Divisions of Pneumology and Cardiovascular Research, Departments of Research and Internal Medicine, University Hospital Basel, CH-4031 Basel, Switzerland
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ABSTRACT |
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Increased collagen and extracellular matrix
(ECM) deposition within the lung is a characteristic feature of lung
fibrosis. Transforming growth factor (TGF)- isoforms play a pivotal
role in the production of collagen and ECM. In this study, we
investigated the effects of TGF-
1 and TGF-
3 on the main processes
controlling ECM deposition using primary human lung fibroblasts. We
analyzed 1) collagen metabolism by
[3H]proline
incorporation, 2) matrix
metalloproteinase (MMP) expression by substrate gel zymography, and
3) tissue inhibitor of
metalloproteinases (TIMP) expression by Western blot analysis. TGF-
1
and TGF-
3 increased the percentage of secreted collagens in
supernatants of primary fibroblasts from 8.0 ± 1.2 (control) to
23.6 ± 4.6 and 22.3 ± 1.3%, respectively. The collagen
percentage in deposited ECM was increased from 5.8 ± 0.3 (control) to 9.0 ± 0.5 and 8.8 ± 0.5% by TGF-
1 and
TGF-
3, respectively. Secretion of MMP-1 (interstitial collagenase)
by fibroblasts was reduced by both TGF-
isoforms, whereas secretion
of MMP-2 (gelatinase A) was unaffected by either of the two isoforms.
Both TGF-
isoforms increased TIMP-1 protein expression, whereas
TIMP-2 protein was decreased. We thus conclude that TGF-
1 and
TGF-
3 are equally potent in increasing ECM deposition. Their
fibrotic effect in lung fibroblasts results from
1) an increase in the secretion and
deposition of total ECM and collagens,
2) a decrease in MMP-1 secretion,
and 3) an increase of TIMP-1 expression.
lung fibrosis; collagens; matrix metalloproteinase; tissue inhibitor of metalloproteinases
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INTRODUCTION |
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THE COMPOSITION OF THE EXTRACELLULAR MATRIX (ECM) of the human lung is critical in the maintenance of normal lung functions, ventilation, and gas exchange. The lung fibroblast is the main producer of pulmonary ECM, which consists of collagens, elastins, and proteoglycans (4, 13-15). The pulmonary ECM, classically thought to be inert, is subjected to a continuous turnover of >10% of the total ECM per day (14, 30, 32). Thus a dynamic equilibrium between synthesis and degradation of the pulmonary ECM is maintaining the physiological balance. This balance is tightly controlled by three regulatory mechanisms: 1) de novo synthesis and deposition of ECM components such as collagens, 2) proteolytic degradation of existing ECM by matrix metalloproteinases (MMPs), and 3) inhibition of MMP activity by specific endogenous antiproteases, the tissue inhibitors of metalloproteinases (TIMPs) (13-15, 30, 34).
During the pathogenesis of lung fibrosis, the homeostasis deteriorates,
resulting in a net increase in deposited ECM and collagen content of
the lung. This altered ECM is responsible for the severe loss of lung
function associated with lung fibrosis (13, 14, 32). During the
pathogenesis of lung fibrosis, local overexpression of cytokines and/or
growth factors stimulates resident lung fibroblasts to synthesize
increased amounts of ECM. In this respect, considerable evidence
suggests that transforming growth factor (TGF)- isoforms are key
mediators responsible for the ECM changes seen in lung fibrosis (4, 5,
13, 21, 22, 28, 30, 32).
The TGF- isoforms belong to a superfamily of polypeptides including
1) TGF-
isoforms themselves,
2) activins, and
3) a complex third subfamily
consisting of morphogenic proteins (bone morphogenic proteins, nodal,
Xenopus Vg-1,
Drosophila dpp, and screw). Three distinct TGF-
isoforms, TGF-
1, TGF-
2, and TGF-
3, are
expressed in mammalian species, all occurring as homodimeric proteins
of 25 kDa each (5, 6, 12, 21). On most cell types studied, three
classes of receptors, type I (T
RI), type II (T
RII), and type III
(T
RIII), that specifically recognize TGF-
isoforms have been
characterized (6, 46). Biological responses to TGF-
isoforms occur
when TGF-
isoforms bind to T
RII, which induces a
heterooligomerization between T
RI and T
RII. On complex formation,
T
RI is phosphorylated by the constitutively active kinase domain of
T
RII, and downstream signaling is initiated (6, 46). Interestingly,
specificity of the actions of TGF-
isoforms in different cell types
seems to be determined by the expression and/or activation of
intracellular signaling molecules as well as by distinct expression of
the T
R subtypes (6, 9, 46). Studies investigating the biological
effects of different TGF-
isoforms demonstrated a considerable
overlap of their activities. However, evidence exists that TGF-
1 and
TGF-
3 have distinct effects and/or potencies in vitro (9, 23, 31,
33).
