Tissue inhibitor of metalloproteinases-1 (TIMP-1)
can be regulated by gp130 cytokines such as IL-6 and oncostatin M
(OSM). Polymerase chain reaction deletion analysis of the murine TIMP-1 proximal promoter in chloramphenicol acetyltransferase reporter gene
constructs identified an AP-1 element (
59/
53) that allows maximal
responsiveness to OSM in HepG2 cells. Fos and Jun nuclear factors bound
constitutively to this site as identified by supershift analysis in
electrophoretic mobility shift assays, and oncostatin M (but not IL-6)
induced an additional "complex 2" that contained c-Fos and JunD.
OSM stimulated a rapid and transient increase in c-Fos mRNA and
nuclear protein that coincided with complex 2 formation. Phorbol
13-myristate 12-acetate could also induce c-Fos but could not regulate
the TIMP-1 reporter gene constructs. Transfection studies also showed
that 3'-deletion of sequences downstream of the transcriptional start
site (+1/+47) markedly reduced OSM -fold induction. Nuclear factors
bound to SP1 and Ets sequences were detected, but were not altered upon
OSM stimulation. Although OSM and IL-6 induced STAT (signal transducers
and activators of transcription) factors to bind a high affinity
Sis-inducible element DNA probe, binding to homologous TIMP-1 promoter
sequences was not detected. Thus, OSM (but not IL-6) stimulates c-Fos,
which participates in maximal activation of TIMP-1 transcription,
likely in cooperation with other factors such as SP1 or as yet
unidentified mechanisms involving the +1 to +47 region of the
promoter.
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INTRODUCTION |
Cytokines play a pivotal role as soluble growth factors acting on
immune and non-immune cells to coordinate the progression of and
resolution from an inflammatory response. The interleukin-6 (IL-6)1 family of cytokines
which include IL-6, leukemia inhibitory factor (LIF), oncostatin M
(OSM), IL-11, ciliary neurotropic factor, and cardiotrophin-1 possess
both unique and shared biological activities and utilize a common
signal transducing receptor subunit gp130 (1, 2). In addition, the
polypeptide primary structures of OSM, LIF, and, to a lesser extent
IL-6, ciliary neurotropic factor, and granulocyte colony-stimulating
factor display sequence homology, suggesting a functional relationship
for these cytokines (3). Biochemical studies on signaling by gp130 have
demonstrated tyrosine phosphorylation of a variety of components of the
Ras-MAP kinase cascade (4, 5) and activation of the recently
characterized family of STATs (signal transducers and activators of
transcription) (6, 7). Upon activation, cytosolic STAT proteins homo-
or heterodimerize and translocate to the nucleus where they bind DNA
elements termed
-interferon activation sequence (GAS). One target
gene of STAT proteins is c-fos, which harbors a GAS-like DNA
element, the Sis-inducible element (SIE), within its promoter.
OSM is secreted as a 28-kDa polypeptide by mitogen-activated T cells
and endotoxin-stimulated macrophages (8). It was first characterized by
its ability to specifically inhibit the growth of the A375 melanoma
cell line (9). Subsequently, OSM was demonstrated to stimulate growth
of fibroblasts (10), aortic endothelial cells (11), and hematopoietic
cells (12), as well as promote leukemic cell differentiation (3), while
suppressing embryonic stem cell differentiation (13) and several tumor
cell lines including the HTB10 lung carcinoma (14). OSM elicits acute
phase protein production by hepatocytes and HepG2 hepatoma cells (15). In addition, unlike other IL-6-cytokines, OSM also specifically up-regulates low density lipoprotein receptor (16) in HepG2 cells. The
transduction of signals that lead to biological activities unique to
OSM but not other IL-6 family members is not yet defined.
Extracellular zinc-dependent endopeptidases (the matrix
metalloproteinases) can be inhibited by a family of proteins called tissue inhibitors of metalloproteinases (includes TIMP-1, -2, -3, and
-4), which act to modulate extracellular matrix metabolism by matrix
metalloproteinases (17-21). TIMP-1 expression is up-regulated in
fibroblasts by a variety of soluble factors including IL-1 (22), tumor
necrosis factor (23), epidermal growth factor (23), transforming growth
factor-
(23), phorbol esters (24), and retinoic acid (25). We have
shown that OSM potently induces mRNA expression of the TIMP-1 gene
in hepatocyte cell lines and primary fibroblasts (26). Work by Edwards
et al. (27) has suggested that the
95 to +47 region of the
murine TIMP-1 promoter is sufficient to confer serum responsiveness in
mouse cells and this region contains several putative regulatory motifs
including SP1, AP-1, and Ets DNA elements. Here, we examine OSM
regulation of the
95 to +47 TIMP-1 promoter in HepG2 cells and
characterize the participation of c-Fos in the binding of nuclear
factors to an AP-1 site between
62 and
53 that is necessary for
marked OSM (but not IL-6) up-regulation of promoter/CAT reporter gene expression. We examined IL-6- and phorbol 13-myristate 12-acetate (PMA)-induced responses to identify OSM-specific effects on this promoter region and the participation of SP1, Ets, and other promoter sequences in regulation of TIMP-1 expression.
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MATERIALS AND METHODS |
Cell Culture and Reagents--
The human hepatoma cell line
HepG2 (purchased from ATCC) cultured and passaged by standard
techniques in
-minimal essential medium (supplemented with 10%
fetal bovine serum (FBS)). Cytokines used were: purified human
recombinant OSM, expressed in Chinese hamster ovary cells, provided by
M. Hanson (Bristol-Myers Squibb Research Institute, Seattle, WA);
purified recombinant human IL-6, IL-1
, and LIF, kindly provided by
Dr. M. Widmer (Immunex Corp., Seattle, WA); interferon-
, purchased
from Genzyme Corp. (Cambridge, MA). Rabbit polyclonal antibodies
against Fos/Jun (anti-pan-Fos, anti-c-Fos, anti-c-Jun, anti-JunB,
anti-JunD), SP1, STAT3 and Ets-1/Ets-2 nuclear factors, and specific
peptides were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA). Anti-STAT1 (ISGF3) was from Transduction Laboratories (Lexington,
KY). Phenylmethylsulfonyl fluoride, pepstatin A, PMA, puromycin, and
emitine were purchased from Sigma. When added, puromycin and emitine
were used at a concentration of 20 µg/ml. Leupeptin was purchased
from Boehringer Mannheim.
Northern Blots--
Total RNA was prepared from HepG2 cells
according to Chomczynski and Sacchi (28). Subconfluent HepG2 cultures
were washed and replenished in medium containing 2% FBS. Cytokines (at
indicated concentrations) were then added, and cultures were incubated
for the indicated times before RNA isolation. Northern blots were prepared by standard techniques and probed with human TIMP-1 cDNA (gift of Dr. A. J. P. Docherty, Celltech, Slough, United
Kingdom) and rat c-Fos cDNA (kindly provided by Dr. Tony Cruz,
Mount Sinai, Toronto, Canada). The intensity of ethidium
bromide-stained 18 S and 28 S bands on the blots was used to estimate
loading of RNA.
Polymerase Chain Reaction (PCR) Deletion of the TIMP-1 Promoter
and Cloning--
Deletions of the TIMP-1 promoter were carried out by
generating truncated PCR products within
95 to +47. PCR reactions
consisted of Vent polymerase (5 units; New England Biolabs), 100 ng of
TIMP-1
223 to +47/pBLCAT3 (27) template DNA, 150 µM
sense and antisense oligonucleotide primers, 5 mM dNTPs,
and 1 × Vent polymerase New England Biolabs buffer in a 50-µl
final reaction volume. Reaction mixtures were overlaid with 30 µl of
mineral oil and denatured for 1 min at 95 °C, followed by annealing
of primers at 55 °C for 2 min and primer extension at 72 °C for 2 min in a Perkin-Elmer PCR thermal cycler for 35 cycles.
Primers used to generate 5' deletions of the TIMP-1 promoter included:
62/
38 for construct B, which contains AP-1 and
Ets sites
(5'-GAGGCTAAGCTTGGATGAGTAATGCGTCCAGGAAGCC-3');
52/
31 for construct C, which retains Ets site
(5'-GAGGCTAAGCTTTGCGTCCAGGAAGCCTGGAGGC-3'); and
39/
16 for construct D
(5'-GAGGCTAAGCTTCCTGGAGGCAGTGATTTCCCCGCC-3'). 3' PCR
primers (for constructs B-D) included a sequence 3' to the
pBLCAT3 multiple cloning site (+470/+447)
(5'-CTGAAAATTCGCCAAGCTCCTCG-3'). For constructs E and F, 3' primers
were: +1/
18, containing wild type SP1 site
(5'-TCGTACAGATCTGCGAAGGGCGGAGTTGGCG-3') and +1/
18 with a
mutated SP1 site (GC
TT,
5'-TCGTACAGATCTGCGAAGGTTGGAGTTGGCG-3'). Restriction sites for HindIII in 5' primers (AGCTTT) and
BglII in 3' primers (AGATCT), as well as 6 nucleotide
residues 5' to each site (for efficient restriction enzyme digestion of
DNA termini), were included in the designed primers. PCR products were
purified from an 8% polyacrylamide gel, restriction-digested with
HindIII/BglII, re-purified, and ligated into
linearized pBLCAT3 vector
(HindIII/BglII-digested). Colony hybridization
(Colony/Plaque Screen, NEN Research Products) with end-labeled PCR
primers as probes were then used to screen for DH5
bacterial
transformants. DNA sequencing was used to verify the correct
constructs.
Chloramphenicol Acetyltransferase (CAT) Assays--
HepG2 cells
in 100-mm dishes were transfected with 10 µg of CAT reporter plasmid
DNA (co-transfected with 1.4 µg of pSV-
Gal plasmid, Promega) by
the calcium phosphate coprecipitation method. Cells were allowed to
recover overnight and then replated into six-well tissue culture
plates. Prior to cytokine stimulation, cells were serum-starved in
serum-free
-minimal essential medium for 6 h. Cytokines were
then added for 18 h. CAT assays were carried out using cell
lysates according to standard protocols and 14C-labeled
chloramphenicol products quantified using a Molecular Dynamics
PhosphorImager and ImageQuant software. Values were normalized to
-galactosidase activity in lysates (29).
Preparation of Nuclear Extracts--
HepG2 cells were stimulated
with the indicated cytokines for various time periods. Nuclear extracts
were prepared according to Andrews et al. (30) with the
following modifications. Buffers A and C contained 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotonin, and 2 µg/ml
pepstatin A and leupeptin. Cells were resuspended in 400 µl of buffer
A, and nuclear proteins were extracted in 100 µl of buffer C, frozen
in liquid nitrogen, and stored at
70 °C.
Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extract (15 µg) was incubated with 2 µg of poly(dI·dC) and 5 µg
of calf thymus DNA in binding buffer (50 mM Tris-Cl (pH
7.5), 50 mM NaCl, 2 mM EDTA, 2 mM
dithiothreitol, 1 mM spermidine, and 5% glycerol) for 15 min on ice. A total of 105 cpm of 32P-labeled
probe was then added, and the binding reaction (20 µl) was incubated
at room temperature for 20 min. Excess of unlabeled oligonucleotide
(50-100-fold) was added to the reaction for competition assays.
Following the reaction, samples were electrophoresed on 5%
polyacrylamide gels (40:1) containing 1.25% glycerol in 0.25 × TBE (1 × TBE: 89 mM Tris borate, 2 mM
EDTA) at 95 V for 3.5 h and dried prior to autoradiography. For
supershift analysis, antibodies were added following the binding
reaction for 1 h at 4 °C. Sequences of oligonucleotides used
for mobility shift assays are shown in Table
I, and their location in the TIMP-1
promoter is illustrated in Fig. 2. AP-1 and SP1 consensus
oligonucleotides were purchased from Santa Cruz Biotechnology
Inc. Oligonucleotides were annealed by heating to 100 °C for 5 min
in 100 mM MgCl2 and 400 mM Tris-Cl
(pH 8), followed by gradual cooling to 20 °C. TIMP-1 DNA probes were
end-labeled using polynucleotide kinase and the high affinity
Sis-inducible element (hSIE) labeled by the fill-in reaction using
[32P]dCTP and the Klenow enzyme. Probes were then
gel-purified.
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Table I
Oligonucleotides used in EMSA
Oligonucleotides were synthesized, annealed, and purified as indicated
under "Materials and Methods." The sequences correspond to the
TIMP-1 promoter regions as indicated. Defined nuclear factor binding
sites for SP1, AP-1, and Ets have been underlined. Probe 3A contains a
mutated AP-1 site (uAP-1). Probe 4A contains a G at position 49 corresponding to the mouse TIMP-1 sequence, whereas probe 4 has A in
that position corresponding to the human TIMP-1 sequence.
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Western Blots--
Nuclear extracts were electrophoresed on a
8% sodium dodecyl sulfate (SDS)-polyacrylamide gel prior to transfer
of proteins onto an Immobilon nitrocellulose membrane. Membranes were
then blocked overnight in 1 × PBS and 5% milk. Following a
10-min incubation in wash buffer (1 × PBS, 0.5% Tween 20),
membranes were incubated with primary rabbit polyclonal antibody
(anti-c-Fos, anti-pan-Fos, anti-c-Jun, anti-JunB, or anti-JunD;
Santa Cruz Biotechnology Inc.) at a 1:2500 dilution for 1 h
at room temperature. To assess specificity of antibody binding, 2 µg
of peptide was preincubated with antibody for 1 h at 4 °C (as
recommended by Santa Cruz Biotechnology Inc.). Membranes were then
washed three times for 10 min each. Goat anti-rabbit horseradish
peroxidase (Sigma) was added for an additional (1:7500 dilution) 1 h and Immobilon filters were developed by Enhanced Chemiluminescence
Renaissance (NEN Life Science Products).
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RESULTS |
Oncostatin M Induces TIMP-1 and c-Fos Expression--
We have
found previously that OSM elevates TIMP-1 mRNA levels after
overnight stimulation in a variety of cell types including HepG2 (26).
Examination of TIMP-1 mRNA expression in HepG2 cells over a time
course of OSM stimulation (Fig. 1)
revealed a 5-fold increase in TIMP-1 message by 1 h, near maximal
at 2 h (40-fold), and maximal mRNA levels at 6 h
(60-fold). OSM also transiently stimulated mRNA levels of the AP-1
immediate-early gene c-fos (Fig. 1). As demonstrated
previously in response to other stimuli (31), the up-regulation of
c-Fos mRNA occurred early (maximal induction at 30 min (20-fold))
and decreased thereafter. Thus, c-Fos mRNA levels peaked slightly
before the marked increase in TIMP-1 mRNA.

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Fig. 1.
OSM up-regulates TIMP-1 and c-Fos mRNA
levels. HepG2 cells were treated with OSM from 15 min to 18 h
and RNA extracted and probed by Northern analysis for TIMP-1 or c-Fos
mRNA. Ethidium bromide (EtBr) staining of 28 S and 18 S
ribosomal RNA is shown in the bottom panel to show
equivalent loading of RNA samples.
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OSM Regulates the TIMP-1 Proximal Promoter--
The proximal
promoter of the TIMP-1 gene (
95/+47 sequence) can regulate downstream
CAT gene expression in pBLCAT3 chimeric reporter constructs (27). When
transiently transfected into HepG2 cells, the expression of
95/+47CAT
could be elevated upon OSM (5.2-fold) or IL-6 (3.8-fold) stimulation
(Fig. 2, construct A). Several
putative DNA elements can be identified within
95 to +47 region of
TIMP-1, including those for SP1, AP-1 (Fos/Jun), and Ets proteins. To
examine the potential role of these sites in transcription, we
generated 5' deletions of this region and cotransfected HepG2 cells
with constructs A-F (Fig. 2) and pSV-
Gal to normalize for
transfection efficiency. Basal levels of CAT transcription were reduced
in the plasmid lacking
95 to
63 sequences (construct B), and
further reduced in the plasmid that also lacked the
62 to
53
sequences, which contained the AP-1 site (construct C). The
62/+47CAT
chimera (construct B) demonstrated maximal responsiveness to OSM with a
11.4-fold increase in CAT activity over unstimulated cells, whereas
IL-6 induced 4.1-fold increases. Deletion of the AP-1 site within this
region (construct C) markedly reduced responsiveness to OSM (from 11.4- to 2.7-fold), whereas responses to IL-6 decreased from 4.1- to 2-fold.
Thus, a promoter region from
62 to
53 of TIMP-1, containing a
putative AP-1 binding site (at
59/
53), contributes to basal
transcription and to induction of transcription by OSM. Deletion of
this sequence removed any significant difference between OSM and IL-6
activity in this assay. Interestingly, deletion of sequences from +1 to
+47 (construct E versus B) dramatically abrogated the
responsiveness to OSM (from 11.4- to 3.7-fold) and also reduced IL-6
responses (from 4.1- to 2.3-fold). Thus, sequences within +1/+47 may
cooperate with the TIMP-1 AP-1 element within
62/
53 for maximal
responsiveness to OSM. SP1 elements are common within TATA-less
promoters and have been demonstrated to participate in basal
transcription (32). A chimeric plasmid with mutation of the SP1 site
(construct F) showed reduced basal transcription (4-fold) but was still
inducible by OSM (construct E versus F).

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Fig. 2.
Deletion analysis of the TIMP-1
promoter. Deletions of the TIMP-1 promoter spanning 95 to +47
were generated by PCR and cloned into pBLCAT3 to examine their capacity
to regulate CAT reporter gene expression. HepG2 cells were
co-transfected with each of the constructs (schematically illustrated
at left) and pSV- Gal and treated with either OSM (50 ng/ml) or IL-6 (100 ng/ml) for 18 h. Cellular extracts were then
prepared, and CAT activity was measured by standard methods and
normalized to -galactosidase activity. Results are shown as percent
conversion and -fold induction upon cytokine stimulation. Values
represent the mean of three separate experiments (standard deviation in
parentheses), each experiment done in duplicate.
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Nuclear Factors Bound to the TIMP-1 Promoter--
We have examined
a variety of overlapping oligonucleotide probes (probes 1-6; location
shown in Fig. 2) spanning
95 to +1 of the TIMP-1 promoter in EMSA
analysis to identify elements that bind nuclear factors in HepG2 cells.
A specific bandshift with either AP-1 or AP-1-Ets (probes 2 and 3) was
seen in unstimulated cell nuclear extracts that we termed "complex
1" (Fig. 3, A and B). At 12 h of OSM treatment, much higher amounts of
complex 1 are apparent. OSM induced the assembly of a second complex
after 30 min of OSM stimulation with decreased mobility ("complex
2") that persisted for several hours. The mobility of complex 2 appeared to increase following 2 h of OSM stimulation. Promoter
sequences at
49 to
40 displayed an Ets core binding element as well
as homology to STAT DNA binding sites (consensus TTCCNNNAA). Using a
probe spanning this region (probe 4), a very weak binding activity was
detected, which was not altered upon OSM stimulation (Fig. 3C), and similar results were noted using the mouse TIMP-1
sequence (probe 4A), which differs by one base pair (data not shown).
The mobility of this complex appeared similar to that of the
AP-1-complex 1 and no other complexes were detectable. Nuclear factors
binding to probe 4 were specifically competed by cold Ets probe but
supershift experiments using an anti-Ets-1/Ets-2 polyclonal antibody
reactive against a highly conserved DNA binding domain of Ets proteins did not identify Ets-1 or Ets-2 binding to this probe (data not shown).
STAT-1 and 3 activation can be detected using the hSIE (33). Using the
hSIE and the same HepG2 extracts as above, we noted that treatment with
OSM resulted in rapid nuclear factor binding with gel shift mobilities
consistent with those observed for homodimers of STAT3 or STAT1 and the
heterodimer of STAT3 with STAT1 (Fig. 3D). Each of these
complexes were verified using antibodies to STAT3 or STAT1 in EMSA
supershift assays (data not shown) as established previously (33).
STAT1 was detected at early time points, whereas STAT3 persisted from
15 min to 12 h of OSM treatment. However, neither the AP-1-Ets or
Ets sequences (probes 3 and 4) detected complexes with the same
mobility and kinetics as compared with the hSIE in response to OSM.

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Fig. 3.
OSM induces binding of TIMP-1 AP-1 probe
complexes in a time-dependent fashion. Binding of
nuclear factors to oligonucleotides (Table I) spanning the TIMP-1
promoter were tested by EMSA. The probes were as follows: A,
AP-1 (probe 2); B, AP-1-Ets (probe 3); C, Ets
(probe 4); and hSIE (D). HepG2 cells were treated with OSM
from 0 to 12 h, and nuclear extracts were prepared and stored at
70 °C until analysis. EMSA gels were dried and subjected to autoradiography. Longer gel profiles (as seen in Fig. 4) showed no
other specific bands; thus, only the tops of the gels are shown here.
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The TIMP-1 AP-1/Ets (probe 3) complexes and STAT mobility shifts were
specifically competed by the 50-fold addition of unlabeled probe 3 or
hSIE probe, respectively (Fig. 4).
Neither AP-1 complexes 1 or 2 could be eliminated by competition with
unlabeled hSIE probe (Fig. 4) or supershifted with anti-STAT1 or
anti-STAT3 (data not shown). EMSA analysis with probe 3A, containing
mutation of nucleotides in the AP-1 site, dramatically reduced
complexes 1 and 2, and other specific complexes were not detectable
(Fig. 5) over the time course of OSM
stimulation. Thus, we could not detect STAT nuclear factors associated
with the AP-1 complexes 1 and 2, nor binding to sequences immediately
flanking the TIMP-1 AP-1-like element that show partial homology to a
STAT DNA binding site. Further downstream sequences showed weak
homology to GAS (differing from the consensus GAS site by a two
nucleotide insertion); however, probe 5 (
38/
15) elicited no early
or late induced gels shifts similar to STAT complexes (data not shown).
Thus, we could not detect STAT-1/3 nuclear protein binding to TIMP-1
promoter sequences in these cells.

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Fig. 4.
Cold competition for AP-1 and STAT nuclear
factor binding. Nuclear extracts from HepG2 cells stimulated for
0.25 (15 min) or 1 h were probed with AP-1/Ets (probe 3) or the
hSIE probe in EMSA analysis. A 50-fold excess of unlabeled cold
competitor-oligonucleotide was added as indicated. Complexes 1 and 2 were competed by cold probe 3 but not by hSIE, whereas cold hSIE
competed for labeled hSIE. The open circle ( ) indicates
the position of nonspecific complexes.
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Fig. 5.
Mutation of the AP-1 site in the AP-1-Ets
EMSA probe. HepG2 cell nuclear extracts prepared from cells
treated for 0, 0.25, 1, and 12 h were probed with either a wild
type AP-1/Ets probe (probe 3) or mutAP-1/Ets probe containing mutations
in the AP-1 site (probe 3A). The open circle ( )
represents nonspecific binding. Mutation of the AP-1 site removed any
detectable binding of OSM-inducible nuclear factors to this probe. STAT
factor activation in nuclear extracts was confirmed using the hSIE
probe at 0 and 0.25 h time points after OSM stimulation.
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Supershift analysis of TIMP-1 AP-1 bands were carried out to identify
Fos and Jun components of these complexes. Nuclear extracts were
prepared from HepG2 cells treated with OSM for 0, 1, and 12 h and
incubated with polyclonal antibodies specific for AP-1 proteins prior
to EMSA analysis. Using an antibody reactive against all Fos-related
antigens (anti-pan-Fos), complex 1 was obliterated at 0, 1, and 12 h, and a Fos supershift was clearly visible at 1- and 12-h time points
(Fig. 6A). The anti-pan-Fos
antibody also supershifted complex 2 from HepG2 cells treated with OSM
for 1 h (Fig. 6A). However, when an anti-c-Fos specific
antibody was used, only complex 2 was supershifted and complex 1 remained unaffected, suggesting that c-Fos is a prerequisite for the
OSM-stimulated assembly of complex 2. Of the anti-Jun antibodies used
in EMSA binding reactions, anti-JunD and anti-JunB supershifted TIMP-1 AP-1 probe complexes (Fig. 6B). JunD was detected at time 0, 1, and 12 h of OSM treatment. JunB could also be detected in these complexes, whereas c-Jun could not be detected at any time. Together, these data suggest that, whereas complex 1 consists of Fos and Jun,
complex 2 specifically contains c-Fos, which likely complexes with JunD
or JunB.

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Fig. 6.
Detection of c-Fos and Jun nuclear factors
bound to the Timp-1 AP-1 element by supershift analysis. Cells
were stimulated with OSM for 0, 1, or 12 h and nuclear extracts
prepared. A, anti-pan-Fos (reactive against all Fos-related
antigens) or anti-c-Fos antibody was used in supershift analysis of
EMSA using the TIMP-1 AP-1 (probe 2) in binding reactions. EMSAs were
then carried out to resolve gel-shifted and supershifted complexes on a
5% polyacrylamide nondenaturing gel. B, supershift analysis
was carried out using antibodies to c-Jun, JunB, and JunD in binding
reactions with nuclear extract of 1-h OSM-stimulated cells and the AP-1
probe 2. Antibody was added to the reactions without extract
(right three lanes, no extract) to show no nonspecific
binding to probe. The mobility of nonspecific proteins binding probe 2 is indicated by an open circle ( ).
