KLF5 Contributes To Regulating Of Proliferation Induced By AngⅡ In Vascular Smooth Muscle Cells

Multiple lines of experimental and clinical evidence indicate that angiotensin II (Ang II) induces not only hypertension but also directly contributes to pathophysiological vascular thickening, and atherosclerosis in humans. A great number of in vitro and in vivo findings on the functions of Ang II have suggested that Ang II causes cell growth, regulates the gene expression of various bioactive substances, and activates multiple intracellular signaling cascades, such as mitogen-activated protein kinase (MAPK) pathways and various transcription factors in vascular smooth muscle cells (VSMCs).Krüppel-like factor 5 (KLF5), a member of the Sp/KLF family of zinc finger factors, is a key regulator of cardiovascular remodeling through activating many genes. It has been demonstrated that KLF5 plays key role in Ang II–induced cardiac hypertrophy and fibrosis and the control of SMC phenotype. A mechanism by which KLF5 accomplishes its proliferative effect is to transcriptionally activate several cell cycle-promoting genes, including cyclin D1, cyclin B1, and Cdc2. Despite the effect of Ang II on proliferation of VSMCs, the mechanism of Ang II-mediated proliferation is not fully elucidated, and led us to hypothesize that KLF5 might be required for Ang II mediated signaling.1 The effects of Ang II on VSMC proliferationAng II functions as a growth factor in VSMCs. Thus, we tested Ang II effect on growth and migration of VSMCs. The results from MTT, cell number and flow cytometric analysis were used to indirectly and directly determine the cell viability respectively, and were expected to reflect the regulation of cell growth by Ang II.1.1 The effects of Ang II on VSMC proliferation The results of MTT and cell number assays showed a dose-dependent stimulation of cell growth by 24 h Ang II treatment. The significant stimulation was observed from the dose of 10-7 M in VSMCs. The results indicated that Ang II exerts growth-stimulating effect on VSMCs. Furthermore, the proliferative effect of Ang II was verified through evaluating the fate of Ang II-treated VSMCs within the cell cycle. Cells were synchronized with 24 h serum starvation and then induced to re-enter the cell cycle by treatment with Ang II. Flow cytometric analysis was performed after propidium iodide staining. After 24 h treatment, Ang II significantly reduced the proportion of G0/G1-phase cell populations, but increased the number of S-phase cells in a dose-dependent fashion (10-8~ 10-6 M).1.2 The effects of Ang II on VSMC marker gene expressionThe expression of PCNA, a marker of cell growth, were paralleled by the number of re-enter the cell cycle, while the expression of SM22αthat is the marker of differentiated VSMCs, were inversely correlated with it, at both mRNA and protein levels. These results suggest that Ang II promotes cell cycle progression to stimulate growth of VSMCs.2 The role of KLF5 in the expression of cyclin D1 and VSMC proliferation induced by Ang IIKLF5 has previously been shown to increase cell growth and cell cycle progression in NIH3T3 cells by accelerating both the G1/S transition and entry into mitosis. Cyclin D1 is a growth sensor induced by growth factors and accelerate G1 progression. To investigate whether KLF5 is involved in Ang II-induced cyclin D1 expression and cell proliferation, the over-expression and knock-down of KLF5 protein in VSMCs was performed followed Ang II treatment, respectively.2.1 The effects of Ang II on the expression of KLF5 and cyclin D1 in cultured VSMCsWestern blot analysis showed that Ang II increased KLF5 protein levels in a dose-dependent manner, paralleling with Ang II-induced cell proliferation and cyclin D1 protein expression. The level of KLF5 protein began to increase at 3 h, and peaked at an increase of 4-fold at 12 h after Ang II treatment and declined thereafter. Similarly, Ang II induced the expression of cyclin D1 at mRNA level in a time-dependent