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and IL-1 Via Enhancement of the Heat Shock Element Binding Activity of Heat Shock Transcription Factor 1
Departments of
* Internal Medicine (Section 4) and
Clinical Diagnosis Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan; and
Department of Internal Medicine, Kiyota Hospital, Sapporo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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has been
previously reported in rat skeletal myoblasts and mouse osteoblasts,
but not in human diploid fibroblasts. As for IL-1, its effect on
HSP47 has not been elucidated. In the present study, using human
embryonic lung fibroblast cells, we first disclosed that both TGF-
and IL-1 induced HSP47 synthesis. We then revealed that the binding
of the heat shock element (HSE) by heat shock transcription factor 1
(HSF1) was enhanced by both cytokines. We further demonstrated that
trimer formation of HSF1, which is essential for its binding to HSE,
was induced by these cytokines. The enhancement of HSP47 synthesis and
their trimer formation of HSF1 were augmented by using a combination of
both cytokines. Collectively, TGF- and IL-1 were found to induce
trimer formation of HSF1 which in turn bound to HSE of HSP47, resulting
in the enhancement of HSP47 expression. Thus, HSP47 could well be a
good candidate for molecular targeting in controlling tissue fibrosis,
given that both principal fibrinogenetic cytokines (TGF-, IL-1)
are commonly involved in its induction through HSF1
trimerization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recently discovered HSP, HSP47, unlike other HSPs, is a basic protein with an isoelectric point near 9 and a relatively small molecular mass (47 kDa) (5, 6, 7). HSP47 localizes in an endoplasmic reticulum (ER) with the ER retention signal sequence RDEL at its C terminus, where it binds to various types (I–V) of newly synthesized procollagens to transport them from the ER to the Golgi apparatus, facilitating their triple helix formation. During this process, procollagens dissociate from HSP47 as the pH inside ER decreases from 7.0 to 6.3 and are secreted at the cell surface; the freed HSP47 molecules are then cycled back to the ER (6, 8). Accordingly, HSP47 is considered to be a collagen-specific molecular chaperon expressed by collagen-secreting cells such as fibroblasts (6).
With most chronic immunological disturbances, including collagen diseases, autoimmune diseases, chronic infection, allergic reactions, etc., tissue fibrosis is inevitably associated and frequently become a determining factor of clinical course and prognosis of the diseases. In the fibrotic lesions, the expressions of HSP47 mRNA and collagen are reported to correlate (9). For example, increased coexpression of collagen and HSP47 at the fibrotic lesion of rat nephritis induced by antithymocyte serum has also been reported (10). In humans, a close correlation between the expression of HSP47 and that of collagen in the tissues of pulmonary fibrosis or progressive systemic sclerosis has also been demonstrated by immunohistochemical studies (11, 12).
Generally, fibrogenesis is effected by various immune cytokines forming
a complex network, with TGF- and IL-1 being the principal
cytokines (13, 14, 15, 16, 17). Both TGF- and IL-1 induce
synthesis of collagen of fibroblasts. TGF- is known to stimulate the
synthesis of mRNA encoding colligin, a rat homologue of HSP47 in
rat skeletal myoblasts and that in mouse osteoblasts (18, 19). However, the effect of TGF- on expression of HSP47 in
human diploid fibroblast is unknown, and as for IL-1, no such
investigation has been made.
The mechanism of HSP expression in heat stress involves a heat shock transcription factor (HSF) interacting with a highly conserved sequence in heat shock protein genes termed the heat shock element (HSE) (20, 21). By heat treatment, the HSF monomer is activated by conversion to a trimer that is capable of binding to the HSE. Binding of HSF to the HSE induces transcription of heat shock genes (20, 21).
In previous investigations, we reported that endogenous TNF augmented
HSP72 expression via enhancement of the HSE-binding activity of HSF in
mouse tumorigenic fibroblasts (L-M cells) (22, 23, 24),
whereas Yamamura et al. (19) demonstrated that in a
mouse osteoblast cell line, two distinct promoters differing from HSE
drove the HSP47 gene in response to TGF- treatment.
