TRANSFORMING GROWTH-FACTOR-BETA ACTIVATES THE PROMOTER OF CYCLIN-DEPENDENT KINASE INHIBITOR P15(INK4B) THROUGH AN SP1 CONSENSUS SITE

Transforming growth factor beta (TGF-beta) causes growth arrest in the G(1) phase in many cell types. One probable pathway for this growth inhibition is through the TGF-beta-mediated up-regulation of the cyclin-dependent kinase (CDK) inhibitor p15(INK4B), which specifically inhibits the enzymatic activities of CDK4 and CDKG6. An active cyclin D-CDK4/6 complex is required for pRb phosphorylation to allow the cell cycle to progress from G(1) to S phase, To study the molecular mechanism of the p15(INK4B) induction by TGF-beta, we isolated a 780-base pair promoter sequence of the human p15 gene and inserted this fragment upstream of a luciferase reporter gene, When this construct was transiently transfected into HaCaT cells, luciferase activity was induced more than 10-fold upon TGF-beta treatment, indicating that the induction of p15(INK4B) expression by TGF-beta is partly exerted at the transcription level, Promoter deletion analysis revealed that the sequence from -110 to -40 relative to the transcription start site is capable of conferring the 10-fold induction by TGF-beta. Within this region there are three Spl consensus sites. Mutation of one of these sites, GGGGCGGAG, substantially reduced both the induction by TGF-beta and the basal promoter activity, whereas mutations in the other two Spl sites and the spacer sequences had Little effect. In addition, gel mobility shift assay indicates that the transcription factors Spl and Sp3 bind to this Spl site. Taken together, these data suggest that a specific Spl consensus site is involved in the mediation of TGF-beta induction as well as the basal promoter activity of the p15 gene and that Sp1 and Sp3 transcription factors might be involved in this regulation.