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

The contribution of the angiotensin (Ang) II type 2 receptor (AT2R) to card iac hypertrophy is still controversial. Here we examined the effect of over expressing the human AT2R in cultured porcine cardiac fibroblasts (pFib) on proliferation, procollagen I mRNA expression, and - as putatively underlyi ng signal-transduction pathways - on mitogen-activated protein kinase ERK1/ ERK2 and phosphotyrosine phosphatase activities. As quantitated by I-125-(S ar(1),Ile(8))-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 c ells. Proliferation was not significantly lower in AT2R expressing pFib tha n 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 Irbe sartan (97.1 +/- 1.4% vs. 108.0 +/- 5.0; P=0.052) and the partial AT2R anta gonist CGP42112 at the agonistic concentration of 50 nM (92.1 +/- 2.7% vs. 99.8 +/- 3.1%; P=0.053). Procollagen I alpha2 (COLIA2) mRNA levels were qua ntitated by (a) northern blot analysis and (b) reverse transcriptase polyme rase chain reaction. COLIA2/GAPDH (a) and COLIA2/beta -actin (b) ratios rev ealed no differences between AT2R-transduced fibroblasts and antisense cont rols when stimulated with II (1 muM, 24 h) plus Irbesartan and 10 ng/ml tra nsforming growth factor beta (1). Ang II stimulation of the endogenous AT1R increased extracellular signal regulated kinase 1/2 activities. This respo nse was reduced by Irbesartan, but PD123319 had no effect. Time course and magnitude of Ang II stimulated ERK1/ERK2 activation was identical in AT2R-t ransduced and control cells. Also, neither simultaneous nor Ang II pre-stim ulation, suggested to induce gene expression of the MAP kinase phosphatase 1, modulated phorbol myristate acetate-stimulated ERK1/ERK2 activation in A T2R-transduced pFib, in AT2R-transduced human umbilical vein endothelial ce lls, and in PC12W cells. By the use of a tyrosine phosphatase assay we obse rved an inhibition of phosphotyrosine phosphatase activity by 30.8% (P=0.00 9, n=5) after 5 min Ang II stimulation of AT2R-expressing pFib. Immunopreci pitation-tyrosine phosphatase assays revealed that inhibition of phosphotyr osine phosphatase 1B, which regulates insulin signaling, contributed to thi s effect. In conclusion, stimulation of the overexpressed human AT2R in por cine 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 specifi c and/or require the interaction between fibroblasts and cardiomyocytes, pr obably via paracrine factors, or mechanical load.