Adenovirus-mediated overexpression and stimulation of the human angiotensin II type 2 receptor in porcine cardiac fibroblasts does not modulate proliferation, collagen I mRNA expression and ERK1/ERK2 activity, but inhibits protein tyrosine phosphatases

Abstract

The contribution of the angiotensin (Ang) II type 2 receptor (AT2R) to cardiac hypertrophy is still controversial. Here we examined the effect of overexpressing the human AT2R in cultured porcine cardiac fibroblasts (pFib) on proliferation, procollagen I mRNA expression, and - as putatively underlying signal-transduction pathways - on mitogen-activated protein kinase ERK1/ERK2 and phosphotyrosine phosphatase activities. As quantitated by 125I-(Sar1,Ile8)-Ang II binding, transduction of cardiac fibroblasts with the adenoviral AT2R expression vector led to a six- to tenfold higher AT2 than endogenous Ang II type 1 receptor (AT1R) expression. The overexpressed AT2R had the same apparent molecular mass as the endogenous AT2R in rat PC12W cells. Proliferation was not significantly lower in AT2R expressing pFib than in antisense-transduced controls (TA2) upon stimulation with Ang II (AT2R 110.5+/-4.8% vs. TA2 110.2+/-5.5%), Ang II plus the AT1R blocker Irbesartan (97.1+/-1.4% vs. 108.0+/-5.0; P=0.052) and the partial AT2R antagonist CGP42112 at the agonistic concentration of 50 nM (92.1+/-2.7% vs. 99.8+/-3.1%; P=0.053). Procollagen Ialpha2 (COL1A2) mRNA levels were quantitated by (a) northern blot analysis and (b) reverse transcriptase polymerase chain reaction. COL1A2/GAPDH (a) and COL1A2/beta-actin (b) ratios revealed no differences between AT2R-transduced fibroblasts and antisense controls when stimulated with Ang II (1 microM, 24 h) plus Irbesartan and 10 ng/ml transforming growth factor beta1. Ang II stimulation of the endogenous AT1R increased extracellular signal regulated kinase 1/2 activities. This response was reduced by Irbesartan, but PD123319 had no effect. Time course and magnitude of Ang II stimulated ERK1/ERK2 activation was identical in AT2R-transduced and control cells. Also, neither simultaneous nor Ang II pre-stimulation, suggested to induce gene expression of the MAP kinase phosphatase 1, modulated phorbol myristate acetate-stimulated ERK1/ERK2 activation in AT2R-transduced pFib, in AT2R-transduced human umbilical vein endothelial cells, and in PC12W cells. By the use of a tyrosine phosphatase assay we observed an inhibition of phosphotyrosine phosphatase activity by 30.8% (P=0.009, n=5) after 5 min Ang II stimulation of AT2R-expressing pFib. Immunoprecipitation-tyrosine phosphatase assays revealed that inhibition of phosphotyrosine phosphatase 1B, which regulates insulin signaling, contributed to this effect. In conclusion, stimulation of the overexpressed human AT2R in porcine cardiac fibroblasts inhibited tyrosine phosphatase activity but had no significant effect on fibroblast functions related to cardiac fibrosis. It is conceivable that possible antifibrotic AT2R effects are species specific and/or require the interaction between fibroblasts and cardiomyocytes, probably via paracrine factors, or mechanical load.

The Angiotensin II Type 2 Receptor Causes Constitutive Growth of Cardiomyocytes and Does Not Antagonize Angiotensin II Type 1 Receptor–Mediated Hypertrophy

“…In cardiac fibroblasts, the AT 2 receptor inhibited protein tyrosine phosphatases but had no effect on MAPK ERK1/2 activity. 23 Our results show that AT 2 receptors alone do not alter ERK1/2 activity, and ERK inhibition did not alter AT 2 receptor constitutive growth. Thus, the mechanism of the constitutive cardiomyocyte growth remains to be determined, but it is clearly not via the MAPK pathway.…”

“…13 The widely accepted notion that the AT 2 receptor simply counteracts the vasoactive roles of the AT 1 receptor is being increasingly challenged. 14,15 For example, AT 2 receptors have been shown to decrease 7,16 or not affect blood pressure and 17 to inhibit, 18 -20 increase, 21,22 and not affect 23 collagen deposition. They have been shown to be involved in fetal development 24 and cause cellular differentiation.…”

“…They have been shown to be involved in fetal development 24 and cause cellular differentiation. 25,26 AT 2 receptors have been reported to inhibit 7,18,27 or stimulate 28 vascular growth and angiogenesis, inhibit 20,26,27,29 and not affect 23 cellular proliferation, and cause 6,30,31 or not affect 32 apoptosis. Most important, AT 2 receptors have been shown to lead to either stimulation of, 21,22,33 inhibition of, 34 or not affect 18,19,32 cardiac hypertrophy.…”

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…Overexpression of the AT 2 receptor in neonatal cardiomyocytes promoted growth (D'Amore et al, 2005). Overexpression and AngII stimulation of the AT 2 receptor in porcine cardiac fibroblasts inhibited protein tyrosine phosphatases (Warnecke et al, 2001). Observed contradictions further highlight the context-specific effects of the AT 2 receptor overexpression.…”

Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a Gαi3/Ras/Raf/MAPK pathway

“…In addition, several stimuli can induce the expression of eNOS mRNA, ultimately leading to an increase in NO production (10). To date, there are many studies (6,38,40) linking ANG II and NO production; however, the mechanism by which ANG II leads to an increase in NO depends not only on the experimental conditions but also appears to depend on the cell type and signaling pathways present in that cell.We (20,26,27) have recently reported that ANG II stimulates Src tyrosine kinase via a pertussis toxin-sensitive AT 2 receptor, which, in turn, activates the MAPK pathway, resulting in increased NOS protein expression in bovine pulmonary artery endothelial cells (PAECs). ANG II activation of ERK has been previously described in several cell types (7,36) and has been shown to involve the G␤␥-subunits of a pertussis…”