In the present study, we first used human embryonic lung fibroblast
(HEL) to confirm the enhancement of HSP47 expression by TGF- and
IL-1, and we then demonstrate that HSF1 is also involved in HSP47
induction by these cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HEL cells, a human embryonic lung fibroblast cell line, were cultured in DMEM supplemented with 10% FBS (Flow Laboratories, North Ryde, Australia) at 37°C under 5% CO2.
Cytokines
Recombinant human TGF- was purchased from AUSTRAL Biologicals
(San Ramon, CA) and activated in 5 mM HCl before use.
Recombinant human IL-1 was purchased from CISTRON Biotechnology
(Pine Brook, NJ) and dissolved in conditioned medium for use.
Antibodies
Anti-HSP47, anti-HSF1, and anti-HSF2 Abs and HSP47 cDNA were generously provided by Dr. K. Nagata (Department of Cell Biology, Chest Disease Research Institute, Kyoto University, Japan).
Metabolic labeling and immunoprecipitations
Cells (1 x 106) were grown in 35-mm culture dishes (Falcon 3025; BD Biosciences, Mountain View, CA) in DMEM supplemented with 10% FBS and treated under various conditions. Then the cells were washed twice with a methionine-free medium (ICN Biomedicals, Costa Mesa, CA) and labeled with 3.7 MBq/ml [35S]methionine (DuPont/NEN Research Products, Boston, MA) for 1 h. Cells were then washed with PBS and solubilized in lysis buffer (1% Nonidet P-40, 0.15 M NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 2 mM N'-ethylmaleimide, 2 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin) for 30 min at 4°C. After centrifuging lysates at 10,000 x g for 30 min, the supernatant was incubated with the anti-HSP47 mAb or anti-human IgG at 4°C for 18 h and subsequently added to protein A-Sepharose R CL-4B Sepharose beads (Pharmacia Biotech Norden, Sollentuna, Sweden) and allowed to shake at 4°C for 1 h. The immunoprecipitates were washed three times with lysis buffer and eluted by boiling the beads for 3 min in Laemmli’s SDS sample buffer. The samples were then analyzed by 10% SDS-PAGE and autoradiography. HSP47 synthesis levels were quantified by densitometry (dual-wave length TLC scanner CS-910; Shimazu, Kyoto, Japan) at 750 nm.
Gel mobility shift analysis
Cells (5 x 106) were grown in culture dishes (Falcon 3025; BD Biosciences) in DMEM supplemented with 10% FBS and treated under various conditions. Cells were harvested, centrifuged, and rapidly frozen in liquid N2. The frozen pellets were suspended in a buffer containing 20 mM HEPES (pH 7.9), 5% (v/v) glycerol, 50 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT and centrifuged at 100,000 x g for 5 min. The supernatants were frozen in liquid N2 and stored at -80°C. The protein concentration was estimated with an assay kit (Bio-Rad Laboratories, Hercules, CA). DNA-binding reaction mixtures contained, in a final volume of 10 µl, 10 µg protein of cell extracts, 1 µg poly(dI-dC) (Pharmacia Biotech Norden), 0.25 µg pUC19, 0.75 ng of an HSE -oligonucleotide (upper strand, 5'-GATCTCGGCTGGAATATTCCCGACCTGGCAGCCGA-3') end labeled with 32P (DuPont/NEN Research Products), and a buffer composed of 10 mM Tris (pH 7.8), 5% glycerol, 50 mM NaCl, 1 mM EDTA, and 0.5 mM DTT. For competition experiments, unlabeled competitor HSE was added to the reaction mixture to a 100-fold molar excess over the labeled probe. Reactions were incubated for 30 min at room temperature and analyzed on 4% polyacrylamide gels. The gels were dried and autoradiographed. The Ab-binding reaction mixtures for supershift analysis contained, in a final volume of 10 µl, 10 µg protein of cell extracts, 1/10 diluted anti-HSF 1 or anti-HSF 2 Ab, and PBS. The mixtures were incubated on ice for 15 min before the DNA-binding reaction.
