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Molecular Endocrinology 20(12):3376–3388
Copyright © 2006 by The Endocrine Society
doi: 10.1210/me.2006-0177
Leptin Increases Tissue Inhibitor of
Metalloproteinase I (TIMP-1) Gene Expression by a
Specificity Protein 1/Signal Transducer and
Activator of Transcription 3 Mechanism
Songbai Lin, Neeraj K. Saxena, Xiaokun Ding, Lance L. Stein, and Frank A. Anania
Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine,
Atlanta, Georgia 30322
Leptin has properties of a profibrogenic cytokine.
In liver, the activated hepatic stellate cell (HSC) is
responsible for a net production of extracellular
matrix. A key molecule synthesized is the tissue
inhibitor of metalloproteinase I (TIMP-1), which
acts to inhibit the activity of matrix metalloproteinases. The purpose of the present study was to
determine how leptin, a gp130 cytokine, orchestrates the regulation of TIMP-1 gene activation and
expression. Transient transfection of primary
HSCs revealed that leptin significantly increased
luciferase activity of a 229-bp TIMP-1 promoter
construct (TIMP-1–229). An EMSA revealed that
leptin enhanced specificity protein 1 (Sp1) binding.
Site-directed mutagenesis for Sp1 reduced the enhancing effect of leptin on TIMP-1 transcriptional
activation, and this effect was dose dependent on
the number of Sp1 sites mutated. Chromatin im-
munoprecipitation revealed that leptin enhanced
binding of Sp1; however, inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation by AG490 also blocked Sp1 phosphorylation and significantly reduced leptin-associated
TIMP-1–229 promoter activity, indicating that one
mechanism for leptin-increased transcriptional activity is via phosphorylation of Sp1 and subsequent
promoter binding. Finally, we demonstrate that
leptin also results in intranuclear pSTAT3 binding
to Sp1. We propose a novel mechanism whereby
leptin-mediated TIMP-1 transcription employs a
Sp1/pSTAT3-dependent mechanism, one of which
is a noncanonical association between Sp1 and
pSTAT3. These data provide a new molecular mechanism whereby the adipocytokine leptin plays a role
in complications of the metabolic syndrome. (Molecular Endocrinology 20: 3376–3388, 2006)
L
LX-2. Their data indicate that TIMP-1 promoter activity
was increased via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling
pathway, which we have previously shown is critical to
leptin-enhanced collagen production (1, 2) and perpetuation of HSC proliferation and inhibition of apoptosis (3).
There are four major TIMPs (TIMP-1, TIMP-2,
TIMP-3, and TIMP-4) that suppress extracellular matrix (ECM) degradation by forming an inhibitory 1:1
complex with matrix metalloproteinases (MMPs) (4). In
addition to acting as an inhibitor of most of the known
MMPs, TIMP-1 has several important biological functions. TIMP-1 is a key regulator of fibrogenic events in
the liver; the extent of carbon tetrachloride-induced
liver fibrosis is elevated in mice that over express
TIMP-1 from a liver-specific TIMP-1 transgene (5). The
major cellular source of TIMP-1 in the injured liver is
the activated HSC (6). HSCs are found in the normal
liver in a quiescent state in which their major function
appears to be the storage of vitamin A. After injury,
HSCs activate or trans-differentiate into proliferating
myofibroblast-like cells that produce abundant levels
of fibrillar collagen and TIMP-1 (7). Secretion of abundant levels of TIMP-1 by activated HSCs leads to
decreased hepatic collagenase activity and thereby
EPTIN, A 16-kDa hormone, has an array of biological effects. Recently, leptin has been shown by
several groups to be critical in the development of
hepatic fibrosis. In relation to tissue inhibitor of metalloproteinase 1 (TIMP-1), Cao et al. (59) recently demonstrated that leptin directly increased TIMP-1 in a
human activated hepatic stellate cell (HSC) cell line
First Published Online August 24, 2006
Abbreviations: AP-1, Activating protein-1; BDL, bile duct
ligation; ChIP, chromatin immunoprecipitation assay; ECM,
extracellular matrix; HSC, hepatic stellate cell; Jak, Janus
kinase; FBS, fetal bovine serum; FFB, ZDF bile-duct ligated;
FFN, ZDF-sham operated; GAPDH, glyceraldehyde phosphate dehydrogenase; LLB, lean bile-duct ligated; LLN, leansham operated; MMP, matrix metalloproteinases; NASH,
nonalcoholic steatohepatitis; Ob-R, leptin receptor; Ob-Rb,
leptin receptor-long form; Ob-Ra, leptin receptor-short form;
PI-3K, phosphatidylinositol-3 kinase; RT-qPCR, real-time
quantitative RT-PCR; SBE, Stat-binding element; SHP-2, domain containing tyrosine phosphatase 2; SF, serum-free;
SOCS3, suppressor of cytokine signaling 3; Sp1, specificity
protein 1; SRE, serum-response element; STAT3, signal
transducer and activator of transcription; TIMP-1, tissue inhibitor of metalloproteinase 1; UTE-1, upstream TIMP-1 element 1.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
3376
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Mol Endocrinol, December 2006, 20(12):3376–3388 3377
promotes a net increase in ECM. TIMP-1 also promotes hepatic fibrogenesis by inhibiting apoptosis of
activated HSCs, and thereby allows perpetuation of
the fibrogenic process. The hypothesis that progression of liver fibrosis is associated with inhibition of
matrix degradation in liver is strongly supported by
studies of the relevant MMPs and TIMPs in cultured
HSCs (7–9).
In chronic liver disease, and in animal models of liver
fibrosis, significant increases in TIMP-1 and TIMP-2
expression have also been observed. Studies of human liver explants (obtained from patients undergoing
liver transplantation) have found increased expression
of TIMP-1, TIMP-2, or both in patients with primary
sclerosing cholangitis, biliary atresia, primary biliary
cirrhosis, and autoimmune chronic active hepatitis (8,
10) These changes in mRNA for TIMP-1 were accompanied by proportional changes in TIMP-1 protein,
which was increased approximately 5-fold in fibrotic
vs. normal human liver as determined by ELISA.
TIMP-1 promoter activity is induced during HSC
culture-activation (11). The TIMP-1 promoter is structurally conserved between rodents and humans and
contains functional binding sites for activating protein-1
(AP-1) (Fos/Jun), and STATs located within a 22-bp
serum-response element (SRE) and motifs for specificity protein 1 (Sp1) and BLP-1 transcription factors
(12). A regulatory motif called the upstream TIMP-1
element [UTE-1] (12), is required for high level promoter activity including activated HSCs. Recent work
(13) indicates that RUNX1A interacts directly with
JunD, but RUNX1B failed to establish such an interaction. JunD and RUNX factors assemble at the adjacent SRE and UTE-1 sites on the TIMP-1 promoter
and form functional interactions that stimulate transcription (14). Activated STAT proteins translocate to
the nucleus to stimulate activation of the TIMP-1 promoter as has been demonstrated previously (13, 15).
