(Received for publication, December 21, 1994; and in revised form, June 6, 1995)
From the
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-B 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-
B activators,
12-O-tetradecanoylphorbol-13-acetate, and tumor necrosis
factor-
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.
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) ()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.
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-B 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-B 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).
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
-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).
Figure 5:
Induction of TIMP-3 expression by TPA and
TNF- in preneoplastic JB6 cells. Cells at 70% confluency were
subjected to TPA (10 ng/ml, panelA) or TNF-
(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.
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-
B) 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-
-induced killing, (19) , upperpanel) or TNF-
(150 units/ml) for H-RT101 (sensitive
to TPA but resistant to TNF-
-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.
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-
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-
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-
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- 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.
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-
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). 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.
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-), tumor promoter (TPA), and
anti-inflammatory agent (dexamethasone)(7, 12) . We
found that in JB6 preneoplastic cells, TPA, and TNF-
(another
cytokine) induced rapid, transient expression of TIMP-3. The induction
is likely mediated through the AP-1 or NF-
B activations,
respectively, as suggested by the TIMP-3 promoter sequence. Indeed,
TIMP-3 promoter activity decreased dramatically in an
AP-1-less/NF-
B-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-
. This is consistent with the transient
transactivation experiment in which there is basal but not
TPA/TNF-
-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-
B. An alternative is that AP-1 and NF-
B sites are
saturated in untreated neoplastic cells, but something else is limiting
for TPA or TNF-
response.
Since both TPA and TNF- 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-
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.
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].