manner, with a maximum of 4-fold increase at 12 hours and sustaining this level at least for 24 hours, with concurrent changes in the level of cyclin D1 protein. Immunocytochemistry analysis indicated that KLF5 protein located in VSMC nucleus.2.2 The effects of KLF5 overexpression on cyclin D1 gene expression and cell proliferation and migration in VSMCTo confirm the role of KLF5 in Ang II-induced cyclin D1 expression and cell proliferation, VSMCs were transfected with Ad-KLF5 to overexpress KLF5. The overexpression of KLF5 increased endogenous cyclin D1 protein level. MTT and Flow cytometric analysis showed that KLF5 overexpression resulted in increase in proliferation and reduced the proportion of G0/G1-phase cell populations, but increased the number of S-phase cells, compared with empty vector controls. In addition, the migration of VSMC increased by KLF5 overexpression.2.3 The effects of KLF5 knockdown on cyclin D1 gene expression and proliferation and migration of VSMCIn response to Ang II, the expression of KLF5 mRNA and protein was significantly attenuated in VSMCs transfected with KLF5-specific siRNA as compared to cells transfected with non-specific siRNA (NS-siRNA). Similarly, KLF5-specific siRNA abolished Ang II–induced cyclin D1 mRNA and protein expression. The knockdown of KLF5 expression reduced cell growth and the number of S-phase cells dramatically even after Ang II treatment. However, these changes were not observed in the cells treated with NS-siRNA. These findings indicate that KLF5 is required for Ang II-induced cyclin D1 expression and VSMC proliferation.3 Interaction between KLF5 and c-Jun mediates Ang II-induced cyclin D1 gene expressionTo study the involvement of the cyclin D1 promoter in cyclin D1 gene activation, a cyclin D1 promoter fragment spanning positions -1745 to +134 was analyzed by sequence comparison. The data showed that the cyclin D1 promoter contains multiple KLF5 and AP-1 regulatory elements that may be involved in the transcriptional regulation of the gene.3.1 The effects of KLF5 on cyclin D1 promoter-reporter gene expressionThe construct pGL3-CD1-Luc containing cyclin D1 promoter (from -1745 to +134) was transfected into COS-7 cells, and the promoter activities were analyzed following Ang II treatment. Ang II induced the promoter activity which was significantly higher than that of the untreated cell by Ang II, and an increase in cyclin D1 promoter activity was further induced after cotransfection with pEGFP-KLF5.3.2 Ang II promotes cyclin D1 expression related to the interaction between KLF5 and c-JunTo confirm how KLF5 activates the expression of cyclin D1 gene, the activity of KLF5 binding to the promoter of cyclin D1 gene was detected by ChIP assay. The results showed that Ang II significantly increased KLF5 binding to cyclin D1 promoter. The two distal KLF5 binding elements of cyclin D1 gene promoter (-538~-801 and -868~-1094) were amplified by PCR in the immunoprecipitates pulled by anti-KLF5 antibody, and a DNA fragment contained two AP-1 binding site and one proximal KLF5 binding element (-10~-245) could be amplified when DNA immunoprecipitated using anti-c-Jun but not anti-KLF5 antibody. These observations were consistent with above results of the reporter gene assay.To determine whether the KLF5 and AP-1 bound to the promoter of cyclin D1 gene interact with each other, cross-coimmunoprecipitation was performed using anti-KLF5 and anti-c-Jun antibodies, respectively. Western blot analysis clearly showed the presence of c-Jun among the immunoprecipitated proteins with anti-KLF5 antibody, likewise, KLF5 protein was also detected in the pellets immunoprecipitated with anti-c-Jun antibody.3.3 GST pull-down analysis for interaction between KLF5 and c-Jun in vitroThe interaction between KLF5 and c-Jun was independently verified by GST pull-down assay in vitro. KLF5 in whole cell lysates could be pulled down by using GST-c-Jun fusion proteins. Treatment of VSMCs with Ang II for 1 h resulted in an increase in the interaction of