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

Abstract

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 ␣-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. The principal function of vascular smooth muscle (VSM) 2 cells in a developed vascular system is the regulation of blood pressure and flow. In certain diseased states, however, VSM cells can undergo a phenotypic modulation toward a proliferative and secretory phenotype or by reverting toward the nonproliferative contractile phenotype (1). The transition from a differentiated phenotype to a fibroblast-like proliferative state is observed during the onset or progression of atherosclerosis (2-3), one of the most common diseases in developed countries (4 -5). Under these conditions, the VSM cell phenotype is reminiscent of that observed during vascular development, where VSM cells play a key role in morphogenesis of the blood vessel and exhibit a high proliferative index, migrate, and produce extracellular matrix components (6). Conditions that promote the proliferative phenotype include the combined action of growth factors, proteolytic enzymes, and exposure to extracellular matrix proteins (7).The reciprocal process has been observed upon completion of wound healing or during formation and organization of a fibrous cap. Under these conditions, VSM cells are exposed to various stimuli, including macrophage-and lymphocyte-derived cytokines and serum components that are currently being discussed as critical regulators of plaque stability.On the molecular level, phenotypic modulation of VSM cells depends on the activation of mitogen-activated protein (MAP) kinases (8). The epidermal growth factor (EGF) receptor is transactivated after G protein-coupled receptor (GPCR) activation and recruits the guanine nucleotide exchange factor (Sos) through adaptor proteins, Shc and Grb2, thereby initiating the canonical Ras/Raf/MEK/ERK cascade (9 -10). Activated MAP kinases of the extracellular signal-regulated kinase (ERK1/2) family in turn translocate to the nucleus and phosphorylate nuclear transcription factors or transcriptional coactivators (11).Within the family of EGF receptor ligands, heparin-binding EGF (HB-EGF) has been implicated in vascular remodeling because it is a potent mitogen, acts as a chemotactic factor for VSM cells, and is abundantly expressed in vascular lesions such as atherosclerosis (12). Upon membrane insertion and signal peptide cleavage, pro-HB-E...

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Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells

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