title: Structure and transcriptional regulation of the human cystatin A gene. The 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element-2 site (-272 to -278) on cystatin A gene is critical for TPA-dependent regulation.
abstract: Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to -2595 in the 5'-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5' upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between -272 and -278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-alpha expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5'-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.
authors.affiliation: Department of Dermatology, Asahikawa Medical College, 3-11 Nishikagura, Asahikawa 078, Japan. ht@asahikawa-med.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Takahashi
authors.forename: K
authors.initials: K
authors.lastname: Asano
authors.forename: M
authors.initials: M
authors.lastname: Kinouchi
authors.forename: A
authors.initials: A
authors.lastname: Ishida-Yamamoto
authors.forename: K D
authors.initials: KD
authors.lastname: Wuepper
authors.forename: H
authors.initials: H
authors.lastname: Iizuka
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002461:Cell Line, Transformed; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015891:Cystatins / Q000235:genetics*; D004247:DNA; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000235:genetics; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D015854:Up-Regulation
chemical_list: D015891:Cystatins; D007527:Isoenzymes; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate