From the Department of Dermatology, Asahikawa Medical College, 3-11 Nishikagura, Asahikawa 078, Japan and the § Department of
Dermatology, The Oregon Health Sciences University, Oregon 97201
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-
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.
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INTRODUCTION |
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 (2-4). 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 (7-9).
CE is a highly insoluble structure formed beneath the plasma membrane
of keratinocytes during terminal differentiation (7-9). 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 5'-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 5'-flanking
region of the cystatin A gene.
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EXPERMENTAL PROCEDURES |
Cell Culture--
SV40-transformed human keratinocytes (SVHK
cells) (24) 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.
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 [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 Cloning Strategies--
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 (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 pCRTM 2.1 vector (Invitrogen, San
Diego, CA).
DNA Sequence--
The isolated clone (pcHCA) was digested with
EcoRI 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 (26).
Plasmid Constructs--
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 p648
T1 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 and
XbaI, were inserted into the promoterless 0-CAT plasmid. The
p648
T2 vector was constructed using the AP-2, HCA10, HCA11, and HCA2
oligonucleotides. Using the p648
T1 vector, we performed PCR with the
AP-2, HCA10, HCA11, and HCA2 oligomers and constructed the p648
T12
vector. The
-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) (27).
Transfection and the CAT Assay--
Transfection of plasmid DNA
into cells was performed by the liposome method using Lipofectin (28).
Typically, 5 µg of reporter plasmid and 2 µg of
-galactosidase
plasmid were co-transfected into 1 × 105 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
240 to
266 fragment, which includes the TRE-2 site (see under
"Results and Discussion").
Materials--
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
[
-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-
B, and
anti-Fra-1 antibodies were purchased from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA).
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RESULTS AND DISCUSSION |
Identification and Structure of the Human Cystatin A Gene--
A
human genomic phage library was screened with a
32P-labeled, full-length human cystatin A cDNA (6). 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 (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).

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Fig. 1.
Structure of the human cystatin A gene.
The structure of the gene is schematically represented by the
bar at the top of the diagram. Exons are indicated by
boxes. pcHCA, pTA648, pTA2672, and pTA4k are the names of
clones obtained by the library screening or by the PCR procedure (see
"Experimental Procedures").
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Fig. 2.
Nucleotide sequence of the human cystatin A
gene. The deduced amino acid sequence is shown below the
nucleotide sequence. The transcription start site is shown as
boldface letter A. The putative polyadenylation site, TRE-1,
TRE-2, TRE-3, and the AP2 site are underlined. The
star indicates the stop codon. The numbers on the
left and right indicate the nucleotide and amino
acid numbers, respectively. The nucleotides corresponding to the
reported cDNA sequences are underlined.
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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) (31-34). 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.
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 +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.

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Fig. 3.
Transcriptional activity of the cystatin A
promoter. CAT expression vectors with various lengths of the
promoter region of the cystatin A gene (5 µg each) were
co-transfected into 1 × 105 SVHK cells with an
internal control vector, pSV- -galactosidase (2 µg). Forty-eight
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.
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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) (35). 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.
The TRE-2 Site (
272 to
278) 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 +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.
5A).
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.
5B).

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Fig. 4.
Effect of TPA on cystatin A promoter
activity. Various CAT expression vectors were co-transfected into
SVHK cells and incubated for 24 h. The transfected cells were then
cultured in the presence ( ) or absence ( ) of TPA (10 ng/ml) for
24 h, and the level of CAT activity was measured. The average CAT
activities relative to the promoterless vector 0-CAT vector were
obtained from at least three independent experiments.
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Fig. 5.
Effect 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.
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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.

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Fig. 6.
Effects of TPA on various TRE-deleted p648CAT
vectors. Various TRE-deleted p648CAT vectors were transfected into
SVHK cells, and the cells were treated with TPA (10 ng/ml) for 24 h. p648CAT T1CAT, TRE-1 site ( 189 to 198)-deleted p648CAT vector;
p648CAT T2CAT, TRE-2 site ( 272 to 278)-deleted p648CAT
vector; p648CAT T12CAT, TRE-1, TRE-2-deleted p648CAT vector. The
average CAT activities relative to the promoterless vector 0-CAT vector
were obtained from at least three independent experiments. , medium
alone; , TPA (10 ng/ml).
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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 (
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.
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 (
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).

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Fig. 7.
Gel shift analysis of nuclear extracts from
TPA-treated SVHK cells. The nuclear extracts from TPA-treated SVHK
cells were reacted with the synthesized oligomer that contained the
TRE-2 region. Lane 1, probe without nuclear extract;
lane 2, nuclear extract from TPA-treated SVHK cells reacted
with the synthesized oligomer containing the TRE-2 region; lane
3, nuclear extract from TPA-treated SVHK cells plus the oligomer
containing the TRE consensus sequence; lane 4, nuclear
extract from TPA-treated SVHK cells with an excess of 100 moles of
unlabeled probe; lane 5, nuclear extract from TPA-treated
SVHK cells with the oligomer containing the NF- B binding consensus
sequence; lane 6, nuclear extract from TPA-treated SVHK
cells with the anti-c-Jun antibody; lane 7, nuclear extract
from TPA-treated SVHK cells with the anti-c-JunB antibody; lane
8, nuclear extract from TPA-treated SVHK cells with the anti-JunD
antibody; lane 9, nuclear extract from TPA-treated SVHK
cells with the anti-c-Fos antibody; lane 10, nuclear extract
from TPA-treated SVHK cells with the anti-Fra-1 antibody; lane
11, nuclear extract from TPA-treated SVHK cells with the
anti-NF- B antibody. The arrows indicates the supershifted
bands.
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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.

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Fig. 8.
Effects of AP-1 family proteins on cystatin A
promoter activity. Various AP-1 family expression vectors (3 µg)
were transfected into SVHK cells with the p648CAT vector and incubated
for 48 h. The concentration of TPA was 10 ng/ml. The average CAT
activities relative to the promoterless vector 0-CAT vector were
obtained from at least three independent experiments.
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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 calcium- and
diacylglycerol-dependent. PKC-
, -
, -
, -
, and
-µ are the novel PKCs, which do not require calcium for activation.
PKC-
and -
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 (41-43). 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.

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Fig. 9.
Effects of PKC isozyme expression vectors on
cystatin A promoter activity. Various PKC isozyme expression
vectors (3 µg) were transfected into SVHK cells with the p648CAT
vector and incubated for 24 h. The transfected cells were then
cultured in the presence or absence of TPA (10 ng/ml) for 24 h.
C, control vector; , PKC- ; , PKC- ; , PKC- ;
, PKC- ; , PKC- . The average CAT activities relative to the
promoterless vector 0-CAT vector were obtained from at least three
independent experiments. , medium; , TPA (10 ng/ml).
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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
(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.
The technical assistance of Y. Kotani and K. Takahashi and the secretarial assistance of Y. Maekawa are greatly
appreciated.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB007773 and AB007774.