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Because sequences within
95 to +1 contained two putative SP1 binding
sites, both of these were examined for the binding of HepG2 nuclear
factors. Oligonucleotides spanning
95 to
66 (probe 1) or
19 to +2
(probe 6) constitutively bound two specific complexes, which remained
unchanged in response to OSM treatment (Fig.
7, A and B). Both
complexes were competed by cold SP1 probe (Fig. 7A) and are
consistent with the mobility of SP1 nuclear factor binding to a
consensus SP1 DNA element (data not shown). Anti-SP1 antibody
supershifted the slower migrating complex but did not appear to affect
the faster migrating specific complex (Fig. 7C). This may
indicate the binding of other SP1 family members (34). Binding of SP1
to the
95/
66 oligonucleotide was only detectable following a one
week exposure of EMSA gels (data not shown), although weak binding to
this probe may be due to insufficient sequence flanking the 5' end of
the SP1 site. Thus, SP1 nuclear factors constitutively occupy binding
sites within the TIMP-1 promoter and OSM does not appear to affect the
SP1 DNA binding.

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Fig. 7.
SP1 nuclear factor binding to the TIMP-1
promoter. A, an excess of cold specific unlabeled competitor
oligonucleotides (cold SP1) or irrelevant competitor (cold AP-1/Ets,
probe 3) were used to identify specific binding of SP1 (probe 6)
gel-shifted complexes. Free probe migrates at the bottom of the gel.
B, nuclear extracts from cells stimulated with OSM for 0, 0.25, 1, or 12 h were incubated with a TIMP-1 putative SP1 DNA
binding element (probe 6) binding examined by EMSA analysis.
C, anti-SP1 antibody was used to supershift SP1 binding to
probe 6. Supershifts were inhibited by preincubation of anti-SP1
antibody with SP1 peptide but not by an irrelevant peptide (Fos
peptide). The open circle ( ) represents the position of
nonspecific complexes.
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OSM, but Not Other Cytokines, Induces Complex 2--
Given the
overlapping biological functions of OSM and IL-6, these and other
cytokines were examined together for their ability to induce DNA
binding of complexes to the TIMP-1 AP-1 probes in EMSAs. Interestingly,
OSM was the only cytokine examined that strongly induced complex 2 (Fig. 8A), whereas both OSM
and IL-6 markedly induced activation of STAT binding to the hSIE probe (Fig. 8B). Thus, within the same assay, OSM and IL-6 utilize
shared (STAT) and distinct (AP-1-complex 2) nuclear signaling pathways leading to protein-DNA interactions.

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Fig. 8.
Selective induction of complex 2 formation
and c-Fos up-regulation by OSM and PMA. Induction of complex 2 formation (and STAT activity) by OSM (50 ng/ml), was compared with IL-6 (100 ng/ml), interferon- (500 units/ml), IL-1 (5 ng/ml), and PMA
(50 nM). Stimulation of DNA binding activity was tested
using probe 3, which contains the TIMP-1 AP-1 sequence (A),
or the hSIE probe (B). Protein levels of c-Fos in HepG2
nuclear extracts (15 µg) of cells treated with the above cytokines or
PMA were examined by Western blotting using an anti-c-Fos specific
primary antibody and a goat anti-rabbit horseradish peroxidase
secondary antibody (C). c-Fos protein was detected by
enhanced chemiluminescence followed by autoradiography.
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PMA has been shown to activate AP-1 (34) and stimulate TIMP-1
expression (24). When tested in HepG2 cells, PMA potently stimulated
complex 2 formation (AP-1 probe 2) and to a greater extent than OSM
(Fig. 8A). To examine the activity of PMA on transcription, we compared the effectiveness of PMA to OSM or IL-6 in stimulating CAT
activity of the TIMP-1
62/+47 reporter gene construct B. Table
II shows that PMA alone (or 20% FBS) was
unable to up-regulate CAT activity through the TIMP-1
62/+47 promoter
element whereas OSM induced 10-fold increases and IL-6 induced 4-fold
changes. When used in combination with IL-6, PMA also failed to
stimulate CAT activity beyond that of IL-6 alone. Thus, despite
equivalent STAT activation and c-Fos induction as in OSM-treated cells,
the combination of IL-6 and PMA was not sufficient to induce similar levels of transcriptional activation. OSM may induce qualitative differences in AP-1 factors distinct from that by PMA and requires additional regulatory elements for the pronounced induction of the
TIMP-1
62/+47 reporter gene construct beyond that induced by
IL-6.
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Table II
Regulation of 62/+47CAT promoter activity by OSM and IL-6 but not
PMA
HepG2 cells were transfected with 10 µg of 62/+47CAT, allowed to
recover overnight and replated in six-well Costar plates. The cultures
were then serum-starved for 6 H and stimulated with OSM, IL-6, PMA,
IL-6 and PMA, or 20% FBS for 18 H in serum-free conditions. CAT
activity was assayed as described in "Materials and Methods." -Fold
change was calculated from phosphorimagery results and averaged from at
least three separate experiments ± S.D.