In the normal human lung, TGF- isoforms are frequently expressed in
bronchiolar epithelial cells and interstitial fibroblasts, with
TGF-
1 being the most abundant isoform in the lung (3, 10). During
the development of lung fibrosis, altered expression patterns of
TGF-
isoforms are found compared with those in control lungs.
Overexpression of TGF-
1 in macrophages and mesenchymal, endothelial,
and mesothelial cells of the lung can be detected in fibrotic lungs,
but the expression level of TGF-
3 was unchanged (11). However, the
mechanisms leading to increased ECM deposition by TGF-
isoforms in
this disease state have not fully been addressed.
The aim of this study was to assess and compare the effects of two
TGF- isoforms, TGF-
1 and TGF-
3, on ECM metabolism in primary
human lung fibroblasts. We analyzed the three major regulatory mechanisms that control ECM turnover, which are
1) secretion and deposition of
collagens, 2) expression of MMP-1
and MMP-2, and 3) expression of
TIMP-1 and TIMP-2. We demonstrate that both isoforms are equally potent
in increasing the ECM accumulation in primary human lung fibroblasts.
In this respect, TGF-
1 and TGF-
3 led to an increase in secretion
and deposition of total ECM and collagens, a decrease in MMP-1
secretion, and an increase in TIMP-1 expression.
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MATERIALS AND METHODS |
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Cell culture. Cell cultures of primary human lung fibroblasts were established from lung tissue obtained from patients undergoing lobectomy due to lung cancer as previously described (39). Lung tissue was taken from the peripheral lung distant from the place where tumor growth was evident and kept overnight at 4°C in phosphate-buffered saline (PBS; Seromed, Biochrom, Berlin, Germany). The following day, the tissue was cut into small pieces (5 × 5 mm) and placed into cell culture flasks (Falcon, Basel, Switzerland) precoated with 10% fetal bovine serum (FBS; GIBCO BRL, Life Technologies, Basel, Switzerland). After fibroblasts had grown out from the tissues, the slices were removed by aspiration, and the cells were allowed to reach confluence. Confluent fibroblasts were then passaged by trypsin treatment and used for the experiments between passages 2 and 5. No antibiotics or antimycotics were added to the culture medium at any time. The procedure for generating primary human lung cell cultures from biopsies obtained during surgery has been approved (M75/97) by the ethical committee of the Faculty of Medicine, University of Basel (Basel, Switzerland).
For all experiments, confluent primary human lung fibroblasts (100%
density) were serum deprived for 24 h with low-serum medium [RPMI
1640 medium supplemented with 0.1% FBS and 20 mM HEPES
(Seromed)] before stimulation with the indicated concentrations
of TGF-1 or TGF-
3.