Leptin signals as a gp130 cytokine; however, there is
controversy related to exactly how gp130 signal transduction by IL-6 and oncostatin directs STAT binding
along the TIMP-1 promoter (16) because induction of
STAT binding to homologous TIMP-1 promoter sequences has not been detected. Hence, gp130 activation of TIMP-1 may likely involve cooperation with
other factors such as Sp1 (17). Recently, Sp1 was
reported to be critical to TIMP-1 promoter activity
because mutations of the two Sp1 sites in the fulllength promoter reduced TIMP-1 promoter transcription activity. In addition, the cotransfection with an
antisense Sp1 oligonucleotides decreased the promoter activity, suggesting that the transcription of the
TIMP-1 promoter is mediated by Sp1 (17).
Finally, STAT3 can activate transcription without
binding itself to a palindromic enhancer element (18).
Leptin, functioning as a gp130 cytokine (19), has been
proved by several investigators to be a critical profibrogenic molecule in hepatic fibrosis (1, 20–26). The
purpose of the present study, therefore, was to elucidate the transcriptional mechanisms responsible for
leptin-increased TIMP-1 gene expression via examination of STAT activation and Sp1 activity. We demonstrate a dual mechanism whereby leptin mediates
enhanced TIMP-1 mRNA expression: that upstream
Sp1 binding is enhanced by leptin via Sp1 phosphorylation and, pSTAT3 activation assembly with Sp1,
along but not directly on the TIMP-1 promoter, is a
second mechanism for leptin-enhanced TIMP-1 promoter activation and gene expression.
RESULTS
TIMP-1 mRNA Increased in Vivo from Livers of
Bile Duct Ligation (BDL)-Treated Lean But Not
fa/fa Rats
BDL was performed in fa/fa rats and respective lean
littermates, as was sham operation. The fatty (fa/fa, or
Zucker diabetic fatty) rat is defective in leptin receptor
(Ob-R) as evidenced by chromosome mapping in a
region of the rat chromosome 5 (27, 28). The fa mutation is a missense mutation in the extracellular domain of OB-R, which results in a glutamine269 to proline269 amino acid substitution (29, 30). In practical
terms, the homozygous genotype (fa/fa) represents a
dysfunctional leptin signal transduction apparatus because prior work demonstrates that the fa mutation
results in a nearly 10-fold reduction in the cell surface
localization of the OB-R (31). Hence, we examined, by
real-time RT-PCR, genes known to be involved with
ECM remodeling and found that fibrotic livers from
lean rats subjected to BDL had a 3.5-fold increase in
TIMP-1 mRNA (Fig. 1A) than did sham-operated animals or fa/fa rats that also underwent BDL.
Dose-Response and Time-Course Studies Reveal
TIMP-1 mRNA Increased in Activated HSCs
in Vitro
Activated HSCs treated with leptin (Fig. 1B) also resulted in a significant increase in TIMP-1 mRNA, which
was maximally sustained with 100 ng/ml leptin (P ⬍
0.01). Importantly, this effect was seen beginning at
3 h and continuing for 48 h after treatment (Fig. 1C). To
determine whether leptin served to stabilize TIMP-1
mRNA transcripts and was not responsible for a nascent increase in TIMP-1 mRNA, we performed an
mRNA stability assay using actinomycin D (Fig. 1D).
These studies failed to indicate that leptin increased
the half-life of TIMP-1 mRNA transcripts. When taken
together, both the in vivo and in vitro results prompted
us to examine the role leptin played in the regulation
of TIMP-1 promoter regulation, and to determine
whether this was related to an AP-1-dependent process, which has been previously reported as a consequence of HSC activation (7, 32).
3378 Mol Endocrinol, December 2006, 20(12):3376–3388
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Deletion Analysis of the Human TIMP-1
Promoter: cis-Regulatory Elements Associated
with Increased Leptin Activity Exists between
ⴚ108 and ⴚ229 bp from the Start Site of
Transcription
To determine whether leptin acted via the AP-1-dependent mechanism cited previously, we constructed
six deletion mutants of the TIMP-1 promoter (Fig. 2A)
and transiently transfected these into freshly isolated
but culture-activated HSCs. Hence, these cells by default already had basal TIMP-1 promoter activity.
When compared with basal activity of the TIMP-1–229
and other deletion constructs, the luciferase activity of
TIMP-1–108 decreased more than 6-fold and no significant effect of leptin treatment on TIMP-1–108 promoter activity was observed. Leptin treatment of HSCs
transfected with the TIMP-1–229 or larger deletion
constructs resulted in a 60–140% increase in TIMP-1
luciferase activity when compared with basal activity
of the TIMP-1–229 construct (Fig. 2B), indicating that
one cis-binding element critical to leptin-enhanced
TIMP-1 expression would be found between ⫺108 and
⫺229 bp upstream from the start site of transcription.
Leptin Increased the Binding of Two
Oligonucleotides Containing Sp1 Sites to HSC
Nuclear Protein Extracts
Fig. 1. TIMP-1 Gene Expression Is Elevated by Leptin Both
in Vivo and in Vitro
A, Total RNA extracted from whole livers from the four
treatment groups was used to determine mRNA expression
of TIMP-1 as described in Materials and Methods by RTqPCR. Level of mRNA expression, observed in sham-operated lean littermates was set at 100% of control. Results are
means ⫾ SE and represent 12 reactions for each treatment
condition. *, P ⬍ 0.01 compared with lean sham-operated
values. B, Subconfluent activated rat HSCs were serum
starved for 16 h with 0.1% FBS in DMEM and then exposed
to increasing concentrations of leptin for 24 h and analyzed
for TIMP-1 mRNA expression by RT-qPCR. Values are
means ⫾ SE, expressed as fold change relative to the control
(no leptin). *, P ⬍ 0.01. The results are representative of three
independent experiments. C, Activated rat HSCs were
treated with leptin (100 ng/ml) for the times indicated and
analyzed for TIMP-1 mRNA expression by RT-qPCR. The
To elucidate the molecular mechanism related to the
effect of leptin on TIMP-1–229 promoter activation, we
synthesized six oligonucleotides (probes 1–6) spanning
the 229-bp promoter region constructed in the previous
studies (Fig. 3A) and performed EMSA with nuclear extracts from leptin-treated HSCs (Fig. 3B). The data indicate that probe 1 and 3 bound nuclear protein from
leptin-treated HSCs with higher affinity than to extracts
from untreated HSCs; however, complexes formed with
probe 3 (P3) were much stronger than those with probe
1[P1] (Fig. 3B). Binding activity was also enhanced with
probe 4 (P4), but this did not change in the presence of
leptin. Sequence analysis revealed probes 1 and 3 (P1
and P3) contained Sp1 consensus binding sites (Fig. 3A);
and leptin appeared to enhance both Sp1 and Sp3 bind-
results, representative of three independent experiments, are
compared with respect to control (no leptin) at the respective
time points). *, P ⬍ 0.01. D, Activated HSCs were grown in
DMEM with 0.1% FBS for 16 h pretreated with leptin (0 or 100
ng/ml) for 4 h and subsequently treated with actinomycin D [5
␮g/ml]. Cells were harvested for total RNA at time points
indicated after addition of actinomycin D. TIMP-1 mRNA was
quantitated using RT-qPCR and normalized to 18 S rRNA.
