From INSERM, Unité 145, Institut
Fédératif de Recherche (IFR) 50, 06107 Nice, France,
¶ INSERM, Unité 526, IFR 50, 06107 Nice, France, and
Institut de Génétique et de Biologie
Moléculaire, Unité Mixte de Recherche 7104, 67404 Illkirch, France
Received for publication, September 9, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Matrix metalloproteinases (MMPs)
are essential for proper extracellular matrix remodeling, a
process that takes place during obesity-mediated adipose tissue
formation. Here, we examine expression profiles and the potential role
of MMPs and their tissue inhibitors (TIMPs) in adipose tissue
remodeling during obesity. Expression patterns are studied by Northern
blot and real-time PCR in two genetic models of obesity
(ob/ob and db/db mice) and in a diet-induced model of obesity (AKR mice). Of the MMPs and TIMPs studied, mRNA levels for MMP-2, MMP-3, MMP-12, MMP-14, MMP-19, and TIMP-1 are strongly induced in obese adipose tissues compared with lean tissues. In contrast, MMP-7 and TIMP-3 mRNAs are markedly decreased in obesity. Interestingly, enzymatic activities of MMP-12 and of a new
identified adipocyte-derived 30-kDa metalloproteinase are enhanced in
obese adipose tissue fractions, demonstrating that MMP/TIMP balance is
shifted toward increased matrix degradation in obesity. Finally, we
analyze the modulation of MMP-2, MMP-19, and TIMP-1 during 3T3-L1
preadipocyte differentiation, and we explore the effect of inhibition
of MMP activity on in vitro adipogenesis. We find that the
synthetic MMP inhibitor BB-94 (Batimastat) decreases adipose conversion
of 3T3-L1 and primary rat preadipocytes. BB-94 represses
differentiation without affecting mitotic clonal expansion but prevents
the early expression of CCAAT/enhancer-binding protein Obesity is a nutritional disorder characterized by excess
of adipose tissue (1). Adipose tissue mass is a reflection of the
number of adipocytes and their amount of fat stored. The development of
obesity is associated with coordinated cellular processes, including
adipocyte hypertrophy followed by recruitment of adipocyte precursors,
and new fat cell differentiation (2, 3). These processes are also
accompanied by neovascularization that is essential for generation and
proper function of the tissue (4). It is generally accepted that such
multiple events include dynamic changes of cell-matrix interactions and
extensive extracellular matrix (ECM)1 remodeling, and that
modifications in proteolytic activities within the adipose
microenvironment might occur during the development of the fat depot.
Although numerous studies have examined the regulation and
intracellular events that occur during adipogenesis, only limited
information is available about molecular mechanisms underlying ECM
remodeling, proteolytic events, and cell-matrix interactions during
obesity-related fat mass development. In a course of differential gene
expression analysis between lean and obese adipose tissues, we recently
identified osteonectin/SPARC, a protein involved in dynamic
interactions between cell and matrix, up-regulated in obesity (5). This
suggested that alteration in the expression level of matrix proteins
may contribute to the development of obesity-associated adipose
tissue growth.
A balance between the opposing activities of proteinases and their
inhibitors controls pericellular proteolytic events. Among enzymes
implicated in the degradation of matrix molecules and in the generation
of bioactive factors, the matrix metalloproteinase (MMP) family is
considered to be primarily responsible for these processes (6). MMPs
comprise a large family of structurally related
zinc-dependent proteinases that has been classified into subgroups on the basis of their structure, substrate specificity, and
cellular localization. These subgroups are collagenases, gelatinases, stromelysins, membrane-type MMPs (MT-MMPs), and other MMPs (7, 8). MMPs
participate in many physiological and pathological processes such as
embryonic development, angiogenesis, wound repair, reproductive
cycling, and metastasis (6, 7). In addition to their direct influence
on the degradation of structural matrix molecules, MMPs have also been
implicated in the generation of bioactive molecules. They can mediate
the release and/or activation of sequestered growth factors and the
cleavage of cell surface adhesion receptors (7). The activity of MMPs
is regulated at the level of gene transcription, proenzyme activation,
and via inhibition of their activity by endogenous inhibitors, the
tissue inhibitors of MMPs (TIMPs) (9). TIMPs are a family of four secreted proteins (TIMP-1 to TIMP-4) that selectively inhibit MMPs in a
1:1 stoichiometric manner. Interestingly, TIMPs can exert biological
activities independent of their MMP-blocking action. The balance
between MMPs and TIMPs is a critical determinant of ECM integrity and
function, and alterations in MMP/TIMP-mediated proteolysis may
contribute to many pathological states.
The role of matrix remodeling and proteolytic pathways that occur
during adipogenesis and adipose tissue formation has just begun to
unfold. Recent studies have indicated that matrix degradation might be
essential for adipogenesis. It has been proposed that serine proteases
of the plasminogen system positively regulated adipocyte
differentiation in vitro, as well as in vivo
during mammary gland involution (10). Other studies have demonstrated that mature fat cells and cultured adipocytes secreted gelatinases A
and B (MMP-2 and MMP-9, respectively), and that their proteolytic activities were induced during adipocyte differentiation (11, 12).
Further, elevated expression of MMP-2 was reported in adipose tissue of
obese mice (13). Interestingly, treatment of 3T3-F442A preadipocytes
with synthetic MMP inhibitors and by neutralizing antibodies decreased
differentiation, suggesting that MMP activities were required for
adipocyte conversion (12). In contrast, another study showed that
addition of TIMP-1 or MMP inhibitor GM6001 accelerated 3T3-L1 adipocyte
differentiation (14). In addition, this study also demonstrated that
stromelysin-1 (MMP-3) determined the rate of adipose reconstitution
during mammary gland involution in mice (14). Moreover, on a high fat
diet, stromelysin-3 (MMP-11)-deficient mice developed adipocyte
hypertrophy compared with wild-type mice (15). Together, these reports
revealed a novel function for MMPs as modulators of adipogenesis.