Determination of collagen and ECM
metabolism. Collagen secretion and actual deposition
into the ECM as well as total secreted and total deposited
proteinaceous ECM were assessed by proline incorporation assays
originally developed by Peterkofsky and Diegelmann (36) and described
in detail earlier (1, 7, 27, 40, 43). Confluent, serum-deprived
cultures of primary human lung fibroblasts were seeded into 12-well
plates (Falcon) and treated with the indicated concentrations of
TGF-1 or TGF-
3 in the presence of 0.5 µCi/ml of
[3H]proline (Amersham
Life Sciences) and 10 µg/ml of ascorbic acid. Determination of total
secreted proteins or collagens was performed in the supernatants from
fibroblasts collected at the indicated time points. These measurements
reflect the amount of collagens released into the supernatants by
fibroblasts in the respective previous time frame. Four
hundred-microliter aliquots of supernatant from each well were
incubated with 100 µl of collagenase assay buffer (50 mM
Tris · HCl, pH 7.5, 5 mM
CaCl2, and 2.5 mM
N-ethylmaleimide) containing 30 U/ml
of collagenase (from Clostridium
histolyticum, Sigma, Buchs, Switzerland)
for 4 h at 37°C. In parallel, a second 400-µl aliquot was
incubated in assay buffer without collagenase. Then, 50 µl of FBS and
100 µl of TCA (Sigma) were added to the samples and incubated on ice
for 30 min to precipitate protein fractions. Precipitates were applied
onto filter units (Whatman, Kent, UK) and washed three times with 2 ml
of TCA and two times with 2 ml of 80% ethanol. Each filter was placed
into 4 ml of liquid scintillation fluid (Zinsser Analytic, Frankfurt,
Germany), and radioactivity was determined in a scintillation counter.
Amounts of total secreted proteins were calculated as disintegrations per minute in supernatants without collagenase. Secreted collagens were
calculated as disintegrations per minute in supernatants without
collagenase
disintegrations per minute in supernatants with
collagenase as previously described (27, 36).
Determination of de novo deposition of total proteins and collagens was performed in deposited ECM after the culture supernatants were removed and the fibroblasts were lysed with 25 mM NH4OH for 10 min at room temperature (RT). Data obtained from these measurements reflect the amount of collagens deposited into ECM over the respective time period and represent the net result of production and turnover of ECM. ECM was ethanol fixed (70% ethanol, two times for 15 min at RT) and washed twice with 50 mM Tris · HCl, 1 mM CaCl2, and 1 mM proline, pH 7.5. The ECM was then incubated for 4 h at 37°C in assay buffer either with or without collagenase as described above. The supernatants were removed after 4 h and residual ECM was solubilized by overnight incubation in 0.3 M NaOH-1% SDS. Equal aliquots of supernatants and solubilized residual ECM were subjected to liquid scintillation counting.
Calculations were made as follows:
1) disintegrations per minute in
solubilized matrix without collagenase = total deposited proteinaceous
ECM, 2) (disintegrations per minute
in supernatant without collagenase × 100)/(disintegrations per
minute in supernatant without collagenase + disintegrations per minute
in solubilized matrix without collagenase) = percent background,
3) disintegrations per minute in
supernatant with collagenase percent background = disintegrations per minute in deposited collagen, and
4) (disintegrations per minute in
ECM collagen × 100)/[disintegrations per minute in collagen + 5.4(disintegrations per minute in supernatant with collagenase + disintegrations per minute in solubilized matrix with collagenase
disintegrations per minute in ECM collagen)] = percent
collagen content of total ECM as previously described (1, 7, 27, 36).
The formula used for the calculation of percent collagen contains the
factor 5.4 to correct for the 5.4-fold higher proline or hydroxyproline
content of collagens compared with that of other proteins.
Zymography. Expression of MMP by
unstimulated or TGF--stimulated lung fibroblasts was assessed by
gelatine or
-casein zymography as described earlier (17, 38). In
brief, gelatinolytic or caseinolytic activity within supernatants of
fibroblasts was determined with zymographic analysis under denaturing
but nonreducing conditions. Aliquots of each sample were applied onto
denaturing 8% SDS-polyacrylamide gels polymerized in the presence of
either 0.1% gelatine or 0.5%
-casein. Electrophoresis was
performed at 25-mA constant current for 2 h at RT, followed by a 1-h
equilibration of the gels in 2.5% Triton X-100 to remove SDS. The gels
were incubated in enzyme buffer (50 mM Tris · HCl, pH
7.3, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) for 18 h at 37°C. Bands of enzymatic activity were visualized by negative staining with standard Coomassie brilliant blue
dye solution. Molecular sizes of the bands displaying enzymatic activity were identified by comparison to prestained standard proteins
(Sigma) and to purified MMP (Anawa Trading, Wangen, Switzerland). The
nature of the proteolytic bands was further characterized by incubation
of identical zymograms in 1) regular
enzyme buffer; 2) 0.1 mg/ml of
phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor;
3) 0.1 mg/ml of Pefabloc, an
irreversible serine protease inhibitor; or
4) 10 mM EDTA, a selective MMP
inhibitor as described earlier (17).
Enzyme-linked immunoassay. Concentrations of secreted MMP-1 and MMP-2 were determined by enzyme-linked immunoassay (EIA; Amersham Life Sciences) in culture supernatants of fibroblasts. Aliquots of the supernatants were removed at the indicated time points, and measurement of MMP-1 and MMP-2 was carried out according to the manufacturer's instructions.
Western blot analysis. For Western
blot analysis, the cells were seeded onto gelatine-coated 150-mm cell
culture dishes (Fakola) and allowed to reach confluence. After 24 h of
serum starvation, the cultures were stimulated with the indicated
concentrations of TGF-1 or TGF-
3, and cytosolic extracts of
primary human lung fibroblasts were prepared by low-salt extraction. In
brief, the cells were washed two times with ice-cold PBS (Seromed) and
harvested by scraping into 1 ml of PBS. The samples were centrifuged
for 30 s at 6,000 g, and the cell
pellets were resuspended in 50 µl of a low-salt buffer [20 mM
HEPES, pH 7.9, 10 mM KCl, 0.1 mM
NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2%
Nonidet P-40, and 10% glycerol, supplemented with a set of proteinase
inhibitors (Complete, Boehringer Mannheim, Rotkreuz,
Switzerland)]. After 10 min of incubation on ice, the samples
were centrifuged at 13,000 g for 1 min, and the supernatants were taken as cytosolic cell extracts.
Protein concentrations of the samples were determined with the standard
Bradford assay (Bio-Rad, Glattbrugg, Switzerland). Expression of TIMP-1
or TIMP-2 in cytosolic extracts of fibroblasts was determined by
Western blot analysis with gradient SDS-PAGE gels (4-15%;
Bio-Rad). Aliquots of cytosolic extracts were applied to the gels and
run at 25-mA constant current for 4 h at RT. After electrophoresis, the
proteins were electroblotted on Immobilon-P transfer membranes
(Millipore, Volketswil, Switzerland) for 90 min at 1 mA/cm2 at RT. The membranes were
blocked in 5% skimmed milk in Tris-buffered saline-Tween 20 (TBS-T; 10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH
8.0) for 1 h at RT. After being blocked, the membranes were incubated
with specific antibodies to TIMP-1 or TIMP-2 (Oncogene Science, Paris,
France) at 4°C overnight. The following day, the membranes were
washed three times with TBS-T and incubated with the secondary
peroxidase-coupled antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
at a dilution of 1:5,000 for 1 h at RT. The membranes were washed three
times in TBS-T, and specific bands were visualized with the enhanced
chemiluminescence system from Amersham Life Sciences according to the
manufacturer's instructions. To quantify protein expression, the bands
were scanned on a DOS-based image-analysis system installed by Raytest
(Straubenhardt, Germany).
Statistical analysis. All experiments were performed in triplicate with at least two independent sets of experiments. All data were obtained from at least five different cell lines of primary human lung fibroblasts. Homogeneity of groups was analyzed by two-tailed Student's t-test for each time point or concentration.
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RESULTS |
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Effects of TGF-1 and
TGF-
3 on total secreted proteins and secreted
collagens. All experiments analyzing the impact of
TGF-
isoforms on ECM metabolism by
[3H]proline
incorporation were done on confluent cultures of human lung
fibroblasts. In this condition, TGF-
isoforms have no effect on cell
proliferation or thymidine incorporation (data not shown). As depicted
in Fig. 1, both TGF-
isoforms
significantly increased total secreted proteins (Fig.
1A) and secreted collagens (Fig. 1B) in a dose-dependent manner over
a concentration range from 0.1 to 5 ng/ml. After 40 h, TGF-
1 and
TGF-
3 (at 1 ng/ml each) had significantly increased total secreted
proteins compared with those in unstimulated conditions. TGF-
1 led
to a 267% increase (47,500 ± 2,000 vs. 17,800 ± 500 dpm), and TGF-
3 led to a 239% increase (42,600 ± 2,800 vs.
17,800 ± 500 dpm; P < 0.001). No effect on total protein secretion was observed when either of the two
TGF-
isoforms was used at concentrations ranging from 0.01 to 0.05 ng/ml (Fig. 1).
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Figure 1B depicts the effects of both
isoforms on secreted collagens as assayed by collagenase digestion of
cell supernatants. Here, a similar dose response was observed. TGF-1
(at 1 ng/ml) increased secretion of collagens by lung fibroblasts from
5,900 ± 700 to 25,500 ± 2,000 dpm, corresponding to a
432% increase. TGF-
3 increased secretion of collagens to 25,900 ± 1,300 dpm, corresponding to a 442% increase. Summarizing these
data, increases in total protein secretion were 267 and 239% for
TGF-
1 and TGF-
3, respectively, at 1 ng/ml, whereas corresponding
increases in secreted collagens were 432 and 442% and thereby
significantly higher (P < 0.05).
This indicates a collagen selectivity of these growth factors compared
with overall protein synthesis.
Effects of TGF-1 and
TGF-
3 on total deposited ECM and deposited
collagens. Secreted ECM molecules such as collagens are either rapidly degraded or deposited and cross-linked into existing ECM. We assessed the actual deposition of total proteins and collagens into ECM by analyzing fibroblast ECM after selective removal of culture
supernatants and cell layers. As demonstrated in Fig. 2A, the
dose response to TGF-
1 and TGF-
3 by assaying deposition of total
proteins is similar to the dose response observed for secretion of
total proteins (Fig. 1A). The
submaximal response to the growth factors was again obtained when the
TGF-
isoforms were used at 1 ng/ml. At this concentration, deposited
total proteinaceous ECM increased by 394% in response to TGF-
1
(66,200 ± 5,700 vs. 16,800 ± 600 dpm) and by 377% in response
to TGF-
3 (63,400 ± 3,200 vs. 16,800 ± 600 dpm) compared with
that in unstimulated control cells (P < 0.001; Fig. 2A).
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We then analyzed deposition of collagens within the total ECM by
collagenase digestion of ECM. Using this method, we found that collagen
deposition increased to 541 and 555% of control values in response to
TGF-1 and TGF-
3, respectively (23,700 ± 1,800 and 24,300 ± 1,000, respectively, vs. 4,400 ± 100 dpm; P < 0.001; Fig.
2B). As observed in the
supernatants, the increases in collagens were again higher than the
increases in total deposited proteins (541 and 394% increases,
respectively, for TGF-
1 and 555 and 377% increases, respectively,
for TGF-
3; P < 0.05).
As yet, no investigations have analyzed the percentages of collagens
within secreted or deposited total proteins by primary human lung
fibroblasts. We therefore calculated the collagen percentage of total
secreted proteins in culture supernatants (Fig.
3A) and the collagen percentage of total deposited ECM (Fig.
3B). The underlying formulas for
these calculations are well described and have been published earlier
(1, 7, 27, 36, 40, 43). In supernatants of unstimulated lung
fibroblasts, soluble collagens constituted 8.0 ± 1.0% of all
secreted proteins. This collagen percentage rose to 23.6 ± 4.6 and 22.3 ± 1.3% in fibroblast cultures stimulated
with TGF-1 and TGF-
3, respectively (at 1 ng/ml each;
P < 0.001; Fig.
3A). These increases correspond to 295 and 279% increases of the collagen percentage in culture
supernatants for TGF-
1 and TGF-
3, respectively.
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The collagen percentage of total deposited ECM in unstimulated human
lung fibroblasts was 5.8 ± 0.2%. This percentage was increased to
9.0 ± 0.5 and 8.8 ± 0.5% by TGF-1 and TGF-
3,
respectively (P < 0.005; Fig.
3B). Thus the increases in the
collagen percentages of deposited ECM were 155 and 152% for TGF-
1
and TGF-
3, respectively (P < 0.001).
Effects of TGF-1 and
TGF-
3 on MMP expression. We performed
zymographic analyses and EIAs of culture supernatants from fibroblasts to assess whether TGF-
1 or TGF-
3 influenced the expression of MMPs, a family of proteases that is essentially responsible for degradation and turnover of ECM. We initially screened fibroblast cultures derived from 10 different patients for the secretion of MMP
isoforms into culture supernatants. Each fibroblast culture investigated secreted two distinct proteases, a 70-kDa protease visible
on gelatine zymography and a 55-kDa protease visible on casein
zymography (data not shown). To characterize the nature of these
proteases, their response to distinct protease inhibitors was
investigated as described earlier (17). Four identical gels containing
aliquots of supernatants from different fibroblast cultures were
subjected to gelatine zymography, processed identically, and incubated
either in regular enzyme buffer (Fig.