Western blot analysis
Cells (1 x 106) were grown in culture dishes in DMEM
supplemented with 10% FBS and treated under various conditions. Cells
were then washed with PBS and solubilized in lysis buffer for 30 min at
4°C. After lysates were centrifuged at 10,000 x g
for 30 min, the supernatant was used as whole cell extract. To prepare
subcellular fraction, cells were lysed on ice in hypotonic buffer (20
mM Tris-HCl (pH 8.0), 0.5 mM CaCl2, 25 mM NaCl, 2
mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin), followed by
Dounce homogenization (40 strokes with a loose-fitting pestle). The
extract was then centrifuged at 10,000 x g for 10 min
at 4°C, and the supernatant solution was used as the cytoplasmic
extract. The pelleted material was washed with hypotonic buffer and
resuspended in lysis buffer. After sonication, the extract was
centrifuged at 15,000 x g for 10 min at 4°C, the
supernatant solution representing the soluble nuclear fraction was
removed. Then each extract was subjected to SDS-PAGE using 7.5%
polyacrylamide gels. After electrophoresis, the fractionated protein
was transferred to a nylon membrane (Schleicher & Schuell, Dassel,
Germany). The membrane was incubated for 60 min at 37°C with a 1/500
dilution of the HSF1 Ab or a 1/2000 dilution of -actin Ab
(Sigma-Aldrich, St. Louis, MO). The immunoblot was developed using a
1/2000 dilution of HRP-labeled donkey anti rabbit IgG secondary Abs
(Amersham, Buckinghamshire, U.K.) and an enhanced by the ECL
system (Amersham).
Northern blot analysis
Total RNA was isolated with the guanidinium isothiocyanate method from HEL cells (1 x 107). The RNA (5 µg/lane) was fractionated in a 1.0% formaldehyde agarose gel and then transferred to a nylon filter membrane. The membrane was hybridized with a 32P-labeled HSP47 cDNA probe or GAPDH cDNA for 24 h at 42°C and autoradiographed. The bands were quantified using densitometry at 750 nm.
Cross-linking assay
Whole cell extracts were prepared by quick-freezing pellets of
PBS-washed cells in liquid N2. The pulverized
pellet material was thawed and resuspended in 
2 packed-cell volume
of buffer A (HEPES (pH 7.9), 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 2
mg/ml leupeptin, 2 mg/ml pepstatin, and 25% glycerol). After 10 min
incubation on ice, the extract was centrifuged at 10,000 x
g. For the cross-linking assay experiment shown, 15-µl
aliquots of the supernatant were incubated for 10 min at room
temperature with 2 mM glutaraldehyde. Reactions were stopped by the
addition of 1 µl of 2 M lysine, and aliquots were electrophoresed on
a 5% polyacrylamide SDS-PAGE gel. Immunoblotting was performed as
described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, or a
combination of the two
We first examined the effects of TGF- (0, 0.1, 1.0, 5.0, and
10.0 ng/ml), IL-1 (0, 0.1, 1.0, 5.0, and 10.0 ng/ml) and a
combination of the two on HSP47 protein expression in HEL cells, using
metabolic labeling and immunoprecipitation methods. After 24 h of
treatment, expression of HSP 47 was dose dependently induced by TGF-
alone and IL-1 alone, reaching a plateau at a dose of 5.0 ng/ml
(Fig. 1
, a and b).
We therefore conducted the following experiments by using 5.0 ng/ml of
each cytokine when used for the noncombination experiments. However,
when we examined the combined effect of these cytokines, we used a
concentration of 1.0 ng/ml for each cytokines, avoiding the plateau
effect found with 5.0 ng/ml. As shown in Fig. 1c, this
1.0-ng/ml combination did in fact have an apparent augmenting.
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and
IL-1
To study the time course of HSP47 induction, we used methods
similar to those in Fig. 1
. With both TGF- (5.0 ng/ml) and IL-1
(5.0 ng/ml), HSP47 began to increase after 6 h and peaked at
24 h followed by a slight decrement. (Fig. 2).
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or IL-1
We then examined the effects of TGF- or IL-1 on expression
of HSP47 mRNA in HEL cells by Northern blotting. The intensity of the
bands corresponding to HSP47 mRNA began to increase after 3 h of
treatment and reached a plateau at 12 h (Fig. 3), which precedes the peaking time (24
h) of HSP47 induction (Fig. 2).
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To determine whether the HSF binding to HSE is mediated in the
activation of HSP47 gene transcription by TGF- or IL-1, we
performed a gel-mobility shift analysis using a
5'-32P-labeled synthetic oligonucleotide
containing the human HSE sequence (Fig. 4). The intensity of the band
corresponding to the HSE-HSF complex was substantially greater in HEL
cells treated for 6 h with 5.0 ng/ml TGF- or IL-1 than in
that of untreated cells. Then, to elucidate which type of HSFs (HSF1 or
HSF2) is involved in enhancement of HSP47 gene expression, we conducted
a supershift assay using anti-HSF1 and anti-HSF2 Abs. The band
intensity of the HSE-HSF Ab complex showed an apparent dose-dependent
increase in the fractions treated with the anti-HSF1 Ab but not in
those with the anti-HSF2 Ab.
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and IL-1 on HSF1 expression
To examine the effect of TGF- or IL-1 on HSF1 expression, we
treated HEL cells for 6 h with 5.0 ng/ml TGF- or IL-1 and
analyzed HSF1 by Western blotting. As shown in Fig. 5, the expression level of HSF1 in whole
cell extracts was not altered but became lower in cytosolic fraction
and higher in nuclear fraction upon cytokine stimulation.
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and IL-1, and their
combination
To explore monomer to trimer conversion of HSF1 by TGF-,
IL-1, or their combination, we cross-linked whole cell extracts with
glutaraldehyde and subjected them to Western analysis using the
anti-HSF1 Ab. Trimer formation became evident after 1 h,
peaking at 3 h for both TGF- and IL-1 treatments with a
reciprocal change of monomers (Fig. 6a). When the cytokines were
combined, a more prominent monomer-trimer conversion was observed (Fig. 6b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1, principal
fibrosis-causing cytokines. That is to say, HSP47 expression in HEL was
maximally enhanced by stimulation with 5.0 ng/ml TGF- or IL-1 to
almost the same degree, 3 times that in nonstimulated cells. This
concentration, 5.0 ng/ml for both TGF- and IL-1, is fairly close
to those detected in patients with tissue fibrosis. For example, in
patients with liver cirrhosis and with pulmonary fibrosis, plasma
concentrations of TGF- and IL-1 were reported to be 3.7 ±
2.1 and 0.1–2.0 ng/ml, respectively (25, 26).
In this regard, 1.0 ng/ml of each these two cytokines, which is within
the range of actual plasma concentrations of the above mentioned
pathological conditions, synergistically and substantially enhanced
HSP47 synthesis when they were combined (Fig. 1c). In other
words, the synergistic effect suggests that TGF- and IL-1 may
cooperatively and readily evoke fibrosis because they are quite often
simultaneously secreted from immune competent cells in inflammatory
lesions, even if each concentration is not high enough for either by
itself to induce HSP47 (25, 26). We then proved that
induction of HSP47 was brought about by the binding of HSF1 to HSE of
the HSP47 gene.
Possible mechanisms by which the HSE-binding activity of HSF1 could be
enhanced by TGF- and IL-1 include the following: 1) TGF- or
IL-1 may enhance HSF1 synthesis; 2) TGF- or IL-1 may induce a
conformational change (trimer formation) of HSF1 as seen with heat
treatment. The first possibility is highly doubtful because no
difference was demonstrated in the HSF1 levels of whole cell extracts
between TGF-- or IL-1-treated cells and untreated cells by
Western analysis. Furthermore, the fact that most HSF1 shifted from
cytoplasmic fraction to nuclear fraction by cytokine stimulation
favored the second possibility because a similar phenomenon is observed
with heat treatment (20, 21) To examine the second
possibility, we performed a cross-linking assay using the anti-HSF1
Ab and found that HSF1 is converted from a monomeric form to a trimeric
form. The time course analysis in response to treatment with TGF- or
IL-1 showed an increment of HSF1 trimer formation, which peaked at
3 h and decreased at 6 h. Thus, HSF1 is initially converted
from a monomer to a trimer with TGF- or IL-1 treatment, then
synthesis of HSP47 mRNA starts by interaction between HSF1 and HSE. The
amount of HSP47 mRNA gradually increased by accumulation of newly
synthesized HSP47 mRNA. Incidentally, the concept of a synergistic
effect of cytokine combination on trimer formation (Fig. 1c)
is compatible with the results of HSP47 induction (Fig. 6b).