Results are expressed as fold change relative to the samples
at time zero. The entire experiment was repeated three separate times in triplicate. TIMP-1 mRNA stability was evaluated by computing the best fit linear curve on a linear plot of
the mRNA fold change vs. time for leptin and nonleptin treatment groups. SF, Serum free.
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Mol Endocrinol, December 2006, 20(12):3376–3388 3379
pendent, we created a series of 2-bp substitution mutants in which the two Sp1 sites contained in this
mutant construct were eliminated (Fig. 4A). We demonstrated that the effect of leptin on TIMP-1–229 was
reduced to basal levels (Fig. 4B) when either Sp1
cis-binding region was mutated (⌬T-229MA or ⌬T229MB). Luciferase activity was reduced further by the
mutation of both Sp1 sites (⌬T-229MAB). We did not
observe any difference in leptin-treated or basal luciferase activity for either the TIMP-1–108 deletion mutant or the ⌬T1–108MA).
Leptin Modulates Sp1 and Sp3 Phosphorylation
Western blot analysis demonstrated that leptin resulted in the phosphorylation of STAT3 at 30 min; this
is in keeping with the kinetics of Jak/STAT phosphorylation (Fig. 5A). Western blot also revealed that protein levels of Sp1 or Sp3 were not changed in HSCs
exposed to leptin (data not shown). Because Sp1 and
Sp3 can be activated as phosphoproteins, we investigated whether leptin might modulate the phosphorylation of Sp1 and Sp3. Serum-free or leptin-treated
cells were harvested, and Sp1 or Sp3 was immunoprecipitated by antibody and protein A/G-agarose
beads. The immunoprecipitates were subjected to
SDS-PAGE (33) and probed with antiphosphoserine
antibody. Phosphorylation of Sp1 was greater at 4 h
when compared with Sp3 after leptin treatment (Fig.
5B). As anticipated, this activity diminished by 24 h.
Fig. 2. Localization of a Human TIMP-1 Promoter Region
(⫺1482 to ⫹88) Mediating the Effect of Leptin on TIMP-1
Transcription in Activated Rat HSCs
A, Various lengths of human TIMP-1 promoter sequences
were cloned upstream from luciferase reporter gene as described in Materials and Methods. The schematic diagram
represents six deletion mutants: TIMP-1–1482, TIMP-1–989,
TIMP-1–582, TIMP-1–369, TIMP-1–229, and TIMP-1–108
used for promoter analysis. B, Transient transfection of activated HSCs with respective promoter activities by firefly luciferase assay. The assay was repeated three times in triplicate and represent the mean ⫾ SE; *, P ⬍ 0.01 with respect
to TIMP-1–229. SF, Serum free.
ing to probes 1 and 3 based on supershift analysis (Fig.
3C, lanes 5 and 6). Competition analysis with excess
cold oligonucleotide P3 confirmed that Sp1 and Sp3
formed specific complexes (Fig. 3C, lane 4). These data
prompted further examination of potential Sp1 sites, and
to determine whether leptin enhanced Sp1-TIMP-1–229
promoter binding.
Substitution Mutation of Sp1 Sites Abolished
Increased Leptin Activity of the
TIMP-1–229 Promoter
To demonstrate that leptin-mediated transcriptional
activation of the TIMP-1–229 promoter was Sp1 de-
Chromatin Immunoprecipitation (ChIP) Assay
Revealed that Leptin Enhanced Only Sp1—But
Not Sp3—Binding to the TIMP-1 Promoter
To determine whether leptin-enhanced TIMP-1 promoter
activation was dependent on Sp1 or Sp3 we performed
a ChIP assay. Sp1 and Sp3 bind TIMP-1–229, but in the
presence of leptin only Sp1 binding was significantly
increased (Fig. 6, A and B). These data are consistent
with published reports that Sp3 may compete as an
inhibitory factor to cis-binding regions associated with
Sp1-enhanced transcriptional activation (34). OB-Rb signals as a gp130 cytokine, but we also failed to identify
pSTAT3 binding to the TIMP-1–229 promoter (Fig. 6A).
Hence, when taken together, the data indicate that leptin
increased only Sp1—but not Sp3 or pSTAT3—binding to
the TIMP-1–229 promoter.
Inhibition of STAT3 Phosphorylation Blocked
Sp1 Phosphorylation and TIMP-1–229
Promoter Activity
The chemical inhibitor AG490 was used to block leptin-associated Jak2 phosphorylation of STAT3. AG490
abolished leptin-associated phosphorylation of both
STAT3 (Fig. 7A) and Sp1 (Fig. 7B); and, AG490 also
abolished leptin-enhanced luciferase activity of TIMP1–229 (Fig. 7C). The presence of AG490 alone did not
altar basal activity of the TIMP-1–229 promoter indi-
3380 Mol Endocrinol, December 2006, 20(12):3376–3388
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Fig. 3. The Effect of Leptin on the Binding of HSC Nuclear Extracts to Oligonucleotides Spanning the 229-bp TIMP-1 Promoter
A, Schematic diagram of six oligonucleotides spanning the 229 bp TIMP-1 promoter were synthesized, annealed and
radiolabeled as probes for EMSA. B, Nuclear extracts (5 ␮g) were prepared from activated primary rat HSCs, serum starved for
16 h, and exposed to leptin (100 ng/ml) for 24 h. Leptin enhanced binding of nuclear extracts prepared from treated HSCs to
probes 1 and 3 when compared with binding activities of untreated extracts. C, To identify complexes formed for probes 1 and
3, competition and antisera reactions were performed as described in Materials and Methods. FP, Free probe; SF, serum-free,
untreated control; L, leptin treatment; Cold 50⫻, competition assay from leptin-treated nuclear extracts in the presence of 50-fold
excess unlabeled probe 3 (P3). Sp1 and Sp3 antisera were added to reaction mixtures for supershift assay in lanes 5 and 6,
respectively, and supershift complexes were formed (long arrow). The assay is representative of three independent experiments.
cating that basal expression of TIMP-1 in activated
HSCs is not associated with gp130 signal transduction. Overexpression of suppressor of cytokine signaling 3 (SOCS-3), a physiological inhibitor of pSTAT3 (4),
blocked Sp1 phosphorylation enhanced by leptin (Fig.
