From the Division of Plastic and Reconstructive Surgery, University of Southern California School of Medicine, Los Angeles, California 90033 and the § Department of Surgery, Childrens Hospital of Los Angeles, University of Southern California School of Medicine, Los Angeles, California 90027
Received for publication, November 30, 2000, and in revised form, March 26, 2001
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ABSTRACT |
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Both cytokines and matrix metalloproteinases
(MMPs) are active during physiologic and pathologic processes such as
cancer metastasis and wound repair. We have systematically studied
cytokine-mediated MMP regulation. Cytokine-mediated proteinase
induction and activation were initially investigated in organ-cultured
human skin followed by determination of underlying cellular and
molecular mechanisms using isolated skin cells. In this report we
demonstrate that tumor necrosis factor- Cytokines have been shown to be involved in many physiologic and
pathologic processes. Tumor necrosis factor- Compelling evidence has documented the role of matrix
metalloproteinases in the remodeling of connective tissue during
angiogenesis, tumor metastasis, and tissue repair (13). Of the growing
family of MMPs, MMP-2 (gelatinase A, 72-kDa type IV collagenase, EC
3.4.24.24) and MMP-9 (gelatinase B, 92-kDa type IV collagenase, EC
3.4.24.35) are unique for their fibronectin-like collagen binding
domains (14). MMP-2 and MMP-9 are thought to be responsible for
detaching basal keratinocytes from the basement membrane and thus
promote their migration to cover exposed connective tissue (15, 16). This notion is based on their restricted expression pattern at the
wound edge and their substrate preference for basement membrane, type
IV collagen. In addition, the type IV collagenases may also degrade
type VII collagen, the major collagen component of anchoring fibrils
essential for the attachment of the epidermis to the dermis, (17, 18).
Although the actual functions of the type IV collagenases in normal
physiologic processes are not clear, accumulated evidence has linked
them to many diseases. Excessive type IV collagenase activity is
associated with non-healing chronic wounds where it is thought that
type IV collagen is over-digested during re-epithelialization (19, 20).
Consistent with this fact, low levels of MMP-9 were found in
hypertrophic scars where collagen is over-deposited (21).
The expression of pro-MMP-9 is regulated by many soluble mediators such
as TNF- Because multiple cytokines are coordinately present in wound sites, we
have studied the interaction of multiple cytokines on MMP-9 induction
and its proteolytic activation in organ-cultured human skin. In the
second phase of the study we dissected the cytokine-mediated MMP-9
regulation at the cellular level. We isolated dermal fibroblasts and
keratinocytes from human skin and examined the effect of cytokines on
MMP-9 regulation. Finally, we investigated the cytokine-responsive
cis-elements in the promoter of the human MMP-9 gene to
identify the molecular target sites of such regulation.
In a previous report we demonstrated that exposure of human skin to
TNF- Materials and Reagents
Vitrogen containing 95% type I collagen was purchased from
Cohesion Technologies (Palo Alto, CA). Cytokines were purchased from R
& D Systems (Minneapolis, MN). The antibodies against MMP-9 (AB805)
were purchased from Chemicon International (Temecula, CA). The
Immobilon-P was purchased from Millipore (Bedford, MA). The enhanced
chemiluminescence (ECL) was purchased from Amersham Pharmacia Biotech.
The gelatin was from Sigma. Gelatin-Sepharose 4B was purchased from
Amersham Pharmacia Biotech. RNA was extracted by Trizol from Life
Technologies, Inc. The reagents for reverse transcriptase-PCR were
purchased from Promega (Madison, WI). The Platinum Taq DNA
polymerase and oligodeoxynucleotide primers were from Life
Technologies, Inc. KGM and KBM were from Clonetics. Quick-change
site-directed mutagenesis kit was purchased from Stratagene (La Jolla,
CA). DNA sequencing was carried out at the University of Southern
California/Norris Comprehensive Cancer Center.
Organ Culture and Cytokine Stimulation of Human Skin
Normal human skin was obtained from patients undergoing
reconstructive or aesthetic surgery (University of Southern California IRB 999061). The full thickness skin was decontaminated by incubation in 2× antibiotic containing DMEM (200 units/ml penicillin G sodium, 200 units/ml streptomycin sulfate, and 0.5 µg/ml amphotericin B) at
4 °C overnight. Then the skin was cut into equal sizes with 0.5 cm
on each side and incubated in DMEM at 37 °C with 5% CO2 for 8 h. To decrease the effects of endogenous soluble factors in
the skin induced by the harvesting process, the medium was changed
three times during the 8-h incubation. Finally, the skin piece was
immersed in 2 ml of DMEM with specific cytokines and was maintained at
37 °C with 5% CO2. The conditioned media were sampled
at the indicated times for gelatinolytic zymogram assay and Western
blot as mentioned below.
Preparation and Culture of Human Dermal Fibroblasts and
Keratinocytes
Dermal fibroblasts and keratinocytes were isolated from full
thickness human skin (3). The isolated fibroblasts were cultured in
DMEM containing 10% fetal bovine serum with antibiotics. The keratinocytes were grown in complete KGM. Before exposure to cytokines the medium was replaced with serum-free DMEM for fibroblasts and KBM,
the basal medium, for keratinocytes. For some experiments the
fibroblasts were embedded in collagen lattices (29).
Purification of Pro-MMP-9
The transformed human keratinocytes (kindly provided by Dr.
David Woodley, University of Southern California) were cultured in KGM
to confluence. The cells were stimulated by 10 ng/ml TNF- Activation of Pro-MMP-9 in Human Skin
The explants of full thickness skin were stimulated with or
without TNF- Gelatinolytic Zymogram
The conditioned media were mixed with SDS-PAGE sample buffer in
the absence of reducing agent and electrophoresed in 10%
polyacrylamide gel containing 0.1% (w/v) gelatin. Electrophoresis was
performed at 4 °C with 120 V for 16 h. After electrophoresis,
SDS in the gel was removed by incubation with 2.5% Triton X-100.
Gelatinolytic activities were developed in buffer containing 5 mM CaCl2, 150 mM NaCl, and 50 mM Tris, pH 7.5, for 16 h at 37 °C. The
gelatinolytic activities were visualized by staining with Coomassie
Blue R-250.
Western Blot
The gelatinase from conditioned media was enriched by binding it
to gelatin-conjugated Sepharose 4B. Briefly, 1 ml of conditioned media
was incubated with 40 µl of gelatin-Sepharose 4B (Amersham Pharmacia
Biotech) for 3 h. The beads were washed four times with 0.4 M NaCl in 50 mM Tris, pH 7.5. The bound protein
was eluted with 1× SDS-PAGE sample buffer at the reducing condition.
