©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Mouse Tissue Inhibitor of Metalloproteinases-3 and Its Promoter
SPECIFIC LACK OF EXPRESSION IN NEOPLASTIC JB6 CELLS MAY REFLECT ALTERED GENE METHYLATION (*)

(Received for publication, December 21, 1994; and in revised form, June 6, 1995)

Yi Sun (1) (2)(§) Glenn Hegamyer (1) Hyungtae Kim (3) Kavitha Sithanandam (3) Hua Li (3) Rebecca Watts (1) Nancy H. Colburn (1)

From the  (1)Cell Biology Section, Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center (NCI FCRDC), Frederick, Maryland 21702, the (2)Department of Cancer Research, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105, and the (3)Biological Carcinogenesis and Development Program, Program Resources Inc./Dyn Corp, NCI FCRDC, Frederick, Maryland 21702

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mouse tissue inhibitor of metalloproteinases-3 (mTIMP-3), a gene specifically not expressed in neoplastic JB6 cells, has been isolated recently through the use of the mRNA differential display technique (Sun, Y., Hegamyer, G., and Colburn, N. H.(1994) Cancer Res. 54, 1139-1144). We report here the full-length mTIMP-3 cDNA sequence, the promoter sequence and partial characterization, expression and induction of TIMP-3, and the possible molecular basis for the lack of mTIMP-3 expression in neoplastic JB6 cells. There are three transcripts arising from alternative polyadenylation of mouse TIMP-3 gene, having sizes of 4.6, 2.8, and 2.3 kilobase pairs, respectively. All three TIMP-3 transcripts are expressed in preneoplastic but not neoplastic JB6 cells. Computer analysis of cloned TIMP-3 promoter revealed six AP-1 binding sites, two NF-kappaB sites, a c-Myc site, and two copies of a p53 binding motif separated by eight base pairs with two mismatches at the second motif, along with many other cis elements. TIMP-3 gene expression was inducible by AP-1 and NF-kappaB activators, 12-O-tetradecanoylphorbol-13-acetate, and tumor necrosis factor-alpha only in preneoplastic cells with an induction peak at 2 h post-treatment, suggesting classification of mTIMP-3 as a member of the immediate early gene family. Southern blot, mutational analysis, and transient transcriptional activation experiments revealed that the lack of expression of mTIMP-3 in neoplastic JB6 cells was due neither to gross deletion nor to promoter mutation of the gene, nor was there a lack of transcription factors required for transcriptional activation. Instead, the lack of TIMP-3 expression in neoplastic JB6 cells may reflect an abnormal methylation of the gene. Both hyper- and hypomethylation of the mTIMP-3 gene are associated with complete down-regulation of gene expression in neoplastic JB6 cell lines. Treatment of neoplastic cells with the methylase inhibitor 5-azacytidine caused reexpression of the mTIMP-3 gene in a tumor cell line that showed hypermethylation but not in another that showed hypomethylation of the gene, suggesting a complex role for methylation in the silencing of gene expression.


INTRODUCTION

The mouse JB6 cell model system has been widely used for tumor promotion/progression studies in attempts to understand the molecular mechanism of multistage carcinogenesis(1, 2) . This model consists of three phenotypically distinct variants including P variants that are resistant to tumor promoter-induced neoplastic transformation, P variants that are sensitive to tumor promoter-induced neoplastic transformation, and neoplastic derivatives. These three variants therefore represent earlier-to-later stages of tumor promotion and progression(1, 2, 3, 4) . Studies of genes differentially expressed among the three variants could lead to the isolation of critical gene(s) whose expression is either causally related to the establishment of distinct phenotypes or required for the maintenance of these phenotypes. Studies of the mechanism under which differential expression of these genes occurs among the variants can lead to a better understanding of multistage carcinogenesis and provide a molecular basis for chemoprevention and/or chemotherapy of cancer.

Since the molecular mechanism for preneoplastic-to-neoplastic progression in the JB6 model is only partly understood(4, 5) , we have recently employed a newly developed technique, mRNA differential display (6) in an attempt to isolate differentially expressed genes that may be responsible for preneoplastic-to-neoplastic progression. Five differentially expressed clones were isolated(5) . One of these clones that is specifically not expressed in neoplastic JB6 cells appeared to be the mouse version of human tissue inhibitor of metalloproteinases-3 (TIMP-3) (^1)considering the 96% amino acid sequence identity(5) , and was later shown to be mouse TIMP-3 with a 100% sequence identity to mTIMP-3 in the open reading frame of the gene(7) .

There are three members of the TIMP family of genes, TIMP-1, TIMP-2, and TIMP-3. TIMPs are related but distinct genes functioning as naturally occurring inhibitors of matrix metalloproteinases(8) . TIMPs therefore regulate matrix degradation and play a role in preventing tumor cell invasion and metastasis(9) . Unlike TIMP-1 and TIMP-2, which are secretory proteins, TIMP-3 is secreted from cells but binds to the extracellular matrix(7, 10) . Mouse and human TIMP-3 have been recently cloned and sequenced by several laboratories(5, 7, 11, 12, 13, 14, 15) . TIMP-3 has been found to be down-regulated in neoplastic JB6 cells(5) ; to inhibit matrix metalloproteinase activity(7) ; and to be regulated during G1 progression or by mitogenic stimulation, differentiation, and senescence(12) . Furthermore, mutation of hTIMP-3 has been linked to Sorsby's fundus dystrophy, a fully penetrant autosomal dominant macular disorder(16) . Although mouse TIMP-3 has been recently cloned (5, 7, 11) , the reported sequence contains only open reading frame and a short part of the 5`- and 3`-untranslated regions(7, 11) . Moreover, the study of mTIMP-3 regulation has been limited due to the lack of available gene promoter. We report here the full-length sequence of mTIMP-3 cDNA, cloning, and partial characterization of the TIMP-3 promoter and TIMP-3 expression (both basal and induced) and a possible molecular mechanism by which expression in neoplastic cells is switched off.


MATERIALS AND METHODS

Cell Culture and Drug Treatment

Mouse JB6 cells were cultured in Eagle's minimal essential medium with 5% fetal calf serum. The typical doubling time for P, P, and neoplastic cells is 28, 24, and 16 h, respectively. For treatment, subconfluent cells were incubated with TPA (10 ng/ml, 1.6 10M, Chemicals for Cancer Research, Inc., Eden Prairie, MN) or TNF-alpha (150 units/ml, Boehringer Mannheim Biochemicals, Indianapolis, IN) for various times up to 24 h, or 5-azacytidine (20 µM, Sigma) for 24 h.

cDNA and Genomic Library Screening and DNA Sequencing

Two cDNA libraries (JB6 P cells and mouse lung) were screened initially using a 200-bp fragment (nucleotides 4353-4553) generated by mRNA differential display (5) . The overlapping cloning yielded a nearly full-length mouse TIMP-3 clone (4548 bp)(5) . A 950-bp cDNA fragment (sun.orf2, nucleotides 53-1006) containing the entire open reading frame of TIMP-3 was used as probe to screen a JB6 P genomic library constructed in Lambda Dash II by Strategene (San Diego, CA). Eighteen positive clones with insert sizes ranging from 9 to 18 kb were obtained after three rounds of screening. One of these clones (clone 7, insert size, 16 kb), which hybridized with a 256-bp fragment at the 5`-untranslated region (sun.5utr, nucleotides 75-331) was selected. The recombinant phage DNA was isolated as described(17) , digested with XbaI, transferred onto Zetabind membrane (Cuno, Inc. Meriden, CT), and hybridized with sun.5utr. A positive subfragment (3.4 kb, most likely containing the TIMP-3 promoter) was subcloned into the expression vector pcDNA3 (Invitrogen, San Diego) and sequenced using Sequenase 2.0 (U. S. Biochemical Corp.).

Primer Extension

To define the transcriptional start of the mTIMP-3 gene, primer extension analysis was performed. The samples included poly(A) RNA (0.6 µg, from JB6 P only) or total RNA (10 µg) from JB6 P cells, an SV40-transformed mouse liver line ((18) , which expresses a very high level of TIMP-3 mRNA), (^2)Ha-ras-transformed NIH3T3 cells (also expresses a high level of TIMP-3),^2 and yeast tRNA as a negative control. RNAs were reverse-transcribed using Moloney murine leukemia virus reverse transcriptase with supplied 5 buffer (Life Technologies, Inc.) after annealing to primer 5E.02E (5`-GGGTCGCTGGAACACGAGTC, nucleotides 134-115) end-labeled with [P]ATP (2 10^5 cpm/reaction) by polynucleotide kinase (Boehringer Mannheim). The RT reaction was conducted for 90 min at 42 °C and terminated by sodium hydroxide. The RT products were phenol extracted, ethanol precipitated, run on a 6% sequencing gel, and exposed to x-ray film.

Northern and Southern Blot Analysis

Total RNA or genomic DNA was isolated from subconfluent JB6 variants and subjected to Northern and Southern analysis as described earlier(19, 20) . Briefly, total RNA (15 µg) was size-fractionated by electrophoresis on 1.2% agarose/ formaldehyde gels, transferred onto Zetabind membrane and hybridized to [P]dCTP-labeled cDNA probes. These hybridizing probes include sun.orf1 (nucleotides 53-1794), sun.orf2 (nucleotides 53-1006), and sun.6 (nucleotides 2665-4591) for TIMP-3. The probes for the other members of TIMP family of genes were generated by RT-PCR with the total RNA from JB6 Cl30.7b (P) cells as templates as described previously(4, 21) . For TIMP-1, the PCR primers were designed based upon the published mouse TIMP-1 sequence (22) . They are mTIMP1.01, 5`-GGAATTCAGAGATACCATGATGGCC, and mTIMP1.02, 5`-GGAATTCCTGGGGGAAGGCTTC, generating a 658-bp fragment. For mTIMP-2, primers are TIMP2.03, 5`-GGAATTCTGCAGCTGCTCCCCGGT, and TIMP2.02, 5`-GGAATTCCTGCTTATGGGTCCTCG, yielding a 602-bp fragment(23) . For Southern analysis, 10 µg of genomic DNA were digested overnight by restriction enzymes including EcoRI, HindIII, BamHI, BglII, MspI, and HpaII (Boehringer Mannheim) to completion at a ratio of 1 µg of DNA/10 units of enzyme. The digested DNA was then electrophoresed on a 0.8% agarose gel, transferred onto Zetabind membrane, and hybridized with either sun.orf2 probe or 2.9 kb (-2846 to +58) of promoter fragment as described above.

Antibody Production, Western Blot Analysis, and Immunoprecipitation

Rabbit anti-peptide TIMP-3 antibody was generated by Macromolecular Resources (Colorado State University, Fort Collins, CO). Briefly, an 18-amino-acid peptide corresponding to the carboxyl-terminal end of the TIMP-3 protein (CYRGWAPPDKSISNATDP) was synthesized using t-butoxycarbonyl chemistry. The purified peptide (90% homogeneity) was conjugated to keyhole limpet hemocyanin (Macromolecular Resources). The conjugated peptide was then used as an antigen to immunize rabbits. Rabbits were boosted with antigen 14 days after the initial shot and every 3 weeks thereafter. The antisera were collected every 2 weeks starting 4 weeks post-initial immunization and used as anti-TIMP-3 antibody without further purification. For Western blot analysis, extracellular matrix (ECM) where TIMP-3 was localized (7, 10) was first isolated. The cells at 90-95% confluency were incubated with a solution containing 10 mM EDTA and 10 mM EGTA at 37 °C for 10-15 min. After cell detachment by periodically rapping against the counter top, ECM was harvested into SDS sample buffer using a rubber policeman and boiled for 3-5 min before loading onto a 15% polyacryamide gel. ECM proteins on the gel were electrically transferred onto nitrocellulose membrane for 1 h at 180 mA. The membrane was first blocked with milk overnight at 4 °C in a shaking platform to eliminate nonspecific binding, incubated with anti-TIMP-3 peptide antibody (1:800 dilution with phosphate-buffered saline, 0.05% Tween 20) for 1 h at room temperature, and finally incubated with horseradish peroxidase-labeled goat anti-rabbit antibody (Kirkeguard & Perry Laboratory, Gaithersburg, MD) for 1 h at room temperature. The TIMP-3 protein was visualized by a chemiluminescence reaction as specified in the kit (ECL Western blotting kit, Amersham Corp.). Immunoprecipitation was performed as described earlier (4, 24) to determine the specificity of TIMP-3 antibody using unlabeled TIMP-3 peptide as a competitor. Briefly, JB6 Cl41.5a (P) cells were metabolically labeled with [S]methionine (0.2 mCi/ml, Amersham Corp.), and cell lysate was prepared(4, 24) . Cell lysates containing 4 10^7 trichloroacetic acid-precipitable cpm were incubated overnight at 4 °C with TIMP-3 peptide antibody alone or with the antibody that had been preincubated for 60 min with TIMP-3 peptide at ratios of 1:10 and 1:100 (antibody/peptide). The immunoprecipitates were pelleted with protein A-Sepharose (Pharmacia Biotech Inc.), washed, and loaded onto 15% SDS-polyacrylamide gel electrophoresis. The dried gel was exposed to x-ray film.

Luciferase Reporter Construction, Transient Transfection, and Luciferase Assays

Two mTIMP-3 promoter fragments AP0 (-491 to +23) and AP6 (-2846 to +58) were generated by PCR amplification (25) with cloned 3.4 kb of mTIMP-3 promoter as the template. The primers used for AP0 were Meth.05 (5`-TGAGGGAAGCTGATGTCCAGCGGTTTCCT, nucleotides -491 to -463) and Meth.02 (5`-ACTGTGAGCGAGAGTCCAGGGCTGCAGAGT, nucleotides +23 to -7), and for AP6 they were GTIP3.01 (5`-TCTAGAAAAGTTCCAACAGAGG, nucleotides -2846 to -2825) and 5E.04E (5`-AGAAGATTCTTGGCGCTGGC, nucleotides +58 to +39). For AP0, the PCR fragment (514 bp) was blunt-ended and ligated into a luciferase reporter, pGL2-basic (Promega), which has been predigested with SmaI. For AP6, the PCR fragment (2904 bp) was ligated into pCR II (Invitrogen, San Diego, CA, used as a shuttle vector for cloning) and then digested with KpnI and Xhol. The gel-purified insert was ligated into pGL2-Basic, which had been predigested with the same restriction enzymes. The resulting luciferase reporter constructs, named AP0 (containing neither AP-1 nor NF-kappaB sites) and AP6 (containing 6 AP-1 and 2 NF-kappaB sites), respectively, were sequenced (completely for AP0 and partially for AP6) to ensure the correct orientation and freedom from PCR-introduced mutation. The constructs, along with AP-1 and NF-kappaB positive luciferase reporter controls (26, 27) were transiently transfected into subconfluent neoplastic JB6 L-RT101 or H-RT101 cells seeded in 24-well plates by a calcium phosphate method, and luciferase activity was assayed as described previously(28) .


RESULTS

Molecular Cloning of the Full-length mTIMP-3

We have previously cloned and sequenced 4548 bp of mTIMP-3 cDNA by screening two cDNA libraries and performing overlapping cloning (5) . The size of mTIMP-3 mRNA has been reported to be 5.2 or 4.5 kb, respectively(5, 7) . Due to its large size, this variation could result from error in the comparison of mTIMP-3 mRNA with the RNA size markers. A primer extension experiment was conducted in an attempt to define the transcriptional initiation site of the gene using the primer near the 5`-end of the clone. As shown in Fig. 1, extension of primer 5E.02E using the total RNA as template yielded a major product of 134 bp from JB6 P cells, SV40-Tx mouse liver cells, and NIH3T3-ras-Tx cells, but not in a negative control (yeast tRNA). The same size product was seen when poly(A) RNA from JB6 P cells was used as a template (data not shown). The slightly slower migration of this major product seen in SV40-Tx liver cells and NIH-3T3-ras-Tx cell was due to slightly uneven electrophoresis of this particular gel. In addition to this major product, several other minor products were also revealed having sizes larger (230 bp) or smaller (118 bp or less) than the 134-bp major product. These minor products were clearly seen in SV-40-Tx mouse liver cells (Fig. 1), which express a very high level of mTIMP-3.^2 The results indicate that there are multiple transcriptional starts in the mTIMP-3 gene with the major site located 134 bp upstream of primer 5E.02E (including the 20 nucleotides of the primer).


Figure 1: Definition of transcriptional initiation site in the mTIMP-3 gene. Total RNA was isolated from JB6 P cells (Cl41.5a), SV40-Tx mouse liver cells, and Ha-ras-transformed NIH3T3 cells by the RNAzol solution. Ten µg of total RNA was subjected to primer extension analysis as detailed under ``Materials and Methods.'' Yeast tRNA (10 µg) was used as a negative control. A known size DNA fragment was sequenced and loaded in the adjacent lane to serve as a size marker. Multiple primer extension products were seen with a major product of 134 bp.



To reveal the 5`-most end sequence including the transcription initiation site of mTIMP-3 gene, we screened the genomic library and isolated a 3.4-kb fragment (Cl7.Xba-4). DNA sequencing revealed that this fragment consisted of a 2846-bp promoter region (-2846 to -1), a 437-bp exon 1 (+1 to +437), and part of intron 1 (133 bp) of the TIMP-3 gene (+438 to +570) (Fig. 2). There is 100% sequence identity of exon 1 between this genomic clone and the cDNA clone (sun clone, (5) ), except 1 nucleotide at the 5`-most end of the cDNA clone, resulting from a cDNA cloning artifact. Based upon the sequence of the genomic clone, we defined the major transcription initiation site of the TIMP-3 gene at the nucleotide adenine, 134 bp upstream of the primer 5E.02E. The genomic sequence data were confirmed by a 5`-racing technique using a mouse brain cDNA library constructed with random priming (data not shown). The full-length cDNA sequence of mouse TIMP-3 has been deposited in Genbank with an accession number of Z30970. It is 4591 bp in size consisting of 316 bp in the 5`-end untranslated region, a 636-bp open reading frame (encoding a 211-amino-acid peptide), and a very long (3639 bp) 3`-untranslated region. The in vitro transcription and translation (using Promega's kit) of the sun6.ext clone (nucleotides 53-2809) produces a 24-kDa peptide, agreeing with the peptide size predicted by open reading frame (data not shown). In the 3`-untranslated region, there is a GA repeat sequence (nucleotides 2667-2699) and the length of repeats varies among the cDNA libraries (data not shown), indicating a dinucleotide polymorphism. The cDNA sequence contains two polyadenylation sites at nucleotides 2446-2451 and 4148-4153, respectively, and a poly(A) tail (nucleotides 4541-4591).


Figure 2: Cloned mTIMP-3 promoter sequence. A 950-bp cDNA fragment containing the entire open reading frame of TIMP-3 was used as a probe to screen a genomic library, and a 3.4-kb fragment was isolated as detailed under ``Materials and Methods.'' This 3.4-kb fragment contains a 2.9-kb promoter sequence. The underlined consensus sequences include a TATA box at the -10 position relative to the transcription initiation site; a GC box, two SP-1 sites; six AP-1 sites, two NF-kappaB sites, one c-Myc site and one p53 binding site among many others. It also contains a CA repeat and a GT repeat at nucleotides -1043 to -998 and -552 to -505, respectively. The sequence has been deposited in the Genbank with an accession number of U19462.



The sequence of the 3.4-kb genomic clone containing mTIMP-3 promoter is shown in Fig. 2. The sequence contains six AP-1 binding sites (-1958 to -1950; -1348 to -1342; -1117 to -1111; -763 to -754; -720 to -714; and -617 to -611, (29, 30, 31, 32, 33, 34) ); two NF-kappaB binding sites (-1921 to -1912 and -1483 to -1475, (35) ); two copies of the p53 binding motif separated by eight base pairs with two mismatches at the second motif (-648 to -620, (36) ); and one c-Myc binding site (-2278 to -2272, (37) ), along with three TATA boxes at -10, -80, and -165, two Sp1 sites (-57 to -52; and -115 to -106) and a GC box (-388 to -379) among many other sites including PEA3.CS, c-fos.SRE, GR.uteroglob, MyoD-mck, AP-2, AP-3, and so on. Interested readers can retrieve the sequence (accession number U19462) and identify the consensus sequence of interest by using the GCG sequence analysis software. The mouse TIMP-3 promoter sequence was compared with the published mouse TIMP1 and the human TIMP-2 promoter sequences (38, 39) by using the GCG Wordsearch program. There was no significant sequence similarity (data not shown).

Expression of TIMP Family Genes in Mouse JB6 Epidermal Variants

We used the open reading frame sequence of mTIMP-3 (sun.orf2, nucleotides 53-1006) as a probe to reexamine the expression pattern in JB6 cells to confirm our previous finding that had utilized a short 3`-end fragment (nucleotides 4353-4553). As shown in Fig. 3A, three transcripts of sizes 4.6, 2.8, and 2.3 kb were detected. All three were detected in four out of four independent P cell lines and in three out of three P lines but in none out of six Tx cells, validating our previous observation that mTIMP-3 is specifically not expressed in neoplastic JB6 cells. Among the three transcripts, the largest messenger RNA (4.6 kb) is the most abundant one in agreement with previous results using a 3`-end fragment generated by the mRNA differential display(5) . To determine the nature of the other two transcripts, we performed the same Northern analysis using poly(A) RNA. All three transcripts are detected (data not shown), indicating that they are all being polyadenylated. Since the two shorter transcripts can be detected neither with the 3`-end fragment (5) nor with the probe sun.6 (nucleotides 2665-4591) (data not shown) and since there is a polyadenylation site at nucleotides 2446-2451, they are most likely alternative polyadenylation products of the TIMP-3 gene.


Figure 3: Expression of TIMPs in mouse JB6 epidermal cells. Total RNA was isolated from JB6 variants with the RNAzol solution. Fifteen µg of total RNA was subjected to Northern analysis as detailed under ``Materials and Methods.'' PanelA shows the expression of TIMP-3, and panelB shows the expression of TIMP-1 and TIMP-2. The beta-actin was used as an internal standard to normalize the loading.



Since TIMP-3 is specifically not expressed in neoplastic JB6 cells, we asked whether this differential expression extended to other TIMPs and examined the expression pattern of TIMP-1 and TIMP-2 in JB6 variants by Northern analysis using as probes the corresponding cDNAs generated by RT-PCR. As shown in Fig. 3B, all JB6 variants, regardless of the stage of neoplastic progression, express both TIMP-1 and TIMP-2 at similar levels. Taken together, the results indicate that TIMP-3 is the only member of the TIMP family whose lack of expression may be implicated in preneoplastic-to-neoplastic progression in the mouse JB6 model.

To extend our observation of TIMP-3 expression from the RNA to the protein level, we measured TIMP-3 protein concentrations among selected JB6 variants by Western blot analysis using rabbit anti-TIMP-3 C-terminal peptide antibody. As shown in Fig. 4(upperpanel), TIMP-3 protein was expressed in two of two P cells, two of two P cells and none of two Tx cell lines, consistent with the Northern finding, indicating that regulation is primarily pretranslational. To confirm the specificity of the anti-TIMP-3 antibody, we used unlabeled peptide (used as antigen for antibody production) in an immunoprecipitation assay to see whether it could competitively block detection of TIMP-3 protein. The result is shown in Fig. 4(bottompanel). Indeed, a 10 times excess of cold peptide completely blocked the detection of the TIMP-3 band, indicating specificity of the 24 kDa band.


Figure 4: The level of TIMP-3 protein in JB6 variants. The extracellular matrix was isolated from representive JB6 variants showing distinct phenotype as indicated. Cells at 90-95% confluency were detached by incubating with the solution containing 10 mM EDTA and 10 mM EGTA. The ECM was harvested and subjected to Western analysis as described under ``Materials and Methods.'' A size marker as indicated was also loaded to define the size of TIMP-3 protein (upper panel). To examine the specificity of the peptide anti-TIMP-3 antibody, a competition assay was conducted as described under ``Materials and Methods.'' Shown is the specific blockage of TIMP-3 detection by 18-mer TIMP-3 peptide (bottom).



Inducibility of the TIMP-3 Gene

DNA sequencing of the TIMP-3 promoter region revealed AP-1 and NF-kappaB sites (Fig. 2), We therefore examined the inducibility of the TIMP-3 gene by TPA (a known AP-1 activator) and TNF-alpha (an activator of the NF-kappaB transcription factor)(40, 41, 42) . Both reagents are tumor promoters in JB6 P cells(1, 2, 3, 4, 43) , and TPA is also a mitogen in JB6 cells (44) . As shown in Fig. 5, TPA and TNF-alpha each induced TIMP-3 expression in preneoplastic P and P cells. TIMP-3 expression was rapidly induced, reaching a peak at 2 h and returning to basal level 24 h after treatment. This result suggests that mTIMP-3 belongs to the immediate early gene family subject to tumor promoter/mitogen induction. We have also examined the inducibility of TIMP-3 by TPA and TNF-alpha in neoplastic JB6 cells (L-RT101, H-RT101) and found that in addition to the lack of basal level expression, neither TPA nor TNF-alpha treatment induced TIMP-3 expression (data not shown), suggesting a possible defect in the promoter region of the gene or a defect in transcription factor activation in neoplastic cells. To test these possibilities, we have conducted a PCR single strand conformation polymorphism direct sequencing analysis ( (25) and (45) -47) of the entire 2.9-kb promoter region in these two neoplastic JB6 lines and have found no point mutations (data not shown). The results exclude the possibility that the lack of basal as well as induced expression of the TIMP-3 gene in neoplastic JB6 cells was due to the point mutations in the promoter region tested.


Figure 5: Induction of TIMP-3 expression by TPA and TNF-alpha in preneoplastic JB6 cells. Cells at 70% confluency were subjected to TPA (10 ng/ml, panelA) or TNF-alpha (150 units/ml, panelB) treatment for the indicated time up to 24 h. Total RNA was isolated and subjected (15 µg) to Northern analysis. The ribosomal 7 S was used as a loading control. The densitometric quantitation of TIMP-3 induction was performed in an LKB Ultrascan XL laser densitometer. Comparison was made by arbitrarily choosing the untreated cells as 1. The band density after normalization is shown on the bottom of each panel.



