Angiotensin II induces Fat1 expression/activation and vascular smooth muscle cell migration via Nox1-dependent reactive oxygen species generation

Abstract

Fat1 is an atypical cadherin that controls vascular smooth muscle cell (VSMC) proliferation and migration. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) is an important source of reactive oxygen species (ROS) in VSMCs. Angiotensin II (Ang II) induces the expression and/or activation of both Fat1 and Nox1 proteins. This study tested the hypothesis that Ang II-induced Fat1 activation and VSMC migration are mediated by Nox1-dependent ROS generation and redox signaling. Studies were performed in cultured VSMCs from Sprague–Dawley rats. Cells were treated with Ang II (1 μmol/L) for short (5 to 30 min) or long term stimulations (3 to 12 h) in the absence or presence of the antioxidant apocynin (10 μmol/L), extracellular-signal-regulated kinases 1/2 (Erk1/2) inhibitor PD98059 (1 μmol/L), or Ang II type 1 receptor (AT1R) valsartan (1 μmol/L). siRNA was used to knockdown Nox1 or Fat1. Cell migration was determined by Boyden chamber assay. Ang II increased Fat1 mRNA and protein levels and promoted Fat1 translocation to the cell membrane, responses that were inhibited by AT1R antagonist and antioxidant treatment. Downregulation of Nox1 inhibited the effects of Ang II on Fat1 protein expression. Nox1 protein induction, ROS generation, and p44/p42 MAPK phosphorylation in response to Ang II were prevented by valsartan and apocynin, and Nox1 siRNA inhibited Ang II-induced ROS generation. Knockdown of Fat1 did not affect Ang II-mediated increases in Nox1 expression or ROS. Inhibition of p44/p42 MAPK phosphorylation by PD98059 abrogated the Ang II-induced increase in Fat1 expression and membrane translocation. Knockdown of Fat1 inhibited Ang II-induced VSMC migration, which was also prevented by valsartan, apocynin, PD98059, and Nox1 siRNA. Our findings indicate that Ang II regulates Fat1 expression and activity and induces Fat1-dependent VSMC migration via activation of AT1R, ERK1/2, and Nox1-derived ROS, suggesting a role for Fat1 downstream of Ang II signaling that leads to vascular remodeling.

Figure 1. Ang II increases Fat1 mRNA and protein expression

Time-course (3 to 12 h) effect of 10⁻⁶ mol/L Ang II on Fat1 gene (A) and protein (B) expression in VSMCs. (C) Effect of Ang II (12 h) on Fat1 protein expression in the absence and in the presence of apocynin or valsartan. (D) Effect of Ang II (12 h) on Fat1 protein expression in non-transfected and Nox1 siRNA-transfected VSMCs. Representative immunoblots: Fat1, Nox1, and β-actin. In this and subsequent figures, immunoblots probed initially for Fat1 or Nox1 were reprobed with a reference protein, as indicated, to assess loading. Results are mean ± SEM of 4 experiments. *P<0.05, vs vehicle.

Figure 2. Fat1 subcellular localization and Fat1 content in membrane fractions

Top panels, representative images of anti-Fat1 staining in VSMCs stimulated with 10⁻⁶ mol/L Ang II or vehicle. VSMCs were double-labeled with antibody to Fat1 (Alexa Fluor 488, anti-rabbit secondary antibody, green fluorescence) and Texas red-X phalloidin. DAPI was used for fluorescent staining of DNA. Arrows indicate Fat1 staining at cellular free edges (B) VSMCs were subjected to membrane and cytosol fractionation after stimulation with 10⁻⁶ mol/L Ang II (5 and 30 min). Membrane to cytosol ratio of Fat1 protein content is expressed as % of vehicle. Representative immunoblots: Fat1and β-actin. Results are mean ± SEM of 5 experiments. *P<0.05, vs vehicle.

Figure 3. Ang II increases Nox1 mRNA and protein expression

Time-course (3 to 12 h) effect of 10⁻⁶ mol/L Ang II on Nox1 mRNA (A) and protein (B) expression in VSMCs. (C) Effect of Ang II (12 h) on Nox1 protein expression in the absence or presence of apocynin and valsartan. (D) Effect of Ang II (12 h) on Nox1 protein expression in VSMCs, non-transfected or transfected with Fat1 siRNA. Representative immunoblots: Nox1, Fat1, and β-actin. Results are mean ± SEM of 3 experiments. *P<0.05, vs vehicle.

Figure 4. Ang II-induced ROS generation in VSMCs is inhibited by AT1R antagonist, antioxidants, and Nox1 siRNA

ROS generation was evaluated by lucigenin chemiluminescence and DHE fluorescence. VSMCs were stimulated with 1 μmol/L Ang II for short (A) and long (B) term in the absence or in the presence of apocynin (10 μmol/L). (C) VSMCs non-transfected and transfected and with Fat1 or Nox1 siRNA. (D) p47phox translocation was evaluated in VSMCs subjected to membrane and cytosol fractionation after stimulation with 10⁻⁶ mol/L Ang II. Membrane to cytosol ratio of p47phox protein content is expressed as % of vehicle. Top panels, representative images of DHE fluorescence. Representative immunoblot: p47phox. Results are mean ± SEM of 6 experiments. *P<0.05, vs vehicle.

Figure 5. Ang II-induced Fat1 expression/activation via ERK1/2 phosphorylation

ERK1/2 MAPK phosphorylation was evaluated in VSMCs stimulated with 1 μmol/L Ang II for short (A) and long (B) term in the absence or in the presence of apocynin (10 μmol/L). The effects of Ang II on Fat1 expression (C) and translocation to the cell membrane (D) were evaluated in the absence or in the presence of PD98059 (1 μmol/L). Representative immunoblots: ERK1/2 MAPK, ERK1/2 MAPK [Thr²⁰²/Tyr²⁰⁴], Fat1, β-actin. Results are mean ± SEM of 6 experiments. *P<0.05, vs vehicle.

Figure 6. Ang II induces VSMC migration through redox signaling and Fat1-dependent mechanisms

VSMC migration in response to Ang II stimulation (1 μmol/L, 12 h) was determined using a Boyden chamber assay. Ang II-induced VSMC migration was determined in the absence or in the presence of apocynin (10 μmol/L), valsartan (10 μmol/L), PD98059 (1 μmol/L), and in cells transfected with siRNA for Nox1 or Fat1. Results are mean ± SEM of 4 experiments. *P<0.05, vs vehicle.

Figure 7. Signalling scheme linking Ang II and ROS generation to Fat1 and VSMC migration

In VSMCs, Ang II induces activation of the AT1R, which triggers Nox1 activation and ROS generation; ROS activates ERK1/2 MAPK, leading to increased Fat1 cadherin activity and expression, which is required for Ang II-induced cellular migration.