KLF5 with GST-c-Jun fusion protein.3.4 KLF5 and c-Jun synergically transactivate the expression of cyclin D1 geneThe construct pGL3-CD1-Luc containing cyclin D1 promoter (from -1745 to +134) was transfected into COS-7 cells, and the promoter activities were assayed following Ang II treatment. Ang II induced the promoter activity which was significantly higher than that of the untreated cell by Ang II, and an increase in cyclin D1 promoter activity was further induced after cotransfection with pEGFP-KLF5 or pcDNA3.1-c-Jun, respectively. However, the cotransfection of KLF5 with c-Jun expression vectors significantly increased the activity of cyclin D1 promoter 5-fold over than that seen with the reporter alone following Ang II treatment.4 Signal transduction mechanisms of Ang II-induced VSMC proliferationProtein phosphorylation plays a major role in modulating the activity of transcription factors. The mitogen-activated protein kinase (MAPK) signaling cascades are involved in transducting extracellular signals to nucleus by phosphorylating and activating a variety of transcription factors. KLF5 has been shown to be phosphorylated by ERK 1/2. To determine whether ERK 1/2 is involved in KLF5 phosphorylation in VSMCs stimulated by Ang II, the role of three MAPKs ERK, JNK and p38MAPK in the signal transduction responses to Ang II was analyzed by Western blot for their phosphorylated forms using specific anti-phospho-kinase antibodies.4.1 Ang II induces KLF5 phosphorylationTo determine the mechanism of KLF5-mediated cyclin D1 expression in response to Ang II, the phosphorylation of KLF5 was first tested by cross-immunoprecipitation using anti-phospho-Ser and anti-KLF5 antibodies, respectively. The results showed that Ang II effectively increased the level of phosphorylated form of KLF5, and unchanged KLF5 total protein amounts under the same conditions. 4.2 Ang II induces activation of ERK 1/2 and p38MAPK pathwaysTo determine whether MAPK pathways are involved in KLF5 phosphorylation in VSMCs stimulated by Ang II, the role of three MAPKs, ERK, JNK and p38MAPK, in the signal transduction responses to Ang II was analyzed by Western blot for their phosphorylated forms using specific anti-phospho-kinase antibodies. The phosphorylated ERK 1/2 and phosphorylated p38MAPK increased to a maximum at 15 min and then decreased gradually within 30 min after treatment of VSMCs with Ang II, while phosphorylated JNK had almost no changes. Importantly, the blockade of ERK and p38MAPK using PD98059, an activated ERK inhibitor, and SB203580, a p38MAPK inhibitor, depressed both Ang II–induced KLF5 phosphorylation and cyclin D1 protein expression, indicating that Ang II induces KLF5 phosphorylation via ERK 1/2 and p38MAPK signaling pathways.4.3 ERK 1/2 and p38MAPK pathways mediates Ang II-induced VSMC proliferation and cyclin D1 gene expressionTo confirm the relationship between activation of ERK 1/2 and p38MAPK pathways and VSMC proliferation induced by Ang II, the effects of PD98059 and SB203580 on VSMC proliferation induced by Ang II were detected. Ang II-stimulated cell proliferation was abolished by PD98059 and SB203580 which block ERK 1/2 and p38MAPK signaling. Reporter gene assay showed that Ang II-induced the cyclin D1 promoter activity was decreased following PD98059 and SB203580 treatment. These results indicated that ERK 1/2 and p38MAPK pathways play important role in cyclin D1 gene expression and VSMC proliferation induced by Ang II.Conclusions1 Ang II stimulates the expression level of KLF5 in time- and dose-dependent manner with concurrent acceleration of the cell cycle progression in VSMCs. 2 KLF5 plays a crucial role in Ang II-induced cyclin D1 expression and proliferation of VSMCs.3 Ang II increased the activity of KLF5 by promoting both its expression and phosphorylation via extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways.4 KLF5 and c-Jun synergistically transactivate cyclin D1 gene transcription through interaction with each other in VSMCs induced with Ang II.