Involvement of Sphingosine 1-Phosphate (SIP)/S1P3 Signaling in Cholestasis-Induced Liver Fibrosis
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090037
ISSN1525-2191
AutoresChangyong Li, Xiangming Jiang, Lin Yang, Xihong Liu, Shi Yue, Liying Li,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoBioactive sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) have been implicated in many critical cellular events, including inflammation, cancer, and angiogenesis. However, the role of S1P/S1PR signaling in the pathogenesis of liver fibrosis has not been well documented. In this study, we found that S1P levels and S1P3 receptor expression in liver tissue were markedly up-regulated in a mouse model of cholestasis-induced liver fibrosis. In addition, the S1P3 receptor was also expressed in green fluorescent protein transgenic bone marrow (BM)-derived cells found in the damaged liver of transplanted chimeric mice that underwent bile duct ligation. Silencing of S1P3 expression significantly inhibited S1P-induced BM cell migration in vitro. Furthermore, a selective S1P3 receptor antagonist, suramin, markedly reduced the number of BM-derived cells during cholestasis. Interestingly, suramin administration clearly ameliorated bile duct ligation-induced hepatic fibrosis, as demonstrated by attenuated deposition of collagen type I and III, reduced smooth muscle α-actin expression, and decreased total hydroxyproline content. In conclusion, our data suggest that S1P/S1P3 signaling plays an important role in cholestasis-induced liver fibrosis through mediating the homing of BM cells. Modulation of S1PR activity may therefore represent a new antifibrotic strategy. Bioactive sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) have been implicated in many critical cellular events, including inflammation, cancer, and angiogenesis. However, the role of S1P/S1PR signaling in the pathogenesis of liver fibrosis has not been well documented. In this study, we found that S1P levels and S1P3 receptor expression in liver tissue were markedly up-regulated in a mouse model of cholestasis-induced liver fibrosis. In addition, the S1P3 receptor was also expressed in green fluorescent protein transgenic bone marrow (BM)-derived cells found in the damaged liver of transplanted chimeric mice that underwent bile duct ligation. Silencing of S1P3 expression significantly inhibited S1P-induced BM cell migration in vitro. Furthermore, a selective S1P3 receptor antagonist, suramin, markedly reduced the number of BM-derived cells during cholestasis. Interestingly, suramin administration clearly ameliorated bile duct ligation-induced hepatic fibrosis, as demonstrated by attenuated deposition of collagen type I and III, reduced smooth muscle α-actin expression, and decreased total hydroxyproline content. In conclusion, our data suggest that S1P/S1P3 signaling plays an important role in cholestasis-induced liver fibrosis through mediating the homing of BM cells. Modulation of S1PR activity may therefore represent a new antifibrotic strategy. Fibrosis is a wound-healing response that engages a range of cell types and mediators to encapsulate injury from multiple causes ranging from viral infection, alcohol abuse, and drug or chemical toxicity to autoimmune and metabolic disorders. Cirrhosis is the advanced stage of fibrosis, typically characterized by nodule formation and organ contraction.1Friedman SL Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2160) Google Scholar It is worth noting that cholestatic liver disorders are a serious clinical problem and often require liver transplantation due to cirrhosis.2Keeffe EB Liver transplantation: current status and novel approaches to liver replacement.Gastroenterology. 2001; 120: 749-762Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar The most common cholestatic liver diseases affecting adults are primary biliary cirrhosis and primary sclerosing cholangitis, which may present diagnostic and therapeutic difficulties.3Kim WR Ludwig J Lindor KD Variant forms of cholestatic diseases involving small bile ducts in adults.Am J Gastroenterol. 2000; 95: 1130-1138Crossref PubMed Google Scholar Generally, the pathogenesis of fibrosis/cirrhosis is characterized by the excessive deposition and histological redistribution of extracellular matrix components in the tissue as consequences of chronic liver injury. Cytokines regulating the scarring responses to injury modulate hepatic fibrogenesis in vivo and in vitro. Strategies with the aim of disrupting the cytokine synthesis and/or signaling pathways markedly decreased hepatic fibrosis in experimental models.4Bataller R Brenner DA Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4065) Google Scholar Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated by sphingomyelin metabolism that acts almost ubiquitously, influencing many key biological parameters including cell proliferation, differentiation, motility, and survival and the regulation of immune function.5Alvarez SE Milstien S Spiegel S Autocrine and paracrine roles of sphingosine-1-phosphate.Trends Endocrinol Metab. 2007; 18: 300-307Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 6Hannun YA Obeid LM Principles of bioactive lipid signalling: lessons from sphingolipids.Nat Rev Mol Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2417) Google Scholar S1P is generated by phosphorylation of sphingosine catalyzed by sphingosine kinases (SphKs). Two isoforms of mammalian SphK have been cloned and characterized: SphK types 1 and 2 (SphK1 and SphK2).7Spiegel S Milstien S Sphingosine-1-phosphate: an enigmatic signalling lipid.Nat Rev Mol Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1748) Google Scholar SphK1 is slightly more efficient than SphK2 in phosphorylating their primary intracellular substrate, sphingosine, whereas SphK2 is significantly more efficient toward unnatural substrates such as the immunomodulatory drug FTY720.8Billich A Bornancin F Devay P Mechtcheriakova D Urtz N Baumruker T Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases.J Biol Chem. 2003; 278: 47408-47415Crossref PubMed Scopus (392) Google Scholar The concentration of S1P in cells is normally low and is regulated tightly by the equilibrium between its formation, catalyzed by SphK, and its degradation, catalyzed by S1P lyase and S1P phosphatase. SphK is stimulated by numerous external stimuli resulting in increased intracellular S1P concentration and increased release from certain cell types.7Spiegel S Milstien S Sphingosine-1-phosphate: an enigmatic signalling lipid.Nat Rev Mol Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1748) Google Scholar Of note, most of the characterized actions of S1P are mediated through a family of five G protein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5, originally referred to as endothelial differentiation gene-1, 5, 3, 6, and 8, respectively.7Spiegel S Milstien S Sphingosine-1-phosphate: an enigmatic signalling lipid.Nat Rev Mol Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1748) Google Scholar The different activities triggered by S1P depend on the pattern of expression of S1P receptors (S1PRs) in each cell type. Among the S1PRs, S1P1-3 are widely expressed in various tissues, whereas the expression of S1P4 is confined to lymphoid and hematopoietic tissue and that of S1P5 to the central nervous system.9Sanchez T Hla T Structural and functional characteristics of S1P receptors.J Cell Biochem. 2004; 92: 913-922Crossref PubMed Scopus (407) Google Scholar From this pattern of expression, it is possible to understand the pathophysiological role of each S1PR. Indeed, the role of S1P and its receptors has been reported in various disease-related models including lung fibrosis10Kono Y Nishiuma T Nishimura Y Kotani Y Okada T Nakamura S Yokoyama M Sphingosine kinase 1 regulates differentiation of human and mouse lung fibroblasts mediated by TGF-β1.Am J Respir Cell Mol Biol. 2007; 37: 395-404Crossref PubMed Scopus (124) Google Scholar, 11Schwab SR Pereira JP Matloubian M Xu Y Huang Y Cyster JG Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients.Science. 2005; 309: 1735-1739Crossref PubMed Scopus (644) Google Scholar, 12Sekiguchi M Iwasaki T Kitano M Kuno H Hashimoto N Kawahito Y Azuma M Hla T Sano H Role of sphingosine 1-phosphate in the pathogenesis of Sjögren's syndrome.J Immunol. 