After SDS-PAGE, the protein was transferred to Immobilon-P (Millipore). The protein blot was exposed to anti-human MMP-9 antibodies and followed by horseradish peroxidase-conjugated secondary antibodies that
were detected by enhanced chemiluminescence.
Reverse Transcriptase-Polymerase Chain Reaction
Stimulation--
The dermal fibroblasts were grown in 10-cm
dishes until subconfluent. To induce quiescence in the cells, the
dishes were washed with DMEM and incubated in serum-free DMEM for
4 h. For the titration experiment, the dishes were stimulated with
TNF- Total RNA Preparation--
After the dishes were stimulated for
the indicated time, they were washed with phosphate-buffered saline,
and the total RNA was extracted by Trizol. RNA was quantified by
measuring the adsorption at 260 nm.
Reverse Transcription and PCR--
2 µg of total RNA was
reverse-transcribed using avian myeloblastosis virus-reverse
transcriptase with 1 µg of random deoxynucleotide hexamer in the
presence of 4 mM MgCl2 and 2.5 mM
dNTP. After completion of the 1-h incubation at 37 °C, the reaction
was terminated via heating to 94 °C for 3 min. The annealing
temperature for amplification of human MMP-9 and Plasmid Constructs
DNA fragments containing the 5'-region of the human
MMP-9 gene and the NF- The plasmid containing the wild type 670-bp upstream region of human
MMP-9 gene was a gracious gift of Drs. Sato and Boyd (33,
34). To facilitate reconstruction, the entire 670-bp promoter was
inserted into pBLCAT2 and named as pM9-670-CAT. Briefly, the 670-bp
promoter was amplified by PCR with the forward primer 5'-AAG CTT CTA
GAG GCT ACT GTC CCC-3' and the reverse primer 5'-TCT AGA GGT GTC TGA
CTG CAG GTG-3'. The PCR product was cloned into pCR2.1-TOPO, an
intermediate vector (Invitrogen). Finally, the insert from the TOPO
vector was inserted into pBLCAT2.
A p65 NF- Transient Transfection, Cytokine Stimulation, and CAT Assay
The early passages of human dermal fibroblasts were seeded in
6-cm dishes. Transfection was conducted with 0.5-µg plasmid and
LipofectAMINE-PLUS according to the manufacturer's instructions (Life
Technologies, Inc.). After incubation for 3 h the plasmid complex
was removed and replaced with 0.5% fetal bovine serum/DMEM, and then
cytokines were added (TNF- Cytokine-mediated Induction and Activation of MMP-9 in Human
Skin--
To establish the profile of cytokine-exerted MMP-9 induction
and activation, we first utilized organ culture of human skin. Normal
human skin, discarded after reconstructive surgery, was cultured in
serum-free DMEM and exposed to TNF-
Clinical investigations have outlined a temporal pattern for pro-MMP-9
expression and activation in normal healing cutaneous wound sites.
Persistent elevation of MMP-9 was found in association with chronic
wounds (36-38). However, causal factors to induce and activate the
proteinase in human tissue at wound sites are still obscure. Our
findings reported here show a specific role for the inflammatory
cytokines in the induction and activation of MMP-9 in human tissue.
Characterization of the TNF- Cytokine-mediated Regulation of MMP-9 in Human Dermal Fibroblasts
and Epidermal Keratinocytes--
To understand the cellular mechanism
of the cytokine-mediated induction and activation of pro-MMP-9 from
intact skin, we studied the MMP profile in isolated dermal fibroblasts
and epidermal keratinocytes. The dermal environment was simulated by
embedding the fibroblasts in three-dimensional type I collagen
lattices. Individual cytokines including TNF-
To complete the dissection we also analyzed the effect of these
cytokines on the proteinase from the keratinocytes. The early passages
of isolated adult keratinocytes cultured as monolayers were stimulated
by TNF-
These experiments demonstrated that the synergistic induction of
pro-MMP-9 in human skin by TNF-
As we demonstrated previously, TNF- Time Course of Co-induction Pro-MMP-9--
To investigate the
kinetics of pro-MMP-9 induction, we performed a time course experiment
using human dermal fibroblasts either embedded in collagen lattices or
cultured as monolayers. This experimental design could also delineate
the role of collagen in the cytokine-mediated MMP-9 expression and
activation. The collagen lattices and monolayers were stimulated with
combinations of TNF- The Minimal Concentration of TNF- TNF- TNF- One of the Two Potential NF-
To elucidate whether the TNF- A TGF- The specific mechanisms whereby the early, inflammatory stages of
wound healing progress to the later, synthetic phases are not well
understood. TNF- This report provides the first evidence that cytokines can induce and
proteolytically activate MMP-9 in intact human skin. Specifically, we
found that TGF- Previous reports showed that TGF- The molecular mechanism for proteolytic activation of pro-MMP-9
in vivo is not clarified. In vitro experiments
show that many proteinases including cathepsin G, trypsin,
The regulatory elements in the 5'-flanking region of human
MMP-9 gene have been analyzed previously (35, 42).
The TNF- A consensus TGF- This is the second report from our laboratory indicating that
the activity of a particular inflammatory cytokine, TNF- There are several unanswered questions from the current literature.
What is the specific role of MMP-9 in the initial phase of normal wound
healing? What does MMP-9 do in chronic wounds to prevent healing to
progress? Will blocking TNF- In conclusion, our results provide integrated studies of multiple
cytokines on MMP-9 induction and activation initiated from human skin
and extended to the isolated skin cells. The analysis of the
cytokine-response elements in the promoter of human MMP-9 gene gives us enriched information for future analysis of abnormal MMP-9 regulation in cancer and aberrant wound healing. These findings may provide an in-depth knowledge of type IV collagenase expression and
activation in wound healing or tumor cell metastasis where cytokines
play a central role to orchestra a multilevel of ECM and MMP gene
expression, ECM deposition, and degradation.
(TNF-
) and transforming
growth factor-
(TGF-
) synergistically induce pro-MMP-9 in human
skin as well as isolated dermal fibroblasts and epidermal
keratinocytes. Furthermore, TNF-
promotes proteolytic activation of
pro-MMP-9 by conversion of the 92-kDa pro-MMP-9 to the 82-kDa active
enzyme. This activation occurred only in skin organ culture and not by
either isolated fibroblasts or keratinocyte, although the pro-MMP-9
activation could be measured in a cell-free system derived from
TNF-
-activated skin. The cytokine-mediated induction of pro-MMP-9 in
dermal fibroblasts was evident by increased mRNA. At the
transcription level, we examined the cytokine-mediated transactivation
of the 5'-region promoter of the human MMP-9 in dermal fibroblasts. The
results demonstrated that TNF-
and TGF-
could independently
stimulate the 5'-flanking 670-base pair promoter. A TGF-
-response
element (
474) and an NF-
B-binding site (
601) were identified to
be the cis-elements for TGF-
or TNF-
activation, respectively. Taken together, these findings suggest a specific mechanism whereby multiple cytokines can regulate MMP-9 expression/activation in the
cells of human skin. These results imply roles for these cytokines in
the regulation of MMP-9 in physiologic and pathologic tissue remodeling.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
)1 is thought to be
essential for macrophage-mediated normal wound healing (1, 2). Whereas
elevated levels of TNF-
have been linked to deficient wound healing
(3, 4), a lack of TNF-
was found to be associated with hypertrophic
scars (5, 6). This suggests that misregulated TNF-
results in
disordered wound healing. Similarly, transforming growth factor-
(TGF-
) is a component of normal wound healing (7, 8). Increased
TGF-
is associated with hypertrophic scar and fibrosis (9, 10). In
addition, accumulating evidence has suggested a role for inflammatory cytokines in promoting tumor metastasis, although the mechanism is not
clarified (11, 12). The roles for these mediators in the regulation of
extracellular matrix may relate to the spectrum of genes they induce
and appear to share a common theme of tissue remodeling.
, IL-1
, TGF-
, the ECM, oncogenes, and tumor promoters
(22-25). After secretion into the ECM, the activity of MMP-9 can be
further regulated by specific tissue inhibitors and by proteolytic
activation via removal of the amino-terminal inhibitory domain. Most of
the previous investigations on cytokine-mediated MMP-9 expression
utilized tumor or transformed cell lines in which the mitogenic
signaling and the cell cycle machinery are constitutively active. In
chronic wounds and some invasive cancer tissue the 92-kDa pro-MMP-9 is
processed into the active 82-kDa form (26-28). Very little is known
about the molecular regulation of expression and proteolytic activation
of MMP-9 in these pathologic situations.
led to activation of the pro-MMP-2, and such proteolytic activation could be reconstructed by embedding the dermal fibroblasts in collagen lattices (29). In the present study we extend our findings
by showing that the 92-kDa pro-MMP-9 is induced in human skin by
TGF-
, and this induction is additively enhanced by a second signal
from TNF-
. In addition, we found that the 92-kDa pro-MMP-9 is
converted to the 82-kDa active form when organ-cultured skin is treated
with TNF-
. Furthermore, we show here that the TNF-
-mediated
pro-MMP-9 activation is caused by an unidentified factor that is
tightly associated with skin tissue. Our cellular dissection
experiments demonstrate that cytokine-mediated pro-MMP-9 induction in
the human skin is due to dermal fibroblasts and epidermal keratinocytes. At the molecular level, we provide evidence showing that
these two cytokines target their response elements in the 5'-promoter
of the human MMP-9 gene. These findings represent the first
demonstration for additive roles of TNF-
and TGF-
on the
induction and activation of MMP-9 in human skin.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in KBM for
72 h in standard culture condition. In these conditions most of
the gelatinase secreted in the medium is the 92-kDa pro-MMP-9. The
conditioned media from 10 10-cm dishes were collected, and particulate
debris was removed by centrifugation at 4,000 × g for
10 min. The conditioned media were passed through a 1-ml
gelatin-Sepharose 4B column followed by washing with 400 mM
NaCl in 50 mM Tris, pH 7.5. The bound gelatinase was eluted
by 100 mM HCl and immediately neutralized by Tris base to
pH 7.5.
at 10 ng/ml for 70 h in DMEM at 37 °C with 5%
CO2. To test whether the TNF-
-mediated pro-MMP-9
activation occurred inside the skin tissue or by secreted factors, the
explants were washed and re-cultured in fresh DMEM without cytokine.
The original conditioned media from the 70-h stimulation and the
conditioned media from the re-culture were incubated at 37 °C for
additional 8-20 h and analyzed by gelatinolytic zymography. In another
experiment, the TNF-
-treated skin explant was minced and then
extracted using a buffer containing 2% Triton X-100, CaCl2
at 0.2 g/liter, KCl at 0.4 g/liter, MgSO4 at 0.1 g/liter,
NaCl at 6.4 g/liter, and 50 mM Tris, pH 7.5. After a 5-h
incubation at room temperature the Triton-soluble and -insoluble
fractions were obtained by centrifugation at 12,000 × g for 10 min. Purified pro-MMP-9 was added to these fractions and incubated at 37 °C for 20 h followed by the
zymogram analysis.
(10 ng/ml) and TGF-
(1 ng/ml) individually or in combination
in DMEM for 20 h. For the time course experiment the cells were
stimulated by a combination of TNF-
with TGF-
and harvested 0, 2, 4, 6, 12, and 20 h later.
-actin was 64 °C
(ROBOCYCLER, Stratagene). The PCR amplification was performed with
platinum Taq DNA polymerase for 30 cycles. The product was
resolved in agarose gel (1.8%), followed by staining with ethidium
bromide, and recorded by digital camera. The relative density of the
products was quantitated by the Alpha imaging system. The
oligonucleotide primers for PCR were adapted from a previous report
(30, 31). The predicted PCR product size for
-actin and for MMP-9 is
548 and 479 bp, respectively. The identity of the PCR product for MMP-9
was confirmed by DNA sequencing (Norris Cancer Center, University of
Southern California).
B-response elements were inserted
into pBLCAT2 (32). The wild type NF-
B reporter plasmid,
pNF
B3x-CAT, was constructed by inserting an enhancer element into
pBLCAT2 at HindIII/BamHI sites. The insertion
fragment consists of a triple tandem repeat of the NF-
B consensus
binding site with the sequence of 5'-AGC TTG GGA CTT TCC GGG ACT TTC
CGG GAC TTT CCG GAT CC-3' (Promega). An NF-
B enhancer mutant,
pNF
Bm3x-CAT, was constructed by insertion of the adapter (5'-AGC TGT
ACA CTT TCC TAC ACT TTCC TAC ACT TTC CG-3') into pBLCAT2.
