p16 蛋白質抑制間質金屬蛋白酶-2的轉錄調節機制探討

p16 is an important negative regulator of the cell cycle. At G1 phase, it reduces the Cyclin D-CDK4/6 complex activity. As a result, cells are arrested at G1 phase. Besides the negative regulatory role in cell cycle, p16 has been reported to inhibit the invasion and metastasis ability of tumor cell. In our study, we use A549 (human lung adenocarcinoma cell), which is a p16 gene deletion cell line to address the role of p16 in the inhibition of tumor invasion. In the promoter activity assay, we found that p16 can inhibit the expression of MMP-2. Then we use the different MMP-2 promoter deletion and site-directed mutation mutants to analysis the target sequence in MMP-2 promoter. We find that the inhibition of MMP-2 caused by p16 is through the transcription factor, Sp1. In order to check if the DNA binding affinity of Sp1 is regulated by p16, we use DNA affinity precipitation assay (DAPA) and Chromatin immunoprecipitation assay (ChIP) to solve this question. Our data show that the DNA binding affinity of Sp1 is inhibited by p16. Besides we find the phosphorylation level of Sp1 is reduced. We next study the ERK, Akt, and cyclin A/CDK2 kinases which have been reported to phosphorylate Sp1. Our findings document that p16 inhibits Sp1-cyclin A binding affinity and reduces Sp1 phosphorylation which causes reduction of Sp1-DNA binding affinity and down-regulation of MMP-2 transcription.