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Published ahead of print on January 14, 2005, doi:10.1165/rcmb.2004-0288OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 342-349, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0288OC

Tumor Necrosis Factor-{alpha} Induces Transforming Growth Factor-ß1 Expression in Lung Fibroblasts Through the Extracellular Signal–Regulated Kinase Pathway

Deborah E. Sullivan, MaryBeth Ferris, Derek Pociask and Arnold R. Brody

Department of Pathology and Laboratory Medicine and the Lung Biology Program, Tulane University Health Sciences Center, New Orleans, Louisiana

Correspondence and requests for reprints should be addressed to Deborah E. Sullivan, Ph.D., Department of Pathology and Laboratory Medicine SL-79, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112. E-mail: dsulliva{at}tulane.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Increased expression of transforming growth factor (TGF)-ß1 and tumor necrosis factor (TNF)-{alpha} are thought to play important roles in the development of pulmonary fibrosis. We recently reported that TNF-{alpha} upregulates TGF-ß1 expression in primary mouse lung fibroblasts (MLFs), a key cell population in fibrogenesis. In the present study, we have investigated signal transduction pathways involved in TNF-{alpha} upregulation of TGF-ß1 in both primary MLFs and the Swiss 3T3 fibroblast cell line. Treatment of fibroblasts with TNF-{alpha} resulted in a significant increase in TGF-ß1 protein as measured by ELISA. The increase in protein was preceded by a 200–400% increase in TGF-ß1 mRNA detected by quantitative, real-time, reverse transcriptase–polymerase chain reaction. Western blot analysis showed that TNF-{alpha} activated the extracellular signal–regulated kinase (ERK), and inhibitors of the ERK-specific mitogen-activated protein kinase pathway (PD98059 or U0126) blocked TNF-{alpha} induction of TGF-ß1 mRNA and protein. mRNA stability experiments showed that TNF-{alpha} increased the half-life of TGF-ß1 mRNA to more than 24 h compared with ~ 15 h in unstimulated cells. Expression of constitutively active MEK1 that selectively phosphorylates ERK was sufficient for TGF-ß1 mRNA stabilization in Swiss 3T3 fibroblasts. These results indicate that TNF-{alpha} activates the ERK-specific mitogen-activated protein kinase pathway leading to increased TGF-ß1 production in fibroblasts, primarily via a post-transcriptional mechanism that involves stabilization of the TGF-ß1 transcript.

Key Words: lung fibroblasts • transforming growth factor-ß1 • tumor neurosis factor-{alpha} • extracellular signal-regulated kinase


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Interstitial pulmonary fibrosis (IPF) afflicts millions of individuals worldwide. The process is caused by a number of drugs, infectious agents, and diverse inhaled environmental agents including ozone and asbestos fibers (1). There are no effective treatments and few therapeutic approaches (2). This situation is due in large part to a lack of understanding of the fundamental molecular mechanisms that mediate the fibrogenic process. We and a number of other investigators are asking if transforming growth factor (TGF)-ß1 and tumor necrosis factor (TNF)-{alpha} are playing significant roles in the development of IPF (3–6).

Accumulating evidence suggests that pulmonary fibrosis is a consequence of TNF-{alpha} expression in the lung. TNF-{alpha} expression is increased in alveolar macrophages and proliferating type II pneumocytes of humans with acute fibrotic changes in the lung (7). TNF-{alpha} is an important proinflammatory cytokine. However, in the lung, TNF-{alpha} may also increase fibroblast proliferation, differentiation, and extracellular matrix deposition, as well as promote induction of matrix metalloproteinases (MMPs) that enhance basement membrane disruption and can facilitate fibroblast migration (8). Furthermore, a TNF-{alpha} promoter polymorphism seems to confer increased risk of developing IPF (9). Finally, agents with anti–TNF-{alpha} properties such as pirfenidone and etanercept have shown promise in treatment of patients with IPF (2). Thus, it appears that TNF-{alpha} plays a key role in the development of interstitial inflammation and fibrosis. However, overexpression of TNF-{alpha} has been reported to diminish pulmonary fibrosis in several animal models of IPF (6), and there is clear evidence that TNF-{alpha} represses the type I collagen promoter (10) and induces the expression of MMPs that participate in the dissolution of matrix (11). Thus, TNF-{alpha} has both fibrotic and antifibrotic properties.

TGF-ß1 is one of the key cytokines in extracellular matrix production and acts at different levels to increase lung collagen deposition (12). There is evidence that TNF-{alpha} influences the expression of TGF-ß1 (3), and an earlier study showed that an anti–TNF-{alpha} antibody reduced bleomycin-induced lung injury in mice (13). These investigators showed decreased expression of TGF-ß1 in the treated mice, a finding correlating with our data showing that TNF-{alpha} receptor knockout mice (TNF-{alpha}RKO) that lack both TNF-{alpha} receptor 1 (p55) and receptor 2 (p75) are protected from the fibroproliferative effects of inhaled asbestos fibers and have reduced levels of TGF-ß1 after exposure (14). These observations are also consistent with our study showing that inbred mouse strains that fail to develop fibroproliferative lesions consequent to asbestos exposure have reduced TNF-{alpha} and TGF-ß1 expression in their lungs (15). Most important is a recent paper showing that adenovirus transduction of TNF-{alpha} expression in lungs of normal rats induces TGF-ß1 production and interstitial fibrogenesis (3). We recently reported that TNF-{alpha} rapidly upregulates TGF-ß1, PDGF A, and PDGF B mRNAs in both primary mesenchymal and epithelial cells of the lung. TGF-ß1, in turn, increased PDGF B levels in bronchiolar alveolar epithelial cells (16). These results suggest that TNF-{alpha} may initiate or perpetuate a cytokine cascade that has been shown to be involved in the development of fibrosis. Clearly, the regulatory mechanisms underlying TNF-{alpha} induction of TGF-ß1 could be central to understanding the pathogenesis of IPF.

A key feature of IPF is the "fibroblastic focus" composed of fibroblasts and fibroblast-like cells (2). There is ample evidence suggesting that fibroblasts are the main cellular source of extracellular matrix deposition that typifies fibrosis, and it is clear that fibroblasts respond to factors produced by epithelial cells and themselves produce a number of factors including TGF-ß1 (17).

Mitogen-activated protein kinases (MAPKs) transduce extracellular signals to intracellular responses in fibroblasts and other cells (see review in Ref. 18). TNF-{alpha} has been observed to induce phosphorylation of MAPKs, which may contribute to both activation of transcription factors and mRNA stability for cytokine and other growth factor genes (19). MAPKs are a group of evolutionarily conserved serine and threonine kinases. The MAPKs are activated by dual-specificity (threonine and tyrosine) protein kinases called MAPK kinases or MEK. When phosphorylated, the MAPKs are capable in turn of phosphorylating a variety of diverse targets such as effector kinases and transcription factors. To date, three major types of MAPK cascades have been reported in mammalian cells that respond synergistically to different upstream signals. The extracellular signal–regulated kinases (ERKs) are implicated in the control of cell growth and division and cell differentiation. ERKs become active in response to cytokines, phorbol esters, and growth factors, whereas the JNK and p38 MAPKs are active primarily in response to interleukin (IL)-1ß, TNF-{alpha}, osmotic stress, and ultraviolet light (18).

The goal of the study presented here is to elucidate the mechanism(s) by which TNF-{alpha} induces TGF-ß1 expression in lung fibroblasts. To determine which signal transduction pathways regulated TGF-ß1 levels, fibroblasts were treated with various inhibitors of protein kinases followed by real-time RT-PCR to measure TGF-ß1 mRNA levels and ELISA for protein analysis. The results indicate that TNF-{alpha} activation of the ERK-specific MAPK signal transduction pathway leads to increased steady-state levels of TGF-ß1 mRNA that precedes an increase in TGF-ß1. Furthermore, we show that the increase in TGF-ß1 mRNA is primarily due to increased stabilization of TGF-ß1 mRNA when the cells are treated with TNF-{alpha}.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents and Antibodies
Recombinant, murine TNF-{alpha} and recombinant, active, human TGF-ß1 were purchased from Peprotech (Rocky Hill, NJ). MEK inhibitors PD98059 and U0126, as well as specific inhibitors of p38 kinase (SB203580), c-jun NH2-terminal kinase (JNK) (SP600125) and protein kinase C (PKC) (GF109203X), were purchased from Calbiochem Co. (La Jolla, CA). The antibodies to phospho-p44/42 (ERK) MAPK (Thr202/Tyr204), total p44/42 (ERK) MAPK, phospho-p38 MAPK (Thr180/Tyr182), total p38 MAPK, phospho-JNK (Thr183/Tyr185), and ß-actin were from Cell Signaling (Beverly, MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture
Primary cultures of mouse lung fibroblasts (MLFs) were established as previously described (16). In this study, we used MLF cultures at passages 2 and 3. Swiss 3T3 fibroblasts were obtained from American Type Culture Collection (no. CCL-92; ATCC, Manassas, VA). Swiss 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% bovine calf serum (Invitrogen, Carlsbad, CA), L-glutamine, nonessential amino acids, penicillin (100 U/ml), and streptomycin (100 µg/ml). Fresh growth media was added every 3–4 d and cells were kept subconfluent until used for experiments. All experiments using Swiss 3T3 fibroblasts were done with cells at passage 5–13. Confluent monolayers were rendered quiescent in media containing 1% bovine calf serum for Swiss 3T3 fibroblasts or 1% fetal bovine serum for MLFs for 48 h before use in experiments. All experiments were performed in media containing 1% serum.

Transfection
DNA constructs encoding a mutant form of MEK1 with aspartic acid substituting for Ser218 and Ser222 and tagged at the N terminus with the influenza virus hemagglutinin (HA) epitope (pHA-MEK1 S218/222DD) (20) were kindly provided by A. Catling (Louisiana State University Health Sciences Center, New Orleans, LA). Transient transfections were performed with appropriate controls using the Amaxa nucleofection technology (Amaxa, Koeln, Germany). Cells were resuspended in solution from nucleofector kit R, following the Amaxa guidelines for cell line transfection. Briefly, 100 µl of 1 x 106 cell suspension mixed with 2 µg plasmid DNA was transferred to the provided cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa). Cells were transfected using the U-30 pulsing parameter and were immediately transferred into wells containing 37°C prewarmed culture medium in 6-well plates. After transfection, cells were cultured for 5 h, washed twice with PBS, and then cultured a further 48 h in media containing 1% calf serum before analyzing by Western blot or quantitative real-time RT-PCR.

Analysis of TGF-ß1 Protein
Total TGF-ß1 in the cell culture supernatant was measured by specific enzyme-linked immunosorbent assay (human TGF-ß1 DuoSet ELISA Development System; R&D Systems, Minneapolis, MN) of cell culture supernatant samples collected from quiescent fibroblasts, stimulated under conditions of low serum. This assay cross-reacts with murine TGF-ß1 and has < 1% cross-reactivity for TGF-ß2 and TGF-ß3. To activate latent TGF-ß1 to the immunoreactive form, cell culture supernatants were acidified and then neutralized according to the manufacturer's recommendations. TGF-ß1 concentration was normalized to total protein as determined by Micro BCA Protein Assay Kit (Pierce, Rockford, IL). Data are expressed as pg TGF-ß1/ml per mg of protein. The background level of latent TGF-ß1 in control medium was less than 150 pg/ml and no active TGF-ß1 was detected.

Analysis of Steady-State mRNA Levels and Stability
TGF-ß1 mRNA was quantified by real-time RT-PCR. Total RNA was isolated employing the UltraSpec RNA isolation reagent (Biotecx Laboratories, Houston, TX) according to the manufacturer's recommendations. Reverse transcription of 250 ng total RNA was performed in a total volume of 20 µL using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's recommendations. One microliter of cDNA was PCR-amplified in 20-µl reactions containing primers at 250 nM in iQ SYBR Green Supermix (Bio-Rad). Aliquots of cDNA were diluted 1:100 for 18S rRNA amplification. PCR was performed for 35 cycles consisting of 95°C for 15 s and 60°C for 45 s using an iCycler iQ Real Time Detection System (Bio-Rad). Sequences of primers used for PCR are presented in Table 1 (21, 22). Dilution curves showed that PCR efficiency was 96–100% for all primer sets used. All samples were run in triplicate for initial experiments and duplicate for confirmatory experiments on the same plate for each primer set. Negative controls, such as cDNA reactions without reverse transcriptase or RNA, and PCR mixtures lacking cDNA were included in each PCR to detect possible contaminants. After amplification, specificity of the reaction was confirmed by melt curve analysis. Relative quantitation was determined using the comparative CT method with data normalized to 18S rRNA and calibrated to the average {Delta}CT of untreated controls. The assay is highly sensitive, detecting as little as 1 x 10–5 picograms of in vitro transcribed TGF-ß1 RNA, and displays linearity over 5 logs (1 pg–1 x 10–5 pg).


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  		TABLE 1. Pcr primer sequences

 
For quantitation of the rate of decay of TGF-ß1 mRNA, quiescent fibroblasts were stimulated with 10 ng/ml TNF-{alpha} for 12 h. Transcription was then inhibited by the addition of the RNA polymerase II–specific inhibitor, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB; Sigma-Aldrich) at a final concentration of 50 µM. At various times after the addition of DRB, medium was removed and total RNA was isolated as described above. The rate of mRNA degradation was subsequently determined using real-time RT-PCR as previously described (23).

Western Blot Analysis
Cell lysates were prepared in lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) and sonicated briefly. After centrifuging for 10 min at 14,000 x g, supernatants were collected and the protein concentration estimated using Micro BCA Protein Assay Reagent Kit (Pierce). Lysates were mixed with 4x SDS reducing sample buffer (62.5 mM Tris-HCl ph 6.8, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, 0.06% bromophenol blue), heated in a boiling water bath for 5 min and analyzed on 12% SDS-polyacrylamide gels. Proteins were transferred to 0.45 µm PVDF membranes by semi-wet electrophoretic transfer in an Invitrogen XCell II Blot Module at 30 volts for 1 h. The PVDF membranes were blocked for 1 h in TBST (20 mM Tris pH 7.6, 137 mM NaCl, 0.1% Tween 20) containing 5% bovine serum albumin and then hybridized overnight with the primary antibody. The primary antibodies were detected with anti-rabbit horseradish peroxidase–conjugated secondary antibody and developed by using enhanced chemiluminescense (ECL) system (Amersham, Piscataway, NJ). Incubation with the primary antibodies diluted 1:1,000 in blocking buffer was overnight at 4°C followed by incubation with the secondary antibody diluted 1:5,000 for 1 h. The ECL-treated membranes were exposed to X-ray film, stripped for 30 min at 50°C in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl (pH 6.8), and rehybridized.

Statistical Analysis
All values are expressed as means ± SEM where n = 3 or 4. An unpaired Student's t test was used to assess the difference between two groups. ANOVA was performed when more than two groups were compared with a single control, and Tukey post tests were used to assess differences between individual groups within the set. Differences were considered statistically significant when P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
TNF-{alpha} Induces Expression of TGF-ß1 mRNA and Protein in Fibroblasts
We previously showed that treatment of primary mouse lung fibroblasts (MLFs) and bronchiolar-alveolar epithelial cells with TNF-{alpha} results in upregulation of TGF-ß1 mRNA expression (16). To further study the kinetics of TNF-{alpha} induction of TGF-ß1 mRNA in fibroblasts, we treated MLFs isolated from adult C57 mice with 5 ng/ml recombinant murine TNF-{alpha} for the indicated times and then measured TGF-ß1 mRNA levels by quantitative real-time RT-PCR. Figure 1 shows that treatment of MLFs with 5 ng/ml TNF-{alpha} results in increased levels of TGF-ß1 mRNA as early as 2 h after exposure, and significantly increased by 4 h, reaching 200% of untreated controls by 12 h, and TGF-ß1 levels remained elevated for 24 h, in agreement with our previous data showing TNF-{alpha} induction of TGF-ß1 gene expression at these time points using Northern blot analysis (16).



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  		Figure 1. TNF-{alpha} induces expression of TGF-ß1 mRNA in primary mouse lung fibroblasts as early as 2 h after treatment. Fibroblasts isolated from the lungs of adult C57 mice were rendered quiescent and treated with 5 ng/ml TNF-{alpha} (solid bars) or media alone (open bars). At the indicated times, total RNA was isolated and TGF-ß1 mRNA measured by quantitative, real-time RT-PCR. TGF-ß1 mRNA levels were significantly increased by 2 h after treatment and remained elevated for more than 24 h. Data are expressed as the mean ± SEM of duplicate plates of each treatment group at each time point from three separate experiments. *P < 0.05 versus media alone at each time point.

 
The specificity of the response to TNF-{alpha} was investigated by using MLFs isolated from mice that are genetically engineered to lack both TNF-{alpha} receptor 1 and receptor 2 (TNF-{alpha}RKO) mice (Jackson Laboratory, Bar Harbor, MA). These cells were treated with 5 ng/ml TNF-{alpha} or, as controls, recombinant, active, human TGF-ß1 and phorbol 12-myristate 13-acetate (PMA), which are known inducers of TGF-ß1 expression (24). Real-time RT-PCR analysis of total RNA isolated 12 h after initiation of treatment shows that MLFs from TNF-{alpha}RKO mice fail to upregulate TGF-ß1 mRNA in response to TNF-{alpha}, but respond to TGF-ß1 or PMA similar to MLFs from normal C57 mice (Figure 2). These results show that MLFs from TNF-{alpha}RKO mice have intact signaling responses capable of upregulating their endogenous TGF-ß1 gene, therefore the observed induction of TGF-ß1 mRNA in normal MLFs is due to specific interaction between TNF-{alpha} and its membrane-bound receptors. It is not clear from these experiments if one or both TNF-{alpha} receptors are required to mediate TNF-{alpha} induction of TGF-ß1 expression.



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  		Figure 2. TNF-{alpha} signaling through TNF receptors is required for TNF-{alpha} induction of TGF-ß1 expression. Fibroblasts isolated from the lungs of TNF-{alpha}RKO (open bars) or C57 (solid bars) mice were rendered quiescent and then treated with TNF-{alpha}, PMA, or TGF-ß1, each at a concentration of 5 ng/ml, for 12 h. As a negative control, cells were treated with media alone (control). TGF-ß1 mRNA was measured by quantitative real-time RT-PCR. Fibroblasts from TNF-{alpha}RKO mice failed to upregulate TGF-ß1 mRNA in response to TNF-{alpha}, but responded to PMA and TGF-ß1 similar to fibroblasts from C57 mice. Data are expressed as the mean ± SEM of measurements from three plates for each treatment group. *P < 0.05 versus media alone, {dagger}P < 0.05 versus TNF-{alpha}RKO + TNF-{alpha}.

 
Studies to dissect the molecular mechanism(s) of TNF-{alpha} regulation of TGF-ß1 are facilitated by using a cell line rather than primary cells, and we have found that Swiss 3T3 fibroblasts are sufficiently similar to MLFs for the experiments described here. For example, Figure 3A shows that TNF-{alpha} induces TGF-ß1 mRNA expression in Swiss 3T3 fibroblasts in a dose-dependent manner by 6 h after treatment, similar to that observed in MLFs (Figure 1). TGF-ß1 mRNA expression was increased at 6 h after treatment with as little as 1 ng/ml TNF-{alpha} and further increased when 5 ng/ml TNF-{alpha} was added. TGF-ß1 mRNA levels reached 400–500% of control levels and remained significantly elevated for 48 h in cells treated with 10 ng/ml TNF-{alpha}.




