Structure and Transcriptional Regulation of the Human Cystatin A Gene

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 (1), but it has also been isolated from the spleen, liver, and epidermis (2)(3)(4). The primary structure of cystatin A consists of a polypeptide chain of 98 amino acid residues that is mainly distributed intracellularly (5). We have recently reported that cystatin A is identical to keratolinin, one of the precursor proteins of cornified cell envelope (CE) 1 (6), which is formed during terminal differentiation of keratinocytes (7)(8)(9).
CE is a highly insoluble structure formed beneath the plasma membrane of keratinocytes during terminal differentiation (7)(8)(9). This structure is 15-20 nm thick and is stabilized by cross-link formation of precursor proteins by N-(␥-glutamyl-)lysine isodipeptide bonds and disulfide bonds, which are catalyzed by transglutaminase(s) and sulfhydryl oxidase, respectively (7,8,10).
In addition to cystatin A, several proteins have been implicated as precursors of CE, which include involucrin (11), loricrin (12), small proline-rich protein(s) (13), elafin (14), and envoplakin (15). Recent evidence suggests that involucrin is an early component of CE and provides a scaffold for the incorporation of other precursor proteins (14,16).
TPA, which is a potent activator of protein kinase C (PKC), induces terminal differentiation of keratinocytes (17,18). Recent studies have revealed that involucrin, loricrin, and transglutaminase 1 genes contain a TRE(s) in their 5Ј-flanking regions and that these TREs induce increased expression of these protein transcripts by TPA (19 -23). We have previously shown that the mRNA level of cystatin A is also stimulated by TPA in SV40-transformed human keratinocytes (SVHK cells) (6).
SVHK cells are a well-established, immortalized cell line sharing features of normal human keratinocytes (24,25). 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 5Ј-flanking region of the cystatin A gene.
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 g/ml streptomycin and incubated at 37°C with 5% CO 2 .
Screening of the Human Genomic DNA Library-A human phage library was purchased from CLONTECH (Palo Alto, CA). A 448-bp human cystatin A cDNA was digested with EcoRI (6) and labeled with [ 32 P]dCTP by the random priming method. Filter hybridization was used to screen 1 ϫ 10 6 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 5ϫ Denhardt's solution (1ϫ 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 2ϫ SSC (1ϫ SSC: 0.15 M NaCl, 0.0015 M sodium citrate), 0.05% SDS and twice for 1 h at 65°C with 1ϫ SSC, 0.1% SDS. The filters were then exposed to Kodak XAR V film at Ϫ70°C for 2 days.
PCR Cloning Strategies-PCR was performed to analyze the promoter, the first exon, and the first intron of cystatin A using the Promoter Finder TM 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 (5Ј-AGTGGCGGGTTTGGCCTCAGATAAGCCTGG-3Ј; ϩ63 to ϩ95). Subsequently, the second PCR was performed with the AP2 primer (as described in the kit) and the HCA2 primer (5Ј-AGATAAGCCTCCAGG-TATCATTTTGCG-3Ј; ϩ51 to ϩ77). To isolate the first exon and the first intron, we performed the first long PCR with the AP3 primer (5Ј-GC-CCTATAGTGAGTCGTATTAGGATGG-3Ј) and the HCA3 primer (5Ј-C-GCAAAATGATACCTGGAGGCTTATCT-3Ј; ϩ63 to ϩ95) The second PCR was performed with the AP4 primer (5Ј-ACCACGCGTGCCCTA-TAG-3Ј) and the HCA4 primer (5Ј-GGAGGCTTATCTGAGGCCAAAC-CCGCCACT-3Ј; ϩ66 to ϩ95). The HCA 1-4 primers were determined by cDNA analysis of keratolinin (6). 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 pCR TM 2.1 vector (Invitrogen, San Diego, CA).
DNA Sequence-The isolated clone (pcHCA) was digested with EcoRI and ligated into the pGEM-3Zf(ϩ) 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 (26).
Transfection and the CAT Assay-Transfection of plasmid DNA into cells was performed by the liposome method using Lipofectin (28). Typically, 5 g of reporter plasmid and 2 g of ␤-galactosidase plasmid were co-transfected into 1 ϫ 10 5 SVHK cells. The ␤-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 (29). The enzyme activity level of ␤-galactosidase in the transfected cell extracts was measured spectrophotometrically (26). 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-Nuclear extraction and gel retardation analyses were performed as described previously (30). The oligonucleotide probe that was used corresponds to the Ϫ240 to Ϫ266 fragment, which includes the TRE-2 site (see under "Results and Discussion").

