Requirement of an intermediate gene expression for biphasic ERK1/2 activation in thrombin-stimulated vascular smooth muscle cells.

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.