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  		Figure 3. Swiss 3T3 fibroblasts also upregulate expression of TGF-ß1 in response to TNF-{alpha}. Quiescent Swiss 3T3 cells were treated with TNF-{alpha} (1, 5 or 10 ng/ml) for the indicated times. As a negative control, cells were treated with media alone (control). (A) TGF-ß1 mRNA was then measured by quantitative real-time RT-PCR. (B) TGF-ß1 in cell culture supernatants was measured by ELISA. TGF-ß1 mRNA was significantly increased in Swiss 3T3 cells within 6 h after treatment with TNF-{alpha} and remained elevated for 48 h. Increased levels of secreted TGF-ß1 were also detected after treatment with TNF-{alpha} for 24 or 48 h. Data are expressed as the mean ± SEM of duplicate plates for each treatment group at each time point from two separate experiments. *P < 0.05 versus media alone at each time point.

 
To determine if TNF-{alpha} induction of TGF-ß1 mRNA translates into increased TGF-ß1 protein production, TGF-ß1 in cell culture supernatants was measured by ELISA. Before acidification, conditioned media from either control or TNF-{alpha}–treated cells produced undetectable levels of TGF-ß1; however, after acidification the conditioned medium from control cells contained low levels of detectable TGF-ß1 and treatment with 1 ng/ml TNF-{alpha} significantly increased the levels of TGF-ß1 in a dose-dependent manner (Figure 3B). Therefore, the rapid TNF-{alpha}–mediated induction of TGF-ß1 mRNA is paralleled by an increase in TGF-ß1 protein that was not detected until 24 h after TNF-{alpha} treatment.

TNF-{alpha} Activates MAPK Pathways in Swiss 3T3 Fibroblasts
TNF-{alpha} has been shown to activate numerous signaling cascades including ERK1/ERK2, JNK, and p38 MAP kinases (19). TNF-{alpha} activation of these kinases in Swiss 3T3 fibroblasts was assessed by Western blotting using the corresponding phosphospecific and nonphosphospecific antibodies. The phosphospecific antibodies detect the phosphorylated (activated) forms of ERK1/ERK2, p38, and JNK, whereas the corresponding nonphosphospecific antibodies detect total protein, independent of phosphorylation status. As shown in Figure 4A, ERK1 (p44) and ERK2 (p42) are transiently phosphorylated as early as 5 min after treatment with 5 ng/ml TNF-{alpha}, with maximal phosphorylation evident by 15 min and decaying thereafter. The time course of activation is indistinguishable between ERK1 and ERK2. Figures 4B and 4C demonstrate activation of p38 and JNK, respectively, with similar kinetics. Two isoforms of JNK, p54 (JNK1) and p46 (JNK2), are evident and correspond to the specificity of the antibody.



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  		Figure 4. TNF-{alpha} activates MAPKs in Swiss 3T3 fibroblasts. Quiescent Swiss 3T3 cells were treated with TNF-{alpha} (5 ng/ml) for the indicated periods. Activities of ERK1/2, p38 MAPK, and JNK were determined by Western blotting whole cell lysates using antibodies specific for phosphorylated, activated forms (top panels) of ERK1/2 (A), p38 (B), and JNK (C). In the corresponding bottom panels, blots were stripped and reprobed with antibodies to total ERK (A), total p38 (B), or ß-actin (C) to show equal amounts of loaded proteins. TNF-{alpha} induced phosphorylation of ERK1/2, p38, and JNK within 5 min and reached peak levels between 15 and 30 min.

 
PD98059 and UO126 Inhibit TNF-{alpha} Induction of TGF-ß1 in Fibroblasts
Having shown that MAP kinases are phsophorylated after TNF-{alpha} treatment, we asked whether these kinases are required for TNF-{alpha} induction of TGF-ß1 expression. Swiss 3T3 fibroblasts were pre-treated with increasing doses of selective kinase inhibitors before addition of 10 ng/ml TNF-{alpha} for an additional 12 h. PD98059 (10–50 µM), a selective inhibitor of ERK activation, inhibited the induction of TGF-ß1 mRNA by TNF-{alpha} in a dose-dependent fashion (Figure 5A). In agreement with this result, U0126 (0.1–10 µM), a mechanistically different inhibitor of ERK activation, also inhibited TNF-{alpha} induction of TGF-ß1 mRNA in a dose-dependent fashion (Figure 5B). Neither PD98059 nor U0126, at the doses used in this study, were toxic to the cells as measured by MTT assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI), nor were cellular levels of 36B4 or ß-actin mRNA affected. PD98059 and U0126 also blocked the accumulation of TGF-ß1 protein in cell culture supernatants of cells treated with TNF-{alpha} (Figure 5C). In contrast, specific inhibitors of p38 (SB203580) and JNK (SP600125) had no effect on TNF-{alpha}–induced expression of TGF-ß1 (data not shown), even though each of these inhibitors effectively blocked activation of their targets as demonstrated by Western blot. TNF-{alpha} has also been reported to activate PKC (25), and activation of PKC has been linked to induction of TGF-ß1 expression (24); therefore, we tested the ability of GF109203X (1–50 µM), a highly selective, cell-permeable inhibitor of PKC{alpha}, ßI, ßII, {gamma}, {delta}, and {epsilon}, but found no inhibition of TNF-{alpha}–induced TGF-ß1 expression (data not shown). DMSO (0.1%), the vehicle in which the inhibitors were dissolved, had no effect on TGF-ß1 mRNA or protein levels. We confirmed the relevance of these results in primary MLFs. PD98059 and U0126 effectively blocked TNF-{alpha}–induced increases in TGF-ß1 expression in primary MLFs when added at 10 µM (Figure 5D). These results highlight an essential role for the ERK-specific MAPK pathway in TNF-{alpha}–induced expression of TGF-ß1.






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  		Figure 5. Specific inhibitors of ERK activation block TNF-{alpha} induction of TGF-ß1 in fibroblasts. Quiescent Swiss 3T3 fibroblasts were treated with increasing concentrations of PD98059 (A) or U0126 (B) or 0.1% DMSO as vehicle control, for 30 min before stimulation with TNF-{alpha} (10 ng/ml) for 12 h. Total RNA was isolated and TGF-ß1 mRNA measured by quantitative real-time RT-PCR. (C) The effect of PD98059 (PD, 50 µM) or U0126 (U, 2.5 µM) on TNF-{alpha} induction of TGF-ß1 in supernatants from Swiss 3T3 cells was measured by ELISA. (D) Quiescent primary MLFs were treated with PD98059 (PD) or U0126 (U), each at 10 µM, or 0.1% DMSO as vehicle control for 30 min before stimulation with TNF-{alpha} (10 ng/ml) for 12 h and then analyzed as described above. Inhibition of ERK activation blocked TGF-ß1 expression in both MLFs and Swiss 3T3 fibroblasts. Data are expressed as the mean ± SEM of measurements from three individual plates at each concentration in one experiment. These results are typical of three separate experiments. *P < 0.05 versus untreated control, {dagger}P < 0.05 versus + TNF-{alpha}, {ddagger}P < 0.05 PD98059 + TNF-{alpha} versus UO126 + TNF-{alpha}.

 
TNF-{alpha} Stabilizes TGF-ß1 mRNA
To examine if TNF-{alpha} also affects TGF-ß1 transcript stability, cells that had been grown in 10 ng/ml TNF-{alpha} for 12 h were treated with DRB (50 µM) for an additional period up to 24 h to inhibit gene transcription. At the indicated times, TGF-ß1 and MMP9 (as control) mRNA levels were measured by real-time RT-PCR. DRB treatment resulted in a progressive decay of TGF-ß1 mRNA over time (Figure 6A) in control cells with a half-life of ~ 15 h. However, there was no decline in TGF-ß1 mRNA levels in TNF-{alpha}–treated cells over the duration of the experiment. In contrast, MMP9 mRNA levels decayed at approximately the same rate in control and TNF-{alpha}–treated cells (Figure 6B). These results suggest that TNF-{alpha} selectively stabilizes TGF-ß1 mRNA in fibroblasts.




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  		Figure 6. TNF-{alpha} stabilizes TGF-ß1 mRNA. Quiescent Swiss 3T3 cells were stimulated with (solid line) or without TNF-{alpha} (dashed line) for 12 h and further incubated with DRB (50 µM) through a 24-h time period. Total RNA was sequentially extracted at the time points indicated and TGF-ß1 (A) and, as control, MMP9 (B) mRNA abundance was measured by quantitative real-time RT-PCR. The results are expressed as the percentage mRNA remaining relative to the corresponding level before addition of DRB. TNF-{alpha} increased the stability of TGF-ß1 mRNA but not MMP9 mRNA. Data are expressed as the mean ± SEM of measurements from four individual plates for each treatment group at each time point in one experiment and are typical of three separate experiments.

 
ERK Activation Is Sufficient for TGF-ß1 mRNA Stabilization
To demonstrate whether ERK activation was sufficient for TGF-ß1 mRNA stabilization, we transfected Swiss 3T3 fibroblasts with a cDNA construct encoding a constitutively active form of MEK1 (HA-MEK1 S218/222DD, caMEK1) that selectively activates ERK. Cells were also transfected with pmaxGFP (Amaxa) as transfection control. Fluorescence microscopy revealed that ~ 70–80% cells were transfected. Western blot analyses showed that caMEK1 was efficiently expressed in transfected cells and resulted in persistent phosphorylation of ERK1/2 (Figure 7A). The effect on TGF-ß1 mRNA stability and levels was analyzed 48 h after transfection as described above. TGF-ß1 mRNA levels were increased by 275% in transfected cells, whereas MMP9 and 36B4 levels were unaffected (Figure 7B). Importantly, TGF-ß1 mRNA was stabilized in caMEK1-transfected cells but not in GFP-transfected cells (Figure 7C).





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  		Figure 7. TGF-ß1 mRNA is stabilized in Swiss 3T3 fibroblasts transfected with constitutively active MEK1. Swiss 3T3 fibroblasts were transfected with 2 µg pHA-MEK1 S218/222DD (lane 1) or pmaxGFP (lane 2) and 5 h later serum starved for an additional 48 h. (A) Whole cell lysates were analyzed by Western blotting using antibodies specific for the HA tag (top panel) or phosphorylated (activated) ERK1/2 (middle panel). The blot was stripped and reprobed with antibodies to total ERK to show equal amounts of loaded proteins (bottom panel). HA-MEK1 S218/22D was efficiently expressed and persistently phosphorylated ERK1/2. (B) Total RNA was isolated and TGF-ß1, MMP9, and 36B4 mRNAs were measured by quantitative real-time RT-PCR. Persistent activation of ERK1/2 increased the levels of TGF-ß1 mRNA but not MMP9 mRNA or 36B4 mRNA. (C) Serum-starved cells were further incubated with DRB (50 µM) through a 24-h time period. Total RNA was sequentially extracted from pHA-MEK1 S218/222DD (solid line) or pmaxGFP (dashed line) transfected cells at the time points indicated, and TGF-ß1 mRNA abundance was measured by quantitative real-time RT-PCR. The results are expressed as the percentage mRNA remaining relative to the corresponding level before addition of DRB. Persistent activation of ERK1/2 increased the stability of TGF-ß1 mRNA. Data are expressed as the mean ± SEM of measurements from three individual plates at each time point. *P < 0.05 versus pmaxGFP-transfected cells.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Despite the growing evidence that TNF-{alpha} influences TGF-ß1 expression, no information apparently was available to explain the molecular basis through which TNF-{alpha} regulates TGF-ß1. In the present investigation, we provide evidence that TNF-{alpha} activates the ERK-specific MAPK signal transduction pathway resulting in stabilization of TGF-ß1 mRNA that leads to increased TGF-ß1 production by primary mouse lung fibroblasts and a fibroblast cell line.

TGF-ß1 production may be controlled at the levels of transcription, translation, secretion of preformed protein and activation of the latent protein to its active form as addressed by a number of investigators (12, 26, 27). Data presented here show for the first time that TNF-{alpha} rapidly (at 4–6 h) upregulates expression of TGF-ß1 mRNA resulting in increased production of TGF-ß1 protein in MLFs and Swiss 3T3 cells, an embryonic fibroblast cell line. As expected, TNF-{alpha} failed to upregulate TGF-ß1 in fibroblasts from lungs of TNF-{alpha}RKO mice, demonstrating that TGF-ß1 upregulation was not due to manipulation of the cells or a contaminating substance in the TNF-{alpha} preparation. These data agree with a number of studies that demonstrated TNF-{alpha} induction of TGF-ß1 mRNA in a variety of other cell types including microglial cells (28), mature adipocytes (29), human proximal tubular cells (30), and rat pulmonary artery endothelial cells (31). Yet, there are several studies that reported no increase in (or reduced) TGF-ß1 expression consequent to TNF-{alpha} treatment (32, 33). These conflicting data may be explained by differences in the experimental protocol or in the cell types used as regulation of TGF-ß1 may be cell type–specific.

We typically see a 200–400% increase in steady-state TGF-ß1 mRNA level in fibroblasts after 6–12 h exposure to TNF-{alpha} (Figures 1 and 3A). The steady-state level of mRNA results from a combination of transcriptional and post-transcriptional events. In preliminary studies, we performed transient transfections of Swiss 3T3 fibroblasts with cDNA constructs consisting of the mouse or human TGF-ß1 promoter driving expression of luciferase and failed to show TNF-{alpha} activation of the TGF-ß1 promoter. In concert with these findings, Kwong and colleagues (32) have recently reported no activation of the TGF-ß1 promoter in transiently transfected cultures of human alveolar epithelial A549 cells after treatment with TNF-{alpha}. These data suggest that TNF-{alpha} does not activate transcription from the TGF-ß1 promoter but instead may upregulate TGF-ß1 by a post-transcriptional mechanism. Alternatively, the promoter-reporter assay may not accurately reflect regulation of the endogenous TGF-ß1 gene, where chromatin effects may represent another level of control. Additional experiments such as nuclear run-on assays are needed to clarify the role of transcription in TNF-{alpha} induction of TGF-ß1.

In the present study, we observed significant stabilization of the TGF-ß1 mRNA in response to TNF-{alpha}. In untreated cells, the half-life of TGF-ß1 mRNA was ~ 15 h, similar to what has been reported elsewhere (34), whereas in TNF-{alpha}–treated cells there was no detectable degradation of TGF-ß1 mRNA over the 24-h duration of the experiment. Studies measuring mRNA half-life are usually performed using actinomycin D as the inhibitor of transcription; however, in our experiments the combination of TNF-{alpha} and actinomycin D was very toxic, making time courses of more than 6 h impossible. The mechanism responsible for TNF-{alpha}–induced stabilization of TGF-ß1 mRNA could be general downregulation of mRNA decay, although this is unlikely because MMP9 mRNA stability was not altered by TNF-{alpha}.

TNF-{alpha} is a pleiotropic cytokine, and most cells show at least some TNF-{alpha} responsiveness (19). After binding of plasma membrane receptors by TNF-{alpha}, a series of TNF-{alpha}–related proteins are recruited to cytoplasmic domains of the receptors, triggering cell-specific signal transduction pathways resulting in regulation of a large spectrum of cellular genes by both transcriptional and post-transcriptional mechanisms (19). Several groups have demonstrated a major role for JNK (35), p38 MAPK (36), and ERK in the regulation of mRNA stabilization (37, 38). By Western blot analysis, we observed a strong and rapid activation of ERK1/ERK2, p38, and JNK MAPKs in fibroblasts treated with TNF-{alpha}. To determine if MAPKs are involved in TNF-{alpha} regulation of TGF-ß1, we used a panel of well-characterized pharmacologic inhibitors. Inhibitors of the p38 and JNK MAPK pathways had no effect on TNF-{alpha} induction of TGF-ß1. In contrast, inhibition of the ERK pathway by PD98059 or UO216 markedly reduced TNF-{alpha} induction of TGF-ß1 (Figures 5A–5D). PD98059 blocks the activation of the upstream kinase (MEK1/2) that phosphorylates and activates ERK1/ERK2, whereas U0126 inhibits MEK1/2 and consequently prevents activation of ERK1/ERK2 (39). U0126 exhibited a stronger inhibitory effect than PD98059 in the cells used in this study. Moreover, expression of constitutively active MEK1 was sufficient to stabilize TGF-ß1 mRNA and induce TGF-ß1 expression (Figure 7). Thus, our data strongly support involvement of the ERK pathway in TNF-{alpha} upregulation of TGF-ß1. Involvement of the MEK/ERK pathway has been implicated in the induction of TGF-ß1 by other factors such as Angiotensin II in vascular smooth muscle cells (40), high glucose (41) or reactive oxygen species (42) in mesangial cells, and TGF-ß1 in intestinal epithelial cells (43). Together, these observations suggest that the MEK/ERK pathway plays an important role in the induction of TGF-ß1 expression in a variety of cell types by multiple stimulants.

The PKC pathway that has been shown to be involved in TNF-{alpha} upregulation of several genes (25) and activation of PKC has been implicated in upregulation of TGF-ß1 by PMA, Angiotensin II, and high glucose in mesangial cells (24). Therefore, we tested whether an inhibitor of PKC would interfere with TNF-{alpha} induction of TGF-ß1, and found that bisindolylmaleimide I (GF109203X), a selective inhibitor of the {alpha}, ß I, ß II, {gamma}, {delta}, and {epsilon} isoforms of PKC, had no effect on TNF-{alpha} induction of TGF-ß1. However, we cannot rule out the possibility that other PKC isoforms such as PKC-{zeta} may be involved because the inhibitor does not affect this isoform. Bisindolylmaleimide I may also inhibit protein kinase A (PKA) when used at higher concentrations (Ki = 2 µM). However, even when used at 10 µM we did not observe any inhibition of TNF-{alpha} induction of TGF-ß1, suggesting that PKA is likewise not involved.

Many cytokines are regulated at the post-transcriptional level, but the precise mechanisms leading to either enhanced RNA stabilization or decay remain unclear. The mRNA decay rates are regulated by cis-acting sequence determinants, mRNA-binding proteins, endo- and exo-RNases, and translation (44). TNF-{alpha} has been shown to upregulate a number of genes by stabilizing mRNA through the modulation of RNA-binding proteins (45, 46). Furthermore, ERK signaling has been associated with increased expression of mRNA-binding proteins such as nucleolin (47) and Hur and increased binding of these proteins to adenylate, uridylate-rich (AU-rich) instability elements (AREs). The presence of AREs in the 3' UTRs of mRNAs correlates with the short half-lives observed for transiently expressed genes such as cytokines, growth factors, and transcription factors (44). The TGF-ß1 mRNA has a single AUUUA motif, yet no ARE has been identified in its 3'UTR, which could account for TGF-ß1's relatively long half-life, even under unstimulated conditions. Sequences within the coding region termed "coding region instability determinant (CRD)" (48) and stem-loop structures formed by the 3'UTR (44) of some mRNAs have also been shown to act as instability elements. Although a number of secondary structures can theoretically form within the TGF-ß1 3'UTR, thus providing binding sites for regulatory proteins, it remains unclear whether these, or sequences in the coding region, are either necessary or sufficient for increasing stability or whether specific RNA binding proteins are involved. We are currently working to identify the cis elements involved in regulation of TGF-ß1 mRNA stability and investigating the possibility that TNF-{alpha} activation of ERK signaling upregulates specific mRNA-binding proteins that modulate TGF-ß1 mRNA stability.

It is becoming clear that expression of TGF-ß1 plays an important role in the pathogenesis of IPF, and the data presented here along with other pertinent studies (3) implicate TNF-{alpha} as a key cytokine in this process. Several studies have also implicated the MEK/ERK pathway in the pathogenesis of IPF (49, 50). Our findings provide evidence that the fibrotic properties of TNF-{alpha} may be mediated at least in part by its ability to upregulate TGF-ß1 through a complex mechanism involving the ERK-specific MAPK signaling pathway. This increased understanding of the mechanisms involved in TNF-{alpha} regulation of TGF-ß1 may lead to new targeting strategies for treatment of IPF.


   Footnotes
 
This work was supported by grants NIH/NIEHS ES06766 and NIH/NHLBI HL60532 (A.R.B.).

Conflict of Interest Statement: D.E.S. has no declared conflicts of interest; M.F. has no declared conflicts of interest; D.P. has no declared conflicts of interest; and A.R.B. has no declared conflicts of interest.

Received in original form September 13, 2004

Received in final form January 12, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Morris G, Brody A. Molecular mechanisms of particle induced lung disease. In: Rom WN, editor. Environmental and occupational medicine, 3rd ed. Philadelphia: Lippincort-Raven; 1998. pp. 305–333.
  2. Selman M, Thannickal VJ, Pardo A, Zisman DA, Martinez FJ, Lynch JP III. Idiopathic pulmonary fibrosis: pathogenesis and therapeutic approaches. Drugs 2004;64:405–430.[CrossRef][Medline]
  3. Sime PJ, Marr RA, Gauldie D, Xing Z, Hewlett BR, Graham FL, Gauldie J. Transfer of tumor necrosis factor-{alpha} to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-b1 and myofibroblasts. Am J Pathol 1998;153:825–832.[Abstract/Free Full Text]
  4. Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-ß1 induces prolonged severe fibrosis in rat lung. J Clin Invest 1997;100:768–776.[Medline]
  5. Warshamana GS, Pociask DA, Fisher KJ, Liu JY, Sime PJ, Brody AR. Titration of non-replicating adenovirus as a vector for transducing active TGF-beta1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice. Int J Exp Pathol 2002;83:183–201.[CrossRef][Medline]
  6. Fujita M, Shannon JM, Morikawa O, Gauldie J, Hara N, Mason RJ. Overexpression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor-beta. Am J Respir Cell Mol Biol 2003;29:669–676.[Abstract/Free Full Text]
  7. Pan LH, Ohtani H, Yamauchi K, Nagura H. Co-expression of TNF alpha and IL-1 beta in human acute pulmonary fibrotic diseases: an immunohistochemical analysis. Pathol Int 1996;46:91–99.[Medline]
  8. Allen JT, Spiteri MA. Growth factors in idiopathic pulmonary fibrosis: relative roles. Respir Res 2002;3:13.[CrossRef][Medline]
  9. Whyte M, Hubbard R, Meliconi R, Whidborne M, Eaton V, Bingle C, Timms J, Duff G, Facchini A, Pacilli A, et al. Increased risk of fibrosing alveolitis associated with interleukin-1 receptor antagonist and tumor necrosis factor-{alpha} gene polymorphisms. Am J Respir Crit Care Med 2000;162:755–758.[Abstract/Free Full Text]
  10. Verrecchia F, Wagner EF, Mauviel A. Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha. EMBO Rep 2002;3:1069–1074.[CrossRef][Medline]
  11. Hozumi A, Nishimura Y, Nishiuma T, Kotani Y, Yokoyama M. Induction of MMP-9 in normal human bronchial epithelial cells by TNF-alpha via NF-kappa B-mediated pathway. Am J Physiol Lung Cell Mol Physiol 2001;281:L1444–L1452.[Abstract/Free Full Text]
  12. Bartram U, Speer CP. The role of transforming growth factor ß in lung development and disease. Chest 2004;125:754–765.[Abstract/Free Full Text]
  13. Piguet PF, Collart MA, Grau GE, Kapanci Y, Vassalli P. Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. J Exp Med 1989;170:655–663.[Abstract/Free Full Text]
  14. Liu J-Y, Brass DM, Hoyle GW, Brody AR. TNF-{alpha} receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. Am J Pathol 1998;153:1839–1847.[Abstract/Free Full Text]
  15. Brass D, Hoyle G, Poovey H, Liu J-Y, Brody A. Reduced TNF-{alpha} and TGF-ß1 expression in the lungs of inbred mice that fail to develop fibroproliferative lesions consequent to asbestos exposure. Am J Pathol 1999;154:853–862.[Abstract/Free Full Text]
  16. Warshamana GS, Corti M, Brody AR. TNF-alpha, PDGF, and TGF-beta(1) expression by primary mouse bronchiolar-alveolar epithelial and mesenchymal cells: TNF-alpha induces TGF-beta(1). Exp Mol Pathol 2001;71:13–33.[CrossRef][Medline]
  17. Phan SH. Fibroblast phenotypes in pulmonary fibrosis. Am J Respir Cell Mol Biol 2003;29:S87–S92.
  18. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002;298:1911–1912.[Abstract/Free Full Text]
  19. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ 2003;10:45–65.[CrossRef][Medline]
  20. Catling AD, Schaeffer HJ, Reuter CW, Reddy GR, Weber MJ. A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function. Mol Cell Biol 1995;15:5214–5225.[Abstract]
  21. Simpson DA, Feeney S, Boyle C, Stitt AW. Retinal VEGF mRNA measured by SYBR green I fluorescence: a versatile approach to quantitative PCR. Mol Vis 2000;6:178–183.[Medline]
  22. Lambert V, Munaut C, Jost M, Noel A, Werb Z, Foidart JM, Rakic JM. Matrix metalloproteinase-9 contributes to choroidal neovascularization. Am J Pathol 2002;161:1247–1253.[Abstract/Free Full Text]
  23. Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 2000;285:194–204.[CrossRef][Medline]
  24. Weigert C, Brodbeck K, Klopfer K, Haring HU, Schleicher ED. Angiotensin II induces human TGF-beta 1 promoter activation: similarity to hyperglycaemia. Diabetologia 2002;45:890–898.[CrossRef][Medline]
