Ang II-induced Expression Of Fibronectin Research Articles

Angiotensin II increases fibronectin and collagen I through the β-catenin-dependent signaling in mouse collecting duct cells.

The contribution of angiotensin II (ANG II) to renal and tubular fibrosis has been widely reported. Recent studies have shown that collecting duct cells can undergo mesenchymal transition suggesting that collecting duct cells are involved in interstitial fibrosis. The Wnt/β-catenin signaling pathway plays an essential role in development, organogenesis, and tissue homeostasis; however, the dysregulation of this pathway has been linked to fibrosis. In this study, we investigated whether AT1 receptor activation induces the expression of fibronectin and collagen I via the β-catenin pathway in mouse collecting duct cell line M-1. ANG II (10(-7) M) treatment in M-1 cells increased mRNA, protein levels of fibronectin and collagen I, the β-catenin target genes (cyclin D1 and c-myc), and the myofibroblast phenotype. These effects were prevented by candesartan, an AT1 receptor blocker. Inhibition of the β-catenin degradation with pyrvinium pamoate (pyr; 10(-9) M) prevented the ANG II-induced expression of fibronectin, collagen I, and β-catenin target genes. ANG II treatment promoted the accumulation of β-catenin protein in a time-dependent manner. Because phosphorylation of glycogen synthase kinase-3β (GSK-3β) inhibits β-catenin degradation, we further evaluated the effects of ANG II and ANG II plus pyr on p-ser9-GSK-3β levels. ANG II-dependent upregulation of β-catenin protein levels was correlated with GSK-3β phosphorylation. These effects were prevented by pyr. Our data indicate that in M-1 collecting duct cells, the β-catenin pathway mediates the stimulation of fibronectin and collagen I in response to AT1 receptor activation.

Angiotensin II-mediated activation of fibrotic pathways through ERK1/2 in rat peritoneal mesothelial cells

Introduction: Peritoneal fibrosis is a common complication of peritoneal dialysis (PD) although the pathway involved is unclear. Of this article, angiotensin II (Ang II)-mediated upregulation of mitogen-activated protein kinase (MAPK) pathway as well as their downstream profibrotic genes including transforming growth factor (TGF)-β1 and fibronectin (FN) was investigated. Methods: Rat peritoneal mesothelial cells (RPMCs) were obtained by enzymatic digestion from the colic omentum. After incubated with Ang II, real-time PCR, ELISA, and Western blot analysis were used to determine RPMCs cellular and secretory (supernatants) levels of TGF-β1, FN, tissue inhibitor of metalloproteinase-1 (TIMP-1), and plasminogen activator inhibitor-1 (PAI-1) as well as the phosphorylation of extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK). We also determined the downstream pathways using the specific inhibitors including PD98059 (ERK1/2), SB230580 (p38 MAPK), SP600125 (JNK), and losartan [Ang II type-1 (AT1] receptor blocker). Results: Ang II increased mRNA and protein levels of TGF-β1, FN, TIMP-1, and PAI-1 in a time- and dose-dependent manner in RPMCs. Ang II induced a 1.5–2-fold increase in both mRNA and protein levels of the above molecules at 10 nmol/L. Ang II also upregulated the phosphorylation of ERK1/2 and p38 but not of JNK. Finally, inhibition of either AT1 or ERK1/2 was able to suppress Ang II-induced expression of FN. Conclusion: In cultured RPMCs, Ang II upregulated profibrotic signaling pathways through AT1-mediated ERK1/2 phosphorylation.

Angiotensin II induces fibronectin expression in human peritoneal mesothelial cells via ERK1/2 and p38 MAPK

