©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overexpression of Phospholipase C-1 in Colorectal Carcinomas Is Associated with Overexpression of Factors That Bind Its Promoter (*)

Seung-Jae Lee , Sang Do Lee , Jae-Gahb Park (1), Chang-Min Kim (2), Sung Ho Ryu , Pann-Ghill Suh (§)

From the (1)Department of Life Science and Basic Science Research Center, Pohang University of Science and Technology, Pohang 790-784, Korea, the Laboratory of Cell Biology, Cancer Research Institute, Seoul National University, Seoul 110-799, Korea, and the (2)Department of Internal Medicine, Korea Cancer Center Hospital, Seoul 139-240, Korea

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 5`-upstream sequence of the phospholipase C-1 (PLC-1) gene contains several transcriptional regulatory regions. We have studied one of the regions (-551 to -480, named GPE1) which exhibits a strong positive regulatory activity. GPE1 stimulated the transcription when fused to heterologous TATA element in an orientation-dependent manner. The region between -536 and -470 was identified as the protein binding site in GPE1 by the DNase I footprinting method. Electrophoretic mobility shift assays with several competitors revealed three protein binding sites in this region, designated as GES1, GES2, and GES3. The binding sites were -535 GGAGGGGGCG -524, -512 TGTCACTCA -504, and -491 CAATCCA -485, respectively. Mutational analyses suggested that GPE1 binding proteins cooperate with each other to activate the transcription of the PLC-1 gene. Additionally, immunoblot analyses revealed that the level of PLC-1 expression was considerably higher in 9 of 11 colorectal carcinomas than in adjacent normal colorectal tissues. In 7 of 9 cases of colorectal carcinomas which express higher level of PLC-1, the DNA binding activities to GES1, GES2, and GES3 sites also increased when compared with normal tissues. These results suggest that the GPE1 binding proteins might be attributed to the elevated expression of PLC-1 in colorectal carcinomas and may play important roles in proliferation of colorectal carcinoma cells.


INTRODUCTION

Many kinds of extracellular stimuli trigger the target cell responses through activation of phospholipase C (PLC)()which hydrolyzes phosphatidylinositol 4,5-bisphosphate into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol(1, 2) . At least nine distinct isozymes were cloned from mammals. Comparison of the deduced amino acid sequences has indicated that PLCs are divided into three types (PLC-, PLC-, and PLC-) and each type contains more than one subtype(1, 3) . Although there are two regions with significant similarity in amino acid sequence, designated as X and Y, the different PLC types have characteristic features in their primary structures(1) . Especially, src homology domains, SH2 and SH3, and split pleckstrin homology domains reside between X and Y regions of the PLC- type(4, 5, 6) . These domains have been found in a growing number of proteins that are involved in the regulation of cell proliferation and differentiation(6, 7) .

As expected from their sequence differences, the various PLC isozymes appear to be activated by distinct groups of receptors through different mechanisms(3) . PLC- is known to be activated by Gq class (8, 9) as well as subunit of G-protein(10, 11) . PLC-, on the other hand, is activated through a direct interaction with growth factor receptor tyrosine kinases, such as epidermal growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor(3) .

PLC-1 was also suggested to be involved in the process of cellular transformation: (a) microinjection of PLC-1 into NIH 3T3 cells caused a dose-dependent transformation, but injection of anti-PLC-1 antibody blocked the serum- or ras-induced transformation(12, 13) ; (b) PLC-1 has been found to be overexpressed in human breast carcinomas(14) , human colorectal cancer (15), familial adenomatous polyposis(16) , and human skins in hyperproliferative conditions(17) . In addition, immunohistochemical studies suggested that expression of PLC-1 was regulated during development and differentiation(18) . The expression of PLC-1 changed during the differentiation of F9(19) , C2C12(20) , and U937 cells(21) . Although the expression of PLC-1 was shown to be linked to various physiologic events such as cell growth and differentiation, little is known about the mechanisms controlling PLC-1 expression.

The transcriptional rate of each gene is largely controlled by the interactions of various regulatory factors to the transcriptional control regions or by the modification of regulatory factors(22) . Therefore, identification and characterization of these cis- and trans-acting factors are needed to understand how transcription of a gene is regulated. We have previously reported the cloning of the promoter of PLC-1 gene(23) . Deletion analysis identified several regions which affect transcriptional activity of the promoter: positive regulatory regions from -551 to -480 (GPE1) and from -90 to -52 (GPE2), a negative regulatory region from -371 to -305 (GNE1) relative to the transcriptional initiation site.

