Abstract
Many of the fibrogenic effects of transforming growth factor-β (TGF-β) might be mediated by connective tissue growth factor (CTGF). The present study investigates the role of mitogen-activated protein (MAP) kinase in the expression of CTGF mRNA in the human lung fibroblast line, HFL-1. TGF-β1 enhanced CTGF mRNA levels in a time- and concentration-dependent manner, and this enhancement was also dependent upon transcription. Inhibition of p38 MAP kinase or extracellular signal–regulated kinase (ERK) activation did not affect TGF-β1–induced CTGF expression. On the other hand, specific inhibitors of phosphatidylinositol 3-kinase (PI3K) suppressed TGF-β1–induced CTGF expression in a concentration-dependent manner. TGF-β1 activated c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, but not ERK in HFL-1 cells. PI3K inhibitors dose-dependently suppressed TGF-β1–induced JNK, but not p38 MAP kinase activation. Finally, JNK1 and JNK2 antisense oligonucleotides attenuated cellular levels of JNK1 and JNK2 protein, respectively, and repressed TGF-β1–induced CTGF expression. These results suggest that TGF-β1–induced CTGF mRNA expression is mediated through the JNK-dependent pathway, whereas p38 MAP kinase and ERK pathways minimally contribute.
Connective tissue growth factor (CTGF) is a potent and ubiquitous 38-kD protein that belongs the CCN family (CYR61, CTGF, and NOV) (1). It plays a unique role in fibroblast proliferation, cell adhesion, and the stimulation of extracellular matrix production (2, 3). CTGF was originally identified in conditioned medium from human umbilical vein endothelial cells (4), and its expression is selectively stimulated by transforming growth factor-β (TGF-β) in cultured fibroblasts (5). Due to its mitogenic action on fibroblasts and its ability to induce expression of the extracellular matrix molecules, collagen type 1, fibronectin, and integrin α5 (2), CTGF is presumed to play an important role in connective tissue cell proliferation and extracellular matrix deposition as mediators of TGF-β activation (6). CTGF seems to be an important player in the pathogenesis of various fibrotic disorders, because it is overexpressed in bleomycin-induced lung fibrosis (7), idiopathic pulmonary fibrosis (8), and other fibrotic disorders (9).
TGF-β signaling is initiated upon its binding to two cell membrane receptors termed type I (TβRI) and type II (TβRII). Both receptors are serine/threonine kinases, and binding to TGF-β results in their phosphorylation. SMAD proteins are the primary substrates of phosphorylated TGF-β receptors. The phosphorylation of SMAD2 or SMAD3 by TGF-β receptor causes its association with SMAD4. The SMAD complexes translocate into the nucleus, where they bind to cofactors that determine the choice of the target gene (10). Holmes and coworkers (11) have recently identified a functional SMAD binding site in the promoter of the human CTGF gene.
In addition to Smad proteins, mitogen-activated protein (MAP) kinases are involved in TGF-β signaling (12, 13). The MAP kinases are a family of serine-threonine protein kinases that are activated in response to a variety of extracellular stimuli. Extracellular signal–regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase constitute three major subfamilies of MAP kinases that appear to mediate cellular responses, including proliferation, differentiation, and apoptosis (14). ERK plays a major role in cell proliferation and differentiation, as well as in survival mediated by various growth factors (15). On the other hand, JNK and p38 MAP kinase are activated by various inflammatory cytokines and environmental stressors, and they play important roles in apoptosis and cytokine production (16, 17). However, recent studies suggest that ERK also plays an important role in the signal cascades leading to the induction of cytokines and chemical mediators (18). Thus, the MAP kinase superfamily regulates numerous cellular functions. However, the role of the MAP kinase superfamily in TGF-β–induced CTGF expression has not been determined.
The present study examines the roles of JNK, p38 MAP kinase, and ERK in TGF-β1–induced CTGF mRNA expression in human lung fibroblasts. In addition, we investigated effects of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), on TGF-β1–induced MAP kinase activation and CTGF expression.
Recombinant human TGF-β1 was obtained from R&D Systems (Minneapolis, MN). Actinomycin D was purchased from Sigma Chemical Co. (St. Louis, MO). Wortmannin and LY294002 to specifically inhibit PI3K activity (19, 20), SB203580 to specifically inhibit p38 MAP kinase activity (21), and PD 98059 to specifically inhibit mitogen-activated protein kinase kinase (MEK)-1 activity (22) were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA) and dissolved in dimethylsulfoxide (DMSO; Sigma). SP600125 to specifically inhibit JNK activity (23) was obtained from BIOMOL Research Labs., Inc. (Plymouth Meeting, PA) and dissolved in DMSO.
Human fetal lung fibroblasts (HFL-1) purchased from the American Type Culture Collection (Rockville, MD) were cultured in Ham's F12K medium (American Type Culture Collection) supplemented with 50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate (GIBCO BRL, Life Technologies, Inc., Rockville, MD) and 10% fetal bovine serum (FBS; Equitech-Bio. Inc., Ingram, TX) and maintained in humidified 5% CO2 at 37°C. After reaching confluence, the serum content of the medium was reduced to 0.4% FBS for 24 h. The cells were then stimulated with 1 ng/ml TGF-β1 to produce CTGF.
A human CTGF cDNA fragment containing residues 996–1,584 (Gen Bank accession no. NM 001901) (4) was amplified using the polymerase chain reaction (PCR). The synthesized sense and antisense PCR primers for CTGF were 5′-GTGGAGTATGTACCGACGGCC-3′ and 5′-ACAGGCAGGTCAGTGAGCACGC-3′ (Kurabo, Osaka, Japan) (24). The PCR products were fractionated on agarose gels, then cloned into the pGEM-t Easy vector (Promega Corp., Madison, WI). Sequencing analysis (Perkin Elmer Corp., PE Applied Biosystems, Foster City, CA) confirmed the identity of the amplified DNA. Human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was cloned as described (25). Complementary RNA (cRNA) probes for human CTGF and G3PDH were synthesized using [α-32P] UTP (ICN Biomedicals, Inc., Aurora, OH) and T7 RNA polymerase (Promega).
Northern blots proceeded as described (25). Total RNA was extracted from HFL-1 cells by a modification of the acid guanidinium thiocyanate-phenol-chloroform method (26). Aliquots of total RNA (5 μg) were resolved by electrophoresis in 1.4% agarose/0.66 M formaldehyde gels, and transferred onto nylon membranes (Hybond-N; Amersham Pharmacia Biotech, Tokyo, Japan). The membranes were hybridized with human CTGF and G3PDH cRNA probes at 60°C overnight. Autoradiographic signals were visualized by exposure to X-ray film (Hyperfilm; Amersham Pharmacia Biotech) at −70°C. The mRNA level was quantified by densitometry using NIH Image Version 1.62, and the optical density of the CTGF band was corrected by comparison with G3PDH mRNA in the same blot.
Western blotting proceeded as described (27). Briefly, cell lysates were electrotransferred, immunoblotted against primary antibodies, then specific reactive proteins were detected using enhanced chemiluminescence. The primary antibodies selectively recognized phosphorylated forms of JNK (anti–phospho-SAPK/JNK, Thr183/Tyr185), p38 MAP kinase (anti–phospho-p38 MAP kinase, Thr180/Tyr182), ERK (anti–phospho-p44/42 MAP kinase, Thr202/Tyr204), and SMAD2 (anti–phospho-SMAD2, Ser 465/467; all from Cell Signaling Technology, Inc., Beverly, MA). To determine the amounts of precipitated JNK, p38 MAP kinase, ERK, and SMAD2 blots were stripped and reprobed using phosphorylation-state independent anti-JNK (SC-571; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-p38 MAP kinase (Cell Signaling Technology), anti-ERK (Cell Signaling Technology), and anti-SMAD2 (Cell Signaling Technology) antibodies.
The kinase activity of MAP kinase–activated protein kinase (MAPKAPK2) was measured as previously described using MAPKAPK2 substrate peptide (KKLNRTLSVA), and anti-MAPKAPK2 polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) was used for immunoprecipitation (28).
JNK phosphorothioate antisense oligonucleotides were synthesized and purified by high-performance liquid chromatography (Kurabo) as described (29). The specific antisense oligonucleotides were as follows: antisense JNK1 (AS-JNK1) 5′-CTCTCTGTAGGCCCGCTTGG-3′ and antisense JNK2 (AS-JNK2) 5′-GTCCGGGCCAGGCCAAAGTC-3′. Control oligonucleotides consisted of the scrambled (Scr) version of each antisense sequence as follows: Scr-JNK1, 5′-CTTTCCGTTGGACCCCTGGG-3′ and Scr-JNK2, 5′-GTGCGCGCGAGCCCGAAATC-3′. Oligonucleotides (3 μg) were added to 94 μl of Ham's F12K medium containing 6 μl FuGene 6 transfection reagent (Roche, Indianapolis, IN). After a 30-min incubation at room temperature, the mixture was added to cells at 50–70% confluence in flasks with Ham's F12K medium containing 0.4% FBS, and incubated in humidified 5% CO2 at 37°C for 36 h. JNK1 and JNK2 contents were then determined by Western blotting whole cell lysates using a commercial antibody raised against JNK1 (SC-571) that also recognizes JNK2. To show the equality of cytoplasmic protein contents, blots were stripped and reproved using anti–β-actin antibody (Sigma).
All values are expressed as means ± SEM of the indicated numbers of experiments. Data were compared using Student's t test with the Bonferroni correction for multiple comparisons. A P value of < 0.05/m (where m is the number of comparisons) was considered statistically significant in the Bonferroni method.
We examined the expression of CTGF by TGF-β1 in the human lung fibroblast cell line, HFL-1. Figure 1A
Figure 1. CTGF mRNA expression induced by TGF-β1 in human lung fibroblasts, HFL-1. (A) Time-dependent induction of CTGF mRNA after stimulation with 1 ng/ml TGF-β1. (B) Dose-dependent induction of CTGF mRNA after 4 h stimulation with TGF-β1. (C) Effect of actinomycin D (5 μg/ml) on CTGF mRNA expression induced by TGF-β1 (1 ng/ml, 4 h). Total RNA was extracted and hybridized with human CTGF and G3PDH cRNA probes. Loading control was human G3PDH probe.
[More] [Minimize]We determined the roles of the three MAP kinases in TGF-β1 signal transduction by detecting their dually phosphorylated (Thr/Tyr) forms by Western blotting against specific anti–phospho kinase antibodies. Amounts of phosphorylated threonine and tyrosine of JNK significantly increased, reached a maximum at 30 min, and remained elevated for up to 120 min after adding TGF-β1 (Figure 2A)
Figure 2. Effect of TGF-β1 on JNK, p38 MAP kinase and ERK activation in human lung fibroblasts, HFL-1. HFL-1 cells were stimulated with TGF-β1 (1 ng/ml) for indicated periods. Activities of JNK, p38 MAP kinase, and ERK were determined by Western blotting whole cell lysates using antibodies specific for phosphorylated, activated forms of JNK (A, top), p38 MAP kinase (B, top), and ERK (C, top). Corresponding bottom panels show Western blots using antibodies to total JNK (A, bottom), p38 MAP kinase (B, bottom) and ERK (C, bottom) to show amounts of each precipitated enzyme. Two identical experiments independently performed yielded similar results.
[More] [Minimize]To further understand the mechanism of TGF-β1–induced CTGF expression, we investigated the roles of MAP kinases using selective inhibitors of MAP kinases. We blocked the p38 MAP kinase pathway by incubating human lung fibroblasts with SB203580, a specific inhibitor of p38 MAP kinase activity, for 30 min before adding TGF-β1. Figure 3A
Figure 3. Effects of SB203580 and PD98059 on TGF-β-induced CTGF expression. (A, D) HFL-1 cells were cultured with 0.1% DMSO (control vehicle), SB203580 (specific inhibitor of p38 MAP kinase; A) or PD98059 (specific inhibitor of MEK-1; D) for 30 min before stimulation with TGF-β1 (1 ng/ml) for 30 min. (A) MAPKAPK2 activity was measured in an in vitro kinase reaction using MAPKAPK2 substrate peptide, and results are expressed as ratio (%) of control value (unstimulated HFL-1 cells). Values represent mean ± SEM of three experiments. (D) ERK activity was determined by Western blotting as described in the legend to Figure 2. (B, E) HFL-1 cells were cultured with 0.1% DMSO (control vehicle), SB203580 (B) or PD98059 (E) for 30 min before stimulation with TGF-β1 (1 ng/ml) for 4 h. Total RNA was extracted and hybridized with human CTGF and G3PDH cRNA probes. (C, F) Messenger RNA levels were quantified by densitometry and optical density of CTGF mRNA band was corrected for G3PDH, and results are expressed as ratio (%) of control value (TGF-β1-stimulated HFL-1 cells). Values represent mean ± SEM of four experiments.
[More] [Minimize]Next, we examined effects of wortmannin and LY294002, specific inhibitors of PI3K activity, on TGF-β1–induced CTGF expression in human lung fibroblasts. In contrast to SB203580 and PD98059, wortmannin dose-dependently suppressed CTGF mRNA expression by HFL-1 cells stimulated with TGF-β1 (Figure 4A)
Figure 4. Effects of wortmannin and LY294002 on TGF-β-induced CTGF expression. (A, C) HFL-1 cells were cultured with 0.1% DMSO (control vehicle), wortmannin (A) or LY294002 (C) for 30 min before stimulation with TGF-β1 (1 ng/ml) for 4 h. Total RNA was extracted and hybridized with human CTGF and G3PDH cRNA probes. (B) Messenger RNA levels were quantified by densitometry as described in legend to Figure 3. Values represent mean ± SEM of four experiments. *P < 0.05 compared with TGF-β1-stimulated cells.
[More] [Minimize]We reported that wortmannin inhibits JNK and p38 MAP kinase activation induced by antigen crosslinking in a mouse mast cell line passively sensitized with ovalbumin-specific IgE (30). Here, we investigated whether or not wortmannin and LY294002 affect TGF-β1–induced MAP kinase activities in human lung fibroblasts. Figure 5A
Figure 5. Effects of wortmannin and LY294002 on JNK, p38 MAP kinase and ERK activation. HFL-1 cells were cultured with 0.1% DMSO (control vehicle), wortmannin, or LY294002 for 30 min before stimulation with TGF-β1 (1 ng/ml) for 30 min. Activities of JNK (A, B), p38 MAP kinase (D, E) and ERK (F) were determined by Western blotting as described in legend to Figure 2. Two identical experiments independently performed yielded similar results. (C) Phosphorylated JNK protein levels were quantified by densitometry. The optical density of the p46 (open bars) and p54 (filled bars) phosphorylated protein band were corrected for total p46 and p54 protein, respectively, and results are expressed as ratio (%) of control value (TGF-β1-stimulated HFL-1 cells).
[More] [Minimize]To further confirm the role of JNK in TGF-β1–induced CTGF expression in human lung fibroblasts, we performed experiments using JNK1 and JNK2 antisense oligonucleotides. JNK has apparent molecular masses of 46 and 54 kD that are largely, but not exclusively, composed of the JNK1 and JNK2 isoforms, respectively (31). JNK1 antisense (AS-JNK1) and JNK2 antisense (AS-JNK2) oligonucleotides distinctly attenuated the 46- and 54-kD components, respectively (Figures 6A and 6B)
Figure 6. Effects of JNK1 and JNK2 antisense oligonucleotides on TGF-β–induced CTGF expression. (A) HFL-1 cells were transfected with JNK1 and JNK2 antisense oligonucleotides (AS) alone and in combination. Each control oligonucleotides, scrambled sequence oligonucleotides (Scr), were transfected alone and in combination. After 36 h transfection, JNK1 and JNK2 contents were determined by Western blotting whole cell lysates using a commercial antibody raised against JNK1 (SC-571) that also recognizes JNK2. The corresponding bottom panels are Western blots using anti–β-actin antibody to demonstrate the equality of cytoplasmic protein contents. (B) JNK protein levels were quantified by densitometry. The optical density of the p46 (open bars) and p54 (filled bars) protein band were corrected for β-actin, and results are expressed as ratio (%) of control value. (C) Cells were transfected with AS or Scr for 36 h, then stimulated with TGF-β1 (1 ng/ml) for 4 h. Total RNA was extracted and hybridized with human CTGF and G3PDH cRNA probes. (D) Messenger RNA levels were quantified by densitometry as described in legend to Figure 3. Results are expressed as ratio (%) of control value. Values represent mean ± SEM of four experiments. *P < 0.05 compared with the results of control oligonucleotides.
[More] [Minimize]TGF-β factors initiate signaling by assembling receptor complexes that activate SMAD proteins (10). Finally, we investigated whether the activation of JNK would affect SMAD signaling pathway by detecting dually phosphorylated (Ser 465/Ser 467) form of SMAD2 by Western blotting against specific anti–phospho kinase antibody. In HFL-1 cells, amounts of phosphorylated SMAD2 significantly increased, reached a maximum at 60 min after adding TGF-β1 (Figure 7A)
Figure 7. (A, B, C) Effects of JNK antisense oligonucleotides and SP600125 on TGF-β–induced SMAD2 phosphorylation in human lung fibroblasts, HFL-1. (A) HFL-1 cells were stimulated with TGF-β1 (1 ng/ml) for indicated periods. (B) Cells were transfected with AS or Scr for 36 h, then stimulated with TGF-β1 for 60 min. (C) Cells were cultured with 0.1% DMSO (control vehicle) or SP600125 for 30 min, and then stimulated with TGF-β1 for indicated periods. Phosphorylation of SMAD2 was determined by Western blotting whole cell lysates using a antibody specific for phosphorylated forms of SMAD2. Corresponding bottom panels show Western blots using antibodies to total SMAD2 to show amounts of each precipitated enzyme. Two identical experiments independently performed yielded similar results. (D, E) Effect of SP600125 on TGF-β–induced CTGF expression. (D) HFL-1 cells were cultured with 0.1% DMSO (control vehicle) or SP600125 for 30 min before stimulation with TGF-β1 (1 ng/ml) for 4 h. Total RNA was extracted and hybridized with human CTGF and G3PDH cRNA probes. (E) Messenger RNA levels were quantified by densitometry as described in legend to Figure 3. Values represent mean ± SEM of four experiments. *P < 0.05 compared with TGF-β1–stimulated cells.
[More] [Minimize]The main finding of the present study is that TGF-β1 induces CTGF mRNA expression in human lung fibroblasts through the JNK-dependent pathway, but not through the p38- or ERK- dependent pathways. This is the first report to describe the role of JNK in regulating TGF-β1–induced CTGF expression in human lung fibroblasts.
Collectively, fibroproliferative diseases result in significant morbidity and mortality. Although the underlying causes are diverse and the pathogenic mechanisms are complex, many of the harmful aspects are probably mediated by the diverse effects of TGF-β as the final universal pathway. TGF-β has been implicated in many fibrotic disorders affecting various targets including the lungs, liver, skin, and kidneys (32). Many of the effects of TGF-β on fibroblast proliferation and extracellular matrix production are probably mediated by CTGF. Namely, CTGF appears to play a critical role in mediating many of the important fibroproliferative effects of TGF-β, including the pathogenesis of fibrotic disorders (7–9). Therefore, it is of key importance to define the mechanisms through which TGF-β upregulates CTGF expression.
