Role of calcium-independent phospholipase A2beta in high glucose-induced activation of RhoA, Rho kinase, and CPI-17 in cultured vascular smooth muscle cells and vascular smooth muscle hypercontractility in diabetic animals

Abstract

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A(2)beta (iPLA(2)beta) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA(2)beta mRNA, protein, and iPLA(2) activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA(2)beta protein up-regulation. Inhibiting iPLA(2)beta activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA(2)beta in iPLA(2)beta-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA(2)beta inhibition. Moreover, increases in iPLA(2) activity and iPLA(2)beta protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA(2)beta, but not iPLA(2)gamma, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA(2)beta up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.

FIGURE 1.

High glucose increases iPLA₂β mRNA, protein expression, and enzymatic activity in a time-dependent manner. 70–80% confluent rat (A–E) or mouse (F) aortic VSMC were incubated in 10% FBS medium containing NG (5.5 mm), HG (25 mm), or mannitol (Mann, 5.5 mm glucose plus 19.5 mm mannitol) for various time periods as indicated. The medium was changed every 12 h. A, a representative DNA acrylamide gel of real time PCR products (NTC, no template control). B, summary of data shown in A; C, a representative Western blot of iPLA₂β and β-actin; D, summary of data of shown in C; E, summary of data of iPLA₂ assay in rat VSMC; F, summary of data of iPLA₂ assay in mouse VSMC. Each experiment was repeated at least three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus NG. NS, not significant versus iPLA₂β-KO/NG/6 h or iPLA₂β-KO/HG/1 h.

FIGURE 2.

BEL inhibition or genetic deletion of iPLA₂β does not affect high glucose-induced PKCβII phosphorylation, whereas inhibition of PKC attenuates high glucose-induced iPLA₂β protein up-regulation. 70–80% confluent rat (A, B, E, and F) and mouse (C and D) aortic VSMC were incubated with 10% FBS medium containing NG or HG in the presence of BEL (3 μm) for 24 h (A and B), GF109203X (GF, 3 μm) for 12 h (E and F), and vehicle (Veh, Me₂SO), respectively. A, C, and E, representative Western blots of iPLA₂β, total PKCβII (PKCβII-T), and phosphorylated PKCβII (PKCβII-P); B, D, and F, summary of data shown in A, C, and E, respectively. Each experiment was repeated at least three times. ***, p < 0.001 versus NG/vehicle in B, NG/WT in D, and NG/vehicle in F; ###, p < 0.001 versus NG/vehicle in B, NG/WT in D, and HG/vehicle in F; NS, not significant, HG/vehicle versus HG/BEL in B; HG/WT versus HG/KO in D.

FIGURE 3.

BEL inhibition or genetic deletion of iPLA₂β abolishes high glucose-induced CPI-17 phosphorylation, whereas reconstitution of iPLA₂β in iPLA₂β-deficient VSMC restores it. 70–80% confluent VSMC were incubated with FBS-free medium containing NG for 24–48 h. Quiescent rat (A and B), mouse WT or iPLA₂β-deficient (iPLA₂β KO) aortic VSMC (C and D), and iPLA₂β adenovirus-infected mouse iPLA₂β-KO VSMC (E and F) were incubated with 10% FBS medium containing NG or HG in the presence of BEL (3 μm) or vehicle (Veh, Me₂SO) for 48 h. iPLA₂β adenoviral expression was induced by doxycycline (1 μg/ml). A, C, and E, representative Western blots Western blots of total CPI-17 (CPI-17-T) and phosphorylated CPI-17 (CPI-17-P). B, D, and F, summary of data shown in A, C, and E, respectively. Each experiment was repeated at least four times. ***, p < 0.001 versus NG/vehicle in B, WT/NG in D, and WT/NG in F; ##, p < 0.01 versus iPLA₂β-KO/NG in F; ###, p < 0.001 versus HG/vehicle in B and WT/HG in D; NS, not significant, versus NG/vehicle and HG/BEL in B, WT/NG and iPLA₂β-KO/HG in D, and iPLA₂β-KO/NG in F.

FIGURE 4.

