5-AZA-2′-deoxycytidine and interleukin-1 cooperate to regulate matrix metalloproteinase-3 gene expression

Article (PDF Available)inInternational Journal of Cancer 129(9):2083-92 · November 2011with36 Reads
DOI: 10.1002/ijc.25865 · Source: PubMed
Abstract
Members of the matrix metalloproteinase (MMP) family of enzymes play a critical role in extracellular matrix remodeling in a number of normal and pathologic processes. Accordingly, activation of MMP gene expression is tightly regulated at the level of transcription by specific transcription factors, most notably following exposure to inflammatory cytokines. Recent studies with 5-aza-2'-deoxycytidine (5-aza-dC), a specific DNA methylase inhibitor, also suggest that epigenetic processes contribute to the regulation of MMP expression. Although inflammation-related aberrant patterns of DNA methylation have been described, a mechanistic link between inflammation and epigenetic alterations in the control of MMP expression remains unclear. Here, we provide evidence that increased MMP-3 expression by 5-aza-dC is modulated by interleukin-1 (IL-1). More specifically, we found that stimulation with IL-1, but not with IL-6 or TNFα, significantly increased the hypomethylation status of the MMP-3 promoter to a level similar to that found in dnmt1/dnmt3b-deficient HCT116 (DKO) cells. Furthermore, we showed that increased MMP-3 expression by 5-aza-dC was associated with increased expression and activity of specific transcription factors known to regulate MMP-3 expression. In fact, treatment with 5-aza-dC was obligatory for some transcription factors to trigger an increase in MMP-3 expression, such as Ap-1. In contrast, CCAAT enhancer-binding proteins and E-twenty six were capable of inducing MMP-3 alone. Overall, these findings provide a novel perspective of the collaborative role of 5-aza-dC and inflammatory cytokines with specific transcription factors that are normally involved in MMP-3 expression.
5-Aza-2
0
-deoxycytidine and interleukin-1 cooperate to regulate
matrix metalloproteinase-3 gene expression
Julie Couillard
1
, Pierre-Olivier Este
`
ve
2
, Sriharsa Pradhan
2
and Yves St-Pierre
1
1
INRS-Institut Armand-Frappier, Universite
´
du Que
´
bec, Laval, Que
´
bec, Canada
2
New England Biolabs Incorporated, 240 County Road, Ipswich, MA
Members of the matrix metalloproteinase (MMP) family of enzymes play a critical role in extracellular matrix remodeling in a
number of normal and pathologic processes. Accordingly, activation of MMP gene expression is tightly regulated at the level of
transcription by specific transcription factors, most notably following exposure to inflammatory cytokines. Recent studies with
5-aza-2
0
-deoxycytidine (5-aza-dC), a specific DNA methylase inhibitor, also suggest that epigenetic processes contribute to the
regulation of MMP expression. Although inflammation-related aberrant patterns of DNA methylation have been described, a
mechanistic link between inflammation and epigenetic alterations in the control of MMP expression remains unclear. Here, we
provide evidence that increased MMP-3 expression by 5-aza-dC is modulated by interleukin-1 (IL-1). More specifically, we found
that stimulation with IL-1, but not with IL-6 or TNFa, significantly increased the hypomethylation status of the MMP-3 promoter
to a level similar to that found in dnmt1/dnmt3b-deficient HCT116 (DKO) cells. Furthermore, we showed that increased MMP-3
expression by 5-aza-dC was associated with increased expression and activity of specific transcription factors known to
regulate MMP-3 expression. In fact, treatment with 5-aza-dC was obligatory for some transcription factors to trigger an increase
in MMP-3 expression, such as Ap-1. In contrast, CCAAT enhancer-binding proteins and E-twenty six were capable of inducing
MMP-3 alone. Overall, these findings provide a novel perspective of the collaborative role of 5-aza-dC and inflammatory
cytokines with specific transcription factors that are normally involved in MMP-3 expression.
Matrix metalloproteinases (MMPs) are a family of zinc-de-
pendent proteolytic enzymes that are capable of degrading
practically all components of the extracellular matrix or other
extracellular targets, including other MMPs. They contribute
to many physiological processes, including embryogenesis,
wound healing and tumor progression. Transcriptional dysre-
gulation of MMP expression plays a prominent role in sev-
eral diseases that involve tissue destruction. For example, ele-
vated levels of stromelysin-1 (MMP-3) have been implicated
in the progression of Crohn’s disease,
1
cancer,
2
rheumatoid
arthritis
3
and coronary diseases.
4
MMP expression is normally low in cells under regular
physiological conditions and is controlled by strict regulatory
mechanisms. The expression of MMPs dramatically increases
following exposur e to a variety of inflammatory mediators.
For example, interleu kin-1 (IL-1) plays a central role in con-
trolling MMP-3 expression, most notably via activation of ac-
tivator protein-1 (AP-1) and members of the E-twenty six
(ETS) family of transcription factors.
5,6
The transcription fac-
tor CCAAT enhancer-binding protein (C/EBP) has also been
implicated in IL-1-mediated MMP-3 expression.
7
Recent studies have shown that the methylation status of
cytosines in CpG dinucleotides located in the MMP promoter
also plays a role in controlling MMP gene expression, most
notably in cancer cells.
8–13
An abnormal DNA methylation
pattern is one of the hallmarks of cancer cells, which normally
harbor widespread DNA hypomethylation of tumor-promoting
genes along with site-specific DNA hypermethylation of tumor
suppressor genes.
14
Although studies have shown that deme-
thylating agents, through the induction of tumor suppressor
gene expression, can decrease tumorigenesis, a possible con-
cern regarding hypermethylation therapy is that it would result
in the activation of tumor-promoting genes, such as MMP
genes. Whether such epigenetic mechanisms alter IL-1-induced
MMP expression, however, remains unclear.
In our work, we report that IL-1 and 5-aza-2
0
-deoxycytidine
(5-aza-dC), a specific DNA methylase inhibitor, cooperate to
induce hypomethylation of the MMP-3 gene at the promoter lev el
in human colorectal cancer cells. Furthermore, 5-aza-dC signifi-
cantly upregulated the activity of transcription factors involved in
MMP-3 expression, thereby contributing to its increased expres-
sion level. Taken together, these results provide a mechanistic link
between IL-1-mediated inflammatory processes and epigenetic
alterations in the control of MMP gene expression.
Key words: matrix metalloproteinase, MMP-3, IL-1, DNA
methylation, gene expression
Additional Supporting Information may be found in the online
version of this article.
Conflict of interest: The authors declare no competing interests
Grant sponsors: National Sciences and Engineering Council of
Canada (NSERC), Canadian Institutes for Health Research
DOI: 10.1002/ijc.25865
History: Received 16 Sep 2010; Accepted 30 Nov 2010; Online 17
Dec 2010
Correspondence to: Yves St-Pierre, INRS-Institut Armand-Frappier,
531 Boul. des Prairies, Laval, Que
´
bec, Canada H7V1B7, Tel.:
þ450-686-5354, Fax: þ450-686-5501, E-mail: yves.st-pierre@iaf.inrs.ca
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International Journal of Cancer
IJC
Material and Methods
Cell lines and reagents
The HeLa cell line was obtained from the American Type
Culture Collection (ATCC, Manassas, VA) and maintained in
Dulbecco’s modified Eagle’s medium [supplemented with
10% (v/v) FCS, 2 mmol/L
L-glutamine and 10 mmol/L HEPES
buffer]. HCT-116 colon cancer cells and double
Dnmt1
/
Dnmt3b
/
(DKO) HCT116 cells were generous
gifts from Dr. Bert Vogelstein (Johns Hopkins Kimmel Compre-
hensive Cancer Center, Baltimore, MD). Cells were grown in
McCoy’s 5A complete medium. All cell culture products were
from Life Technologies (Burlington, ON, Canada). 5-Aza-dC
was purchased from Sigma chemicals (St. Louis, MO).
Recombinant human IL-1, TNFa and IL-6 were from Invitro-
gen (Burlington, ON, Canada).
RNA isolation and semiquantitative RT-PCR
Total cellular RNA was isolated from HCT116, DKO or
HeLa cells using TRIzol reagent (Life Technologies) accord-
ing to the manufacturer’s instructions. First-strand cDNA
was prepared from 2 lg of cellular RNA in 20 lL of reaction
volume using the preamplification system reverse transcrip-
tase RTomniscript (QIAGEN, Mississauga, ON, Canada).
After reverse transcription, hMMP-3 (gene ID 4314) (sense
primer: 5
0
-AGA GGT GAC TCC ACT CAC AT-3
0
and anti-
sense: 5
0
-GGT CTG TGA GTG AGT GAT AG-3
0
), product
size: 310 bp; hMMP-9 (gene ID 4318) (sense: 5
0
-CAA CAT
CAC CTA TTG GAT CC-3
0
and antisense : 5
0
-CGG GTG
TAG AGT CTC TCG CT-3
0
), product size: 479 bp and
GAPDH (gene ID 2597) (sense: 5
0
-CGG AGT CAA CGG
ATT TGG TCG TAT-3
0
and antisense: 5
0
-CAG AAG TGG
TGG TAC CTC TTC CGA-3
0
), product size: 307 bp, cDNAs
were amplifier using the following conditions: 94
C for 0.5
min, 58
C for 1 min and 72
C for 1 min, followed by a final
extension step at 72
C for 10 min. Thirty-five cycles of
amplification were performed in a thermal cycler (MJ
Research, Watertown, MA). The amplified products were an-
alyzed by electrophoresis in 1% agarose gels using SYBR Safe
DNA gel (Life Technologies) staining and UV illumination.
Plasmid construction and site-directed mutagenesis
An 877-bp PCR-amplified genomic DNA fragment encoding
the 5
0
flanking promoter of the human MMP-3 gene was
obtained from genomic DNA isolated from HCT116 cells
using the following primers: sense: 5
0
-TAG GGA GGA GGG
GAA A-3
0
and antisense: 5
0
-TTC TCT CAA CCT TCC
CAAT-3
0
. The promoter was further subcloned into the lucif-
erase gene (reporter vector pGL3-Basic) (Promega, Madison,
WI). Oligonucleotide-directed site-specific mutagenesis of
AP-1 and C/EBP consensus sites was carried out with the
QuikChange Mutagenesis Kit from Stratagene (La Jolla, CA).
The AP-1 site was mutated using the following primers:
sense: 5
0
-GCA AGG ATG TTT CAA GCTG-3
0
and antisense:
5
0
-CAG CTT GAA ACA TCC TTGC-3
0
. The C/EBP site was
mutated using the following primers: sense: 5
0
-ACT TTG
AAT TTC CTG TGT TTC CTG CAG GTCC-3
0
and anti-
sense: 5
0
-GGA CCT GCA GGA AAC ACA GGA AAT TCA
AAGT-3
0
. The ETS-mutated MMP-3 promoter was a gener-
ous gift from Dr. M. Aumer cier (Institut de Biologie de Lille,
Institut Pasteur de Lille, Lille Cedex, France).
15
The expres-
sion vector encoding full-length c-Jun was a generous gift
from Dr. Michael J. Birrer (National Institutes of Health,
MD). All other cDNA expression vectors were purchased
from OriGene Technologies (Rockville, MD).
Transient transfection and luciferase assay
HCT116 cells were seeded the day before treatment with
5-aza-dC (5 lM) for 48 hr. Cells were then washed twice
with PBS and transfected with 0.1–1.0 lgofplasmidDNA
using DNAfectin (Applied Biological Materials, Richmond, BC,
Canada) according to the manufacturer’s protocol. The culture
medium was changed to complete McCoy’s medium 5 hr after
transfection. Forty-eight hours after transfection, recombinant
IL-1 (10 ng/mL) was added, and cells were incubated for an
additional 20 hr. The transfection efficiency was monitored
using the pCMV/b-gal reporter vector (Promega). Luciferase ac-
tivity was measured using the Luciferase Assay System protocol
(Promega) and a luminometer (Lumat LB 9507, Berthold). The
b-galactosidase activity was detected using a colorimetric
enzyme assay using the Luminescent b-Galactosidase Detection
Kit II (Clontech Laboratories, Mountain View, CA).
Preparation of genomic DNA and bisulfite treatment
HCT116 or HeLa cells were treated with 5-aza-dC (5 lM)
for 72 hr with or without 10 ng/mL of human recombinant
IL-1 for the last 20 hr before harvesting the cells. Genomic
DNA was then isolated and trea ted with sodium bisulfite
using the EpiTect kit (QIAGEN) according to the manufac-
turer’s instructions. The methylation status of the MMP-3
promoter region was analyzed by PCR amplification by mix-
ing bisulfite-treated DNA with specific oligoprimers using the
Taq DNA Polymerase Kit (QIAGEN). The samples were
incubated for 2 min at 95
C, followed by 40 cycles as follows:
1 min at 95
C; 2 min at 56
C; 1 min at 72
C and a final
extension at 72
C for 10 min. The product of the first round
of PCR was then subjected to a second round of amplifica-
tion with a set of nested primers under the same PCR condi-
tions. PCR products were separated by gel electrophoresis
and cloned into the pCR4-TOPO vector (Life Technologies)
using the TOPO-TA Cloning Kit (Life Technologies). Ten to
twenty clones were subjected to sequencing. The sequences of
the primers used to amplify the human MMP-3 promoter
from bisulfite-treated DNA are available upon request.
Western blot analysis
HCT116 cells were washed with PBS and lysed at 4
C for
20 min with a lysis buffer containing 50 mM Tris-Cl, pH 8.0,
150 mM NaCl, 0.02% sodium azide, 100 lg/mL phenylmethyl-
sulfonyl fluoride, 1% Nonidet P-40 and a protease inhibitor
mixture (Sigma). The protein concentrations in the supernatants
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were determined using the Bio-Rad protein assay reagent based
on the Bradford colorimetric method. After electrophoresis in
an 11% sodium dodecyl sulfate-polyacrylamide gel (Life Tech-
nologies), the proteins were transferred onto Protran
V
R
pure
nitrocellulose membranes (Schleicher and Schuell). After over-
night electroblotting at 4
C, the membranes were incubated
with Tris-buffered saline containing 0.1% Tween-20, 5% nonfat
dry milk (Bio-Rad) and a rabbit polyclonal antibody against
DNMT1 (New England Biolabs), c-Jun (Cell Signaling Technol-
ogies; CST) and rabbit polyclonal antibody against actin
(Sigma). Anti-mouse or anti-rabbit secondary antibodies conju-
gated to horseradish peroxidase were used, and the bands were
visualized with the chemiluminescence detection system (CST).
Results
IL-1 and 5-aza-dC synergize to induce MMP-3
expression in HCT116 colon carcinoma cells
Our previous studies have shown that methylation inhibitors,
such as 5-aza-d C or zebularine, induce MMP-3 expression in
HCT116 cells.
9
As this induction was the highest observed
among the MMP tested, and because MMP-3 is involved in
the progression of colorectal cancer and colorectal chronic
inflammation diseases,
16,17
we have further investigated the
mechanisms regu lating its expression. Because IL-1 is known
to induce MMP expression and because epigenetic chan ges
have been linked to inflammatory processes, we investigated
whether DNA hypomethylation could influence IL-1-induced
MMP-3 expression. Our results showed that 5-aza-dC signifi-
cantly increased IL-1-induced MMP-3 expression in
HCT116 when compared to cells treated with IL-1 or 5-aza-
dC alone (Fig. 1a). The combined effect of IL-1 and hypome-
thylation was also observed using Dnmt1/Dnmt3b double-
knockout (DKO) HCT116 cells, which demonstrated higher
MMP-3 expression levels following IL-1 stimulation than did
control wild-type HCT116. Independent dose-response
experiments (1–50 ng/mL) showed that maximal MMP-3
expression was reached at a dose of 5 ng/mL of IL-1 (Data
known). Kinetic analysis showed that the ability of 5-aza-dC
to increase IL-1-induced MMP-3 expression was higher at
4–12 hr poststimulation (Fig. 1b). Nevertheless, a similar syn-
ergistic effect between 5-aza-dC and IL-1 was also discovered
when we examined the expression levels of MMP-9, another
member of the MMP family that is regulated by DNA hypo-
methylation
8
(Supporting Information).
IL-1 increases demethylation of the MMP-3 promoter
Sequencing of bisulfite-converted genomic DNA was used to
determine the methylation status of the MMP-3 promoter in
5-aza-dC-treated HCT116 cells that were stimulated or not
with IL-1. As expected, 5-aza-dC induced MMP-3 promoter
hypomethylation when compared to nontreated cells (Fig. 2).
Interestingly, however, we found that stimulation with IL-1
significantly increased the hypomethylation status of the pro-
moter induced by 5-aza-dC to a level similar to that found in
control DKO cells. A small but significant effect of IL-1 alone
on the methylation status of HCT116 cells was also observed.
This cooperative effect of IL-1 and 5-aza-dC on hypomethy-
lation and MMP-3 expression was also observed using HeLa
cells (Fig. 3) and was specific because no significant increase
was observed with IL-6 or TNFa. Similar results were
obtained for MMP-9 (Supporting Information).
5-aza-dC cooperates with c-Jun to upregulate
MMP-3 expression in HCT116 cells
Consensus sequences located within the 800-bp region of the
transcription initiation site have been shown to be essential
for full promoter MMP-3 activity
18
(Supporting Information).
This promoter region contains consensus-binding sites for the
AP-1, ETS and C/EBP transcription factors. Therefore, we
examined whether 5-aza-dC and IL-1 cooperated with these
factors to induce MMP-3 expression. We initially focused on
AP-1, which plays a central role in the activation of most of
the MMP genes.
