Cloning of the Human Phospholipase C-gamma 1 Promoter and Identification of a DR6-type Vitamin D-responsive Element*

(Received for publication, October 4, 1996, and in revised form, December 9, 1996)

Zhongjian Xie and Daniel D. Bikle Dagger

From the Endocrine Unit, Veterans Affairs Medical Center, University of California, San Francisco, California 94121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The 5'-flanking region of the human phospholipase C-gamma 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-gamma 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-gamma 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-gamma 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.


INTRODUCTION

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-beta , PLC-gamma , and PLC-delta ), and each type contains several subtypes (3, 4). PLC-gamma 1, unlike the other PLC isoenzymes, contains a src homology 2 domain through which PLC-gamma 1 interacts with various tyrosine kinase growth factor receptors (5-8). PLC-gamma 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-gamma 1 protein is higher in neoplastic keratinocyte cell lines than in normal keratinocytes (13). Calcium-induced differentiating keratinocytes express over 2-fold more PLC-gamma 1 protein than undifferentiated keratinocytes (14). These observations suggest that PLC-gamma 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-gamma 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-gamma 1 (24). To understand the molecular mechanism of this regulation, we cloned the 5'-flanking region of the human PLC-gamma 1 gene that confers promoter activity and identified within the 5'-flanking region a DR6-type vitamin D-responsive element (VDRE).


MATERIALS AND METHODS

Isolation of Genomic Clone

The subclones containing phospholipase C-gamma 1 genomic DNA were obtained from a human P1 genomic DNA library using as probe a 5-kb PLC-gamma 1 cDNA (Genome Systems). To isolate the 5'-flanking region of the PLC-gamma 1 gene, the subclones were further screened by colony hybridization using the oligonucleotide (5'-CGTTGCGCTTGCTCCCGGGC-3') from the 5'-untranslated region of PLC-gamma 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.

Construction of Plasmids

The XhoI-StylI fragment was subcloned into a pGL-3-basic vector (Promega). The PLC-gamma 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.

Cell Culture

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 Assay

PLC-gamma 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 pRSVbeta -gal (27), a beta -galactosidase expression vector that contains a beta -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 beta -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.

S1 Nuclear Protection Assay

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-gamma 1 5'-flanking region cloned into the pGL-3-basic vector, using Klenow and [alpha -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.

Primer Extension Analysis

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-gamma 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.

DNA Mobility Shift Assay

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).


RESULTS

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-gamma 1 gene were isolated from the random subclones by screening 96 subcultures using an oligonucleotide from the untranslated region of the human PLC-gamma 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-gamma 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-gamma 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).


Fig. 1. Restriction map and sequence of 5'-flanking region of the human PLC-gamma 1 gene. A, restriction map of the genomic clones showing the restriction sites in the 9-kb HindIII fragment. H, HindIII; Xb, XbaI; Xh, XhoI; S, StyII. B, nucleotide sequence of the 1011-bp XhoI-StylI fragment with part of the coding region. The amino acid sequence is printed under the nucleotide sequence of the translated portion using the single-letter code. The transcription start site is designated as +1. The potential SP1 and AP2 sites, and CCAAT box are underlined and labeled respectively by SP1, AP1, and CCAAT. Two direct repeats are presented in bold type.
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Both S1 nuclease protection assay and primer extension analysis were performed to determine the transcriptional start site for the human PLC-gamma 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.


Fig. 2. Determination of the transcriptional start site. A, in a nuclease protection assay, an antisense probe spanning 212 bp upstream from the translation start codon in the human PLC-gamma 1 genomic DNA was hybridized with human keratinocyte poly(A) RNA and then digested by S1 nuclease. The size of the protected fragment was 135 bp. Lanes G, A, T, and C are sequence reactions of the antisense probe used as a size marker. B, a primer extension experiment was carried out using as primer a 5'-labeled oligonucleotide corresponding to the antisense strand of the region 40-60 bp downstream from the translation start codon in the human PLC-gamma 1 gene and human keratinocyte poly(A) RNA as template. The size of the extension fragment was 195 bp, confirming the transcription start site as 135 bp upstream of the translation start codon. M, size marker.
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In an attempt to delineate the sequences essential for human PLC-gamma 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-gamma 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-gamma 1 gene are essential for transcriptional initiation.


Fig. 3.

The induction of human PLC-gamma 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 beta -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 beta -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.


