Krüppel-Like Factor 5 And C-Jun Cooperatively Inhibit P21 Expression And VSMC Proliferation

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. It is well known that angiotensin II (Ang II), via AT1 receptor, activates multiple intracellular signaling cascades (MAPK, PKC, JAK/STAT, etc), ultimately leads to changes in gene expression that contribute to VSMC proliferation and vascular remodeling.Krüppel-like factors (KLFs) belong to the transcription factor family containing zinc-finger, and are mainly involved in regulating cell growth, differentiation, proliferation and apoptosis process. Krüppel-like factor 5 (KLF5, also known as BTEB2/IKLF) was identified as a transcription factor involved in regulation of the expression of genes responsible for cell proliferation. The c-Jun gene encodes a basic region-leucine zipper (bZIP) transcription factor implicated in many cellular processes including proproliferative effect. Ang II has been shown to modulate the expression of KLF5 and c-Jun. p21 is a member of the Cip/Kip family of cyclin dependant kinase inhibitors (CKIs), which can induce G0/G1 arrest by inhibiting DNA synthesis. On the basis of earlier findings, we presumed that KLF5 and c-Jun which may be involved in Ang II-triggered p21 inhibition, promote VSMC proliferation. Thus, we investigated crucial mechanisms of the interaction between KLF5 and c-Jun in the transcriptional activation of p21 gene, that will contribute to penetrate the cardiovascular disease.Methods:The protein extracts from VSMC were used to detect the expression of KLF5, c-Jun, p21, and phosphorylation of various signaling molecules by Western blotting. Co-immunoprecipitation assay was done to examine the interaction between KLF5 and c-Jun. Interaction between KLF5 and c-Jun was detected by reporter gene assay. Expression and localization of KLF5 in VSMC induced by Ang II were determined by cell immunofluorescence analysis. Flow cytometric/cell cycle analysis was used to reveal the distribution of VSMC in the various phases of the cell cycle.Results:1 Ang II promotes cell cycle progression in VSMC Flow cytometric/cell cycle analysis showed that Ang II treatment resulted in a statistically significant increase in the S population but a decrease in the G1 population relative to the serum-starved VSMC.these results suggested that Ang II can promote progression of VSMC cell cycle through the G1 phase to the S phase, and subsequently induce proliferation in VSMC.2 Ang II induces KLF5, c-Jun expression and inhibits p21 expression in VSMC Western blot analysis showed that the expression of KLF5 and c-Jun was induced by Ang II in dose- and time-dependent manners. On the contrary, p21 expression was reduced by Ang II treatment, especially after 12 h. These results suggested that KLF5, c-Jun and p21 may be involved in Ang II-triggered VSMC proliferation. Cell immunofluorescence analysis indicated that KLF5 was located in the nucleus and kytoplasm in quiescent VSMC, but located mostly in the nucleus in VSMC treated with Ang II for 12 and 24 h. Reporter gene assay showed that KLF5 overexpression resulted in a 37% decrease in p21 promoter activity, indicating that KLF5 inhibits p21 gene expression induced by Ang II.3 KLF5 and c-Jun cooperatively inhibit p21 promoter activityThe above findings suggested that Ang II induced KLF5 and c-Jun expression. Whether these two transcription factors have synergistical function on p21 expression induced by Ang II? To determine this, co-immunoprecipitation assay was done to examine the interaction between KLF5 and c-Jun. The results showed that treatment of VSMC with Ang II resulted in a time-dependent increase in the interaction of KLF5 with c-Jun, which reached a maximum after 1 h, and then began to reduce after 3h. Reporter gene assay showed that KLF5 overexpression resulted in a 37% decrease in p21 transcriptional activity, while KLF5 and c-Jun overexpression resulted in a 60% decrease in p21 promoter activity, suggesting that KLF5 and c-Jun synergistically repress the p21 promoter activity. 4 Ang II increases the interaction between KLF5 and c-Jun by inducing KLF5 phosphorylationTo understand whether Ang II affects KLF5 phosphorylation, immunoprecipitation assay was performed. The results suggested that Ang II stimulation rapidly induced the phosphorylation of KLF5 within 0.5 h, and reached the maximum after 1 h and persisted for up to 3 h. Western blot analysis was used to detect the signaling pathways, and found that Ang II increased rapidly the level of phospho-ERK1/2 and MEK.5 ERK1/2 inhibitor PD98059 prevents Ang II-induced phosphorylation of KLF5To further testify that activated ERK may be involved in Ang II-induced KLF5 phosphorylation that subsequently leads to the increase in the interaction between KLF5 and c-Jun in VSMC, VSMC was pretreated for 2 h with PD98059 and then stimulated with Ang II. Immunoprecipitation assay revealed that the phosphorylation of KLF5 induced by Ang II was significantly inhibited by PD98059 pretreatment. Infection of VSMC with MEK expression plasmid substantially increased the KLF5 phosphorylation. Co-immunoprecipitation Assay showed that treatment of VSMC with PD98059 markedly diminished the interaction between KLF5 and c-Jun induced by Ang II, suggesting that activation of ERK pathway is involved in Ang II-induced KLF5 phosphorylation that subsequently leads to the increase in the interaction between KLF5 and c-Jun in VSMC.6 Activation of the ERK pathway is involved in Ang II-triggered p21 inhibitionReporter gene assay revealed that the strongest suppression was observed when all three expression vectors for KLF5, c-Jun, and MEK were cotransfected. The p21 promoter activity returned to baseline level after PD98059 treatment. The results suggest that Ang II inhibited p21 transcriptional activity through ERK pathway.Conclusions:1 Ang II induces KLF5, c-Jun expression and inhibits p21 expression in VSMC.2 KLF5 and c-Jun cooperatively inhibit p21 promoter activity.3 Ang II increases the interaction between KLF5 and c-Jun by inducing KLF5 phosphorylation.4 Ang II inhibits p21 transcriptional activity through ERK pathway.