Fibronectin is produced in different isoforms by osteoblasts during differentiation. In contrast to the circulating Isoform, which lacks extra domains, the fibronectin laid down by the osteoblasts in the matrix contains one or both extra-domains called EDA and EDB. We had found that deletion of fibronectin in osteoblasts decreased BMD. This is mediated by diminished osteoblast differentiation and cannot be corrected by the circulating isoform of fibronectin, suggesting a role for EDA and/or EDB in modulating osteoblast behavior.
To evaluate the role of EDA in osteoblasts, wildtype newborn murine calvarial osteoblasts were transiently transfected with a fibronectin construct containing EDA. This enhanced osteoblast differentiation (von Kossa staining, alkaline phosphatase and osteocalcin). In contrast, siRNA directed against EDA suppressed differentiation. This effect was mediated via rac1 and enhancement of wnt signaling. Because EDA increased integrin-mediated signaling as evidenced by increased pFAK and can affect α4β1, α9β1or α5β1 integrins, we deleted β1-integrin in osteoblasts. This prevented EDA-mediated enhancement of differentiation. Inhibiting α4β1 diminished pFAK and osteoblast differentiation. Finally, a peptide called CS1 that activates α4β1 increased total BMD in treated mice.
Similar studies were performed for EDB. EDB enhanced differentiation and its silencing using siRNA inhibited differentiation. However, EDB effects are mediated by β3 integrin, because the absence of β3 prevented EDB effects. Adding echistatin activated β3 in osteoblasts and enhanced differentiation. This was mediated by the RGD sequence because transfecting EDB-fibronectin lacking RGD prevented stimulation by EDB. Furthermore, knockout of β3 in one allele is associated with a decrease in BMD in 5-week-old β3+/− heterozygote mice, while homozygote loss is not, presumably because β3-integrin is critical for osteoclastic resorption.
In summary, EDA activates α4β1 integrin, while EDB activates β3 integrin. The presence of either domain enhanced osteoblast differentiation. Thus we have clarified the role of two isoforms of fibronectin in osteoblasts.
14 May 2016 - 17 May 2016