Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta 1 and TGF-beta 3

Oliver Eickelberg, Eleonore Köhler, Frank Reichenberger, Sybille Bertschin, Thomas Woodtli, Paul Erne, André P. Perruchoud, and Michael Roth

Divisions of Pneumology and Cardiovascular Research, Departments of Research and Internal Medicine, University Hospital Basel, CH-4031 Basel, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Increased collagen and extracellular matrix (ECM) deposition within the lung is a characteristic feature of lung fibrosis. Transforming growth factor (TGF)-beta isoforms play a pivotal role in the production of collagen and ECM. In this study, we investigated the effects of TGF-beta 1 and TGF-beta 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-beta 1 and TGF-beta 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-beta 1 and TGF-beta 3, respectively. Secretion of MMP-1 (interstitial collagenase) by fibroblasts was reduced by both TGF-beta isoforms, whereas secretion of MMP-2 (gelatinase A) was unaffected by either of the two isoforms. Both TGF-beta isoforms increased TIMP-1 protein expression, whereas TIMP-2 protein was decreased. We thus conclude that TGF-beta 1 and TGF-beta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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)-beta isoforms are key mediators responsible for the ECM changes seen in lung fibrosis (4, 5, 13, 21, 22, 28, 30, 32).

The TGF-beta isoforms belong to a superfamily of polypeptides including 1) TGF-beta 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-beta isoforms, TGF-beta 1, TGF-beta 2, and TGF-beta 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 (Tbeta RI), type II (Tbeta RII), and type III (Tbeta RIII), that specifically recognize TGF-beta isoforms have been characterized (6, 46). Biological responses to TGF-beta isoforms occur when TGF-beta isoforms bind to Tbeta RII, which induces a heterooligomerization between Tbeta RI and Tbeta RII. On complex formation, Tbeta RI is phosphorylated by the constitutively active kinase domain of Tbeta RII, and downstream signaling is initiated (6, 46). Interestingly, specificity of the actions of TGF-beta 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 Tbeta R subtypes (6, 9, 46). Studies investigating the biological effects of different TGF-beta isoforms demonstrated a considerable overlap of their activities. However, evidence exists that TGF-beta 1 and TGF-beta 3 have distinct effects and/or potencies in vitro (9, 23, 31, 33).

In the normal human lung, TGF-beta isoforms are frequently expressed in bronchiolar epithelial cells and interstitial fibroblasts, with TGF-beta 1 being the most abundant isoform in the lung (3, 10). During the development of lung fibrosis, altered expression patterns of TGF-beta isoforms are found compared with those in control lungs. Overexpression of TGF-beta 1 in macrophages and mesenchymal, endothelial, and mesothelial cells of the lung can be detected in fibrotic lungs, but the expression level of TGF-beta 3 was unchanged (11). However, the mechanisms leading to increased ECM deposition by TGF-beta 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-beta isoforms, TGF-beta 1 and TGF-beta 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-beta 1 and TGF-beta 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.


    MATERIALS AND METHODS
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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-beta 1 or TGF-beta 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-beta 1 or TGF-beta 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-beta -stimulated lung fibroblasts was assessed by gelatine or beta -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% beta -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-beta 1 or TGF-beta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Effects of TGF-beta 1 and TGF-beta 3 on total secreted proteins and secreted collagens. All experiments analyzing the impact of TGF-beta isoforms on ECM metabolism by [3H]proline incorporation were done on confluent cultures of human lung fibroblasts. In this condition, TGF-beta isoforms have no effect on cell proliferation or thymidine incorporation (data not shown). As depicted in Fig. 1, both TGF-beta 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-beta 1 and TGF-beta 3 (at 1 ng/ml each) had significantly increased total secreted proteins compared with those in unstimulated conditions. TGF-beta 1 led to a 267% increase (47,500 ± 2,000 vs. 17,800 ± 500 dpm), and TGF-beta 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-beta isoforms was used at concentrations ranging from 0.01 to 0.05 ng/ml (Fig. 1).


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Fig. 1.   Effect of transforming growth factor (TGF)-beta isoforms on total secreted proteins (A) and secreted collagens (B). Human lung fibroblasts were stimulated with indicated concentrations of TGF-beta 1 (solid bars) or TGF-beta 3 (open bars) in presence of 0.5 µCi/ml of [3H]proline, and protein and collagen secretion were assessed in culture supernatants after 40 h. Con, unstimulated control fibroblasts. Values are means ± SD from 3 independent experiments with 1 cell line. Similar results were obtained in 4 different cell lines of primary derived human lung fibroblasts. * Significant difference compared with unstimulated control fibroblasts, P < 0.005.

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-beta 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-beta 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-beta 1 and TGF-beta 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-beta 1 and TGF-beta 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-beta 1 and TGF-beta 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-beta isoforms were used at 1 ng/ml. At this concentration, deposited total proteinaceous ECM increased by 394% in response to TGF-beta 1 (66,200 ± 5,700 vs. 16,800 ± 600 dpm) and by 377% in response to TGF-beta 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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Fig. 2.   Effect of TGF-beta 1 and TGF-beta 3 on total deposited proteinaceous ECM (A) and deposited collagens (B). Human lung fibroblasts were stimulated with indicated concentrations of TGF-beta 1 (solid bars) or TGF-beta 3 (open bars) in presence of 0.5 µCi/ml of [3H]proline, and protein and collagen secretion were assessed after 40 h in deposited ECM. Values are means ± SD from 3 independent experiments with 1 cell line. Similar results were obtained in 4 different cell lines. * Significant difference compared with unstimulated control fibroblasts, P < 0.005.

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-beta 1 and TGF-beta 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-beta 1 and 555 and 377% increases, respectively, for TGF-beta 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-beta 1 and TGF-beta 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-beta 1 and TGF-beta 3, respectively.


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Fig. 3.   Collagen percentages were calculated within secreted proteins (A) and deposited ECM (B) of human lung fibroblasts. Calculations were carried out in unstimulated, quiescent cells as well as in cultures stimulated with indicated amounts of TGF-beta 1 (solid bars) or TGF-beta 3 (open bars). Values are means ± SD from 3 independent experiments with 1 cell line. Similar results were obtained in 4 different cell lines of primary fibroblasts. * Significant difference compared with unstimulated control fibroblasts, P < 0.02.

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-beta 1 and TGF-beta 3, respectively (P < 0.005; Fig. 3B). Thus the increases in the collagen percentages of deposited ECM were 155 and 152% for TGF-beta 1 and TGF-beta 3, respectively (P < 0.001).

Effects of TGF-beta 1 and TGF-beta 3 on MMP expression. We performed zymographic analyses and EIAs of culture supernatants from fibroblasts to assess whether TGF-beta 1 or TGF-beta 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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Fig. 4.   Inhibition profile of matrix metalloproteinase (MMP)-2 secreted by human lung fibroblasts. Aliquots of supernatants from 2 different cell lines (lanes 1 and 2) along with purified, activated MMP-2 were applied to 4 identical gelatine zymography gels. Gels were processed either in regular enzyme buffer (A); in enzyme buffer plus 0.1 mg/ml of phenylmethylsulfonyl fluoride, a serine protease inhibitor (B); in enzyme buffer plus 0.1 mg/ml of Pefabloc, an irreversible serine protease inhibitor (C); or in enzyme plus 10 mM EDTA, a metalloproteinase inhibitor (D). Activated MMP-2 exhibits 2 distinct bands characteristic of latent (72-kDa) and activated (66-kDa) forms of enzyme. Culture supernatants from fibroblasts consistently exhibited only latent MMP-2.

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-beta 1 or TGF-beta 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-beta 1 and TGF-beta 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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Fig. 5.   TGF-beta 1 and TGF-beta 3 reduce MMP-1 secretion by human lung fibroblasts. Aliquots of supernatants from cultures treated with and without TGF-beta 1 (lanes 1-4) or TGF-beta 3 (lanes 5-8) were subjected to casein zymography, and MMP-1 activity was visualized by negative staining with Coomassie brilliant blue dye solution (A). MMP-1 activity was scanned densitometrically, and percentages of control value were calculated from 3 independent experiments with different fibroblast lines (B). S, purified standard MMP-1 migrating at 55 kDa; lane 1, unstimulated control fibroblasts; lane 2, with TGF-beta 1 at 0.5 ng/ml; lane 3, with TGF-beta 1 at 1 ng/ml; lane 4, with TGF-beta 1 at 5 ng/ml; lane 5, unstimulated control fibroblasts; lane 6, with TGF-beta 3 at 0.5 ng/ml; lane 7, with TGF-beta 3 at 1 ng/ml; lane 8, with TGF-beta 3 at 5 ng/ml. Significant difference compared with unstimulated control fibroblasts: * P < 0.02; ** P < 0.01.

In contrast, secretion of MMP-2 by fibroblasts was unaffected by the addition of either TGF-beta 1 or TGF-beta 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-beta 1 (Fig. 6, lanes 1-4) or TGF-beta 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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Fig. 6.   TGF-beta 1 and TGF-beta 3 do not affect MMP-2 secretion by human lung fibroblasts. Stimulation and harvesting procedures were identical to those in Fig. 5 with the exception that aliquots were subjected to gelatine zymography to visualize MMP-2 activity (A). Proteolytic bands were scanned densitometrically, and percentages of control value were calculated from 3 independent experiments with different fibroblast lines (B). S, purified latent MMP-2 migrating at 72 kDa; lane 1, unstimulated control fibroblasts; lane 2, with TGF-beta 1 at 0.5 ng/ml; lane 3, with TGF-beta 1 at 1 ng/ml; lane 4, with TGF-beta 1 at 5 ng/ml; lane 5, unstimulated control fibroblasts; lane 6, with TGF-beta 3 at 0.5 ng/ml; lane 7, with TGF-beta 3 at 1 ng/ml; lane 8, with TGF-beta 3 at 5 ng/ml.

To further quantify control MMP-1 and MMP-2 expression and to quantify the effects of TGF-beta 1 and TGF-beta 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-beta -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-beta isoforms (1 ng/ml each) at 20 h: by 43% (20.3 ± 0.7 ng/ml) with TGF-beta 1 and by 39% (21.8 ± 1 ng/ml) with TGF-beta 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-beta isoforms (Fig. 7).


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Fig. 7.   Effects of TGF-beta 1 and TGF-beta 3 on MMP-1 (A) and MMP-2 (B) expression as assessed by enzyme-linked immunoassay (EIA) of culture supernatants from human lung fibroblasts. Culture supernatants from control cells or from cells stimulated with TGF-beta 1 or TGF-beta 3 (at 1 ng/ml each) were removed at 20 h and analyzed by EIA specific for human MMP-1 or MMP-2. Values are means ± SD of 2 independent experiments from 1 cell line analyzed in duplicate. Similar results were obtained in 3 different fibroblast lines. * Significant difference compared with unstimulated control fibroblasts, P < 0.001.