In this study, the GPE1 region, which might be important for the transcriptional regulation of the PLC-1 gene during the physiologic events such as proliferation, has been further characterized. DNase I footprinting and electrophoretic mobility shift assay revealed three protein binding sites (GES1, GES2, and GES3) in this region. Mutational analyses of the protein binding sites revealed that GES1, GES2, and GES3 binding proteins cooperate each other to activate the transcription through the GPE1 region. GES1, GES2, and GES3 binding proteins seemed to be overexpressed in colorectal carcinomas, suggesting that these proteins might be attributed to the elevated level of PLC-1 in cancer tissues.


MATERIALS AND METHODS

Cell Culture Conditions

C2C12 cells (mouse skeletal myoblast line) were maintained at low density to prevent fusion in Dulbecco's modified Eagle's medium (Life Technologies Inc., Gaithersburg, MD) supplemented with 15% fetal bovine serum (HyClone Laboratories Inc., Logan, UT) and 1 mM sodium pyruvate (Life Technologies Inc.). Cells were grown in a humidified incubator at 37 °C in 5% CO.

Construction of CAT Plasmids

The XhoI-ApaI fragment which contains the GPE1 region was isolated from pPLC551CAT plasmid (23) and blunt-ended with T4 DNA polymerase (New England Biolabs Inc., Beverly, MA). It was then inserted into the upstream E1b-TATA box of E1bCAT (24) in both orientations and named pGPE1F and pGPE1R. The construction of pPLC551CAT and pPLC497CAT was described by Lee et al. (23). Oligonucleotides, F1, F2, F2M, were inserted into the upstream E1b-TATA box of E1bCAT. F1 was inserted into the XhoI site. F2 and F2M were inserted into the HindIII site. The resulting plasmids were named pF1, pF2, pF2M, respectively. F1 oligonucleotide was inserted into the XhoI site of pF2 to construct pF1F2.

DNA Transfection and Chloramphenicol Acetyltransferase (CAT) Assay

Reporter CAT plasmids were transfected into C2C12 cells (approximately 30-50% confluent) by the calcium phosphate coprecipitation method(25) . Ten µg of plasmid DNA was cotransfected with 3 µg of pCH110 (Pharmacia LKB Biotechnology Inc., Uppsala, Sweden), a -galactosidase expression vector which was used as an internal control to normalize transfection efficiency. After exposure to the DNA precipitate for 12-16 h, cells were washed and fresh media was then added. The cells were harvested 48 h later, and CAT activities were measured in the cell lysates(26) . One-tenth of cell lysate was used to determine -galactosidase activity. For each CAT reaction, cell extract was combined with 4 µl of [C]chloramphenicol (Amersham Corp., Buckinghamshire, United Kingdom, 55 mCi/mmol, 25 µCi/ml), 10 µl of 20 mg/ml acetyl-coenzyme A, and 250 mM Tris, pH 7.8, to a final volume of 150 µl. The mixtures were incubated at 37 °C for 2 h, and the reactions were stopped by extraction with 1 ml of ethyl acetate. Acetylated and nonacetylated chloramphenicol were separated by thin-layer chromatography.

Preparation of Nuclear Extract

Nuclear proteins were extracted according to the procedure described by Dignam et al.(27) . Cells were harvested in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl, 10 mM KCl, and 0.5 mM DTT) and lysed using Dounce homogenizer. The homogenate was centrifuged to collect nuclei, and the pellet was resuspended in buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT), incubated at 4 °C for 30 min with gentle stirring, and centrifuged for 30 min at 15,000 rpm in a Sorvall SS-34 rotor. The supernatant was dialyzed several hours against 200 volumes of buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT), frozen in liquid nitrogen, and stored at -70 °C.

