Angiotensin II–Induced Transactivation of Epidermal Growth Factor Receptor Regulates Fibronectin and Transforming Growth Factor-b Synthesis via Transcriptional and Posttranscriptional Mechanisms

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of a growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein–coupled receptors. Here, we report the involvement of EGF-R in Ang II–induced synthesis of fibronectin and transforming growth factor-b (TGF-b) in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose dependently, with a maximal increase ('5-fold) observed after 12 hours of incubation. Fibronectin synthesis induced by Ang II or calcium ionophore was completely abolished by tyrosine kinase inhibitors and intracellular Ca chelating agents. Ang II–induced fibronectin mRNA was not affected by protein kinase C inhibitors or protein kinase C depletion, whereas specific inhibition of EGF-R function by a dominant negative EGF-R mutant and tyrphostin AG1478 abolished induction of fibronectin mRNA. We isolated the rat fibronectin gene, including the 59-flanking region, and found that the activator protein-1 (AP-1) binding site present in the promoter region was responsible for the Ang II responsiveness of this gene. A gel retardation assay revealed the binding of nuclear protein to the AP-1 site, which was supershifted with anti–c-fos and anti–c-jun but not anti–activating transcription factor (ATF)-2 antibodies. Conditioned medium from Ang II–treated cells contained TGF-b bioactivity, and addition of neutralizing TGF-b antibody modestly (46%) inhibited induction of fibronectin. Ang II–induced synthesis of TGF-b was also abolished by inhibition of EGF-R function. The effect of TGF-b was exerted by stabilizing fibronectin mRNA without affecting the promoter activity and required de novo protein synthesis. We concluded that Ang II–induced expression of fibronectin and TGF-b is mediated by downstream signaling of EGF-R transactivated by Ca-dependent tyrosine kinase and that Ang II–induced fibronectin mRNA expression is regulated by 2 different mechanisms, which are transcriptional control by binding of the c-fos/c-jun complex to the AP-1 site and posttranscriptional control by mRNA stabilization due to autocrine or paracrine effects of TGF-b. Thus, this study suggests that the action of Ang II on extracellular matrix formation should be interpreted in association with the EGF-R signaling cascade. (Circ Res. 1999;84:1073-1084.)