B consensus binding site at
601 was characterized
previously (35). Scanning the 670-bp promoter for potential transcription factor binding sites, we noticed a consensus p50 NF-
B-binding site located at
328 from the transcription start site
(MatInspector version 2.2, GSF-National Research Center for Environment and Health). A p65 NF-
B enhancer deletion construct, pM9-590-CAT, that contains the 590-bp region upstream from the transcription start site was created. This construct was generated by
PCR with a forward primer 5'-AAG CTT AGC CTT GCC TAG CAG AGC CCA TTC-3'
and a backward primer 5'-TCT AGA GGT GTC TGA CTG CAG GTG-3'. Another
deletion construct, pM9-460-CAT, was generated by deleting the p65
NF-
B and the potential TGF-
-response element (TRE) (
474). This
plasmid was constructed by digesting pM9-670-CAT with
HindIII and EcoR V, filling-in, and self
re-ligation. A mutation construct in the TRE, pM9-670-mTRE-CAT, was
generated by PCR-based mutagenesis. Briefly, the TRE mutation was
generated by PCR using sequencing grade Taq DNA polymerase
with a forward primer 5'-AAG CTT CTA GAG GCT ACT GTC CCC-3' and reverse
primer 5'-GTC AGA TAT CCT CCC CTG ATC ACT CCC CAC ACT-3'. In this TRE
mutant the wild type sequence 5'-AGGTTTGGGGA-3' was substituted by
5'-TGATCAGGGGA-3' (the mutant bases
are underlined). The PCR product was ligated to pCR2.1-TOPO
vector. Then the 200-bp HindIII/EcoRV fragment from the wild type pM9-670-CAT was replaced by the mutant version. Finally, we created a site-directed mutant at the potential p50 NF-
B-binding region (
328/
319) and named it pM9-670-mp50-CAT. This was accomplished by the QuickChange Site-directed Mutagenesis Kit
(Stratagene) with the following primer: 5'-TCA GAC CAA GGG ATG
AAG GAT AAC TCC AGC TTC ATC CCC CTC CC-3' (the
mismatched four nucleotides are underlined). The insertion fragments of
the wild type, the mutant pM9-670mTRE-CAT, and mutant
pM9-670-mp50-CAT were confirmed by DNA sequencing (Norris Cancer
Center, University of Southern California).
at 10 ng/ml and TGF-
1 ng/ml). For the
time course experiment the cells were harvested at 24, 48, and 60 h post-transfection. For promoter analysis experiments most of the
transfection times were 62 h. The cells were washed by
phosphate-buffered saline and harvested in 400-µl 0.25 M
Tris at pH 8.0 buffer. The cells were lysed via three rounds of quick freeze and thaw followed by heating for 10 min at 60 °C. The
extracts were briefly centrifuged, and the supernatant was harvested
for the CAT assay. The reaction was performed in a 125-µl system with 50 µl of lysate, 1 µl of [14-C]chloramphenicol
(PerkinElmer Life Sciences, 1.9 MBq/ml), 5 µl of 10 mM
butyl-CoA (Roche Molecular Biochemicals), and 69 µl of the 0.25 M Tris, pH 8.0. After incubating at 37 °C for 14 h, the lipid phase was extracted by chloroform. The products were resolved
by silica gel TLC (Whatman) with 3% methanol and 97% chloroform. The
acetylated products were detected by PhosphorImaging (Molecular Dynamics).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and TGF-
either individually
or combined (10 ng/ml for TNF-
and 1 ng/ml for TGF-
). The
conditioned media were sampled at the indicated time points. The
resultant conditioned media were assayed for type IV collagenase activity by gelatinolytic zymography. In the absence of exogenous cytokine, minimal 92-kDa gelatinolytic activity was present in the
conditioned medium after culturing for 72 h (Fig.
1A). Treatment of the skin
with TNF-
induced a small increase of the 92-kDa gelatinolytic
activity. Remarkably, in the presence of TNF-
, the 92-kDa
gelatinolytic activity progressively disappeared with a concomitant
increase in the 82-kDa gelatinolytic activity. The 92- and 82-kDa
gelatinolytic activities were confirmed as MMP-9 by Western blot (Fig.
1B). In contrast, exposure of the skin to TGF-
markedly
induced the 92-kDa pro-MMP-9. Unlike the response to TNF-
, TGF-
failed to promote MMP-9 proteolytic activation to the 82-kDa form. When
both cytokines were applied, a synergistic induction of pro-MMP-9 was
detectable at 48 h. Furthermore, simultaneous exposure to the two
cytokines led to substantial conversion of the pro-MMP-9 to the active
form. After cultivation with the two cytokines for 96 h, most of
the pro-MMP-9 was converted to the active 82-kDa active form.
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Fig. 1.
Cytokine-mediated induction and activation of
MMP-9 in organ-cultured human skin. Normal full thickness human
skin was cultured in serum-free DMEM and stimulated by TNF- (10 ng/ml) and TGF-
(1 ng/ml) either individually or combined. Skin
samples from eight distinct individuals were examined for
cytokine-mediated induction and activation of MMP-9. All showed similar
results. A, conditioned media were sampled at the indicated
time points and analyzed for gelatinolytic activities by zymogram. The
92- and 82-kDa gelatinolytic activities are indicated by
arrows. B, identities of the 92- and 82-kDa
gelatinolytic activities as pro-MMP-9 and active MMP-9 were determined
by Western blot. The total gelatinase from 500-µl conditioned media
derived from 72-h culture was enriched by gelatin-conjugated Sepharose
4B matrix. The bound protein was resolved by SDS-PAGE and detected by
Western blot using polyclonal anti-MMP-9 antibodies.
-induced Proteolytic Activation of
Pro-MMP-9--
The next question was dissecting the nature of the
TNF-
-induced activation of pro-MMP-9 in the organ-cultured human
skin and whether activation is performed by a skin-associated factor or
a secreted soluble factor. The skin explants were stimulated with or
without TNF-
for 70 h, and the conditioned media were collected. The treated explants were washed and re-plated in fresh DMEM
without additional cytokine. The previous conditioned medium and the
re-plated culture were incubated at 37 °C and sampled at 0, 8, and
20 h (Fig. 2). As shown by
zymography, the 82-kDa MMP-9 was generated by direct contact with the
skin tissue but not by incubation with the conditioned medium. Next, we
attempted to extract the pro-MMP-9 activator from the TNF-
-treated
skin. The TNF-
-treated explant was extracted by nonionic detergent, Triton X-100. Purified pro-MMP-9 was added to the Triton-soluble and
-insoluble fractions and incubated for 20 h (Fig.
3). The results show that pro-MMP-9 is
processed to the 82-kDa form in the Triton-insoluble fraction, which
suggests that the unidentified pro-MMP-9 activator is tightly
associated with the tissue structure.
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Fig. 2.
TNF- -mediated
pro-MMP-9 activation occurs in the skin tissue but not in the
medium. Human skin was cultured in DMEM with or without TNF-
.
After 70 h of incubation the conditioned media were collected. The
skin tissues were washed and replanted in the fresh DMEM. The original
conditioned media and the replanted skin tissue in DMEM were incubated
at 37 °C, and media were sampled at the indicated time points. MMP-9
activities were measured by gelatinolytic zymogram.
