Pulmonary Center and Department of Biochemistry, Boston University School of Medicine, Boston 02118-2394; and Boston Veterans Affairs Medical Center, Boston, Massachusetts 02130
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ABSTRACT |
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Transforming
growth factor- (TGF-
) stimulates
1(I) collagen mRNA synthesis in
human lung fibroblasts through a mechanism that is partially sensitive
to cycloheximide and that may involve synthesis of connective tissue
growth factor (CTGF). Northern blot analyses indicate that TGF-
stimulates time- and dose-dependent increases in CTGF mRNA. In
TGF-
-stimulated fibroblasts, maximal levels of CTGF mRNA (3.7-fold
above baseline) occur at 6 h. The TGF-
-stimulated increase in CTGF
mRNA was not blocked by cycloheximide. Nuclear run-on analysis
indicates that TGF-
increases the CTGF transcription rate. The
TGF-
-stimulated increases in CTGF transcription and steady-state
levels of CTGF mRNA are attenuated in prostaglandin E2
(PGE2)-treated fibroblasts.
PGE2 fails to attenuate luciferase activity induced by TGF-
in fibroblasts transfected with the TGF-
-responsive luciferase reporter construct p3TP-LUX. In amino acid-deprived fibroblasts, PGE2
and insulin regulate
1(I)
collagen mRNA levels without affecting CTGF mRNA levels. The data
suggest that the regulation of
1(I) collagen mRNA levels by
TGF-
and PGE2 may function
through both CTGF-dependent and CTGF-independent mechanisms.
transforming growth factor-; insulin; amino acid deficiency; type I collagen; human lung fibroblast
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INTRODUCTION |
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IN HUMAN LUNG FIBROBLASTS, transforming growth
factor- (TGF-
) stimulates an increase in
1(I) collagen mRNA (10) that is
attenuated by prostaglandin E2
(PGE2) and forskolin (9, 11).
This rise in
1(I) collagen mRNA
is due to increased transcription and increased mRNA stability (50,
53). Investigations into the mechanism by which TGF-
stimulates
collagen synthesis indicate that TGF-
induces the synthesis of an
autocrine factor that is directly responsible for activating
1(I) collagen transcription (41). This putative autocrine factor has been tentatively identified as
connective tissue growth factor (CTGF) (27, 51).
CTGF, a member of the CCN (CTGF, Cyr61/Cef10, Nov) family, is a
cysteine-rich, heparin-binding, 349-amino acid polypeptide (2). Other
members of the CCN family include the serum-induced immediate-early
genes cyr61 and
Fisp12/BIGM2
(4, 48), a v-src-induced peptide
(Cef10) (49), a putative protooncogene
(nov) (30), and a potential tumor
suppresser (Elm1) (23). The high degree of amino acid homology
(50-90%) among CCN members is distinguished by conservation of 38 cysteine residues. In addition, all CCN proteins possess a secretory
signal peptide and four distinct protein modules: an insulin-like
growth factor binding domain, a von Willebrand factor type C repeat, a
thrombospondin type 1 repeat, and a COOH-terminal module (1). TGF-
but not epidermal growth factor, fibroblast growth factor, or
platelet-derived growth factor stimulates CTGF transcription in NRK
fibroblasts (35). In adult mammals, CTGF is expressed in high levels
during wound repair and at sites of connective tissue formation in a
variety of fibrotic disorders (15, 25, 26, 29, 46). Recombinant CTGF
stimulates fibroblast proliferation and extracellular matrix protein
synthesis (14, 35).
In these studies, we examined the effects and interactions of TGF-
and PGE2 on the steady-state
levels of CTGF and
1(I) collagen mRNA. We report that PGE2
inhibits transcription of the CTGF gene. TGF-
and
PGE2 appear to affect steady-state
levels of
1(I) collagen mRNA
via CTGF-dependent and -independent mechanisms.
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METHODS |
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Tissue culture. Human embryonic lung fibroblasts (IMR-90, Institute for Medical Research, Camden, NJ) were grown in Dulbecco's modified Eagle's medium supplemented with 0.37 g sodium bicarbonate/100 ml, 10% (vol/vol) fetal bovine serum, 100 U penicillin/ml, 10 µg streptomycin/ml, 0.1 mM pyruvate, and 0.1 mM nonessential amino acids. After confluence, the serum content of the medium was reduced to 0.4% fetal bovine serum for 24 h. Cell numbers were determined by triplicate cell counts with an electronic particle counter (Coulter Counter ZM).
RNA isolation and Northern analysis.