However, their expression profile and role in the cellular
microenvironment during obesity-mediated adipose tissue development
remain poorly defined. Here, we examine the cellular regulation of MMPs
and their endogenous inhibitors in white adipose tissue from two rodent
genetic models of obesity and from a diet-induced model of obesity, and
we further explore the effect of pharmacological inhibition of
metalloproteinases on adipogenesis. Our data show that obesity is
associated with profound changes in the MMP/TIMP balance and support a
potential role for these matrix proteins in the control of remodeling
events and adipogenesis during obesity-mediated fat mass development.
Animals and Protocols--
Care of animals was performed in
accordance with institutional guidelines. Male C57BL/6,
ob/ob, db/db, and AKR mice
were obtained at 6 weeks of age from Harlan (Gannat, France).
Male Wistar rats (150-200 g) were from Iffa-Credo (L'Arbresle,
France). Animals were maintained in a temperature-controlled
facility (22 °C) on a 12-h light-dark cycle with regular
unrestricted diet. Induction of obesity by high fat diet in AKR mice
was carried out as described previously (5). Animals were killed by
cervical dislocation, and epididymal fat pads were rapidly dissected
and processed for RNA or protein analysis.
Total RNA Extraction and Northern Blot--
Total RNA from
epididymal white adipose tissue or cells was isolated using the TRIzol
reagent following the instructions from the manufacturer (Invitrogen).
Northern blotting was conducted as previously described (5). Briefly,
10 µg of denatured RNA was resolved by electrophoresis on 1.2%
agarose gels and transferred to positively charged nylon membrane.
Blots were hybridized overnight at 42 °C with specific
32P-labeled cDNA probes. Membranes were exposed to
phosphor screen for 4-24 h, and the signals were scanned with a STORM
840 and quantified using ImageQuant 5.0 software (Molecular Dynamics, Amersham Biosciences). Blots were stripped and rehybridized with an 18 S oligonucleotide probe as an indicator of RNA integrity and loading.
In some experiments data were normalized to the signal generated from
18 S probe.
Real-time Quantitative PCR--
After treatment with DNase I, 1 µg of total RNA was reverse transcribed using random priming and
Superscript II reverse transcriptase (Invitrogen), according to the
instructions from the manufacturer. Quantitative PCR was performed by
monitoring in real time the increase in fluorescence of the SYBR Green
dye on an ABI PRISM 7000 Sequence Detector System (Applied Biosystems)
according to the instructions from the manufacturer. Gene-specific
primers (Invitrogen) were designed using the Primer Express software
from Applied Biosystems (Table I).
Relative expression level of the target gene in obese adipose tissue
was plotted as -fold change compared with lean control. 18 S rRNA was
used for normalization. Each real-time quantitative PCR assay was
performed twice using triplicate samples.
cDNA--
Mouse cDNA clones for matrilysin (MMP-7),
stromelysin-2 (MMP-10), and MMP-19 were gifts from T. Ny (Umeå
University, Umeå, Sweden). cDNA probes for collagenase-2 (MMP-8)
and murine collagenase-like A and B (Mcol-A and -B, respectively) were
gifts from C. López-Otín (University of Oviedo, Oviedo,
Spain). Rat cDNA fragments for gelatinase A (MMP-2), gelatinase B
(MMP-9), stromelysin-1 (MMP-3), stromelysin-3 (MMP-11), MT1-MMP
(MMP-14), collagenase-3 (MMP-13), TIMP-1, TIMP-2, and TIMP-3 were used
to probe for the corresponding mRNAs. cDNA probes for mouse
metalloelastase (MMP-12) and TIMP-4 were obtained by reverse
transcriptase-PCR from mouse epididymal white adipose tissue RNA as
described for other probes (5). cDNA for aP2 was provided by C. Dani (CNRS, UMR 6543, Nice, France). cDNAs were labeled to high
specific activity by random priming and used as probes for Northern
blot analysis.
Adipose Tissue Fractionation--
Epididymal fat pads from male
Wistar rats or from male C57BL/6 and ob/ob mice were minced
and digested with the Liberase Blendzyme 3 (Roche Molecular
Biochemicals) as previously described (5). Isolated adipocytes were
separated from stromal-vascular cells (S-V) by centrifugation. The
floating top layer of adipocytes and the S-V pellet were washed three
times in Krebs-Ringer bicarbonate Hepes pH 7.4 buffer and processed for
RNA or protein preparation. Adequate cellular fractionation was
confirmed by analyzing the expression of S-V and adipogenic markers
(Wnt-10b and aP2, respectively). For primary rat preadipocyte culture,
the S-V fraction was filtered through a 30-µm nylon mesh, centrifuged
at 500 × g for 5 min, and plated at 50% confluence in
the presence of Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum and antibiotics.
Substrate Zymography--
Freshly isolated adipocytes or S-V
cells were lysed in a buffer containing 1% Triton X-100, 150 mM NaCl, and 20 mM Tris, pH 7.4, supplemented
with a protease inhibitor mixture (Complete EDTA-free, Roche Molecular
Biochemicals) at 4 °C under agitation for 30 min. Lysates were
clarified by brief spinning, and protein concentration was evaluated by
bicinchoninic acid technique (BCA protein assay kit, Pierce). 40 µg
of nonreduced protein sample was loaded on 10% SDS-polyacrylamide gels
containing 1 mg/ml Adipocyte Differentiation--
3T3-L1 preadipocytes (American
Type Culture Collection) and primary rat preadipocytes prepared as
described above were propagated in DMEM supplemented with 10% fetal
calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin and
allowed to reach confluence. After 2 days (day 0), the differentiation
was initiated by addition of a hormonal mixture composed of 2 µM insulin, 1 µM dexamethasone, and 0.25 mM isobutylmethylxanthine in DMEM plus 10% fetal bovine serum with or without 10 µM BB-94. Three days later (day
3), the induction medium was replaced by DMEM supplemented with 10%
fetal bovine serum plus insulin only, and cells were then fed every 2 days. Adipogenesis was scored by analysis of the expression of
adipocyte-specific genes (aP2 and peroxisome proliferator-activated receptor
Cell number at various stages of differentiation was determined by
trypsinizing cell monolayers from six-well culture plates followed by
counting with a Coulter Counter (Coulter Electronics). Data are
expressed as the mean ± S.D.
