N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

Abstract

Objective-The extracellular N terminus of the endothelin B (ET B ) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ET B receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs). Methods and Results-VSMCs expressing either the full-length ET B receptor or an N-terminally truncated ET B receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ET B receptor, IRL1620 (an ET B -selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22␣, and ␣-actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of G i proteins. In contrast, in VSMCs expressing N-terminally truncated ET B receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression. Key Words: transactivation Ⅲ EGF receptor Ⅲ ERK1/2 Ⅲ differentiation E ndothelin-1 (ET-1), one of the most potent vasoactive peptides, acts on 2 G protein-coupled receptors (GPCRs): the ET A receptor mainly expressed in vascular smooth muscle cells (VSMCs) and the endothelin B (ET B ) receptor predominantly expressed in endothelial cells. 1,2 Whereas VSMCs normally express only low levels of ET B receptors, a dramatic upregulation occurs during atherosclerosis, hypercholesterolemia, and in models of focal ischemia. [3][4][5][6] In atherosclerotic plaques, VSMCs close to foam cells show a predominant expression of ET B receptors and an enhanced immunoreactivity of ET-1. It was therefore suggested that ET-1 and ET B receptors may play a role in atherogenesis. 4 A protective role for the ET B receptor has been reported for the monocrotalin-induced pulmonary hypertension in mice. 7 Similarly, inhibition of the ET B receptor deteriorated remodeling after vascular injury in mice, 8 pointing to a protective role of the ET B receptor in atherosclerosis. Conclusions-ThisHowever, the precise role of ET B receptors in VSMCs remains elusive.VSMCs in native vessels are characterized by a slow proliferation rate and an abundant expression of contractile proteins, such as smooth muscle (SM) myosin and SM ␣-actin. On vascular injury, VSMCs show a loss of contractile protein expression and increased proliferative and migratory responses to mitogens or cytokines. 9,10 However, within 3 to 6 months after vascular injury, VSMCs may regain a contractile phenotype through yet unknown mechanisms. 11 It is notable that thrombin via the protease-activated receptor 1 (PAR1) 12,13 and rapamycin, a blocker of the mammalian target of rapamycin, induce differentiation of VSMCs b...

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Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor

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