4A), in
enzyme buffer plus 0.1 mg/ml of PMSF (Fig.
4B), in enzyme buffer plus 0.1 mg/ml
of Pefabloc (Fig. 4C), or in enzyme
buffer plus 10 mM EDTA (Fig. 4D).
Figure 4 demonstrates that only the selective MMP inhibitor EDTA
completely inhibited proteolysis on gelatine zymography, whereas the
serine protease inhibitors PMSF or Pefabloc did not affect the
appearance of proteolytic bands. Similar data were obtained with casein
zymography. Thus these data confirm that the proteases secreted by
fibroblasts are of metalloproteinase origin.
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Figure 5A
depicts a characteristic casein zymography obtained from culture
supernatants of lung fibroblasts, demonstrating proteolysis due to
MMP-1 at the expected size of 55 kDa. Twenty hours after stimulation
with TGF-1 or TGF-
3 at the indicated concentrations, the
secretion of MMP-1 by fibroblasts was significantly downregulated. As
seen from the densitometric scan of three different zymographies (Fig.
5B), both TGF-
1 and TGF-
3
inhibited MMP-1 secretion by primary human lung fibroblasts over a
concentration range from 0.1 to 5 ng/ml.
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In contrast, secretion of MMP-2 by fibroblasts was unaffected by the
addition of either TGF-1 or TGF-
3. Lung fibroblasts secrete high
amounts of MMP-2 at the expected size of 70 kDa (Fig. 6A,
arrow). However, we did not observe an effect of TGF-
1 (Fig. 6,
lanes
1-4) or
TGF-
3 (Fig. 6, lanes
5-8) on the
secretion of MMP-2 by fibroblasts. This was confirmed by densitometric
scans of three independent zymographies (Fig.
6B).
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To further quantify control MMP-1 and MMP-2 expression and to quantify
the effects of TGF-1 and TGF-
3 on MMP-1 secretion, we performed
EIAs specific for human MMP-1 and MMP-2 using culture supernatants of
human lung fibroblasts. Figure 7
demonstrates the values of baseline and TGF-
-stimulated secretion of
MMP-1 and MMP-2 into culture supernatants. In supernatants of quiescent fibroblasts, the MMP-1 concentration was 35.5 ± 0.7 ng/ml. This secretion was significantly inhibited by both TGF-
isoforms (1 ng/ml
each) at 20 h: by 43% (20.3 ± 0.7 ng/ml) with TGF-
1 and by 39%
(21.8 ± 1 ng/ml) with TGF-
3 (P < 0.001; Fig. 7). The concentration of MMP-2 in supernatants of
quiescent fibroblasts was 121.2 ± 12.5 ng/ml, thereby 3.4-fold the
concentration of MMP-1. As noted on zymography, MMP-2 secretion was not
influenced by either of the two TGF-
isoforms (Fig. 7).
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Effects of TGF-1 and
TGF-
3 on TIMP-1 and TIMP-2 expression.
To analyze whether TGF-
1 or TGF-
3 affected expression of the endogenous MMP inhibitors TIMP-1 or TIMP-2, cytosolic extracts of
fibroblasts were analyzed by Western blot analyses with antibodies specific for human TIMP-1 or TIMP-2. Figure
8A
represents a characteristic Western blot demonstrating expression of
TIMP-1 at 28 kDa in human lung fibroblasts. In quiescent fibroblasts,
TIMP-1 was detected at low levels (Fig.
8A, 0 h). Both TGF-
1 and TGF-
3
(at 1 ng/ml each) rapidly upregulated the baseline expression of
TIMP-1, with TGF-
1 exhibiting a more sustained action than TGF-
3.
TGF-
1 led to a maximal increase in TIMP-1 at 20 h, whereas the
maximal increase in TIMP-1 expression by TGF-
3 was observed as early as 8 h, with a decline thereafter (see densitometric scan of three independent blots in Fig.
