Vasodilator-stimulated phosphoprotein regulates proliferation and growth inhibition by nitric oxide in vascular smooth muscle cells

Abstract

Objective: Vasodilator-stimulated phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition.

Methods and results: Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed.

Conclusions: Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

Figure 1

Expression of wt-VASP and S239A-VASP in VASP^−/− mouse vascular SMCs stimulates DNA synthesis and cell growth, whereas expression of S157A-VASP is inhibitory. VASP^−/− vascular SMCs were transfected with VSV-G–tagged wt-VASP or S157A-VASP or S239A-VASP using a retroviral vector (LXSN). Three clones (cl1, cl2, and cl3) with different wt-VASP expression levels were selected (see lanes 3, 4, and 5 in insert). S157A-VASP and S239A-VASP expressing clones with similar VASP expression level as that in C57BL/6 SMCs were selected (see lane 1, 6, and 7 in insert). DNA synthesis (A) and cell growth (B) were measured in wt-VASP, S157A-VASP, or S239A-VASP expressing clones and LXSN controls. Data are expressed as fold over starved. *P<0.05 (n=3). Protein lysates of SMCs expressing various VASP constructs were subjected to Western blotting. Blots were probed with antibody against VASP (insert in A). Ln 1: C57BL/6; 2: vector (LXSN); 3, 4, and 5: wt-VASP expressing clones 1, 2, and 3 (Cl1–3); Ln 6: S157A-VASP; Ln 7: S239A-VASP.

Figure 2

SNAP and cGMP analogues inhibit 15% FBS-induced DNA synthesis in VASP^−/− mouse vascular SMCs expressing wt-VASP, but not in SMCs expressing S239A-VASP. DNA synthesis was measured in SMCs expressing wt-VASP and S239A-VASP in the presence of different concentrations of SNAP (A), db-cGMP (B, left panel), and 8-bromo-cGMP (B, right panel), respectively. Data are expressed as percent of serum-stimulated control. *P<0.05 (n=3).

Figure 3

The effects of expression of wt-VASP, S157A-VASP, and S239A-VASP on DNA synthesis and NO-mediated growth inhibition in rat vascular SMCs. Rat SMCs were transfected with VSV-G-tagged wt-VASP, S157A-VASP, and S239A-VASP, respectively, using the Tet-inducible expression system. Protein lysates of SMCs expressing various VASP constructs in the presence or absence of Tet (1 μg/mL) were subjected to Western blotting. Blots were probed with antibodies against VASP and VSV-G tag. The absence of Tet induces ectopic expression of wt-VASP, S157A-VASP, and S239A-VASP (A). DNA synthesis in response to 10% FBS was measured in SMCs expressing wt-VASP, S157A-VASP, and S239A-VASP in the presence (Tet⁺) or absence (Tet⁻) of Tet, respectively (B). DNA synthesis in response to 10% FBS was also measured in SMCs expressing wt-VASP and S239A-VASP in the presence of different concentrations of SNAP in the presence (Tet⁺) or absence (Tet⁻) of Tet (C). Data are expressed as the percent of serum-stimulated control. *P<0.05 (n=3).

Figure 4

Function of VASP in regulating SMC proliferation. VASP functions as a modulator of SMC growth by integrating positive and negative signals that target different phosphorylation sites of VASP. Serum-induced VASP phosphorylation at serine157 by PKC promotes SMC proliferation, whereas NO-induced VASP phosphorylation at serine239 by PKG is required for the inhibitory effects of NO on SMC proliferation.