锌指蛋白ZFP580在全反式维甲酸调节大鼠血管平滑肌细胞迁移中的作用及机制

The role of ZFP580 in the regulation of rat VSMCs migration by ATRA and its mechanism

目的:探讨锌指基因ZFP580在全反式维甲酸(ATRA)调节VSMCs迁移功能中的作用及其机制。方法:分离,培养并鉴定大鼠主动脉VSMCs;分别予以0、5、10、20μmol/L ATRA刺激VSMCs 24h,以0μmol/L ATRA组为对照组,观察不同溶度ATRA刺激不同时间对VSMCs迁移能力的影响或给予0、20μmol/L ATRA刺激VSMCs 24、48、72h,观察ATRA刺激不同时间对VSMCs迁移能力的影响;QPCR及Western blot检测ATRA刺激VSMCs后ZFP580的mRNA和蛋白表达变化;应用ERK抑制剂PD98059抑制ERK的蛋白表达,观察ERK信号蛋白表达变化对ATRA刺激后ZFP580蛋白表达的影响;腺病毒转染技术获得过表达或低表达ZFP580的VSMCs,QPCR及Western blot检测MMP-2和MMP-9、ZFP580蛋白和mRNA表达水平。结果:分离的VSMCs在培养10d后,免疫荧光显示平滑肌细胞特异性标记物SM22α抗体阳性。与对照组相比,5、10、20μmol/L ATRA预刺激分别降低了32%、43%和59%的VSMCs迁移能力;20μmol/L ATRA刺激VSMCs与对照组相比,在24、48、72h分别降低49%、36%和22%细胞迁移能力。ZFP580的mRNA和蛋白表达随着ATRA刺激溶度的增加和刺激时间的延长而升高。ERK在ATRA刺激15min即显著升高,运用ERK抑制剂PD98059(20μmol/L)预处理抑制ERK蛋白表达并降低了ATRA诱导ZFP580的蛋白表达。过表达ZFP580降低MMP-2和MMP-9的mRNA和蛋白表达,反之,低表达ZFP580则上调了MMP-2和MMP-9的mRNA和蛋白表达。结论:ATRA可通过ERK信号通路上调ZFP580的表达,而ZFP580通过调控MMP-2和MMP-9的表达参与ATRA对VSMCs迁移的抑制作用。

Objective:To investigate the probable roles of the novel C2H2 zinc finger transcription factor ZFP580 on all-transretinoic acid(ATRA)-regulated VSMCs migration and underlying mechanisms.Methods:Rat aortic VSMCs were isolated,cultured and identified.VSMCs were treated with ATRA at the concentrations of 0,5,10 or 20μmol/L for 24 hours.The migration ability of VSMCs was observed in each group and compared with control group which was treated by 0μmol/L ATRA.The mRNA and protein expression levels of ZFP580 were detected by QPCR and Western blot.ZFP580 protein expression in VSMCs was detected under ATRA stimulation when ERK inhibitor PD98059 was used to inhibit the protein expression of ERK.Adenovirus transfection technology was used to obtain VSMCs with overexpression or low expression of ZFP580,and QPCR and Western blot were used to detect the mRNA and protein levels of MMP-2,MMP-9 and ZFP580.Results:On the 10th day of VSMCs culture,immunofluorescence showed that SM22 alpha antibody,as a specific marker of smooth muscle cells,was positive.Compared to the control group,VSMCs migration was reduced by 32%,43%,and 59%in the group of 5,10,and 20μmol/L ATRA pretreatment.Compared with the control group,VSMCs treated by 20μmol/L ATRA reduced the cell migration by 49%,36%and 22%at 24,48 and 72 h.The mRNA and protein expression levels of ZFP580 were increased with the increase of ATRA stimulation solubility and the extension of stimulation time.ERK was increased significantly after 15 min of ATRA stimulation.Pretreatment with ERK inhibitor PD98059(20μmol/L)inhibited the expression of ERK protein and reduced the expression of ATRA-induced ZFP580 protein.Overexpression of ZFP580 inhibited the expressions of MMP-2 and MMP-9,whereas down-expression of ZFP580 promoted the expressions of MMP-2 and MMP-9.Conclusion:ATRA increased the expression of ZFP580 through the ERK signaling pathway,while ZFP580 was involved in ATRA's inhibition of VSMCs migration by affecting the expression of downstream MMP-2 and MMP-9.