8B).
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In contrast to TIMP-1, the expression of TIMP-2 was clearly
downregulated by both TGF- isoforms (Fig.
9). Downregulation of TIMP-2 occurred as
early as 8 h after stimulation with the growth factors, and the extent
of TIMP-2 inhibition was similar for both TGF-
isoforms (Fig. 9).
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DISCUSSION |
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In the present study, we investigated the effects of two isoforms of
the TGF- superfamily, TGF-
1 and TGF-
3, on ECM deposition by
primary human lung fibroblasts. We assessed the impact of TGF-
1 and
TGF-
3 on three mechanisms that are primarily responsible for the
accumulation and/or turnover of ECM in fibrotic disease states:
1) de novo production and deposition
of ECM and collagens; 2) secretion
of MMP-1 and MMP-2, the metalloproteinases expressed by human lung
fibroblasts capable of degrading ECM; and
3) expression of TIMP-1 and TIMP-2,
two antiproteases that counteract ECM degradation through inhibition of
MMP activity. Overall, both TGF-
1 and TGF-
3 were equally potent
in increasing the synthesis and deposition of ECM molecules by human
lung fibroblasts. We found no significant difference between the
effects or efficacies of the two TGF-
isoforms in any of the
experiments. The net accumulation of ECM coincided with
1) a net increase in de novo
secretion and deposition of ECM molecules, especially collagens;
2) a decrease in MMP-1 secretion;
and 3) an increase in the expression
of TIMP-1.
During the development of lung fibrosis, TGF- isoforms are generally
recognized as key mediators responsible for the accumulation of ECM (4,
13, 19, 21, 22, 28, 32). This observation originates from multiple in
vivo and in vitro findings; TGF-
isoforms are consistently
overexpressed in biopsies from fibrotic lungs, especially in areas of
active fibrosis (5, 11, 20, 24, 41, 45). In animal models of
bleomycin-induced lung fibrosis, overexpression of TGF-
isoforms
precedes the increased expression of collagens (41). Accordingly, local
overexpression of TGF-
1 in rat lungs leads to increased deposition
of collagens (41, 45). Neutralizing antibodies to TGF-
1 inhibit
collagen accumulation in this animal model (20), thus causally linking TGF-
overexpression to the development of fibrosis.
In vitro, it is well established that TGF- isoforms upregulate mRNA
and protein levels of collagens, fibronectins, and laminins in a
variety of cell types (4, 5, 8, 11). However, increased de novo
synthesis of ECM molecules does not necessarily lead to increased
deposition of ECM and does not exclusively contribute to the
pathogenesis of fibrosis. Rather, fibrosis is regarded as a disturbed
balance between degradation and accumulation of ECM in favor of
accumulation. In the lung, the physiological turnover of ECM is
estimated to be >10% of the total ECM per day (14, 30). Any insult
affecting this physiological balance between synthesis and degradation
thus leads to altered composition of the ECM. Such insults generally
act at the following levels: 1) expression of ECM molecules (14, 30);
2) expression and/or activation of
ECM-degrading proteases, especially MMPs (4, 14, 26, 30, 34); or
3) expression of inhibitors of these proteases (TIMPs) (4, 14, 34). It is therefore reasonable to
characterize the overall effects of TGF-
isoforms on each of these
mechanisms in an in vitro model of TGF-
-induced fibrosis with
primary fibroblasts of human lungs.
The interstitial fibroblast of the lung accounts for 40% of all lung
cells and is the cell type primarily responsible for the synthesis of
the most proteinaceous and nonproteinaceous components of the pulmonary
ECM (14, 15). From an analysis of the expression of multiple ECM
molecules, TGF-1 has been shown to increase synthesis of the
fibrillar collagen types I, II, III, and V at mRNA and protein levels
(8, 22, 29, 35, 37, 42). However, de novo synthesis does not
necessarily reflect cross-linking and aggregation of collagen fibrils
into existing ECM. We therefore assessed the effects of TGF-
1 and
TGF-
3 on both the secretion and deposition of collagens, both of
which were similarly affected by the two TGF-
isoforms.
