Requirement of an Intermediate Gene Expression for Biphasic ERK1/2 Activation in Thrombin-stimulated Vascular Smooth Muscle Cells

Alejandra Pérez Sastre,Solveig Grossmann,H Peter Reusch,Michael Schaefer

doi:10.1074/jbc.m800949200

Alejandra Pérez Sastre, Solveig Grossmann + Show 2 more

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https://doi.org/10.1074/jbc.m800949200

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The expression of contractile proteins in vascular smooth muscle cells is controlled by still poorly defined mechanisms. A thrombin-inducible expression of smooth muscle-specific alpha-actin and myosin heavy chain requires transactivation of the epidermal growth factor (EGF) receptor and a biphasic activation of ERK1/2. Here we demonstrate that the sustained second phase of ERK1/2 phosphorylation requires de novo RNA and protein synthesis. Depolymerization of the actin cytoskeleton by cytochalasin D or disruption of transit between the endoplasmic reticulum and the Golgi apparatus by brefeldin A prevented the second phase of ERK1/2 phosphorylation. We thus conclude that synthesis and trafficking of a plasma membrane-resident protein may be critical intermediates. Analysis of the expression of protease-activated receptor 1, heparin-binding EGF (HB-EGF), and the EGF receptor revealed that pro-HB-EGF is significantly up-regulated upon thrombin stimulation. The kinetic of HB-EGF expression closely matched that of the second phase of ERK1/2 phosphorylation. Because inhibition of matrix metalloproteases or of the EGF receptor strongly attenuated the late phase of ERK1/2 phosphorylation, the second phase of ERK1/2 activation is primarily relayed by shedding of EGF receptor ligands. The small interfering RNA-mediated knockdown of HB-EGF expression confirmed an important role of HB-EGF expression in triggering the second phase of ERK1/2 activation. Confocal imaging of a yellow fluorescent protein-tagged HB-EGF construct demonstrates the rapid plasma membrane integration of the newly synthesized protein. These data imply that the hormonal control of contractile protein expression relies on an intermediate HB-EGF expression to sustain the signaling strength within the Ras/Raf/MEK/ERK cascade.

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The principal function of vascular smooth muscle (VSM)2 cells in a developed vascular system is the regulation of blood
We tested the hypothesis that an intermediate gene expression may underlie the delayed appearance of a second wave of ERK1/2 activation, and we provide evidence for a functionally relevant de novo expression of HB-epidermal growth factor (EGF) that contributes to the second phase of ERK1/2 activation and subsequent SM-␣-actin expression in newborn rat aortic VSM cells
To analyze whether both phases of activation depend on the phosphorylation of preexisting signaling molecules or whether de novo mRNA transcription and synthesis of proteins may be required, VSM cells were pretreated with actinomycin D (4 ␮M), an inhibitor of mRNA polymerase activity, prior to thrombin stimulation

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Introduction

The principal function of vascular smooth muscle (VSM)2 cells in a developed vascular system is the regulation of blood. In thrombin-stimulated VSM cells, the protease-activated receptor 1 (PAR1) induces a biphasic and long lasting activation of ERK1/2 and, subsequently, promotes the expression of contractile proteins and differentiation markers such as smooth muscle-specific ␣-actin and smooth muscle-specific myosin heavy chain [18].

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