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Fig. 3.
Proteolytic activation of pro-MMP-9 in the
cell-free skin extract. Adult human skin was stimulated with
TNF- (10 ng/ml) in DMEM for 70 h. The tissue was washed and
minced following extraction using 2% Triton X-100 (see "Experimental
Procedures"). The detergent-soluble and -insoluble fractions were
separated by centrifugation. Purified pro-MMP-9 was added or not to
these fractions and incubated at 37 °C for 20 h. MMP-9
activities were measured by gelatinolytic zymogram.
, TGF-
1, PDGF-AB,
EGF, IL-8, and IL-6 were applied at 10 ng/ml in the serum-free culture
medium. In another panel, a combination of TGF-
with other cytokines
was applied to the cells. After culturing for 68 h, the
conditioned media were analyzed for gelatinolytic activity (Fig.
4A). The results show that
TNF-
alone could moderately induce the 92-kDa gelatinolytic
activity, and the other cytokines in the list did not induce the 92-kDa gelatinolytic activity. Remarkably, the combination of TNF-
with TGF-
led to a synergistic induction of the 92-kDa gelatinolytic activity in the fibroblasts. The identity of the 92-kDa gelatinolytic activity was confirmed to be pro-MMP-9 by Western blot (Fig.
4B). In combination with TGF-
, the other cytokines
tested, PDGF-AB, EGF, IL-6 and IL-8, all failed to induce the MMP-9. We
also tested the combination of these cytokines with TNF-
and found
no induction of MMP-9 (data not show). Taken together, these
experiments demonstrated that two signals, TNF-
and TGF-
, are
required for maximal induction of the pro-MMP-9 in the dermal
fibroblasts.
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Fig. 4.
Cytokine-mediated induction of pro-MMP-9 in
human dermal fibroblasts and epidermal keratinocyte. A,
dermal fibroblasts were isolated from the adult human skin, and early
passages were embedded into type I collagen. The cells were cultured in
serum-free DMEM and stimulated with TNF- , TGF-
, PDGF-BB, EGF,
IL-8, and IL-6 at 10 ng/ml. In another panel the cells were stimulated
with a combination of TGF-
with TNF-
, PDGF, EFG, IL-8, or IL-6,
all at 10 ng/ml. After cultivation for 68 h the conditioned media
were analyzed for gelatinolytic zymography. The 92-kDa MMP-9 and 72- and 62-kDa MMP-2 are indicated. B, the identity of the
92-kDa gelatinase activity was confirmed as MMP-9. The total gelatinase
from 500-µl conditioned media was enriched by gelatin-conjugated
Sepharose 4B matrix. The bound protein was resolved by SDS-PAGE and
detected for MMP-9 by Western blot. C, the keratinocytes
were isolated from the epidermal portion of adult human skin and the
early passages were used for the experiment. The confluent monolayers
were stimulated by TNF-
and TGF-
individually or combined in
basal medium (KBM) for 70 h. A typical zymography is
presented.
and/or TGF-
. Similar to the results with fibroblasts,
TNF-
alone could induce pro-MMP-9 expression, and this effect was
enhanced by TGF-
exposure (Fig. 4C). In contrast to
fibroblast cultures where TGF-
alone failed to induce the MMP-9,
TGF-
alone was sufficient to induce the proteinase in the keratinocytes.
and TGF-
could be reproduced by
both dermal fibroblasts and keratinocytes. However, neither TNF-
alone nor in combination with other cytokines could promote the
proteolytic conversion of pro-MMP-9 to the 82-kDa active enzyme in the
isolated primary cells. We then hypothesized that an additional skin
cell such as dendritic cells or the interaction of several different
skin cells might be required for the pro-MMP-9 activation found in
intact skin. We tested the effect of the cytokine on the proteinase
activation by monocyte and granuocyte colony-stimulating factor-stimulated dendritic cells (kindly provided by Dr.
Jeffery Webber, University of Southern California). Neither of these
cells activated the pro-MMP-9 by TNF-
stimulation. In addition, we performed co-culture of the keratinocytes and fibroblasts. In all these
experiments we failed to reconstruct the proteolytic activation of
pro-MMP-9 from the human skin.
stimulated fibroblasts in a
collagen environment to convert the 72-kDa pro-MMP-2 into the active
62-kDa MMP-2 (29). In this report, we show that TGF-
can also
partially stimulate the activation of pro-MMP-2 (Fig. 4A).
Other cytokines such as PDGF, IL-8, IL-6, and EGF had no significant
effect on the activation of pro-MMP-2 in this system. Another
difference between the fibroblast and keratinocyte responses is that
the 72-kDa pro-MMP-2 is constantly expressed in fibroblasts, whereas it
is induced by TGF-
in the keratinocyte.
and TGF-
both at 10 ng/ml. The conditioned
media were sampled at the indicated time points. Results show that in
the absence of cytokine co-stimulation, neither the 92-kDa pro-MMP-9 nor the 82-kDa form was expressed in either culture condition. This
result indicates that collagen per se does not contribute to
MMP-9 induction (Fig. 5). As we reported
previously, these data reiterate that the pro-MMP-2 activation, as
measured by accumulation of 62-kDa MMP-2, is moderately enhanced by
collagen. Exposure of the monolayers or the lattices simultaneously
with TNF-
and TGF-
led to an equal and substantial induction of
92-kDa pro-MMP-9. The time course experiment shows that significant
amounts of pro-MMP-9 protein are accumulated in the conditioned medium
between 6 and 20 h after exposure of the fibroblasts to these
cytokines.
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Fig. 5.
Time course of co-induction of pro-MMP-9 by
TNF- and TGF-
.
The early passages of human dermal fibroblasts were cultured as
monolayers or embedded in type I collagen. The cells were stimulated by
a combination of TNF-
(10 ng/ml) and TGF-
(10 ng/ml) in DMEM.
A, the fibroblast monolayers were stimulated by the combined
cytokines, and conditioned media was sampled at the indicated time
points and analyzed for gelatinolytic activities. B, the
fibroblasts embedded in collagen lattices were stimulated by the
cytokines, and conditioned media were assayed for gelatinolytic
activities.
and TGF-
on Pro-MMP-9
Induction--
Because collagen apparently had no significant effect
on the cytokine-mediated co-induction of pro-MMP-9, we measured the efficacy of TNF-
and TGF-
on pro-MMP-9 expression in monolayer cultures. Since both cytokines are required for the induction of the
proteinase in the fibroblasts, we fixed the concentration of one
cytokine at 10 ng/ml and varied the other one to determine the minimal
efficacy. The results show that at a fixed concentration of TNF-
at
10 ng/ml, TGF-
stimulated the fibroblasts to express pro-MMP-9 in a
dose-dependent manner (Fig.
