Structure and Transcriptional Regulation of the Human Cystatin A Gene
1998; Elsevier BV; Volume: 273; Issue: 28 Linguagem: Inglês
10.1074/jbc.273.28.17375
ISSN1083-351X
AutoresHidetoshi Takahashi, Kazuhiro Asano, Motoshi Kinouchi, Akemi Ishida‐Yamamoto, Kirk D. Wuepper, Hajime Iizuka,
Tópico(s)Renal and related cancers
ResumoCystatin 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-α 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. 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-α 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. Cystatin A is a cysteine proteinase inhibitor that belongs to family 1 of the cystatin superfamily. Cystatin A was originally isolated from polymorphonuclear granulocytes (1Brzin J. Kopitar M. Turk V. Machleidt W. Hoppe-Seyler's Physiol. Chem. 1983; 364: 1475-1480Crossref PubMed Scopus (103) Google Scholar), but it has also been isolated from the spleen, liver, and epidermis (2Brzin J. Kopitar M. Turk V. FEBS Lett. 1982; 138: 193-197Crossref PubMed Scopus (18) Google Scholar, 3Green G.D.J. Kembhavi A.A. Davies M.E. Biochem. J. 1984; 218: 939-946Crossref PubMed Scopus (139) Google Scholar, 4Jarvinen M. J. Invest. Dermatol. 1978; 71: 114-118Abstract Full Text PDF PubMed Scopus (101) Google Scholar). The primary structure of cystatin A consists of a polypeptide chain of 98 amino acid residues that is mainly distributed intracellularly (5Barrett A.J. Trends Biochem. Sci. 1987; 12: 193-196Abstract Full Text PDF Scopus (264) Google Scholar). We have recently reported that cystatin A is identical to keratolinin, one of the precursor proteins of cornified cell envelope (CE) 1The abbreviations used are: CE, cornified cell envelope; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; PKC, protein kinase C; SVHK, SV40-transformed human keratinocyte; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA responsive element; bp, base pair(s). (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar), which is formed during terminal differentiation of keratinocytes (7Simon M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 275-292Google Scholar, 8Hohl D. Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (173) Google Scholar, 9Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. J. Invest. Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar). CE is a highly insoluble structure formed beneath the plasma membrane of keratinocytes during terminal differentiation (7Simon M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 275-292Google Scholar, 8Hohl D. Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (173) Google Scholar, 9Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. J. Invest. Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar). This structure is 15–20 nm thick and is stabilized by cross-link formation of precursor proteins by N-(γ-glutamyl)lysine isodipeptide bonds and disulfide bonds, which are catalyzed by transglutaminase(s) and sulfhydryl oxidase, respectively (7Simon M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 275-292Google Scholar, 8Hohl D. Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (173) Google Scholar, 10Yamada K. Takamori K. Ogawa H. Arch. Dermatol. Res. 1987; 279: 194-197Crossref PubMed Scopus (24) Google Scholar). In addition to cystatin A, several proteins have been implicated as precursors of CE, which include involucrin (11Eckert R.L. Green H. Cell. 1986; 46: 583-589Abstract Full Text PDF PubMed Scopus (321) Google Scholar), loricrin (12Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. J. Biol. Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar), small proline-rich protein(s) (13Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar), elafin (14Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar), and envoplakin (15Ruhrberg C. Hajibagheri M.A. Simon M. Dooley T.P. Watt F.M. J. Cell Biol. 1996; 134: 715-729Crossref PubMed Scopus (152) Google Scholar). Recent evidence suggests that involucrin is an early component of CE and provides a scaffold for the incorporation of other precursor proteins (14Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 16Ishida-Yamamoto A. Kartasova T. Matsuo S. Kuroki T. Iizuka H. J. Invest. Dermatol. 