Effects of TGF-beta 1 and TGF-beta 3 on TIMP-1 and TIMP-2 expression. To analyze whether TGF-beta 1 or TGF-beta 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-beta 1 and TGF-beta 3 (at 1 ng/ml each) rapidly upregulated the baseline expression of TIMP-1, with TGF-beta 1 exhibiting a more sustained action than TGF-beta 3. TGF-beta 1 led to a maximal increase in TIMP-1 at 20 h, whereas the maximal increase in TIMP-1 expression by TGF-beta 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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Fig. 8.   TGF-beta 1 and TGF-beta 3 increase tissue inhibitor of metalloproteinases (TIMP)-1 expression by human lung fibroblasts. Cytosolic extracts from control and TGF-beta 1- or TGF-beta 3-stimulated (+) cells (at 1 ng/ml each) were prepared at indicated time points, separated on SDS-PAGE gels, blotted onto nitrocellulose membranes, and hybridized with an antibody specific for TIMP-1 at a titer of 1:500. Recombinant (rec) TIMP-1 migrating at 28 kDa was used as a positive control. A: characteristic Western blot. B: densitometric analyses of TIMP-1 expression averaged from 3 independent Western blots with different fibroblast lines. ** Significant difference compared with control fibroblasts, P < 0.001.

In contrast to TIMP-1, the expression of TIMP-2 was clearly downregulated by both TGF-beta 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-beta isoforms (Fig. 9).


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Fig. 9.   TGF-beta 1 and TGF-beta 3 reduce TIMP-2 expression by human lung fibroblasts. Samples were processed as described in Fig. 8 but hybridized with an antibody specific for TIMP-2 at a titer of 1:500. Recombinant TIMP-2 migrating at 21 kDa was used as a positive control. A: characteristic Western blot. B: densitometric analyses of TIMP-2 expression averaged from 3 independent Western blots with different fibroblast lines. Significant difference compared with control fibroblasts: * P < 0.01; ** P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we investigated the effects of two isoforms of the TGF-beta superfamily, TGF-beta 1 and TGF-beta 3, on ECM deposition by primary human lung fibroblasts. We assessed the impact of TGF-beta 1 and TGF-beta 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-beta 1 and TGF-beta 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-beta 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-beta 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-beta 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-beta isoforms precedes the increased expression of collagens (41). Accordingly, local overexpression of TGF-beta 1 in rat lungs leads to increased deposition of collagens (41, 45). Neutralizing antibodies to TGF-beta 1 inhibit collagen accumulation in this animal model (20), thus causally linking TGF-beta overexpression to the development of fibrosis.

In vitro, it is well established that TGF-beta 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-beta isoforms on each of these mechanisms in an in vitro model of TGF-beta -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-beta 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-beta 1 and TGF-beta 3 on both the secretion and deposition of collagens, both of which were similarly affected by the two TGF-beta 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-beta 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-beta isoforms on the protease-antiprotease system of MMPs and TIMPs were as yet less well characterized. Although TGF-beta 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-beta 1 and TGF-beta 3 significantly decreased MMP-1 expression but had no effect on MMP-2 expression. Several investigations (16, 47) have demonstrated that TGF-beta 1 decreased protease expression in transformed cell lines, e.g., MMP-1. In contrast, TGF-beta increased antiprotease expression, such as TIMPs (16, 47) or plasminogen activator inhibitors (25). In fibroblasts derived from the periodontal ligament, TGF-beta 1 reduced MMP-1 expression by 50% but had no effect on TIMP-1 (2). In addition, the effects of TGF-beta even differed between distinct MMP isoforms. Immortalized fibroblasts increased MMP-13 (collagenase 3) but decreased MMP-1 expression in response to TGF-beta 1 (44). Thus the effects of TGF-beta 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-beta isoforms on MMP expression, expression of TIMPs in response to TGF-beta is also regulated in an isoform-specific manner. Our results demonstrate the divergent effects of TGF-beta 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-beta 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-beta 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-beta isoforms on MMP isoforms, the underlying mechanism for the responses of TIMP isoforms to TGF-beta isoforms may reside within the promoter structures and possible TGF-beta response elements of the TIMP genes.

In summary, we sought to characterize the effects of two TGF-beta isoforms, TGF-beta 1 and TGF-beta 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-beta 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.


    ACKNOWLEDGEMENTS

We are indebted to Victoria Bruce for unmatched enthusiasm and critical help during the preparation of this manuscript.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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