An apparent inconsistency regarding the effect of various cytokine
concentrations that evoked the synergistic effect in Fig. 1c
(1 ng/ml for each cytokines) and Fig. 6b (5 ng/ml for each
cytokines) may be interpreted by assuming that there is a maximum
amount of active HSF1 (trimer form) that can interact with HSE to
induce HSP47 mRNA because the level of HSP47 induced by combined 5
ng/ml TGF- and 5 ng/ml IL-1 was almost the same as that induced
by each cytokine of the same dose. Nonetheless, the fact that the
effect and TGF- or IL-1 was synergistic but not antagonistic
suggests the possibility that there may be dissimilar mechanisms
(signal routes) to induce HSF1 trimer for each cytokine.
The mechanism of trimer formation of HSF1 hitherto described is
hyperphosphorylation of serine/threonine residues of HSF1 by heat
treatment to undergo conformational change (20, 21).
Hyperphosphorylation prolongs the effective half-life of the HSF1
trimer, which facilitates HSF1 binding activity (27). This
mechanism is most likely true for IL-1 because phosphorylation of
HSF1 was indeed proved to occur in HEL cells treated with this cytokine
(data not shown). These results were consistent with the previous
findings by Schett et al. (28) that IL-1 induced
activation of HSF1 with phosphorylation and augmented HSP70 expression
in synovial tissue from rheumatoid arthritis.
Further, stress-activated protein kinase/c-Jun N-terminal kinase, a mitogen-activated protein kinase that phosphorylates HSF1 after heat stress, is also activated by IL-1 (29, 30), suggesting the involvement of stress-activated protein kinase/c-Jun N-terminal kinase in the phosphorylation of HSF1.
In contrast, phosphorylation of HSF1 was not observed in the cells
treated with TGF-. In this context, the previous assumption of
distinct mechanisms for TGF- and IL-1 may be plausible. To
explore an alternative mechanism, we investigated the interaction of
HSF1 with Smad3, Smad4, and TAK1, well-known mediators of TGF-
signaling (31, 32), by a cross-linking assay; however,
none of these transducers bound to HSF1 (data not shown). Elucidation
of the mechanism for trimer formation of HSF1 by TGF- treatment is
needed in the future.
HSF1 is a rather ubiquitous transcription factor for various HSPs other
than HSP47 (20, 21, 28). Therefore, it was assumed that
TGF- may induce, in addition to HSP47, other types of HSP. However,
when we examined the expression of HSP72 in HEL cells treated with
TGF-, no increment was observed, suggesting some counteracting
factors against the induction of HSP72. Further elucidation is
needed.
Nevertheless, taken together the present results suggest that both
TGF- and IL-1 activate HSF1, which in turn stimulates the
transcription of HSP47 mRNA, resulting in increased expression of HSP47
protein.
Tissue fibrosis, as the result of various chronic immunological
disturbances, modifies the outcome of disorders (9, 13, 14, 15). Therefore, development of the modalities to inhibit
fibrosis has been long desired. In this regard, the present results
suggest that HSP47, a molecule commonly induced by both cytokines, may
be an appropriate candidate target to be inhibited rather than the
molecules unique for each signal transduced by TGF- or IL-1.
Further, because HSP47 acts as a universal molecular chaperone for all
types of collagen (6), inhibition of HSP47 is thought to
interfere with the secretion of various collagens simultaneously. In
fact, we have recently confirmed that transfection of HEL cells with
HSP47-specific ribozyme brought about almost complete inhibition of
collagen secretion from these cells. Therefore, a modality to attenuate
the expression of HSP47 could be a promising approach to preventing
fibrosis.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HSP, heat shock protein; HSF, heat shock factor; HSE, heat shock element; ER, endoplasmic reticulum; HEL, human embryonic lung fibroblast.
Received for publication November 21, 2001. Accepted for publication March 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(I) and 1(III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. J. Clin. Invest. 94:2481.
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1 in patients with hepatocellular carcinoma: comparison with chronic liver disease. Cancer 73:2275.[Medline]
and tumor necrosis factor- release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure. J. Immunol. 150:4188.[Abstract]
signalling from cell membrane to nucleus through SMAD proteins. Nature 390:456.
signal transduction. Science 270:2008.This article has been cited by other articles:
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