 5B, top row, examples are shown in the bottom row)
The main findings of this work are that 1) two isoforms of fibronectin, one containing EDA and one containing EDB, stimulate osteoblast differentiation, and 2) EDA-containing fibronectin activates integrin ␣41, and EDB-containing fibronectin binds through RGD to 3-containing integrin, activating it
This study was initiated because the circulating isoform of fibronectin lacking the EDA and the EDB domains was unable to support osteoblast differentiation in transgenic mice lacking fibronectin production by the osteoblast [2]
Summary
The two extradomains EDA and EDB are produced by osteoblasts [2]. We generated expression constructs consisting of the whole fibronectin cDNA and including either the EDA (FN-EDA) or the EDB domain (FN-EDB) (Fig. 1A). The decrease is limited, it is expected, based on the low expression of osterix in primary calvarial osteoblasts (roughly 5–10% by flow cytometry based on fluorescent protein expression in the promoter) (Fig. 1F) [17] Transfection of these cells with either the FN-EDA or FN-EDB constructs enhanced osteocalcin mRNA expression already 3 days after transfection compared with the control fibronectin construct (FN), again indicating boosted differentiation of osteoblasts in the presence of EDA- or EDB-containing fibronectin, as was the case in wildtype osteoblasts shown, C and D (Fig. 1G). Silencing either EDA- or EDB-containing fibronectin confirmed suppression of osteoblast differentiation in vitro (Fig. 2D), as evidenced by nodule formation, alkaline phosphatase protein activity, or osteocalcin mRNA expression in line with the stimulatory effect of either domain on osteoblasts. Changes in the levels of the relevant fibronectin isoforms are summarized numerically on day 4 of overexpression together with the associated changes in mineralization at the end of in vitro differentiation. (Summary of data presented in Figs. 1 (C–E) and 3 (A and B).) Note that only EDB protein concentration in the conditioned medium in the double transfection is not significantly increased at day 4, whereas all other values are
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