19
Our results showed that although all AP-1
Figure 1. IL-1 and 5-aza-dC synergize to induce MMP-3 expression
in HCT116 colon carcinoma cells. (a)MMP-3 expression levels in
HCT116 and DKO cells following treatment with 5-aza-dC (5 lM, 72
hr) and/or IL-1 (20 hr). The DKO cells were only treated with IL-
1 because of the already unmethylated status of the cells, and
they were used as control of hypomethylated cells. (b) Kinetic
analysis showing changes in MMP-3 gene expression following
treatment with 5-aza-dC and/or IL-1, as measured by real-time
quantitative PCR. The results represent at least three independent
experiments. GAPDH was used as loading control. Two-way ANOVA
was done for statistical analysis p values; untreatedvs. 5-aza-dC:
p < 0.001 (between 12 and 20 hr); untreatedvs. IL-1: p < 0.05 (at
20 hr); untreatedvs. 5-aza-dC þ IL-1: p < 0.001 (between 4 and
20 hr); 5-aza-dC vs. IL-1: p < 0.01 (between 12 and 20 hr); 5-aza-
dC vs. 5-aza-dC þ IL-1: p < 0.001 (between 4 and 12 hr) and IL-1
vs. 5-aza-dC þ IL-1: p < 0.001 (between 4 and 12 hr).
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isoforms alone were mostly ineffective in inducing MMP-3
gene expression in HCT116 cells, each was capable of induc-
ing such expression in the presence of 5-aza-dC, although c-
Jun was the most effective among the isoforms to induce
strong and constitutive MMP-3 expression (Fig. 4a and data
not shown). A similar cooperativ e effect between c-Jun and
Figure 2. IL-1 and 5-aza-dC synergize to demethylate the MMP-3 promoter in HCT116 cells. (a) Bisulfite DNA sequence analysis showing the
methylation status of CpG dinucleotides located in the proximal region of the human MMP-3 promoter in HCT116 cells following treatment with
IL-1 and/or 5-aza-dC. Bisulfite sequencing has also been done in DKO cells as demethylated model. (b) Schematic representation showing the
overall effect of IL-1 on 5-aza-dC-induced hypomethylation within the MMP-3 promoter, as determined by bisulfite sequencing in (a).
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5-aza-dC was also observed under transient transfection con-
ditions using a reporter construct containing the MMP-3 pro-
moter (Fig. 4b). This ability of 5-aza-dC was most likely
linked to its ability to significantly increase c-Jun expression
and nuclear activity ( p < 0.05) (Figs. 4c and 4d). In contrast,
IL-1 could not induce a similar increase in AP-1-dependent
nuclear activity, consistent with its inability to augment exog-
enous MMP-3 promoter activation.
Functional interaction between 5-aza-dC and other
transcription factors in the regulation of
MMP-3 promoter activity
Our results showing that de novo expression of AP-1 iso-
forms alone was insufficient to induce MMP-3 and required
treatment with 5-aza-dC are consistent with previous studies
showing that AP-1-induced MMP expression is dependent
on the presence of other transcription factors.
5,20,21
For
example, using reporter vectors, Westerm arck et al. have
shown that MMP-1 promoter activity is dependent on func-
tional interactions between AP-1 and ETS factors.
20
There-
fore, we investigated whether other transcription factors
could cooperate with 5-aza-dC, most notably C/EBP, which
acts as a positive transcriptional activator of MMP.
22,23
Our
results showed that in contrast to AP-1 isoforms, each C/
EBPa and b isoform alone could induce endogenous MMP-3
gene expression in HCT116 cells (Fig. 5a). This induction by
C/EBP increased significantly following treatment with 5-aza-
dC (p < 0.001). Such a functional interaction between 5-aza-
dC and C/EBP was also observed using the MMP-3 reporter
construct (Fig. 5b). This finding was consistent with the abil-
ity of 5-aza-dC, but not IL-1, to significantly increase nuclear
C/EBP-dependent activity (p < 0.001) (Fig. 5c). However, a
similar cooperative response between 5-aza-dC and transcrip-
tion factors was not observed with ETS, although ETS alone
could upregulate MMP-3 expression (Fig. 6a). This finding is
consistent with our results showing that, in contrast to c-Jun-
Figure 3. Cooperative effect of IL-1 and 5-aza-dC on endogenous MMP-3 gene expression in HeLa cells. (a) MMP-3 expression levels in
HeLa cells induced by 5-aza-dC alone or in the presence of IL-1, IL-6 or TNFa (10 ng/mL). GAPDH was used as a loading control. The
results represent at least three independent experiments. (b) Schematic representation showing the overall effect of IL-1 on 5-aza-dC-
induced hypomethylation within the MMP-3 promoter in HeLa cells, as determined by bisulfite sequencing.