7D). Hence, leptin induced STAT3 and Sp1 phosphorylation, which if abolished prohibited leptin-associated
TIMP-1–229 promoter activation.
Leptin Enhanced TIMP-1–229 Promoter Activity
Also Involves a Direct Interaction between
pSTAT3 and Sp1
Finally, we investigated whether another molecular
mechanism was responsible for leptin-induced TIMP1–229 promoter activation. We overexpressed Sp1
and immunoprecipitated STAT3-Sp1 complexes by
using Sp1 antibody and detected the coimmunoprecipitated STAT3-Sp1 complex by immunoblotting with
anti-pSTAT3 antibody (Fig. 8A) or phosphoserine an-
tibodies to detect Sp1. Only leptin-treated HSC lysates immunoprecipitated with antiphosphoserine antibody were detected with the anti-pSTAT3 antibody.
In the EMSA, the addition of the anti-STAT3 antibody
decreased the binding of Sp1 or Sp3 to the oligonucleotide (Fig. 8B), whereas antibodies against STAT1
did not. When taken together, these data reveal that
Sp1 and pSTAT3 have a physical relationship that
binds the TIMP-1 promoter to mediate leptin activation
of this gene.
DISCUSSION
Hepatic fibrosis was historically thought to be a passive and irreversible process due to the collapse of the
hepatic parenchyma and its substitution with collagen-rich tissue (35, 36) Currently, hepatic fibrosis is
considered to be a dynamic model of the woundhealing response to chronic liver injury (37). Nonalco-
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Fig. 4. Mutation of the Sp1 Sites Abolishes Effect of Leptin
on TIMP-1 Promoter Activity
A, Four substitution mutation constructs for the TIMP-1–
229 deletion mutant were synthesized as described in Materials and Methods. B, Subconfluent primary activated HSCs
were transiently transfected with these constructs to determine the role of Sp1 in mediating leptin-enhanced TIMP-1
promoter activity as assessed by firefly luciferase activity.
Relative promoter activity from three independent experiments performed in triplicate expressed as mean ⫾ SE. *, P ⬍
0.01 with respect to basal TIMP-1–229 promoter activity. SF,
Serum free.
holic steatohepatitis (NASH) has been recognized as a
major cause of liver fibrosis (38). First described by
Ludwig et al. (39), it is considered a part of the spectrum of nonalcoholic fatty liver disease that ranges
from steatosis to cirrhosis and can eventually lead to
hepatocellular carcinoma. NASH is an important component of the metabolic syndrome, which is characterized by obesity, type 2 diabetes mellitus, and dyslipidemia, with insulin resistance as a common feature.
Because the prevalence of obesity is rapidly increasing, a rise in the prevalence of NASH is anticipated.
Hepatic fibrosis is the result of the wound-healing
response to repeated injury and is associated with
major alterations in both the quantity and composition
of ECM (40). Accumulation of ECM results from both
increased synthesis and decreased degradation (41).
Decreased activity of ECM-removing matrix metalloproteinases (MMPs) is mainly due to an overexpression of their specific inhibitors, TIMPs—in particular
TIMP-1. Strong evidence has been put forth that adipocytokines, or adipokines, clearly regulate liver fibrosis. Adipokines are cytokines primarily derived from
adipose tissue. We and others (24, 26) have shown
that leptin is required for HSC activation, type I collagen synthesis, and perpetuation of the activated HSC
phenotype—which are all associated with a net increase of hepatic ECM. Leptin-promoting events for
increased ECM include increased activated HSC proliferation, impedance of HSC death by apoptosis, and
Mol Endocrinol, December 2006, 20(12):3376–3388 3381
Fig. 5. Leptin Increases Phosphorylation, But Not Protein
Level, of Sp1 and Sp3
A, Subconfluent primary activated HSCs were serum
starved for 16 h, and then cultured in the presence of leptin
(100 ng/ml) or vehicle for the times indicated before cellular
proteins were harvested. The Western blots were performed
as described in Materials and Methods in triplicate using
antibody to pSTAT3. B, Primary activated HSCs were grown
and starved as described above, and then cultured in the
presence of leptin (100 ng/ml) or vehicle for the times indicated. Sp1 and Sp3 proteins were immunoprecipitated (IP)
and the immunoprecipitates were subjected to SDS-PAGE.
The membranes were probed with antiphosphoserine antibody to evaluate the phosphorylation status of Sp1 and Sp3.
The membranes were also probed with anti-Sp1 and -Sp3
antibody to monitor the quantity of Sp1 or Sp3. The gels
shown were representative of three independent experiments. Maximal phosphorylation occurs for Sp1 at 4 h with
minimal phosphorylation of Sp3. IB, Immunoblot.
increased synthesis of type I collagen (1–3). Leptin has
also been associated with the fibrogenic response in
kidney (42, 43), fertility and blastocyst implantation in
the female reproductive tract (44–46), and in woundhealing of the epidermis of leptin-deficient, ob/ob mice
(47).
Our in vivo data from BDL-operated rats with a
competent leptin signaling axis, as well as our in vitro
data, indicate that leptin increases TIMP-1 mRNA
transcripts. Such increases are not due to prolongation of the TIMP-1 mRNA half-life as a result of posttranscriptional modification (Fig. 1D). The prevailing
data concerning TIMP-1 gene regulation are based on
HSC activation and subsequent activation of AP-1
binding in the proximal portion of the TIMP-1 promoter
as discussed previously. Long-term activated rat
HSCs persistently express, among other proteins, Jun
D. UTE-1 is a regulatory DNA motif essential for TIMP1
promoter activity in a variety of cell types including
HSCs; and the relationship between the UTE-1 site
and its adjacent upstream SRE in the promoter are
critical to TIMP-1 gene regulation (14). The key regulatory sequence within the SRE is an AP-1 site that in
HSC directs high level transcription via its interaction
with JunD (14). Importantly, the TIMP-1 STAT element
has a much lower affinity in comparison to AP-1 (13);
hence, simultaneous binding of AP-1 and JunD is
probably necessary to confer responsiveness after
3382 Mol Endocrinol, December 2006, 20(12):3376–3388
Fig. 6. ChIP Assay Reveals that Leptin Enhances the Binding of Sp1, But Not Sp3 or pSTAT3, to the Rat TIMP-1
Promoter
A, Subconfluent primary activated HSCs were serum
starved for 16 h, and then cultured in the presence of leptin
(100 ng/ml) or vehicle for 24 h before the cells were lysed and
sonicated. Formaldehyde cross-linked chromatin from HSCs
was incubated with Sp1, Sp3, pSTAT3, or control antisera.
Immunoprecipitated DNA was analyzed by PCR with primer
to proximal promoter of rat TIMP-1 gene or glyceraldehyde
phosphate dehydrogenase (GAPDH) gene. Total input DNA
at a 1:100 dilution was used as a positive control of the PCRs.
DNA prepared from chromatin that was immunoprecipitated
with nonimmune rabbit sera (⫹IgG) was used as a negative
control for assays. The lanes are designated either according
to the antisera used for the ChIP assay or by “Input,” which
indicates the preimmunoprecipitated chromatin used in the
PCR. The gel shown is representative of three independent
experiments. B, Protein-DNA complexes immunoprecipitated were measured by real-time PCR. Results, expressed
as fold change differences of DNA (amplified promoter) between leptin and vehicle samples were calculated relative to
GAPDH as described in Materials and Methods. *, P ⬍ 0.01,
leptin treatment vs. untreated control. Results are means ⫾
SE and represent three independent studies. SF, Serum free.
stellate cell activation, and activation of the TIMP-1
proximal promoter. To date, only one other group has
examined the regulation of TIMP-1 by leptin and reports that MAPK activity increases hydrogen peroxide
production as a potential mechanism for activating the
TIMP-1 promoter (48). We have not found that leptin
alone will up-regulate the UTE or SRE in the proximal
promoter of the TIMP-1 gene. One potential reason for
the lack of responsiveness of the ⫺108 promoter in
these studies is that leptin up-regulation of the TIMP-1
promoter may be transient, and these studies were
conducted in the activated HSC phenotype in which
activation of TIMP-1 via AP-1 has already occurred.
Recent evidence also corroborates what we have
found—insofar as the role of gp130 signal transduc-
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
tion—which exploits Sp1-TIMP-1 promoter binding to
activate transcription (18). Hence, there may be several mechanisms of TIMP-1 activation in HSCs including one associated with transactivation, oxidative
stress, and Sp1 phosphorylation by the Jak2/STAT 3
pathway with subsequent nuclear translocation and
TIMP-1 promoter binding.
The data presented here clearly indicate that leptinmediated increase in TIMP-1 promoter activity is
linked to Sp1 promoter binding. The mechanism of
leptin-enhanced Sp1 TIMP-1 promoter activation is
the consequence of two events. Except for the TIMP1–108 mutant, the other deletion mutant constructs
revealed significant differences under leptin-treated
conditions (Fig. 2B). Although we did note that Sp3
can also bind the putative Sp1 sites employed in
EMSA analysis (Fig. 3C), ChIP analysis revealed that
leptin enhanced only the binding of Sp1, and not Sp3,
to the TIMP-1 promoter employed in these studies
(Fig. 6). Importantly, leptin resulted in minimal phosphorylation activity when compared with Sp1 (Fig. 5B).
Even though the principle leptin signaling pathway is
via Jak2/STAT3 phosphorylation ,we also failed to detect pSTAT3 binding to the promoter by ChIP assay.
Conversely, AG490, which inhibits Jak2 phosphorylation of STAT3 (Fig. 7A), not only inhibited Sp1 phosphorylation (Fig. 7B) but also markedly diminished leptin-enhanced TIMP-1 promoter activity (Fig. 7C). Sp1
belongs to the family of zinc finger proteins that are
ubiquitously expressed (49). In our studies, mutation
of either Sp1 cis-binding elements resulted in a reduction in leptin-mediated TIMP-1–229 activity; importantly, the Sp1 binding site at ⫺121 and ⫺128 bp
appear to be indispensable for leptin-stimulated TIMP1–229 promoter activity (Fig. 4B; ⌬T-229MA).
Activated STAT3 can alter transcription by dimerization, and subsequent translocation to the nucleus
with binding to enhancer elements of target genes,
thereby inducing transcriptional activation (50). However, we did not find this to be the case by ChIP assay
for the TIMP-1–229 promoter. Instead we found that
leptin resulted in phosphorylation of Sp1 (Figs. 5B and
7B) and was required for leptin-enhanced TIMP-1 promoter activation (Fig. 7C); and Sp1, but neither Sp3
nor pSTAT3, was found to enhance TIMP-1 promoter
binding by ChIP assay (Fig. 6). Hence, one mechanism
whereby leptin acts is via phosphorylation of Sp1 and
not by direct pSTAT3 binding. Although AG490 and
SOCS-3 are respective chemical and biological inhibitors of Jak2 phosphorylation and STAT3 phosphorylation, these inhibitors could affect other signaling
pathways and affect the activity of other hormones
such as insulin. Recently it has been shown that domain-containing tyrosine phosphatase 2 (SHP-2) and
SOCS3 do not just regulate the pathways that they are
known to be associated with (SHP-2 with MAPK and
SOCS-3 with JAK/STAT), but also have a strong effect
on the other pathway (51). Hence, future work in assessing SOCS-3 overexpression in inhibiting alternate
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Mol Endocrinol, December 2006, 20(12):3376–3388 3383
Fig. 7. AG490 Inhibited Leptin-Stimulated STAT3 and Sp1 Phosphorylation and Abolished the Leptin-Enhanced TIMP-1 Promoter Activity
A, Subconfluent primary activated HSCs were serum starved for 16 h, and then cultured with leptin (100 ng/ml) or vehicle with
or without AG490 (10 ␮M) for 4 h before cellular proteins were harvested. IL6 (10 ng/ml) was used as positive control for STAT3
activation. pSTAT3 and STAT3 were assayed by immunoblot (IB) analysis as shown. Equal loading of proteins in each lane was
confirmed by probing membranes for ␤-actin. B, Identical experimental design to A was conducted to probe to determine whether
AG490 blocked phosphorylation of Sp1 after immunoprecipitation and probed with anti-phosphoserine antibody to evaluate the
phosphorylation status of Sp1. The membranes were also probed with anti-Sp1 to monitor the quantity of Sp1. The present study
is representative of two independent experiments. C, Subconfluent primary activated HSCs were transfected with TIMP-1–229,
serum starved for 16 h, and then cultured as described above for 24 h before cell lysates were harvested for measurement of
luciferase activity. Relative promoter activity from three independent experiments performed in triplicate are expressed as
means ⫾ SE; *, P ⬍ 0.01. D, 90% confluent primary activated HSCs were transfected with FLAG-SOCS-3 expression vector or
corresponding empty vector as described elsewhere. The cells were serum starved for 16 h, and then cultured with leptin (100
ng/ml), IL-6 (10 ng/ml) or vehicle for 4 h before cellular proteins were harvested. The lysates were immunoprecipitated using Sp1
antibody or control anti-sera. Total input protein was assessed to show the successful transfection of FLAG-SOCS-3 expression
vector using flag antibody.
signaling pathways and leptin-mediated gene regulation will be required.
More than a decade ago, Jackson and Tjian (52)
published the first study on Sp1 phosphorylation. Because the first report many other studies on this topic
have been published using various cell systems and
promoters (53) and Sp1 phosphorylation has been
stimulated during viral infection (54), and organ development in the lung and liver (55, 56). Leptin has been
shown to be a growth factor (3, 44, 47). To our knowledge, this is the first report indicating that leptin can
potentiate transcription by phosphorylation of Sp1. In
short, these data provide a direct molecular link, i.e.
JAK is directly responsible for the phosphorylation of
Sp1 via pSTAT3; but it does not exclude the possibility
that another intermediary signaling molecule (between
pSTAT3 and Sp1) is ultimately responsible for Sp1
phosphorylation. Hence, additional studies will be
needed to elucidate such signaling details.
We also have provided evidence that leptin drives
TIMP-1 expression via both pSTAT3 activation and
Sp1 in a novel noncanonical way. This hypothesis was
substantiated by the fact that STAT3, but not STAT1,
antibodies blocked the leptin-induced binding of Sp1
to the Sp1 oligonucleotide sequence from the TIMP-1
promoter (Fig. 8B); and, by overexpression of Sp1, we
have convincingly demonstrated that only leptintreated lysates immunoprecipitated with anti-Sp1 antibodies reveal pSTAT3 when subjected to immunoblot with antibodies to pSTAT3 or antiphosphoserine
antibodies to detect Sp1 (Fig. 8A). There is evidence
that transcription factors can activate transcription
without DNA binding. STAT3 and Sp1 cooperatively
activate the CAAT-enhancer-binding protein ␦ promoter (57), albeit through adjacent Sp1 and STAT3
binding sites. However, the TIMP-1 promoter region
examined in the EMSA here contained only Sp1 binding sites, but no STAT3 binding elements (SBEs). We
describe here a novel mechanism (Fig. 9) for the transcriptional regulation of TIMP-1 by which leptin not
only activates Sp1 phosphorylation via STAT3 activation but also facilitates STAT3 association with Sp1
bound to the SBE-less TIMP-1 promoter. The characterization of this mechanism may be relevant for the
design of novel treatment strategies, particularly for
liver fibrosis related to NASH.
3384 Mol Endocrinol, December 2006, 20(12):3376–3388
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Fig. 8. Sp1 and STAT3 Complex Was Detected and STAT3 Antibodies Disrupt the Leptin-Induced Binding of Sp1 to the TIMP-1
Promoter
A, Primary activated HSCs were transfected with 24 ␮g of Sp1 expression vector (pN3-Sp1FL-complete), serum starved for
16 h, and treated with or without leptin for 24 h. Nuclear protein was extracted and 200 ␮g of protein were subjected to
immunoprecipitation (IP) using an antibody against Sp1 or antirabbit IgG (nonimmune sera). The immunoprecipitated proteins
were separated and subjected to immunoblot analysis with either anti-pSTAT3, or anti-phosphoserine. Twenty micrograms of
nuclear protein were input for detecting Sp1 expression. Immunoblot (IB) with either anti-pSTAT3 or anti-phosphoserine
antibodies of leptin-treated primary activated HSC nuclear protein lysate immunoprecipitated with anti-Sp1 antibody revealed
pSTAT3-Sp1 complexes. COIP, Coimmunoprecipitation. B, To identify a physical mechanism whereby STAT3 would complex
with Sp1, EMSA was carried out using 5 ␮g nuclear extracts prepared from leptin-treated HSCs for all lanes. Lane 1, Free probe;
lane 2, serum free (SF) exposed HSC nuclear extracts; lane 3, leptin-treated primary activated HSC nuclear extract with probe
3 (cf. Fig. 3) only (Leptin); lane 4, supershift assay with nonimmune serum IgG; lane 5, antibody to Sp1 resulting in supershift of
Sp1-olignonucleotide complex (Sp1); lane 6, STAT3 antisera disrupted Sp1 or Sp3 binding to the oligonucleotide, but STAT1
antisera (lane 7) did not result in nuclear protein-oligonucleotide disruption. This EMSA is representative of three independent
experiments.
MATERIALS AND METHODS
Experimental Animals
The BDL protocol was approved by the Institutional Animal
Care and Use Committee of Emory University, and all animals
received human care and were euthanized in accord with
Institutional and Ethical Guidelines of the American Veterinary
Association.
Animal Model for in Vivo Studies
Four-week-old fa/fa rats and their lean littermates were purchased from Charles River Laboratories (Wilmington, MA).
Animals were housed for four additional weeks in a temperature-controlled environment (20–22 C) with a 12-h light, 12-h
dark cycle, and fed ad libitum with Laboratory Chow (Ralston
Purina, St. Louis, MO) and water. Rats were assigned to one
of four groups (n ⫽10): BDL (lean, or LLB), BDL (fa/fa, or
FFB), sham-operated (lean, LLN) and sham-operated (fa/
fa, or FFN). BDL, or sham operation was performed as
described elsewhere (58). Twenty-one days after surgery,
total hepatectomy was performed, and the animals were
exsanguinated. Livers harvested were snap-frozen in liquid
nitrogen.
Isolation and Culture of Primary Hepatic Stellate Cells
Quiescent stellate cells were isolated as described in detail
elsewhere. HSCs were isolated from Sprague Dawley rats
(Charles River). All rats received humane care, and the
Institutional Animal Care and Use Committee of Emory
University approved the HSC isolation protocol. In brief, in
situ perfusion of the liver was performed with 20 mg/dl
Pronase (Boehringer Mannheim, Indianapolis, IN), followed
with collagenase (Crescent Chemical, Hauppauge, NY).
Dispersed cell suspensions were layered on a discontinu-
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
Mol Endocrinol, December 2006, 20(12):3376–3388 3385
Fig. 9. Potential Mechanism for Leptin Activation of STAT3 and Sp1 in the Regulation of the TIMP-1 Promoter
Based upon the data presented leptin activates STAT3 phosphorylation via Jak2. This process also results in the phosphorylation of Sp1 as well as nuclear translocation and binding of phosphorylated Sp1 to the TIMP-1 promoter. Based on ChIP
analysis (Fig. 5) and coimmunoprecipitation and EMSAs (Fig. 8), we propose that STAT3 nuclear translocation results in the
physical association of pSTAT3 and phosphorylated Sp1.
ous density gradient of 8.2% and 15.6% Accudenz (Accurate Chemical and Scientific, Westbury, NY). The resulting
upper layer consisted of more than 95% HSCs. Cells were
plated in modified medium 199 OR containing 20% fetal
bovine serum (FBS) (Flow Laboratories, Naperville, IL). The
purity of cells was assessed by immunolocalization of
smooth muscle ␣ actin in the monolayer as well as by
intrinsic autofluorescence. The viability of all cells was
verified by phase-contrast microscopy as well as the ability
to exclude propidium iodide. Cell viability of cultures used
for experiments was greater than 95%. Subconfluent activated cells in culture (75%) 7–10 d after isolation were
washed twice with PBS and serum-starved for 16 h with
0.1% FBS and 1% penicillin-streptomycin in DMEM. The
concentration of leptin was determined previously and its
use is described elsewhere (1).
