Transactivation of EGF receptor induced by angiotensin II regulates fibronectin and TGF-beta gene expression via transcriptional and post-transcriptional mechanisms

The signaling cascade elicited by angiotensin II (Ang II) resembles that ch aracteristic of growth factor, and recent evidence indicates transactivatio n of epidermal growth factor receptor (EGF-R) by G protein-coupled receptor s. Here, we report the involvement of EGF-R in Ang II-induced synthesis of fibronectin and TGF-beta in cardiac fibroblasts. Ang II stimulated fibronec tin mRNA levels dose-dependently with a maximal increase (similar to5-fold) observed after 12 h of incubation. Ang II-, or calcium ionophore-induced f ibronectin synthesis was completely abolished by tyrosine kinase inhibitors and intracellular Ca2+ chelating agents. Ang II-induced fibronectin mRNA w as not affected by PKC inhibitors or PKC depletion, whereas specific inhibi tion of EGF-R function by a dominant negative EGF-R mutant and tyrphostin A G1478 abolished induction of fibronectin mRNA. We isolated the rat fibronec tin gene including the 5'-flanking region and found that the AP-1 binding s ite present in the promoter region was responsible for the Ang II responsiv eness of this gene. Gel retardation assay revealed the binding of nuclear p rotein to the AP-1 site, which was supershifted with anti-c-fos and anti-c- jun but not anti-ATF-2 antibodies. Conditioned medium from Ang II-treated c ells contained TGF-beta bioactivity and addition of neutralizing TGF-beta a ntibody modestly (46%) inhibited induction of fibronectin. Ang II-induced s ynthesis of TGF-beta was also abolished by inhibition of EGF-R function. Th e effect of TGF-beta was exerted by stabilizing fibronectin mRNA without af fecting the promoter activity and required de novo protein synthesis. We co ncluded that Ang II-induced expression of fibronectin and TGF-beta is media ted by downstream signaling of EGF-R transactivated by Ca2+-dependent tyros ine kinase, and that Ang II-induced fibronectin mRNA expression is regulate d by two different mechanisms ; transcriptional control by binding of c-fos /c-jun complex to the AP-1 site, and post-transcriptional control by mRNA s tabilization due to autocrine and/or paracrine effects of TGF-beta. Thus, t his study suggested that the action of Ang II on extracellular matrix forma tion should be interpreted in association with the EGF-R signaling cascade.