  25. Gorospe M, Kumar S, Baglioni C. Tumor necrosis factor increases stability of interleukin-1 mRNA by activating protein kinase C. J Biol Chem 1993;268:6214–6220.[Abstract/Free Full Text]
  26. Kim JK, Romeo D, Young DY, Park K. Transforming growth factor-ß: expression in normal and pathological conditions. Horm Res 1994;42:5–8.[Medline]
  27. Pociask DA, Sime PJ, Brody AR. Asbestos-derived reactive oxygen species activate TGF-beta1. Lab Invest 2004;84:1013–1023.[CrossRef][Medline]
  28. Chao CC, Hu S, Sheng WS, Tsang M, Peterson PK. Tumor necrosis factor-{alpha} mediates the release of bioactive transforming growth factor-ß in murine microglial cell cultures. Clin Immunol Immunopathol 1995;77:358–365.[CrossRef][Medline]
  29. Samad F, Uysal KT, Wiesbrock SM, Pandey M, Hotamisligil GS, Loskutoff DJ. Tumor necrosis factor alpha is a key component in the obesity-linked elevation of plasminogen activator inhibitor 1. Proc Natl Acad Sci USA 1999;96:6902–6907.[Abstract/Free Full Text]
  30. Phillips AO, Topley N, Steadman R, Morrisey K, Williams JD. Induction of TGF-ß1 synthesis in D-glucose primed human proximal tubular cells: differential stimulation by the macrophage derived pro-inflammatory cytokines IL-1ß and TNF{alpha}. Kidney Int 1996;50:1546–1554.[Medline]
  31. Phan SH, Gharaee-Kermani M, McGarry B, Kunkel SL, Wolber FW. Regulation of rat pulmonary artery endothelial cell transforming growth factor-ß production by IL-1ß and tumor necrosis factor-{alpha}. J Immunol 1992;149:103–106.[Abstract]
  32. Kwong KY, Literat A, Zhu NL, Huang HH, Li C, Jones CA, Minoo P. Expression of transforming growth factor beta (TGF-beta1) in human epithelial alveolar cells: a pro-inflammatory mediator independent pathway. Life Sci 2004;74:2941–2957.[CrossRef][Medline]
  33. Hodge S, Hodge G, Holmes M, Flower R, Scicchitano R. Interleukin-4 and tumour necrosis factor-alpha inhibit transforming growth factor-beta production in a human bronchial epithelial cell line: possible relevance to inflammatory mechanisms in chronic obstructive pulmonary disease. Respirology 2001;6:205–211.[CrossRef][Medline]
  34. Bascom CC, Wolfshohl JR, Coffey RJ Jr, Madisen L, Webb NR, Purchio AR, Derynck R, Moses HL. Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors beta 1 and beta 2. Mol Cell Biol 1989;9:5508–5515.[Abstract/Free Full Text]
  35. Chen CY, Del Gatto-Konczak F, Wu Z, Karin M. Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science 1998;280:1945–1949.[Abstract/Free Full Text]
  36. Winzen R, Kracht M, Ritter B, Wilhelm A, Chen CY, Shyu AB, Muller M, Gaestel M, Resch K, Holtmann H. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J 1999;18:4969–4980.[CrossRef][Medline]
  37. Esnault S, Malter JS. Granulocyte macrophage-colony-stimulating factor mRNA is stabilized in airway eosinophils and peripheral blood eosinophils activated by TNF-alpha plus fibronectin. J Immunol 2001;166:4658–4663.[Abstract/Free Full Text]
  38. Andoh A, Shimada M, Bamba S, Okuno T, Araki Y, Fujiyama Y, Bamba T. Extracellular signal-regulated kinases 1 and 2 participate in interleukin-17 plus tumor necrosis factor-alpha-induced stabilization of interleukin-6 mRNA in human pancreatic myofibroblasts. Biochim Biophys Acta 2002;1591:69–74.[Medline]
  39. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000;351:95–105.[CrossRef][Medline]
  40. Hamaguchi A, Kim S, Izumi Y, Zhan Y, Yamanaka S, Iwao H. Contribution of extracellular signal-regulated kinase to angiotensin II-induced transforming growth factor-beta1 expression in vascular smooth muscle cells. Hypertension 1999;34:126–131.[Abstract/Free Full Text]
  41. Isono M, Cruz MC, Chen S, Hong SW, Ziyadeh FN. Extracellular signal-regulated kinase mediates stimulation of TGF-beta1 and matrix by high glucose in mesangial cells. J Am Soc Nephrol 2000;11:2222–2230.[Abstract/Free Full Text]
  42. Grewal JS, Mukhin YV, Garnovskaya MN, Raymond JR, Greene EL. Serotonin 5–HT2A receptor induces TGF-beta1 expression in mesangial cells via ERK: proliferative and fibrotic signals. Am J Physiol 1999;276:F922–F930.
  43. Yue J, Mulder KM. Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway. J Biol Chem 2000;275:30765–30773.[Abstract/Free Full Text]
  44. Guhaniyogi J, Brewer G. Regulation of mRNA stability in mammalian cells. Gene 2001;265:11–23.[CrossRef][Medline]
  45. Nabors LB, Suswam E, Huang Y, Yang X, Johnson MJ, King PH. Tumor necrosis factor alpha induces angiogenic factor up-regulation in malignant glioma cells: a role for RNA stabilization and HuR. Cancer Res 2003;63:4181–4187.[Abstract/Free Full Text]
  46. Esnault S, Malter JS. Hyaluronic acid or TNF-alpha plus fibronectin triggers granulocyte macrophage-colony-stimulating factor mRNA stabilization in eosinophils yet engages differential intracellular pathways and mRNA binding proteins. J Immunol 2003;171:6780–6787.[Abstract/Free Full Text]
  47. Westmark CJ, Malter JS. Up-regulation of nucleolin mRNA and protein in peripheral blood mononuclear cells by extracellular-regulated kinase. J Biol Chem 2001;276:1119–1126.[Abstract/Free Full Text]
  48. Wisdom R, Lee W. The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors. Genes Dev 1991;5:232–243.[Abstract/Free Full Text]
  49. Yoshida K, Kuwano K, Hagimoto N, Watanabe K, Matsuba T, Fujita M, Inoshima I, Hara N. MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis. J Pathol 2002;198:388–396.[CrossRef][Medline]
  50. Robledo RF, Buder-Hoffmann SA, Cummins AB, Walsh ES, Taatjes DJ, Mossman BT. Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice. Am J Pathol 2000;156:1307–1316.[Abstract/Free Full Text]



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