1997; 108: 12-16Abstract Full Text PDF PubMed Scopus (34) Google Scholar). TPA, which is a potent activator of protein kinase C (PKC), induces terminal differentiation of keratinocytes (17Hawley-Nelson P. Stanley J.R. Schmidt J. Gullino M. Yuspa S. Exp. Cell Res. 1982; 137: 155-167Crossref PubMed Scopus (106) Google Scholar, 18Mafson R.A. Steinberg M.L. Defendi V. Cancer Res. 1982; 42: 4600-4605PubMed Google Scholar). Recent studies have revealed that involucrin, loricrin, and transglutaminase 1 genes contain a TRE(s) in their 5′-flanking regions and that these TREs induce increased expression of these protein transcripts by TPA (19Takahashi H. Iizuka H. J. Invest. Dermatol. 1993; 100: 10-15Abstract Full Text PDF PubMed Google Scholar, 20Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 21Lopez-Bayghen E. Vega A. Cadena A. Granados S.E. Jave L. Gariglio P. Alvares-Saras L.M. J. Biol. Chem. 1996; 271: 512-520Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22DiSepio D. Jones A. Longley M.A. Bundman D. Rothnagel J.A. Roop D.R. J. Biol. Chem. 1995; 270: 10792-10799Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 23Yamanishi K. Inazawa J. Liew F.-W. Nonomura K. Ariyama T. Yasuno H. Abe T. Doi H. Hirano J. Fukushima S. J. Biol. Chem. 1992; 267: 17858-17863Abstract Full Text PDF PubMed Google Scholar). We have previously shown that the mRNA level of cystatin A is also stimulated by TPA in SV40-transformed human keratinocytes (SVHK cells) (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar). SVHK cells are a well-established, immortalized cell line sharing features of normal human keratinocytes (24Steinberg M.L. Defendi V. J. Invest. Dermatol. 1983; 81: 131S-136SAbstract Full Text PDF PubMed Scopus (58) Google Scholar, 25Takahashi H. Tamura T. Tsutsui M. Iizuka H. J. Dermatol. 1990; 17: 457-464Crossref PubMed Scopus (16) Google Scholar). These cells express relatively high levels of cystatin A as compared with other cell lines, such as A432 and SCC13 (data not shown). In the present study, we have identified the structure of the human cystatin A gene by screening a human genomic library and by using the polymerase chain reaction (PCR). We have also analyzed the regulation of cystatin A promoter activity by using a CAT reporter vector, which was connected to the 5′-flanking region of the cystatin A gene. SV40-transformed human keratinocytes (SVHK cells) (24Steinberg M.L. Defendi V. J. Invest. Dermatol. 1983; 81: 131S-136SAbstract Full Text PDF PubMed Scopus (58) Google Scholar) were a generous gift from Dr. M. L. Steinberg (Department of Chemistry, City College of the City University of New York, NY). The cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin and incubated at 37 °C with 5% CO2. A human phage library was purchased from CLONTECH (Palo Alto, CA). A 448-bp human cystatin A cDNA was digested withEcoRI (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar) and labeled with [32P]dCTP by the random priming method. Filter hybridization was used to screen 1 × 106 clones with a probe at 65 °C overnight in a solution composed of 1 m NaCl, 50 mm Tris-HCl (pH 8.0), 10 mm EDTA, 0.1% SDS, and 5× Denhardt's solution (1× Denhardt's solution: 0.002% polyvinylpyrrolidone, 0.002% Ficol, 0.02% bovine serum albumin). Subsequently, the filters were washed three times for 10 min at room temperature with 2× SSC (1× SSC: 0.15 m NaCl, 0.0015 m sodium citrate), 0.05% SDS and twice for 1 h at 65 °C with 1× SSC, 0.1% SDS. The filters were then exposed to Kodak XAR V film at −70 °C for 2 days. PCR was performed to analyze the promoter, the first exon, and the first intron of cystatin A using the Promoter FinderTM DNA Walking Kit (CLONTECH, Palo Alto, CA). To isolate the promoter and the first exon, we performed the first long PCR with the AP1 primer (as described in the kit) and the HCA1 primer (5′-AGTGGCGGGTTTGGCCTCAGATAAGCCTGG-3′; +63 to +95). Subsequently, the second PCR was performed with the AP2 primer (as described in the kit) and the HCA2 primer (5′-AGATAAGCCTCCAGGTATCATTTTGCG-3′; +51 to +77). To isolate the first exon and the first intron, we performed the first long PCR with the AP3 primer (5′-GCCCTATAGTGAGTCGTATTAGGATGG-3′) and the HCA3 primer (5′-CGCAAAATGATACCTGGAGGCTTATCT-3′; +63 to +95) The second PCR was performed with the AP4 primer (5′-ACCACGCGTGCCCTATAG-3′) and the HCA4 primer (5′-GGAGGCTTATCTGAGGCCAAACCCGCCACT-3′; +66 to +95). The HCA 1–4 primers were determined by cDNA analysis of keratolinin (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar). The long PCR kit was purchased from Takara Shuzo Co. (Otsu, Japan). The DNA was amplified for 35 cycles on a DNA cycler (Perkin-Elmer Corp., Norwalk, CT) at 98 °C for 20 s and then 68 °C for 15 min. The PCR products (pTA648, pTA2672, and pTA4.0) were subcloned into the pCRTM 2.1 vector (Invitrogen, San Diego, CA). The isolated clone (pcHCA) was digested withEcoRI and ligated into the pGEM-3Zf(+) vector. The constructed plasmids were denatured with 0.2 m sodium hydroxide. The single-stranded DNA was sequenced by the dideoxy chain termination method using the SP6 and T7 promoter primers (26Maniatis T. Fritsch E.H. Sambrook T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Habor, NY1989Google Scholar). The HindIII,XbaI-digested fragments from the pTA2672 and pTA648 vectors were inserted into the promoterless 0-CAT plasmids (p2.5CAT and p648CAT, respectively). Deleted vectors p478CAT, p238CAT, and p68CAT were generated by PCR using oligonucleotides HCA5 (5′-CTGTATGTTAAACATTTCCAG-3′; −478 to −457), HCA6 (5′-CATTGTCAAAGAGAATGCAG-3′; −239 to −221), HCA7 (5′-GCTGTTTGTGGAAAATAAAG-3′; −69 to −50), and HCA2. Each TRE-deleted fragment was generated by PCR using the overlap extension method with oligonucleotides HCA8 (5′-CATCCTGTTTCTGAATTATGAAATC-3′; −274 to −244, deleted TRE-2 region), HCA9 (5′-TAATTCAGAACAGGATGGAACCAT-3′; −267 to −236, deleted TRE-2 region), HCA10 (5′-GCAAGTAGATGTCCTAACAAGCAT-3′; −211 to −180, deleted TRE-1 region), and HCA11 (5′-GTTAGGACATCAACTTGCCCACTTG-3′; −204 to −173, deleted TRE-1 region). To construct the p648ΔT1 vector, we performed two PCR amplifications using the AP-2 and HCA8 oligomers or the HCA9 and HCA2 oligomers with the p648CAT vector as the template. Next, we performed a PCR with the AP-2 and HCA2 oligomers using mixed products derived from the initial PCR. The second PCR product was subcloned into the pCRTM 2.1 vector. Using the T7 promoter oligomer, we performed sequence analysis to confirm the orientation of the promoter and to confirm the absence of the deleted portion. The deleted fragments, which were isolated by digestion with HindIII andXbaI, were inserted into the promoterless 0-CAT plasmid. The p648ΔT2 vector was constructed using the AP-2, HCA10, HCA11, and HCA2 oligonucleotides. Using the p648ΔT1 vector, we performed PCR with the AP-2, HCA10, HCA11, and HCA2 oligomers and constructed the p648ΔT12 vector. The β-galactosidase expression vector was kindly supplied by Dr. T. Watanabe (Medical Institute of Bioregulation, Kyushu University, Japan). The PKC expression vectors were generous gifts of Dr. S. Ohno (Department of Molecular Biology, Yokohama City University School of Medicine) (27Ohno S. Akita Y. Konno Y. Imajoh S. Suzuki K. Cell. 1988; 53: 731-741Abstract Full Text PDF PubMed Scopus (299) Google Scholar). Transfection of plasmid DNA into cells was performed by the liposome method using Lipofectin (28Felgner P.L. Gadek T.R. Holm M. Roman R. Chan H.W. Wenz M. Northrop J.P. Ringold G.M. Danielsen M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7413-7417Crossref PubMed Scopus (4369) Google Scholar). Typically, 5 μg of reporter plasmid and 2 μg of β-galactosidase plasmid were co-transfected into 1 × 105 SVHK cells. The β-galactosidase plasmid was used as the internal standard to normalize each transfection efficacy. After 48 h, cells were collected, and the CAT assay was performed (29Neuman J.R. Morency C.A. Russian K.D. Biotechniques. 