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Oncostatin M Stimulates c-Fos Protein Nuclear
Accumulation--
Because the data implicate c-Fos in gel-shifted
complex 2, and OSM markedly stimulated early transient expression of
c-Fos mRNA (Fig. 1), we examined c-Fos protein levels in nuclear
extracts from HepG2 cells by Western blots (Fig. 8C). In the
same extracts used for EMSA, OSM and PMA markedly up-regulated c-Fos
protein levels in HepG2 cell nuclei. Expression of c-Fos protein was
abrogated in the presence of protein synthesis inhibitors (puromycin
and emitine) (Fig. 9B), and in
addition, these agents blocked complex 2 formation in response to OSM
(Fig. 9A). Complex 1 formation was not affected at 0.25 or
1 h; however, the inhibitors did reduce complex 1 at 12 h,
which suggested newly synthesized proteins are involved in complex 1 at
this later time point. OSM stimulated nuclear accumulation of c-Fos by
1 h, which persisted to 12 h of OSM treatment (Fig.
9B). Interestingly, the electrophoretic mobility of c-Fos
was reduced at 12 h when compared with 1 h of OSM
stimulation. This difference in mobility is first observed after 2 h of OSM treatment (data not shown), persists for up to 12 h, and
may represent a change in the phosphorylation status of c-Fos.

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Fig. 9.
Effect of OSM on TIMP-1 AP-1 complexes and
nuclear c-Fos, JunB, and JunD protein accumulation and the influence of
protein synthesis inhibitors. AP-1 complexes (probe 2) were
examined by EMSA analysis (A) and the same extracts assessed
by Western blots for c-Fos (B), JunB (C), and
JunD (D) protein levels from HepG2 nuclear extracts of cells
treated with OSM (50 ng/ml) for the indicated times above. A
requirement for new protein synthesis of TIMP-1 gel-shift complexes and
nuclear AP-1 protein levels was assessed by the addition of the protein
synthesis inhibitors puromycin and emitine (P/E), each used
at 20 µg/ml. Antibody specificity in Westerns was assessed by using
relevant peptide competitors as noted under "Materials and
Methods." Asterisks denote the position of bands that were
competed by relevant peptide but not irrelevant peptides for c-Fos,
JunB, and JunD.
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In contrast to c-Fos, and consistent with their presence in AP-1
gel-shifted complexes, both JunB and JunD were constitutively present
and JunD was moderately up-regulated at the 1 h time point of OSM
treatment (Fig. 9, C and D). Puromycin and
emitine had no effect on JunB at early time points of OSM stimulation
and only affected JunD levels after 12 h of treatment. Detection
of c-Fos, JunB, and JunD in Western blots was confirmed by specific competition of relevant peptides for each of the antibodies used, and
c-Jun was undetectable from the same nuclear protein extracts (data not
shown). Thus, OSM stimulates the nuclear accumulation of c-Fos protein,
whereas JunB and JunD nuclear factors are constitutively resident
within the nucleus and appear largely unaffected by OSM at the level of
new protein synthesis.
 |
DISCUSSION |
The transcription factor c-Fos is one of several participants in
various Fos/Jun complexes that bind AP-1 sites of gene promoters to
regulate transcription (31). We have shown here that, upon stimulation
by OSM in HepG2 cells, c-Fos is highly induced at the mRNA and
protein level and takes part in an AP-1 complex (complex 2) that is
separate from that constitutively present (complex 1) using the
AP-1-containing sequence (
59/
53) of TIMP-1 as a probe. OSM induced
rapid and transient expression of c-Fos (peak at 30 min) and nuclear
accumulation, which bound the AP-1 site in complex with Jun proteins
(peaking at 1-2 h). Nuclear c-Fos protein levels coincided with the
induction of complex 2 by OSM. Protein synthesis inhibitors (puromycin
and emitine) inhibited c-Fos protein expression and formation of
complex 2. Thus, c-Fos is prerequisite to complex 2 formation, and its
nuclear accumulation in response to OSM is dependent upon new protein
synthesis. Such AP-1 activation may contribute to effects of OSM on
growth regulation or expression of other genes. Previous work has shown
OSM-mediated induction of other immediate early genes such as
egr-1, c-jun, and c-myc in fibroblasts
(35), which also suggests a broad range of gene products could in turn
be regulated by OSM stimulation.
Although functional redundancy within the IL-6-type family of cytokines
is a common observation, OSM manifests both shared and distinct
biological activities with other family members (IL-6, LIF, IL-11,
ciliary neurotropic factor, and cardiotrophin-1), which could be
attributed to differential expression of receptor complexes on
different cells. Alternatively, the subunits unique to individual
receptor complexes may contribute to the biological activities of these
cytokines. OSM binds and activates cells through the LIF receptor (type
I OSM receptor) and a newly identified complex of gp130 and OSM
receptor
chain (type II OSM receptor) (36) in human cells. Like
other related family members, OSM stimulates components of the Ras-MAP
kinase pathway, such as Ras, Raf-1, Grb2, Shc, and the p42 MAP kinase
(4, 37), and the JAK-STAT pathway (6, 7). Other signaling pathways have
also been implicated in mediating signals transduced by OSM, including those affecting phosphatidylinositol 3'-kinase and the canonical Src
kinase (38). We have here confirmed that OSM potently induces mRNA
expression of the TIMP-1 gene in HepG2 cells, and shown that OSM
strongly enhances transcription of a CAT reporter gene flanked by
62
to +47 of TIMP-1 promoter construct. Up-regulation of CAT activity was
most strongly induced by OSM treatment, less so by IL-6, and not at all
by 20% FBS, PMA, or the combination of IL-6 and PMA. Taken together,
this implicates OSM in activating a signaling event(s) distinct from
IL-6 or serum factors that likely act on target DNA elements within the
TIMP-1 promoter for up-regulating its transcription and expression.
