Induction of disease-associated keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun.
Overexpression of keratin 16 has been observed in keratinocytes in those skin diseases characterized by hyperproliferation such as psoriasis. Therefore, keratin 16 is usually referred to as a disease-associated keratin. In the present study, we found that epidermal growth factor (EGF) increased the expression of keratin 16 mRNA and protein synthesis in a time-dependent manner in HaCaT cells. Reporter assays revealed that the EGF response region was in the range of -162 to -114 bp. Disruption of the Sp1 site (-127 to -122 bp) and the AP1 site (-148 to -142 bp) of the keratin 16 promoter by site-directed mutagenesis significantly inhibited keratin 16 promoter activity induced by EGF. Furthermore, keratin 16 gene expression induced by Ras activation was also regulated in the same manner as the EGF response. By using the DNA affinity precipitation assay in HaCaT and SL2 cells, Sp1 directly interacted with the Sp1 site of the promoter, and c-Jun and c-Fos precipitated with the Sp1 oligonucleotide was attributable to the interaction between the Sp1 and AP1 proteins. Moreover, cotransfection assays revealed that Sp1 acted synergistically with c-Jun to activate keratin 16. The coactivators p300/CBP could collaborate with Sp1 and c-Jun in the activation of keratin 16 promoter, and EGF-induced promoter activation was blocked by the viral oncoprotein E1A. Taken together, these results suggest that Sp1 and AP1 sites in the essential promoter region are critical for EGF response, and Sp1 showed a functional cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional regulation of EGF-induced keratin 16 gene expression.
Wang
Ying-Nai
YN
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.
Chang
Wen-Chang
WC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D050881:E1A-Associated p300 Protein; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007633:Keratins / Q000096:biosynthesis*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D009687:Nuclear Proteins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D004815:Epidermal Growth Factor; D007633:Keratins; D004247:DNA; D008156:Luciferases; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse