This Article Abstract Full Text (PDF) All Versions of this Article: 2007-0065OCv1 37/4/395    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kono, Y. Articles by Yokoyama, M. Search for Related Content PubMed PubMed Citation Articles by Kono, Y. Articles by Yokoyama, M.

Sphingosine Kinase 1 Regulates Differentiation of Human and Mouse Lung Fibroblasts Mediated by TGF-1

¹ Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, and ² Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Japan

Correspondence and requests for reprints should be addressed to Yoshihiro Nishimura, M.D., Ph.D., Division of Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: nishiy{at}med.kobe-u.ac.jp

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS Discussion References

 
Transforming growth factor (TGF-) contributes to the progression of pulmonary fibrosis through up-regulation of –smooth muscle actin (–SMA) as lung myofibroblast differentiation. Bioactive sphingosine 1-phosphate (S1P) has been shown to mimic TGF- signals; however, the function of S1P in lung fibrotic process has not been well documented. We found, in a mouse model of bleomycin lung fibrosis, that SPHK1 and –SMA were colocalized within lung fibrotic foci and that these expressions were significantly increased in primary cultured fibroblasts. Using human lung fibroblasts WI-38, we explored the rationale of sphingosine kinase (SPHK) with TGF-1 stimulation. SPHK inhibitors and small interference RNA (siRNA) targeted SPHK1 decreased –SMA and fibronectin expression up-regulated by TGF-1. In the meantime, SPHK1 inhibition did not affect smad2 phosphorylation in response to TGF-1. Then we examined whether S1P receptors transactivation may affect TGF- signals. siRNA against S1P₂ and S1P₃, but not S1P₁, reduced –SMA expression as well as Y-27632, Rho kinase inhibitor. We also detected activation of Rho GTPase upon stimulation of TGF-1 on the cell membrane where S1P₂ or S1P₃ was overexpressed. These data suggested that SPHK1 activation by TGF-1 leads to Rho-associated myofibroblasts differentiation mediated by transactivated S1P receptors in the lung fibrogenic process.

Key Words: S1P • SPHK1 • fibroblast • Rho • -SMA

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS Discussion References   This research shows a novel mechanism that sphingosine kinase 1 activation by TGF-1 leads to Rho-associated myofibroblast differentiation mediated by transactivated S1P receptors. Our data will help provide new insights into controlling clinical fibrotic diseases.

 
Myofibroblasts play a central role in the pathogenesis of lung fibrosis. The presence of myofibroblasts in fibrotic lesion increases extracellular matrix deposition and collagen synthesis capacity, resulting in structural remodeling and destruction of alveolocapillary units during and after lung injury (1). These cells are commonly identified by its expression of –smooth muscle actin (-SMA) and by features that are intermediate phenotype between the smooth muscle cells and the fibroblasts (1). The origin of the myofibroblast is not clearly established; however, in general the resident intrapulmonary fibroblasts respond to a variety of stimuli during fibrogenic responses and differentiate into myofibroblasts (2). It is well documented that myofibroblast differentiation occurs after exposure to TGF-, which results in smad2 and smad3 activation, translocation of the smad complex into the nucleus, and increased –SMA transcription (3, 4).

Sphingosine kinase (SPHK) catalyzes the phosphorylation of sphingosine to generate a bioactive lipid mediator, sphingosine 1-phosphate (S1P). Two distinct isoforms of SPHK, SPHK1 and SPHK2, have been cloned and characterized. SPHK1 is activated in response to various agonists such as TNF-, vascular endothelial growth factor (VEGF), and FcRI, and promotes cell growth, cytoskeletal rearrangement, cell movement, and suppression of apoptosis (5, 6). On the other hand, little is known about the role of SPHK2, although it has recently been shown that SPHK2 suppresses cell growth and enhances apoptosis (7–9). Recent reports showed that platelet-derive growth factor (PDGF), FcRI, and NGF induced SPHK1 activation and translocation to plasma membrane, leading to spatially restricted formation of S1P, which then activates S1P receptors (10–13). Furthermore, it has been shown that S1P activates smad signaling and mimics TGF-–induced cell responses in epithelial dendritic cells (14), keratinocytes (15), and rat mesangial cells (16). Although we previously showed that S1P induced differentiation of fibroblasts into myofibroblasts (17), molecular mechanisms of fibrotic process are largely unknown.

In the current study, we hypothesized that S1P production by SPHK activation may play some role in lung fibrotic process involving myofibroblast differentiation. Using SPHK inhibitors or small interference RNA (siRNA)-mediated silencing treatment, we clarified how SPHK-S1P activation can affect the downstream signaling of TGF-.

	MATERIALS AND METHODS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS Discussion References

 
Materials
Recombinant human TGF-1 was purchased from Wako (Osaka, Japan). S1P, N'-N'-dimethylsphingosine (DMS), pertussis toxin, mouse monoclonal anti––SMA antibody, and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody were obtained from Sigma-Aldrich (St. Louis, MO). Anti-smad2 antibody was from Cell Signaling Technology (Beverly, MA). Anti–phospho-smad2 and anti–-tubulin antibody were from Upstate Biotechnology (Lake Placid, NY). Mouse anti-fibronectin antibody was purchased from BD Biosciences Pharmingen (San Jose, CA). Anti-GST antibody was from GE Healthcare Bio-Science Corp (Piscataway, NJ). Sphingosine kinase inhibitor (SK-I, 2-(p-Hydroxyanilino)-4-(p-chlorop henyl) thiazole), Y27632 and goat anti-SPHK1 antibody were from Calbiochem (La Jolla, CA). SB431542 was from Tocris Cookson Inc. (Ellisville, MO). Horseradish peroxidase–linked anti-mouse IgG and anti-rabbit IgG antibodies were from Amersham Biosciences (Piscataway, NJ). Anti-goat antibody Alexa594 was from Molecular Probes (Carlsbad, CA).

A rabbit polyclonal anti-mouse SPHK1 antibody was raised against the synthetic peptide GSRDAPSGRDSRRGPPPEEP (amino acid residues 362–381) conjugated to glutathione S-transferase. The antibody was affinity-purified by using the immunogen-immobilized Sepharose 4B.

Cell Culture
Human fetal lung fibroblast cell line, WI-38, was obtained from Riken Cell Bank (Tsukuba, Japan). Cells were grown and maintained in 6-well dishes with MF-medium (Toyobo Co., Ltd., Osaka, Japan) containing 1% fetal calf serum (FCS). In a preliminary study, we maintained human fibroblasts with conditioning media containing 10% FCS. Subculture using this media caused higher –SMA expression by generations (data not shown). Since 0.8 ng/ml TGF was detected in the media by enzyme-linked immunosorbent assay, we used low serum media (1% FCS) to maintain cells. At 3 to 6 passages, 80 to 90% confluent cells were serum-starved for 24 hours and then stimulated with TGF-1 for the indicated time. Inhibitors were pretreated 1 hour before TGF-1 stimulation.

Mouse Model of Bleomycin-Induced Lung Fibrosis
All animal experiments proceeded according to the Guidelines for Animal Experimentation at Kobe University Graduate School of Medicine. Seven- to eight-week-old female C57/BL6 mouse (Nippon CLEA, Tokyo, Japan) weighing 22 to 25 g were anesthetized by pentobarbital (50 mg/kg intraperitoneally; Abbott Laboratories, Abbott, IL), and the tracheas were exposed by neck incisions. Bleomycin (Nippon Kayaku, Tokyo, Japan), at the dose of 2.5 U/kg in 50 µl of PBS or PBS alone as a control, was administered into the trachea through a 30-gauge needle. After 14 days the mice were killed and their lungs were removed to perform immunohistochemical analysis and primary fibroblasts culture.

Mice Primary Fibroblast Isolation
We used a modified protocol of the mice fibroblast isolation method described previously (18). Briefly, mouse lungs were cut into small pieces, minced, and digested enzymatically by collagenase type III and DNase I (Worthington Biochemical, Lakewood, NJ) in Dulbecco's modified Eagle's medium with 5% FCS for 90 minutes. After filtration, cells were centrifuged, washed, and cultured in 6-cm dishes in MF-medium containing 1% FCS for 10 days. Confluent cells at first passage were used for Western blot analysis.

Western Blot Analysis
Cells were harvested and lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 15 mM NaF, 1% Triton X-100, 5 mM EDTA, 1 mM sodium orthovanadate, and protease inhibitor cocktails (Sigma-Aldrich). Cell lysates were prepared in SDS-sample buffer and subjected to SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and immunostained with antibodies against –SMA, fibronectin -tubulin, smad2, phospho-smad2, or SPHK1. Bands were visualized by the enhanced chemiluminescence method and their intensities were quantified by MultiGauge software (Fujifilm, Tokyo, Japan).

RNA Isolation and Real-Time Quantitative RT-PCR Analysis
Total RNA was extracted using ISOGEN reagent (Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized from 1 µg of total RNA by using ExScript RT reagent kits (Takara, Otsu, Japan) and random hexamer primers. Quantitative PCR was performed using real-time SYBR Green PCR technology and an ABI PRISM 7500 Sequence Detection system (Applied Biosystems, Foster City, CA). The following primers were used. For human SPHK1: forward primer, 5'-AGCTTCCTTGAACCATTATGCTG-3', and reverse primer, 5'-AGGTCTTCATTGGTGACCTGCT-3'; for human SPHK2: forward primer, 5'-CTGTCTGCTCCGAGGACTGC-3', and reverse primer, 5'-CAAAGGGATTGACCAATAGAAGC-3'; for human –SMA: forward primer, 5'-GACAATGGCTCTGGGCTCTGTAA-3', and reverse primer, 5'-ATGCCATGTTCTATCGGGTACTT-3'; for human S1P₁: forward primer, 5'-TATCATCGTCCGGCATTACA-3', and reverse primer, 5'-GAACACCACCGAGGTAGTT-3'; for human S1P₂: forward primer, 5'-GCGCCATTGTGGTGGAAAA-3', and reverse primer, 5'-CATTGCCGAGTGGAACTTGCT-3'; for human S1P₃: forward primer, 5'-GGTGATTGTGGTGAGCGTGTT-3', and reverse primer, 5'-AGGCCACATCAATGAGGAAGA-3'; and for human GAPDH: forward primer, 5'-GGCCTCCAAGGAGTAAGACC-3', and reverse primer, 5'-AGGGGTCTACATGGCAATG-3'. Amplification reactions were performed in duplicate with SYBR Premix Ex Taq (Takara), and the thermal cycling conditions were as follows: 10 seconds at 95°C, 40 cycles of 5 seconds at 95°C, and 34 seconds at 60°C. The expression of each mRNA was normalized to GAPDH mRNA expression.

