Transforming growth factor beta activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site.
Transforming growth factor beta (TGF-beta) causes growth arrest in the G1 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 p15INK4B, which specifically inhibits the enzymatic activities of CDK4 and CDK6. An active cyclin D-CDK4/6 complex is required for pRb phosphorylation to allow the cell cycle to progress from G1 to S phase. To study the molecular mechanism of the p15INK4B 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 p15INK4B 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 Sp1 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 Sp1 sites and the spacer sequences had little effect. In addition, gel mobility shift assay indicates that the transcription factors Sp1 and Sp3 bind to this Sp1 site. Taken together, these data suggest that a specific Sp1 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.
Li
J M
JM
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27708, USA.
Nichols
M A
MA
Chandrasekharan
S
S
Xiong
Y
Y
Wang
X F
XF
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D018797:Cell Cycle Proteins; D002460:Cell Line; D016384:Consensus Sequence; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers; D016329:Sp1 Transcription Factor; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D004247:DNA