1987; 5: 441Google Scholar). The enzyme activity level of β-galactosidase in the transfected cell extracts was measured spectrophotometrically (26Maniatis T. Fritsch E.H. Sambrook T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Habor, NY1989Google Scholar). Relative CAT activity is expressed as the count of acetylated fraction corrected for the activity of the 0-CAT vector. Nuclear extraction and gel retardation analyses were performed as described previously (30Takahashi H. Kobayashi H. Iizuka H. Arch. Dermatol. Res. 1995; 287: 740-746Crossref PubMed Scopus (11) Google Scholar). The oligonucleotide probe that was used corresponds to the −240 to −266 fragment, which includes the TRE-2 site (see under "Results and Discussion"). Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc. Penicillin and streptomycin were obtained from M. A. Bioproducts (Walkersville, MD). The pGEM3Zf(+) vector was purchased from Promega (Madison, WI). The [α-32P]dCTP and deoxycytidine thiotriphosphate (1000 Ci/ml) were purchased from Amersham Pharmacia Biotech. All other chemicals were obtained from Nakarai Chemicals Ltd. (Kyoto, Japan). Anti-c-Jun, anti-Jun B, anti-Jun D, anti-c-Fos, anti-NF-κB, and anti-Fra-1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A human genomic phage library was screened with a32P-labeled, full-length human cystatin A cDNA (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Of the 1 × 106 clones screened, one clone (pcHCA) was identified that intensely hybridized with the cystatin A cDNA probe. DNA sequence analysis revealed that pcHCA contained the second and the third exon of the cystatin A gene (Figs. 1 and 2). In order to isolate the first exon and the promoter region, PCR was performed with the AP1/HCA1 primer pair and the AP2/HCA2 primer pair (see "Experimental Procedures"). Two fragments were obtained, one 648 bp and the other 2672 bp. These fragments were subcloned into pCRTM 2.1 vector (pTA648 and pTA2672; see Fig. 1), and DNA sequence analysis was performed. These fragments contained the first exon with the 5′-untranslated region (Figs. 1 and 2). In order to isolate the first exon and the first intron, PCR was performed with the AP3/HCA3 primer pair and the AP4/HCA4 primer pair. A fragment of 4 kb (pTA4k) was amplified, and DNA sequence analysis revealed that the fragment contained the first exon with a portion of the first intron. From these results, cystatin A was shown to contain three exons and two introns. The first, second, and third exons consisted of coding sequences of the cystatin A gene that were 66, 102, and 126 bp in length (Fig. 2). The second intron was approximately 3.6 kb in length, and the first intron was 14 kb in length, as determined by PCR using oligomers coding the first and the second exon (data not shown). We have previously shown that TPA increases the level of mRNA in SVHK cells (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Therefore, the transcription initiation site of the cystatin A gene was determined by the primer extension method using RNA prepared from TPA-treated SVHK cells. The transcription initiation site was located 55 bp upstream from the first translation site (Fig. 2,A).Figure 2Nucleotide sequence of the human cystatin A gene. The deduced amino acid sequence is shown below the nucleotide sequence. The transcription start site is shown asboldface letter A. The putative polyadenylation site, TRE-1, TRE-2, TRE-3, and the AP2 site are underlined. Thestar indicates the stop codon. The numbers on theleft and right indicate the nucleotide and amino acid numbers, respectively. The nucleotides corresponding to the reported cDNA sequences are underlined.View Large Image Figure ViewerDownload (PPT) Cysteine proteinase inhibitors have been subdivided into three families based on primary structure, molecular weight, number of disulfide bonds, and subcellular localization (5Barrett A.J. Trends Biochem. Sci. 1987; 12: 193-196Abstract Full Text PDF Scopus (264) Google Scholar). Family 1 cystatins (cystatins A and B) consist of approximately 100 amino acid residues (11–12 kDa) and lack disulfide bonds. Family 2 cystatins (cystatins C, S, and D) are approximately 120 amino acids in length (13–14 kDa) and contain two disulfide bonds. Family 3 cystatins, also known as the kininogen family, contain nine disulfide bonds. The human cystatin A gene consists of three exons and two introns similar to cystatin B (family 1), cystatin C (family 2), cystatin S (family 2), and cystatin D (family 2) (31Pennacchio L.A. Lehesjoki A.