title: 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.
authors.affiliation: Department of Internal Medicine/Cardiology, Charité, Campus Virchow Clinic, Humboldt University, Forschungshaus 37, Augustenburger Platz 1, 13353 Berlin, Germany. warnecke@charite.de
authors.forename: C
authors.initials: C
authors.lastname: Warnecke
authors.forename: D
authors.initials: D
authors.lastname: Kaup
authors.forename: U
authors.initials: U
authors.lastname: Marienfeld
authors.forename: W
authors.initials: W
authors.lastname: Poller
authors.forename: C
authors.initials: C
authors.lastname: Yankah
authors.forename: M
authors.initials: M
authors.lastname: Gräfe
authors.forename: E
authors.initials: E
authors.lastname: Fleck
authors.forename: V
authors.initials: V
authors.lastname: Regitz-Zagrosek
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen; D024042:Collagen Type I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008957:Models, Genetic; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D054645:Protein Phosphatase 1; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000378:metabolism*; D015398:Signal Transduction; D013552:Swine; D013997:Time Factors; D014161:Transduction, Genetic; D014443:Tyrosine / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D012333:RNA, Messenger; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D014443:Tyrosine; D003094:Collagen; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D054645:Protein Phosphatase 1; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases