Abstract
Background: In essential hypertension, angiotensin (Ang) II induces arterial remodeling that is dependent on the activation of ERK1/2 pathway in vascular smooth muscle cells (VSMC). Arterial remodeling is also dependent on transglutaminase 2 (TG2), a protein with an emerging role in vascular pathophysiology. Beside its effect on extracellular matrix, TG2 is a multifunctional protein with GTPase activity and as such, could potentially be a signaling molecule. We hypothesized that TG2 mediates Ang II-induced ERK1/2 activation in VSMC during arterial remodeling. Methods and Results: To determine the role of TG2 in Ang II type 1 receptor (AT 1 R) signaling, His 6 tagged-TG2 was overexpressed in the human embryonic kidney (HEK) 293 cell line stably overexpressing HA tagged-AT 1 R. Protein and phosphorylation levels were determined by Western blot. When stimulated for 2-60 min, TG2 overexpression potentiated 100 nM Ang II-induced ERK1/2 phosphorylation compared to control cells overexpressing green fluorescent protein (GFP, P <0.05, n=4). Maximal potentiation was observed after 2 min of stimulation at which time TG2 transfected cells showed an increase of 64±31% compared to GFP transfected cells. The role of TG2 in Ang II-induced phosphorylation of ERK1/2 was studied by TG2 siRNA knockdown in mouse vascular smooth muscle cell line (MOVAS). TG2 siRNA decreased TG2 expression by 83±5% compared to cells treated with control siRNA (n=4). Transfection of TG2 siRNA did not significantly change cell viability when compared to ctrl siRNA (n=3). Ten min stimulation with 1 and 100 nM Ang II increased ERK1/2 phosphorylation by 38±18% and 60±29%, respectively, and this was prevented by TG2 knockdown ( P <0.01). Using western blots, HA-AT 1 R dimer/monomer ratio was increased by co-expression of His 6 -TG2 compared to HA-AT1R expressed alone. Conclusion: These results suggest that TG2 mediates ERK1/2 activation by Ang II through a mechanism involving TG2-induced AT 1 R dimerization. Overall, our results demonstrate that TG2 is involved in the activation of ERK1/2 in response to Ang II and could participate in arterial remodeling induced by Ang II.
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