-E. Stone N.E. Willour V.L. Virtaneva K. Miao J. D'Amato E. Ramirez L. Faham M. Koskiniemi M. Warrington J.A. Norio R. Chapelle A. Cox D.R. Myers R.M. Science. 1996; 271: 1731-1734Crossref PubMed Scopus (494) Google Scholar, 32Abrahamson M. Olafsson I. Palsdottir A. Ulvback M. Lundwall A. Grubb J.A. Biochem. J. 1990; 268: 287-294Crossref PubMed Scopus (522) Google Scholar, 33Saitoh E. Kim H.-S. Smithies O. Maeda N. Gene. 1987; 61: 329-338Crossref PubMed Scopus (57) Google Scholar, 34Freije J.P. Abrahamson M. Olafsson I. Velasco I. Grubb A. Lopez-Otin C. J. Biol. Chem. 1991; 266: 20538-20543Abstract Full Text PDF PubMed Google Scholar). DNA sequence analyses showed that the 5′-flanking region of the human cystatin A gene did not contain a CAAT box or a TATA box. The other cystatin genes, except for cystatin C, also do not contain these sites. In order to determine the basal promoter region of the human cystatin A gene, six deletion fragments spanning from +77 to −2595 in the 5′-flanking region were fused with the CAT gene and transfected into SVHK cells (Fig. 3). The construct containing the +77 to −2595 fragment (p2672CAT) expressed a CAT activity level 13 times as high as the reverse-oriented construct (Fig. 3) or the constructs with vector but with no flanking region (data not shown). These data indicate that the 5′-flanking region of the human cystatin A gene contains a sequence that confers promoter activity. Deletion of the p2672CAT fragment up to −238 demonstrated minimal loss in basal activity. When the 5′-flanking region was deleted to the −68 position, the CAT activity was markedly depressed, suggesting that the most proximal −238 bp of the 5′-flanking region is essential for the basal transcription. Within −68 to −238, the cystatin A promoter contains an AP-2-like sequence (TCCCCATGCC; −75 to −84). AP-2 is an enhancer-binding protein that has been purified and cloned from HeLa cells, and this protein specifically interacts with the consensus sequence (T/C)C(C/G)CC(A/C)N(GCG/CGC) (35Imagawa M. Chiu R. Karin M. Cell. 1987; 51: 251-260Abstract Full Text PDF PubMed Scopus (1030) Google Scholar). Preliminary analysis showed that deletion of the AP-2 region decreased the basal promoter activity by one-third. The AP-2-like site might contribute to the basal transcription of human cystatin A gene. Cystatin S, which is highly expressed in the salivary gland, is induced by the β-adrenergic agonist isoprotererol (36Shaw P.A. Cox J.L. Barka T. Naito Y. J. Biol. Chem. 1988; 263: 18133-18137Abstract Full Text PDF PubMed Google Scholar). Previously, we have reported that cAMP and TPA increases the mRNA level of cystatin A in SVHK cells (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar). So far, there is no evidence for the TPA-dependent induction of other cystatins. To determine whether transcription of the human cystatin A gene is stimulated by TPA, five deletion constructs were transfected into SVHK cells in the presence or absence of TPA. The results showed that the construct containing the fragment +77 to −478 responded to TPA stimulation. The CAT activity level increased 3-fold following a 24-h exposure to TPA (Fig. 4). Consistent with the fact that the effect of TPA is mediated by PKC, the TPA-dependent cystatin A promoter activity was mimicked by other PKC activators, 1-oleoyl-2-acetylglycerol and mezerein (Fig. 5 A). 