Lack of Defect in Transcription Factor Activation in Neoplastic JB6 Cells

We next tested the hypothesis that lack of expression (both basal and induced levels) of mTIMP-3 in neoplastic JB6 cells may result from lack of activated transcription factors required for TIMP-3 expression. Two constructs, AP0 and AP6 (luciferase reporters driven by different lengths of mTIMP-3 promoter sequence, see ``Materials and Methods'') were transiently transfected into L-RT101 or H-RT101 cells. If transcription factors required for TIMP-3 expression and/or activation are defective in these cells, one should see no luciferase activity. As shown in Fig. 6, there was substantial luciferase activity in both neoplastic cell lines (L-RT101, upperpanel, H-RT101, bottompanel) when AP6, a luciferase reporter driven by the 2.9-kb mTIMP-3 promoter was used. The level was 10-fold greater than the positive controls that contain either the 4 AP-1 consensus sequence or human immunodeficiency virus promoter containing two NF-kappaB binding sites at the promoter regions of the luciferase reporters(26, 27) . The luciferase activity was also detectable using AP0, which is driven by an AP-1- and NFkappaB-less TIMP-3 promoter sequence. Neither TPA nor TNF-alpha significantly induced the luciferase activity of any of the luciferase reporters used. We have also transiently transfected these constructs into P cells (Cl41.5a), which showed both basal and induced expression of TIMP-3 by Northern analysis (see Figs. 3 and 5). The level of luciferase activity in Cl41.5a cells is less than in neoplastic cells under both uninduced and induced conditions (data not shown), consistent with the fact that neoplastic JB6 cells have a higher level of AP-1 activity(48) . The lower level in P cells might alternatively reflect competition of endogenous TIMP-3 promoter for activated transcription factors. The results indicate that 1) the cloned 2.9-kb TIMP-3 promoter fragment is a functional promoter; 2) TIMP-3 down-regulation in neoplastic JB6 cells is not explainable by lack of transcription factors; and 3) the basal level of activated transcription factors appears to be relatively high in neoplastic JB6 cells.


Figure 6: TIMP-3 promoter activity assayed in neoplastic JB6 cells. Subconfluent L-RT101 and H-RT101 cells in 24-well plates were transiently transfected with AP0 or AP6 construct, along with positive (Ps-Contr, 4 AP-1 or 2 NF-kappaB) and negative (Vector, pGL2-Basic) controls by a calcium phosphate method as described under ``Materials and Methods.'' Thirty h post-transfection, cells were treated with TPA (10 ng/ml) for L-RT101 (resistant to TPA but sensitive to TNF-alpha-induced killing, (19) , upperpanel) or TNF-alpha (150 units/ml) for H-RT101 (sensitive to TPA but resistant to TNF-alpha-induced killing, (19) , bottompanel) for 12 h. After treatment, cells were lysed on the plate with the lysis buffer (Promega, Madison, WI) and luciferase activity was assayed in a Monolight luminometer (Analytic Luminescence Laboratory, San Diego, CA) as described previously(28) . Shown are the mean ± S.E. from three independent transfections. RLU stands for relative light units.



Altered Methylation of mTIMP-3 in Neoplastic JB6 Cells

Lack of expression of mTIMP-3 in neoplastic cells could result from certain other mechanisms including 1) structural alterations of the gene, such as gene rearrangement, or 2) abnormal gene methylation, since DNA methylation of cytosine residues at specific sequences has been found to be associated with inactive states of genes(49) . To investigate these two possibilities, we performed genomic Southern analysis using the sun.orf2 (nucleotides 53-1006, flanking the entire encoding region) clone as a probe. When genomic DNA from 4 JB6 variants, P (Cl30.7b), P (Cl41.5a), and two neoplastic lines (L-RT101 and H-RT101) was cut with EcoRI, BamHI, HindIII, and BglII, the hybridization band patterns in all four variants were identical (not shown). This result excluded the possibility of structural alterations derived from gross rearrangement or deletion of mouse TIMP-3 gene. To determine the possible involvement of gene methylation in TIMP-3 expression, we used a methylation-sensitive enzyme HpaII. HpaII digestion gave rise to three distinct hybridizing patterns (Fig. 7A). One was seen in the two preneoplastic lines, the other two were seen in each of two neoplastic lines (lanes1-3 and 5). Since HpaII only digests unmethylated CCGG, the more digested the bands, the less gene methylation, and vice versa. In the two neoplastic lines analyzed, one was hypermethylated (L-RT101, lane3) with fewer hybridizing bands, and the other was hypomethylated (H-RT101, lane5) with more bands in the TIMP-3 gene. We next tested the methylation status of the TIMP-3 promoter in neoplastic cells as compared with preneoplastic cells by HpaII digestion/genomic Southern hybridized with the 2.9-kb promoter sequence. As shown in Fig. 7B, again three different digestion patterns were revealed. The two preneoplastic lines showed identical hybridization patterns (two bands of 6.5 and 5.6 kb, lanes1 and 2). The hypermethylated L-RT101 line showed a 6.6-kb hybridizing band and an unclear 5.6-kb band (lane3). The hypomethylated H-RT101 line showed five hybridizing bands (lane5). These results indicate that both the TIMP-3 gene and its promoter sequence are abnormally methylated and suggest that this abnormal methylation (either hyper- or hypo-) of the TIMP-3 gene may be associated with down-regulation of the gene in the neoplastic JB6 cells.


Figure 7: Altered methylation pattern in both TIMP-3 gene and promoter in JB6 tumor cell lines. Subconfluent L-RT101 and H-RT101 cells were subjected to 5-azacytidine (20 µM) treatment for 24 h. The treated and untreated tumor cells, along with preneoplastic P and P cells were subjected to genomic DNA isolation followed by HpaII digestion and Southern analysis probed with either clone sun.orf2 (nucleotides 53-1006) flanking the entire open reading frame of TIMP-3 (A) or the 2.9 kb promoter fragment (B). The size of hybridization bands is shown.



