The Study On The Mechanism Of Glutathione S-transferase Pi Regulating Proliferation And Migration Of Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) proliferate, migrate to the intima, contribute to thickening the artery wall and exacerbating atherosclerotic lesions. Angiotensin Ⅱ (Ang Ⅱ)-elicited excessive proliferation, hypertrophy and migration of VSMCs are vital to atheroclerosis related vascular remodeling. Signal transducer and activator of transcription3(STAT3) is thought to participate in Ang Ⅱ-induced cell proliferation and migration. Under the stimulation by Ang Ⅱ. STAT3is phosphorylated at tyrosine705, dimerizes, translocates to the nucleus, binds to specific DNA elements in the promoters, activates transcription of proliferation and migration associated gene. Blocking STAT3signaling effectively abolishes the proliferative response to Ang Ⅱ. Glutathione S-transferases (GSTs) are identified as a multigene family of isozymes that catalyse the nucleophilic attack of the sulfur atom of glutathione (GSH) on electrophilic groups of substrate molecules. Glutathione S-transferase pi (GSTpi) exists extensively in various kinds of cells and protects cells against different stresses. Upregulating GSTpi expression is the underlying mechanism to protect cells and organisms from toxic insults. Some studies show that GSTpi contributes to the regulation of cell proliferation. However, knowledge remains limited about what GSTpi acts in the proliferation of VSMCs.We investigated that VSMC proliferated in thoracic aortas of HFD fed rats, in which GSTpi protein level is upregulated. In vitro, Ang II upregulated GSTpi protein level in VSMC. These data suggested that upregulation of GSTpi might regulate the Ang Ⅱ-induced atheroclerosis related vascular remodeling. Overexpression and ribonucleic acid interference (RNAi) experiments demonstrated that GSTpi inhibited Ang Ⅱ-induced proliferation, hypertrophy and migration of VSMCs and arrested progression of cell cycle from G0/G1to S phase. Immunoprecipitation, mass spectrometry and confocal microscopy analyses showed that GSTpi directly associated with STAT3to prevent Ang Ⅱ-triggered binding of Src to STAT3and thus suppressed Ang Ⅱ-stimulated phosphorylation and nuclear translocation of STAT3, as well as cyclin D1expression. In contrast, GSTpi didn’t affect Ang Ⅱ-activated extracellular signal-regulated kinase (ERK1/2). Since GSTpi directly interacted with STAT3to exert its inhibitory effect in STAT3signalling, it inhibited both Ang Ⅱ and Platelet-derived growth factor (PDGF)-induced proliferation and activation of STAT3in VSMCs.In the present study, catalytically inactive mutant GSTpi (Y7F, phenylalanine replaced tyrosine in the seventh amino-terminal position) also inhibited the proliferation of VSMCs and prevented the activation of STAT3. Thus GSTpi inhibited Ang Ⅱ-induced VSMC proliferation via its non-catalytic ligand-binding activity. Moreover, Catalytically active GSTpi potentiates S-glutathionylation of proteins. S-glutathionylation is a novel post-translational modification through disulfide linkages between GSH and redox-sensitive cysteine residues within proteins. Ang Ⅱ led to S-glutathionylation of STAT3in VSMCs. However, GSTpi didn’t influence Ang Ⅱ induced.S-glutathionylation of STAT3.In conclusion, GSTpi serves a regulatory role in modulating signaling cascade through its ligand-binding properties to prevent Ang Ⅱ-triggered proliferative signaling in VSMCs, suggesting that it may protect vessels against the stresses associated with atherosclerosis formation.