Role of integrins in angiotensin II-induced proliferation of vascular smooth muscle cells

Abstract

Angiotensin II (AII) binds to G protein-coupled receptor AT(1) and stimulates extracellular signal-regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix, which occurs via integrins. To study cross-talk between G protein-coupled receptor- and integrin-induced signaling, we hypothesized that integrins are involved in AII-induced proliferation of VSMC. Using Oligo GEArray and quantitative RT-PCR, we established that messages for α(1)-, α(5)-, α(V)-, and β(1)-integrins are predominant in VSMC. VSMC were cultured on plastic dishes or on plates coated with either extracellular matrix or poly-d-lysine (which promotes electrostatic cell attachment independent of integrins). AII significantly induced proliferation in VSMC grown on collagen I or fibronectin, and this effect was blocked by the ERK inhibitor PD-98059, suggesting that AII-induced proliferation requires ERK activity. VSMC grown on collagen I or on fibronectin demonstrated approximately three- and approximately sixfold increases in ERK phosphorylation after stimulation with 100 nM AII, respectively, whereas VSMC grown on poly-d-lysine demonstrated no significant ERK activation, supporting the importance of integrin-mediated adhesion. AII-induced ERK activation was reduced by >65% by synthetic peptides containing an RGD (arginine-glycine-aspartic acid) sequence that inhibit α(5)β(1)-integrin, and by ∼60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α(1)β(1). Furthermore, neutralizing antibody against β(1)-integrin and silencing of α(1), α(5), and β(1) expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α(5)β(1) and α(1)β(1)) in AII-induced proliferation of VSMC.

Fig. 1.

mRNA and protein expression of integrins in vascular smooth muscle cells (VSMC). A: expression of mRNAs for specific integrins was assessed by the Oligo GEArray, as described in experimental procedures. B: Western blot analyses of VSMC lysates (40 μg of total protein) were performed with commercially available anti-integrin antibodies to demonstrate the expression of these integrins at the protein level.

Fig. 2.

VSMC proliferation and angiotensin II (AII)-induced ERK activation is dependent on integrin-mediated anchorage. A: VSMC were seeded (2 × 10⁵/well) on control plastic six-well plates or on six-well plates coated with poly-d-lysine, collagen I, or fibronectin and cultured in complete growth media. After 48 h, VSMC were trypsinized, and the number of cells in each well was counted with a hemocytometer. Results are presented as no. of cells/well (average of three independently counted wells). *P < 0.05 vs. samples from plastic plates. B: VSMC grown onto plastic, poly-d-lysine, collagen I, or fibronectin-coated plates, were stimulated with 100 nM AII for 5 min, lysed, and analyzed by Western blotting for ERK activation. Results are presented as intensities of phospho-ERK bands relative to total ERK and expressed as fold of basal (cells without AII treatment) phosphorylation. Results are means + SE of at least three independent experiments performed in duplicate. *P < 0.05, **P < 0.01 vs. vehicle.

Fig. 3.

AII-induced proliferation of VSMC is dependent on integrin-mediated adhesion and ERK activity. A: VSMC grown onto poly-d-lysine-, collagen I-, or fibronectin-coated plates were preincubated for 1 h with 10 μM PD-98059 or vehicle before stimulation with 100 nM AII or 10 ng/ml EGF (positive control) or vehicle for 24 h. Cell proliferation was assessed by the VisionBlue fluorescence cell viability assay kit, as described in experimental procedures. Results are presented as %relative fluorescence units (RFU) (means + SE) of at least six independent experiments. B: VSMC cultured on plates coated with collagen I were pretreated for 2 h with 200 μM inactive peptides or lysine-threonine-serine (KTS) peptides and stimulated with AII for 24 h in the presence of peptides. C: cells were cultured on plates coated with fibronectin, pretreated for 2 h with 200 μM inactive peptides or cyclic arginine-glycine-aspartic acid (RGD) peptides, and stimulated with AII for 24 h in the presence of peptides. Cell proliferation was assessed by the VisionBlue fluorescence cell viability assay kit, as described in experimental procedures. **P < 0.01, ***P < 0.001 vs. vehicle-treated samples.

Fig. 4.

AII-induced ERK phosphorylation is integrin dependent. Cells were cultured on plates coated with collagen I (A) or fibronectin (B), grown to 80% confluence, and serum starved for 36 h. Cells were pretreated for 2 h with 200 μM inactive peptides, cyclic RGD, or KTS peptides before stimulation with 100 nM AII for 5 min. ERK activity was assessed by Western blotting with phospho-specific ERK antibody. Values are expressed as fold of basal (vehicle-treated cells). C: quiescent VSMC were pretreated for 2 h with 100 μg/ml of anti-integrin antibodies or normal mouse or goat IgG before stimulation with vehicle or with 100 nM of AII for 5 min. Cells were lysed and analyzed by Western blotting for ERK activation. Results are presented as intensities of phospho-ERK bands relative to total ERK and expressed as fold of basal (cells without AII treatment) phosphorylation (means + SE) of at least three independent experiments performed in duplicate. *P < 0.05, ***P < 0.001 vs. vehicle-treated samples.

Fig. 5.

Transfection of VSMC with integrin short interfering RNAs (siRNAs) decreases AII-induced ERK activation. A, top and middle: VSMC were nucleofected with 100 nM of siRNA for integrin α₁ (-α₁) or β₁ (-β₁) alone, or with combinations of both siRNA (-α₁β₁), or with the same amount of scrambled siRNA (control). Transfections were performed as described in experimental procedures. Bars represent intensities of phospho-ERK (p-ERK) bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed at least 3 times in duplicate. Values are means + SE. ANOVA-AII treated (-α₁ or -β₁) compared with AII treated (-α₁β₁) was not significant. B, top and middle: VSMC were nucleofected either with 100 nM of siRNA for integrin α₅ (-α₅) or β₁ (-β₁) alone, with combinations of both siRNA (-α₅β₁), or with the same amount of scrambled siRNA (control). Forty-eight hours postnucleofection, cells were stimulated with vehicle or 100 nM AII for 5 min, lysed, and analyzed for ERK phosphorylation. **P < 0.01 vs. vehicle-treated samples. ANOVA-AII treated (-α₅ or -β₁) compared with AII treated (-α₅β₁) was not significant. Bottom: Western blot analyses of lysates of VSMC cells transfected with scrambled (Scr) siRNA or siRNAs for α₁-, α₅-, and β₁-integrins (40 μg of total protein) were performed with commercially available antibodies against α₁-, α₅-, and β₁-integrin subunits, to demonstrate downregulation of these proteins. Blots were stripped and reprobed with an antibody against GAPDH to control the specificity of silencing and protein loading.