Redox regulation of epidermal growth factor receptor signaling during the development of pulmonary hypertension

Abstract

The development of pulmonary hypertension (PH) involves the uncontrolled proliferation of pulmonary smooth muscle cells via increased growth factor receptor signaling. However, the role of epidermal growth factor receptor (EGFR) signaling is controversial, as humans with advanced PH exhibit no changes in EGFR protein levels and purpose of the present study was to determine whether there are post-translational mechanisms that enhance EGFR signaling in PH. The EGFR inhibitor, gefinitib, significantly attenuated EGFR signaling and prevented the development of PH in monocrotaline (MCT)-exposed rats, confirming the contribution of EGFR activation in MCT induced PH. There was an early MCT-mediated increase in hydrogen peroxide, which correlated with the binding of the active metabolite of MCT, monocrotaline pyrrole, to catalase Cys377, disrupting its multimeric structure. This early oxidative stress was responsible for the oxidation of EGFR and the formation of sodium dodecyl sulfate (SDS) stable EGFR dimers through dityrosine cross-linking. These cross-linked dimers exhibited increased EGFR autophosphorylation and signaling. The activation of EGFR signaling did not correlate with pp60src dependent Y845 phosphorylation or EGFR ligand expression. Importantly, the analysis of patients with advanced PH revealed the same enhancement of EGFR autophosphorylation and covalent dimer formation in pulmonary arteries, while total EGFR protein levels were unchanged. As in the MCT exposed rat model, the activation of EGFR in human samples was independent of pp60src phosphorylation site and ligand expression. This study provides a novel molecular mechanism of oxidative stress stimulated covalent EGFR dimerization via tyrosine dimerization that contributes into development of PH.

BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes

“…It is still unknown how this interaction affects signaling of either receptor, with the mechanism and/or binding domains that facilitate heteromer formation largely unknown. One possible mechanism that has not been tested directly is the reported contribution of reactive oxygen species (83,84) and their potential oxidation of the EGFR on dimers formation (85).…”

DNA Damage and Pulmonary Hypertension

“…In addition, oxidative stress can also promote vasoconstriction via increased production of endothelin-1 [ 158 ] and thromboxane A2 [ 159 ], decreased production of prostacyclin [ 160 , 161 ], and increased hypoxic cytosolic Ca 2+ concentration in PASMCs [ 162 , 163 ]. In agreement with the crucial role of oxidative stress in the pathogenesis of PAH [ 164 , 165 , 166 , 167 , 168 ], antioxidant therapy was reported to have beneficial effects in animal models of the disease [ 169 , 170 , 171 , 172 ].…”

Inhibition of Anaplerosis Attenuated Vascular Proliferation in Pulmonary Arterial Hypertension

“…A total of 20-40 mg of lung tissue was lysed in permeabilization buffer mixed with Halt TM Protease and Phosphatase Inhibitor Cocktail (78444, Thermo Fisher Scientific, Rockford, IL, USA) using a FisherBrand Homogenizer-850. Then centrifuged the homogenate at 10,000 g for 10 min, and the supernatant was collected [12]. Membrane and cytosolic fractions were isolated using the FractionPREP TM cell fraction kit (Biovision, Milpitas, CA, USA) according to the manufacturer's instruction.…”