Electrophoretic Mobility Shift Assay (EMSA)

From the sequence around the footprinted region, double-stranded DNA fragments were prepared by annealing of complement oligonucleotides. The sequences of sense strands of the double stranded oligonucleotides used in this study are: F1: 5`-CTGTGGGGAGGGGGCGTGGCGGCGTGCTGTCACTCACT-GCCC (-539/-498); F1M: 5`-CTGTGGTTCATATCACTGGCGGCGTGCTGTCACTCACTGCCC; F1a: 5`-CTGTGGGGAGGGGGCGTGGC (-539/-520); F1b: 5`-TGGCGGCGTGCTGTCACTCACTGCCC (-523/-498); M1: 5`-TTTATGCGTGCTGTCACTCACTGCCC; M2: 5`-TGGCGGATGTCTGTCACTCACTGCCC; M3: 5`-TGGCGGCGTGCGTGAACTCACTGCCC; M4: 5`-TGGCGGCGTGCTGTCAAGACCTGCCC; M5: 5`-TGGCGGCGTGCTGTCACTCACGTAAC; F2: 5`-TGCCCGCTAGCCAA-TCCACGGGTGCGCCCCTCCCCGGAGAGGG (-502/-460); F2M: 5`-TG-CCCGCTAGCGTCCGTTCGGGTGCGCCCCTCCCCGGAGAGGG; GES1: 5`-TGGGGAGGGGGCGTGG (-536/-521); GES2: 5`-TGCTGTCACTC-ACT (-515/-502); GES3: 5`-TAGCCAATCCACG (-495/-483); Sp1: 5`-ATTCGATCGGGGCGGGGCGAGC; AP2: 5`-GATCGAACTGACCGCCCGCGGCCCGT; AP1: 5`-CGCTTGATGAGTCAGCCGGAA; NF1/CTF: 5`-CCTTTGGCATGCTGCCAATATG. Mutated sequences are underlined and the locations of the sequences in the PLC-1 gene are indicated at the end of each sequence. Oligonucleotides were labeled with [-P]dCTP by filling in reaction using Klenow fragment. F1a and F1b were 5`-end labeled by T4 polynucleotide kinase. The binding reactions for gel shift assay were preincubated for 5 min on ice in a total volume of 13 µl containing 12% glycerol, 12 mM HEPES, pH 7.9, 4 mM Tris, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 1 µg of poly(dI-dC) poly(dI-dc) as nonspecific competitor, and 1-2 µg of nuclear extract with or without specific competitor indicated. After addition of P-labeled probe (2 10 cpm), the mixtures were incubated for an additional 30 min at 15 °C, then loaded onto 5% polyacrylamide nondenaturing gels and subjected to electrophoresis for 2 h at 150 V in 0.25 Tris borate-EDTA buffer(28) .

DNase I Footprinting

A 510-base pair DNA fragment corresponding to the positions from -644 to -135 in the PLC-1 gene was 3`-end labeled with [-P]dCTP using Klenow fragment on the antisense strand. In the binding reaction, 20-50 µg of C2C12 nuclear extract and 5 10 cpm of P-labeled probe were used in the same condition as EMSA in a total volume of 100 µl. The reactions were chilled on ice and treated with 1-2 units of DNase I (Ambion Inc., Austin, TX) in 100 µl of 2 assay buffer containing 80 mM Tris, pH 7.4, 12 mM MgCl, 4 mM CaCl, and 100 mM KCl for 2 min on ice. DNase I reactions were terminated by adding 50 µl of stop solution (1 mg/ml proteinase K, 0.5% SDS, 125 mM EDTA, and 0.25 mg/ml tRNA) and incubated at 55 °C for 1 h, and then extracted with phenol/chloroform (1:1, v/v). Resulting DNA fragments were analyzed on 6% denaturing polyacrylamide gels with M13 sequencing ladder as size marker. For the footprinting with Sp1 protein, we used vaccinia-expressed and purified human Sp1 protein (Promega Corp., Madison, WI).

Mutagenesis at Protein Binding Sites

To prepare the templates in a phagemid form, the XhoI/HpaI fragment of pPLC551CAT which contains the -551/+75 region of the PLC-1 gene and CAT gene were inserted into pBluescriptII KS (Stratagene, La Jolla, CA) digested by XhoI and EcoRV. The plasmid was named pKS551 and used for preparing single stranded template. pKS479 was constructed with the fragment from pPLC479CAT by the same strategy. To generate the mutations at protein binding sites in GPE1, the oligonucleotides, F1M, M3, and F2M, were used as mutagenic oligonucleotides for mutations at GES1, GES2, and GES3 sites, respectively. Uracil-containing DNA templates were prepared by the method of Kunkel(29) . Mutagenic oligonucleotides were phosphorylated at 5`-ends by T4 polynucleotide kinase (New England Biolabs Inc.) and annealed with templates. In vitro primer extension was performed using 1 unit of T4 DNA polymerase (New England Biolabs Inc.) and 3 units of T4 DNA ligase (Amersham Corp.) for 5 min on ice, for 5 min at room temperature, and 90 min at 37 °C. After the incubation, Escherichia coli strain DH5 was transformed by 1/20 of the reaction mixture. Double and triple mutations were generated using single and double mutated templates, respectively. Mutations were confirmed by DNA sequence analysis.