Nuclear Extracts and Western Blot--
Nuclear extracts from
3T3-L1 cells were prepared at the indicated times according to the
method of Schreiber et al. (16) with the following
modifications. Cell monolayers were washed twice in ice-cold
phosphate-buffered saline, pH 7.4, scraped, and collected by
centrifugation at 1500 × g for 5 min. Cell pellet was
lysed in cold hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and protease inhibitor mixture)
supplemented with 10% IGEPAL CA-630 (Sigma). After 15 min on ice, the
homogenate was centrifuged at 3000 × g for 30 s.
Nuclear pellet was resuspended in ice-cold hypertonic buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, and
protease inhibitor mixture) and vortexed every 5 min during 30 min on
ice. The supernatant was collected after centrifugation at 12,000 × g for 5 min. The resulting nuclear extracts (30 µg)
were separated by SDS-polyacrylamide gel electrophoresis and analyzed
by immunoblot using a polyclonal antibody to C/EBP Expression of MMP and TIMP mRNAs in Genetic Models of
Obesity--
To test the hypothesis that obesity-linked adipose tissue
growth might be associated with alterations in MMP/TIMP balance, we
first evaluated expression of 13 MMPs in epididymal white adipose tissue of genetically obese ob/ob and
db/db mice and their lean controls (see Table
II for MMP nomenclature). The extreme
obesity of these two strains results from leptin signaling disruption, and they represent well studied models of obesity.
ob/ob mice lack functional leptin, whereas
db/db mice have no functional leptin receptor
(17, 18). Northern blot analysis showed that MMP-2, MMP-3, MMP-12,
MMP-14, and MMP-19 mRNA levels were markedly increased in adipose
tissue of both ob/ob and
db/db mice compared with their lean littermates
(C57BL/6) (Fig. 1A). In
contrast, MMP-7 transcript was strongly reduced in obese adipose
tissues. MMP-9, MMP-11, and MMP-13 transcripts were detected in fat
tissues, but levels were similar between lean and obese mice (data not shown). MMP-8, Mcol-A, Mcol-B, and MMP-10 transcripts were not detected
in adipose tissue by Northern blot analysis (data not shown). Because
activity of mature MMPs and activation of proenzymes are regulated by
their physiological inhibitors, the TIMPs, we compared expression
pattern of the four members of the TIMP family (TIMP-1 to TIMP-4)
described to date in adipose tissue of lean and obese mice. As
previously documented, all TIMPs were found to be expressed in white
fat (19). TIMP-1 mRNA expression level was strongly elevated in
adipose tissue of ob/ob and
db/db mice, whereas TIMP-3 mRNA level was
decreased (Fig. 1B). No significant changes in TIMP-2 and
TIMP-4 transcripts were observed between lean and obese adipose
tissues.
Expression of MMP and TIMP mRNAs in Diet-induced
Obesity--
To further characterize the regulation of MMPs and TIMPs
in obesity, we analyzed their expression in wild-type AKR mice that developed obesity on high fat diet. In the following experiments, we
focused mainly on MMP-2, MMP-7, MMP-12, MMP-19, TIMP-1, and TIMP-3 that
were the most differentially expressed in fatty tissues studied. AKR
mice were fed a high fat diet or control chow for 12 weeks. Fat pads
were dissected and weighed (Fig.
2A). As observed previously,
fat pads from fat-fed mice weighed ~4.5-fold more than those of
control mice (Fig. 2A) (5). Northern blot analysis showed
that MMP-12, MMP-19, and TIMP-1 transcripts were elevated in adipose
tissue of fat-fed mice as compared with chow-fed, whereas MMP-7
mRNA level strongly decreased in adipose tissue from obese mice
(Fig. 2B). In parallel experiments, TIMP-2 and TIMP-4
transcripts remained constant, whereas MMP-2 and TIMP-3 transcripts
were below the detection level in these tissues (data not shown). Using
a more sensitive assay, we examined the amount of MMP-2 and TIMP-3 mRNAs in diet-induced obesity. Adipose tissue from fat-fed mice showed a 2.5-fold increase in MMP-2 mRNA level and a 2-fold
decrease in TIMP-3 mRNA level, as assessed by real-time
quantitative PCR (Fig. 2C).
These data demonstrate that expression of MMP-2, MMP-7, MMP-12, MMP-19,
TIMP-1, and TIMP-3 was modulated in mice with diet-induced obesity. Our
observations are consistent with those obtained in ob/ob and db/db mice in
whom obesity is inherited trait (Fig. 1). These findings collectively
show that obesity is associated with profound changes in expression of
a subset of MMPs and TIMPs in adipose tissue.
Expression of MMP and TIMP mRNAs in Adipocytes and
Stromal-Vascular Cells Isolated from Lean and Obese Adipose
Tissue--
In addition to mature fat cells, white adipose tissue
contains adipocyte precursors and a variety of other cell types such as
fibroblasts, smooth muscle cells, endothelial cells, and macrophages. We next determined the source of MMPs and TIMPs expression in adipose
tissue. Fat pads from lean (C57BL/6) and ob/ob
mice were divided into adipocytes and non-adipose cells. Total RNA of
these two fractions was analyzed by Northern blot and real-time
quantitative PCR (Fig. 3). Adequate
separation of the two cell fractions was confirmed by quantitative PCR
assay for the S-V marker Wnt-10b, and by Northern blotting for the
adipocyte marker aP2 (Fig. 3C) (20, 21). All transcripts
tested were detected in both adipocytes and their associated S-V cells.