12/15-Lipoxygenases(s) are selectively involved in high glucose-induced CPI-17 phosphorylation. A, 70–80% confluent mouse WT and iPLA₂β-deficient VSMC were labeled with [³H]AA and then incubated with NG or HG for 6 h. The medium [³H]AA and cellular [³H]phospholipids were analyzed by thin layer chromatography and quantified by liquid scintillation spectrometry. B, D, E, and F, quiescent rat (B, D, and E) and mouse (F) aortic VSMC were incubated with NG or HG for 24 h (E) or 48 h (B, D, and F) in the presence of vehicle (Veh, Me₂SO), NDGA (30 μm), indomethacin (Indo, 50 μm),17-octadecynoic acid (ODA, 10 μm), MK886 (MK, 1 μm), baicalein (Bai, 10 μm), and luteolin (Lut, 50 μm), respectively. Phosphorylated CPI-17 (CPI-17-P) and total CPI-17 (CPI-17-T) were determined by Western blot (B, D, and F). The medium 15(S)-HETE was measured by a specific 15(S)-HETE EIA Kit (E). C, LO isoform expressions were determined by real time PCR in rat VSMC. The results are expressed as the means ± S.E. from three experiments. **, p < 0.01 versus WT/NG in A versus NG/vehicle in E. ***, p < 0.001 versus NG/vehicle in E. ###, p < 0.001 versus WT/HG in A and HG/vehicle in E.

FIGURE 5.

BEL inhibition or genetic deletion of iPLA₂β diminishes high glucose-induced MYPT1 Thr-853 phosphorylation. Quiescent rat (A and B) or mouse WT or iPLA₂β-KO aortic VSMC (C and D) were incubated with 10% FBS medium containing NG or HG in the presence or absence of BEL (3 μm) or vehicle (Veh, Me₂SO) for 48 h. A and C, representative Western blots of total MYPT1 (MYPT1-T) and phosphorylated MYPT1 (MYPT1-P). B and D, summary of data shown in A and C, respectively. Each experiment was repeated at least three times. **, p < 0.01 versus NG/vehicle in B and WT/NG in D; ###, p < 0.001 versus HG/vehicle in B and WT/HG in D; NS, not significant, versus NG/vehicle and HG/BEL in B and WT/NG or iPLA₂β-KO/HG in D.

FIGURE 6.

BEL inhibition or genetic deletion of iPLA₂β alleviates high glucose-induced RhoA activation. Quiescent rat (A and B), mouse WT, or iPLA₂β-KO (KO) aortic VSMC (C and D) were incubated with 10% FBS medium containing NG or HG in the presence of BEL (3 μm) or vehicle (Veh, Me₂SO) for 48 h. A and C, representative Western blots of total RhoA and GTP-bound RhoA (GTP RhoA). B and D, summary of data shown in A and C, respectively. Each experiment was repeated at least three times. ***, p < 0.001 versus NG/vehicle in B or NG/WT in D; ###, p < 0.001 versus HG/vehicle in B or HG/WT in D; NS, not significant, versus NG/vehicle and HG/BEL in B or NG/WT and HG/KO in D.

FIGURE 7.

iPLA₂β is activated in the diabetic vessel wall and is involved in diabetes-associated vascular smooth muscle hypercontractility. Aorta and/or mesentery artery were isolated from db/db and C57BL/KsJ control mice or STZ and C57BL/6J control mice or rats (Sprague-Dawley). A and B, summary of data of iPLA₂ assays. C and D, representative Western blots of iPLA₂β and β-actin. E–H, summary of data for isometric tension measurement. Intact thoracic aorta (E), α-toxin permeabilized mesenteric artery (F), and intact abdominal aorta (G and H) were incubated with BEL (10 μm, 30 min), Y-27632 (10 μm), (R)-BEL or (S)-BEL (each 10 μm, 60 min), or vehicle (Veh, Me₂SO) for 60 min, respectively, prior to 5-HT (1 μm), GTPγS (50 μm), and PE (10 μm) stimulation, respectively. Each experiment was repeated at least three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control in A, B, E, F, and G and WT/control in H; ##, p < 0.01; ###, p < 0.001 versus vehicle in E and F; NS, not significant, in G (vehicle versus (R)-BEL) and in H (iPLA₂β-KO/control versus iPLA₂β-KO/STZ).

FIGURE 8.

Model for diabetes-induced iPLA₂β protein up-regulation and the role of iPLA₂β in diabetes-induced RhoA/ROCK/CPI-17 activation in VSMC and diabetes-associated vascular smooth muscle hypercontractility. The pharmacological, molecular, and genetic approaches used in this study are also indicated.