Preparation of Whole Cell Extract from Tissue Specimens and Immunoblot Analysis

Surgical total colorectomy specimens were obtained from the Department of Surgery at the Seoul National University Hospital and the Korea Cancer Center Hospital. Fresh specimens of normal and neoplastic tissues were immediately frozen in liquid nitrogen and stored at -80 °C for further study. For preparation of whole cell extract, tissue specimens were minced with a razor blade and resuspended in extraction buffer (20 mM HEPES, pH 7.9, 0.35 M NaCl, 20% glycerol, 1 mM MgCl, 1% Nonidet P-40, 1 mM DTT, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml aprotinin) and incubated on ice. After centrifugation, supernatant was frozen in liquid nitrogen and stored at -80 °C. For immunoblot analysis, the whole cell extract was separated by 8% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred onto nitrocellulose membranes and were probed with the anti-PLC-1 monoclonal antibody (B-16-5, 1 µg/ml) (30) for 4 h. Immunoreactive bands were visualized by ECL system (Amersham Corp.) using peroxidase-conjugated goat anti-mouse IgG + IgA + IgM antibody.


RESULTS

GPE1 Activates Heterologous Promoter in an Orientation-dependent Manner

We have previously reported the 5`-region of the rat PLC-1 gene and demonstrated that it contains promoter activity. The promoter regions important for gene expression were found to be located at -551 to -480, -371 to -305, and -90 to -52 base pairs from the transcriptional initiation site, designated as GPE1, GNE1, and GPE2, respectively(23) . The ability of GPE1 to activate a basic heterologous promoter was investigated by inserting it upstream of the E1b-TATA box which is a basic promoter of the adenovirus E1b gene. When the activity was measured in C2C12 cells, GPE1 was observed to stimulate the transcription directed by the E1b-TATA box (Fig. 1). This activity of GPE1 is orientation-dependent, since the activation disappeared when it was reversely oriented.


Figure 1: Effect of GPE1 on heterologous promoter. GPE1 region was isolated from the PLC-1 gene and inserted into the upstream E1b-TATA box in right (pGPE1F) and reverse (pGPE1R) orientations. Transient transfection assays were performed in C2C12 cells. For each transfection, 20 µg of plasmids were used and after 48 h of expression, the CAT activities were measured. The graph represents fold increase relative to the activity of E1bCAT. Similar results were obtained from several independent experiments.



Several Proteins Bind to GPE1 Region

Nuclear extracts from C2C12 cells conferred a pattern of DNase I-protection encompassing the region from -536 to -470 base pairs (Fig. 2A) which roughly matches with GPE1 region. Two double-stranded oligonucleotides, F1 and F2 which correspond to the sequences, [-539 to -498 and -502 to -460, respectively, were used for the competition assays. F1 and F2 competed for binding of nuclear proteins to the corresponding regions (Fig. 2B).


Figure 2: Identification of GPE1 binding protein. DNA fragment corresponding to the region -644/-135 of PLC-1 gene was labeled at lower strand and used as a probe for DNase I footprinting analysis. A, the probe was incubated with 20 or 50 µg of C2C12 nuclear extract, and then 1 or 2 units of DNase I was treated for 2 min on ice. The resulting fragments were analyzed on 6% denaturing acrylamide gel. Lanes A and C represent M13 sequencing ladder. The sequence of protected region and its position are shown on the left. B, the cold oligonucleotides (100-fold molar excess), F1 and F2, were added to the binding reactions as competitors. The sequences of the oligonucleotides are shown in Fig. 3.