However, except for MMP-19, expression of all transcripts was mainly
associated with the S-V compartment. Fig. 3A shows that in
ob/ob-derived cells MMP-7 mRNA was decreased by a
1.9-fold (adipocytes) and by a 4.6-fold (non-adipose cells) compared
with lean counterparts, whereas MMP-12 transcript was strongly induced
in the two obese fractions (4.4- and 17.8-fold in adipocytes and S-V
cells, respectively). TIMP-1 transcript was, respectively, 2.6- and
4.3-fold higher in obese adipocytes and S-V cells than in lean ones.
MMP-19 transcript was increased approximately by 1.4-fold in the two
fractions isolated from ob/ob mice. Fig.
3B shows real-time PCR analyses of MMP-2 and TIMP-3 transcript distribution in different cell fractions. MMP-2 mRNA level was increased by 2.2-fold in obese adipocytes, whereas the amount
was decreased by 3-fold in S-V cells isolated from obese fat tissue. In
ob/ob-derived cells, TIMP-3 transcript was
markedly decreased in adipocytes (3.6-fold) and in S-V cells (8.2-fold) compared with lean controls. Note that modulation of MMP-7, MMP-12, TIMP-1, and TIMP-3 expression was to a greater degree in S-V cells than
in adipocytes.
These results indicate that the S-V fraction of adipose tissue is the
main source of MMP and TIMP expression, and that modulation of their
expression profile in obesity occurs mainly in these cells.
MMP Enzymatic Activities Associated with Adipocytes and
Stromal-Vascular Cells Isolated from Lean and Obese Adipose
Tissue--
We next examined MMP activities in adipocytes and
non-adipose cells from lean (C57BL/6) and ob/ob
adipose tissue to determine whether modulation of mRNA steady state
levels is reflected by changes of corresponding proteins.
Cell-associated metalloproteinase activities were assessed by type I
collagen and
Together, these data confirm at the protein level the
overexpression of MMP-12 in obese tissue and allow the observation of a ~30-kDa metalloproteinase activity in obese adipose tissue.
Synthetic MMP Inhibitor BB-94 Prevents Adipocyte
Differentiation--
In an effort to understand the role of MMPs in
adipose tissue mass formation in vivo, we investigated the
expression pattern of MMP-2, MMP-7, MMP-12, MMP-19, TIMP-1, and TIMP-3
during adipocyte differentiation in vitro. Growth-arrested
3T3-L1 preadipocytes (day 0) were induced to differentiate by addition
of adipogenic mixture. Adipose conversion was monitored by the analysis
of the adipocyte lipid-binding protein aP2 expression. Northern blot analysis showed that MMP-2 is strongly expressed in committed preadipocytes (Fig. 5A). After
a strong decline within 1 day after induction of differentiation,
expression of MMP-2 was induced at day 2, reached maximal level at day
3, and then returned to a low level at day 6 after induction. In
contrast, MMP-19 mRNA level increased throughout the
differentiation process, whereas TIMP-1 transcript is highly expressed
in preadipocytes and decreased during adipocyte conversion. Expression
of MMP-7, MMP-12, and TIMP-3 mRNAs was below the detection level
(data not shown).
These observations prompted us to examine the effect of pharmacological
inhibition of MMPs on adipogenesis in vitro. Modulation of
preadipocyte differentiation by MMP inhibitors has been reported recently (12, 14). However, the precise molecular mechanisms involved
remain unknown. We wanted to extend the above findings by first
studying the effect of BB-94, a broad spectrum MMP inhibitor on 3T3-L1
and primary rat preadipocyte differentiation. Cell differentiation was
induced by the addition of the adipogenic medium in the presence or
absence of 10 µM BB-94 (Fig. 5B).
Differentiation was assessed by staining with oil red O to detect
intracellular triglyceride deposit accumulation and by
adipocyte-specific gene induction analysis. Undifferentiated
preadipocytes cultured for the same period and stained with oil red O
is shown for comparison. Cells cultured in the presence of the
induction medium accumulated lipid droplets and adopted morphological
characteristics of adipocytes. In contrast, BB-94 treatment throughout
the differentiation protocol severely decreased the number of 3T3-L1
and primary differentiated cells (Fig. 5B). The effect of
another MMP inhibitor, GM6001, was also investigated. Similar to our
observations with BB-94, 3T3-L1 preadipocyte differentiation was
inhibited in presence of GM6001 (data not shown).
We also examined the expression of adipocyte markers in differentiating
3T3-L1 exposed to BB-94. Northern blot analysis was performed using the
late adipogenic marker aP2 as probe (Fig. 5C). In
vehicle-treated cells, aP2 transcript was induced at day 3 and reached
maximal level at day 6 after induction of differentiation (Fig.
5C). Expression of aP2 was delayed in BB-94-treated cells, and the level of expression of this mRNA never achieved that of vehicle-treated cells. The expression of PPAR Synthetic MMP Inhibitor BB-94 Blocks Early Induction of
C/EBP Rosiglitazone Rescues the Effect of BB-94 on Adipocyte
Differentiation--
Finally, we tested whether rosiglitazone could
rescue differentiation of 3T3-L1 in presence of BB-94. Rosiglitazone is
a potent activator of PPAR In this study we have described the expression pattern and
activation of a set of MMPs in the adipose tissue during obesity, and
we have provided further evidence for involvement of metalloproteinase activities in adipocyte differentiation. From our observations, we
propose that the MMP/TIMP system might play an integral role in
obesity-mediated adipose tissue growth. By studying differences between
obese and lean fat tissues, we found that the expression of five MMPs
(MMP-2, MMP-3, MMP-12, MMP-19, MMP-14) and one TIMP (TIMP-1) was
up-regulated in obese adipose tissues, whereas that of MMP-7 and TIMP-3
was down-regulated. We describe the altered expression profile of MMPs
and TIMPs in several models of experimental obesity, including genetic
models of obesity caused by mutations in the leptin receptor or leptin
itself. Leptin is an adipocyte-derived hormone that plays an important
role in the regulation of energy balance (26). More recently, this
hormone has also been implicated in angiogenesis and in matrix
remodeling by modulating MMP and TIMP expression (27, 28). However, our
data clearly demonstrate that regulation of MMP and TIMP expression in
adipose tissue during obesity is independent on a functional leptin
signaling system. However, this does not preclude the possibility that,
in diet-induced obesity, leptin could be involved in MMP/TIMP
regulation. Moreover, although all MMPs and TIMPs transcripts analyzed
were detected in the adipocyte fraction of the adipose tissue, we found
that, except for MMP-19, they were predominantly expressed by
surrounding S-V cells. This observation suggests that obesity-linked
matrix remodeling events are associated with the expression of a
complex mix of MMPs that is mainly orchestrated by the S-V compartment of the adipose tissue. Further studies are required to precisely define
sites of MMPs release in remodeling adipose tissue during obesity.
Interestingly, a similar situation is observed during tumor
establishment, where it is not only tumor cells that overexpress MMPs,
but also surrounding, normal stromal cells.
During the preparation of this manuscript, one study reported the
analysis of MMPs and TIMPs expression in a model of nutritionally induced obesity (29). Similar results were reported concerning the
increased amount of MMP-3, MMP-12, MMP-14, and TIMP-1 and decreased
amount of MMP-7 in obesity. However, this work also reported increased
levels of MMP-11 and MMP-13 mRNAs and diminished levels of MMP-9
and TIMP-4 mRNAs. In contrast, we found no significant modulation
of these latter transcripts associated with obesity. Whether these
differences in expression pattern are related to different experimental
approaches (semiquantitative reverse transcriptase-PCR versus Northern blot and real-time quantitative PCR in our
present study) or reflect differences in models of obesity studied
remains to be established.
Using a combination of collagen and In this report, MMP and TIMP expression was primarily studied at the
transcript level, which does not necessarily reflect the level of
protein and/or activity. However, the study of MMP activity in
vivo is complex, because there is a lack of sensitive and specific
assays and the activated form of some MMPs can be lost during adipose
tissue extraction and sample preparations. However, in the case of
MMP-12, the induction of its transcript correlated with the induction
of caseinase activity, as shown by zymography. In agreement with
previous studies, we found that adipocytes produced high levels of
MMP-2 gelatinase activity (12, 13). However, the level of its activity
was not modulated in obesity, whereas MMP-2 transcript was up-regulated
at least in the adipocyte fraction. Note that the presence of active
MMP-2 in both adipocyte and S-V cells is associated with a
corresponding expression of MMP-14 (MT1-MMP) and TIMP-2, which are
required for MMP-2 activation (7). Together, our data suggest that
changes in expression of MMPs associated with obesity lead to an
increase of proteolytic activities, and that release and activation of MMPs are likely to create a proteolytic environment where different molecules can be degraded.
Of the MMPs studied, MMP-7 (also known as matrilysin) exhibits a unique
expression pattern in obesity. MMP-7 is an epithelial-specific MMP that
has been involved in promoting tumorigenesis and epithelial cell
apoptosis (32). Expression of two natural inhibitors of metalloproteinases was also modulated in adipose tissue during obesity.
The differential regulation of TIMP-1 and TIMP-3 might have biological
significance in adipose tissue homeostasis. Indeed, TIMP-1 and TIMP-3
have divergent effects on cell proliferation and death. TIMP-1 has
growth factor-like activity in a variety of cell types and can inhibit
apoptosis through a non-MMP inhibitory pathway (33, 34). In contrast to
TIMP-1 and depending on the cell types examined, TIMP-3 promotes
apoptosis (35). Thus, it is tempting to speculate that the concomitant
modulation of TIMP-1, TIMP-3, and MMP-7 in obesity would result in
suppression of apoptosis and might be involved in maintenance of
adipose tissue homeostasis. In addition, low level of TIMP-3 might
increase MMP activities and thus be involved in maintaining a high
proteolytic index within the adipose microenvironment. Alternatively,
TIMP-3 suppression might influence the activity of the ADAM (a
disintegrin and metalloprotease) family of metalloproteinases because
reports indicated that TIMP-3 exhibits inhibitory activity against
several ADAMs that are not inhibited by other TIMPs, such as tumor
necrosis factor Because of the multitude of functional properties attributed to the MMP
system, it is possible that MMP activities affect multiple processes,
acting in concert to lead to the development of adipose mass. MMPs
could increase matrix plasticity, thereby facilitating adipose tissue
remodeling and/or adipocyte hypertrophy. Indeed, adipocytes produce a
complex mixture of ECM and are enveloped by a basement membrane (37).
It is likely that MMP-2, MMP-3, MMP-14, and MMP-19 participate in the
degradation of the adipocyte ECM, as they have been shown to degrade
collectively basement membrane components. Further, it is conceivable
to suggest that other factors may constitute physiological targets of
MMP-mediated cleavage within the adipose microenvironment during
obesity. MMPs might control the release of transforming growth
factor- Finally, MMPs may have additional functions in obesity-associated
adipose tissue growth such as supporting the differentiation of adipose
precursor cells. Recent studies have documented the change of MMP gene
expression and activities during adipocyte differentiation (11, 12,
14). Consistent with these data, we showed that MMP-2 and TIMP-1 are
differentially regulated during adipocyte differentiation. Moreover, we
demonstrated that MMP-19 mRNA expression is detectable only in
mature 3T3-L1 adipocytes. This prompted us to investigate the role of
metalloproteinase activity in adipocyte conversion. In agreement with
previous studies performed in 3T3-F442A preadipocytes (12, 29), we
found that BB-94 prevents the differentiation of 3T3-L1 and primary
preadipocyte cultures, suggesting that MMP activities are likely
important for adipocyte differentiation. We cannot rule out the
possibility that the peptide hydroxamate BB-94 may also influence
adipocyte differentiation by inhibiting other related
metalloproteinases such as members of the ADAM family (7). However, it
has been shown that preadipocyte treatment with MMP-2 and MMP-9
neutralizing antibodies also decreased adipocyte differentiation (12).
In addition, a recent study has revealed that daily injection of galardin, a synthetic MMP inhibitor, impaired adipose tissue
development in mice (41). These observations further support the
hypothesis that the proteolytic activity of MMPs is critical for
adipose tissue development. More interestingly, we observed that BB-94 acted early in the differentiation program, at the level of C/EBP In conclusion, we have revealed that obesity is associated with
specific changes in the MMP/TIMP balance and that MMP activities modulate adipocyte differentiation at an early stage of the program. Our data illustrate a potential role for these matrix proteins in the
control of proteolytic events within the adipose tissue.
, a
transcription factor that is thought to play a major role in the
adipogenic program. Such findings support a role for the MMP/TIMP
system in the control of proteolytic events and adipogenesis during obesity-mediated fat mass development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Primers used for amplification of MMP-2, TIMP-3, and Wnt-10b PCR
products
-casein (Sigma) or type I collagen prepared from
rat tail tendon. Following electrophoresis, proteins were renatured by
incubating gels in 2.5% Triton X-100 for 2 h at 37 °C. Gels
were then washed three times in distilled water and incubated in
substrate buffer (50 mM Tris, pH 7.4, and 5 mM
CaCl2) at 37 °C for 24 h (collagen zymography) or
48 h (
-casein zymography) with gentle shaking. Gels were
stained with 0.1% Coomassie Blue R-250 (Sigma) and destained in 7%
acetic acid. Enzymatic activities appear as cleared bands in a dark
background. In separate experiments, gels were incubated at 37 °C in
substrate buffer containing the hydroxamate MMP inhibitor BB-94
(British Biotech, Oxford, United Kingdom; 10 µM) or EDTA
(1 mM) to verify that zymogen activities were attributable
to MMPs.
(PPAR
)) and by lipid accumulation using microscopic analysis or oil red O staining. In standard conditions, cytoplasmic lipid droplets were visible by day 4, and cells were fully
differentiated by day 6. For BB-94 treatment, control cells were
exposed to an identical concentration of vehicle (Me2SO).
Where indicated, cell differentiation was initiated in the presence of
rosiglitazone (0.01 µM).
, or a monoclonal
antibody to PPAR
from Santa Cruz Biotechnology, Inc. Immunoreactive
proteins were revealed by enhanced chemiluminescence using
ECLTM (Amersham Biosciences). Western blotting was also
performed on adipocytes and S-V cell lysates to evaluate MMP-12
expression. Each cellular fraction was mixed with Laemmli buffer (3%
SDS, 70 mM Tris, pH 7, 11% glycerol) and protein
concentration was assayed by bicinchoninic acid technique (BCA protein
assay kit, Pierce). Samples were separated by SDS-polyacrylamide gel
electrophoresis and analyzed by immunoblot using a monoclonal antibody
against MMP-12 (R&D Systems, Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Nomenclature of MMPs studied
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Fig. 1.
Expression of MMP and TIMP mRNAs in
genetic models of obesity. 10 µg of total RNA isolated from
epididymal fat pads of 17-week-old ob/ob mice
(n = 3), 24-week-old db/db mice
(n = 3), and their lean controls (C57BL/6)
(n = 8) was analyzed by Northern blot. mRNA level
of the indicated MMPs (A) and TIMPs (B) was
measured using 32P-labeled appropriate cDNA probe. 18 S
rRNA is shown as a control for loading and integrity of RNA. Data shown
are representative of at least three independent experiments.
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Fig. 2.
Expression of MMP and TIMP mRNAs in
diet-induced obesity. The obesity-prone AKR strain was fed with
regular diet (chow) or high fat diet (high
fat). A, after 12 weeks, animals
(n = 5/group) were euthanized and the epididymal fat
pads were removed and weighed. B, total RNA was extracted
and expression of MMP-7, -12, -19, and TIMP-1 was analyzed by Northern
blot. 18 S rRNA is shown as a control for loading and integrity of RNA.
C, mRNA expression level of MMP-2 and TIMP-3 was
analyzed by real-time quantitative PCR. mRNA expression data was
normalized to 18 S rRNA level in the corresponding sample. Values are
the mean ± S.D. of three independent experiments.
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Fig. 3.
Expression of MMP and TIMP
mRNAs in adipocytes and S-V cells of adipose tissue from lean and
obese mice. Adipocytes (adip) from 13-week-old
ob/ob mice (n = 4) and lean controls
(C57BL/6) (n = 10) were separated from stromal-vascular
fraction and total RNA was prepared from each cellular fraction.
A, quantification of mRNA levels for MMP-7, -12, and -19 and TIMP-1 analyzed by Northern blot. The intensity of hybridization
signal was calculated from the ImageQuant program and normalized
against the relative level of 18 S rRNA in the corresponding sample.
B, mRNA level for MMP-2 and TIMP-3 was determined by
real-time quantitative PCR. mRNA expression data were normalized to
18 S rRNA level in the corresponding sample. C, expression
of adipocyte and S-V cell-specific markers. Wnt-10b mRNA was
analyzed by quantitative PCR, and aP2 mRNA was measured by Northern
blot. Values are the mean ± S.D. of three independent
experiments.
