Abstract

Backgroundα2-adrenoceptors are important regulators of vascular tone and blood pressure. Regulation of cell proliferation is a less well investigated consequence of α2-adrenoceptor activation. We have previously shown that α2B-adrenoceptor activation stimulates proliferation of vascular smooth muscle cells (VSMCs). This may be important for blood vessel development and plasticity and for the pathology and therapeutics of cardiovascular disorders. The underlying cellular mechanisms have remained mostly unknown. This study explored pathways of regulation of gene expression and intracellular signaling related to α2B-adrenoceptor-evoked VSMC proliferation.ResultsThe cellular mechanisms and signaling pathways of α2B-adrenoceptor-evoked proliferation of VSMCs are complex and include redundancy. Functional enrichment analysis and pathway analysis identified differentially expressed genes associated with α2B-adrenoceptor-regulated VSMC proliferation. They included the upregulated genes Egr1, F3, Ptgs2 and Serpine1 and the downregulated genes Cx3cl1, Cav1, Rhoa, Nppb and Prrx1. The most highly upregulated gene, Lypd8, represents a novel finding in the VSMC context. Inhibitor library screening and kinase activity profiling were applied to identify kinases in the involved signaling pathways. Putative upstream kinases identified by two different screens included PKC, Raf-1, Src, the MAP kinases p38 and JNK and the receptor tyrosine kinases EGFR and HGF/HGFR. As a novel finding, the Src family kinase Lyn was also identified as a putative upstream kinase.Conclusionsα2B-adrenoceptors may mediate their pro-proliferative effects in VSMCs by promoting the activity of bFGF and PDGF and the growth factor receptors EGFR, HGFR and VEGFR-1/2. The Src family kinase Lyn was also identified as a putative upstream kinase. Lyn is known to be expressed in VSMCs and has been identified as an important regulator of GPCR trafficking and GPCR effects on cell proliferation. Identified Ser/Thr kinases included several PKC isoforms and the β-adrenoceptor kinases 1 and 2. Cross-talk between the signaling mechanisms involved in α2B-adrenoceptor-evoked VSMC proliferation thus appears to involve PKC activation, subsequent changes in gene expression, transactivation of EGFR, and modulation of kinase activities and growth factor-mediated signaling. While many of the identified individual signals were relatively small in terms of effect size, many of them were validated by combining pathway analysis and our integrated screening approach.

Highlights

  • The α2-adrenoceptors, a subclass of the family of Gprotein coupled receptors (GPCRs), are targets for cardiovascular drug development because they mediate important actions of noradrenaline and adrenaline in the regulation of vascular tone and blood pressure

  • Proliferation and migration of VSMCs are involved in the development of many major cardiovascular diseases, Fig. 6 Protein tyrosine kinase (PTK, panel a) and serine/threonine kinase (STK, panel b) activity profiles at 5 min, 30 min, 2 h and 24 h in A7r5α2Bvascular smooth muscle cells treated with 100 nM dexmedetomidine (DEX) or vehicle

  • The aim of this study was to shed light on the mechanisms involved in the α2B-adrenoceptor-evoked enhancement of proliferation in A7r5 VSMCs by exploiting DNA and kinase activity profiling microarrays and kinase/phosphatase inhibitor library screening

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Summary

Introduction

The α2-adrenoceptors, a subclass of the family of Gprotein coupled receptors (GPCRs), are targets for cardiovascular drug development because they mediate important actions of noradrenaline and adrenaline in the regulation of vascular tone and blood pressure. Regulation of cell proliferation is a less well investigated consequence of α2-adrenoceptor activation. Inhibition of cholangiocarcinoma and pheochromocytoma cell proliferation by α2-adrenoceptor agonists has been reported [15, 16]. In many of these studies, the investigated cells expressed all three α2-adrenoceptor subtypes, making it impossible to specify the subtype(s) involved. Little is known about the effects of α2-adrenoceptor activation on the proliferation of vascular smooth muscle cells (VSMCs). Our own previous results indicated that activation of α2B-adrenoceptors would have prominent stimulatory effects on the proliferation of cultured A7r5 rat VSMCs [17]

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