4-O-methyl-phorbol 12-myristate 13-acetate, a very weak PKC activator, produced much less effect on the promoter activity. Furthermore, the effect of TPA was inhibited by the PKC inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (Fig. 5 B).Figure 5Effect of PKC activators and inhibitor on cystatin A promoter activity. p648CAT vector was transfected into SVHK cells, and the cells were treated with 1-oleoyl-2-acetylglycerol (100 μg/ml), mezerein (1 μm), or 4-O-methyl-phorbol 12-myristate 13-acetate (200 ng/ml) (A) or with TPA (10 ng/ml) or 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (100 μm) for 24 h (B). The average CAT activities relative to the promoterless vector 0-CAT vector were obtained from at least three independent experiments.View Large Image Figure ViewerDownload (PPT) There are two putative TPA responsive elements (TRE-1, −189 to −195; TRE-2, −272 to −278) and one AP-2 responsive site (−74 to −83) within the +77 to −478 region of the cystatin A gene (Fig. 2). In order to determine the critical region of the TPA regulatory site, three TRE-deleted constructs were transfected into SVHK cells. Deletion of the T-2 region (−272 to −278) or the T-2 plus T-1 region (−189 to −195) completely abolished the TPA responsiveness (Fig. 6). Conversely, deletion of T-1 showed no significant loss in TPA responsiveness. These results indicate that the sequence −272 to −278 (TRE-2) is responsible for the TPA stimulation. In keratinocytes, TPA is a potent inducer of differentiation and increases the expression of CE precursor protein(s), as well as transglutaminase. TRE sites have been identified in a number of differentiation-related genes, such as loricrin, involucrin, small proline-rich protein(s), and transglutaminase 1 (19Takahashi H. Iizuka H. J. Invest. Dermatol. 1993; 100: 10-15Abstract Full Text PDF PubMed Google Scholar, 20Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 21Lopez-Bayghen E. Vega A. Cadena A. Granados S.E. Jave L. Gariglio P. Alvares-Saras L.M. J. Biol. Chem. 1996; 271: 512-520Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22DiSepio D. Jones A. Longley M.A. Bundman D. Rothnagel J.A. Roop D.R. J. Biol. Chem. 1995; 270: 10792-10799Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 23Yamanishi K. Inazawa J. Liew F.-W. Nonomura K. Ariyama T. Yasuno H. Abe T. Doi H. Hirano J. Fukushima S. J. Biol. Chem. 1992; 267: 17858-17863Abstract Full Text PDF PubMed Google Scholar). Our study revealed that the TRE-2 region (−272 to −278) of the cystatin A gene was critical for the TPA-induced promoter activity. This is consistent with a common controlling mechanism for the expression of CE precursor proteins, as well as their cross-linking enzyme, transglutaminase 1. The AP-1 protein, which is a complex consisting of Jun and Fos family proteins, binds to TREs and regulates the TPA-inducible genes. In order to determine the binding protein(s) in the TRE-2 region of the human cystatin A gene, a 38-bp synthetic oligonucleotide representing the TRE-2 region (−272 to −278 bp) was evaluated using a DNA gel shift assay. Incubation of the oligonucleotide with the nuclear extract from TPA-treated SVHK cells yielded three DNA-protein binding complexes (Fig. 7, lane 2). The specificity of the binding was verified by a competition assay using the same (Fig. 7,lane 4) or unrelated (Fig. 7, lane 5) unlabeled oligonucleotides in excess of 100 moles. Furthermore, anti-c-Jun, anti-JunD, and anti-c-Fos antibodies decreased the specific bands, whereas supershifted bands appeared near the top of the lane (Fig. 7,lanes 6, 8, and 9). A supershifted band was not detected by the addition of anti-JunB, anti-Fra-1, or anti-NF-kB antibodies (Fig. 7, lanes 7, 10, and 11). In order to determine the effects of these AP-1 related proteins on the transcription of cystatin A, various expression vectors of the Jun and Fos family proteins were transfected into SVHK cells. Co-transfection of the p648CAT vector with the c-Jun and c-Fos expression vectors or the JunD and c-Fos expression vectors resulted in an increase in the CAT activity level (Fig. 8). This finding suggests that the nuclear proteins that bind to the TRE-2 region are most likely composed of c-Jun and c-Fos or of JunD and c-Fos. It has been reported that cystatin A is expressed in the upper spinous layer to the granular layer of the normal epidermis (37Tezuka T. Qing J. Saheki M. Kushida S. Takahashi M. Dermatology. 1994; 188: 21-24Crossref PubMed Scopus (36) Google Scholar). Immunohistochemistry of the normal epidermis revealed that the c-Fos protein is localized in the upper spinous and granular cell layers, whereas the c-Jun protein is localized in the granular cell layer. Fra-1 is expressed in all of the epidermal cell layers except for the basal cell layer. Conversely, JunB and JunD are present in all of the epidermal cell layers (38Welter J.F. Eckert R.L. Oncogene. 