| Objective:The proliferation and directed migration of vascular smooth muscle cells (VSMCs) from the media into the intima are key events in pathogenic vascular injuries, such as hypertension, atherosclerosis and restenosis. Platelet-derived growth factor (PDGF) is a potent mitogenic agent, which induces the phenotype of VSMC from contractile/differentiated to synthetic/proliferative status and initiates a multitude of biological effects through the activation of intracellular signal transduction pathways that contributes to VSMC proliferation, migration, and the development of atherosclerotic lesions. Five different dimeric isoforms of PDGF have been described: AA, AB, BB, CC and DD. As a receptor tyrosine kinase, PDGF receptor (PDGFR) modulates PDGF signal transduction and activates transcription factors.Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor, and plays important roles in cell growth, proliferation, differentiation, embryogenesis as well as carcinogenesis. The expression of KLF4 is upregulated in growth arrest cells and terminally differentiated cells, and has an inhibition effect on cell proliferation. In the present study, we verified the different effects of PDGF-BB on the expression of proliferation and differentiation markers of VSMCs for understanding the physiological and pathobiological roles of KLF4 in phenotypic switching and proliferation of VSMCs.Methods and results:1 Effect of PDGF-BB on proliferation activity in VSMCs. Results of MTT assay showed that PDGF-BB significantly increased the proliferation activity of VSMCs in dose- and time-dependent manners.2 Effect of PDGF-BB on cell cycle in VSMCs. Flow cytometric analysis showed that PDGF-BB treatment resulted in a statistically significant increase in the S and G2/M phase, but a decrease in the G0/G1 phase compared with the serum-starved group. These results suggested that PDGF-BB promotes cell cycle progression in VSMCs.3 Effect of PDGF-BB on migration in VSMCs.Cell migration was assayed by wound healing analysis. Compared to the control, the number of migration cells increased 5.83 fold, suggesting that VSMC migration is significantly induced by PDGF-BB.4 Effect of PDGF-BB on the expression of phenotypic markers in VSMCs.Western blot analysis showed that PDGF-BB induced the expression of proliferation cell nuclear antigen (PCNA), but reduced the level of CDK inhibitor p27 and differentiation marker SM22 alpha (SM22α). The changes of phenotypic markers consist with the increased cell proliferation.5 Effect of PDGF-BB on the phosphorylation and expression of KLF4.Western blot showed that PDGF-BB induced the expression of KLF4. Immunoprecipitation using anti-phosphoserine antibody showed that the level of phosphorylated KLF4 was markedly increased at 15 min in VSMCs treated with 10 ng/ml of PDGF-BB, and reached its highest level at 90 min. These results suggested that PDGF-BB upregulates the activity of KLF4.6 Effect of PDGF-BB on the interaction between KLF4 and other transcription factors.The lysates from PDGF-BB-stimulated VSMCs were immunoprecipited with anti-KLF4 antibody, and then immunoblotted with anti-NF-κB, anti-smad2, anti-smad3, anti-HDAC2 and anti-HDAC5 antibodies. The results showed that KLF4 interacted with NF-κB, smad2, smad3 and HDAC2, respectively. Moreover, the interaction of KLF4 with NF-κB was increased and it with smad3 or HDAC2 decreased in a time-dependent manner in PDGF-BB-stimulated VSMCs.Conclusions:1 PDGF-BB induces VSMC proliferation and migration.2 PDGF-BB upregulates the expression of proliferation-associated protein PCNA, and downregulates the expression of proliferation inhibitor p27 and differentiation-associated protein SM22α.3 PDGF-BB induces the phosphorylation and expression of KLF4. PDGF-BB promotes the interaction between KLF4 and NF-κB, and inhibits the interaction between KLF4 and smad3 or HDAC2.
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