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

Abstract

—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-β (TGF-β) 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 Ca²⁺ 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 5′-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-β bioactivity, and addition of neutralizing TGF-β antibody modestly (46%) inhibited induction of fibronectin. Ang II–induced synthesis of TGF-β was also abolished by inhibition of EGF-R function. The effect of TGF-β 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-β is mediated by downstream signaling of EGF-R transactivated by Ca²⁺-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-β. 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.