To analyze the interactions of GPE1 with nuclear factors in more detail, several oligonucleotides were designed and used in electrophoretic mobility shift assay (Fig. 3). F1 covers the 5`-part of the DNase I-protected region and F1M contains a mutation around the GC-rich sequence which is related to AP-2 and Sp1-binding sites(23) . F2 covers the 3`-part of the protected region and F2M contains a mutation in the CCAAT box-like sequence. In the presence of C2C12 nuclear extracts, F1 forms four different DNA-protein complexes, A, B, C, and D from the most slowly-migrating complex as shown in Fig. 3A. Addition of excess unlabeled F1 competitor abolished formation of the complexes with labeled probe. To determine which regions of F1 are responsible for the formation of the protein-DNA complexes, F1M, F1a, and F1b were used as competitors. F1a and F1b correspond to the 5`- and the 3`-part of F1, respectively. In the presence of F1M, only complex B was formed with labeled F1 probe, which suggests that the sequence, -533 GGAGGGGGCG -524, is responsible for the formation of complex B. Consistently, unlabeled F1a which contains the sequence, -533 GGAGGGGGCG -524, but not the 3`-part of F1 abolished the complex B. On the other hand, F1b containing the 3`-part of F1 but not the 5`-part, sequestered the factors forming complexes C and D with the F1 probe. To further localize the site responsible for formation of complexes C and D, P-labeled F1b probe and M1-M5 competitors were used in competition assay. Whereas F1b, M1, M2, and M5 worked as effective competitors for the F1b probe, M3 and M4 (especially M3) did not (Fig. 3B). These results suggest that the factor(s) forming the complexes C and D bind to the sequence, -512 TGTCACTCA -504. Only a single shifted band, complex E, was detected in the assay with F2 probe (Fig. 3A). Complex E persisted in the presence of excess unlabeled F2M, whereas cold F2 itself effectively competed for the formation of complex E. Therefore, as summarized in Fig. 4, the protein binding sites in the GPE1 region appear to be -533 GGAGGGGGCG -524, -512 TGTCACTCA -504, and -491 CAATCCA -485 which were designated as GES1, GES2, and GES3, respectively.


Figure 3: Determination of the protein binding sites in the GPE1 region. The sequences of the oligonucleotides which were used as probes and/or competitors in EMSA are shown below. The mutated sequences are shown in boxes. Two µg of C2C12 nuclear extracts were incubated with 2 10 cpm of probe and analyzed on 5% acrylamide gel. The DNA-protein complexes are indicated by an arrowhead.




Figure 4: Summary of protein binding sites in GPE1 region. The sequence of the protected region in DNase I footprinting is displayed. The protein binding sites identified by EMSA are shown in boxes and the names of the binding sequences are shown above the boxes.



GES1 Binding Protein Might be a Member of the Sp1 Family

GES1 site is homologous to Sp1- and AP2-binding sites(23, 31, 32) . Therefore, we examined whether GES1 binding protein was able to bind to Sp1- or AP2-binding sites by adding the oligonucleotide competitors containing Sp1- or AP2-binding sequences to EMSA. Sp1 competitor abolished formation of complex on the GES1 site (Fig. 5A), whereas GES2 and GES3 binding proteins did not bind to the Sp1 site (data not shown). The specificity of the interaction between GES1 binding protein and Sp1 sequence appeared to be high, since 50 molar excess of competitors was enough for complete competition. In contrast, the AP2 sequence could not compete with the GES1 site (Fig. 5A). It was also examined whether the Sp1 protein was capable of binding to the GES1 site. As revealed by DNase I footprinting, purified Sp1 protein bound to the GES1 site in proportion to the amount of the protein added (Fig. 5B). These results suggest that GES1 binding protein specifically binds to the Sp1 site and also that Sp1 protein binds to the GES1 site.


Figure 5: Sp1 binds to GES1 site. A, EMSA with various competitors. Two µg of C2C12 nuclear extract were preincubated without or with the indicated amounts of the oligonucleotides which contain Sp1, AP2, AP1, or NF1/CTF binding sequences before adding the labeled probes. The amount of competitor is written in molar excess. B, DNase I footprinting with purified Sp1 protein. The -644/-135 probe described in Fig. 3 was incubated with the indicated amounts of Sp1 proteins. According to the manufacturer, 1 fpu (footprint unit) is the amount of protein required to give full protection against DNase I on SV40 early promoter DNA. After digestion with DNase I, the fragments were analyzed on 6% sequencing gel. The sequence of the protected region is shown on the left and the GES1 site is underlined.



GES2 site has weak homology to AP1 site(33) . However, in the competitive EMSA, GES2 binding protein was not able to bind to AP1 site, indicating that GES2 binding protein may not be related to a family of AP1 transcription factor (Fig. 5A). On the other hand, the GES3 site is homologous to CCAAT box. From the EMSA using the binding site for NF1/CTF, one of the CCAAT box binding protein(34) , as a cold competitor, it was proved that GES3 binding protein did not bind to the NF1/CTF site (Fig. 5A).