Total cellular RNA was isolated with the single-step method employing
guanidine thiocyanate-phenol-chloroform extraction as described by
Chomczynski and Sacchi (6). RNA was quantified by absorbance at 260 nm.
Purity was determined by absorbance at 280 and 310 nm. RNA (10 µg)
was electrophoresed through a 1% agarose-6% formaldehyde gel and
transferred to a nitrocellulose filter. RNA loading was assessed by
ethidium bromide staining of ribosomal bands and by cohybridization
with Gs, a constitutively expressed mRNA that codes for a GTP binding
protein (31), or glyceraldehyde-3-phosphate dehydrogenase (GADPH).
Hybridization was performed with 0.5-1.0 × 106
counts · min1 · lane
1-labeled
probe (specific activity, 4-10 × 108
counts · min
1 · µg
1),
and the filter was washed according to methods previously described (13). The filter was exposed to X-ray film for autoradiography at
several different times to ensure that the bands could be quantified by
densitometry within the linear range. The
1(I) collagen probe came from a
rat
1(I) collagen cDNA that
specifically binds human
1(I)
collagen mRNA (16).
Nuclear run-on assay. Confluent, quiescent IMR-90 fibroblast cultures in 150-mm dishes were washed twice with Puck's saline and scraped into a Nonidet P-40 lysis buffer. After two low-speed spins, the pellet was reconstituted in a glycerol buffer. In vitro labeling of nascent RNA and hybridization with cDNA immobilized on nitrocellulose filters were performed according to the methods previously reported (19, 21). No hybridization occurred to filters containing plasmids without inserts.
Western blotting. PAGE was performed
under reducing conditions with 7.5% polyacrylamide minigels as
previously described (33). Samples (100 µg) for SDS-PAGE and Western
blotting were prepared from the cell layer of quiescent confluent
IMR-90 fibroblasts grown in 35-cm tissue culture dishes. The cells were
stimulated with 1 ng/ml of TGF- and harvested after 6 h. The cell
layer was dissolved in radioimmunoprecipitation assay buffer at 4°C and centrifuged 14,000 g for 10 min.
Proteins were transferred to nitrocellulose membranes, blocked for 2 h
at room temperature with 10% evaporated milk in phosphate-buffered
saline with 0.1% Tween (33), and incubated with a 1:1,000 dilution of
rabbit anti-CTGF-(247
260) peptide antiserum overnight at 4°C.
Anti-CTGF antibody was generously provided by David R. Brigstock
(Wexner Institute for Pediatric Research, Columbus, OH).
Statistics. Student's t-test was used for means of unequal size. Probability values < 0.05 were considered significant.
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RESULTS |
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In TGF--stimulated fibroblasts, increases in
1(I) collagen mRNA were
detected after ~4-6 h, with maximal
1(I) collagen mRNA levels
detected after 18 h (12). The time-course studies of TGF-
-stimulated
increases in CTGF mRNA indicated that increases in CTGF mRNA preceded
increases in
1(I) collagen
mRNA. Increased CTGF mRNA appears after 2 h, with a peak response
(3.7-fold above baseline) appearing 6 h after TGF-
stimulation
(P < 0.05;
n = 3 experiments; Fig.
1A).
In contrast, insulin induces an increase in
1(I) collagen mRNA (18) without
increasing CTGF mRNA levels (Fig.
1B). It appears that increases in
1(I) collagen mRNA may occur
through CTGF-dependent and CTGF-independent pathways.
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TGF- (at concentrations of 0.5 ng/ml or greater) stimulates an
increase in
1(I) collagen mRNA
and protein (12). Dose-response studies indicate that comparable
concentrations of TGF-
are required to stimulate increases in CTGF
mRNA levels (Fig. 2). The
TGF-
-stimulated increases in
1(I) collagen mRNA are composed
of cycloheximide-sensitive and cycloheximide-insensitive components (37). As expected of a TGF-
-induced immediate-early gene, increases in CTGF mRNA were insensitive to cycloheximide (5 µM; Fig.
3A). At
4 h, cycloheximide alone did not affect CTGF mRNA levels or the
TGF-
-induced increase in CTGF mRNA (Fig.
3B).
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Fine and Goldstein (8) have previously shown that
activation of protein kinase A (PKA) by
PGE2 attenuates the
TGF--stimulated increase in
1(I) collagen mRNA and protein.
To demonstrate that PGE2 blocks
the TGF-
-stimulated increase in CTGF mRNA levels, we incubated
TGF-
-stimulated fibroblasts with varying concentrations of
PGE2 (Fig.
4A). The
TGF-
-stimulated increase in CTGF mRNA was reduced in fibroblasts
treated with 100 nM PGE2 (73%
reduction; P < 0.05;
n = 3 experiments).
