(Received for publication, October 4, 1996, and in revised form, December 9, 1996)
From the Endocrine Unit, Veterans Affairs Medical Center, University of California, San Francisco, California 94121
The 5-flanking region of the human phospholipase
C-
1 gene was isolated from a human P1 genomic DNA library. The
S1-nuclease mapping and primer extension analysis revealed that there
is a single transcriptional start site located at 135 bases upstream from the translation start codon in the human phospholipase C-
1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA box. The
fragment +135 to
877 in the 5
-flanking region of the human phospholipase C-
1 gene was subcloned into a luciferase reporter vector. The chimeric gene produced a high level of luciferase activity
and responded to 1,25-(OH)2D3 in transiently
transfected human keratinocytes. Deletion and mutation studies of the
fragment +135 to
877 demonstrated a vitamin D-responsive element that contains a motif arranged as two direct repeats separated by 6 bases
(DR6), AGGTCAgaccacTGGACA, located between
786 and
803 base pairs.
Incubation of the oligonucleotide containing the DR6 with keratinocyte
nuclear extracts produced a specific protein-DNA complex that shifted
to a higher molecular weight form upon the addition of an antibody
specific to the 1,25-(OH)2D3 receptor. Therefore, the 5
-flanking region of the human phospholipase C-
1 gene confers promoter activity and contains a DR6-type vitamin D-responsive element that mediates, at least in part, the enhanced expression of this gene in human keratinocytes by
1,25-(OH)2D3.
Phospholipase C (PLC)1 is a family of
isoenzymes that cleave phosphatidyl inositol bisphosphate to two second
messengers, inositol triphosphate and diacylglycerol, in response to a
transmembrane signal (1, 2). Diacylglycerol is the physiological
activator of protein kinase C, and inositol triphosphate causes the
release of calcium from the endoplasmic reticulum. PLCs can be divided into three types (PLC-, PLC-
, and PLC-
), and each type
contains several subtypes (3, 4). PLC-
1, unlike the other PLC
isoenzymes, contains a src homology 2 domain through which
PLC-
1 interacts with various tyrosine kinase growth factor receptors
(5-8). PLC-
1 is overexpressed in primary human breast carcinoma
(9), human colorectal cancer (10), familial adenomatous polyposis (11), and hyperproliferative epidermal diseases (12). The amount of PLC-
1
protein is higher in neoplastic keratinocyte cell lines than in normal
keratinocytes (13). Calcium-induced differentiating keratinocytes
express over 2-fold more PLC-
1 protein than undifferentiated keratinocytes (14). These observations suggest that PLC-
1 might be
involved in the regulation of cell proliferation and
differentiation.
The differentiation of normal human keratinocytes is induced by
extracellular calcium and 1,25-(OH)2D3
(15-20). The mechanism underlying the regulation by
1,25-(OH)2D3 is thought to include changes in
intracellular calcium, PLC, and protein kinase C activation. PLC-1
is one of the major PLC isoenzymes that mediate cellular signal
transduction. Treatment with 1,25-(OH)2D3
dramatically up-regulates the protein and mRNA expression of
PLC-
1 (24). To understand the molecular mechanism of this
regulation, we cloned the 5
-flanking region of the human PLC-
1 gene
that confers promoter activity and identified within the 5
-flanking
region a DR6-type vitamin D-responsive element (VDRE).
The subclones containing
phospholipase C-1 genomic DNA were obtained from a human P1 genomic
DNA library using as probe a 5-kb PLC-
1 cDNA (Genome Systems).
To isolate the 5
-flanking region of the PLC-
1 gene, the subclones
were further screened by colony hybridization using the oligonucleotide
(5
-CGTTGCGCTTGCTCCCGGGC-3
) from the 5
-untranslated region of
PLC-
1 cDNA as probe. From the selected subclone, a 1.1-kb
XhoI fragment was resubcloned into a pBluscript SK(
)
vector (Stratagene). The nucleotide sequence of the insert was
sequenced using the dideoxy chain termination method. The sequence of
each strand was confirmed by repeating the sequencing in both
directions at least three times. The sequence of the GC-compressed
region was confirmed using dITP instead of dGTP.