GPE1 Binding Factors Seem to Cooperate with Each Other Functionally

In order to understand the function of each protein binding in the GPE1 region, the effects of mutations at the protein binding sites were assessed in C2C12 cells (Fig. 6). For site-directed mutagenesis, pKS551 plasmid was used as a template (see ``Materials and Methods''). Transcriptional activities were denoted as percentage of that of pKS551. The activity of pGES123 which has mutations at all protein binding sites in GPE1 was 20.2% relative to that of pKS551. This is consistent with the fact that the relative activity of pKS479 to pKS551 is 25.2%, implying that GES1, GES2, and GES3 sites can represent the whole GPE1 region. Mutation at the GES1 (pGES1) or GES2 (pGES2) sites did not reduce the transcriptional activity, whereas GES3 mutation (pGES3) resulted in a apparent reduction in CAT expression. In the case of the double mutation in which the GES3 site was mutated together with GES1 (pGES13) or GES2 (pGES23) sites, the transcriptional activity of the GPE1 region was completely abolished even though GES2 or GES1 sites still remained intact in each case. On the contrary, pGES12 which has mutations at GES1 and GES2 sites showed twice as high activity as pGES13 or pGES23. From these results, it seems that the GES3 site and at least one of the GES1 and GES2 sites are required for ultimate activity of the GPE1 region. Therefore, transcriptional activation function of the GPE1 region might arise from functional cooperation of the GPE1 binding proteins identified in this study.


Figure 6: Mutational analysis of protein binding sites in the GPE1 region. The GPE1 region of each plasmid is displayed on the left. Wild-type protein binding sites are shown as open boxes and mutated binding sites are shown as hatched boxes. The plasmids were transfected into C2C12 cells and relative CAT activities are shown on the right. -Galactosidase expression vectors were cotransfected to normalize the transfection efficiencies. The activities are expressed as percentage to that of pKS551 where it is defined as 100. The mean values of at least five independent experiments are shown.



The functions of GPE1 binding proteins were further analyzed by oligonucleotide reconstitution in the upstream basic promoter, E1b-TATA box (Fig. 7). F2, which contains GES3 site, showed stimulated transcription from E1b-TATA box about 17-fold as compared to E1bCAT, whereas F1 which possesses GES1 and GES2 sites showed no effect. The stimulation by F2 was eliminated by the mutation at the GES3 site, which confirmed the involvement of GES3 binding protein in transcriptional activation. Moreover, pF1F2 was even more active than pF2, indicating that the activating function of F2 was further potentiated by F1 oligonucleotide. These results imply the functional interactions among GPE1 binding proteins.


Figure 7: Oligonucleotide reconstitution analysis. The oligonucleotides, F1 which has GES1 and GES2 sites, and F2 which has GES3 site, were inserted in the upstream E1b-TATA box. The structures of the plasmids were displayed at the left. These plasmids were transfected into C2C12 cells. The resulting CAT activities are shown at the right. Transfection efficiencies were normalized by the activities from cotransfected -galactosidase expression vector. The result shown here is representative of three independent experiments.



GES1, GES2, and GES3 Binding Proteins Were Highly Detected in Colorectal Carcinomas

In recent studies, it was reported that human carcinomas contained higher levels of PLC-1 than normal cells (14, 15). Since the GPE1 region is a strong positive transcriptional regulatory element, we examined the amount of proteins bound to GES1, GES2, and GES3 sites in colorectal cancer and normal tissue extracts to demonstrate whether PLC-1 expression may be related to GES1, GES2, and GES3 binding proteins. As observed in previous studies by Noh et al.(15) , the content of PLC-1 was higher in 9 out of 11 cases of colorectal cancer tissues than in adjacent normal tissues (Fig. 8). Electrophoretic mobility shift assay revealed that the amounts of GES1, GES2, and GES3 binding proteins increased in 7 of 9 cases where PLC-1 was overexpressed, when compared with normal tissues. On the contrary, there was no significant increase in PLC-1 content in 2 out of 11 cases and these cases consistently showed no difference in the GES1, GES2, and GES3 binding proteins compared with normal tissues (data not shown). These results indicate that the increase of PLC-1 content seems to be well associated with the increase of GES1, GES2, and GES3 binding activities in colorectal cancer tissues.