-casein substrate gel zymography (Fig.
4). Collagen zymography allowed the
detection of enzymatic activities at ~82-92 and 62-72 kDa that are
consistent with latent proforms and active forms of MMP-9 and MMP-2,
respectively (Fig. 4A). Activities were high in both cell
fractions, and there was no significant difference between lean and
obese cells. Note that a similar pattern of activities was observed in
gelatin substrate zymography (data not shown). Using
-casein, a
preferential substrate for MMP-12 (22), we showed that S-V cells
contained a caseinolytic activity migrating at 22 kDa. The molecular
mass of this activity corresponds to that expected for the active form
of MMP-12. This activity was detectable only in the non-adipose
fraction and was strongly increased in obese cells compared with lean
ones (Fig. 4B). Under conditions of sample preparation used
here, we did not observe the activity of the proenzyme form of MMP-12.
Immunoblot analysis further confirmed the identity of this caseinolytic
band as MMP-12 (Fig. 4C). In addition, a prominent ~30-kDa
caseinase was detected in both adipocytes and S-V cells (Fig.
4D). Interestingly, this activity was higher in adipocytes
from ob/ob mice compared with that of lean mice,
suggesting that increase of this activity is adipocyte-specific.
Further, the 30-kDa caseinase was inhibited by BB-94 (Fig.
4D, lower panel), which is consistent
with a metalloproteinase activity.
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Fig. 4.
MMP enzymatic activities in adipocytes and
S-V cells of adipose tissue from lean and obese mice. Epididymal
fat pads from 13-week-old obese ob/ob mice
(n = 3) and their lean control (C57BL/6)
(n = 8) were divided into adipocytes and stromal cells.
40 µg of nonreduced protein extract from each sample was fractionated
on 10% SDS-polyacrylamide gel copolymerized with 1 mg/ml type I
collagen (A) or -casein (B and D),
and subjected to substrate gel zymography as described under
"Materials and Methods" or were separated on 13.5%
SDS-polyacrylamide gel for immunoblot analysis using a monoclonal
antibody to MMP-12 (C). In D, gel was divided
into sections for digestion in substrate buffer in absence
(top) or presence of the MMP inhibitor BB-94 (10 µM) (bottom). Representative Coomassie Blue
staining of gels is shown. Bands corresponding to specific MMPs are
shown. Asterisks indicate active forms of MMP-2 and MMP-9.
These data are representative of two independent experiments.
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Fig. 5.
Inhibition of MMP activities prevents
adipocyte differentiation. A, 2-day postconfluent
3T3-L1 preadipocytes were induced to differentiate with medium
containing dexamethasone, isobutylmethylxanthine, and insulin
(MIX). Total cellular RNA was isolated at different days
after the induction of differentiation, and 10 µg of RNA was
subjected to Northern blot analysis using cDNA probe for MMP-2,
MMP-19, TIMP-1, and aP2. B, lipid accumulation in
differentiating preadipocyte cells. Primary rat preadipocytes or 3T3-L1
preadipocytes were differentiated or not with the standard mix in
presence or absence of BB-94 (10 µM). Cells were stained
with oil red O and analyzed microscopically (magnification, ×200) on
day 6 of the differentiation program. Representative fields of primary
cells (top) and 3T3-L1 cells (bottom) are shown.
C and D, expression of adipocyte markers.
C, 2-day postconfluent (day 0) 3T3-L1 preadipocytes were
induced to differentiate with the standard mix in absence or presence
of BB-94 (10 µM). aP2 mRNA expression was analyzed by
Northern blot. 18 S rRNA is shown as a control for loading and RNA
integrity. D, 3T3-L1 preadipocyte differentiation was
induced in absence (control) or presence of BB-94 (10 µM)
or rosiglitazone (0.1 µM). At different days after the
induction of differentiation, nuclear extracts were prepared and
examined for PPAR expression by Western blot and enhanced
chemiluminescence detection. Data are representative of at least three
independent experiments.
, a master regulator of
adipocyte gene expression, was also analyzed (Fig. 5D).
Addition of the thiazolidinedione rosiglitazone, a synthetic agonist
for PPAR
, during the differentiation protocol is shown as a positive control for PPAR
expression. Nuclear extracts were prepared from undifferentiated and differentiated cells and analyzed by Western blot.
As expected, PPAR
expression markedly increased in control and
rosiglitazone-treated cells at day 3 and was maximal at day 6 after
induction. In BB-94-treated cells, expression of PPAR
was
undetectable at day 3 and was ~80% lower compared with control cells
at the end of the protocol. These results indicate that BB-94 alters
expression of adipocyte-specific proteins, and are consistent with the
visualized inhibition of fat cell differentiation. Further, the
anti-adipogenic effect of BB-94 on 3T3-L1 and primary culture of
preadipocytes is in total agreement with those observed in 3T3-F442A
preadipocyte cell line (12).
but Not Mitotic Clonal Expansion--
To gain
further insight into the mechanism by which BB-94 affects adipogenesis,
we next examined the effect of BB-94 on two early
differentiation-associated events, mitotic clonal expansion and
C/EBP
expression (23). During the first 2 days after induction, 3T3-L1 preadipocytes synchronously undergo mitotic clonal expansion that is an essential step of the differentiation program (24, 25). As
illustrated in Fig. 6A, this
process did not appear to be altered by BB-94 treatment. The increase
in cell number following induction of differentiation was the same
whether BB-94 was present or not. This demonstrates that BB-94 induces
inhibition of adipogenesis without affecting mitotic clonal expansion.
By contrast, 24 h after the induction of differentiation,
expression of the two nuclear C/EBP
forms (18 and 38 kDa) was
markedly reduced by BB-94 treatment (Fig. 6B). This
observation indicates that MMP inhibitor represses adipocyte
differentiation by inhibiting early induction of the C/EBP
transcription factor.
