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

Angiotensin II (AII) binds to G protein-coupled receptor AT1 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 α1-, α5-, αV-, and β1-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 α5β1-integrin, and by ∼60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α1β1. Furthermore, neutralizing antibody against β1-integrin and silencing of α1, α5, and β1 expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α5β1 and α1β1) in AII-induced proliferation of VSMC

Catalytically inactive or folate-binding deficient GNMT mutants are capable of the antiproliferative effect.

<p><b>A</b>. Crystal structure of GNMT tetramer (RCSB Protein Data Bank 3ths; subunits are shown in different colors) with bound 5-MTHF monoglutamate (two molecules shown in spacefill mode are bound per tetramer). <b>B</b>. Positions of amino acid residues in the GNMT catalytic center (from RCSB Protein Data Bank 1XVA). Acetate (Ac) is the competitive inhibitor of Gly and presumably occupies the same position in the active center. Glu 15 (E15*) is from a different subunit. Dotted lines indicate hydrogen bonds. <b>C</b> and <b>D</b>. The enzyme activities and CD spectra of GNMT mutants, analyzed in this study. <b>E</b>. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or corresponding mutants. <i>Error bars</i> represent ± S.D., <i>n =3</i>. <b>F</b>. Folate binding site at the GNMT subunit interface (as shown in panel <b>A</b>); Selected for mutagenesis are residues within close distance to 5-MTHF molecule (these residues are from all four subunits, which are denoted in parentheses). <b>G</b>. Binding of 5-MTHF by GNMT mutants. <i>Error bars</i> represent ± S.D., <i>n =2</i>. <b>H</b>. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or folate-binding deficient mutants mutants. <i>Error bars</i> represent ± S.D., <i>n =3</i>. <b>I</b>. The supplementation with excessively high media folate or SAM does not rescue cells from the GNMT antiproliferative effect. <i>Error bars</i> represent ± S.D., <i>n =3</i>.</p

Effect of GNMT transient transfection on cellular proliferation.

<p>Cell viability was assessed by MTT assay (absorbance at 570 nm reflects the number of live cells). <i>Error bars</i> represent ± S.D., <i>n =3</i>. Insets show levels of GNMT (Western blot) at different time points after transfection.</p

Subcellular localization-specific effects of GNMT.

<p><b>A</b>. Sequences used to target GNMT to cytosol or nucleus. B. Distribution of GNMT fusion constructs between cytosol and the nucleus. All constructs included GFP tag at the C-terminus of GNMT. Respective subcellular targeting sequences were introduced at the C-terminus of the GFP tag. <b>C</b>. Levels of corresponding GNMT constructs after transient transfection (Western blot assay). <b>D</b>. MTT assay of cells transfected with GNMT constructs. <i>Error bars</i> represent ± S.D., <i>n =2</i>; *, <i>p < 0</i>.<i>05</i>.</p

Disposition of GNMT in cellular metabolism.

<p>GNMT converts SAM to SAH, methylating glycine to sarcosine. This reaction regulates SAM/SAH ratio and shuttles methyl groups, from activated methyl cycle back to the folate pool. Inhibitory effect of 5-CH3-THF (5-MTHF) on GNMT catalysis is indicated. Hcy, homocysteine; Sarc, sarcosine; THF, tetrahydrofolate.</p

Cellular responses to GNMT expression.

<p>A. Distribution of GNMT-expressing cells (<i>right panel</i>) between cell cycle phases (propidium iodide staining) compared to control (<i>left panel</i>) GNMT-deficient cells. <b>B</b>. Assessment of DNA damage in GNMT expressing cells by the Comet assay. <b>C</b>. Apoptotic cells assessed by Annexin V/propidium iodide staining after GNMT expression (<i>bottom right quadrant</i>, early apoptotic cells; <i>upper right quadrant</i>, late apoptotic cells); only green cells (expressing GFP-GNMT) were evaluated. <b>D</b>. Calculation of apoptotic cells from C. <b>E</b>. Activation of ERK phosphorylation in response to GNMT expression. <b>F</b>. zVAD-fmk, but not ERK inhibitor PD98059, partially rescues cells from the antiproliferative effect of GNMT (data for A549 cells are shown).</p