Figure 8: GES1, GES2, and GES3 binding activities in human colorectal cancer tissues. A, Western blot analysis of PLC-1 protein in colorectal cancer and normal tissues. Three hundred µg of whole cell extracts were separated on 8% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed by monoclonal anti-PLC-1 antibody (B-16-5). Lanes N and C indicate normal and cancer tissues, respectively. STD indicates the purified PLC-1 standard. B, GES1, GES2, and GES3 binding activities were examined by gel retardation assays using the same extracts shown in panel A. Fifteen µg of whole cell extracts were used in each assay.




DISCUSSION

The expression of the PLC-1 gene is regulated during various physiologic events such as carcinogenesis and differentiation(14, 15, 16, 17, 20, 21) . However, the molecular mechanism underlying the regulation of PLC-1 expression is still obscure. In this study, we have characterized the GPE1 region which is one of the transcriptional regulatory regions upstream of the PLC-1 gene along with the nuclear factors that bind to this region.

By DNase I footprinting and EMSA, the proteins bound to GPE1 were identified and their binding sites were determined. Formation of four different complexes with F1 probe was observed and named complexes A, B, C, and D. Complex B appeared to be formed by interaction of the protein to the sequence, -533 GGAGGGGGCG -524, designated as GES1. Complexes C and D seem to be generated with the same DNA sequence, -512 TGTCACTCA -504, designated as GES2. The two GES2 binding proteins might be two different proteins derived from different genes or two different forms of a protein from a single gene. The two GES2 binding proteins shared several physical properties, including optimal temperature and optimal salt concentration for DNA binding, and thermostability (data not shown). It is, therefore, more likely that GES2 binding proteins are the products of a single gene, which could be generated by an alternative splicing or protein modification.

The complex A is considered to be a multicomplex, which includes GES1 and GES2 binding proteins, by the following reasons: 1) complex A was detected only under the conditions that both GES1 and GES2 sites are occupied. Complex A disappeared when binding of either GES1 or GES2 was hindered (Fig. 3A). 2) When the amount of nuclear protein increased gradually in EMSA, more complex A was formed. However, the formation of complex B, C, and D reached the peak at 5 µg and then decreased with additional nuclear proteins (data not shown). 3) Inhibition of the protein binding to GES1 site by competition with F1a increased complexes C and D, compared with the reaction without competitors (Fig. 3A). Inhibition of the binding on GES2 site, on the other hand, increased formation of complex B.

GES1 site is similar to AP2 and Sp1 sites. Purified Sp1 proteins recognized the GES1 site, and GES1 binding protein also bound to the oligonucleotide containing the Sp1-binding site. An oligonucleotide containing the AP2-binding site, on the contrary, was not recognized by GES1 binding protein. These data suggested that C2C12 nuclear protein factor binding to the GES1 site shared sequence specificity with Sp1 protein. Sp1 enhances the transcription of epidermal growth factor receptor(35) , and in cases with the overexpression of Sp1, the mRNA of epidermal growth factor receptor was expressed in high levels in human gastric carcinomas(36) . Interestingly, it has been shown that PLC-1 was overexpressed and colocalized with epidermal growth factor receptor in human breast cancer(14) . Based on these findings and the fact that Sp1 can bind to the GPE1 region of the PLC-1 gene, it can be suggested that Sp1 may be a transcriptional regulator of PLC-1 gene as well as epidermal growth factor receptor gene. However, several transcription factors which belong to the Sp1 family have been cloned, and some of them share the binding specificity with Sp1 protein (37-39). Therefore, it is also possible that GES1 binding protein might be a member of the Sp1 family rather than Sp1 itself.

On the other hand, although the GES2 site is weakly homologous to the AP1 site, the GES2 binding protein did not bind to the AP1 site. This result suggests that GES2 binding protein might be a novel DNA-binding protein. The identity of GES3 binding protein is also obscure. As the GES3 site is similar to the CCAAT box, it could be one of the CCAAT box binding proteins or could be a novel kind of transcription factor. Among CCAAT box binding proteins, NF1/CTF does not seem to be the GES3 binding protein, since the GES3 binding protein was not able to bind to the NF1/CTF site.

