Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

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Abstract

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT1/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT1 and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT1/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT1/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.

Introduction

Angiotensin II (Ang II) is a multifunctional hormone that has various effects on vascular smooth muscle cells (VSMCs). It has been reported previously that in Ang II-treated VSMCs, cyclin-dependent kinase (CDK) activity was suppressed, leading to G1-phase arrest and cell hypertrophy [1], [2], [3], [4]. Mounting evidence shows that Ang II activation contributes to pathological vascular remodeling, largely by stimulating VSMCs hypertrophy [4], [5]. However, the downstream signal transduction mechanisms by which Ang II stimulates VSMCs hypertrophy have not been defined.

ERK5 has recently been identified as a new member of the MAPK family, ERK5 is twice the size of ERK1/2, and similar to ERK1/2, ERK5 has the Thr–Glu–Tyr (TEY) activation motif [6], the pertinent phosphorylation sites of ERK5 have been mapped to Thr218/Tyr220. Activated ERK5 phosphorylates multiple substrates including myocyte enhancer factor 2 (MEF2) which induces the reprogramming of gene expression [6], [7]. Our previous studies have indicated that ERK5 phosphorylation contributed to MEF2C activation and played an important role in the hypertrophy of HASMCs induced by Ang II [8]. However, the upstream signaling molecules which mediate ERK5 activation in response to Ang II remain elusive.

Protein kinase D (PKD) is a founding member of a new protein kinase family within the CAMK group and separate from the previously identified PKCs [9]. Accumulating evidence demonstrates that PKD plays an important role in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling [10]. Most recently, it has been proposed that PKD may control hypertrophy of VSMCs via the AT1/PKCδ pathway by Ang II [11]. PKD alters the signal pathway of the MAPK family ERK1/2 and JNK [12], [13], [14]. However, the effect of PKD on ERK5 activation in HASMCs is not well understood.

The aim of this study was to determine whether and how Ang II activates ERK5 in HASMCs and to examine the potential role of ERK5 in Ang II-mediated signal transduction. The results presented here demonstrated that Ang II rapidly induces activation of ERK5 via the AT1/PKC/PKD pathway, which subsequently leads to ERK5 translocation, MEF2C activation and HASMCs hypertrophy. Based on our findings, we suggest that AT1/PKC/PKD/ERK5 pathway is implicated in Ang II-induced HASMCs hypertrophy.

Section snippets

Materials and methods

Materials. All basic laboratory reagents were from Sigma–Aldrich (St. Louis, MO). Ang II from Sigma. Antibodies against MEF2C, ERK5, phospho-MEF2C, phospho-ERK5 were from Santa Cruz Biotechnology; antibodies against PKD, ERK1/2, phospho-PKD, phospho-ERK1/2 from Cell Signaling Technology (Beverly, MA). SDS–polyacrylamide gels were from Pierce (Rockford, IL), PVDF and proteingel apparatus were from Bio-Rad (Hercules, CA). HASMCs were purchased from ScienCell (San Diego, CA). Minimal essential

Activation of MAPKs by Ang II in HASMCs

To determine the signaling molecules that are specifically involved in the hypertrophic response of cells pre-treated with Ang II, we first examined the potential role of ERK1/2 and ERK5 in HASMCs in response to Ang II stimulation. Ang II (100 nmol/L) induced phosphorylation of ERK5 after 5 min, with peak phosphorylation between 15 and 30 min, which returned to base line after 60 min. However, the phosphorylation of ERK1/2 was earlier than that of ERK5 (Fig. 1A). The phosphorylation response of

Discussion

The experiments presented here were designed to elucidate the underlying mechanisms of hypertrophy in HASMCs. Ang II-induced ERK5 activation and nuclear translocation was mediated by AT1/PKC/PKD pathway in HASMCs, which results in phosphorylation of MEF2C and consequent HASMCs hypertrophy. Our data show that Ang II-stimulated ERK5 phosphorylation in a time- and dose-dependent manner in HASMCs. Inhibition of AT1 and PKC by their pharmacological inhibitors significantly reduced ERK5 activation by

Acknowledgments

We gratefully acknowledge the technical assistance from Dr. Xuping Wang, Dr. Hong Jiang, Dr. Jinbo Feng and Dr. Chunxi Liu in histopathological and cellular molecular biological analysis. This study was supported by National Basic Research Program of China (973 Program) (No. 2007CB512003).

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    These authors contributed equally to this work.

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