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A Peptide from a ras Effector-Domain Blocks ras-Dependent Cardiac Hypertrophy in Myocytes

Address correspondence to Nabil El-Sherif, M.D., Cardiology Service, NY Harbor VA Healthcare System, 800 Poly Place, Brooklyn, NY 11209, USA; tel 718 630 3740; fax 718 630 3740; e-mail nelsherif{at}aol.com; or to Matthew R. Pincus, M.D., Ph.D., Department of Pathology & Laboratory Medicine, NY Harbor VA Medical Center, 800 Poly Place, Brooklyn, NY 11209, USA; tel 718 630 3688; fax 718 630 2960; e-mail matthew.pincus2{at}med.va.gov.

PNC-2 is a peptide corresponding to an effector domain (residues 96–110) of ras-p21 that strongly and specifically blocks mitogenic signal transduction by oncogenic but not activated, normally-expressed wild-type ras-p21 protein. Since myocardial hypertrophy can be induced both by oncogenic and overexpressed wild-type ras-p21, we investigated whether PNC-2 can block norepinephrine (NE)-induced, ras-dependent myocardial hypertrophy in cardiac myocytes. Since PNC-2 blocks oncogenic ras-p21-induced activation of JNK and ERK, we further determined whether this peptide blocks activation of these kinases in NE-treated myocytes. Using cultured neonatal rat ventricular myocytes (NRVM), we found that NE alone significantly increased NRVM surface area, ³H-leucine uptake, protein/DNA ratio, and atrial nartiuretic factor (ANF) mRNA levels in these cells. However, pretreatment of the NRVM with PNC-2 linked on its carboxyl terminal end to a transmembrane-penetrating leader sequence (PNC-2-leader) resulted in strong inhibition of NE-mediated cell growth and ³H-leucine uptake and in significantly lower protein/DNA ratios. Induction of ANF mRNA levels was likewise inhibited by PNC-2-leader. In contrast, no inhibition of any of these NE-induced events was observed with a negative control peptide, X13-leader. Western blot analysis showed that JNK and ERK1/2 activity, but not p38 activity, was increased in NRVM within 5 min of exposure to NE (2 µM). Pretreatment with PNC-2-leader decreased ERK1/2 and JNK activity to basal levels. We conclude that a synthetic peptide designed to block oncogenic ras can also counter the effects of NE-induced hypertrophy associated with overexpression of ras p21 by blocking JNK/ jun and ERK activation. PNC-2 may provide a prototype for novel therapy in cardiac conditions associated with activation of NE.

Keywords: myocardial hypertrophy, ras-p21, jun-N-terminal kinase (JNK), ERK, epinephrine-induced hypertrophy, atrial natriuretic factor (ANP), RNase protection assay

Abbreviations: ANF = atrial natriuretic factor; Ang II = angiotensin II; EP = epinephrine; ERK = extracellular growth factor-responsive kinase also called mitogen-activated protein kinase; ET-1 = endothelin-1; GPCR = G-protein-coupled receptors; JNK = jun N-terminal kinase; NE = norepinephrine; NRVM = cultured neonatal rat ventricular myocytes; p38 = p38 MAP kinase (MAPK); PE = phenylephrine; PNC-2-leader = ras peptide containing amino acids 96-110 attached at its carboxyl terminus to transmembrane-penetrating leader sequence; RPA = ribonuclease protection assay; X13-leader, control peptide from cytochrome P450 attached at carboxyl terminus to transmembrane-penetrating leader sequence