TGF-β uses the SMAD signaling pathway (10). Other studies have shown that the regulation of CTGF expression is dependent on the action of a specific TGF-β response element (TβRE) that is a feature of human and murine CTGF promoters (33). A functional SMAD-binding site has recently been identified in the human CTGF promoter (11), suggesting that the SMAD signaling pathway plays a critical role in the regulation of TGF-β–induced CTGF expression. However, several other downstream signaling pathways are also activated by TGF-β, including PI3K (34), MAP kinases such as JNK (12) and p38 MAP kinase (13, 35), and RhoA (36). Furthermore, in human lung fibroblasts, Kucich and coworkers (37) have shown that TGF-β–induced CTGF expression requires the activity of a phosphatidylcholine-specific phospholipase C, protein kinase C, and a member of the Ras superfamily of small GTPases. In the present study, we found that exposing human lung fibroblasts to TGF-β1 resulted in the activation of JNK (Figure 2A) and p38 MAP kinase (Figure 2B), but not of ERK (Figure 2C). Our studies showed that a decrease in cellular amounts of JNK in human lung fibroblasts caused by JNK1 and JNK2 antisense oligonucleotides (Figures 6A and 6B) potently inhibited the induction of CTGF mRNA expression by TGF-β1 (Figures 6C and 6D). In contrast, blocking the p38 MAP kinase signaling pathway by SB203580 and the ERK signaling pathway by PD98059 did not obviously affect the induction of CTGF expression by TGF-β1 (Figure 3), indicating that activation of the p38 MAP kinase and the ERK pathway is not essential for the enhancement of CTGF expression in human lung fibroblasts. Furthermore, we demonstrated that wortmannin and LY294002, as specific inhibitors of PI3K, suppressed the activation of JNK (Figures 5A, 5B, and 5C), but not of p38 MAP kinase (Figures 5D and 5E), ERK (Figure 5F), or the induction of CTGF mRNA expression by TGF-β1 (Figure 4). We considered that the inhibitory effects of these PI3K inhibitors on TGF-β1–induced JNK activation and CTGF expression were not attributed to its toxicity, because these PI3K inhibitors did not affect p38 MAP kinase activity induced by TGF-β1. These results indicate that TGF-β1–induced CTGF mRNA expression is mediated through the JNK-dependent pathway, and that PI3K may act upstream of JNK. We are the first to show that the PI3K-JNK pathway plays a crucial role in the expression of CTGF induced by TGF-β1 in human lung fibroblasts.
A recent report has demonstrated that the human CTGF promoter has two putative AP1 sites, and that activation of this promoter depends on c-Jun, which is the target substrate of JNK, and c-Fos (38). In addition, SMAD3 and SMAD4 cooperate with c-Jun/c-Fos to mediate TGF-β–induced transcription, suggesting that SMAD signaling and JNK signaling converge at AP1-binding promoter sites (39). Conversely, it was reported that TβRI-mediated phosphorylation of SMAD3 requires JNK activity, and SMAD3 phosphorylation by JNK facilitates its nuclear accumulation (12). We also demonstrated in this study that the selective blockade of JNK inhibited phosphorylation of SMAD2 induced by TGF-β1 (Figures 7B and 7C). These results suggest that JNK regulates SMAD-mediated signaling and transcription in human lung fibroblasts.
In conclusion, we demonstrated that TGF-β1–induced CTGF mRNA expression is mediated through the JNK-dependent pathway, whereas p38 MAP kinase and ERK pathways contribute only minimally in human lung fibroblasts. TGF-β exerts not only fibroproliferative, but also anti-inflammatory actions in the pathogenesis of fibrotic disorders (40). In contrast, CTGF mediates only the fibroproliferative effects of TGF-β. Therefore, understanding the mechanisms regulating the expression of CTGF increased by TGF-β is of great importance to inhibit the progress of fibrotic disorders. Our findings of the role of the JNK-dependent pathway in TGF-β1–induced CTGF expression are essential to providing new therapeutic options for regulating connective tissue cell proliferation, migration, and collagen synthesis in pulmonary fibrosis as well as in other fibrotic disorders.
The authors thank Osamu Araki and Masami Murakami (Gunma University) for their expert technical support, and Akihiro Yoshii and Katsuaki Endou (Gunma University) for valuable discussion.
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