Interestingly, the collagen percentage of total proteins was higher in
the secreted (8.0 ± 1.2%) than in the deposited fraction (5.8 ± 0.3%) of quiescent fibroblasts. Furthermore, increases in
collagen percentages in response to TGF-
isoforms were clearly
higher in secreted proteins than in deposited proteins. Thus these
observations reveal different collagen amounts between the soluble
(supernatants) and the deposited (ECM) fractions synthesized by primary
human lung fibroblasts. In this respect, it would be interesting to
assess whether there is a corresponding differential distribution of
collagen isoforms between secreted and deposited proteins.
In contrast to collagen metabolism, the effects of TGF- isoforms on
the protease-antiprotease system of MMPs and TIMPs were as yet less
well characterized. Although TGF-
isoforms have consistently led to
increased collagen synthesis in most cell types studied, their effects
on MMP and TIMP expression are controversial and highly tissue-type
specific. In our model, TGF-
1 and TGF-
3 significantly decreased
MMP-1 expression but had no effect on MMP-2 expression. Several
investigations (16, 47) have demonstrated that TGF-
1 decreased
protease expression in transformed cell lines, e.g., MMP-1. In
contrast, TGF-
increased antiprotease expression, such as TIMPs (16,
47) or plasminogen activator inhibitors (25). In fibroblasts derived
from the periodontal ligament, TGF-
1 reduced MMP-1 expression by
50% but had no effect on TIMP-1 (2). In addition, the effects of
TGF-
even differed between distinct MMP isoforms. Immortalized
fibroblasts increased MMP-13 (collagenase 3) but decreased MMP-1
expression in response to TGF-
1 (44). Thus the effects of TGF-
isoforms on MMP expression are highly cell-type and isoform specific,
and it would be intriguing to unravel the signal transduction
mechanisms underlying these specificities.
Similar to the divergent effects of TGF- isoforms on MMP expression,
expression of TIMPs in response to TGF-
is also regulated in an
isoform-specific manner. Our results demonstrate the divergent effects
of TGF-
isoforms on TIMP-1 (increase) and TIMP-2 (decrease). Whereas
increased TIMP-1 expression could directly contribute to increased ECM
accumulation through inhibition of ECM degradation, the biological
significance of decreased TIMP-2 expression in response to TGF-
isoforms remains unclear. However, similar observations were made in
the case of oncostatin M. Oncostatin M-stimulated synovial cells
upregulated TIMP-1 but downregulated TIMP-3 (18). Furthermore, TGF-
induced TIMP-1 expression in dermal fibroblasts, but TIMP-2 has not
been analyzed in that model (42). As for the opposite effects of
TGF-
isoforms on MMP isoforms, the underlying mechanism for the
responses of TIMP isoforms to TGF-
isoforms may reside within the
promoter structures and possible TGF-
response elements of the TIMP genes.
In summary, we sought to characterize the effects of two TGF-
isoforms, TGF-
1 and TGF-
3, on mechanisms associated with ECM
deposition in a cell culture model using primary derived human lung
fibroblasts. Our investigations demonstrate that both isoforms equally
affect three distinct regulatory mechanisms controlling ECM
composition. These are de novo secretion and deposition of ECM, MMP-1
secretion, and expression of TIMP-1. All of these mechanisms potentially contribute to the accumulation of ECM and could explain the
potent fibrogenic potency of TGF-
isoforms observed both in vivo and
in vitro. Our observations thus characterize the effects of both growth
factors in primary human lung fibroblasts and emphasize the biological
role of these growth factors during the pathogenesis of lung fibrosis.
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ACKNOWLEDGEMENTS |
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We are indebted to Victoria Bruce for unmatched enthusiasm and critical help during the preparation of this manuscript.
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FOOTNOTES |
---|
This study was supported by the Swiss National Science Foundation (No. 32-39'446.93) and the Swiss Heart Foundation.
O. Eickelberg is presently a Feodor-Lynen Fellow supported by the Alexander von Humboldt Association (Department of Pathology, Yale University School of Medicine, New Haven, CT).
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 and present address of O. Eickelberg: Yale Univ. School of Medicine, Dept. of Pathology, 310 Cedar St., LB 08, New Haven, CT 06520-8023 (E-mail: oliver.eickelberg{at}yale.edu).
Received 24 June 1998; accepted in final form 27 January 1999.
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