RESULTS AND DISCUSSION
Identification and Structure of the Human Cystatin A Gene-A human genomic phage library was screened with a 32 P-labeled, full-length human cystatin A cDNA (6). Of the 1 ϫ 10 6 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 pCR TM 2.1 vector (pTA648 and pTA2672; see Fig. 1), and DNA sequence analysis was performed. These fragments contained the first exon with the 5Ј-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 (6). 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).
Cysteine proteinase inhibitors have been subdivided into three families based on primary structure, molecular weight, number of disulfide bonds, and subcellular localization (5). 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) (31)(32)(33)(34). DNA sequence analyses showed that the 5Ј-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.

Identification of the Basal Promoter Region of the Human
Cystatin A Gene-In order to determine the basal promoter region of the human cystatin A gene, six deletion fragments spanning from ϩ77 to Ϫ2595 in the 5Ј-flanking region were fused with the CAT gene and transfected into SVHK cells (Fig.  3). The construct containing the ϩ77 to Ϫ2595 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 5Ј-flanking region of the human cystatin A gene contains a sequence that confers promoter activity. Deletion of the p2672CAT fragment up to Ϫ238 demonstrated minimal loss in basal activity. When the 5Ј-flanking region was deleted to the Ϫ68 position, the CAT activity was markedly depressed, suggesting that the most proximal Ϫ238 bp of the 5Ј-flanking region is essential for the basal transcription. Within Ϫ68 to Ϫ238, the cystatin A promoter contains an AP-2-like sequence (TCCCCATGCC; Ϫ75 to Ϫ84). AP-2 is an enhancer-binding protein that has been purified and cloned CAT expression vectors with various lengths of the promoter region of the cystatin A gene (5 g each) were co-transfected into 1 ϫ 10 5 SVHK cells with an internal control vector, pSV-␤-galactosidase (2 g). Fortyeight hours after the transfection, the cells were harvested, and extracts were assayed for CAT and ␤-galactosidase activity. T1, T2, and T3 indicate the position of the TRE sites. The average CAT activities relative to the promoterless vector 0-CAT vector were obtained from at least three independent experiments 48 h posttransfection.
from HeLa cells, and this protein specifically interacts with the consensus sequence (T/C)C(C/G)CC(A/C)N(GCG/CGC) (35). Preliminary analysis showed that deletion of the AP-2 region decreased the basal promoter activity by one-third. The AP-2like site might contribute to the basal transcription of human cystatin A gene.
The TRE-2 Site (Ϫ272 to Ϫ278) Is Critical for the Up-regulation of the Cystatin A Gene by TPA-Cystatin S, which is highly expressed in the salivary gland, is induced by the ␤-adrenergic agonist isoprotererol (36). Previously, we have reported that cAMP and TPA increases the mRNA level of cystatin A in SVHK cells (6). 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 ϩ77 to Ϫ478 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. 5A). 4-O-methyl-phorbol 12-myristate 13acetate, 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)-2methyl piperazine dihydrochloride (Fig. 5B).
There are two putative TPA responsive elements (TRE-1, Ϫ189 to Ϫ195; TRE-2, Ϫ272 to Ϫ278) and one AP-2 responsive site (Ϫ74 to Ϫ83) within the ϩ77 to Ϫ478 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 (Ϫ272 to Ϫ278) or the T-2 plus T-1 region (Ϫ189 to Ϫ195) 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 Ϫ272 to Ϫ278 (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 (19 -23). Our study revealed that the TRE-2 region (Ϫ272 to Ϫ278) 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.
c-Jun, JunD, and c-Fos Bind to the TRE-2 Region and Increase the Cystatin A Promoter Activity-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 (Ϫ272 to Ϫ278 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  (37). 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 (38). 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.
Transcription of the Cystatin A Gene Is Increased by Transfection of the PKC-␣ Expression Vector-PKC is a large family of proteins consisting of at least 11 isozymes (39). PKC-␣, -␤I, -␤II, and -␥ are the classical PKC proteins that are calciumand diacylglycerol-dependent. PKC-␦, -⑀, -, -, and -are the novel PKCs, which do not require calcium for activation. PKCand -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-␣, -␦, -⑀, -, and - (40).
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-␦, -⑀, -, or -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-␦, -⑀, -, or -, the increase was not statistically significant as compared with that of the cells transfected with the control vector ( Fig. 9, ␦, ⑀, , and ). Cystatin A promoter activity, however, was significantly stimulated by co-transfection of p648CAT and the PKC-␣ vectors, which was further stimulated by TPA (Fig. 9, ␣). These results suggest that PKC-␣ 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 (41)(42)(43). In normal skin, PKC-is expressed in the uppermost granular layer (43), whereas PKC-␣ mRNA is expressed from the basal to the spinous layers (42). 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 5Ј-upstream region. The cystatin A gene was shown to be regulated by PKC pathway, similar to other CE precursor proteins (6,44). 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.