KLF5 And HhLIM Cooperatively Activate Pdgf-BB-induced Expression Of Vascular Smooth Muscle Proliferative Marker Gene

Objective:The proliferation of vascular smooth muscle cells (VSMC) is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and postangioplasty restenosis. Many growth factors and cytokines contribute to this process, among them, platelet-derived growth factor BB (PDGF-BB) possesses the most potent mitogenic effect.Kruppel-like factor 5(KLF5), a zinc finger transcription factor belongs to a family known as the Sp/KLF factors, which is associated with cell proliferation, apoptosis and development. Previous studies have shown that the expression of KLF5 was increased in the neointimal, which could activate the expression of platelet-derived growth factor-A chain. As a transcriptional factor, KLF5 interacts with many other transcription factors/cofactors, such as c-jun, RARα, CREB binding protein (CBP), PPAR-δ, HDAC2 and p300 and regulates the expression of many genes involved in cell proliferation.hhLIM contains two LIM domains, the latter is a cysteine-rich zinc-finger motif found in a large family of proteins and is now recognized as a key component of the regulatory machinery of the cell by mediating protein-protein interactions. Recent studies have indicated that hhLIM, as a cofactor, have diverse cellular roles as regulators of gene expression, cytoarchitecture, cell adhesion, cell motility and signal transduction. However, the mechanisms whereby KLF5 and hhLIM regulate VSMC gene expression are not known.A mechanism by which KLF5 accomplishes its proliferative effect is to transcriptionally activate several cell cycle-promoting genes. Despite the effect of PDGF-BB on proliferation of VSMCs, the mechanism of PDGF-BB mediated proliferation is not fully elucidated. Thus, we investigated crucial mechanisms of the interaction between KLF5 and hhLIM in the transcriptional activation of cyclinE, which will contribute to penetrate the cardiovascular disease.Methods:VSMC was isolated from the thoracic aorta of Sprague-Dawley rats. In all experiments, only cell passages 3~5 were used. Co-immunoprecipitation assay was done to examine the interaction between KLF5 and hhLIM. The expression of KLF5 and hhLIM and CyclinE were examined by Western blotting. Flow cytometric/cell cycle analysis was used to reveal the distribution of VSMC in the various phases of the cell cycle. Reproter gene assay was done to detect the effect of KLF5 and hhLIM on the expression of CyclinE; Immunohistochemistry were performed to detect the expression of the KLF5, hhLIM and CyclinE in the different model groups.Results:1 PDGF-BB accelerates cell cycle progression of VSMC Flow cytometric/cell cycle analysis showed that PDGF-BB treatment resulted in a significant increase in the S population but a decrease in the G1 population relative to the control. These results suggested that PDGF-BB can promote progression of VSMC cell cycle through the G1 phase to the S phase, and subsequently induce proliferation in VSMC. Cell counting analysis also showed that cell proliferation increased in dose-dependent manner, indicating that PDGF-BB could promotes cell cycle progression.2 PDGF-BB promote the expression of hhLIMWestern blot analysis showed that the expression of hhLIM was induced by PDGF-BB in dose-dependent manners. On the contrary, hhLIM expression was not reduced by PDGF-BB treatment. These results suggested that hhLIM may be involved in PDGF-BB triggered VSMC proliferation.3 PDGF-BB promote the interaction between KLF5 and hhLIMSince the expression of KLF5 was not reduced by PDGF-BB, we next to detect that whether PDGF-BB affect the interaction between KLF5 and hhLIM. Treating cells with PDGF-BB significantly increased KLF5 levels in precipitates pulled down with anti-hhLIM antibody, which suggest that PDGF-BB promote the interaction between KLF5 and hhLIM. 4 KLF5 and hhLIM cooperatively promote cell proliferationKLF4 and KLF5 adenovirus expression system was established to investigate the effect of KLF5 and hhLIM on neointimal hyperplasia after balloon injury. At 14 days after balloon injury, the injured vessel (injured group) showed neointimal hyperplasia. (I/M ratio, injured versus sham, 3.33+0.26 versus 0.12±0.03, p < 0.05). There was no significant difference between hhLIM-transfected group (I/M ratio, 3+0.26) and the injured group. However, KLF5 overexpression significantly increased neointimal hyperplasia and I/M ratio (I/M ratio, pAd versus pAd-KLF5, 3.33+0.26 versus 4.12±0.23, p < 0.05) compared with the pAd group. The most interesting thing is that neointimal formation and I/M ratio after transfer of KLF5 and hhLIM (6.40+0.23)which significantly increased as compared with model group(3.33+0.26), hhLIM group(3+0.26) and KLF5 group (4.12±0.23). PCNA immunostaining showed a significantly higher positve rate in the intimal and medial layers of the hhLIM and KLF5 group (70+2.3%) as compared with those in the model group (26+3.7%) and hhLIM group (33+1.7%). These results showed that KLF5 and hhLIM cooperactively promote the neointimal hyperplasia after balloon injury.5 KLF5 and hhLIM cooperatively stimulates VSMC proliferation and accelerates cell cycle progressionCell counting analysis showed that overexpression of KLF5 (14+2×104) resulted in in a statistically significant increase compared with that overexpression of pAd or pAd-hhLIM. The number of cells transfected with hhLIM and KLF5 together (17±3×104) further increased compared with pAd-hhLIM or pAd-KLF5 group (14±2×104). Cell cycle analysis showed markedly increased S-phase bulging after KLF5 and hhLIM co-transfection 24h in VSMCs. Flow cytometric/cell cycle analysis also showed that hhLIM and KLF5 cotransfection for 24 h resulted in a statistically significant increase in the S population but a decrease in the G1 population relative to the KLF5 group.These results suggested that KLF5 and hhLIM cooperatively stimulates VSMC proliferation and accelerates cell cycle progression. 6 KLF5 and hhLIM cooperatively promotes CyclinE promoter activityTo further investigate the molecular mechanisms of KLF5 and hhLIM promote cell proliferation, expression levels of Cyclins and some cellular factors were analyzed by Western blot in both VSMCs and rat carotid arteries.Western Blotting result showed that overexpression of KLF5 could induce the expression of Cyclins or other kinases, such as, CDK2,CyclinD,CyclinE. Overexpression of hhLIM has no effect on the expression of these genes. But co-overexpression of KLF5 and hhLIM could cxooperactively the expression of CyclinE. These results prompt us to believe that KLF5 and hhLIM could increase cell proliferation by promoting the expression of cyclinE cooperatively. The injured arteries caused by balloon were harvested 14 days following injury, and protein extracts were prepared and subjected to Western blotting assay. The hhLIM and KLF5 cotransfected group showed significantly increased the expression of CyclinE than the control and model groups. Apparently, KLF5 and hhLIM could increase cell proliferation by promoting the expression of cyclinE cooperatively at the cellular and tissue levels.7 KLF5 and hhLIM activate cyclinE promoter cooperativelyTo investigate the molecular mechanisms of KLF5 and hhLIM promote the expression of cyclinE, CHO cells were transfected with cyclinE promoter-reporter plasmids and pRL-TK, pGL3-Basic, pMT-KLF5 or/and pEGFP-hhLIM expression plasmids. Reporter gene assay showed that KLF5 -overexpression resulted in an increase in CyclinE transcriptional activity, while KLF5 and hhLIM overexpression resulted in significantly decrease in CyclinE promoter activity compared with KLF5 group, suggesting that KLF5 and hhLIM synergistically activates the CyclinE promoter activity. To further investigate the molecular mechanism of this interaction, Oligo pull down was performed. There are two TCE sites on the CyclinE promoter. The binding activity of KLF5 on the TCE was decreased after silencing the expression of KLF5. However, the binding activity of KLF5 on the TCE was also decreased, but especially TCE1 after silencing the expression of hhLIM with specific small interfering RNA. The results above suggest that hhLIM and KLF5 cooperatively activates cyclinE promoter by promoting the binding of KLF5 on the TCE1 of the CylinE promoter.8 KLF5 and hhLIM promote cell proliferation cooperatively via up-regulation the expression of cyclinEWestern blotting showed that whether treated with PDGF-BB or not, silencing the expression of KLF5 markly suppressed the expression of cyclinE. However, silencing the expression of hhLIM had no effect on the expression of CyclinE, which suggest that hhLIM promote the expression of CyclinE by interacting with KLF5.At 14 days after balloon injury, KLF5 SiRNA group significantly reduced neointimal hyperplasia and I/M ratio (I/M ratio, 0.5±0.5 versus 3.3±0.26, p < 0.05) compared with the Model group. Immunohistochemistry showed PCNA expression was decreased in KLF5 SiRNA group. Western Blotting resulted showed that the expression of CyclinE was also decreased significantly. However, Knockdown the expression of hhLIM has a little increase on the neointimal hyperplasia and I/M ratio compared with the Model group (I/M ratio, 5.0±1.7 versus 3.3±0.26, p < 0.05).Conclusions:1 PDGF-BB promotes cell cycle progression of VSMC.2 PDGF-BB increases the expression of hhLIM and the interaction between KLF5 and hhLIM.3 KLF5 and hhLIM cooperatively promote VSMC proliferation.4 KLF5 and hhLIM cooperatively promote the expression of cyclinE.5 hhLIM promote cell proliferation via up-regulation the expression of cyclinE mediated by KLF5.