To further investigate the possible association of the mTIMP-3 gene methylation with the lack of gene expression in neoplastic cells, we treated the two neoplastic lines with 5-azacytidine, a known inhibitor of DNA methylase (50) followed by Southern and Northern analysis. We first examined whether 5-azacytidine treatment changes the HpaII digestion pattern in the TIMP-3 gene. As shown in Fig. 7, A and B, lanes4 and 6, azacytidine treatment did induce hypomethylation of the gene as well as the gene promoter region as evidenced by increased numbers of hybridizing bands in both neoplastic lines, although the hybridizing pattern is still different from that of preneoplastic P and P cells. The same treatment was conducted in both neoplastic lines to determine the effect of methylase inhibitor on the basal as well as the inducible levels of TIMP-3 gene expression. Subconfluent cells were treated with 20 µM 5-azacytidine for 24 h. Cells were divided into two groups. Group I cells were either left untreated for 2 h or treated immediately with TPA or TNF-alpha for 2 h (the duration showing the maximal induction of TIMP-3, see Fig. 5) and were then harvested. The group II cells were cultured for an additional 24 h before being subjected to 2 h of TPA or TNF-alpha treatment. Total RNA was isolated from the cells, and Northern analysis was performed. As shown in Fig. 8, 5-azacytidine did cause basal reexpression of the TIMP-3 gene in the neoplastic line (L-RT101), which showed hypermethylated status, but not in the other (H-RT101), which had a hypomethylated TIMP-3 gene. The reexpression of the gene was readily seen in group II cells, although slight reexpression of the gene can be visualized in the group I cells. Furthermore, in contrast to preneoplastic P and P cells, TPA or TNF-alpha treatment (following 5-azacytidine exposure) of neoplastic cells did not significantly elevate the expression of TIMP-3. These results indicate that abnormal methylation (either hyper- or hypo-) of the TIMP-3 gene contributes to the down-regulation of TIMP-3 gene expression in neoplastic JB6 cells.


Figure 8: Methylase inhibitor 5-azacytidine causes reexpression of the mTIMP-3 gene in L-RT101 but not H-RT101 tumor cells. Following 24 h of treatment with 5-azacytidine, tumor cells were either immediately or after 24 h in culture treated with TPA or TNF-alpha for 2 h and then subjected to total RNA isolation and Northern analysis with sun.orf2 as probe. The ribosomal RNA 28 S and 18 S were included (on the bottom) to show an approximately equal loading of the total RNA.




DISCUSSION

We have shown here the full-length cDNA sequence of the TIMP-3 gene. An interesting feature of mouse TIMP-3 messenger RNA is that it contains a short open reading frame with a very long 3`-untranslated region. Although it has been suggested that this region may reflect a regulatory function or an additional level of control over mRNA translation(7) , the biological significance of this long 3`-untranslated region is at present unclear, as is the expression of three different transcripts. Several lines of evidence suggest that the two shorter (2.8 and 2.3 kb) transcripts are likely to be alternative polyadenylation products of mTIMP-3 gene that have a common transriptional start site. Among these are 1) 3`-end probes including the clone sun.6 (nucleotides 2665-4591) do not detect them, 2) there are two polyadenylation sites with one located in nucleotides 2446-2451, suitable for shorter transcripts; 3) all three transcripts can be detected when poly(A) RNA is used, indicating that all contain the poly(A) tail; and 4) all three transcripts are subject to TPA and TNF-alpha induction (in P, P cells) and all are not expressed in neoplastic cells, indicating they are subject to similar regulation.

The sequence homology and cysteine residue conservation of mouse TIMP-3 to TIMP-3 isolated from other species (human and chicken) and to the other members of TIMP family (TIMP-1 and TIMP-2) have been discussed in detail(7) . The open reading frame of mouse TIMP-3 predicts a 23-amino-acid signal peptide followed by a mature protein consisting of 188 amino acids. For secretory proteins such as TIMP-3, the signal peptide on the newly made protein directs the ribosome to the endoplastic reticulum membrane and across it where the rest of the protein is synthesized. The signal peptide is cleaved by a peptidase, and mature protein synthesized is secreted through the Golgi vesicle (51) . An interesting finding that is consistent with previous observations (7) is that the TIMP-3 located in ECM (both mouse and human) migrates as a 24-kDa protein by Western analysis instead of 21-kDa as predicted by the amino acid composition of the mature protein (Fig. 4).^2 DNA transfection of expression constructs containing either the entire open reading frame or the sequence encoding only mature protein of TIMP-3 revealed that the former but not the latter leads to TIMP-3 expression in ECM, indicating that the N-terminal 23-amino-acid signal peptide is required for localization of TIMP-3 protein into ECM.^2 The apparent size difference seen likely reflects a post-translational modification such as phosphorylation or glycosylation, although we cannot exclude the possibility that TIMP-3 in ECM still contains the signal sequence giving a molecular mass of 24 kDa.

We report here also the cloning of the 2.9 kb of mTIMP-3 transcriptional promoter sequence and evidence indicating that it is a functional promoter. This is the longest cloned promoter among the TIMP family genes(38, 39) . Computer analysis of the sequence revealed many consensus binding sites for a variety of transcription factors. The presence of cis elements provides a basis for expecting TIMP-3 regulation by known modulators such as serum, growth factors, mitogens, tumor promoters, cytokines, hormones, and stress factors. They also imply a possible regulation of TIMP-3 by c-Myc and p53. This is currently under investigation. Overall, the high density of response elements in the TIMP-3 promoter suggests the importance of this gene in the cellular responses to many environmental stimuli.