Real-Time Quantitative RT-PCRs (RT-qPCR) from
Whole Liver Post-Bile Duct Ligation and Cultured HSCs
to Detect Matrix Gene Expression
Total RNA from either whole liver from BDL-injured rats or
HSCs was obtained using Trizol Reagent (Invitrogen, Carlsbad, CA). Total RNA (1␮g) was reverse-transcribed in a 20-␮l
reaction containing random primers and iScript reverse Transcriptase (Bio-Rad, Hercules, CA). Real-time PCR was performed with iCycler IQ Real-time detection system (Bio-Rad)
using IQ SYBR Green super mix (Bio-Rad). The primers used
for the amplification of the rat TIMP-1 (accession no.
NM_053819) were: forward 5⬘-CCAGCCATGGAGAGCCTCTG-3⬘, and reverse 5⬘-TCAGATTATGCCAGGGAACC-3⬘.
All samples were run in triplicate. The relative fold increase of
specific RNA was calculated by the comparative cycle of
threshold detection method and values were normalized to
18 S rRNA. Final results, expressed as n-fold differences of
mRNA between different tissues relative to the 18 S rRNA,
were calculated with the following formula that is based on
two replications in each cycle: n ⫽ fold ⫽ 2(c ⫺ t)Target gene/
2(c ⫺ t) 18S rRNA; c represents cycle threshold (Ct) for target
gene or 18 S rRNA gene detection in control tissues; t represents Ct for target gene or 18 S rRNA gene detection in
other tissues.
TIMP-1 mRNA Stability Assay
Primary HSCs are grown in DMEM with 0.1% FBS for 16 h,
treated with leptin (0 or 100 ng/ml) for 4 h and subsequently
treated with actinomycin D (5 ␮g/ml) in 100-mm culture
dishes. HSCs were harvested for RNA at 0, 3, 8, 24, and 48 h
after the addition of actinomycin D. The leptin concentrations
used in these studies has been previously used (3, 22, 48, 59).
Total RNA was prepared using Trizol (Invitrogen) and isolated
according to the manufacturer’s protocol. TIMP-1 mRNA and
18 S rRNA were quantitated for each sample relative to the
sample at 0 h using RT-qPCR. The quantity of TIMP-1 mRNA
was normalized to the amount of 18 S rRNA and the results
were expressed as fold change. The entire experiment was
repeated three separate times. The mRNA stability of TIMP-1
mRNA was evaluated by drawing the best-fit linear curve on
a linear plot of the TIMP-1 mRNA fold change vs. time.
TIMP-1 Promoter Constructs
Human genomic DNA was extracted from the human MCF-7
cell line by using the QIAamp DNA blood kit (QIAGEN, Valencia, CA). TIMP-1 promoters were amplified from human
genomic DNA using the PCR. Forward primers, which anneal
at ⫺1482, ⫺989, ⫺582, ⫺369, ⫺229, and ⫺108 bp upstream
from the transcription start site, were used with a 21-bp
reverse primer ending at position ⫹88 bp of the human
TIMP-1 promoter. XhoI and HindIII restriction sites were introduced for subsequent cloning into the XhoI/HindIII-digested pGL3 basic luciferase reporter vector (Promega, Madison, WI). Primer sequences were: forward primers 5⬘-CTCGAGTAGATTTAGAGGGTACAGCTGCTG-3⬘, 5⬘-CTCGAGTGGGGTGGGGATTAGTTTTC-3⬘, 5⬘-TCGAGCCATGTATTGACTCTGTGATCC-3⬘, 5⬘-CTCGAGGAAGGGCTGAACTAATTTGGG-3⬘, 5⬘-CTCGAGAGGCGGCTTGGAAGGAATAG-3⬘, 5⬘-
3386
Mol Endocrinol, December 2006, 20(12):3376–3388
CTCGAGGTGGGTGGATGAGTAATGCA-3⬘ and reverse
primer 5⬘-AAGCTTCAGCTCCGGTCCCTG-3⬘. One nanogram of human genomic DNA and 1.25 pmol of each primer
were used for the amplification of the TIMP-1 promoter.
Annealing of primers was performed at 60 C for 1 min followed by 72 C in 30 1-min cycles and elongation at 72 C for
10 min using a High Fidelity PCR kit (Invitrogen). The PCR
was analyzed by agarose gel electrophoresis and the expected products were extracted and purified using the QIAquick gel extraction (QIAGEN). The purified product was used
for ligation into the TOPO TA vector (Invitrogen). After XhoI/
HindIII digestion of the TIMP-1, inserts were ligated into the
TOPO TA vector and extracted by using the Gel extraction kit
from QIAGEN. Vectors were recloned in the pGL3 basic
vector to yield TIMP-1–1482, TIMP-1–989, TIMP-1–582,
TIMP-1–369, TIMP-1–229, and TIMP-1–108. The correct size
and orientation of the cloned insert were analyzed by sequencing the plasmid DNA following standard plasmid preparation procedure by alkaline lysis.
Site-Directed Mutagenesis
The Sp1 mutant constructs were generated using the
QuikChange site directed mutagenesis kit (Stratagene, La
Jolla, CA) using TIMP-1–229 and TIMP-1–108 constructs as
templates. Substitution mutation at both Sp1 sites, i.e. ⫺34
and ⫺35 bp, and ⫺124 and ⫺125 bp upstream from the start
site of transcription were created with the following sequences: Sp1 mutant A sense, 5⬘-CCGCTGCCACCCCCATCCCTAGCGTGGAC-3⬘, Sp1 mutant A antisense, 5⬘-GTCCACGCT AGGGATGGGGGTGGCAGC GG-3⬘, Sp1 mutant B
sense, 5⬘-CCTTTCGTCGGCCATCCCCT TGGCTTCTGC-3⬘,
Sp1 mutant B antisense, 5⬘-GCAGAAGCCAAGGGGATGGC
CGACGAA ACGG-3⬘. The mutations were confirmed by DNA
sequencing.
Transient Transfections and Reporter Assays
Activated HSCs, after no more than two passages, were
grown in DMEM containing 10% FBS, penicillin (100 U/ml),
and streptomycin (100 ␮g/ml), until cells were 60–80% confluent. Transient transfection assays were performed using
Lipofectamine Plus reagent (Invitrogen,) with 1 ␮g of the
TIMP-1 reporter plasmid plus 200 ng of Renilla luciferase
plasmid (Promega) as an internal control for transfection efficiency. Four hours after transfection, the cells were supplemented with complete media. HSCs were serum starved
overnight before stimulation with leptin (100 ng/ml). Luciferase activity in cell extracts was measured using a Dynatech
luminometer (Dynatech Laboratories, Chantilly, VA). Firefly
luciferase activity was normalized to Renilla luciferase
activity.