PGE2 alone did not alter CTGF mRNA levels.
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CTGF is found associated with the cell layer as well as released to the
medium (52). We assayed the fibroblast cell layer for CTGF by Western
blotting (Fig. 4B). With the use of
an antibody directed against amino acids 247-260 of CTGF, a single
protein with an apparent molecular mass of 38 kDa was
detected in the cell layer from TGF--stimulated fibroblasts. A
low-intensity band was detected in control fibroblasts and fibroblasts
stimulated with both PGE2 and
TGF-
.
PGE2 inhibits TGF--stimulated
increases in
1(I) collagen mRNA
through increased adenylate cyclase activity (8). In fibroblasts pretreated with the adenylate cyclase activator forskolin or
PGE2, TGF-
failed to increase
CTGF mRNA levels (Fig. 5). Furthermore, PGE2 and forskolin did not inhibit
the TGF-
-stimulated increase in endogenous TGF-
type I receptor
mRNA.
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Recent advances in the signal transduction mechanism of TGF-
establish that phosphorylation of Smad2 and Smad3 precedes
translocation to the nucleus and activation of gene transcription (24,
39, 43, 44, 55). We used the luciferase reporter construct p3TP-LUX to
further assess the repression of
PGE2 on TGF-
signaling.
p3TP-LUX was engineered for maximal TGF-
responsiveness with three
31-nucleotide activator protein-1 sites concatenated to a region
(
636 to
740 bp) of the plasminogen activator inhibitor
promoter (54). In fibroblasts transfected with p3TP-LUX, TGF-
stimulated a 17.8-fold increase in luciferase activity above that in
control fibroblasts (P < 0.05;
n = 3 experiments; Fig.
6A).
Preincubation with PGE2 did not
affect the TGF-
-induced increase in luciferase activity. When
adenylate cyclase was activated directly by forskolin, the results were
comparable. In forskolin-treated fibroblasts, the TGF-
-stimulated
luciferase activity was not significantly different from the luciferase
activity observed in TGF-
-stimulated control fibroblasts
(Fig. 6B).
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Nuclear transcriptional run-on assays were performed to determine the
mechanism resulting in increased CTGF mRNA. Nuclei were isolated 5 h
after TGF- stimulation in the presence and absence of
PGE2. The rates of transcription
for the CTGF,
1(I) collagen, TGF-
type I receptor, and GADPH genes were determined (Fig.
7). We found that TGF-
stimulates
transcription of the CTGF gene and that this increase was blocked by
PGE2. At this early time point, we
did not detect a change in the rate of transcription of
1(I) collagen, TGF-
type I
receptor, or GADPH genes with either TGF-
or
PGE2.
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Finally, to demonstrate that the
PGE2-mediated reduction of
1(I) collagen mRNA is mediated
by mechanisms other than those involving CTGF, we used fibroblasts
cultured in amino acid-deficient medium. In amino acid-deprived
fibroblasts, steady-state levels of
1(I) collagen mRNA were
decreased compared with those in fibroblasts maintained in complete
medium (38). Furthermore, these fibroblasts responded to insulin,
resulting in increased levels of
1(I) collagen mRNA. We found
that PGE2 further decreased
1(I) collagen mRNA in
fibroblasts incubated in amino acid-deficient medium (Fig. 8). CTGF mRNA was not affected by
PGE2 or insulin in amino
acid-deprived fibroblasts.
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DISCUSSION |
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TGF- induces the transcription of several immediate-early genes
including fibronectin (28), JunB (5, 22, 32, 42), and CTGF (20, 35). In
addition, TGF-
treatment of fibroblasts induces an increase in
extracellular matrix components, especially type I collagen (28).
Depending on the cell type, TGF-
increases
1(I) collagen mRNA levels by
increasing the transcription rate or stability of the transcript (50,
53). Our findings suggest that increases in CTGF mRNA precede increases
in
1(I) collagen mRNA and that
comparable doses of TGF-
stimulate synthesis of CTGF and
1(I) collagen. This is
consistent with the hypothesis that CTGF mediates the
TGF-
-stimulated increase in
1(I) collagen mRNA.
The regulation of 1(I) collagen
expression by TGF-
and other effectors is complex (37). Our data
indicate that a portion of the TGF-
-stimulated increase in
1(I) collagen mRNA expression is not dependent on protein synthesis (independent of CTGF production) (17). In addition, other effectors can increase
1(I) collagen mRNA levels
through CTGF-independent mechanisms. For example, insulin increases
1(I) collagen mRNA levels
without affecting CTGF expression in fibroblasts incubated in amino
acid-deficient medium.
