Angiotensin II Stimulates p21-Activated Kinase in Vascular Smooth Muscle Cells

Role in Activation of JNK

From the Department of Medicine, Division of Cardiology, University of Washington, Seattle (U.S., T.I., M.I., J.S., B.C.B.), and Kinetek Pharmaceuticals Inc, Vancouver, British Columbia, Canada (M.I.H., S.P.).

Correspondence to Bradford C. Berk, Division of Cardiology, University of Washington, 1959 NE Pacific St, Seattle, WA 98195. E-mail bcberk{at}u.washington.edu

Abstract—Angiotensin II (Ang II) has been previously shown to stimulate the extracellular signal–regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase family members. Little is known regarding the upstream signaling molecules involved in Ang II–mediated JNK activation. Ang II has been shown to activate the Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT) pathway, suggesting similarities to cytokine signaling. In response to cytokines such as interleukin-1 and tumor necrosis factor-, the p21-activated kinase (PAK) has been identified as an upstream component in JNK activation. Therefore, we hypothesized that PAK may be involved in JNK activation by Ang II in vascular smooth muscle cells (VSMCs). PAK activity was measured by myelin basic protein phosphorylation in rat aortic VSMCs. In response to Ang II, PAK was rapidly stimulated within 1 minute, with a peak (5-fold increase) at 30 minutes. PAK stimulation preceded activation of JNK in VSMCs. Ang II–mediated activation of both PAK and JNK was Ca²⁺ dependent and inhibited by downregulation of phorbol ester–sensitive protein kinase C isoforms (by pretreatment with phorbol 12,13-dibutyrate) but not by pretreatment with GF109203X. Activation of both PAK and JNK was partially inhibited by tyrosine kinase inhibitors but not by specific Src inhibitors, suggesting regulation by a tyrosine kinase other than c-Src. Finally, introduction of dominant negative PAK markedly reduced the JNK activation by Ang II in both Chinese hamster ovary and COS cells stably expressing the Ang II type 1 receptor (AT1R). Our data provide evidence for PAK as an upstream mediator of JNK in Ang II signaling and extend the role of Ang II as a proinflammatory mediator for VSMCs.

Key Words: angiotensin II • mitogen-activated protein kinase • vascular smooth muscle cell • c-Jun kinase

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