Interleukin-6 also regulates the TIMP-1 promoter (Fig. 2) but appears
not to activate c-Fos or form complex 2; nor did IL-6 regulate
AP-1-containing promoter/CAT (Fig. 2) constructs to as great a degree
as OSM. This is consistent with previous work, which showed a similar
effect on the regulation of the rat TIMP-1 promoter, as well as an AP-1
site requirement for maximal responses (39), although AP-1 activation
was not identified. A role for AP-1 in TIMP-1 promoter regulation has also been implicated in F9 cells overexpressing AP-1 genes (40). Because OSM is more effective at stimulating acute phase protein production by HepG2 cells than other gp130 cytokines (26), and regulates low density lipoprotein receptors on these cells (16), we
suggest that c-Fos activation is an additional component of OSM
signaling which contributes to differential effects. Alternatively, OSM
or IL-6 may confer differences in Fos/Jun complexes through posttranslational modifications such as phosphorylation or alter the
composition of associated dimers of AP-1.
In addition to the AP-1 site (27, 40, 41), the proximal TIMP-1 promoter
also contains putative regulatory motifs including an Ets binding
sequence (27, 40) with homology to STAT elements, and SP1 elements
(27). Both the Ets site (putative STAT site) and SP1 site appeared to
contribute somewhat to basal transcription levels based on deletion
analysis (Fig. 2); however, binding of factors to these sites was not
altered upon OSM stimulation. Previous studies have shown that Ets can
cooperate with AP-1 in regulating transcription in other cells (42,
43). Others have shown that STATs can bind AP-1/Ets sequences of the
rat TIMP-1 in HepG2 cells (39), and human TIMP-1 promoter in astrocytes
(44) and that this site also contributes to transcription by OSM. Our
results in HepG2 cells did not detect binding of STATs to this sequence despite the presence of activated STAT-1 and STAT-3 (hSIE binding) in
the nuclear extracts and long exposures of gels. In addition, mutation
of the AP-1 site in the AP-1-Ets probe completely eliminated detection
of any OSM-inducible nuclear factors capable of binding this probe
(Fig. 5). Differences between the levels of STAT proteins expressed in
HepG2 cells and astrocytes could account for this result. The
prominence of AP-1 nuclear factor binding to this sequence that we
observe is consistent with previous studies (44), wherein AP-1 binding
appeared dramatically greater than STAT binding in EMSA assays with an
equivalent of an AP-1/Ets probe. We suggest that this abundance
of AP-1 binding reflects a physiological importance among other factors
participating in the regulation of this proximal TIMP-1 promoter.
Although deletion of the AP-1 motif (
59/
52) markedly affected OSM
activation, this AP-1 element was not sufficient for full OSM-responsiveness because deletion of sequences between +1 to +47 also
reduced transcription (Fig. 2). The TIMP-1 gene may utilize SP1 binding
sites and a putative initiator element downstream of the transcription
initiation site as described for other TATA-less promoters. We have
observed a putative pyrimidine-rich initiator element immediately 3' to
+1 of the TIMP-1 gene that may be necessary for the assembly of a
competent transcriptional machinery apparatus for transcription
initiation (45). Alternatively, additional responsive elements 3' to
the initiation start site may cooperate with the TIMP-1 AP-1 element at
59/
53 in response to OSM. Interestingly, Logan et al.
(40) have characterized a weak AP-1 binding site within +1/+47, which
may also be a target for OSM signals. We are currently examining this
aspect.
Although c-Fos and its participation in complex 2 could also be
stimulated by PMA (as assessed by supershifts; data not shown), PMA
alone was unable to up-regulate TIMP-1 promoter activity as assayed by
CAT reporter gene expression (Table II). In addition, we found that the
costimulation of HepG2 cells with IL-6 (STAT induction) and PMA (c-Fos
induction) could not appreciably up-regulate the CAT activity driven by
the
62 to +47 TIMP promoter. This also suggests that OSM induces an
additional signal that regulates TIMP-1 in this system. STAT proteins
may interact with SP1 (46) and Jun (47) proteins to cooperate in
elevating transcription in other systems; thus, a similar mechanism
could occur in HepG2 cells. Alternatively, OSM and PMA may differ
qualitatively in affecting posttranslational modification of AP-1
nuclear factors, such as phosphorylation, that influence the
transactivation potential of these transcription factors.
In summary, maximal expression of TIMP-1 by OSM (but not IL-6) may, at
least in part, be attributed to the induction of c-Fos and its complex
with AP-1 sites in the proximal promoter. This may also involve
posttranslational modifications such as phosphorylation to these
factors. Cooperation of nuclear proteins binding to SP1 and Ets DNA
elements is also needed for maximal expression, and OSM signaling may
recruit additional factors that interact with sequences downstream from
+1 to +47. Our results also support the existence of shared and
distinct signaling pathways by OSM and IL-6 in HepG2 cells.
We thank R. Martelli and T. Ferri for
secretarial assistance and D. Gojmerac for technical assistance.