An immediate-early or delayed early up-regulation of TIMP-3 expression has been found in response to serum stimulation, a growth factor (epidermal growth factor), cytokine (TGF-beta), tumor promoter (TPA), and anti-inflammatory agent (dexamethasone)(7, 12) . We found that in JB6 preneoplastic cells, TPA, and TNF-alpha (another cytokine) induced rapid, transient expression of TIMP-3. The induction is likely mediated through the AP-1 or NF-kappaB activations, respectively, as suggested by the TIMP-3 promoter sequence. Indeed, TIMP-3 promoter activity decreased dramatically in an AP-1-less/NF-kappaB-less construct (AP0) compared with AP6, a construct containing the full-length cloned TIMP-3 promoter fragment (see Fig. 6). It is noteworthy that L-RT101 cells re-expressing TIMP-3 after methylase inhibitor treatment are resistant to superinduction by TPA/TNF-alpha. This is consistent with the transient transactivation experiment in which there is basal but not TPA/TNF-alpha-induced reporter activation (Fig. 6) and suggest the possibility that 5-azacytidine-induced endogenous TIMP-3 expression and basal reporter activation may occur independent of AP-1 or NF-kappaB. An alternative is that AP-1 and NF-kappaB sites are saturated in untreated neoplastic cells, but something else is limiting for TPA or TNF-alpha response.

Since both TPA and TNF-alpha are tumor promoters in mouse JB6 P cells(1, 2, 3, 4, 43) , it seems paradoxical that a tumor promoter both induces TIMP-3 expression and induces transformation, whereas transformed cells show a lack of expression of TIMP-3. We hypothesize that in the JB6 model superinduction of TIMP-3 expression is necessary or required for neoplastic transformation as an early event but is not necessary for the maintenance of the tumor cell phenotype, in analogy to ``hit-and-run'' mechanisms postulated for certain DNA tumor virus(52) . As support of this hypothesis, chicken TIMP-3 has been reported to have an oncogenic transforming activity in chicken primary fibroblasts(53) . Alternatively, TIMP-3 could be a tumor suppressor gene whose down-regulation is causally related or associated with neoplastic progression. It seems illogical, but it has precedent that tumor promoters should induce a tumor suppressor gene. We and others have evidence showing that TPA and TNF-alpha induce the tumor suppressor gene WAF-1/CIP-1/p21(28, 54) . We are currently testing these hypotheses by DNA transfection experiments using the JB6 model.

Abnormal DNA methylation (both hypo- and hyper-methylation) has been frequently observed in cancer cells(55) . We have shown, for the first time to our knowledge, that either hyper- or hypomethylation of the TIMP-3 gene including the promoter might be associated with complete down-regulation of the gene in neoplastic JB6 cells. We have further shown that treatment of cells (L-RT101) having hypermethylated TIMP-3 with the methylase inhibitor 5-azacytidine causes a reexpression of the TIMP-3 gene. Due to the lack of available methylase activator, we are unable to test whether reexpression occurs in the cells (H-RT101) having a hypomethylated TIMP-3 after methylase activator treatment. We cannot, however, exclude the possibility of rapid mRNA degradation or promoter nonfunctionality in the chromatin of neoplastic cells. Nevertheless, available evidence suggests that altered methylation contributes to lack of expression.

Altered methylation can silence gene expression (shown here and (56) ) and also plays an important role in the generation of mutations in cancer cells. The high incidence found in the p53 tumor suppressor gene of CT transitions resulting from the spontaneous deamination of 5-methylcytosine residue is a good example(55, 57) . The fact that no point mutation was found in the 2.9-kb promoter region of the TIMP-3 gene excludes the possibility of mutational silencing of gene expression (both at basal and induced levels) in neoplastic cells. Since in neoplastic JB6 cells, 1) there is no point mutation in the promoter region nor is there gross deletion or rearrangement of the gene; 2) a TIMP-3 promoter driven reporter can be transactivated; and 3) the TIMP-3 gene is abnormally methylated and can be re-expressed in response to methylase inhibitor, we conclude that abnormal methylation, rather than point mutation/deletion and/or transcription factor inactivation plays a major role in the silencing of gene expression. Finally, the TIMP-3 silencing by either hyper- or hypomethylation in two neoplastic lines provides a good model to study, using a genomic sequencing techniques(58, 59) , the precise regulation of methylation/demethylation at particular cytidine(s) in the promoter region that leads to down-regulation of gene expression.

In summary, we have cloned and sequenced the full-length mouse TIMP-3 cDNA and a 2.9-kb mTIMP-3 promoter fragment and have characterized the promoter activity. We have also presented evidence suggesting that lack of expression of TIMP-3 in neoplastic JB6 cells may be largely due to abnormal methylation. These data imply an important role for TIMP-3 in neoplastic progression and provide new tools to further study its biological function.


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) Z30970 [GenBank]and U19462[GenBank].

§
To whom correspondence should be addressed: Dept. of Cancer Research, Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 313-996-1959; Fax: 313-996-7158.

(^1)
The abbreviations used are: TIMP-3, tissue inhibitor of metalloproteinase-3; hTIMP-3, human TIMP-3; mTIMP-3, mouse TIMP-3; bp, base pair(s); TPA, 12-O-tetradecanoylphorbol-13-acetate; kb, kilobase pair(s); RT, reverse transcriptase; PCR, polymerase chain reaction; ECM, extracellular matrix; TNF-alpha, tumor necrosis factor; AP, activator protein; TX, transformed.

(^2)
Y. Sun and N. H. Colburn, unpublished observations.


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