ABSTRACT
TGFβ/smad pathway is recognized as an important signal pathway to promote the pathogenesis of atherosclerosis (AS). Peroxisome proliferator-activated receptor γ (PPARγ) activation is considered to be important in modulating AS. Herein, we investigated the regulation of PPARγ on c-Ski, the repressor of TGFβ/smad pathway, in rat AS model and cultured vascular smooth muscle cells (VSMCs). c-Ski mRNA and protein expression were detected by real-time PCR and Western blot, respectively, in vivo and in vitro with treatment of PPARγ agonist rosiglitazone and antagonist GW9662. The proliferation and collagen secretion of VSMCs after c-Ski transfection were investigated. The underlying mechanism was further investigated by online program NUBIScan and luciferase reporter gene analysis. Results showed that both mRNA and protein expressions of c-Ski in the AS lesions was down-regulated in vivo, while in cultured VSMCs, c-Ski transfection significantly suppressed the proliferation and collagen secretion of rat VSMCs. Rosiglitazone significantly up-regulated mRNA and protein levels of c-Ski in VSMCs, which could be blocked by GW9662. Online NUBIScan analysis suggested possible PPARγ binding sites in the promoter region of c-Ski. In addition, luciferase activity of c-Ski reporter gene was also increased obviously in the presence of rosiglitazone. These results indicate that c-Ski is one of the newly found target genes of PPARγ and thus involved in the anti-AS effect of PPARγ.
Subject(s)
Animals , Male , Rats , Anilides , Pharmacology , Atherosclerosis , Cells, Cultured , Muscle, Smooth, Vascular , Cell Biology , Myocytes, Smooth Muscle , Metabolism , PPAR gamma , Physiology , Proto-Oncogene Proteins , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Rats, Wistar , Repressor Proteins , Genetics , Metabolism , Signal Transduction , Smad Proteins , Metabolism , Thiazolidinediones , Pharmacology , Transforming Growth Factor beta , Metabolism , Up-RegulationABSTRACT
Objective To construct the eukaryotic expression plasmid of pEGFPC1uPAR gene and explore the effect on the proliferation and invasion ability of Pam 212 cells. Methods The human uPAR cDNA was cloned by PCR, and inserted into the eukaryotic expression plasmid pEGFPC1. After identification of sequencing, the reconstructive plasmid was transformed transiently into Pam 212 cells, then the cell growth and the invasion ability were evaluated. Results The reconstructive plasmid of pEGFPC1uPAR was validated by sequencing. The reconstructive plasmid can promote the growth of Pam 212 cells and enhance the invasion ability. Conclusion The pEGFPC1uPAR plasmid was constructed successfully and uPAR was confirmed to promote the growth and the invasion ability of Pam 212 cells, which lay the foundation for further studies of uPAR in vivo.