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and IFN-
: Critical Role of STAT-1
1
Department of Cell Biology, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
, cytokines used for
treating some cancers and multiple sclerosis, on MMP-9 expression in
human astroglioma and fibrosarcoma cell lines and primary astrocytes.
Our results demonstrate that IFN- and IFN- significantly inhibit
MMP-9 enzymatic activity and protein expression that is induced by PMA
and the cytokine TNF-
. The inhibitory effects of IFN- and IFN-
on MMP-9 expression correlate with decreased steady state MMP-9 mRNA
levels and suppression of MMP-9 promoter activity. IFN-- and
IFN--mediated inhibition of MMP-9 gene expression is dependent on
the transcription factor STAT-1, since IFN- and IFN- fail to
suppress MMP-9 expression in STAT-1-deficient primary astrocytes and
human fibrosarcoma cells. Reconstitution of human STAT-1
successfully restores the inhibitory effects of IFN- and IFN- on
MMP-9 gene expression. Thus, these data demonstrate the critical role
of STAT-1 in IFN- and IFN- suppression of MMP-9 gene
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1, insulin-like growth factor-binding proteins, and FasL can be
cleaved by MMPs, which is a crucial step in regulating the availability
and function of biologically active forms of cytokines and growth
factors (6, 7, 8, 9). The direct correlation between high levels of MMPs and diseases that feature enhanced proteolytic turnover of the ECM, such as malignant tumors, has been observed both in vivo and in vitro (for reviews, see Refs. 3 and 4). Aberrant expression of MMPs has been documented in head and neck carcinomas, astrogliomas, gastric cancer, and melanomas (4). In addition, serum and tissue MMP levels or profiles of MMP expression are used as prognostic factors in certain types of malignant tumors (5). Two of the gelatinases, 72-kDa type IV collagenase (MMP-2) and 97-kDa type IV collagenase (MMP-9), have important roles in tumor invasion, as MMP-2 and especially MMP-9 levels are strongly correlated with malignant tumor grade (10, 11). MMPs are also implicated in the pathogenesis of inflammatory demyelinating diseases of the CNS, such as multiple sclerosis (MS). MMPs are involved in T cell migration into the CNS, disruption of the brain-blood barrier, breakdown of the myelin sheath, and activation of proinflammatory cytokines (for review, see Ref. 12). Serum MMP-9 levels can be used as surrogate markers of disease activity in relapsing-remitting MS, and MMP-9 levels in cerebrospinal fluid are a valid indicator in evaluating the onset and severity of functional lesions in MS (13, 14).
MMP-9 activity is regulated by several mechanisms, including gene
transcription, mRNA stability, proenzyme activation, and inhibition of
enzyme activity. The MMP-9 gene can be induced by a variety of oncogene
products, cytokines, mitogens, and phorbol ester (4), and
regulation at the posttranscription level (mRNA stabilization) has also
been reported recently (15). Secreted MMP-9 (also called
pro-MMP-9) is associated with tissue inhibitor of metalloproteinase-1
(TIMP-1), and binding by TIMP-1 prevents activating enzymes such as
MMP-2, MMP-3, and trypsin from accessing MMP-9 (4, 16).
MMP-9 also has been shown to associate with tumor cell surface
molecules such as CD44 and the
v6 integrin; this
colocalization may be a critical step in regulating tumor invasion
(17, 18). Recently, MMP-9 was reported to be involved in
the process of tumor angiogenesis by increasing the availability of
vascular endothelial cell growth factor, an important
angiogenesis inducer in malignant tumors (19).
IFNs are multifunctional cytokines that have antiviral,
antiproliferative, and immunomodulatory effects (for review, see Ref.
20). IFNs are used in the clinical management of malignant
tumors, MS, and chronic viral hepatitis (21, 22).
Beneficial effects in attenuating angiogenesis in malignant tumors have
also been reported, although the exact mechanism is still unknown
(23, 24). Type I IFNs (IFN-/IFN-) are synthesized by
virus-infected cells, and type II IFN (IFN-) is produced by
activated T cells and NK cells. The type I IFNs specifically bind to
receptors composed of two subunits, IFN-R1 and IFN-R2. Ligand
binding induces trans-phosphorylation of the Janus kinases
TYK2 and JAK1, which subsequently activate STAT-1 and STAT-2.
Together with p48, STAT-1 and STAT-2 form a heterotrimeric complex
(known as latent cytosolic transcription factor, ISGF3), which
translocates to the nucleus, where ISGF3 binds to IFN-stimulated
response elements (ISREs) in the promoters of target genes. The
receptor for IFN- has two subunits, IFN-R1 and IFN-R2. JAK1
and JAK2 kinases are used to phosphorylate the STAT-1 protein, which
dimerizes, translocates to the nucleus, and induces target gene
transcription by binding to -activated sequences (GAS) in the
promoters of IFN--responsive genes (for reviews, see Refs.
25 and 26). STAT-1 is the common component
in the signaling pathways of type I and type II IFNs. Besides binding
to cis-acting elements, STAT-1 has been shown to interact
with a variety of transcriptional coactivators, including CBP, p300,
pCIP, Nmi, and BRACA1 (for review, see Ref. 27). Genes
that are negatively regulated by IFN- and/or IFN- are far fewer
than those positively induced (for review, see Ref. 27).
Among the negatively regulated ones are MMPs (MMP-1, -2, -13, and
stromelysin), perlecan, bullous pemphigoid Ag-1, cell cycle genes
(c-myc, cyclin D, cyclin A), and thyroid-specific genes (for
review, see Ref. 27). The detailed mechanisms of
transcriptional suppression by IFN- or IFN- are still unclear,
but both STAT-1-dependent and STAT-1-independent processes are
implicated (28).
In this study, we investigated the inhibitory effects of IFN- and
IFN- on MMP-9 expression in a variety of cells, including human
astroglioma cells, human fibrosarcoma cell, and primary astrocytes. The
results indicate that IFN- and IFN- transcriptionally suppress
MMP-9 expression, and the inhibitory effects are dependent on
STAT-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CRT-MG human astroglioma cell line, derived from a
neoplastic frontal lobe lesion, was grown in RPMI 1640 medium
supplemented with 10 mM HEPES (pH 7.2), 2 mM L-glutamine,
100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS, as
previously described (29). U87-MG, U251-MG, and CH235-MG
human astroglioma cell lines were grown in DMEM/Ham’s F-12 medium
supplemented with 10 mM HEPES, 2 mM L-glutamine, 100 U/ml
penicillin, 10 µg/ml streptomycin, and 10% FBS (29).
The STAT-1-deficient U3A cell line (30) (a generous
gift of G. Stark, Cleveland Clinic, Cleveland, OH) and the human
fibrosarcoma cell line 2fTGH were maintained in DMEM medium
supplemented with 10 mM HEPES, 2 mM L-glutamine, 100 U/ml
penicillin, 10 µg/ml streptomycin, and 10% FBS.
Astrocyte cultures
Primary astrocyte cultures were established from neonatal
cerebra of STAT-1-deficient mice (31) and wild-type
mice, as described previously (32). After 2 wk in primary
culture, oligodendrocytes and microglia were removed by mechanical
dislodgment. Astrocytes were harvested by trypsinization (0.25%
trypsin, 0.02% EDTA) and monitored for purity by immunofluorescence.
Astrocyte cultures were routinely >97% positive for glial fibrillary
acidic protein, an intracellular Ag unique to astrocytes. Human primary
adult astrocytes were derived from epilepsy patients, as previously
described (33).
Reagents
Human rIFN- was a generous gift from Biogen (Cambridge, MA);
human rIFN- was generously provided by Berlex Laboratories
(Richmond, CA); and human rTNF- was the generous gift of Genentech
(South San Francisco, CA). Murine IFN- was purchased from R&D
Systems (Minneapolis, MN), and murine IFN- was purchased from
Biosource (Camarillo, CA). PMA and gelatin were purchased from Sigma
(St. Louis, MO). Mouse anti-human MMP-9 mAb was the generous gift
of J. Engler, University of Alabama (Birmingham, AL). Rabbit
anti-human STAT-1 polyclonal Ab and anti-STAT-1
phosphotyrosine polyclonal Ab were purchased from Upstate Biotechnology
(Lake Placid, NY). Mouse anti-human STAT-1 mAb was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse
anti-human TIMP-1 mAb was purchased from Chemicon International
(Temecula, CA). The secondary peroxidase-conjugated Abs and ECL
reagents were from Amersham (Arlington Heights, IL).
Gelatin substrate gel zymography
Zymography was performed as described previously
(29). In brief, cells were incubated until 
80%
confluent; then the media were aspirated and fresh serum-free medium
was added to each dish, with and without PMA and TNF- treatment.
Supernatants were collected after a 24- to 48-h incubation and
concentrated. Concentrated supernatants (500–750 µl) were mixed with
SDS sample buffer without reducing agent, and proteins were subjected
to SDS-PAGE in 8% polyacrylamide gels that were copolymerized with
1–2 mg/ml gelatin. After electrophoresis, the gels were washed several
times in 2.5% Triton X-100 for 1 h at room temperature to remove
the SDS, and then incubated for 12–72 h (dependent on the cell type)
at 37°C in buffer containing 5 mM CaCl2 and 1
µM ZnCl2. The gels were stained with Coomassie
blue (0.25%) for 30 min, then destained for 1 h in a solution of
acetic acid and methanol. Proteolytic activity was evidenced as clear
bands (zones of gelatin degradation) against the blue background of
stained gelatin. Quantification was performed on the Bio-Rad (Richmond,
CA) Gel Doc 1000 using the Molecular Analyst Program.
Immunoprecipitation and immunoblot analysis
The same supernatants obtained for zymography were used in
immunoblot analysis for MMP-9 and TIMP-1 proteins. Concentrated
supernatants (750 µl) were boiled for 5 min in Laemmli’s sample
buffer and electrophoresed in 8% SDS-PAGE gels. Proteins were
transferred to nitrocellulose, and the membrane was then blocked in 1%
BSA in TBS with 0.01% Tween 20 for 1 h. The blots were incubated
with anti-MMP-9 Ab (5 µg/ml) or anti-TIMP-1 Ab (1/500) in Ab
dilution buffer (0.5% Tween 20, 1% BSA, 10% glycerol, 1 M glucose in
TBS) at 4°C overnight. Blots were washed four times in TBS with
0.01% Tween 20, and subsequently incubated in sheep anti-mouse
peroxidase-conjugated Ab (1/3000) in Ab dilution buffer. After a 45-min
incubation at room temperature, the blots were washed four times, and
ECL reagents were used for development. For immunoprecipitation, cell
lysates of treated cells were prepared as described previously
(34). Five hundred micrograms of total protein were
incubated with 4 µg polyclonal antisera to human STAT-1 overnight.
Protein A/G agarose beads were added for 2 h at 4°C, and the
immunoprecipitates were washed five times with lysis buffer, eluted
from the agarose beads by boiling in 2x SDS sample buffer, and
subjected to 8% SDS-PAGE. Proteins were transferred to nitrocellulose,
and the membrane was blocked in 1% BSA in TBS with 0.01% Tween 20 for
1 h. The blots were incubated with anti-STAT-1 mAb (1
µg/ml) in Ab dilution buffer (0.5% Tween 20, 1% BSA, 10% glycerol,
1 M glucose in TBS) at 4°C overnight. The blots were washed and
developed as described above. For reblotting, membranes were stripped
at 56°C in buffer containing 100 mM 2-ME, 2% SDS, 62.5 mM Tris-HCl
(pH 6.7) with occasional shaking, and reprobed with relevant
Abs.
Total RNA isolation and RNase protection assay (RPA)
Total cellular RNA was isolated from confluent monolayers of cells using TRIzol reagent (Life Technologies, Grand Island, NY), according to the manufacturer’s instructions. A 376-bp fragment corresponding to 1751–2127 nt of human MMP-9 cDNA (a generous gift of G. Goldberg, Washington University, St. Louis, MO) was subcloned in the HindIII/PstI polylinker site of pGEM3Z vector (Promega, Madison, WI). The construct was linearized by EcoRI and used to generate a radiolabeled antisense RNA probe of 424 nt with T7 RNA polymerase. A pAMP-1 vector containing a fragment of human GAPDH cDNA (corresponding to 43–531 nt) was linearized with NcoI, and used to generate a radiolabeled antisense RNA of 290 nt with T7 polymerase. Fifteen to 20 µg of total RNA was hybridized with MMP-9 (50 x 103 cpm) and GAPDH (25 x 103 cpm) riboprobes at 42°C overnight. The hybridized mixture was then treated with RNase A/T1 (1/200) at room temperature for 1 h and analyzed by 5% denaturing (8 M urea) PAGE, and the gels were exposed to x-ray film. Quantification of protected RNA fragments was performed using the PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Values for MMP-9 mRNA expression were normalized to GAPDH mRNA levels for each experimental condition. GAPDH mRNA was used as a control gene, as its levels are not affected by PMA or cytokine treatment.
Plasmids, transient transfection, and luciferase/-galactosidase
assays
A luciferase reporter plasmid driven by 670 bp of the human
MMP-9 promoter (35) (a gift of D. Boyd, MD Anderson Cancer
Center, Houston, TX) was used in this study, and the reporter vector
pTK (Clontech, Palo Alto, CA) expressing -galactosidase under
control of the herpes simplex virus thymidine promoter was used as an
internal reference plasmid. The hCIITAp1.7 construct, in which the
luciferase gene is under control of the type IV promoter of the human
class II transactivator (CIITA) gene, was described previously
(36). The human STAT-1 and STAT-1 expression vectors
have been described previously (37) (a gift of J. Darnell,
Rockefeller University, New York, NY). Transient transfection was
performed by electroporation or Lipofectamine (Life Technologies).
Transfected cells were either unstimulated or stimulated for 12 h
with PMA (50 ng/ml), IFN- (500 U/ml), IFN- (500 U/ml), or PMA
plus IFN- or IFN-. Cells were also transfected with a
promoterless vector control (pGL3-basic) and pTK, and stimulated as
described above. Cell extracts were assayed in triplicate for
luciferase and -galactosidase enzyme activities, as previously
described (38). The luciferase activity of each sample was
normalized to -galactosidase activity before calculating the fold
activation value. The luciferase activity from the vector control was
arbitrarily set at 1 for calculation of fold activation.
Stable transfection of STAT-1
STAT-1 stable transfectants were generated by transfecting
STAT-1-deficient U3A cells with the human STAT-1 expression
vector using the Lipofectamine Plus reagent, according to the
manufacturer’s instructions (Life Technologies). Mock transfection was
also performed in U3A cells by transfecting the pcDNA3 vector.
Transfectants were selected in G418 sulfate (500 µg/ml) and screened
for expression of STAT-1 by Western blotting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or IFN- inhibit MMP-9 enzymatic activity and protein
expression
MMP-9 activity can be induced by a variety of oncogenes, mitogens,
cytokines, and phorbol ester. To investigate the effects of IFN- and
IFN- on MMP-9 expression, CRT-MG astroglioma cells were treated with
PMA or TNF-, with or without IFN- and IFN- for 36 h, and
conditioned medium was harvested and subjected to gelatin zymography.
The optimal concentrations of PMA and TNF- for induction of MMP-9
activity were determined by dose-response experiments, as were the
concentrations of IFN- and IFN- for optimal inhibition of MMP-9
activity (data not shown). As shown in Fig. 1
, gelatinolytic activity at 97 kDa
corresponding to the molecular mass of pro-MMP-9 was detected in
conditioned media from PMA- and TNF--stimulated cells
(lanes 2 and 3), with PMA being a much
stronger inducer. There was no constitutively expressed MMP-9 enzymatic
activity in CRT-MG cells treated with serum-free medium
(lane 1), and IFN- or IFN- had no
inducible effects on MMP-9 expression (lanes 4 and
5). The simultaneous addition of either IFN- or IFN-
with PMA or TNF- suppressed MMP-9 enzymatic activity by greater than
50% (Fig. 1, lanes 6-9). The inhibitory effect
of IFN- or IFN- on MMP-9 activity was also observed in other
human astroglioma cell lines (U87-MG, CH235-MG, and U251-MG) and
primary human and murine astrocytes, although the extent of inhibition
varied depending on the cell examined.
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-induced MMP-9 protein expression was inhibited by
IFN- or IFN- treatment (Fig. 2),
with IFN- being a stronger inhibitor. Thus, decreased MMP-9
enzymatic activity correlates with the diminished synthesis of MMP-9
protein upon treatment by IFN- or IFN-. These results were also
replicated in the other cell types indicated above.
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or IFN-
TIMP-1 is an endogenous inhibitor of MMP-9; it can bind to
pro-MMP-9 and prevent activation by MMP-9-activating proteinases
(4). To exclude the possibility that IFN- or IFN-
inhibits MMP-9 enzymatic activity by enhancing TIMP-1 protein
expression, conditioned media from control, PMA-, and TNF--treated
CRT-MG cells in the absence or presence of IFN- or IFN- were
subjected to immunoblotting analysis for TIMP-1 expression. IFN- or
IFN- did not affect TIMP-1 protein levels (data not shown); thus, it
is unlikely that IFN- or IFN- indirectly decreases MMP-9
enzymatic activity by stimulating TIMP-1 protein expression.
Modulation of steady state MMP-9 mRNA levels by IFN- or IFN-
To investigate whether decreased MMP-9 protein expression upon
IFN- or IFN- treatment was due to diminished steady state mRNA
levels, RPA was performed in CRT-MG cells. We initially performed a
kinetic analysis of MMP-9 mRNA expression; the results indicated that
upon stimulation by PMA or TNF-, maximal levels of MMP-9 mRNA were
detected between 12 and 24 h after stimulation (data not shown).