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Fig. 6.
The metalloproteinase inhibitor BB-94 blocks
early induction of C/EBP but not mitotic
clonal expansion. A, analysis of mitotic clonal
expansion in differentiating 3T3-L1 preadipocytes in the absence or
presence of BB-94 (10 µM). At different days after the
induction of differentiation (days 0-5), cells were trypsinized and
counted using a Coulter counter. Values are the mean ± S.D. of
two independent experiments performed in triplicate. B,
3T3-L1 preadipocyte differentiation was induced in absence (control) or
presence of BB-94 (10 µM) for 24 h. Nuclear extracts
(30 µg) from 2-day postconfluent 3T3-L1 preadipocytes (day 0) or from
24-h differentiated cells were analyzed by Western blot using a
C/EBP
antibody and enhanced chemiluminescence detection. Data shown
are representative of three independent experiments. The position of
the two C/EBP
isoforms (38 and 18 kDa) is shown by
arrow.
and thereby forces differentiation by
directly trans-activating the activity of endogenous PPAR
. 3T3-L1
preadipocytes were subjected to the differentiation program in the
presence or absence of BB-94 and rosiglitazone, and the extent of
differentiation was compared between different treatments (Table
III). Addition of rosiglitazone to the
induction medium increased the number of differentiated cells. As
expected, in the absence of rosiglitazone, BB-94 blocked
differentiation triggered by the adipogenic medium. However, induction
of differentiation in presence of rosiglitazone restored adipocyte
differentiation to levels seen in the absence of BB-94. This result
indicates that rosiglitazone can overcome the inhibitory effect of
BB-94 on adipocyte differentiation and confirms that BB-94 inhibits the
differentiation program at a step that precedes PPAR
activation.
Rosiglitazone rescues the effect of BB-94 on adipocyte differentiation
symbol indicates < 5% differentiation by day 7; + symbols
indicate the relative number of preadipocytes differentiated into
adipocytes. ND, not determined. Results are representative of three
individual experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-casein zymography, at least
four activities were detected in adipocytes and S-V cells. On the basis
of apparent molecular sizes, substrate specificities, Western blotting,
and sensitivity to inhibition by BB-94, we concluded that these
activities represent MMPs. We identified MMP-2, MMP-9, MMP-12, and a
novel 30-kDa activity. To our knowledge this 30-kDa caseinase is not
attributable to a previously identified enzymatic activity, and thus
may reflect a novel adipocyte-derived metalloproteinase. Interestingly,
enzymatic level of MMP-12 and of the ~30-kDa metalloproteinase is
elevated in obesity. We are currently investigating the nature of
this adipocyte-associated metalloproteinase activity. MMP-12 (also known as macrophage metalloelastase) is a
macrophage-specific MMP and is required for macrophage-mediated ECM
proteolysis and tissue invasion (30). In the context of adipose tissue,
MMP-12 might be expressed by macrophages, which are cellular components of the tissue, or by preadipocytes, which are known to function as
macrophage-like cells (31). At present, we do not know the biological
significance of elevated MMP-12 activity in obese adipose tissue.
convertase (ADAM-17) (7, 36). In support of this,
Kawaguchi et al. (40) recently found that female transgenic
mice expressing ADAM-12 become obese, demonstrating that an ADAM
metalloproteinase might also regulate adipogenesis.
, an ECM sequestered growth factor, the processing of the
membrane-bound tumor necrosis factor
, the cleavage of the
matricellular protein SPARC, and the bioavailability of adipogenic
factors such as insulin-like growth factor-1 (7). In this regard, it is
interesting to note that some of these molecules have been implicated
in the development and pathophysiology of obesity (5, 38, 39). A
pivotal role of MMPs in angiogenesis has also been documented,
suggesting that MMPs may play an additional role in obesity-related
adipose tissue growth.
expression, and without affecting the mitotic clonal phase of the
program. C/EBP
gene is expressed at a relatively early stage of
adipogenesis and is involved in the activation of the terminal adipogenic transcription factors, C/EBP
and PPAR
(23, 42). Consistent with this, we also found that the thiazolidinedione rosiglitazone can overcome the inhibitory effect of BB-94,
demonstrating that BB-94 inhibits the differentiation program at a step
that precedes PPAR
activation. Our observations are consistent with a role for MMP activities at an early step of adipocyte differentiation and suggest that adipogenesis requires the activation or release of
factors that might play a role in this process.
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ACKNOWLEDGEMENTS |
---|
We thank Georges Manfroni for expertise in animal care, Françoise Cottrez for assistance in real-time PCR analyses, and Marcel Deckert and Anne Johnston for discussion and critical reading. We also thank Christian Dani, Tor Ny, and Carlos López-Otín for providing cDNAs.
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FOOTNOTES |
---|
* This work was supported in part by INSERM, the Association pour la Recherche sur le Cancer, and a grant from Groupe Merck-Lipha (Lyon, France).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.
§ Recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche (France).
** To whom correspondence should be addressed: INSERM Unité 385, Faculté de Médecine, ave. de Valombrose, 06107 Nice Cédex 2, France. Tel.: 33-4-93-37-77-90; Fax: 33-4-93-81-14-04; E-mail: tartare@unice.fr.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M209196200
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ABBREVIATIONS |
---|
The abbreviations used are:
ECM, extracellular
matrix;
MMP, matrix metalloproteinase;
TIMP, tissue inhibitor of
metalloproteinase;
C/EBP, CCAAT/enhancer-binding protein
;
PPAR
, peroxisome proliferator-activated receptor
;
S-V, stromal-vascular;
ADAM, a disintegrin and metalloprotease;
MT-MMP, membrane-type matrix metalloproteinase;
DMEM, Dulbecco's modified
Eagle's medium.
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