Group VIA phospholipase A2 (iPLA2beta) participates in angiotensin II-induced transcriptional up-regulation of regulator of g-protein signaling-2 in vascular smooth muscle cells

Abstract

Rgs2 (regulator of G-protein signaling-2)-deficient mice exhibit severe hypertension, and genetic variations of RGS2 occur in hypertensive patients. RGS2 mRNA up-regulation by angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) is a potentially important negative feedback mechanism in blood pressure homeostasis, but how it occurs is unknown. Here we demonstrate that group VIA phospholipase A2 (iPLA2beta) plays a pivotal role in Ang II-induced RGS2 mRNA up-regulation in VSMC by three independent approaches, including pharmacologic inhibition with a bromoenol lactone suicide substrate, suppression of iPLA2beta expression with antisense oligonucleotides, and genetic deletion in iPLA2beta-null mice. Selective inhibition of iPLA2beta by each of these approaches abolishes Ang II-induced RGS2 mRNA up-regulation. Furthermore, using adenovirus-mediated gene transfer, we demonstrate that restoration of iPLA2beta-expression in iPLA2beta-null VSMC reconstitutes the ability of Ang II to up-regulate RGS2 mRNA expression. In contrast, Ang II-induced vasodilator-stimulated phosphoprotein phosphorylation and Ang II receptor expression are unaffected. Moreover, in wild-type but not iPLA2beta-null VSMC, Ang II stimulates iPLA2 enzymatic activity significantly. Both arachidonic acid and lysophosphatidylcholine, products of iPLA2beta action, induce RGS2 mRNA up-regulation. Inhibition of lipoxygenases, particularly 15-lipoxygenase, and cyclooxygenases, but not cytochrome P450-dependent epoxygenases inhibits Ang II- or AA-induced RGS2 mRNA expression. Moreover, RGS2 protein expression is also up-regulated by Ang II, and this is attenuated by bromoenol lactone. Disruption of the Ang II/iPLA2beta/RGS2 feedback pathway in iPLA2beta-null cells potentiates Ang II-induced vasodilator-stimulated phosphoprotein and Akt phosphorylation in a time-dependent manner. Collectively, our results demonstrate that iPLA2beta participates in Ang II-induced transcriptional up-regulation of RGS2 in VSMC.

FIGURE 1. BEL dose dependently inhibits Ang II-induced RGS2 mRNA up-regulation in VSMC

Quiescent VSMC were incubated with different concentrations of BEL, vehicle (Me₂SO), or propranolol (10 μm) for 30 min, respectively. Cells were then stimulated with 100 nm Ang II for 1 h. RGS2 mRNA expression was determined by real-time PCR and normalized to 18 S rRNA and to the basal level. A, a representative DNA acrylamide gel of real-time PCR products. P, positive control, using a RGS2 cDNA as PCR template; N, negative control, no PCR template. B, a dose curve of BEL inhibition on RGS2 mRNA expression. C, BEL, not propranolol, inhibits RGS2 mRNA expression. The summary data are from at least four time independent experiments. ###, p < 0.001 versus basal; ***, p < 0.001 versus Ang II stimulation alone.

FIGURE 2. iPLA₂β antisense oligonucleotide down-regulates iPLA₂β protein and inhibits Ang II-induced RGS2 mRNA up-regulation in VSMC

Cells were transfected with 2 μg of iPLA₂β antisense or sense oligonucleotide. The cells were cultured in serum-free medium for 48 h for quiescence, and then stimulated with 100 nm Ang II for 1 h. The iPLA₂β and cPLA₂ proteins were determined by Western blot using anti-iPLA₂β antibody (Santa Cruz) and anti-cPLA₂ antibody, respectively. RGS2 mRNA was determined by real-time PCR. A, a representative iPLA₂β and cPLA₂ Western blot. B, summary of the Western blot shown in A. C, antisense, not sense, inhibits RGS2 mRNA expression. The summary data are from at least three time independent experiments. ***, p < 0.001 versus the basal sample (non-transfected cells); ##, p < 0.01 versus the basal sample (without Ang II stimulation); *, p < 0.05 versus Ang II stimulation alone.

FIGURE 3. Ang II fails to stimulate RGS2 mRNA expression in iPLA₂β-null VSMC

Aortic vascular smooth muscle cells were isolated from wild-type and iPLA₂β-null mice (iPLA₂β KO). iPLA₂β and RGS2 mRNA expressions were determined by real-time PCR. A, a representative DNA acrylamide gel of real-time PCR products. P, positive control, using an iPLA₂β cDNA as PCR template; N, negative control, no PCR template. B, summary of the real-time PCR shown in A. C, Ang II fails to stimulate RGS2 mRNA expression in iPLA₂β-null VSMC. The summary data are from at least three time independent experiments. ###, p < 0.001 versus basal; ***, p < 0.001 versus basal.

FIGURE 4. Reconstitution of iPLA₂β expression by adenoviral-mediated gene transfer in iPLA₂β-null VSMC dose-dependently restores Ang II-induced RGS2 mRNA up-regulation

iPLA₂β-null cells were infected with a recombinant adenovirus encoding iPLA₂β and green fluorescent protein, and a Tet-on adenovirus encoding a transcriptional regulator in the presence of different concentrations of DOX for 12 h. After removal of adenovirus, cells were cultured in the absence of fetal bovine serum for another 36 h. Over 95% of cells were infected by adenovirus based on % of cells that expressed green fluorescent protein. The cells were then stimulated with Ang II (100 nm, 1 h). The iPLA₂β and β-actin protein expressions were determined by Western blot. RGS2 mRNA was determined by real-time PCR. A, a representative Western blot. B, summary of the Western blot shown in A. C, RGS2 mRNA from each sample was normalized to 18 S rRNA and then to the sample that has neither Ang II stimulation and nor iPLA₂β expression (0 DOX). The summary data are from at least three time independent experiments. ***, p < 0.001.

FIGURE 5. Inhibition of iPLA₂β by BEL or genetic deletion nether affects Ang II-induced VASP protein phosphorylation nor Ang II receptor (AT1R) protein expression

Prior to Ang II stimulation (100 nm, 5 min), wild-type cells were pretreated with BEL (3 μm, 30 min). The VASP phosphorylation levels in both wild-type (A and B) and iPLA₂β-null cells (C and D) were determined using an anti-VASP Ser-157 phospho-specific antibody and normalized to β-actin and then to the basal level. The AT1R protein expression was determined by Western blot using an anti-AT1R antibody (E and F). The summary data are from at least three time independent experiments. ***, p < 0.001 versus basal. KO, knock-out.

FIGURE 6. BEL or genetic deletion of iPLA₂β abolishes PGF2α-induced RGS2 mRNA up-regulation in VSMC

Prior to PGF2α stimulation (30μm, 1 h), wild-type cells (A), but not iPLA₂β-null cells (B), were preincubated with different concentrations of BEL. The RGS2 mRNA expression was determined by real-time PCR. The summary data are from at least seven independent experiments. ###, p < 0.001 versus basal; ***, p < 0.001 versus PGF2α stimulation alone; **, p < 0.01 versus basal.

FIGURE 7. Ang II stimulates iPLA₂β enzymatic activity in wild-type VSMC, but in BEL-pretreated or iPLA₂β-null VSMC

Prior to Ang II stimulation (100 nm, 1 h), wild-type cells (A), but not iPLA₂β-null cells (B), were pretreated with BEL (3 μm, 30 min). Equal amounts of protein from basal and stimulated cells were assayed for iPLA₂ enzymatic activity (in absorbance/mg of protein units) using a commercially available kit. The summary data are from at least three time independent experiments. ##, p < 0.01 versus basal; ***, p < 0.01 versus Ang II stimulation alone.

FIGURE 8. Free AA, LPC, and AA metabolite are involved in Ang II-induced RGS2 mRNA expression in VSMC

Quiescent rat VSMC were incubated for 1 h with different concentrations of AA (A) or LPC (B). Quiescent mouse wild-type (C) or iPLA₂β-null VSMC (D) were stimulated with AA (30 μm) or LPC (30 μm) for 1 h. E and F, quiescent rat VSMC were pretreated with vehicle (Veh, Me₂SO), nordihydroguaiaretic acid (NDGA, 30 μm) (60, 61), indomethacin (Indo, 50 μm) (61, 62), or 17-octadecynoic acid (ODA, 10 μm) (63), MK (MK886, 1 μm) (61), baicalein (Bai,10 μm) (62), or luteolin (Lut,50 μm) (64), respectively, prior to stimulation by Ang II (100 nm)or AA (30 μm) for 1 h. The RGS2 mRNA expression was determined by real-time PCR. Shown in the figure is a summary of three independent experiments. * and #, p < 0.05; and ** and ##, p < 0.01 versus basal. ***, p < 0.001 versus basal/Veh, and ###, p < 0.001 versus Ang II/Veh or AA/Veh.

FIGURE 9. BEL dose-dependently inhibits Ang II-induced RGS2 protein up-regulation in VSMC

A, a representative Western blot. Cells were transfected with 20 nm RGS2 siRNA, control siRNA, or Lipofectamine 2000 only. RGS2 and β-actin protein expressions were blotted by anti-RGS2 and β-actin antibodies, respectively. B and C, quiescent VSMC were incubated with different concentrations of BEL or vehicle (Me₂SO) for 30 min, respectively, before Ang II stimulation (100 nm Ang II, 2 h). RGS2 protein expression was determined by Western blot using an anti-RGS2 antibody and normalized to β-actin and then to the basal level. B, A representative RGS2 Western blot. The summary data are from at least four time independent experiments (C). ##, p < 0.01 versus basal; **, p < 0.01 versus Ang II stimulation alone.

FIGURE 10. Genetic deletion of iPLA₂β partially, but significantly, potentiates Ang II-induced VASP and Akt phosphorylation in a time-dependent manner

Quiescent mouse WT and iPLA₂β-null VSMC were stimulated with Ang II (100 nm) for the indicated intervals. VASP phosphorylation levels (VASP-P, A and B) were determined with anti-VASP Ser-157 phospho-specific antibody and normalized to β-actin and then to the WT basal level. Akt phosphorylation levels (C and D) were determined with an anti-Akt Ser-473 phospho-specific antibody. The phosphorylation level was normalized to the total Akt (Akt-T) level and then to the WT basal level. The representative Western blots (A and C) are from at least three independent experiments and quantitative data are summarized in B and D. **, p < 0.01; ***, p < 0.001 versus WT basal. KO, knock-out.