1995; 11: 2681-2687PubMed Google Scholar). Our finding that the cystatin A gene is regulated by c-Jun and c-Fos, or by c-Jun and JunD, is compatible with the expression pattern of the AP-1 protein family in the epidermis. PKC is a large family of proteins consisting of at least 11 isozymes (39Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4229) Google Scholar). PKC-α, -βI, -βII, and -γ are the classical PKC proteins that are calcium- and diacylglycerol-dependent. PKC-δ, -ε, -η, -θ, and -μ are the novel PKCs, which do not require calcium for activation. PKC-ζ and -τ are the atypical PKCs, which require neither calcium nor diacylglycerol for activation. TPA activates classical and novel PKCs but not atypical PKCs. The epidermal keratinocytes contain PKC-α, -δ, -ε, -η, and -ζ (40Dlugosz A.A. Mischak H. Muchinski J.F. Yuspa H.S. Mol. Cell. Biol. 1992; 12: 286-292Google Scholar). In order to determine the PKC isozyme(s) responsible for cystatin A gene expression, we co-transfected SVHK cells with the p648CAT vector and various PKC isozyme expression vectors. Consistent with TPA-induced activation of endogenous PKC(s), cystatin A promoter activity was increased by TPA in SVKH cells transfected with the control vector (Fig. 9, C). Transfection of PKC-δ, -ε, -η, or -ζ had no effect on the cystatin A promoter activity as compared with the transfection of the control vector. Although TPA also increased the promoter activity of SVHK cells transfected with PKC-δ, -ε, -η, or -ζ, the increase was not statistically significant as compared with that of the cells transfected with the control vector (Fig. 9, δ, ε, η, and ζ). Cystatin A promoter activity, however, was significantly stimulated by co-transfection of p648CAT and the PKC-α vectors, which was further stimulated by TPA (Fig. 9, α). These results suggest that PKC-α is responsible for the stimulation of the human cystatin A promoter activity. There are several reports concerning the localization of PKC isozymes in the epidermis (41Leibersperger H. Gschwendt M. Gernold M. Marks F. J. Biol. Chem. 1991; 266: 14778-14787Abstract Full Text PDF PubMed Google Scholar, 42Wevers A. Wirnitzer U. Schaarschmidt H. Hagemann L. Mahrle G. Arch. Dermatol. Res. 1992; 284: 5-7Crossref PubMed Scopus (9) Google Scholar, 43Koizumi H. Kohno Y. Osada S. Ohno S. Ohkawara A. Kuroki T. J. Invest. Dermatol. 1993; 101: 858-863Abstract Full Text PDF PubMed Google Scholar). In normal skin, PKC-η is expressed in the uppermost granular layer (43Koizumi H. Kohno Y. Osada S. Ohno S. Ohkawara A. Kuroki T. J. Invest. Dermatol. 1993; 101: 858-863Abstract Full Text PDF PubMed Google Scholar), whereas PKC-α mRNA is expressed from the basal to the spinous layers (42Wevers A. Wirnitzer U. Schaarschmidt H. Hagemann L. Mahrle G. Arch. Dermatol. Res. 1992; 284: 5-7Crossref PubMed Scopus (9) Google Scholar). Because cystatin A is expressed in the upper spinous layer and the granular layer, there would be other transcription factors (either stimulatory or inhibitory) that regulate the differentiation-specific expression of cystatin A in the epidermis. In this study, we characterized the structure of the human cystatin A gene, including the 5′-upstream region. The cystatin A gene was shown to be regulated by PKC pathway, similar to other CE precursor proteins (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 44Stanwell C. Denning M.F. Rutberg S.E. Cheng C. Yuspa S.H. Dlugosz A.A. J. Invest. Dermatol. 1996; 106: 482-489Abstract Full Text PDF PubMed Scopus (39) Google Scholar). The characterization of the AP1-dependent signaling pathway via specific PKC isozymes will clarify the nature of the regulation and will elucidate the molecular mechanisms of keratinocyte differentiation. The technical assistance of Y. Kotani and K. Takahashi and the secretarial assistance of Y. Maekawa are greatly appreciated.
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