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To determine if the human PLC-gamma 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-gamma 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-gamma 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.


Fig. 4. DNA mobility shift assay with fragments of the 5'-flanking region of the human PLC-gamma 1 gene. A, binding of vitamin D-responsive region -748 to -828 with the vitamin D receptor. B, binding of the fragment -786 to -803 containing a DR-6 type VDRE with the vitamin D receptor. The arrows indicate VDR-DNA and antibody-VDR-DNA complexes. W1, wild type fragment -748 to -828; M1, mutant fragment -748 to -828; SP1, SP1 consensus oligonucleotide; H, VDRE at region -152 to -172 in the human 24-hydroxylase gene; W2, wild type fragment -786 to -803; M2, mutant fragment -786 to -803.
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DISCUSSION

We have cloned the 5'-flanking region of the human PLC-gamma 1 gene that confers promoter activity when transiently transfected into human keratinocytes. The sequence in the human PLC-gamma 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-beta 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-gamma 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-gamma 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-gamma 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-gamma 1 gene. The transfection experiments showed that the DR6 sequence was completely silent in the human PLC-gamma 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-gamma 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.


Fig. 5. Homologies of the DR6-type VDREs from different genes. Each direct repeat is presented in capital letters. The identical nucleotides between each repeat are presented in bold letters.
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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-gamma 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-gamma 1 gene. However, the isolated human PLC-gamma 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-gamma 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-gamma 1 VDRE, as shown by DNA mobility shift assay, implying that VDR might be binding to the human PLC-gamma 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.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U80983[GenBank].


Dagger    To whom correspondence should be addressed: Endocrine Unit, VA Medical Center, 4150 Clement St. (111N), San Francisco, CA 94121. Tel.: 415-750-2089; Fax: 415-750-6929.
1   The abbreviations used are: PLC, phospholipase C; VDR, vitamin D receptor; VDRE, vitamin D-responsive element; DR, direct repeat; kb, kilobase(s); bp, base pair(s).