GPE1 region could stimulate the transcription from the heterologous promoter, E1b-TATA box (Fig. 1), even though the promoter of PLC-1 gene is TATA-less. Therefore, we propose that GPE1 is a general transcriptional activating element. Moreover, activating function of GPE1 is nearly abolished by orienting it reversely. This suggests elaborate interactions among GPE1 binding proteins and basic transcriptional machinery.

The function of each protein binding site at the GPE1 region was assessed by site-directed mutagenesis. GES3 alone can activate the transcription from the PLC-1 gene, whereas GES1 or GES2 alone were not able to activate transcription by itself. However, GES3 needs to cooperate with GES1 or GES2 for full activity, implying that GES3 functionally cooperates with the other GPE1 binding proteins. Therefore, it is likely that the minimum requirement for GPE1 activity may be GES3 and at least one of GES1 and GES2. In other words, GES1 and GES2 might be redundant for the transcriptional activity of GPE1 since only one of them can make up ultimate activity of GPE1 region by functional cooperation with GES3. In contrast, GES3 seems to be necessary for full activity of GPE1 since mutation at the GES3 site caused reduction of transcriptional activity, whereas mutations at GES1 or GES2 did not. The specific roles of these GPE1 binding proteins are still obscure. One possible explanation is that the GES3 binding protein may influence the transcription by direct interaction with basic transcription machinery, whereas GES1 and GES2 binding proteins may modulate the activity of GES3 binding protein. This explanation is partly supported by the oligonucleotide reconstitution experiment (Fig. 7). F2 oligonucleotide containing GES3 site could stimulate E1b-TATA box, whereas F1 oligonucleotide which possesses GES1 and GES2 sites did not augment the transcription by itself. However, F1 oligonucleotide was able to potentiate the transcriptional activation function of F2 oligonucleotide.

Recent studies showed that human primary breast carcinomas and colorectal carcinomas contained considerably higher levels of PLC-1 than normal tissues(14, 15) . However, the factors that cause the overexpression of PLC-1 in cancer cells remained uncovered. In this study, we have identified the DNA binding proteins to GPE1, one of the transcriptional regulatory regions of the PLC-1 gene, and examined the PLC-1 expression and the amount of GPE1 binding proteins in colorectal cancer tissues and adjacent normal tissues. Immunoblot analysis and electrophoretic mobility shift assay revealed the correlation between PLC-1 expression and the content of GPE1 binding proteins in 9 out of 11 cases; GPE1 binding proteins increased in 7 of 9 cases where PLC-1 increased, and in the other 2 cases where PLC-1 did not increase, the amount of GPE1 binding proteins was not changed either. These results suggest that the increase of GPE1 binding activities might be important for the overexpression of PLC-1 in cancer cells. The cooperation of GPE1 binding proteins (GES1, GES2, and GES3 binding proteins) was suggested by mutational analyses. Since all the binding proteins to GES1, GES2, and GES3 sites were elevated in cancer tissues, the cooperation of GPE1 binding proteins might occur in carcinomas to overproduce PLC-1, which may result in the amplification of the proliferation signal. The isolation of each GPE1 binding protein might be an essential step to understand the process of tumorigenesis.

In conclusion, we have identified and analyzed at least three sites (GES1, GES2, and GES3), to which several transcription factors bind, in the GPE1 region of PLC-1 promoter. GPE1 binding proteins might cooperate with each other to confer trans-regulation to PLC-1 expression. The contents of these GPE1 binding proteins were higher in colorectal cancer tissues than in normal tissues as was that of PLC-1. Therefore, we propose that the increase of GPE1 binding proteins and their cooperation might be attributed to the overexpression of PLC-1 in carcinomas.


FOOTNOTES

*
This work was supported by Non-directed Research Fund, Korea Research Foundation (1994), The Basic Science Research Institute Program, the Korea Science and Engineering Foundation (KOSEF-92-2400-05), and The Ministry of Education Project BSRI-94-4434, Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Life Science, Pohang University of Science and Technology, San 31 Hyoja Dong, Pohang 790-784, Republic of Korea. Tel.: 82-562-279-2293; Fax: 82-562-279-2199.

The abbreviations used are: PLC, phospholipase C; GPE, PLC-1 gene positive element; GNE, PLC-1 gene negative element; SH, src homology; CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay.


ACKNOWLEDGEMENTS

We thank Drs. C. B. Chae and S. K. Jang (POSTECH) for critical review of this manuscript, and Dr. S.-J. Kim (NIH) for advice.


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