Protein kinase C-delta regulates migration and proliferation of vascular smooth muscle cells through the extracellular signal-regulated kinase 1/2

Abstract

Background: Smooth muscle cell (SMC) migration and proliferation are early and crucial events in the pathogenesis of intimal hyperplasia, the primary cause of restenosis after vascular intervention. We tested the hypothesis that protein kinase C-delta (PKCdelta), a ubiquitously expressed intracellular protein kinase, regulates vascular SMC proliferation and migration.

Methods: Exogenous PKCdelta was expressed in cultured SMCs via stable transfection or adenovirus-mediated gene transfer. Conversely, endogenous PKCdelta was inhibited by means of targeted gene deletion (gene knock-out). Cell proliferation and migration were determined by (3)H-thymidine incorporation and 24-well transwell assay, respectively.

Results: We isolated and examined three A10 SMC lines in which PKCdelta was stably transfected. Compared with cells that were transfected with an empty vector, cells transfected with PKCdelta exhibited reduced ability to proliferate. Moreover, PKCdelta transfection inhibited SMC migration toward platelet-derived growth factor-BB. Similar inhibitory effects on proliferation and migration were also observed when PKCdelta was introduced into primary aortic SMCs via an adenoviral vector. Interestingly, SMCs isolated from PKCdelta knockout mice also displayed decreased chemotaxis and proliferation compared with PKCdelta(+/+) littermates, suggesting a complex yet critical role for PKCdelta. We studied the mitogen-activated protein kinase extracellular signal-regulated kinases (ERK) 1/2 as a possible signaling pathway for PKCdelta's inhibitory effect. PKCdelta overexpression diminished ERK1/2 activity. Molecular restoration of ERK activation reversed the inhibitory effect of PKCdelta on SMC proliferation and migration.

Conclusions: We demonstrate that although normal migration and proliferation is lessened in SMCs deficient in PKCdelta, its prolonged activation also diminishes those behaviors. This suggests a dual, critical role for PKCdelta in SMC proliferation and migration, and thus intimal hyperplasia and restenosis.

Figure 1. PKCδ overexpressing A10 SMC lines demonstrate decreased proliferation and chemotaxis

(A) A10, empty vector (PT3, PT4) or PKCδ (PKCδ 3, 4 & 5) cells were seeded into 24-well plates at a density of 10,000 cells per well on day 0 and maintained in 10% FBS. Cells were counted on days 1, 3, 5 and 7. (n=3, * p<0.05, compared to control A10 SMC line) (B) A10, empty vector or PKCδ cells were serum -starved for 48 h and then stimulated for 24 h with or without PDGF-BB (5 ng/ml). Incorporation of ³H-thymidine was measured. (C) A10, empty vector or PKCδ cells were made quiescent and then subjected to the chemotaxis assay as described in Experimental Procedures. (n=3, # p< 0.01, compared to A10 basal; * p< 0.01, compared to A10 treated with PDGF-BB).

Figure 2. PKCδ overexpression in RASMCs, via adenoviral transfection, inhibits proliferation and migration

(A) RASMCs were infected with AdNull or AdPKCδ. Forty-eight hours after infection, cells were lysed and analyzed by immunoblotting. Additional infected cells were re-seeded into 24-well plates at a density of 10,000 cells per well on day 0 and maintained in 10% FBS. Cells were counted on days 1, 2, 5 or 7. (B) Following infection with AdNull or AdPKCδ, RASMCs were serum-starved for 48 h. Basal and PDGF-BB induced DNA synthesis was measured using the 3H-thymidine incorporation. (C) RASMCs were infected with AdNull or AdPKCδ. Forty-eight hours after infection, basal and PDGF-BB (5 ng/ml) induced chemotaxis was evaluated. (n=3, *p< 0.05, compared to AdNull control).

Figure 3. PKCδ selective gene deletion downregulates SMC proliferation and migration

(A) Mouse SMCs were isolated from PKCδ −/− and +/+ littermates. SMCs from both groups were lysed and analyzed by immunoblotting. Additional cells were seeded into 24-well plates at a density of 10,000 cells/well on day 0 and maintained in 10% FBS. Cells were counted on days 1, 2, 4 and 8. (n=3, * p<0.05, compared to PKCδ +/+ control) (B) PKCδ −/− and +/+ SMCs were serum-starved for 48 h and then stimulated for 24 h with or without PDGF-BB (5 ng/ml). The incorporation of ³H-thymidine was measured. (B) PKCδ −/− and +/+ SMCs were serum-starved for 48h. Chemotaxis with and without PDGF-BB (5 ng/ml) was evaluated as described in Experimental Procedures. (n=3, *p< 0.05, compared to PKCδ +/+ control).

Figure 4. PKCδ overexpression inhibits ERK1/2 activity

(A) A10, PKCδ or vector transfected cells were made quiescent by incubation in the low-serum media for 48 h. Cells were stimulated with PDGF-BB (5 ng/ml) for 15 min. Cell lysates were immuno-precipitated with an anti-ERK1/2 antibody. The activity of ERK in the IP complex was measured by its ability to phosphorylate MBP. (B) RASMCs were infected with AdNull or AdPKCδ. Following serum starvation for 48 h and PDGF-BB treatment (5 ng/ml for 15 min) were indicated, cell lysates were immunoprecipitated with an anti-ERK1/2 antibody and ERK activity in the IP complex was measured by its ability to phosphorylate MBP. (C) RASMCs were infected with AdNull or AdPKCδ. Following serum starvation and PDGF-BB treatment (5 ng/ml for 15 min), cell lysates underwent Western blot analysis with anti-ERK1/2 antibodies. (D) Mouse SMCs from PKCδ −/− and +/+ littermates underwent low-serum incubation and stimulation with PDGF-BB (5 ng/ml) for 15 min. Cell lysates were immunoprecipitated with an anti-ERK1/2 antibody and ERK activity in the IP complex was measured by MBP phosphorylation.

Figure 5. PKCδ’s inhibition of migration and proliferation can be overcome by restoring ERK1/2 activity

(A) Following low-serum incubation for 48 h, incorporation of ³H-thymidine was measured in A10 SMCs infected with equal quantities of AdNull, AdPKCδ, or AdMEK (60,000 total viral particles per cell) and stimulated for 24 h with or without PDGF-BB (5 ng/ml). (B) AdNull, AdPKCδ, or AdMEK infected A10 SMCs were serum-starved for 48 h. Chemotaxis with and without PDGF-BB (5 ng/ml) was evaluated. [n=3; *, p < 0.05 as compared to AdNull control]