6). With a sufficient amount of TNF-
,
TGF-
1 at concentrations as low as 0.1 ng/ml could significantly
induce pro-MMP-9 expression, and vice versa, when the TGF-
concentration was fixed at 10 ng/ml, TNF-
stimulated the fibroblasts
to generate pro-MMP-9 with dose dependence. With sufficient TGF-
,
TNF-
at 0.1 ng/ml could clearly stimulate the fibroblasts to express
pro-MMP-9. These concentrations for the efficacy of TNF-
and TGF-
on MMP-9 induction are important because they are similar to
concentrations reported previously of the cytokine required to induce
angiogenesis and wound healing (1, 39-41).
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Fig. 6.
Concentration dependence of the
cytokine-mediated induction of pro-MMP-9. The human dermal
fibroblasts were cultured as monolayers. One set of six wells was
stimulated with TNF- at 10 ng/ml and with varying concentrations of
TGF-
as indicated. Another set of six wells was stimulated by
TGF-
at 10 ng/ml and varying concentrations of TNF-
. After
stimulation for 48 h the conditioned media were analyzed for
gelatinolytic zymography.
- and TGF-
-mediated Induction of Pro-MMP-9 Is Regulated
at mRNA Level--
To address the mechanism of the
cytokine-mediated induction of pro-MMP-9 protein, we measured the
levels of MMP-9 mRNA in response to the cytokines individually or
combined. Results revealed that the MMP-9 mRNA level was
significantly higher after stimulation by TNF-
for 20 h,
whereas TGF-
individually only slightly increased the proteinase
mRNA (Fig. 7A).
Simultaneous stimulation by the cytokines led to an additional increase
of MMP-9 mRNA. A time course experiment was performed by
co-stimulation of the fibroblasts with these two cytokines. As shown in
Fig. 7B, the MMP-9 mRNA is induced at 4 h after
stimulation, and the steady state was reached after stimulation for
6 h (Fig. 7B). As a control, the mRNA level of
-actin, a housekeeping gene, was constitutively expressed and
unchanged in response to the cytokines. These experimental results
suggest that the cytokine-mediated induction of pro-MMP-9 is
up-regulated at the mRNA level. The temporal induction of MMP-9 mRNA level (the half-time for the maximal induction,
t1/2 = 3 h) correlated well with the elevated
MMP-9 protein level (t1/2 = 10 h) as well as
with the pro-MMP-9 induction in the organ-cultured human skin
(t1/2 = 24 h). The rapid induction of MMP-9
mRNA by these cytokines suggests that the regulation is likely
through the direct activation of the transcriptional machinery for the
MMP-9 gene. To support this notion we tested the effects of
the transcriptional inhibitor, actinomycin D, and the protein synthesis
inhibitor, cycloheximide, on the pro-MMP-9 induction. As expected, the
cytokine-mediated induction of pro-MMP-9 was totally attenuated by
actinomycin D at 2.5 µg/ml and cycloheximide at 5 µg/ml (Fig.
8).
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Fig. 7.
The cytokine-mediated induction of MMP-9 is
up-regulated at the mRNA level. A, the human dermal
fibroblasts as monolayers were stimulated by TNF- (10 ng/ml) and
TGF-
(10 ng/ml) individually or combined in serum-free DMEM. After
stimulation for 20 h the total RNA was extracted. The mRNA
levels of MMP-9 and
-actin were determined by RT-PCR. For the
-actin, one-third of PCR product was loaded into the gel as shown.
The identity of PCR product for MMP-9 was confirmed by its expected
size and DNA sequencing. B, time course of MMP-9 mRNA
co-induction by the two cytokines. The mRNA level of MMP-9 and
-actin was measured at the indicated time points, and the relative
amount was quantitated by Alpha-imaging.
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Fig. 8.
Actinomycin D and cycloheximide attenuates
the cytokine-mediated induction of pro-MMP-9 in dermal
fibroblasts. The dermal fibroblasts were embedded in collagen and
stimulated with or without TNF- (10 ng/ml) and TGF-
(1 ng/ml) as
indicated. Actinomycin D (Act D) and cycloheximide
(CHX) were applied at the indicated concentration in the
DMEM. After 70-h culture the conditioned media were analyzed for
gelatinolytic zymography.
and TGF-
Activate the Human MMP-9 Gene
Promoter--
The 5'-regulatory region of the human MMP-9
gene has been documented (34, 35, 42). The reporter assay experiment
shows that the minimal response elements for TNF-
stimulation are
located within
670 bp upstream of the transcription start site.
Therefore we tested the cytokine-mediated MMP-9 promoter
activation in human dermal fibroblasts that have the cytokine
responsiveness of the proteinase at both protein and mRNA levels.
The cells were transiently transfected with a plasmid containing a CAT
reporter gene driven by a segment of 670 bp from the 5'-promoter region
of human MMP-9 gene. After transfection the cells were
stimulated with either an individual cytokine or a combination of two
cytokines. Results showed that TNF-
or TGF-
individually could
activate the 670-bp promoter (Fig.
9A). In fact the promoter
activation could be measured after stimulation for 24 h. The
combination of the two cytokines led to a minor additional stimulation.
The transcriptional activation, as measured by CAT assay, solely
depends on the MMP-9 promoter because these cytokines failed
to activate the vector plasmid that has the thymidine kinase basal
promoter (data not shown). The conditioned media from the transfected
cells were also assayed for MMP-9 activities, which may represent the
endogenous MMP-9 promoter activation (Fig. 9B).
These experiments demonstrate that induction of MMP-9 by TNF-
and
TGF-
is regulated at least in part within the 5'-promoter region of
the human gene.
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Fig. 9.
TNF- and
TGF-
activates the human MMP-9
promoter. The human dermal fibroblasts were transiently
transfected by pMMP9-670-CAT, which contains 670 bp of 5'-promoter of
the human MMP-9 gene. After a 3-h transfection the
cells were stimulated by TNF-
(10 ng/ml) and TGF-
(1 ng/ml) as
indicated for an additional 24, 48, and 68 h. The promoter
activation was measured by CAT assay. A, the CAT activities
expressed in the cells are shown as the percentage of acetylated
products measured by PhosphorImaging and are the average of three
replicates. B, the conditioned medium of the transient
transfected cells was analyzed for gelatinolytic activities, which
represent the endogenous MMP-9 induction.
