Prorenin Induces Intracellular Signaling in Cardiomyocytes Independently of Angiotensin II Jasper J. Saris, Peter A.C. ’t Hoen, Ingrid M. Garrelds, Dick H.W. Dekkers, Johan T. den Dunnen, Jos M.J. Lamers, A.H. Jan Danser Abstract—Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentration- dependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (n=4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (P0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, pro-Anp, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensin- independent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression. (Hypertension. 2006;48:564-571.) Key Words: p38 MAP kinase  actin  microarray  hypertrophy  HSP27 P rorenin, the inactive precursor of renin, circulates in human plasma in excess of renin, at concentrations that are up to 100 times higher. 1 The reasons for this are unknown, but an attractive concept is that circulating prorenin is taken up at tissue sites where it contributes, after its local activation to renin, to tissue angiotensin production. Evidence for this concept comes from studies in transgenic animals displaying (inducible) prorenin expression. 2,3 Prorenin activation at tis- sue sites might involve proteolytic removal of its proseg- ment. 4 Alternatively, activation could occur in a nonproteo- lytic manner, for instance, through binding to a receptor. Indeed, Ichihara et al 5 have proposed recently that human prorenin has so-called gate and handle regions for nonpro- teolytic activation. According to this concept, the handle region interacts with a putative receptor, which subsequently leads to unfolding of the gate region from the renin molecule. In vivo treatment with a decoy peptide corresponding to the gate region reduced the renal content of angiotensin (Ang) I and II in streptozotocin-induced diabetic rats, thereby sup- porting, for the first time, tissue Ang production by endoge- nous prorenin. Interestingly, Nguyen et al 6 have cloned a (pro)renin receptor, which exactly fulfills the above description, because prorenin binding to this receptor allowed prorenin to become fully enzymatically active in a nonproteolytic manner. Unex- pectedly, (pro)renin binding to this receptor in glomerular mesangial cells also induced angiotensin-independent effects, that is, an increase in DNA synthesis, activation of the mitogen-activated protein kinases (MAPKs) extracellular sig- nal regulated kinase 1 (p44)/extracellular signal regulated kinase 2 (p42), and plasminogen-activator inhibitor (PAI)-1 release, 6,7 thus leading the authors to suggest that prorenin acts as an agonist of this receptor. Prorenin, a phosphomannosylated protein, also binds to mannose 6-phosphate/insulin-like growth factor II receptors (IGF2R). Binding to these receptors is followed by internal- Received May 17, 2006; first decision June 5, 2006; revision accepted July 27, 2006. From the Departments of Pharmacology (J.J.S., I.M.G., A.H.J.D.) and Biochemistry (D.H.W.D., J.M.J.L.), Erasmus MC, Rotterdam, The Netherlands; the Center for Human and Clinical Genetics (P.A.C.t.H., J.T.d.D.), and the Leiden Genome Technology Center (J.T.d.D.), Leiden University Medical Center, Leiden, The Netherlands. Correspondence to A.H. Jan Danser, Department of Pharmacology, Room EE1418b, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail a.danser@erasmusmc.nl © 2006 American Heart Association, Inc. Hypertension is available at http://www.hypertensionaha.org DOI: 10.1161/01.HYP.0000240064.19301.1b 564 Cardiac Hypertrophy, Prorenin, ACE2, and Estrogen by guest on September 18, 2015 http://hyper.ahajournals.org/ Downloaded from