The
XhoI-StylI fragment was subcloned into a
pGL-3-basic vector (Promega). The PLC-1 gene was placed 2 bp
upstream from the luciferase gene. Subsequent 5
deletion constructs
were made with restriction enzyme digestion. The constructs containing
the fragment
748 to
828 and the fragment
786 to
803 were made
by ligating the fragments to the heterologous simian virus 40 (SV40)
promoter in the pGL-3-promoter vector. Correct orientation of the
inserts with respect to the luciferase sequence was verified by
restriction enzyme analysis.
Normal human keratinocytes were isolated from neonatal human foreskins and grown in serum free keratinocyte growth medium (Clonetics) (25). Briefly, keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4 °C, overnight), and primary cultures were established in keratinocyte growth medium containing 0.07 mM calcium. Second passage keratinocytes were plated in 60-mm culture dishes with keratinocyte growth medium plus 0.03 mM calcium at 20-30% confluency for the transfection experiments.
DNA Transfection and Luciferase AssayPLC-1 luciferase
chimeric plasmids were transfected into normal human keratinocytes
using a polybrene method 24 h after plating cells in 60-mm culture
dishes (26). Cells were co-transfected with 0.2 µg of pRSV
-gal
(27), a
-galactosidase expression vector that contains a
-galactosidase gene that is driven by a Rous sarcoma virus promoter
and enhancer, which was used as an internal control to normalize for
transfection efficiency. 1,25-(OH)2D3 was added
to the cells 24 h after transfection at a final concentration of
10
9 M. The same amount of ethanol (vehicle)
was added to the control plates. The final concentration of the ethanol
was 1% in the culture medium. Cells were harvested 24 h after the
addition of 1,25-(OH)2D3. The cells were lysed,
and the cell extracts were assayed for luciferase activities using
Luciferase Assay System (Promega). The
-galactosidase activities
were assayed using Galacto-LightTM kit (TROPIX Inc.). A pGL-3-control
vector (Promega) containing SV40 promoter and SV40 enhancer, which are
known to be unresponsive to 1,25-(OH)2D3, was
included in each transfection experiment as a control. Every experiment
was done in triplicate and was repeated at least three times.
Total cellular RNA was isolated
from the first passage of normal human keratinocytes by RNA STAT-60TM
kit (Tel-Test "B" Inc). The poly(A) RNA was obtained using a
poly(A) mRNA isolation kit (Stratagene). The S1-nuclease Protection
assay was carried out using the S1-AssayTM kit from Ambion. An
antisense DNA probe was synthesized from a 212-bp
Bsu36I-StylI fragment upstream of the translation
start codon in the human PLC-1 5
-flanking region cloned into the
pGL-3-basic vector, using Klenow and [
-32P]dCTP. This
was accomplished by use of the antisense GL primer2 primer that bound
the downstream sense GL primer2 primer in the vector such that the
synthesized probe spanned the insert. The probe was coprecipitated with
1 µg of the normal human keratinocyte poly(A) RNA. Hybridization was
performed by dissolving the precipitate in 10 µl of hybridization
buffer at 42 °C overnight. Unprotected DNA was digested with S1
nuclease at 37 °C for 30 min. The resulting fragment was recovered
by ethanol precipitation, denatured, and analyzed on an 8% sequencing
gel with a sequencing ladder as a standard. The sequencing reaction was
performed using the dideoxy chain termination method.
The total RNA and poly(A) RNA
from normal human keratinocytes were isolated in a same way as that for
S1-nuclease protection assay. The primer extension analysis was
performed using the Primer Extension System from Promega. 2 µg of
poly(A) RNA was hybridized with an end labeled primer corresponding to
the region 40-60 bp downstream from the translation start codon of the
antisense strand of the human PLC-1 cDNA. The hybridization
mixture was heated at 75 °C for 15 min and then incubated at
42 °C for 40 min. Actinomycin D was added to the mixture at a final
concentration of 75 ng/µl to inhibit secondary structure formation of
the RNA. The extension products were analyzed on a denatured 6%
polyacrylamide gel.
The nuclear extracts were made from normal human keratinocytes according to the method described by Abmayr and Workman (28). The recombinant vitamin D receptor was from Affinity Bioreagents Inc. Synthetic oligonucleotides used for the DNA mobility shift assay were end-labeled by T4 polynucleotide kinase. The DNA-protein reactions were performed in a total of 17 µl; nuclear extracts (12 µg of protein) were incubated with 2 µg of poly(dI·dC) (Pharmacia Biotech Inc.) and 10,000 cpm of 32P-labeled probe in 10 ml of binding buffer (20 mM HEPES, pH 7.9, 20% glycerol, 50 mM KCl, 0.5 mM dithiothreitol) at 30 °C for 25 min. Unlabeled competitors were added at the preincubation step. In the super gel shift reaction, a polyclonal anti-vitamin D receptor antibody (3 µl from the original stock, Affinity Bioreagents Inc.) was added to the DNA-protein reaction and incubated for an additional 25 min. Protein-DNA complexes were electrophoresed in a 6% nondenaturing polyacrylamide gel in 1 × gel shift running buffer (50 mM Tris, 380 mM glycerin, 2 mM EDTA, pH 8.5).
Three positive clones were obtained from the human P1 genomic DNA
library screening. One of the positive clones was digested by
HindIII, and the random fragments were subcloned into a
pZErO vector (Invitrogen). Three independent subclones that
contain the 5-flanking region of the human PLC-
1 gene were isolated from the random subclones by screening 96 subcultures using an oligonucleotide from the untranslated region of the human PLC-
1 cDNA (see "Materials and Methods"). Restriction analysis
indicated that all three positive subclones contained a 9-kb
HindIII insert spanning more than 8 kb upstream from the
translation start codon in the human PLC-
1 gene. The restriction map
for the 9-kb fragment is shown in Fig. 1A.
The HindIII-StylI (9 kb),
XbaI-StylI (2.5 kb), and
XhoI-StylI (1 kb) fragments were individually
subcloned in a pGL-3-basic vector and transfected into human
keratinocytes. The results showed that the 1-kb
XhoI-StylI fragment construct expressed the
highest luciferase activity (data not shown). Therefore, we focused on
the 1-kb XhoI-StylI fragment in the subsequent
experiments. The sequence analysis revealed that the 1-kb
XhoI-StylI fragment in the 5
-flanking region of
the human PLC-
1 gene was very GC-rich. 16 putative SP1 sites and 8 putative AP2 sites were clustered in the 1-kb
XhoI-StylI fragment. No TATA box was found in
this fragment. There was a putative CCAAT box located between
581 and
585 bp upstream from the transcriptional start site (Fig. 1B).
Both S1 nuclease protection assay and primer extension analysis were
performed to determine the transcriptional start site for the human
PLC-1 gene. The S1 nuclease protection assay was performed using an
antisense probe spanning 212 bp upstream from the translation start
codon. This probe hybridized to the poly(A) RNA isolated from human
keratinocytes. After S1 nuclease digestion, a single protected fragment
of 135 bp was detected (Fig. 2A). The result
suggested that the transcriptional start site is 135 bp upstream from
the translation start codon. The primer extension analysis showed a
195-bp single extension fragment whose 5
end is 135 bp upstream from
the translation start codon (Fig. 2B), confirming the result
obtained with the S1 nuclease protection assay.
In an attempt to delineate the sequences essential for human PLC-1
gene transcription, nine deletional fragments spanning from +135 to
877 bp in the 5
-flanking region were fused with the coding region of
the luciferase gene in the luciferase vector and transfected into
normal human keratinocytes (Fig. 3A). The construct containing the +135 to
877 fragment construct expressed luciferase activity 50-fold higher than that from the vector alone (Fig. 3B). The data clearly indicated that the 5
-flanking
region of the human PLC-
1 gene contains a sequence that confers
promoter activity. Deletional analysis to
200 bp showed little loss
in basal activity. When the 5
deletions reached
39 bp, the
luciferase activities were greatly reduced. The fragment +13 to +135,
which did not contain the transcriptional start site, lost all activity (Fig. 3B). The data suggested that the most proximal 200 bp
of the 5
-flanking region of the human PLC-
1 gene are essential for
transcriptional initiation.
The induction of human PLC-1 promoter by
1,25-(OH)2D3 using transfection experiments
with deletional and mutant constructs. A, nine 5
deletional
fragments spanning from +135 bp to
877 bp were ligated to the
luciferase gene in a pGL-3 basic vector. The fragments
748 to
828
and
786 to
803 were ligated to the SV40 promoter and luciferase
gene in a pGL-3-promoter vector. The constructs were transfected into
human keratinocytes as described under "Materials and Methods."