The 12- to 24-h time points were used to determine the change in steady
state levels of MMP-9 mRNA upon IFN- or IFN- treatment. As shown
in Fig. 3A, MMP-9 mRNA was not
constitutively expresed in CRT-MG cells (lane 1),
while treatment with PMA or TNF- resulted in induction of MMP-9 mRNA
(lanes 2 and 3). IFN- or IFN- had no
inducible effects on MMP-9 mRNA expression (lanes 4
and 5), but inhibited PMA and TNF- induced steady state
MMP-9 mRNA levels (lanes 6–9). Quantification of the
results from three independent experiments is shown in Fig. 3B. Similar results were obtained in U87-MG astroglioma
cells (data not shown). Experiments
(t1/2) were also performed to
assess whether IFN- or IFN- decreases MMP-9 mRNA by destabilizing
the transcripts. Our results indicate that PMA-induced MMP-9 mRNA is
relatively stable and does not decay to any appreciable extent over a
12-h period. IFN- or IFN- inhibited MMP-9 mRNA expression by
50–60%, but did not alter PMA-induced MMP-9 mRNA stability (data
not shown). Collectively, our data indicate that the inhibitory effects
of IFN- or IFN- on PMA-induced MMP-9 expression are not at the
posttranscriptional level, and the main regulatory point is at the
transcriptional level.
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or IFN- suppresses transcription of the MMP-9 gene
To investigate the effects of IFN- or IFN- on MMP-9 gene
transcription, a luciferase reporter driven by the human MMP-9 promoter
sequence was used. U87-MG cells were used for these experiments since
these cells are more amenable to transfection than CRT-MG cells, and
they show a comparable level of IFN- or IFN- inhibition of MMP-9
expression as the CRT-MG cells. The MMP-9 promoter was transiently
transfected into U87-MG cells, and after a 24-h recovery, the cells
were incubated with serum-free medium, PMA, IFN-, IFN-, or PMA,
plus either IFN- or IFN- for 12 h. As illustrated in Fig. 4, MMP-9 promoter activity was negligible
in cells incubated with serum-free medium, IFN-, or IFN-. PMA
induced MMP-9 promoter activity by 30-fold, and the addition of
either IFN- or IFN- inhibited PMA-induced MMP-9 promoter activity
by 45% and 70%, respectively. Luciferase activity in pGL3-basic
construct-transfected cells was relatively constant, and levels were
not affected by PMA, IFN-, or IFN- treatment (data not shown).
These results indicate that IFN- and IFN- inhibit MMP-9 gene
expression by suppressing MMP-9 promoter activity.
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or IFN- does not inhibit MMP-9 expression in
STAT-1-null cells
Because STAT-1 is the common component in both IFN- and
IFN- signaling pathways, we wished to determine the involvement of
STAT-1 in IFN-- and IFN--mediated inhibition of MMP-9 gene
transcription. Evidence for the importance of STAT-1 was obtained
from primary murine astrocytes. As illustrated in Fig. 5, primary astrocytes from wild-type and
STAT-1-deficient mice were transiently transfected with the MMP-9
luciferase reporter construct and stimulated with PMA. In wild-type and
STAT-1-deficient astrocytes, MMP-9 promoter activity was induced by
PMA (3-fold). IFN- or IFN- inhibited MMP-9 promoter activity
by 49% and 30%, respectively, in the wild-type astrocytes,
while in STAT-1-deficient cells, the inhibitory effect of IFN- or
IFN- was abrogated. These results indicate that inhibition of MMP-9
gene transcription by IFN- or IFN- is dependent on STAT-1.
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in U3A cells restores the inhibitory
effects of IFN- or IFN- on MMP-9 gene expression
To further confirm the critical role of STAT-1 in
down-regulation of MMP-9 gene expression, U3A cells, a
STAT-1-deficient cell line generated from 2fTGH cells
(30), were used as a model system to analyze the effects
of IFN- or IFN- on MMP-9 expression in the absence or presence of
STAT-1. Immunoblot analysis confirmed that the STAT-1 protein is
absent in U3A cells (Fig. 6A).
Human STAT-1 was restored into U3A cells by stable transfection
(Fig. 6A). The STAT-1 protein was functional, as IFN-
induced tyrosine phosphorylation of STAT-1 protein in the
U3A-STAT-1 cells (Fig. 6A). We performed MMP-9 promoter
assays using mock-transfected U3A cells and U3A-STAT-1 cells. As
shown in Fig. 6B, PMA treatment resulted in 7-fold
induction of MMP-9 promoter activity in U3A mock-transfected cells. The
addition of either IFN- or IFN- had no significant inhibitory
effect on MMP-9 promoter activity. However, in U3A-STAT-1 cells,
IFN- or IFN- treatment decreased PMA-induced MMP-9 promoter
activity by 49% and 65%, respectively (Fig. 6B). As
a positive control for restoration of STAT-1, we examined the
activity of the type IV CIITA promoter, which is responsive to IFN-
stimulation (36), in U3A and U3A-STAT-1 stably
transfected cells (Fig. 6C). IFN- treatment failed to
induce promoter activity in STAT-1-deficient U3A cells; however,
IFN- treatment resulted in 6.5-fold induction of CIITA promoter
activity in STAT-1-restored cells. IFN- was not tested in this
experiment since it does not induce CIITA expression. These results
demonstrate that the restoration of STAT-1 allows for the inhibitory
effects of IFNs on MMP-9 gene expression, and inducibility of CIITA
gene expression in the same cells.
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in inhibiting PMA-induced endogenous
MMP-9 gene expression, RPA analysis was performed to compare IFN--
and IFN--mediated MMP-9 mRNA suppression in U3A cells and
U3A-STAT-1 stable transfectants. U3A cells were refractory to the
inhibitory effects of IFN- and IFN- (Fig. 7, A and B);
however, in U3A-STAT-1 cells, PMA-induced MMP-9 mRNA expression was
inhibited by IFN- or IFN- (52% and 47%, respectively)
(Fig. 7, C and D).
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can result in
inhibition of endogenous MMP-9 protein synthesis, mock-transfected U3A
cells and U3A-STAT-1 cells were treated with PMA in the absence or
presence of IFN- or IFN- for 24 h. Conditioned media were
subjected to zymography and immunoblotting analysis for examination of
MMP-9 protein. As illustrated in Fig. 8A, IFN- or IFN- failed
to inhibit MMP-9 enzymatic activity in U3A cells, while partial
inhibition was achieved in U3A-STAT-1 cells. Similarly, MMP-9
protein expression was not inhibited by IFN- or IFN- in U3A
cells, while transfection of STAT-1 restored the inhibitory effects
of IFN- or IFN- on MMP-9 protein expression; reconstitution of
STAT-1 resulted in 50% inhibition of MMP-9 protein expression by
IFN- and IFN- (Fig. 8B). Collectively, our data
illustrate that STAT-1 is indispensable for suppression of MMP-9
gene expression by IFN- and IFN-.
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on IFN-- and IFN--mediated suppression
of MMP-9 gene expression
As described above, STAT-1 is the common component in the
signaling pathways of both IFN- and IFN-. STAT-1, which is
missing the last 38 carboxyl-terminal amino acids, is generated from an
alternative transcript (Fig. 9A). STAT-1 is capable of
restoring type I IFN signaling (IFN- and IFN-), but not that of
IFN- in STAT-1-deficient U3A cells (39, 40). In
addition, STAT-1 functions as a dominant-negative form of STAT-1,
thereby inhibiting IFN--inducible gene expression; however, in the
case of IFN-, STAT-1 does not interfere with ISGF3 formation and
subsequent functional effects (39). To further
characterize the mechanism of STAT-1-mediated MMP-9 gene
suppression, we analyzed the influence of STAT-1 in this system. As
shown in Fig. 9B, transient expression of STAT-1 resulted
in 46% and 52% inhibition of PMA-induced MMP-9 promoter
activity by IFN- and IFN- in U3A cells, respectively, comparable
with the results obtained with stable transfection (Fig. 6
B). However, transient overexpression of STAT-1 caused
only 8% inhibition of PMA-induced MMP-9 promoter activity by
IFN- (Fig. 9B), indicating that STAT-1 is not
sufficient to restore IFN--mediated suppression of the MMP-9
promoter activity. For IFN-, overexpression of STAT-1 led to
30% inhibition of PMA-induced MMP-9 promoter activity (Fig. 9B), indicating that STAT-1 can partially restore the
inhibitory effect of IFN- (compared with 52% inhibition with
STAT-1). As STAT-1 has dominant-negative effects on STAT-1, we
further examined the effect of STAT-1 on STAT-1-dependent
suppression of MMP-9 promoter activity by IFNs. The percentage of
IFN- or IFN- inhibition in the presence of STAT-1 was compared
with that in the presence of the pcDNA3 control vector, which was set
at 100%. Overexpression of STAT-1 decreased IFN--mediated
inhibition of MMP-9 promoter activity to 53% and 49% in
wild-type 2fTGH and STAT-1 stably restored U3A cells, respectively
(Fig. 9C), indicating that STAT-1 partially inhibits
IFN--mediated suppression of MMP-9 promoter activity. However,
STAT-1 has a modest inhibitory effect on IFN--mediated
suppression of MMP-9 promoter activity (Fig. 9C), in
agreement with previous published results (39). Thus,
STAT-1 has differential effects on IFN-- and IFN--mediated
suppression of PMA-induced MMP-9 promoter activity, reflective of the
differences in the signaling pathways of the IFNs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IFN- inhibit PMA- and TNF--induced MMP-9 enzymatic activity and
protein synthesis in a variety of tumor cell lines and primary
astrocytes, as determined by gelatin zymography and immunoblotting,
respectively. The inhibition of MMP-9 expression at the protein level
correlates with decreased steady state mRNA levels and promoter
activity; thus, the inhibitory effects of IFN- and IFN- on MMP-9
expression result from attenuated transcription.
The human MMP-9 gene has a 2.2-kb promoter region, but the
proximal 670-bp promoter sequence contains all the essential
transcription-regulatory components. Important cis-acting
elements include two AP-1 sites, NF-
B, Sp1, GT box, and PEA3
elements. It is generally accepted that maximal induction of MMP-9 gene
expression requires all these cis-acting elements, although
the proximal AP-1 site plays an indispensable role in MMP-9 gene
transcription (44). Transcriptional regulation of MMP-9 is
believed to be the most important component for MMP-9 expression
(35, 45, 46). In this case, what is the mechanism by which
IFN- and IFN- inhibit MMP-9 gene transcription?
Initially, we scanned the MMP-9 promoter sequence for GAS and/or ISRE
elements, which are important cis-acting elements for
transcriptional regulation of IFN-/IFN- target genes. Our results
indicated that there were no consensus GAS or ISRE elements located in
the human MMP-9 promoter; thus, it is unlikely that IFN-- and
IFN--activated STAT-1 or ISGF3, respectively, decreases MMP-9
gene expression by directly binding to the human MMP-9 promoter.
Because STAT-1 is involved in the signaling pathway of both IFN-
and IFN-, we examined the effects of IFN- and IFN- on MMP-9
gene expression in STAT-1-deficient cells. Using primary astrocytes
from STAT-1-deficient mice and STAT-1-deficient U3A cells, we
show that IFN-- or IFN--mediated inhibition of MMP-9 gene
expression is a STAT-1-dependent process. Reconstitution experiments
utilizing STAT-1 stable transfectants demonstrated that IFN- and
IFN- inhibition of MMP-9 gene expression was restored, illustrating
the critical role of STAT-1 in this response. STAT-1, which lacks
the 38-aa C-terminal domain of STAT-1, failed to reconstitute
IFN--mediated suppression of MMP-9, but partially restored
IFN--mediated inhibition, in agreement with previously reported
results on STAT-1 (39). Overexpression of STAT-1
inhibited IFN--mediated suppression of MMP-9; however, for IFN-,
the dominant-negative effect was much less pronounced than that for
IFN-. This indicates that STAT-1 is differentially utilized by
IFN- and IFN- to suppress MMP-9; however, for both systems,
STAT-1 is indispensable for the optimal inhibition of MMP-9 gene
expression. Future experiments will address the importance of
individual functional domains of STAT-1 in mediating MMP-9 gene
suppression by IFN- and IFN-.
Among the genes negatively regulated by IFNs, c-myc
expression suppressed by IFN- involves both STAT-1-dependent and
STAT-1-independent pathways; a consensus GAS element in the
c-myc promoter is necessary, but not sufficient for
attenuating c-myc transcription (28). For
perlecan, another gene inhibited by IFN-, transcriptional
suppression is dependent on multiple GAS elements located at the distal
region of its promoter (47). The MMP-9 gene and other
IFN-inhibited genes, including BPAG1, cyclin A, and MMP-2, all lack GAS
elements in their promoter regions (38, 47, 48), so the
possible inhibitory mechanism does not involve direct binding of the
STAT-1 protein to the promoter sequence. More likely, negative
regulation occurs by affecting the function of coactivators and
components of the general transcriptional machinery. Among all the
identified coactivators, CREB-binding protein (CBP)/p300 have been
intensively studied. The two related coactivators have been shown to
interact with a variety of transcription factors, including
c-fos, c-jun, p53, CREB, c-Myb, YY1, STAT-1,
and STAT-2 (for review, see Ref. 49). Through interactions
with these transcription factors, CBP/p300 can potentiate the
transcriptional activity of a wide range of genes. In addition,
CBP/p300 also have intrinsic histone acetyltransferase activity, and
can affect gene transcription by modifying the chromatin structure of
target genes (for reviews, see Refs. 50 and
51). It has been shown that competition for limited
amounts of CBP/p300 between the JAK/STAT and Ras/AP-1 signaling
pathways is the mechanism underlying IFN--mediated inhibition of the
scavenger gene (SR-A) transcription (52). Transcription
factors that are important for MMP-9 gene transcription, such as AP-1
factors and NF-B, have been shown to interact with CBP/p300
(53, 54, 55). In addition, STAT-1 and STAT-2 also associate
with CBP/p300 in gene transcription induced by IFN- or IFN-
(56, 57, 58). Thus, a possible mechanism to explain IFN--
and IFN--mediated inhibition of MMP-9 gene transcription is that
activated STAT-1 recruits CBP/p300 away from the MMP-9
gene promoter transcription complex. Indeed, preliminary experiments
indicate that CBP is important for optimal MMP-9 gene
transcription, and that overexpression of CBP abrogates IFN-- and
IFN--mediated inhibition of MMP-9 gene expression (data not shown).
Experiments are underway to determine possible changes in interactions
between CBP/p300 and AP-1 and NF-B transcription factors in the
context of the MMP-9 promoter upon IFN-/IFN- treatment.
IFN- and IFN- are pleiotropic cytokines affecting various aspects
of astroglioma functions, including proliferation, cytokine/chemokine
production, cell motility, and expression of adhesion molecules and
integrins (59). Effective inhibition of tumor angiogenesis
was observed by introducing type I IFNs, IFN-/IFN-, through
retrovirus vectors (23). Gene transfer of IFN- into
brain tumors or overexpression of the IFN- gene by retrovirus can
successfully repress tumor angiogenesis (24, 60). We have
reported previously that IFN- down-regulates MMP-2 expression in
human astroglioma cells by suppressing gene transcription
(29). In this current report, our results indicate that
IFN- and IFN- transcriptionally suppress MMP-9 gene expression.
Collectively, these data suggest that the antitumor and antiangiogenic
functions of IFN- and IFN- may reflect their inhibitory effects
on MMP-9 and MMP-2 expression. IFN- is also a potent therapeutic
drug for clinical management of MS (61). IFN-
inhibition of migration of activated T lymphocytes across the
blood-brain barrier is dependent on IFN--mediated suppression of
MMP-9 protein expression (62, 63, 64). Thus, suppression of
MMP-9 gene transcription via a STAT-1-dependent mechanism may
contribute to the beneficial effects of IFN- or IFN- in a variety
of diseases, including brain tumors and MS.
Over the past decade, the role of MMPs in tumor invasion and
angiogenesis has been well established. Based on rational design
techniques, several MMP inhibitors have been synthesized and
therapeutic effects examined. Unfortunately, several large-scale
clinical trials of broad-spectrum MMP inhibitors on malignant cancers
failed or were suspended because of intolerable side effects, most
likely due to blockage of MMP functions that are critical for normal
physiological processes (65). Thus, further studies are
required to clarify the role of individual MMPs in tumor invasion and
to elucidate the regulatory mechanisms of individual MMPs. Further
understanding of the mechanism by which STAT-1 inhibits MMP-9 gene
transcription may help to identify more specific therapeutic targets to
attenuate MMP-9 expression.
|
Acknowledgments |
|---|
-deficient mice, Dr. J. Engler (University
of Alabama, Birmingham, AL) for the MMP-9 mAb, Dr. G.
Goldberg (Washington University) for the human MMP-9 cDNA, Dr.
D. Boyd (MD Anderson Cancer Center) for the MMP-9 promoter
construct, and Dr. J. Darnell (Rockefeller University) for
the human STAT-1 and STAT-1 expression vectors. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Etty N. Benveniste, Department of Cell Biology, MCLM 395, University of Alabama, 1918 University Boulevard, Birmingham, AL 35294-0005. E-mail address: tika{at}uab.edu
3 Abbreviations used in this paper: MMP, matrix metalloproteinase; CBP, CREB-binding protein; CIITA, class II transactivator; ECM, extracellular matrix; GAS, -activated sequence; ISGF3, latent cytosolic transcription factor; ISRE, IFN-stimulated response element; JAK, Janus kinase; MS, multiple sclerosis; RPA, RNase protection assay; TIMP, tissue inhibitor of metalloproteinase.
Received for publication May 29, 2001. Accepted for publication August 27, 2001.
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