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and CEBP-mediated activity, no significant increase in nu-
clear ETS-dependent activity was observed following treat-
ment of HCT116 cells with 5-aza-dC (Fig. 6b). Although
mutations in the consensus sequences of these transcription
factors showed that AP-1, ETS and CEBP were all required
to obtain maximal MMP-3 promoter activity (Supporting In-
formation), functional deletion of the AP-1-binding sites in
the MMP-3 reporter vector completely abolished the ability
Figure 4. The role of c-JUN in MMP-3 gene expression in HCT116 cells following treatment with 5-aza-dC. (a) MMP-3 expression levels in
HCT116 determined by RT-PCR analysis following transfection with an expression vector encoding human c-Jun, in the absence or the
presence of 5-aza-dC and/or IL-1. GAPDH was used as a loading control. The results represent at least three independent experiments.
(b)HCT116 cells were transiently transfected with a luciferase reporter plasmid containing 877 bp of the human MMP-3 promoter and a c-
JUN expression vector in the presence of 5-aza-dC and/or IL-1. Translational activity of luciferase mRNA in response to each stimulus was
quantified as the ratio of luciferase activity to b-galactosidase activity. (c) Expression of Dnmt1 and c-Jun in HCT116 cells following
treatment with 5-aza-dC (5 lM) and IL-1 (10 ng/mL), as determined by Western blot analysis. Actin was used as a loading control. The
results represent at least three independent experiments. (d)AP-1-dependent transcriptional activity following treatment with 5-aza-dC and/
or IL-1. AP-1-specific translational activity of luciferase mRNA in response to IL-1 and/or 5-aza-dC was quantified as the ratio of luciferase
activity to b-galactosidase activity. The results represent at least three independent experiments.
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of 5-aza-dC to increase MMP-3 expression (Supporting Infor-
mation), suggesting that AP-1 plays an essential role in
MMP-3 promoter activity in hypomethylated HCT116 cells.
Discussion
In our work, we report that DNA methylation and IL-1
cooperate to induce MMP-3 gene expression. More specifi-
cally, we showed that (i) stimulation with IL-1 significantly
increased the hypomethylation status of the MMP-3 pro-
moter to a level similar to that found in dnmt1/dnmt3b-defi-
cient DKO cells. This effect was observed in both colon
HCT116 carcinoma cells and HeLa cells; (ii) this demethyla-
tion correlated with higher constitutive expression of the
MMP-3;(iii) increased MMP-3 expression by 5-aza-dC was
associated with increased expression and activity of specific
transcription factors, including AP-1 and C/EBP and (iv)
although ETS, AP-1 and C/EBP all play an important role in
MMP-3 expression, they showed distinct abilities to induce
MMP-3 and were differentially sensitive to 5-aza-dC. For
example, although C/EBP and ETS were capable of inducing
MMP-3 alone, de novo expression of AP-1 isoforms required
treatment with 5-aza-dC. Taken together, these results pro-
vide evidence that the regulation of MMP-3 gene expression
by DNA demethylation induced by 5-aza-dC invo lves coop-
erative interactions with IL-1 and specific transcription
factors.
Recently, several studies have shown that the demethyla-
tion of MMP promoters induced by treatment with 5-aza-dC
or by progres sion of diseases ( e.g., osteoarthritis and cancer)
plays an important role in MMP gene expression.
8,9,11–13
Our
study provides a novel perspective of the collaborative effect
of 5-aza-dC and inflammatory cytokines on MMP-3 gene
expression via specific transcriptional pathways. Although
our results could not establish whether 5-aza-dC directly
affected the ability of these transcription factors to bind the
MMP-3 promoter, they suggested that 5-aza-dC acted, at
least in part, by increasing the expression and function of
these factors. Upregulation of MMP-3 expression by the com-
bining effect of 5-aza-dC was also accelerated. For example, a
significant increase in MMP-3 expression was observed
within 4–8 hr using both IL-1 and 5-aza-dC when compared
to 12–20 hr in cells treated with 5-aza-dC or IL-1 alone. This
hypothesis is consistent with previous results showing that
increased c-JUN activity is associated with hypomethylation
Figure 5. Effect of 5-aza-dC on C/EBP- and ETS-dependent transcriptional activity and MMP-3 gene expression. (a) MMP-3 expression levels
in HCT116 determined by RT-PCR analysis following transfection with an expression vector encoding human C/EBPa and C/EBPb isoforms,
in the absence or the presence of 5-aza-dC and/or IL-1. GAPDH was used as a loading control. (b)Luciferase activity of a luciferase reporter
plasmid containing 877 bp of the human MMP-3 promoter following cotransfection with an expression vector encoding human C/EBPa,in
the absence or the presence of 5-aza-dC and/or IL-1. Translational activity of luciferase mRNA in response to each stimulus was quantified
as the ratio of luciferase activity to b-galactosidase activity. (c)CEBPa-dependent transcriptional activity following treatment with 5-aza-dC
and/or IL-1. Translational activity of luciferase mRNA was quantified as the ratio of luciferase activity to b-galactosidase activity. The results
are the average of triplicates and represent at least three independent experiments.