B, the luciferase activities of the nine 5
deletional
constructs were measured following 24 h of exposure to
1,25-(OH)2D3 or vehicle, divided by
-galactosidase activity, and expressed as the percentage of activity
of the +135 to
877 construct in the absence of
1,25-(OH)2D3. The activity obtained with the
pGL-3-basic vector showed the vector background. C, a similar
experiment was performed with constructs containing
748 to
828,
786 to
803, and a mutant construct in which two random sequences
replaced AGGTCA and TGGACA. The results are normalized to
-galactosidase activity. A construct containing the vitamin D-responsive region at
143 to
293 in the human 24-hydroxylase gene
(24-hydroxylase) was used as a positive control. The
activity obtained from the pGL-3 promoter vector showed the vector
background.
To determine if the human PLC-1 gene transcriptionally responds to
1,25-(OH)2D3, the nine deletional constructs
were transfected into human keratinocytes in the presence or the
absence of 1,25-(OH)2D3. The results showed
that the construct containing fragment +135 to
877 was responsive to
1,25-(OH)2D3 stimulation. The luciferase activity was increased over 3-fold after 24 h of exposure to
1,25-(OH)2D3. 5
deletion to
748 bp totally
abolished the responsiveness to 1,25-(OH)2D3
(Fig. 3B), indicating that the responsive region was located
between
748 and
828 bp. To confirm that the
748 to
828 bp
region contains a VDRE, this fragment was subcloned into the
pGL-3-promoter vector. Transfection experiments showed that the
promoter activity was induced over 2-fold in human keratinocytes by
1,25-(OH)2D3 (Fig. 3C). The region
748 to
828 contains an SP1 site and two direct repeats separated by
6 bases (DR6), AGGTCAgaccacTGGACA (named PDR6) (Fig. 1). The PDR6 was
located in the region
786 to
803. The construct containing only the
PDR6 showed the same fold induction by
1,25-(OH)2D3 as did the construct containing the region
748 to
828 (Fig. 3C). A mutant construct
containing the sequence TAGGTAgaccacATGCAT (named MPDR6) gave no
response to 1,25-(OH)2D3. The vitamin
D-responsive region at
143 to
293 in the human 24-hydroxylase gene
(29) subcloned into the pGL-3-promoter vector was used as a positive
control in the transfection experiments. The 24-hydroxylase VDRE
construct showed nearly 3-fold induction by
1,25-(OH)2D3. These results indicate that the
human PLC-
1 gene contains a DR6-type sequence in the region
786 to
803, which is of comparable responsiveness to 1,25-(OH)3
D3 as the VDRE in the 24-hydroxylase gene.
DR6-type VDREs in other genes have been reported to bind the VDR as a
homodimer or heterodimer (30, 31). To determine if the responsive
region in the human PLC-1 gene binds to the VDR in human
keratinocytes, an 80-bp synthetic oligonucleotide (named W1)
representing the vitamin D-responsive region
748 to
828 bp was
evaluated using the DNA mobility shift assay. Incubation of the
oligonucleotide W1 with the nuclear extracts from the human keratinocytes yielded two specific DNA-protein binding complexes (Fig.
4A). The specificity of the binding was
verified by competition with the same or mutant unlabeled
oligonucleotides at 100 molar excess. The results showed that the two
binding complexes were reduced by W1 but not by a mutant fragment
(named M1) containing MPDR6 instead of PDR6 (Fig. 4A). These
data suggest that the two bands are specific complexes of the sequence
PDR6 with the nuclear factors in the human keratinocytes. The bands are
not SP1 complexes because binding was not blocked by an SP1 consensus
oligonucleotide even though there is a putative SP1 site in this region
(Fig. 4A). However, the upper band was competed out by a
21-bp unlabeled oligonucleotide (named H) containing a DR3-type VDRE
(AGGTGAgcgAGGGCG) found in the human 24-hydroxylase gene, suggesting
that the upper band was a VDR-VDRE complex (Fig. 4A). To
narrow down the vitamin D binding region, we repeated the experiment
but used a 38-bp oligonucleotide (named W2) containing the sequence
PDR6 with 10 flanking bases on each side. The results showed that a
single main complex formed after the incubation of the fragment W2 with the nuclear extracts from the human keratinocytes (Fig. 4B).
The binding complex was reduced by the unlabeled oligonucleotides W2
and H but not by a mutant fragment (named M2) containing MPDR6 instead
of PDR6. The binding complex was shifted to a higher molecular weight
form upon the addition of an antibody specific to the VDR. Incubation
of the labeled fragment W2 with the recombinant vitamin D receptor
yielded two shifted binds that were blocked by an unlabeled oligonucleotide W2 (Fig. 4B). The results indicate that the
sequence PDR6 in the region
786 to
803 binds to the VDR in human
keratinocytes.