B-responsive Elements in the MMP-9
Promoter Is Essential for TNF-
to Activate the
Transcription--
An NF-
B-responsive element, TGGAATTCCC, which is
located
601 to
592 bp upstream of the transcription start site, was
previously defined in tumor cells (35). However, the role of this
cis-element in TNF-
-mediated induction of MMP-9 in normal human
cells has not been addressed. We scanned the 5'-promoter region of the
human MMP-9 for potential transcription factor binding sites
and found that another potential NF-
B-response element, GGGGGATCCC,
is located
328 to
319 bp upstream from the transcription start site. The difference between the two NF-
B sites is that the
600/
591 site matches to the subtype p65 NF-
B-binding site,
whereas the
328/
319 site matches to p50 NF-
B-binding site. To
clarify the role of these two potential NF-
B elements in the
induction of MMP-9 promoter, we tested the
601/
592
NF-
B deletion variant, pM9-590-CAT (which retains the
328/
319
NF-
B site) for cytokine-induced transactivation. Our data showed
that deletion of the
601/
592 NF-
B site led to a failure of
response to TNF-
but partially retained the TGF-
responsiveness
in the dermal fibroblasts (Fig. 10A). This result also
indicates that the
328/
319 NF-
B site is not sufficient to
mediate the TNF-
signal for transactivation of the promoter. To
verify this notion we generated a site-directed mutant within the
328/
319 element, pM9-670-mp50-CAT, of which four conservative
nucleotides were replaced (illustrated in Fig. 10B). The CAT
assay results show that this mutant has similar responsiveness to the
cytokines as the wild type (Fig. 10A). Thus these
experimental data indicate that the TNF-
-initiated signal is
targeting the
605/
592 p65 NF-
B site.
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Fig. 10.
One of the two potential
NF- B-responsive elements in the
MMP-9 promoter is essential for
TNF-
-mediated promoter activation. The
dermal fibroblasts were transiently transfected with pM9-670-CAT (wild
type), pM9-590-CAT (p65NF-
B delete), and pM9-670-mp50-CAT
(p50NF-
B site mutant). After transfection for 3 h the cells
were stimulated with the cytokines individually or combined.
A, the CAT activities expressed in the cells were measured.
The values represent the mean of results obtained from three replicate
culture wells. B, illustration of the construction used in
the experiments. The potential p65 and p50 NF-
B-binding sites, and
the mutant version are illustrated.
/TGF-
synergistic induction of MMP-9
is a convergence of the two cytokine-initiated pathways on NF-
B
signaling, we engineered a triple NF-
B-responsive element in the CAT
reporter plasmid (pNF
B3x-CAT). A control plasmid that has the
mutated nucleotides within the NF-
B-response element was also
generated (pNF
Bm3x-CAT). The dermal fibroblasts were transfected by
these plasmids and stimulated with TNF-
and TGF-
individually or
simultaneously. The results demonstrate that TNF-
, but not TGF-
,
activates the NF-
B pathway (data not shown). This also implied that
the TGF-
signaling may directly act on the promoter rather than by
cross-talking to synergy in the NF-
B pathway.
-response Element in the MMP-9 Promoter Is Essential for
the TGF-
-mediated Transactivation--
A 10-base pair sequence,
GGTTTGGGGA, located at
474 to
464 bp in the human MMP-9
promoter was previously recognized by matching it to the consensus
sequence of TGF-
inhibitory element, GnnTTGGnGn (35). The
TGF-
-exerted suppression of gene transcription was often contributed
to this inhibitory element, and the proto-oncogene product c-Fos was
identified in a complex that binds at this element (43). However, in
some cases this cis-element has been shown to mediate the
TGF-
-exerted transcription activation (44). Based on these reports
and our observation that TGF-
stimulated the 670-bp promoter, we
speculated that this consensus TGF-
-response element might mediate
the TGF-
-induced MMP-9 transcription. To demonstrate this, we
generated a mutant version of this element, TGATCAGGGGA, of which four
nucleotides were substituted in the TRE site in the 670-bp promoter,
pM9-mTRE670-CAT (Fig. 11). The dermal
fibroblasts were transiently transfected with this plasmid as well as
the wild type plasmid, pM9-670-CAT. After transfection the cells were
stimulated by TNF-
and TGF-
either individually or
simultaneously. As expected, we found that TGF-
failed to activate
pM9-670-mTRE-CAT, whereas the cytokine could activate wild type
pM9-670-CAT. Consistent with these results, TNF-
could stimulate
the transcription of both wild type and TRE mutant promoter. In
addition, we constructed another plasmid, pM9-460-CAT, in which both
the
601 NF-
B and
474 TRE sites are deleted. The results showed
that TNF-
and TGF-
individually or simultaneously failed to
activate the promoter in this plasmid (Fig. 11). Taken together, these
results indicate the
474 TGF-
-response element is the target for
TGF-
signaling at the human MMP-9 promoter. Also, these
results demonstrate that the TNF-
- and TGF-
-response elements are
distinct entities within the 670-bp promoter.
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Fig. 11.
TGF- activates the
minimal promoter of the human MMP-9 through the
TRE-response site. The human dermal fibroblasts were transiently
transfected by the CAT reporter plasmids, pM9-670-CAT (wild type),
pM9-670-mTRE-CAT (TRE site mutant), and pM9-460-CAT (p65 NF-
B and
TRE delete). The cells were stimulated by cytokines individually or
combined. A, the CAT activities from the cells were
measured. The mean value of triplicate experiments is indicated for
each reporter plasmid. B, the plasmid constructs and the
potential cytokine-response elements are illustrated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is part of the early cytokine cascade after skin
injury. TGF-
is deposited in wounds by platelets trapped in fibrin
clots during hemostasis. MMP-9 is present during normal healing for the
first few days after injury and then disappears. The specific role of
MMP-9 in normal healing is not known. However, the elevated pro-MMP-9
level and more significantly the accumulated active 82-kDa MMP-9 is
associated with non-healing wounds (19, 26, 27, 37). Based on the
substrate preference for type IV collagen, the basement membrane
matrix, the implications for the dysregulation of this proteinase are
also clear. Poorly healing wounds have increased levels of inflammatory
mediators, including TNF-
(45). If normal healing requires the
down-regulation of MMP-9 to be successful at a defined spatial and
temporal manner, then increased TNF-
and TGF-
in the wound
maintains MMP-9 activity, and a potential mechanism for failed wound
healing is evident. In this report we demonstrate that an inflammatory
cytokine, TNF-
, potentiates activation of pro-MMP-9 in cultured
human skin. Based on the facts that both TNF-
and active MMP-9 are
elevated in chronic wounds and invasive tumors, we believe that the
presence of this inflammatory cytokine may be the causal factor for the proteolytic activation of the proteinase, which in turn breaks down
type IV collagen in the basement membrane and thus leads to abnormal
healing or tumor cell invasion.
induces pro-MMP-9 expression, and this induction is
enhanced by TNF-
. In addition, in the presence of TNF-
, pro-MMP-9
is proteolytically converted to the 82-kDa active enzyme. This
TNF-
-mediated proteolytic activation could not be detected in the
isolated fibroblast, keratinocyte, or the dendritic cell cultures.