Transfection with siRNA
SPHK1-targeted siRNA-1 (5'-GGGCAAGGCCUUGCAGCUC-3' and 5'-GAGCUGCAAGGCCUUGCCC-3'), siRNA-2 (5'-GGGCAAGGCCUUGCAGCUC-3' and 5'-GAGCUGCAAGGCCUUGCCC-3'), SPHK2-targeted siRNA (5'-GCUGGGCUGUCCUUCAACCU-3' and 5'-AGGUUGAAGGACAGCCCAGC-3'), S1P₁-targeted siRNA (5'-GGAGAACAGCAUUAAACUG-3' and 5'-CAGUUUAAUGCUGUUCUCC-3'), S1P₂-targeted siRNA (5'-UACCUUGCUCUCUGGCUCU-3' and 5'-AGAGCCAGAGAGCAAGGUA-3'), S1P₃-targeted siRNA (5'-GGUCAACAUUCUGAUGUCU-3' and 5'-AGACAUCAGAAUGUUGACC-3'), and control siRNA (5'-UUCUCCGAACGUGUCACGU-3' and 5'-ACGUGACACGUUCGGAGAA-3') were synthesized (Japan Bio Services Co., Ltd, Saitama, Japan). Fifty to seventy percent confluent human lung fibroblasts in 6-well dishes were prepared. Lipofectamine 2000 and Opti-MEM (Invitrogen, Carlsbad, CA) were mixed and incubated at room temperature for 20 minutes. siRNA–lipofectamine complexes were added to cells for 48 hours. After subsequent serum starvation, cells were treated with or without 5 ng/ml TGF-1 for 48 hours.

Immunofluorescent Cytochemistry
Cells were spread on 2-well glass chamber slides (Nunc, Rochester, NY) and cultured in MF medium for 1 day. After washing with PBS, cells were fixed for 20 minutes at room temperature with acetone-methanol (1:1), and permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. They were blocked with 3% bovine serum albumin for 1 hour and then incubated with anti––SMA monoclonal antibody (1:400) for 1 hour, followed by the incubation with FITC-conjugated anti-mouse antibody (1:200) for 1 hour. Samples were observed and scanned using a confocal laser scanning microscope LSM510 (Carl Zeiss, Jena, Germany).

Immunohistochemistry
Mice lungs were infused at a pressure of 20 cm H₂O with 10% buffered formalin for 24 hours, embedded in paraffin, and sectioned at 5-µm thickness. After deparaffinization, tissue sections were pretreated with 3% hydrogen peroxidase for 10 minutes and blocked with normal goat serum for 30 minutes. Then they were incubated for 1 hour with rabbit anti-SPHK1 (1:100) or anti––SMA (1:100) antibody as a primary antibody. Normal mouse or rabbit serum was used for negative control. After washing with PBS, they were incubated with biotinylated secondary antibodies, washed with PBS, and incubated with VECTASTAIN ABC Regent (Vector Laboratories, Burlingame, CA) for 30 minutes. When mouse monoclonal antibody was used, the Vector M.O.M. Immunodetection Kit (Vector Laboratories) was used according to the manufacturer's protocol. Diaminobenzidine was used as a substrate for the immunoperoxidase reaction. Sections were lightly counterstained with hematoxylin, and analyzed by brightfield microscopy.

The Measurement of Sphingosine 1-Phosphate Release
We modified the method of measuring S1P described previously (19). Confluent human lung fibroblasts in 6-well dishes were stimulated by 5 ng/ml TGF-1 for 20 minutes. After scraping cells with 200 µl of methanol containing 0.5 µl conc. HCl, lipids were extracted by adding 400 µl of chloroform /1 M NaCl (1:1 vol/vol) and 20 µl 3 N NaOH. The basic aqueous phase containing S1P was separated, combined after re-extraction, and incubated with 10 units of alkaline phosphatase (Sigma-Aldrich) at 37°C for 30 minutes in 90 µl of phosphatase buffer (200 mM Tris-HCl [pH 7.4], 75 mM MgCl₂ in 2 M glycine buffer [pH 9.0]). After terminating reaction with 20 µl of HCl, lipids were extracted twice with 300 µl of chloroform. Pooled organic fractions were dried and resuspended with 35 µl of buffer A (20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 15 mM NaF, 0.5 mM 4-deoxypyridoxine, sodium orthovanadate, and protease inhibitor cocktails in 200 mM Tris-HCl [pH 7.4]) containing 0.25% Triton X-100. Ten microliters of recombinant sphingosine kinase 1 (10 µg) was added to the solubilized lipids and reactions were started by addition of 5 µl of [-³²P] ATP (10 µCi, 10 mM) containing 100 mM MgCl₂. After incubation for 30 minutes at 37°C, the reaction was terminated by adding 20 µl of HCl on ice, followed by the addition of 200 µl of chloroform/methanol/HCl (100: 200: 1 vol/vol) and vortexing. After adding 60 µl of chloroform and 60 µl of 2 M KCl and centrifugation at 10,000 x g for 10 minutes, lipids containing the radioactive S1P in organic phase were extracted and separated by thin layer chromatography on silica gel G60 with 1-butanol/methanol/acetic acid/water (80:20:10:20 vol/vol). Visualized spots were quantified with an autoimage analyzer (BAS 2500; Fuji Xerox Co. Ltd., Tokyo, Japan).

Immunofluorescent Detection of GST-Rhotekin and S1P Receptors
The cDNA clone encoding the GST fusion proteins of Rho-binding domain of Rhotekin (7–89) in the pGEX vector was transformed into Escherichia coli and cultured at 22°C. The cells were subsequently disrupted by sonication in lysis buffer and centrifuged at 100,000 x g for 60 minutes. The supernatant was applied to a glutathion-sepharose affinity column and GST-Rhotekin (7–89) was eluted with 10 mM reduced glutathione.

Cells were spread on 4-well glass chamber slides (Nunc) and cultured for 1 day. S1P₁/pECFP, S1P₂/pEGFP, or S1P₃/pEGFP (Clontech, Palo Alto, CA) were mixed with Fugene6 (Roche Diagnostics, Indianapolis, IN) and incubated at room temperature for 15 minutes. DNA complexes were added to cells for 48 hours. After subsequent serum starvation, cells were treated with or without 5 ng/ml TGF-1 for 20 minutes. After washing with PBS, cells were fixed for 10 minutes at room temperature with 2% paraformaldehyde in PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. Cells were incubated with 40 µg/ml of GST-Rhotekin (7–89) for 1 hour. After fixing with paraformaldehyde for 10 minutes, they were incubated with anti-GST antibody (1:400) for 1 hour, following by incubating with anti-goat antibody Alexa 594 (1:400) for 1 hour. Samples were observed and scanned using a confocal laser scanning microscope.

Statistical Analysis
Results are expressed as mean ± SE. Statistical significance was assessed by using paired Student's t test or one-way ANOVA or Kruskal Wallis test with appropriate post hoc analysis (Sheffe) to exclude possible interaction between various variables within subgroups using Statcel software (OMS Publishing Inc., Saitama, Japan). A value of P < 0.05 was considered to be statistically significant.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS Discussion References

 
SPHK1 Expression Is Increased in Bleomycin-Induced Lung Fibrosis Lesions
To study the molecular mechanism underlying the etiology of lung fibrosis, we speculated whether SPHK/S1P signaling is involved in this pathogenesis. We first examined SPHK expression in experimentally induced lung fibrosis lesions.

C57/BL6 mice were administered bleomycin into the tracheas. Fourteen days after treatment, mice were killed and paraffin-embedded lung sections were examined for immunohistochemistry. As shown in Figure 1A, SPHK1 was strongly expressed in fibrotic foci (thick arrows), subepithelial layers of bronchus including smooth muscle cells (thin arrows), and relatively strong in vascular endothelial cells and bronchial epithelial cells. Immunopositive staining of –SMA was evident at smooth muscle cells underlying the bronchus and fibrotic foci. Next, the expression of fibronectin and SPHK1 as well as –SMA in lung fibroblasts from bleomycin-treated mice was quantified and compared with that in the cells from vehicle-treated mice. The expression of SPHK1 protein was significantly increased in the lung fibroblasts from bleomycin-treated mice compared with that in the control cells (0.72 ± 0.16 versus 0.15 ± 0.05, respectively) (Figures 1B and 1E). The bleomycin-treated cells also showed an increased expression of –SMA and fibronectin proteins which is characteristic of myofibroblast differentiation (1.46 ± 0.57 versus 0.46 ± 0.18 and 1.71 ± 0.33 versus 0.20 ± 0.06, respectively) (Figures 1C and 1D). These findings suggest that SPHK1 highly expressed in lung fibroblasts from bleomycin-treated mice may play a role in myofibroblast differentiation.

View larger version (78K): [in this window] [in a new window]   Figure 1. –SMA and SPHK1 are colocalized in fibrotic foci of bleomycin-treated mice lungs. Seven- to eight-week-old C57/BL6 mice were administered bleomycin into the trachea at a dose of 2.5 U/kg. After 14 days mice were killed and their lungs were removed. (A) Paraffin-embedded lung tissue was immunostained with normal rabbit IgG (a and b), anti-SPHK (c and d), and anti––SMA antibody (e and f). SPHK1 and –SMA were strongly expressed in fibrotic foci (thick arrows) and subepithelial layers (thin arrows). Original magnification: x200 (a, c, and e) and x400 (b, d, and f). These are representative pictures of three independent experiments. Primary mouse lung fibroblasts after bleomycin treatment increased –SMA, fibronectin, and SPHK expressions. Fourteen days after bleomycin instillation, mouse lungs were removed and their fibroblasts were isolated. Ten days later, cell lysates were collected and subjected to Western blot analysis (B). The intensity of each band with anti––SMA (C), anti-fibronectin (D), or anti-SPHK1 (E) antibody was quantified and normalized to that with anti–-tubulin antibody. Data are mean ± SE of three independent experiments. *P < 0.05 versus control.

SPHK1 but Not SPHK2 Is Up-Regulated by TGF-1 in Human Fibroblasts

View larger version (32K): [in this window] [in a new window]   Figure 2. TGF-1 up-regulated SPHK1 expression but not SPHK2 in human lung fibroblasts. Human lung fibroblasts WI-38 were treated with TGF-1 for 24 hours at indicated concentrations. Cell lysates were prepared and subjected to Western blot analysis. The intensity of each band with SPHK1 antibody was quantified and normalized to that with anti–-tubulin antibody. Data are mean ± SE of four independent experiments (A). Total RNA was extracted, and SPHK1 (B) and SPHK2 (C) mRNA expressions were quantified using real-time quantitative RT-PCR. Data are mean ± SE of four independent experiments after normalization to GAPDH. *P < 0.05 versus control, **P < 0.01 versus control.

SPHK1 Mediated TGF-1–Induced Myofibroblast Differentiation

View larger version (67K): [in this window] [in a new window]   Figure 3. SPHK inhibitors reduced -SMA and fibronectin expression. (A) Serum-starved fibroblasts were pretreated with indicated inhibitors for 1 hour and then treated with TGF-1 (0, 5, or 10 ng/ml). After 48 hours, cell lysates were prepared and subjected to Western blot analysis. The intensity of each band with anti––SMA (B) or anti-fibronectin (C) antibody was quantified and normalized to that with anti–-tubulin antibody. Data are mean ± SE of five independent experiments. (D) Total RNA was extracted and –SMA mRNA expression was quantified using real-time quantitative RT-PCR. Data are mean ± SE of five independent experiments after normalization to GAPDH. *P < 0.05 versus control, P < 0.05 versus TGF1 (5 ng/ml), P < 0.01 versus TGF-1 (5 ng/ml). (E) SPHK inhibitors diminished actin formation in TGF-–induced differentiation. Cells were spread on 2-well glass chamber slides and stimulated with TGF-1 pretreated with or without inhibitors. Cells were fixed and incubated with anti––SMA antibody, followed by binding with FITC-conjugated anti-mouse antibody. Samples were observed using a confocal laser scanning microscope. a, no stimulation; b, 5 ng/ml TGF-1; c, 5 ng/ml TGF-1 with 5 µM DMS; d, 5 ng/ml TGF-1 with 5 µM SK-I; e, 5 ng/ml TGF-1 with 5 µM SB431542. This is a representative picture of three independent experiments.

Silencing SPHK1 Expression by siRNA Inhibited TGF-1–Induced Myofibroblast Differentiation

View larger version (66K): [in this window] [in a new window]   Figure 4. Transfection of SPHK1 siRNA reduced –SMA and fibronectin expression. (A) Fifty to seventy percent confluent fibroblasts were transfected with control-siRNA, SPHK-siRNA-1, SPHK-siRNA-2, or SPHK2-siRNA. After 48 hours, cells were treated with TGF-1 (5 ng/ml) for 48 hours. Cell lysates were subjected to Western blot analysis. The intensity of each band with anti––SMA (B) or anti-fibronectin (C) antibody was quantified and normalized to that with anti–-tubulin antibody. Data are mean ± SE of five independent experiments. (D) Total RNA was extracted and –SMA mRNA expression was quantified using real-time quantitative RT-PCR. Data are mean ± SE of five independent experiments after normalization to GAPDH. *P < 0.05, **P < 0.01. (E) SPHK siRNA diminished actin formation in TGF-–induced differentiation. Cells were spread on 2-well glass chamber slides and transfeced with control siRNA (a and b), SPHK1 siRNA-1 (c and d), or SPHK2 siRNA (e and f). Forty-eight hours after stimulation with (b, d, and f) or without (a, c, and e) 5 ng/ml TGF-1, cells were fixed and incubated with anti––SMA antibody, followed by binding with FITC-conjugated anti-mouse antibody. Samples were observed using a confocal laser scanning microscope. This is a representative picture of three independent experiments. (F) Control- or SPHK1 siRNA–transfected fibroblasts were pretreated with or without S1P (100 nM) 1 hour before the addition of TGF-1 (10 ng/ml). Cell lysates were subjected to Western blot analysis. The intensity of each band with anti––SMA (G) or anti-fibronectin (H) antibody was quantified and normalized to that with anti–-tubulin antibody. Data are mean ± SE of four independent experiments.

Comparison of SPHK/S1P and smad2 Signaling Pathways Downstream of the TGF-1 Signal
Since it is postulated that smad signal activation mediates TGF-–induced myofibroblast differentiation in lung fibroblasts (4), we assessed the involvement of SPHK in the smad signaling either by inhibiting its activity using inhibitors or by silencing its expression using siRNA. As shown in Figure 5, TGF-1–induced smad2 phosphorylation was not affected by DMS or SK-I. Similarly, TGF-1–induced smad2 phosphorylation was not attenuated in SPHK1-knockdown cells. These results suggested that SPHK/S1P and smad2 signaling pathways function independently in the downstream of TGF- signals, and that SPHK/S1P signaling leads to myofibroblast differentiation because down-regulation of SPHK1 resulted in strong inhibition of TGF-–induced myofibroblast differentiation (Figures 3 and 4).

View larger version (96K): [in this window] [in a new window]   Figure 5. SPHK inhibition did not change TGF-1–induced phosphorylation of smad2. Cells were pretreated with DMS (5 µM), SK-I (5 µM), or SB431542 (5 µM) for 1 hour, or transfected with SPHK1 siRNA-1 for 48 hours. After stimulation with 5 ng/ml TGF-1 or 5 µM S1P for indicating time, cell lysates were prepared and subjected to Western blot analysis using anti–phospho-smad2 and anti-smad2 antibodies. These are representative pictures of four independent experiments.

View larger version (46K): [in this window] [in a new window]   Figure 6. Transfection of human lung fibroblasts with S1P2 and S1P3 siRNA reduced –SMA expression but not S1P1 siRNA. (A) Cells were pretreated with 10 µM Y27632 for 1 hour or transfected with control, S1P1, S1P2, or S1P3 siRNA for 48 hours. Forty-eight hours after stimulation with 5 ng/ml TGF-1, cell lysates were subjected to Western blot analysis. The intensity of each band with anti––SMA (B) or anti-fibronectin (C) antibody was quantified and normalized to that with anti–-tubulin antibody. Data are mean ± SE of five independent experiments. Total RNA was extracted and –SMA (D) or SPHK1 (E) mRNA expression was quantified using real-time quantitative RT-PCR. Data are mean ± SE of five independent experiments after normalization to GAPDH. *P < 0.05, **P < 0.01.

TGF-1 Causes Activation of Rho GTPase through S1P2 and S1P3
Based on our present results, we hypothesized that TGF-1–induced activation of SPHK1 and subsequent production of S1P causes S1P receptor activation which play roles in TGF-–induced myofibroblast differentiation through Rho/Rho kinase–mediated signaling pathways. To demonstrate the activation of S1P receptors by TGF-1 in lung fibroblasts, first we quantified the intracellular amounts of S1P production after TGF-1 stimulation. As shown in Figure 7A, S1P was significantly (2- to 3-fold) increased by TGF- stimulation.

View larger version (72K): [in this window] [in a new window]   Figure 7. TGF-1 increases S1P production and translocates of Rho to bind with S1P2 and S1P3 receptors. (A) Cells were cultured in 6-well plates and treated with TGF-1 (5 ng/ml) for 20 minutes. The amount of intracellular S1P was quantified by the method described in MATERIALS AND METHODS. To detect the activation of Rho, CFP-S1P1 (B and C), GFP-S1P2 (D and E), and GFP-S1P3 (F and G) was transfected to cells on 4-well glass chamber slides. Cells were treated with (C, E, and G) or without (B, D, and F) TGF-1 (5 ng/ml) for 20 minutes and fixed. Each S1P receptor was expressed on the transfected cell membrane (left, green color). Rho bound to GST-Rhotekin was localized in cytosol and translocated to cell membrane after TGF-1 stimulation (center, red color). Rho was co-expression of with S1P2 and S1P3, but not S1P1 (right, yellow color).

	Discussion

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS Discussion References

 
TGF- is a key cytokine in the differentiation of fibroblasts into myofibroblasts as characterized by –SMA expression (1). Myofibroblasts are responsible for active extracellular matrix deposition and are considered as the main cells that cause various aspects of pathogenesis in lung fibrosis (23). Since the persistence or disappearance of the myofibroblasts may cause disease progression or resolution, targeting the myofibroblasts should be one of productive therapeutic strategies.

It was shown that TGF- induces rapid rearrangement of actin filament system, resulting in membrane ruffling via Rho GTPase-dependent pathways (24). Dominant-negative mutants of RhoA and Cdc42 abrogated the TGF-–induced responses such as membrane ruffling, whereas the mutant of smad4 had no influence. Chen and coworkers also described that RhoA signaling is another important pathway for TGF-–induced vascular smooth muscle cell differentiation in addition to smad signaling (25). Since the molecular events of smads and Rho GTPases in the downstream of TGF- have not been cleared, the present studies demonstrated that TGF-–induced transactivation of S1P receptors mediates TGF-–induced myofibroblast differentiation via Rho/Rho kinase signaling pathway, which enabled to form an important paradigm for the understanding of molecular mechanisms of TGF-–induced myofibroblast differentiation.

SPHK1 has been shown to localize primarily in the cytosol, and several growth factors such as TNF- induce its translocation to the plasma membrane. It has been reported that S1P produced by SPHK1 translocates to the close vicinity to its receptor and transactivates cell surface S1P receptors (12, 13). We previously showed that WI-38 human lung fibroblast expressed S1P₁, S1P₂, and S1P₃ receptors (17). All S1P receptor subtypes are coupled to several heterotrimeric G proteins. S1P₁ is coupled with Gi, whereas S1P₂ and S1P₃ coupled with not only G_i but also G_q and G_12/13 (26, 27). Recently Ishii and colleagues generated mice that are null for S1P₂ or for both S1P₂ and S1P₃, and found that these mice displayed significant decrease in Rho activity in mouse embryonic fibroblasts (21). That is compatible with our observations that siRNA of S1P₂ and S1P₃, and Rho kinase inhibitor Y27632 inhibited myofibroblasts differentiation, and that activated Rho by TGF- stimulation translocated to cell membranes where S1P₂ and S1P₃ were localized.

Xin and coworkers reported that exogenous S1P mimicked TGF- signaling such as smads phosphorylation, followed by activation of connective tissue growth factor (16). We also observed S1P-induced smad2 phosphorylarion in lung fibroblasts. In the same way, our study revealed that inhibition of SPHK1 either by inhibition of SPHK activity using inhibitors or by silencing SPHK1 expression using siRNA (Figure 5) did not block TGF-1–induced smad2 phosphorylation. Furthermore, down-regulation of S1P₂ or S1P₃ using siRNA delivery did not affect TGF-1–induced smad2 phosphorylation (data not shown). These results indicate that Rho signaling pathway may be indispensable to TGF-1–induced –SMA expression. However, when either smads or Rho signals were inhibited, the expression of –SMA mRNA and protein decreased after 48 hours of culture. Further studies are necessary to clarify the physiological relevance of these two signaling pathways after TGF-1 stimulation.

Sato and colleagues reported that the overexpression of S1P phosphatase, which converts S1P to sphingosine, stimulates both basal and TGF-–induced collagen promoter activity in human foreskin fibroblasts, whereas exogenous S1P (10–30 µM) treatment or overexpression of SPHK1 did not stimulate its activity (28). Davaille and coworkers reported that S1P (3–10 µM) inhibited growth of liver myofibroblasts (29, 30). In contrast, we previously described that S1P (10–100 nM) increased –SMA expression in lung fibroblasts (17). A possible explanation for these discrepancies may come from that the effect of S1P is different depending on the concentration used. It has been shown that S1P at micromolar concentrations is taken up by intracellular compartments and acts as a second messenger, and that S1P at nanomolar activates S1P receptors (19, 31, 32).

Johnson and coworkers reported that SPHK1 staining was seen in epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, type II alveolar cells, foamy macrophages, and endothelial cells of blood vessels in normal human lung tissue (33). We observed that SPHK1 was expressed in similar areas of mice lungs, and in addition SPHK1 was strongly stained within –SMA–positive fibrosis foci after bleomycin treatment. We confirmed that SPHK protein expression was up-regulated in bleomycin-treated fibroblasts, on the other hand SPHK mRNA expression was decreased (data not shown). We speculated that SPHK mRNA is down-regulated in response to earlier SPHK upregulation. These fibroblasts were isolated at Day 14 after bleomycin instillation, and this time point is in the earlier stage of lung fibrotic process. We showed that TGF-1–induced SPHK1 overexpression may have some roles in lung fibroblast differentiation, and further studies are needed to clarify molecular mechanisms underlying pathogenesis of human lung fibrosis. In this context the role of S1P in other fibrotic diseases, such as skin scleroderma, kidney fibrosis, and liver cirrhosis, needs to be explored.

In summary, we have demonstrated that SPHK1/S1P signaling pathway plays important roles in the regulation of TGF-1–induced differentiation of fibroblasts into myofibroblasts via trans-activation of S1P₂ and/or S1P₃ receptors, leading to Rho/Rho kinase signaling pathway. These data provide insight into novel mechanism with potential therapeutic implication for lung fibrosis.

	Acknowledgments

 
The authors thank Yoshiko Urata, M.D., Ph.D., and Kozo Ninomiya, M.D., Ph.D. (Division of Cardiovascular and Respiratory Medicine, Kobe University Graduate School of Medicine), for research technical advice.

	Footnotes

 
This work was supported by a grant-in-aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS18590848, Y. N.).

Originally Published in Press as DOI: 10.1165/rcmb.2007-0065OC on July 19, 2007

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 February 28, 2007

Accepted in final form May 17, 2007

	References

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS Discussion References

 

1. Phan SH. The myofibroblast in pulmonary fibrosis. Chest 2002;122:286S–289S.[CrossRef][Medline]
2. Willis BC, DuBois RM, Borok Z. Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc 2006;3:377–382.[Abstract/Free Full Text]
3. Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, Sorescu D. NAD(P)H oxidase 4 mediates transforming growth factor 1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res 2005;97:900–907.[Abstract/Free Full Text]
4. Hu B, Wu Z, Phan SH. Smad2 Mediates Transforming growth factor--induced -smooth muscle actin expression. Am J Respir Cell Mol Biol 2003;29:397–404.[Abstract/Free Full Text]
5. Shu X, Wu W, Mosteller RD, Broek D. Sphingosine kinase nediates vascular endothelial gowth factor-induced activation of ras and mitogen–activated protein kinase. Mol Cell Biol 2002;22:7758–7768.[Abstract/Free Full Text]
6. Taha TA, Hannun YA, Obeid LM. Sphingosine kinase: biochemical and cellular regulation and role in disease. J Biochem Mol Biol 2006;39:113–131.[Medline]
7. Igarashi N, Okada T, Hayashi S, Fujita T, Jahangeer S, Nakamura S. Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis. J Biol Chem 2003;47:46832–46839.
8. Maceyka M, Sankala H, Hait NC, Le Stunff H, Liu H, Toman R, Collier C, Zhang M, Satin LS, Merrill AH Jr, et al. SphK1 and Sphk2, sphingosine kinase isoenzymes with opposing function in sphingolipid metabolism. J Biochem (Tokyo) 2005;280:37118–37129.
9. Okada T, Ding G, Sonoda H, Kajimoto T, Haga Y, Khosrowbeygi A, Gao S, Nakamura S. Involvement of N-terminal-extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis. J Biochem (Tokyo) 2005;280:36318–36325.
10. Goparaju SK, Jolly PS, Watterson KR, Bektas M, Alvarez S, Sarkar S, Mel L, Ishii I, Chun J, Milstien S, et al. The S1P₂ receptor negatively regulates platelet-drived growth factor-induced motility and proliferation. Mol Cell Biol 2005;25:4237–4249.[Abstract/Free Full Text]
11. Hobson JP, Rosenfeldt HM, Barak LS, Olivera A, Poulton S, Caron MG, Milstien S, Spiegel S. Sphingosine-1-phospate receptor EDG-1 in PDGF-induced cell motility. Science 2001;291:1800–1803.[Abstract/Free Full Text]
12. Jolly PS, Bektas M, Olivera A, Gonzalez-Espinosa C, Proia RL, Rivera J, Milstien S, Spiegel S. Transactivation of sphingosine-1-phosphate receptors by FcRI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med 2004;199:959–970.[Abstract/Free Full Text]
13. Toman RE, Payne SG, Watterson KR, Maceyka M, Lee NH, Milstien S, Bigbee JW, Spiegel S. Differential transactivation of sphingosine-1-phosphate receptors modulated NGF-induced neurite extension. J Cell Biol 2004;166:381–392.[Abstract/Free Full Text]
14. Radeke HH, Von Wenckstern H, Stoidtner K, Sauer B, Hammer S, Kleuser B. Overlapping signaling pathways of sphingosine 1-phosphate and TGF- in murine langerhans cell line XS52. J Immunol 2005;174:2778–2786.[Abstract/Free Full Text]
15. Sauer B, Vogler R, Von Wenckstern H, Fujii M, Anzano MB, Glick AB, Schäfer-Korting M, Roberts AB, Kleuser B. Involvement of Smad signaling in sphingosine 1-phosphate-mediated biological responses of keratinocytes. J Biol Chem 2004;279:38471–38479.[Abstract/Free Full Text]
16. Xin C, Ren S, Kleuser B, Shabahang S, Eberhardt W, Radeke H, Schäfer-Korting M, Pfeilschifter J, Huwiler A. Sphingosine 1-phosphate cross-activates the Smad signaling cascade and mimics transforming growth factor--induced cell responses. J Biol Chem 2004;279:35255–35262.[Abstract/Free Full Text]
17. Urata Y, Nishimura Y, Hirase T, Yokoyama M. Sphingosine 1-phosphate induces alpha-smooth muscle actin expression in lung fibroblasts via Rho-kinase. Kobe J Med Sci 2005;51:17–27.[Medline]
18. Huaux F, Liu T, McGarry B, Ullenbruch M, Phan SH. Dual roles of IL-4 in lung injury and fibrosis. J Immunol 2003;170:2083–2092.[Abstract/Free Full Text]
19. Edsall LC, Spiegel S. Enzymatic measurement of sphingosine 1-phosphate. Anal Biochem 1999;272:80–86.[CrossRef][Medline]
20. Yamanaka M, Shegogue D, Pei H, Bu S, Bielawska A, Bielawski J, Pettus B, Hannun YA, Obeid L, Trojanowska M. Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor- and mediates TIMP-1 up-regulation. J Biol Chem 2004;279:53994–54001.[Abstract/Free Full Text]
21. Ishii I, Ye X, Friedman B, Kawamura S, Contos JJ, Kingsbury MA, Yang AH, Zhang G, Brown JH, Chun J. Marked perinatal lethality and cellular signaling deficits in mice null for the two sphingosine 1-phosphate (S1P) receptors, S1P₂/LP_B2/EDG-5 and S1P₃/LP_B3/EDG-3. J Biol Chem 2002;277:25152–25159.[Abstract/Free Full Text]
22. Ueda S, Kataoka T, Satoh T. Role of the Sec 14-like domain of Db1 family exchange factors in the regulation of Rho family GTPase in different subcellular sites. Cell Signal 2004;16:899–906.[CrossRef][Medline]
23. King TE Jr, Schwarz MI, Brown K, Tooze JA, Colby TV, Waldron JA Jr, Flint A, Thurlbeck W, Cherniack RM. Idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2001;164:1025–1032.[Abstract/Free Full Text]
24. Edlund S, Landström M, Heldin CH, Aspenström P. Transforming growth factor--induced mobilization of actin cytoskeleton requires signaling by small GTPase Cdc-42 and RhoA. Mol Biol Cell 2002;13:902–914.[Abstract/Free Full Text]
25. Chen S, Crawford M, Day RM, Briones VR, Leader JE, Jose PA, Lechleider RJ. RhoA modulates Smad signaling during transforming growth factor--induced smooth muscle differentiation. J Biol Chem 2006;281:1765–1770.[Abstract/Free Full Text]
26. Radeff-Huang J, Seasholtz TM, Matteo RG, Brown JH. G protein mediated signaling pathways in lysophospholipid cell proliferation and survival. J Cell Biochem 2004;92:949–966.[CrossRef][Medline]
27. Windh RT, Lee MJ, Hla T, An S, Barr AJ, Manning DR. Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3 and H218/Edge-5 to the G₁, G_q and G₁₂ families of heterotrimeric G proteins. J Biol Chem 1999;274:27351–27358.[Abstract/Free Full Text]
28. Sato M, Markiewicz M, Yamanaka M, Bielawska A, Mao C, Obeid LM, Hannun YA, Trojanowska M. Modulation of transforming growth factor- (TGF-) signaling by endogenous sphingolipid mediators. J Biol Chem 2003;278:9276–9282.[Abstract/Free Full Text]
29. Davaille J, Gallois C, Habib A, Li L, Mallat A, Tao J, Levade T, Lotersztajn S. Antiproliferative properties of sphingosine 1-phosphate in human hepatic myofibroblasts. J Biol Chem 2000;44:34628–34633.
30. Davaille J, Li L, Mallat A, Lotersztajn S. Sphingosine 1-phosphate triggers both apoptotic and survival signals for human hepatic myofibroblasts. J Biol Chem 2002;277:37323–37330.[Abstract/Free Full Text]
31. Hla T, Lee MJ, Ancellin N, Liu CH, Thangada S, Thompson BD, Kluk M. Sphingosine -1-phosphate: extracellular mediator or intracellular second messenger. Biochem Pharmacol 1999;15:201–207.
32. Spiegel S, Milstien S. Endogenous and intracellularly generated sphingosine 1-phosphate can regulate cellular processes by divergent pathways. Biochem Soc Trans 2003;31:1216–1219.[Medline]
33. Johnson KR, Johnson KY, Crellin HG, Ogretmen B, Boylan AM, Harley RA, Obeid LM. Immunohistochemical distribution of sphingosine kinase 1 in normal and tumor lung tissue. J Histochem Cytochem 2005;53:1159–1166.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Uhlig and E. Gulbins Sphingolipids in the Lungs Am. J. Respir. Crit. Care Med., December 1, 2008; 178(11): 1100 - 1114. [Abstract] [Full Text] [PDF]

				X. Ye Lysophospholipid signaling in the function and pathology of the reproductive system Hum. Reprod. Update, September 1, 2008; 14(5): 519 - 536. [Abstract] [Full Text] [PDF]

				S. Bu, B. Kapanadze, T. Hsu, and M. Trojanowska Opposite Effects of Dihydrosphingosine 1-Phosphate and Sphingosine 1-Phosphate on Transforming Growth Factor-{beta}/Smad Signaling Are Mediated through the PTEN/PPM1A-dependent Pathway J. Biol. Chem., July 11, 2008; 283(28): 19593 - 19602. [Abstract] [Full Text] [PDF]

				D. A. Lebman and S. Spiegel Thematic Review Series: Sphingolipids. Cross-talk at the crossroads of sphingosine-1-phosphate, growth factors, and cytokine signaling J. Lipid Res., July 1, 2008; 49(7): 1388 - 1394. [Abstract] [Full Text] [PDF]

				K. Takabe, S. W. Paugh, S. Milstien, and S. Spiegel "Inside-Out" Signaling of Sphingosine-1-Phosphate: Therapeutic Targets Pharmacol. Rev., June 1, 2008; 60(2): 181 - 195. [Abstract] [Full Text] [PDF]

				T. Nishiuma, Y. Nishimura, T. Okada, E. Kuramoto, Y. Kotani, S. Jahangeer, and S.-i. Nakamura Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1085 - L1093. [Abstract] [Full Text] [PDF]

				H. P. Kim and A. M. K. Choi Caveolin-1 stops profibrogenic signaling? Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L841 - L842. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 2007-0065OCv1 37/4/395    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Pub