REFERENCES

  1. Berridge, M. J., and Irvine, R. F. (1984) Nature 312, 315-321 [Medline] [Order article via Infotrieve]
  2. Majerus, P. W., Connolly, T. M., Deckmyn, H., Ross, T. S., Bross, T. E., Ishii, H., Bansal, V. S., and Wilson, D. B. (1986) Science 234, 1519-1526 [Medline] [Order article via Infotrieve]
  3. Rhee, S. G., and Choi, K. D. (1992) Adv. Second Messenger Phosphoprotein Res. 26, 35-61 [Medline] [Order article via Infotrieve]
  4. Rhee, S. G., Suh, P.-G., Ryu, S.-H., and Lee, S. Y. (1989) Science 244, 546-550 [Medline] [Order article via Infotrieve]
  5. Stahl, M. L., Ferenz, C. R., Kelleher, K. L., Kriz, R. W., and Knopf, J. L. (1988) Nature 332, 269-272 [CrossRef][Medline] [Order article via Infotrieve]
  6. Suh, P. G., Ryu, S. H., Moon, K. H., Suh, H. W., and Rhee, S. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5419-5423 [Abstract]
  7. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302 [Medline] [Order article via Infotrieve]
  8. Goldschmidt-Clermont, P. J., Kim, J. W., Machesky, L. M., Rhee, S. G., and Pollard, T. D. (1991) Science 251, 1231-1233 [Medline] [Order article via Infotrieve]
  9. Arteaga, C. L., Johnson, M. D., Todderud, G., Coffey, R. J., Carpenter, G., and Page, D. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10435-10439 [Abstract]
  10. Park, J.-G., Lee, Y. H., Kim, S. S., Park, K. J., Noh, D.-Y., Ryu, S. H., and Suh, P.-G. (1994) Cancer Res 54, 2240-2244 [Abstract]
  11. Noh, D.-Y., Lee, Y. H., Kim, S. S., Kim, Y. I., Ryu, S.-H., Suh, P.-G., and Park, J. G. (1994) Cancer 73, 36-41 [Medline] [Order article via Infotrieve]
  12. Nanney, L. B., Gates, R. E., Todderud, G., King, L. E., Jr., and Carpenter, G. (1992) Cell Growth & Differ. 3, 233-239 [Abstract]
  13. Punnonen, K., Denning, M. F., Rhee, S. G., and Yuspa, S. H. (1994) Mol. Carcinogen. 10, 216-225 [Medline] [Order article via Infotrieve]
  14. Punnonen, K., Denning, M., Lee, E., Li, L., Rhee, S. G., and Yuspa, S. H. (1993) J. Invest. Dermatol. 101, 719-726 [Abstract]
  15. Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K., and Yuspa, S. H. (1980) Cell 19, 245-254 [Medline] [Order article via Infotrieve]
  16. Boyce, S. T., and Ham, R. G. (1983) J. Invest. Dermatol. 81, Suppl. 1, 33S-40S
  17. Pillai, S., Bikle, D. D., and Elias, P. M. (1988) J. Biol. Chem. 263, 5390-5395 [Abstract/Free Full Text]
  18. Hosomi, J., Hosoi, J., Abe, E., Suda, T., and Kuroki, T. (1983) Endocrinology 113, 1950-1957 [Abstract]
  19. Smith, E. L., Walworth, N. C., and Holick, M. F. (1986) J. Invest. Dermatol. 86, 709-714 [Abstract]
  20. Pillai, S., and Bikle, D. D. (1991) J. Cell. Physiol. 146, 94-100 [Medline] [Order article via Infotrieve]
  21. Deleted in proofDeleted in proof
  22. Deleted in proofDeleted in proof
  23. Deleted in proofDeleted in proof
  24. Pillai, S., Bikle, D. D., Su, M.-J., Ratnam, A., and Abe, J. (1995) J. Clin. Invest. 96, 602-609 [Medline] [Order article via Infotrieve]
  25. Pillai, S., Bikle, D. D., Hincenbergs, M., and Elias, P. M. (1988) J. Cell. Physiol. 134, 229-237 [Medline] [Order article via Infotrieve]
  26. Jiang, C.-K., Connolly, D., and Blumenberg, M. (1991) J. Invest. Dermatol. 97, 969-973 [Abstract]
  27. Carroll, J. M., and Taichman, L. B. (1992) J. Cell Sci. 103, 925-930 [Abstract/Free Full Text]
  28. Abmayr, S. M., and Workman, J. L. (1994) Current Protocol in Molecular Biology, John Wiley & Sons, Inc., New York
  29. Chen, K.-S., and DeLuca, H. F. (1995) Biochem. Biophys. Acta 1263, 1-9 [Medline] [Order article via Infotrieve]
  30. Carlberg, C., Bendik, I., Wyss, A., Meier, E., Sturzenbecker, L. J., Grippo, J. F., and Hunziker, W. (1993) Nature 361, 657-660 [CrossRef][Medline] [Order article via Infotrieve]
  31. Kahlen, J.-P., and Carlberg, C. (1994) Biochem. Biophys. Res. Commun. 202, 1366-1372 [CrossRef][Medline] [Order article via Infotrieve]
  32. Kim, S.-J., Glick, A., Sporn, M. B., and Roberts, A. B. (1989) J. Biol. Chem. 264, 402-408 [Abstract/Free Full Text]
  33. Ishii, S., Xu, Y. H., Stratton, R. H., Roe, B. A., Merlino, G. T., and Pastan, I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4920-4924 [Abstract]
  34. Sehgal, A., Patil, N., and Chao, M. (1988) Mol. Cell. Biol. 8, 3160-3167 [Medline] [Order article via Infotrieve]
  35. Mitchell, P. J., and Tjian, R. (1989) Science 245, 371-378 [Medline] [Order article via Infotrieve]
  36. Thangue, N. B. L., and Rigby, P. W. J. (1988) in Transcription and Splicing (Hames, B. D., and Glover, D. M., eds), pp. 1-44, IRL Press, Oxford
  37. Liu, M., and Freedman, L. P. (1994) Mol. Endocrinol. 8, 1593-1604 [Abstract]
  38. Kerner, S. A., Scott, R. A., and Pike, J. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4455-4459 [Abstract]
  39. Ozono, K., Liao, J., Kerner, S. A., Scott, R. A., and Pike, J. W. (1990) J. Biol. Chem. 265, 21881-21888 [Abstract/Free Full Text]
  40. Schüle, R., Umesono, K., Mangelsdorf, D. J., Bolado, J., Pike, J. W., and Evans, R. M. (1990) Cell 61, 497-504 [Medline] [Order article via Infotrieve]
  41. Schräder, M., Bendik, I., Becker-André, M., and Carlberg, C. (1993) J. Biol. Chem. 268, 17830-17836 [Abstract/Free Full Text]

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