Nuclear Extract Preparation
Nuclear extracts were prepared as described previously.
Briefly, HSCs were washed and scraped in ice-cold PBS and
transferred to centrifuge tubes. Samples were spun at
4000 ⫻ g for 3 min at 4 C. The pellet was resuspended in
buffer containing 10 mM HEPES (pH 7.0); 1.5 mM MgCl2; and
10 mM KCl on ice for 10 min. The samples were vortexed for
10 sec and spun at 4000 ⫻ g for 3 min. The resulting pellet
was resuspended in buffer containing 20 mM HEPES (pH 7.9),
420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol, and incubated on ice for 20 min. These samples were
spun at 16,800 ⫻ g for 2 min and the supernatant was
transferred to Eppendorf tubes (Eppendorf International,
Hamburg, Germany) and stored at ⫺80 C. Protein concentration was determined by the Bradford assay (Bio-Rad) (60).
All buffers contained protease inhibitor cocktail (Roche, Nutley, NJ) and 1 mM Na3VO4, 20 mM NaF, and 1 mM Na4P2O7.
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
EMSA
Oligonucleotides were annealed and labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and
[␥-32P] deoxy (d)-ATP (DuPont, Wilmington, DE; 3000 Ci/
mmol). Labeled oligonucleotides were used as probes or
remained unlabeled as competitors. A total of 5 ␮g protein
was incubated with 35 fmol of [␥-32P]dATP-labeled probe in
binding buffer [10 mM HEPES (pH 7.9), 1 mM EDTA, 5 mM
MgCl2, 10% glycerol, 5 mM dithiothreitol, protease inhibitor
cocktail and 1 ␮g of poly(deoxyinosine-deoxycytosine)] for 10
min on ice. The [␥-32P]dATP-labeled oligonucleotides were
added and the mixture was incubated on ice for 60 min.
DNA-protein complexes were separated from free DNA by
electrophoresis across a 4% nondenaturing polyacrylamide
gel. All gels were electrophoresed in 0.5⫻ Tris-borate-EDTA
buffer at 200 V for 1–2 h. Gels were dried under vacuum and
exposed to film (Eastman Kodak, Rochester, NY). For competition experiments, a 50-fold molar excess of unlabeled
oligonucleotide was added to the binding reaction. For supershift experiments, 5 ␮g of nuclear protein extract were
incubated 60 min on ice, each with 1 ␮g of the appropriate
antibody in a maximum volume of 40 ␮l before the binding
reaction. Antibodies used were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
ChIP Assay
ChIP analysis was performed using a commercially available
kit (Upstate, Charlottesville, VA) on HSCs treated with or
without leptin for 24 h as detailed elsewhere (61). Proteins
bound to DNA were cross-linked using formaldehyde at a
final concentration of 1% for 20 min at room temperature.
Protein-DNA complexes were immunoprecipitated using primary antibodies for Sp1, Sp3, and STAT3 (Santa Cruz Biotechnology). Sp1- and Sp3-TIMP-1 promoter complexes
were measured by real-time PCR. The primers used for the
amplification of the rat TIMP-1 promoter (accession no.
NM_053819) region between ⫺62⬃⫺230 bp were: forward
5⬘-AGGGGCTTAAGCGGTCTTTA-3⬘, and reverse 5⬘-CACCCACTGCTGAAGTCA AA-3⬘. The samples were electrophoresed using a 2% agarose gel, and visualized by ethidium
bromide staining.
Overexpression of SOCS-3 and Sp1
The mammalian expression vectors harboring SOCS 3 (pEFFLAG-1/mSOCS-3) was a kind gift of Dr. D. Hilton (Victoria,
Australia) (62). The human Sp1 expression vector (pN3Sp1FL-complete), was kindly provided by Dr. Guntram Suske
(Universitat Marburg, Marburg, Germany) (49). Subconfluent
(90%) activated HSCs were transfected with FLAG-SOCS-3
expression vector or corresponding empty vector (PEGFPN1) using Lipofectamine Plus reagent (Invitrogen). The cells
were serum starved for 16 h, and then cultured with leptin
(100 ng/ml), IL-6 (10 ng/ml) or vehicle for 24 h before cellular
proteins were harvested for immunoprecipitation study. For
the overexpression of Sp1, 90% confluent HSCs in were
transfected with 24 ␮g of Sp1 expression vector, serum
starved for 16 h and treated with or without leptin for 24 h.
Nuclear protein was extracted as described above and
200 ␮g of protein were used for immunoprecipitation
experiments.
Coimmunoprecipitation, Immunoprecipitation, and
Western Blot Analysis
For coimmunoprecipitation, HSCs were lysed, and nuclear
proteins were extracted and immunoprecipitated with 5 ␮l of
antiserum per 200 ␮g of nuclear proteins. For immunoprecipitation, HSCs were lysed, and lysates were immunopre-
Lin et al. • Mechanism of Leptin-Induced TIMP-1 Expression
cipitated with 5 ␮l of antiserum per 0.5 ml of cell lysate as
described (24). The immunocomplexes were collected by
adding protein A/G-agarose beads. For Western blots, leptintreated or -untreated HSCs were washed with PBS, lysed in
SDS-PAGE (33) loading buffer (200 ␮l/603-mm dish), boiled
10 min, and clarified by centrifugation (8 min, full speed,
Eppendorf microcentrifuge). Protein extracts were fractionated across 10% acrylamide SDS-PAGE gels. Proteins were
transblotted onto polyvinylidene difluoride membranes. Blots
were probed with anti-pSTAT3, anti-Sp1 and anti-Sp3 antibodies or antiphosphoserine antibody (Sigma, St. Louis, MO)
to investigate the phosphorylation status of Sp1 or Sp3 (63).
Immunoreactive proteins were blotted with a secondary antirabbit horseradish peroxidase (Santa Cruz Biotechnology)
and visualized using chemiluminescence reagents (Pierce,
Rockford, IL) and exposed to x-ray film. Signal intensities
were analyzed using the Fluorchem 800 Advanced Fluorescence Image Analysis System (Alpha Innotech, San Leandro,
CA).
Mol Endocrinol, December 2006, 20(12):3376–3388 3387
9.
10.
11.
12.
Acknowledgments
Received April 26, 2006. Accepted August 16, 2006.
Address all correspondence and requests for reprints to:
Frank A. Anania, Division of Digestive Diseases, Department
of Medicine, Emory University School of Medicine, Room
248, Whitehead Biomedical Research Building, 615 Michael
Street, Atlanta, Georgia 30322. E-mail: [email protected].
This work was supported by National Institutes of Health
Grants DK062092 and DK064399.
Disclosure Statement: The authors have nothing to
disclose.
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