CTGF is present in abundance during wound healing in many tissues
including the lung (15, 25, 29, 40, 46). Recombinant CTGF increases
steady-state levels of 1(I)
collagen mRNA (14). Anti-CTGF antibodies or antisense oligonucleotides
directed against CTGF mRNA block TGF-
-mediated anchorage-independent
growth (35). In addition, CTGF is subject to proteolytic cleavage to
biologically active fragments (3, 52). Full-length CTGF binds to the
extracellular matrix and may act as a matrix signaling protein (14,
52). CTGF may function in a similar manner to Cyr61, another member of
the CCN family (34, 45). Its function may involve cellular activation
as well as interaction with other matrix-associated proteins.
TGF- increases the steady-state level of CTGF mRNA by activating
transcription as assessed by nuclear run-on analysis at 5 h. In
contrast, the steady-state level of
1(I) collagen mRNA is
unaffected by TGF-
at that time. In addition, Northern blot analysis
of TGF-
-stimulated fibroblasts demonstrates that CTGF mRNA levels
are not affected by protein synthesis inhibition with cycloheximide,
whereas
1(I) collagen mRNA
levels are decreased (17). These results indicate that a portion of the
TGF-
-induced increases in
1(I) collagen mRNA may be
mediated by CTGF by posttranscriptional mechanisms.
PGE2 inhibits the TGF--induced
increases in
1(I) collagen mRNA
levels (8). PGE2 also inhibits the
TGF-
-stimulated increase in CTGF transcription as assessed by
nuclear run-on analysis. In contrast, the upregulation of TGF-
type
I receptor mRNA by TGF-
is not affected by treatment with
PGE2. Furthermore,
PGE2 fails to inhibit
TGF-
-induced stimulation of the reporter construct p3TP-LUX. Our
data indicate that PGE2 inhibits
CTGF transcription, perhaps by increasing adenylate cyclase activity.
Kothapalli et al. (36) showed that forskolin and cholera toxin blocked
increases in CTGF mRNA presumably by increasing adenylate cyclase
activity. It is unlikely that the
PGE2-induced decrease in the CTGF
mRNA level is due to phosphorylation of the TGF-
receptor or Smad proteins because PGE2 did not
interfere with TGF-
-stimulated transcription of the TGF-
type I
receptor and activity of the p3TP-LUX reporter. Alternatively, high
levels of cAMP activate PKA or PKA-activated kinases that may
phosphorylate transcription factors to inhibit CTGF transcription.
Transcriptional coactivators CBP/p300 interact with activator protein-1
sites through a complex with Smad proteins (Smad3 and/or Smad2 bound to
Smad4) (7, 47, 56). CTGF transcription may be inhibited by a mechanism that involves PKA phosphorylation of transcriptional coactivators.
Fibroblasts incubated in amino acid-deficient medium downregulate
1(I) collagen mRNA levels (38).
PGE2 further downregulates
1(I) collagen mRNA under these
culture conditions without affecting the steady-state levels of CTGF
mRNA. As noted, insulin increases
1(I) collagen mRNA but has no
effect on CTGF mRNA in amino acid-deprived fibroblasts. Our data with
amino acid-deficient medium suggest that the
PGE2-induced downregulation and
insulin-induced upregulation of
1(I) collagen is independent of
CTGF mRNA expression.
In summary, 1(I) collagen mRNA
levels are regulated by multiple mechanisms in TGF-
-stimulated
fibroblasts. PGE2 inhibits the
TGF-
-stimulated increases in both CTGF and
1(I) collagen mRNA levels. In
contrast, PGE2 decreases
1(I) collagen mRNA levels in
amino acid-deprived fibroblasts in the presence of sustained CTGF mRNA
levels. CTGF expression does not appear to account for the
TGF-
-stimulated, cycloheximide-sensitive increase in
1(I) collagen mRNA levels. In
addition,
1(I) collagen mRNA
levels may be regulated posttranscriptionally through CTGF-independent mechanisms. Our results suggest that the regulation of
1(I) collagen mRNA levels by
TGF-
and PGE2 may function
through both CTGF-dependent and CTGF-independent mechanisms and that
the antifibrotic effects of PGE2
involve inhibition of CTGF transcription.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant P50-HL-56386 and the Veterans Affairs Merit Review Research Program.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. A. Ricupero, The Pulmonary Center, Boston Univ. School of Medicine, 715 Albany St., Boston, MA 02118-2394 (E-mail: ricupero{at}bu.edu).
Received 3 December 1998; accepted in final form 19 July 1999.
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