We have cloned the 5-flanking region of the human PLC-
1 gene
that confers promoter activity when transiently transfected into human
keratinocytes. The sequence in the human PLC-
1 flanking region is
GC-rich and has no TATA box, similar to many genes that are important
in the control of cell proliferation and differentiation such as
transforming growth factor-
1 (32), epidermal growth factor receptor
(33), and nerve growth factor (34). The region between
200 and
39
bp contains several putative SP1 and AP2 binding sites and appears to
contain the promoter because the deletion from
200 to
39 bp
dramatically reduced promoter activity (Fig. 3B). The
transcription initiation of the human PLC-
1 gene could be similar to
other GC-rich genes in which SP1 binding to the GC-boxes, rather than a
TFIID-TATA complex, is able to activate gene transcription (35).
The pentanucleotide CCAAT, which is usually found within 50 to
100
bp upstream from the transcriptional start site in mammalian genes
where it appears to have a role in mediating promoter function (36),
was located between
581 bp and
585 bp in the human PLC-
1 gene.
However, this putative CCAAT box has no clear function because the
deletion from
613 to
551 bp did not remarkably reduce basal promoter activity. Therefore, basal transcription of the human PLC-
1
gene does not appear to require a CCAAT-binding protein.
A DR-6 type VDRE, AGGTCAgaccacTGGACA, has been precisely localized
within the 5-flanking region of the human PLC-
1 gene. The
transfection experiments showed that the DR6 sequence was completely
silent in the human PLC-
1 gene in the absence of
1,25-(OH)2D3 but was activated by the addition
of 1,25-(OH)2D3. In the DNA mobility shift
assays, the DR6 specifically bound to the vitamin D receptor in the
human keratinocytes, as recognized by the vitamin D receptor antibody.
Substitution mutation of the 6-base repeats in the DR6 sequence totally
abolished the response to vitamin D as well as the DNA binding ability,
indicating that 1,25-(OH)2D3 activates the
human PLC-
1 gene through its vitamin D receptor interacting with the
DR6-type VDRE localized in the 5
-flanking region. The sequences of the
two repeats share some homology with the known DR6-type VDREs in the
human osteocalcin gene (30) and rat 24-hydroxylase gene (31) (Fig.
5). Alignment of these DR6-type VDREs showed that
one-third of the nucleotides are identical between each repeat. It
seems that the second and the sixth nucleotides within each repeat are
always G and A, respectively, suggesting that these bases are critical
for DNA-receptor binding and confer transactivation upon vitamin D
stimulation. Single base mutations will be required to define the
precise nucleotides that are essential to mediate the responsiveness to
1,25-(OH)2D3.
Vitamin D receptor and nonreceptor transcriptional factors binding to
distinct sites in a promoter or enhancer region is one mechanism by
which the profound alteration in gene expression can occur from small
changes in the concentration of trans-acting factors (37). A typical
vitamin D receptor and nonreceptor transcriptional factor interaction
model was reported in the human osteocalcin VDRE, which contains an AP1
site; AP1 binding proteins were shown to regulate VDRE function
(38-40). Although no AP1 site was found in the human PLC-1 gene,
SP1 has also been reported to interact with the vitamin D receptor by
independently binding to a different motif (37). We found 16 putative
SP1 sites clustered downstream of the DR6 sequence in the 5
-flanking
region of the human PLC-
1 gene. However, the isolated human PLC-
1
VDRE ligated to a heterologous SV40 promoter did not appear to differ
in the degree of response to 1,25-(OH)2D3 as
the VDRE within its own gene context. The data suggest that the SP1
sites are not involved in the vitamin D-induced human PLC-
1
transcription.
DR6-type VDREs of the human osteocalcin gene (41) and the rat
24-hydroxylase gene have been shown to bind VDR-RAR heterodimers, as
well as VDR-VDR homodimers (31). In this report, we found that
recombinant VDR was able to bind to the human PLC-1 VDRE, as shown
by DNA mobility shift assay, implying that VDR might be binding to the
human PLC-
1 VDRE as a homodimer or monomer. However, the
keratinocyte nuclear extracts give a different pattern of binding to
the DR6 than the recombinant VDR, suggesting that other factors are
also involved in VDR-VDRE binding. Further experiments are needed to
identify these additional factors.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U80983[GenBank].