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of the c-JUN promoter in tumor cells. This hypomethylation
state has been further associated with increased carcinogene-
sis. For instance, carcinogenic environmental contaminants
induce c-JUN mRNA expression following promoter hypome-
thylation.
24
In fact, the hypomethylation of protooncogenes is
one of the first lines of evidenc e of tumorigenic development
associated with epigenetic mechanisms.
25
Although the contributions of ETS and AP-1 to MMP-3
gene expression have been previously documented, our data
further suggest that C/EBPa plays an important role in the
regulation of MMP-3. Although C/EBP-binding sites have
been reported in the promoters of several MMP genes, our
data provide evidence that C/EBP a and C/EBPb are both
strong inducers of MMP-3 transcription activity. This role of
C/EBP in MMP-3 gene activation is further supported by our
results showing that deletion of a C/EBP consensus site in
the MMP-3 promoter significantly reduces its transcriptional
activity. It should be noted, however, that we mutated only
one of three potential C/EBP-binding sites within the MMP-3
promoter, which may explain the higher residual MMP-3 ac-
tivity in the C/EBP mutant when compared to cells harboring
constructs with mutations in the AP-1- or ETS-binding sites.
The determination of whether the other C/EBP consensus
sites are functional requires further investigation.
Our results showing that MMP-3 expression correla ted
with the ability of IL-1 to induce DNA demethylation were
initially unexpected. In contrast to 5-aza-dC, we found that
IL-1 did not induce DNMT1 degradation. Whether IL-1
induces active demethylation via Gadd45, as suggested by
recent studies, remains a possibility.
26,27
Nevertheless, our
observations are consistent with those of Hashimoto et al.,
who recently reported changes in DNA methylation in
human articular chondrocytes following stimulation with
inflammatory cytokines, including IL-1.
28
How IL-1 induces
demethylation of the MMP-3 promoter is currently being
investigated.
Although the clinical use of azacytidine and decitabi ne
has received significant attention, little is known about their
molecular mechanisms of action. Nucleoside analogs clearly
demonstrate cytotoxicity effects and induce DNA hypome-
thylation.
29
Recent studies have shown that DNA damage
response (DDR) is induced by decitabine.
30
Interestingly,
inflammatory cytokines can also induce DDR, most notably
during the cellular senescence process.
31
Chronic inflamma-
tion is associated with aging and plays an important role in
many diseases such as cancer, atherosclerosis and osteoarthri-
tis.
32
Several factors and signaling pathways can induce estab-
lishment of senescence.
33
Among these factors, we found
MMP-3, -10 and -1, IL-1, IL-6, IL-8, ICAM-1 and TIMP-2.
34
These factors are also called senescence-associated secretory
phenotype (SASP). SASP genes, most notably MMP-3 and
IL-1, are constitutively expressed in cancer cells because
of the senescence induction process. Interestingly, we found
that MMP-3 expression is induced and persists in epithelial
cells after a 5-aza-dC/IL-1 treatment. Whether other SASP
genes are concomitantly induced in HCT116 and HeLa cells
remains an interesting possibility.
35–37
Many genes coding for
SASP proteins are physically regrouped in loci of human and
mouse genomes, most notably many MMPs (MMP-1, -3, -10
and -12 and other) or members of CXCL and CCL cytokines
family. These loci constitute important organization units of
chromatin and transcriptional control.
38
From a therapeutical
point of view, if decitabin (or 5-aza-dC) induces DNA dam-
ages/DNA repair along with chronic inflammation, one could
envisage that pharmacological agents such as decitabine could
be added to stimuli-inducing senescence. This would give a
mechanistic explanation by which IL-1 can contribute to
demethylate MMP-3 promoter with 5-aza-dC.
Finally, given their ability to induce the expression of tu-
mor suppressor genes, epigenetic drugs such as 5-aza-dC are
currently used for the treatment of various forms of
cancers.
39,40
Although MMPs have traditionally been associ-
ated with increased aggressiveness, recent studies using genet-
ically engineered MMP-deficient mice have also shown that
MMPs may also prevent or inhibit tumor progre ssion.
41,42
Whether 5-aza-dC may increase this beneficial MMP-de-
pendent antitumoral response or whether its therapeutic effi-
ciency is impaired by protumorigenic activity remains a chal-
lenging question.
Acknowledgements
The authors thank Diane Tremblay for her excellent technical support and
Dr. Edouard F. Potworowski for critical reading of the manuscript. J.C. is
supported by the Canadian Institutes for Health Research.
Figure 6. Effect of 5-aza-dC on ETS-dependent transcriptional
activity and MMP-3 gene expression. (a) MMP-3 expression in
HCT116 after treatment with 5-aza-dC and/or transient transfection
of expression vectors encoding c-JUN or ETS2. GAPDH was used as
a loading control. (b)ETS-dependent transcriptional activity in
HCT116 following treatment with 5-aza-dC. Translational activity of
luciferase mRNA was quantified as the ratio of luciferase activity to
b-galactosidase activity. The results are the average of triplicates
and represent at least three independent experiments.
Cancer Cell Biology
2090 Regulation of MMP-3 gene expression
Int. J. Cancer: 129, 2083–2092 (2011)
V
C
2010 UICC
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    • "Although we did not measure proliferation in the tissue explants, glucose uptake by the tissue explants was measured in culture media and confirmed that tissues remained viable throughout the culture period (Table 1). AZA could be directly interacting with transcription factors [65] or re-organizing chromatin in such a way that regulatory regions within the promoter are exposed666768 , thus facilitating binding of transcription factors which are increased in the presence of infection. We saw little variation in both TIMP-1 promoter-specific and global methylation (data not shown) in response to the explant treatments; therefore the increased TIMP-1 transcription observed in tissues pre-treated with AZA and subsequently cultured with LPS indicates that TIMP-1 activation appears to require an AZA-induced change in chromatin structure, such that DNA binding sites in the promoter region become accessible to transcriptional activators. "
    [Show abstract] [Hide abstract] ABSTRACT: Background An appropriate transcriptional profile in the placenta and fetal membranes is required for successful pregnancy; any variations may lead to inappropriate timing of birth. Epigenetic regulation through reversible modification of chromatin has emerged as a fundamental mechanism for the control of gene expression in a range of biological systems and can be modified by pharmacological intervention, thus providing novel therapeutic avenues. TIMP-1 is an endogenous inhibitor of MMPs, and hence is intimately involved in maintaining the integrity of the fetal membranes until labor. Objective and Methods To determine if TIMP-1 is regulated by DNA methylation in gestational tissues we employed an in vitro model in which gestational tissue explants were treated with demethylating agent 5-aza-2'-deoxycytidine (AZA) and lipopolysaccharide (LPS). Results Quantitative Real-Time PCR (qRT-PCR) revealed that TIMP-1 transcription was significantly increased by combined treatment of AZA and LPS, but not LPS alone, in villous, amnion and choriodecidua explants after 24 and 48 hrs, whilst western blotting showed protein production was stimulated after 24 hrs only. Upon interrogation of the TIMP-1 promoter using Sequenom EpiTyper MassARRAY, we discovered sex-specific differential methylation, in part explained by x-linked methylation in females. Increased TIMP-1 in the presence of LPS was potentiated by AZA treatment, signifying that a change in chromatin structure, but not in DNA methylation at the promoter region, is required for transcriptional activators to access the promoter region of TIMP-1. Conclusions Collectively, these observations support a potential role for pharmacological agents that modify chromatin structure to be utilized in the therapeutic targeting of TIMP-1 to prevent premature rupture of the fetal membranes in an infectious setting.
    Full-text · Article · Dec 2015
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    Article · Nov 2012
  • [Show abstract] [Hide abstract] ABSTRACT: Intrinsic or photo-induced skin aging mainly affects the structural organization of collagen and elastin fibers, the remodeling of which involves proteases such as matrix metalloproteinases (MMPs). Indeed, senescent fibroblasts are characterized by high MMP secretion levels. MMPs display several types of regulation, of which epigenetic modifications may be influenced by the cell environment itself. In addition, amplified oxidative stress following ultraviolet exposure drives MMP expression, leading to transregulation between the dermis and epidermis. Subsequently, the importance of neutrophil elastase in solar elastosis generates a feedback loop, in which elastolysis can mediate collagenolysis through the generation of elastin-derived peptides. Disorganization of dermal collagen and elastin fibers in aged dermis may release peptides containing a GxxPG motif; these are named elastokines as they induce biological effects in a similar way to cytokines. These peptides may have a crucial role in skin inflammaging through the liberation of proinflammatory cytokines, thus leaving the elderly highly susceptible to diseases such as skin cancers, as well as inflammatory and autoimmune diseases.
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