The renin-angiotensin system has been implicated in the pathogenesis of fibrosis in various organs. However, its involvement in peritoneal fibrosis, a crucial complication of peritoneal dialysis, is unclear. Human peritoneal mesothelial cells (HPMC) play a major role in peritoneal fibrosis by producing extracellular matrix (ECM). However, there is scant data regarding the effect of angiotensin II (Ang II) on ECM expression and signal transduction pathways in HPMC. The concentration of Ang II in the peritoneal dialysis effluent was measured by radioimmunoassay. We investigated the expression of Ang II type 1 (AT1) and type 2 (AT2) receptors by HPMC. We also examined the effect of Ang II upon fibronectin production by HPMC, and dissected the receptor and intracellular signaling pathways involved. Ang II levels in the peritoneal dialysis effluent at the onset of peritonitis were 30 times higher than baseline levels. HPMC expression of AT1 and AT2 receptors was confirmed at the mRNA and protein level by reverse transcriptase-polymerase chain reaction (PCR), Western blotting, and immunocytochemistry. Quantitative reverse transcriptase-PCR and Western blotting showed that 10 nmol/L Ang II increased fibronectin mRNA expression followed by secretion of fibronectin protein. This response was completely inhibited by the AT1 receptor antagonist RNH6270, while the AT2 receptor antagonist PD123319 had no effect. Ang II-induced fibronectin expression was mediated by the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK), but not c-Jun N-terminal kinase. These results indicate the potential importance of ERK1/2 and p38 MAPK signaling pathways in Ang II-induced fibronectin expression in HPMC, and suggest the therapeutic potential of AT1 receptor blockers in the prevention or treatment of peritoneal fibrosis in patients on peritoneal dialysis.

Angiotensin II signaling and HB-EGF shedding via metalloproteinase in glomerular mesangial cells

Angiotensin II (Ang II) has been implicated in the development of glomerulosclerosis by stimulating fibronectin (FN) synthesis. The processing and release of heparin binding-endothelin growth factor (HB-EGF) are activated by protein kinase C (PKC) and Ca2+ signaling. We studied the roles of HB-EGF and endothelial growth factor (EGF) receptor (EGFR) in Ang II-induced FN expression using mesangial cells. Mesangial cells were prepared from mouse kidneys by the explant method and cells were used at passages 4 and 5. Ang II stimulated FN mRNA levels dose-dependently with a maximal increase (3.4-fold) after 12 hours of incubation. This action was completely inhibited by PKC inhibitors and slightly blocked by Ca2+ chelating agents. FN mRNA accumulation by Ang II was abolished by tyrosine kinase inhibitors, a specific inhibitor for EGFR (AG1478) and extracellular signal-regulated kinase (ERK) inactivation. Addition of neutralizing anti-HB-EGF antibody, as well as pretreatment with heparin or the metalloproteinase inhibitor batimastat abolished induction of FN expression by Ang II. In mesangial cells stably transfected with a chimeric construct containing HB-EGF and alkaline phosphatase (ALP) genes, ALP activity in incubation medium was rapidly increased by Ang II (1.7-fold at 0.5 min) and reached a 4.1-fold increase at two minutes. Ang II phosphorylated EGFR (maximal at 2 min) and ERK (maximal at 8 min) in a PKC- and metalloproteinase-dependent manner. Ang II stimulated the expression and release of transforming growth factor-beta (TGF-beta) via EGFR-mediated signaling, and the released TGF-beta also contributed to Ang II-mediated FN expression via EGFR transactivation. Ang II-mediated FN expression was regulated by autocrine effects of HB-EGF and TGF-beta, suggesting a novel paradigm for cross-talk between Ang II and growth factor receptor signaling pathways.

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 characteristic of 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 TGF-beta in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose-dependently with a maximal increase (approximately 5-fold) observed after 12 h of incubation. Ang II-, or calcium ionophore-induced fibronectin synthesis was completely abolished by tyrosine kinase inhibitors and intracellular Ca2+ chelating agents. Ang II-induced fibronectin mRNA was not affected by PKC inhibitors or PKC 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 AP-1 binding site present in the promoter region was responsible for the Ang II responsiveness of this gene. 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-ATF-2 antibodies. Conditioned medium from Ang II-treated cells contained TGF-beta bioactivity and addition of neutralizing TGF-beta antibody modestly (46%) inhibited induction of fibronectin. Ang II-induced synthesis of TGF-beta was also abolished by inhibition of EGF-R function. The effect of TGF-beta 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-beta 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 two different mechanisms; transcriptional control by binding of c-fos/c-jun complex to the AP-1 site, and post-transcriptional control by mRNA stabilization due to autocrine and/or paracrine effects of TGF-beta. Thus, this study suggested that the action of Ang II on extracellular matrix formation should be interpreted in association with the EGF-R signaling cascade.

Angiotensin II-induced transactivation of epidermal growth factor receptor regulates fibronectin and transforming growth factor-beta 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-beta (TGF-beta) in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose dependently, with a maximal increase (approximately 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-beta bioactivity, and addition of neutralizing TGF-beta antibody modestly (46%) inhibited induction of fibronectin. Ang II-induced synthesis of TGF-beta was also abolished by inhibition of EGF-R function. The effect of TGF-beta 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-beta 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-beta. 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.