However the data suggest that the unidentified TNF-
-mediated MMP-9
activator is tightly associated to the skin tissue.
and TNF-
individually could
induce MMP-9 (22, 46-48). However, these results came mainly from
tumor-derived cells or lymphocytes, which are not relevant to the
reactions of normal human skin. The synergistic effect of the two
cytokines on pro-MMP-9 induction in the organ-cultured skin correlated
well with the two cytokine-exerted inductions of the enzyme in
fibroblasts and keratinocytes. Based on our data we believe that the
collective response of the cytokine-mediated MMP-9 expression in human
skin is contributed, in part, from both the dermal fibroblasts and
epidermal keratinocytes. Defining the minimal efficacy of TNF-
and
TGF-
on pro-MMP-9 induction at their level of sub-nanogram level is
important because they are within the physiological or pathological
region. Three lines of evidence demonstrated here suggest that the
cytokine-mediated pro-MMP-9 induction is regulated at transcriptional
control. 1) The MMP-9 mRNA is promptly raised after stimulation. 2)
Induction of the enzyme is attenuated by actinomycin D. 3) The
MMP-9 promoter is activated by the cytokines.
-chymotrypsin and stromelysin-1 (MMP-3), collagenase-1, matrilysin,
and MMP-2 can activate this enzyme (49-51). Based on our finding of
TNF-
-mediated MMP-9 activation in the skin, we have surveyed whether
TNF-
can induce MMP-3 (stromelysin-1). Indeed, we found that TNF-
could induce MMP-3 in the skin organ
culture.2 However, in dermal
fibroblast culture we also found elevated MMP-3 levels, whereas the
pro-MMP-9 failed to be activated.2 In fact, in the
stromelysin-1 (MMP-3) knockout mice, pro-MMP-9 could still be
activated, which suggested a potential stromelysin-1-independent pro-MMP-9 activation in mice (52). Thus, the cellular and molecular mechanism for the TNF-
-mediated activation of pro-MMP-9 in the human
skin remains to be elucidated.
-response element was identified within the 670-bp of the
5'-promoter region of the human gene. In this study we confirmed the
importance of the
601 p65 NF-
B for the TNF-
-mediated
transcriptional activation of the MMP-9 gene in the human
dermal fibroblasts. In contrast, the
328 p50 NF-
B site is not
essential for the TNF-
-mediated transcription of the
MMP-9.
-response element was previously recognized at
474
bp in the promoter of the human MMP-9 gene, although the
role of this element was not characterized (35). Our speculation that
this might be the TGF-
-response element in the 670-bp promoter was
demonstrated by mutation and deletion experiments. These showed the
essential role of this site in TGF-
-mediated MMP-9
promoter activation. Although it was previously defined as an
inhibitory element (43), this element can mediate transcription
activation of transglutaminase gene in one cell line and inhibition of
the same promoter in another cell type (44). The choice between activation and inhibition of transactivation seems to rely on other
cellular factors pre-deposited or activated in the particular cell
type. In this study we have demonstrated that TNF-
and TGF-
individually activated their distinct response cis-elements in the
670-bp human MMP-9 promoter. Nevertheless, we did not
observe the full synergistic transactivation by simultaneous
stimulation of the transient transfected cells with TNF-
and
TGF-
, whereas the synergy is overwhelming in the enzyme induction.
Such a difference may derive from the additional hierarchic regulatory
elements in the endogenous promoter of the MMP-9 gene, which
is not included in the 670-bp version of the promoter. Interestingly,
similar co-induction by TNF-
and TGF-
was observed for human type
VII collagen, in which both NF-
B and SMAD factors were found
recruited to the 5'-flanking promoter (53). The signal pathway that
regulates MMP-9 expression is not understood despite some information
suggesting roles for c-Jun amino-terminal kinase and the extracellular
signal-regulated kinase (34).
, is a part
of a complex regulatory system for matrix metalloproteinases. Because
this cytokine is present during the early period after injury, these
findings suggest a role for this mediator in the initial induction and
activation of proteinases in the initial stages of healing. The
specific role of the MMPs in wound healing is, as yet, not fully
understood. Type IV collagen is a substrate for the MMP-9 and an
important component of the epidermal basement membrane (54). We
speculate that properly induced and activated MMP-9 may play a positive
role in tissue repair by promoting keratinocyte detachment from the
basement zone through controlled digestion of the type IV collagen.
Changing the structure of this matrix protein may make it easier for
epidermal cells to detach and begin migrating along the wound margin.
Control of this process would be essential, since continued dissolution
of the basement membrane would prevent a normal epidermal layer from
developing. In the healing wound, TNF-
and TGF-
are seldom both
present beyond the first few days after injury. Because there are
response elements for both cytokines in the promoter of MMP-9,
transcription of this proteinase is more tightly controlled. Validating
this hypothesis will be the focus of future experiments.
decrease the activation of MMP-9 in
chronic wounds? More importantly, will blocking TNF-
cause chronic
wounds to heal? Although TGF-
also appears to be involved in the
up-regulation of MMP-9, it is usually present in multiple phases of
wound healing. This fact makes us conclude that manipulation of TGF-
concentration is less likely to affect MMP-induced abnormal wound
healing. These are questions that will require significant further study.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Peter Laird for valuable editorial assistance and Dr. Susan Downey for supplying of the human skin discarded after reconstructive procedures. We also thank Drs. Nimni and Han for sharing their equipment.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Grant GM 50967.
¶ Supported by National Institutes of Health Grant GM55081.
To whom correspondence should be addressed: 1450 San Pablo
St., Ste. 2000, Division of Plastic and Reconstructive Surgery, University of Southern California School of Medicine, Los Angeles, CA
90033. Tel.: 323-442-6410; Fax: 323-442-6477; E-mail:
wgarner@surgery.usc.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M010839200
2 Y.-P. Han, T.-L. Tuan, M. Hughes, H. Wu, and W. L. Garner, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF-, tumor
necrosis factor-
;
TGF-
, transforming growth factor-
;
bp, base
pair;
MMPs, matrix metalloproteinases;
DMEM, Dulbecco's modified
Eagle's medium;
CAT, chloramphenicol acetyltransferase;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
ECM, extracellular matrix;
TRE, TGF-
-response element;
PDGF, platelet-derived growth factor;
EGF, epidermal growth factor;
Il, interleukin.
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REFERENCES |
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