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Published ahead of print on September 1, 2005, doi:10.1165/rcmb.2005-0184OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 65-72, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0184OC

Lipoxin A4 Inhibits Proliferation of Human Lung Fibroblasts Induced by Connective TissueGrowth Factor

Sheng-Hua Wu, Xiang-Hua Wu, Chao Lu, Ling Dong and Zi-Qing Chen

Department of Pediatrics, Central Laboratory, the First Affiliated Hospital of Nanjing Medical University, Nanjing University of Technology, Nanjing, Jiangsu, People's Republic of China

Correspondence and requests for reprints should be addressed to Sheng-Hua Wu, M.D., Department of Pediatrics, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu 210029, People's Republic of China. E-mail: kad-yc{at}163.com


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Connective tissue growth factor (CTGF) plays an important role in pathways leading to lung fibrosis via the mitogenic action of CTGF on fibroblasts. Studies have shown that lipoxin A4 (LXA4) inhibits proliferation of renal mesangial cells induced by leukotriene D4 or platelet-derived growth factor. This study investigates the regulatory role of LXA4 on proliferation of human lung fibroblasts (HLF) induced by CTGF and mechanisms of LXA4 action. CTGF induced HLF proliferation; enhanced the expression of cyclin D1; phosphorylated extracellular signal-regulated kinase (ERK)1/2, phosphoinositide 3-kinase (PI3-K), protein kinase B (PKB), and DNA-binding activity of signal transducers and activators of transcription-3 (STAT3); and inhibited expression of p27kip1. LXA4 downregulated the CTGF-stimulated HLF proliferation and expression of cyclin D1; and phosphorylated ERK1/2, PI3-K, PKB, and DNA-binding activity of STAT3. CTGF-induced decrement in expression of p27kip1 was ameliorated by LXA4. PI3-K or STAT blockade but not ERK1/2 blockade partially inhibited the CTGF-activated proliferation of HLF. Transfection of the human LXA4 receptor gene into HLF intensified the inhibition of LXA4 on CTGF-induced cell proliferation. These results demonstrate that CTGF induces proliferation of HLF via upregulation of PI3-K/PKB, STAT3, and cyclin D1, and downregulation of p27kip1. LXA4 inhibits these effects of CTGF on HLF.

Key Words: lipoxin A4 • connective tissue growth factor • fibroblasts • lung • proliferation


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Connective tissue growth factor (CTGF), a downstream mediator of the effects of transforming growth factor (TGF)-{beta} on fibroblasts, is a 38-kD, cysteine-rich protein that stimulates in vitro fibroblast growth and matrix deposition (1) and is an important player in the pathogenesis of various fibrotic disorders, including lung fibrosis and idiopathic pulmonary fibrosis (2–5). The molecular mechanisms by which TGF-{beta} induces CTGF expression in human lung fibroblasts (HLF) have been well documented (6). However, the molecular mechanisms by which CTGF stimulates proliferation of HLF remain to be established. Previous studies have demonstrated that CTGF induced the S-phase entry by upregulating cyclin A levels via reduction of p27kip1 levels in cyclic adenosine monophosphate–arrested normal rat kidney fibroblasts (7). In human glomerular mesangial cells, CTGF activated extracellular signal-regulated kinase (ERK)1/2 pathway, cyclin D1 protein (8), phosphoinositide 3-kinase (PI3-K), Jun NH2-terminal kinase, protein kinase C, and protein kinase B (PKB) (9, 10). The present studies were undertaken to explore the mechanisms by which CTGF stimulates proliferation of HLF.

Lipoxin A4 (LXA4) is an endogenously produced eicosanoid with potent anti-inflammatory bioactions (11–13). LXA4 has been shown to inhibit mesangial cell proliferation induced by platelet-derived growth factor (PDGF), epidermal growth factor (14, 15), leukotriene D4 (LTD4) (16), and tumor necrosis factor-{alpha} (12). It was also demonstrated that LXA4 and its analogs were potent inhibitors for cell proliferation in lung adenocarcinoma cell line (17) and umbilical vein endothelial cells stimulated with vascular endothelial growth factor (18). It remains unclear whether LXA4 inhibits CTGF-induced proliferation of fibroblasts. LXA4 exerts its effects through signals generated by binding to a high-affinity, pertussis toxin (PTX)-sensitive, G protein-coupled receptor, LXA4 receptor (ALXR), on a variety of cell types, including human synovial fibroblasts (13). A recent publication established formyl peptide receptor-like 1, a homologous receptor of ALXR, expression on human lung and skin fibroblasts (19). It is speculated that LXA4 could modulate the proliferation of HLF through signals generated by binding to ALXR. The current study were undertaken to find whether LXA4 could modulate the proliferation of HLF induced by CTGF and to examine the involvement of cyclin D1, ERK1/2, PI3-K, PKB, p27kip1, and signal transducers and activators of transcription-3 (STAT3) in CTGF and LXA4 actions.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), and other tissue culture reagents were obtained from Gibco BRL (Grand Island, NY). Formazan, TriZol, Moloney-murine leukemia virus reverse transcription enzyme, dNTP, Oligo (dT)15, RNase, pGEM-T-easy vector, {gamma}-[32P]ATP, T4 polynucleotide kinase, Taq DNA polymerase, and a Gel Shift Assay kit were purchased from Promega (Madison, WI). Advantage 2 DNA polymerase and pcDNA3.1/myc-his vector were obtained from BD Biosciences (La Jolla, CA). Restriction endonuclease HindIII and XhoI were purchased from New England BioLab (Beverly, MA). The Endo-free Plasmid Maxi Kit was obtained from Qiagen (Hilden, Germany). DNA Ligation kit ver. 2.1 was purchased from Takara (Osaka, Japan). Lipofectamine 2000 reagent was obtained from Invitrogen (Carlsbad, CA). Nuclear Protein Extraction kit was purchased from Active Motif (Carlsbad, CA). Rabbit anti-rat threonine/tyrosine diphosphorylated ERK1/2, total ERK1/2, serine phosphorylated PKB, total PKB, tyrosine phosphorylated PI3-K p85, total PI3-K p85, p27kip1 (C19), cyclin D1 (R124), {alpha}-tubulin and 6xhistidine antibodies, goat anti-rabbit IgG, and double-stranded oligonucleotide probes of STAT3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An enhanced chemiluminescence reagent system and polyvinylidene difluoride membranes were obtained from Amersham (Arlington, IL). Recombinant CTGF was purchased from Fibrogen (San Francisco, CA). UO126, LY294002 and parthenolide were obtained from Calbiochem (La Jolla, CA). Dimethyl sulfoxide, PTX, 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT), propidium iodide, and LXA4 was purchased from Sigma (St Louis, MO).

Cell Culture
The HLF cell line was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. HLF monolayers were cultured in DMEM supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and insulin (5 µg/ml) at 37°C in a 5% CO2 incubator. These cells have been extensively characterized and express vimentin, desmin, and fibroblast-specific surface antigen but not keratin. Cell viability was measured by trypan blue exclusion assay in a pilot experiment, and the percentage of viable cells was more than 90% after exposure to LXA4 (10 nmol/l) or CTGF (50 ng/ml) for 24 h, PTX (100 ng/ml) for 18 h, LY294002 (25 µmol/l) for 30 min, UO126 (10 µmol/l) for 1 h, or parthenolide (50 µmol/l) for 2 h.

Cell Proliferation Assay
HLF were seeded in 24-well plates (5 x 104 cells/well). After the cells reached 80% confluence, the cells were growth arrested by incubation in DMEM containing 0.5% FCS for 48 h. Growth-arrested cells were pretreated with or without LXA4 at different concentrations, LY294002 (25 µmol/l) for 30 min, UO126 (10 µmol/l) for 1 h, parthenolide (50 µmol/L) for 2 h, or PTX (100 ng/ml) for 18 h, and the coincubation was continued for 24 h after addition of CTGF (50 ng/ml) in DMEM containing 0.5% FCS. Proliferation of HLF was measured by determining the number of viable cells using an assay in which a tetrazolium salt was reduced by viable cells to aqueous soluble formazan. The quantity of formazan product as measured by spectrophotometry at A450nm was directly proportional to the number of living cells (16). After HLF were incubated with 50 µg of MTT for 4 h, the supernatants were removed, and dimethyl sulfoxide was added to each well. A450nm was measured in six samples of each group.

Cell Cycle Analysis
Cells were trypsinized and suspended and were washed with PBS. Cell pellets were fixed in ethanol and Triton X-100 for 2 h and resuspended in 0.1 mg/ml of RNase for 15 min at room temperature. The single-cell suspension was adjusted to 107 cells/ml and stained by propidium iodide at 4°C for 30 min in the dark. Flow cytometric analysis was performed at a flow rate of 100 events/s by using a dual-laser flow cytometer (Becton Dickinson, San Jose, CA). A total of 10,000 events was counted. Cell debris and clumps were excluded from the analysis by gating single cells in the forward and side light scatters. Propidium iodide was excited using the 488-nm UV line of the argon laser. Data were analyzed with Mac-based software (Tree Star, San Carlos, CA).

RT-PCR Analysis
Cells (2 x 106/well) were harvested, and total RNA extraction was performed using TriZol reagent followed by chloroform-isopropanol extraction and ethanol precipitation. RNA purity (A260/A280 > 1.6) was checked by a spectrophotometer (GeneQuant, Type II; Pharmacia, Uppsala, Sweden), and RNA integrity was confirmed by visualization of 28 and 18 s bands (2:1) on 1% agarose gel. Subsequently, 1 µg of RNA was reverse transcribed using the Moloney-murine leukemia virus reverse transcription system. RT-PCR analysis was performed with the following sets of primers for human ALXR: 5'-CACCAGGTGCTGCT GGCAAG-3' (sense) and 5'-AATATCCCTGACCCCATCCTCA-3' (antisense), amplifying a 1,095-bp fragment including the full open reading frame (13). The {beta}-actin was used as internal controls; 5'-CATG TGCAAGGCCGGCTT-3' (sense) and 5'-GAAGGTGTGGTGCCA GATTT-3' (antisense), generated a 226-bp fragment. PCR reactions were performed in a 50-µl mixture containing 10 x PCR buffer, MgCl2, dNTP, and Taq DNA polymerase. Amplification protocols for ALXR and {beta}-actin consisted of 30 repetitive cycles of predenaturing at 95°C for 4 min, denaturing at 94°C for 30 s, annealing at 60°C for 60 s for ALXR or at 56°C for 30 s for {beta}-actin, extension at 72°C for 80 s, and final extension at 72°C for 5 min. Amplified cDNA was separated by 3% agarose gel electrophoresis and visualized with ethidium bromide. Semiquantitative analysis was performed using UVP-gel densitometry (San Gabriel, CA).

Western Blotting
Quiescent cells were pretreated with or without LXA4 (10 nmol/l) or LY294002 (25 µmol/l) for 30 min, UO126 (10 µmol/l) for 1 h, or parthenolide (50 µmol/l) for 2 h. Coincubation was continued for 30 min (for PI3-K, PKB measurement), 1 h (for ERK1/2 measurement), 3 h (for p27kip1 measurement), or 24 h (for cyclin D1 measurement) after addition of CTGF (50 ng/ml) in DMEM containing 0.5% FCS. The cells were collected, and total protein was extracted by using a Protein Extraction Kit following the manufacturer's instructions. Samples were normalized for total protein assessed by the method of Bradford. The lysates (40 µg of protein/well) were electrophoresed on 10% SDS-PAGE for 4 h before blotting onto polyvinylidene difluoride membranes. Nonspecific sites on the membranes were blocked with 5% nonfat milk. The blots were incubated with primary rabbit anti-rat antibodies against phospho (P)-ERK1/2, P-PI3-K, P-PKB, p27kip1, cyclin D1, or {alpha}-tubulin at 1:2,000 dilution for 1 h followed by washing and incubation for 1 h with a horseradish peroxidase–conjugated goat anti-rabbit IgG antibodies at 1:2,000 final dilution. The equal loading was confirmed by reprobing with the antibodies against total ERK1/2, total PI3-K, or total PKB, respectively (1:1,000 each). After washing, the membranes were incubated with an enhanced chemiluminescence reagent system and exposed to Kodak Biomax films. Semiquantitative analysis was performed by using UVP-gel densitometry.

Electrophoretic Mobility Shift Assay
Growth-arrested cells were pretreated with or without LXA4 (10 nmol/l) or LY294002 (25 µmol/l) for 30 min, UO126 (10 µmol/l) for 1 h, or parthenolide (50 µmol/l) for 2 h. Coincubation was continued for 1 h after addition of CTGF (50 ng/ml) in DMEM containing 0.5% FCS. Nuclear protein was extracted using a Nuclear Protein Extraction Kit. Electrophoretic mobility shift assay (EMSA) was performed by using a Gel Shift Assay Kit following the manufacturer's instructions. The nuclear extracts containing 30 µg of total proteins were preincubated with gel shift binding buffer for 10 min, followed by the addition of 1 µl of a {gamma}-[32P]-labeled, double-stranded oligonucleotide probe of STAT3 and incubation for 20 min. The oligonucleotide pairs of STAT3 were 5'-GATCCTTCTGGGAATTCCTAGATC-3' and 5'-GATCTAGGAA TTCCCAGAAGGATC-3' and were radiolabeled with {gamma}-[32P]ATP by incubation with 10 U of T4 polynucleotide kinase. Formed nuclear protein–DNA complexes were dissolved in 4% nondenaturing polyacrylamide gels, and electrophoresis was performed under 90 V for 2 h. Gels were dried and exposed to Kodak Biomax films at –70°C for 36 h. Semiquantitative analysis was performed by using UVP-gel densitometry. To assess the specificity of the reaction, competition assays were performed with 100-fold excess of unlabeled consensus oligonucleotide pairs of STAT3. The unlabeled probes were added to the binding reaction mixture 10 min before the addition of the labeled probes.

Construction of pcDNA3.1/ALXR
To construct and clone the eukaryotic expression vector of human ALXR, the ALXR was amplified by PCR from cDNA of human monocytic leukemia cell line THP-1 (Shanghai Institute of Cell Biology, Chinese Academy of Sciences). The primer pair of human ALXR was selected based on mRNA sequence of human ALXR from Genbank (NCBI ID: AF054013). The sense primer (5'-ATCTCGAGTATG GAAACCAACTTCTCCAC-3') had an incorporated XhoI site. The antisense primer (5'-GCAAGCTTCGGCATTGCCTGTAACTCAGT CTCTG-3') had an incorporated HindIII site, and the stop codon TCA was removed. After RT-PCR amplification, the ALXR was inserted to the plasmid pGEM-T and transformed into Escherichia coli JM109. Positive clones were amplified, digested by HindIII and XhoI, screened, and identified by sequencing. The eukaryotic expression vector pcDNA3.1/ALXR containing 6xhistidine gene was constructed, transformed into E. coli JM109, amplified, and digested by restriction endonucleases. The sequence of inserted fragment in pcDNA3.1/ALXR-his was analyzed. Sequence analysis showed that the gene homology between ALXR cloned and ALXR reported by GenBank was 100%.

ALXR Transfection and Expression
HLF (5 x 103/well) were seeded in six-well plates and grown to 90% confluence in complete medium without antibiotics. The recombinant plasmid pcDNA3.1/ALXR-his or empty pcDNA3.1 vector was transfected into the cells by using Lipofectamine 2000 transfection reagent following the manufacturer's protocol. Positive clones were collected and amplified after incubation with G418 for 3 wk. The stable expression of ALXR in HLF was demonstrated by Western blot using the antibodies against 6xhistidine (Figure 1B).



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  		Figure 1. Expression of ALXR in HLF. (A) Expression of ALXR mRNA was examined by RT-PCR amplification of total RNA derived from HLF or THP-1 cells. Control lane (HLF) was run without template cDNA. (B) Expression of the transfected ALXR-his protein was examined by Western blot analysis using the antibodies against 6xhistidine in HLF.

 
Statistical Analysis
Results are expressed as mean ± SEM. Experimental data were analyzed using one-way ANOVA followed by S-N-K test (Q test) by Statistical Package for Social Sciences version 10.0 (SPSS, Chicago, IL). Differences were considered to be statistically significant at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Expression of ALXR in HLF
Expression of human ALXR mRNA in HLF was demonstrated by RT-PCR amplification of HLF-derived total RNA (Figure 1A). This result was consistent with a previous study that established formyl peptide receptor-like 1/ALXR expression on human synovial fibroblasts (13) and on human lung and skin fibroblasts (19). Expression of ALXR-his protein in HLF transfected with pcDNA3.1/ALXR-his is shown in Figure 1B. The result that cells transfected with empty pcDNA3.1 vector did not express the ALXR-his indicated the specific expression of ALXR-his in the cells transfected with pcDNA3.1/ALXR-his.

LXA4 Inhibits Proliferation of HLF Induced by CTGF
LXA4 dose dependently inhibits CTGF-stimulated proliferation of HLF assessed by MTT colorimetric assay (Figure 2A). Pretreatment of HLF with PTX blocked LXA4-induced inhibitory effects on CTGF-stimulated proliferation of HLF. Transfection of ALXR into HLF resulted in overexpression of ALXR in HLF and amplified the inhibitory effect of LXA4 on CTGF-induced proliferation of HLF. Similar results were obtained when the cells were studied by flow cytometric analysis (Figure 3). CTGF increased the cells at S and G2/M phase and significantly reduced the cells at G0/G1 phase (Figure 3C) (P < 0.05 compared with G0/G1 in Figure 3A). LXA4 ameliorated CTGF-reduced the cells at G0/G1 phase (Figure 3D) (P < 0.05 compared with G0/G1 in Figure 3C).



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  		Figure 2. Proliferation of HLF assessed by MTT colorimetric assay. Cultured and serum-deprived cells were treated with CTGF for 24 h with or without preincubation with LXA4 or LY294002 (25 µmol/l) for 30 min, UO126 (10 µmol/l) for 1 h, parthenolide (50 µmol/l) for 2 h, or PTX (100 ng/ml) for 18 h. (A) Cells were transfected with empty pcDNA3.1 vector or pcDNA3.1/ALXR-his vector. Data are mean ± SEM of six independent experiments. *P < 0.05 compared with the cells treated with CTGF in 0.5% FCS. {dagger}P < 0.05 compared with the cells treated with 0.5% FCS alone. {ddagger}P < 0.05 as comparison between two columns as shown. (B) *P < 0.05 compared with the cells treated with CTGF in 0.5% FCS. {dagger}P < 0.05 as comparison between two columns as shown.

 
LXA4 Suppressed CTGF-Stimulated Phosphorylation of PI3-K and PKB
In a preliminary experiment, we performed the time courses of CTGF-induced phosphorylation of PI3-K and PKB from 0–180 min. The phosphorylation of PI3-K and PKB was elevated from 10 min and remained elevated until 60 min, similar to results reported previously (9). Thus, HLF were exposed to CTGF for 30 min to allow for measurement of PI3-K and PKB. The phosphorylation of PI3-K and PKB evoked by CTGF was significantly downregulated by LXA4 when the results of Western blot were quantified by densitometry (P < 0.05) (Figure 4). LY294002, a selective PI3-K inhibitor (9), significantly inhibited CTGF-induced proliferation of HLF (Figure 2B) and phosphorylation of PI3-K and PKB (Figure 4).

LXA4 Counter-Regulated CTGF-Induced Alteration of p27kip1 and Cyclin D1
In a pilot experiment, the time course of CTGF-reduced expression of p27kip1 from 0–12 h was assessed. After stimulation of the HLF with CTGF, p27kip1 expression decreased significantly to nearly nondetectable levels by 3 h, similar to the results reported by Kothapall and colleagues (20). Thus, HLF were exposed to CTGF for 3 h for measurement of p27kip1. There was high expression of p27kip1 protein in controlled growth-arrested HLF and low expression of p27kip1 protein in CTGF-treated HLF (Figure 4). However, CTGF-induced decrement in expression of p27kip1 was significantly ameliorated by LXA4 when the results of the Western blot were quantified by densitometry (data not shown, P < 0.05). Pilot studies also assessed the time course of CTGF-stimulated expression of cyclin D1 from 0–48 h. The increment of cyclin D1 activity started from 6 h and peaked at 24 h after addition of CTGF to the culture media, similar to results reported previously (8). In the present studies, HLF were exposed to CTGF for 24 h for measurement of cyclin D1. CTGF stimulated activity of cyclin D1 (Figure 4), and LXA4 significantly inhibited CTGF-induced activity of cyclin D1 in HLF when the Western blot was quantified by densitometry (data not shown, P < 0.05). PI3-K blockade not only inhibited the CTGF-induced expression of PI3-K and PKB but also blocked the CTGF- induced decrement of p27kip1 expression, increment of cyclin D1 expression (Figure 4), and STAT3 activity (Figure 5).

LXA4 Modulated CTGF-Activated Phosphorylation of ERK1/2
In a preliminary experiment, we determined the time courses of CTGF-induced phosphorylation of ERK1/2 from 0–180 min. The expression of phosphorylated ERK1/2 stimulated by CTGF increased from 5 min and remained elevated until 2 h, similar to results reported previously (8, 9). In the current study, HLF were exposed to CTGF for 1 h for measurement of ERK1/2. LXA4 increased the phosphorylation of ERK1/2, as described in a previous study on human renal mesangial cells (16). Similar to that study, LXA4 increased the phosphorylation of ERK1/2 in HLF to a lesser extent of the phosphorylation of ERK1/2 activated by CTGF (Figure 4). Even then, LXA4 partially but significantly inhibited CTGF-induced phosphorylation of ERK1/2 when the results of Western blot were quantified by densitometry (P < 0.05) (Figure 4). UO126, a selective ERK1/2 inhibitor (21), suppressed CTGF-stimulated phosphorylation of ERK1/2 but not PI3-K or PKB (Figure 4), had no effect on CTGF-induced proliferation of HLF (Figure 2B), and partially but significantly ameliorated CTGF-downregulated expression of p27kip1 (Figure 4) and CTGF- upregulated expression of cyclin D1 when the results of the Western blot were quantified by densitometry (data not shown, P < 0.05).

LXA4 Downregulated CTGF-Evoked DNA-Binding Activity of STAT3
Competition assay performed with unlabeled probes demonstrated the specificity of DNA-binding activity of STAT3 measured by EMSA (Figure 5). In pilot experiment, the time course of CTGF-induced activation of STAT3 from 0–180 min was assessed. After stimulation of the HLF with CTGF, activation of STAT3 increased from 10 min and remained elevated until 60 min. HLF were exposed to CTGF for 1 h for measurement of DNA-binding activity of STAT3. CTGF stimulated the DNA-binding activity of STAT3 in HLF (Figure 5), and this enhanced activity of STAT3 was significantly inhibited by treatment of the cells with LXA4 or LY294002 when the results of EMSA were quantified by densitometry (data not shown, P < 0.05). Parthenolide, a potent inhibitor of STAT (22), abrogated the DNA-binding activity of STAT3 (Figure 5) and the proliferation of HLF (Figure 2B) stimulated by CTGF. Pretreatment of the CTGF-treated cells with parthenolide slightly increased the activity of ERK1/2, but this increment did not reach the statistically significant difference as compared with the cells treated with CTGF alone when the results of Western blot were quantified by densitometry (data not shown) (Figure 4). There was slight increment of activity of STAT3 induced by UO126 in HLF stimulated by CTGF (Figure 5), but this increment, when EMSA was quantified by densitometry (data not shown), did not reach the statistically significant difference as compared with the activity of STAT3 in HLF treated with CTGF alone.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The potential role of CTGF in the pathogenesis of lung fibrosis has been well documented (2–6). LXA4 is an eicosanoid (i.e., a 20-carbon derivative of membrane-derived arachidonic acid) and is generated via biochemical pathways catalyzed by lipoxygenase enzymes. LXA4 antagonizes many cell responses evoked by proinflammatory cytokines and acts as endogenous "stop signals" in the inflammatory process (11–13). Recently, several studies have demonstrated an antiproliferative role of LXA4 (12, 14–18). LXA4 inhibits proliferation of human renal mesangial cells induced by PDGF, epidermal growth factor (14, 15), LTD4 (16), and tumor necrosis factor-{alpha} (12). LXA4 and its analogs were potent inhibitors for cell proliferation in a lung adenocarcinoma cell line (17) and umbilical vein endothelial cells stimulated with vascular endothelial growth factor (18). It is possible that LXA4 inhibits proliferation of HLF because expression of ALXR in HLF was demonstrated (Figure 1). In the present study, we provide the first evidence that LXA4 inhibited CTGF-stimulated proliferation of HLF. First, CTGF-induced HLF growth was suppressed by LXA4 in a dose-dependent manner (Figure 2A). Furthermore, LXA4 ameliorated the CTGF-downregulated expression of the cells at the G0/G1 phase (Figure 3). Finally, LXA4 inhibited the expression of the signaling molecules for cellular survival, such as PI3-K, PKB, cyclin D1, and STAT3 (Figures 4 and 5). Pretreatment with PTX blocked the inhibitory effects of LXA4 on CTGF-induced proliferation of HLF, and overexpression of ALXR in HLF enhanced the inhibitory effects of LXA4 on CTGF-induced proliferation of HLF, suggesting ALXR mediated the action of LXA4 on HLF (Figure 2A). The functional interaction between CTGF and LXA4 is attributed to LXA4's modification on the signaling pathway of CTGF rather than the binding of CTGF on the cells because the receptor of CTGF (10) is different from that of LXA4 (16).



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  		Figure 3. Cell-cycle analysis assessed by incorporation of propidium iodide into DNA by fluorescence-activated cell sorting analysis using a dual-laser flow cytometer. (A) Cells were treated with 0.5% FCS alone. (B) Cells were treated with LXA4 (10 nmol/liter) alone for 24.5 h. (C) Cells were treated with CTGF (50 ng/ml) for 24 h. (D) Cells were treated with CTGF (50 ng/ml) for 24 h with preincubation of LXA4 (10 nmol/liter) for 30 min. Data are mean ± SEM of six independent experiments.

 
In the current study, we investigated the molecular mechanisms by which CTGF stimulated proliferation of HLF and LXA4–inhibited, CTGF-induced cell growth. LXA4 downregulated the CTGF-induced phosphorylation of PI3-K, PKB, and expression of cyclin D1 and ameliorated the CTGF-downregulated expression of p27kip1 (Figure 4). These results were consistent with previous studies that demonstrated that CTGF activated PKB, PI3-K, and cyclin D1 in human mesangial cells (9, 10) and downregulated the expression of p27kip1 in cyclic adenosine monophosphate–arrested normal rat kidney fibroblasts (20). LXA4 inhibited PDGF-induced proliferation of mesangial cells via downregulation of PI3-K, Akt/PKB, and cyclin E and upregulation of p27kip1 (14). LXA4 also suppressed LTD4-induced proliferation of mesangial cells via downregulation of PI3-K (16). PKB (also known as Akt) is a key mediator of signal transduction in cell survival and is a downstream kinase of PI3-K (23). PKB downregulates p27kip1 transcription by phosphorylation-dependent inhibition of the Forkhead family of transcription factor (24), phosphorylates p27kip1 at Thr187 and Thr198, and promotes p27kip1 degradation (25). p27kip1, one of the cyclin-dependent kinase (CDK) inhibitors and a potential mediator of antimitogenic signals, causes G1 arrest by inactivating cyclin D/CDK4/6 and cyclin E/CDK2 complexes (25). Our data show that PI3-K blockade by LY294002 inhibited the CTGF-stimulated proliferation of HLF (Figure 2B) and the CTGF-activated phosphorylation of PI3-K, PKB, and expression of cyclin D1 and ameliorated the CTGF-downregulated p27kip1 (Figure 4), suggesting that the PI3-K/PKB/p27kip1/cyclin D1 pathway mediates the CTGF-induced proliferation of HLF. The effects of LXA4 mimicked the inhibition of PI3-K with LY294002 on CTGF (Figure 4), suggesting that the PI3-K/PKB/p27kip1/cyclin D1 pathway also mediates the effects of LXA4 on CTGF-induced proliferation of HLF.



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  		Figure 4. Western blot analysis of phospho (P)-PI3-K, total PI3-K, P-ERK1/2, total ERK1/2, P-PKB, total PKB, p27kip1, cyclin D1. The lowest panel shows Western blotting of {alpha}-tubulin served as a loading control. Cultured and serum-deprived cells were treated with CTGF for 30 min (for PI3-K and PKB measurement), 1 h (for ERK1/2 measurement), 3 h (for p27kip1 measurement), or 24 h (for cyclin D1 measurement) with or without preincubation with LXA4 or LY294002 (25 µmol/l) for 30 min, UO126 (10 µmol/liter) for 1 h, or parthenolide (50 µmol/liter) for 2 h. Results shown are representative of four independent experiments.

 
STAT proteins are latent transcription factors. Activation of STAT proteins is often associated with differentiation and growth regulation (26). STAT3 is a member of the STAT protein family and a key signaling molecule in mediating a survival function (27). In the present studies, the marked DNA-binding activities of STAT3 in parallel with proliferation of HLF induced by CTGF were blocked by parthenolide (Figures 2B and 5), indicating that STAT3 plays a crucial role in CTGF-stimulated proliferation of HLF. PI3-K blockade by LY294002 inhibited the activities of STAT3 stimulated by CTGF (Figure 5), suggesting that CTGF-induced STAT3 activation may be modulated by phosphorylated PI3-K. Similar results have been reported by other investigators. For example, PI3-K blockade by LY294002 decreased the STAT3 activation induced by carbon monoxide in endothelial cells (28). STAT3 did not affect the phosphorylation of PI3-K because STAT3 blockade by parthenolide had no effect on CTGF-stimulated expression of phospho-PI3-K (Figure 4). Pretreatment of HLF with parthenolide also inhibited CTGF-induced activation of PKB and cyclin D1 and ameliorated CTGF-downregulated p27kip1 (Figure 4), suggesting that the PKB/p27kip1/cyclin D1 pathway was modulated by STAT3 in HLF stimulated by CTGF. There are several lines of evidence indicating the requirement of STAT3 in the activation of the PKB/p27kip1/cyclin D1 pathway. During IL-6–induced proliferation of fibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis, activation of STAT3 downregulated p27kip1, resulting in the induction of cyclin D1 and E1 expression and hyperphosphorylation of retinoblastoma protein (29). STAT3-deficient neurons were unable to induce PKB phosphorylation in response to ciliary neutropic factor, suggesting a direct link between the activation of STAT3 and of PKB (27). Transformation of mouse fibroblasts by v-Src resulted in constitutive activation of STAT3, which activated the promotor of cyclin D1 and increased phosphorylation of Rb (30). In the current study, the activation of STAT3 induced by CTGF in parallel with proliferation of HLF were downregulated by LXA4 (Figures 2A and 5), indicating STAT3 mediates the inhibitory effects of LXA4 on CTGF-stimulated proliferation of HLF. Our results were supported by the investigation of Leonard and colleagues (31), who demonstrated that the LXA4 analog increased renal mRNA levels of suppressors of cytokine signaling-1 (SOCS-1) and SOCS-2, which were endogenous inhibitors of JAK/STAT signaling pathways (31).



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  		Figure 5. DNA-binding activities of STAT3 assessed by electrophoretic mobility shift assay. Cultured and serum-deprived cells were treated with CTGF for 1 h with or without preincubation with LXA4 or LY294002 (25 µmol/liter) for 30 min, UO126 (10 µmol/liter) for 1 h, or parthenolide (50 µmol/liter) for 2 h. In competition studies, a 100-fold molar excess of unlabeled oligonucleotide was added to the binding reaction mixture before the addition of the labeled probes of STAT3. The upper arrow denotes the specific STAT3-DNA complexes. Results shown are representative of four independent experiments.

 
Overexpression of phospho-ERK1/2 in mesangial cells stimulated by CTGF has been reported (8–10). Consistent with the previous studies, in the present study CTGF stimulated phosphorylation of ERK1/2 (Figure 4). LXA4 partially but significantly inhibited CTGF-induced phosphorylation of ERK1/2 (Figure 4), implicating the involvement of ERK1/2 in LXA4-induced inhibitory effects on CTGF-stimulated proliferation of HLF. It has been demonstrated that LXA4 inhibited LTD4-induced Ras activation (16). Ras is an upstream GTPase of MEK/ERK1/2. This mechanism may contribute to the inhibition of LXA4 on phosphorylation of ERK1/2 induced by CTGF. In the present study, ERK1/2 blockade by UO126 had no effect on proliferation of HLF stimulated by CTGF (Figure 2B), although ERK1/2 blockade inhibited phosphorylation of ERK1/2 induced by CTGF (Figure 4). Pretreatment of the cells with UO126 partially but significantly inhibited cyclin D1 activation and ameliorated downregulation of p27kip1 but did not affect the expression of PI3-K and PKB induced by CTGF (Figure 4), suggesting that proliferation of HLF stimulated by CTGF was ERK1/2 pathway independent even though ERK1/2 partially modulated the activities of cyclin D1 and p27kip1. A regulatory role of ERK1/2 on the activities of cyclin D1 and p27kip1 has been reported. In fibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis, activation of ERK1/2 induced by IL-6 downregulated p27kip1, resulting in the induction of cyclin D1 and E1 expression (29). Similar findings were reported by the studies on CCL39 lung fibroblasts from Chinese hamster (32) and NIH 3T3 fibroblasts (33). The involvement of PI3-K in ERK1/2 activation has not consistently been found. PI3-K pathway blockade prevented phosphorylation of ERK1/2 in PDGF-induced mesangial cell migration (34), in Toll-like receptor 2-stimulated inflammatory response of neutrophils (35), and in placenta growth factor-induced monocyte activation (36). In contrast, PI3-K blockade by LY294002 failed to inhibit ERK1/2 phosphorylation in human mesangial cells in response to CTGF (9). Consistent with the latter investigation, data in our studies showed that LY294002 did not inhibit ERK1/2 phosphorylation induced by CTGF (Figure 4).

There was the antagonism between ERK1/2 and STAT3 in some cells. Overexpression of phospho-ERK1/2 inhibited the DNA-binding activities of STAT3 in several cell lines stimulated by IL-6 (37). STAT blockade by parthenolide increased the phosphorylation of ERK1/2 in cardiomyocytes treated by cardiotrophin-1 (38). In the present study, UO126 slightly increased the DNA-binding activity of STAT3 (Figure 5), and parthenolide slightly increased the phosphorylation of ERK1/2 (Figure 4). These increments did not reach the statistically significant difference when the results were quantified by densitometry. A concern in explaining the results that showed that ERK1/2 blockade failed to inhibit proliferation of HLF stimulated by CTGF (Figure 2B) is that ERK1/2 blockade did not inhibit the activity of STAT3 stimulated by CTGF and only partially antagonized the effects of CTGF on the expression of p27kip1 and cyclin D1.

In conclusion, our data demonstrate that CTGF induced proliferation of HLF via PI3-K/PKB/p27kip1/cyclin D1 and STAT3 pathway-dependent signal transduction. CTGF increased the phosphorylation of ERK1/2, which may not contribute to the proliferation of HLF induced by CTGF. LXA4 inhibited the proliferation of HLF evoked by CTGF via upregulation of p27kip1 and downregulation of activities of PI3-K, PKB, cyclin D1, and STAT3 but not ERK1/2. Our results further expand on the antifibrotic repertoire of lipoxins, which represents useful tools for the development of a new therapy in treatment of lung fibrosis.


   Footnotes
 
This work was supported by 135 Medical Emphasis Grant (No. 135-45) from the Government of Jiangsu Province, People's Republic of China.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0184OC on September 1, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 15, 2005

Accepted in final form August 23, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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