2008; 180: 1921-1928Crossref PubMed Scopus (41) Google Scholar; in addition, our previous study unraveled a major role of S1P2 receptor in the wound healing response to acute liver injury induced by carbon tetrachloride.13Serriere-Lanneau V Teixeira-Clerc F Li LY Schippers M de Wries W Julien B Tran-Van-Nhieu J Manin S Poelstra K Chun J Carpentier S Levade T Mallat A Lotersztajn S The sphingosine 1-phosphate receptor S1P2 triggers hepatic wound healing.FASEB J. 2007; 21: 2005-2013Crossref PubMed Scopus (72) Google Scholar However, the exact functional role of S1P as well as the therapeutic potential of strategies aimed at S1P/S1PRs signalings in liver fibrogenesis is still unknown. It is well established that myofibroblasts are the principal effector cells that are responsible for the overproduction of extracellular matrix in fibrotic liver.1Friedman SL Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2160) Google Scholar, 4Bataller R Brenner DA Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4065) Google Scholar Myofibroblasts can be produced by the activation of hepatic stellate cells, portal fibroblasts, and fibrocytes, as well as cells derived from epithelial mesenchymal transition.1Friedman SL Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2160) Google Scholar, 4Bataller R Brenner DA Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4065) Google Scholar, 14Kisseleva T Brenner DA Mechanisms of fibrogenesis.Exp Biol Med. 2008; 233: 109-122Crossref PubMed Scopus (369) Google Scholar Recently, several lines of evidence have indicated that a significant proportion of myofibroblasts are of bone marrow (BM) origin in liver fibrosis.15Forbes SJ Russo FP Rey V Burra P Rugge M Wright NA Alison MR A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis.Gastroenterology. 2004; 126: 955-963Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 16Russo FP Alison MR Bigger BW Amofah E Florou A Amin F Bou-Gharios G Jeffery R Iredale JP Forbes SJ The bone marrow functionally contributes to liver fibrosis.Gastroenterology. 2006; 130: 1807-1821Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 17Kisseleva T Uchinami H Feirt N Quintana-Bustamante O Segovia JC Schwabe RF Brenner DA Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis.J Hepatol. 2006; 45: 429-438Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 18di Bonzo LV Ferrero I Cravanzola C Mareschi K Rustichell D Novo E Sanavio F Cannito S Zamara E Bertero M Davit A Francica S Novelli F Colombatto S Fagioli F Parola M Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential.Gut. 2008; 57: 223-231Crossref PubMed Scopus (257) Google Scholar, 19Kallis YN Alison MR Forbes SJ Bone marrow stem cells and liver disease.Gut. 2007; 56: 716-724Crossref PubMed Scopus (125) Google Scholar However, much less is known about the mechanism that governs the mobilization of BM cells after chronic liver injury. Importantly, several reports have demonstrated that S1P strongly stimulated BM stem cells migration in vitro.20Annabi B Thibeault S Lee YT Bousquet-Gagnon N Eliopoulos N Barrette S Galipeau J Beliveau R Matrix metalloproteinase regulation of sphingosine-1-phosphate-induced angiogenic properties of bone marrow stromal cells.Exp Hematol. 2003; 31: 640-649Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Meriane M Duhamel S Lejeune L Galipeau J Annabi B Cooperation of matrix metalloproteinases with the RhoA/Rho kinase and mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase signaling pathways is required for the sphingosine-1-phosphate-induced mobilization of marrow-derived stromal cells.Stem Cells. 2006; 24: 2557-2565Crossref PubMed Scopus (67) Google Scholar, 22Jaganathan BG Ruester B Dressel L Stein S Grez M Seifried E Henschler R Rho inhibition induces migration of mesenchymal stromal cells.Stem Cells. 2007; 25: 1966-1974Crossref PubMed Scopus (80) Google Scholar Along this line, we hypothesized that S1P is involved in liver fibrogenesis through mediation of the migration of BM cells to the damaged liver. In an effort to establish what factors govern BM cells engraftment after chronic liver injury, we have sought to investigate the pathogenesis of liver fibrosis in experimental animals induced by bile duct ligation (BDL). This pattern is rather unique, being sustained by compensatory proliferation of biliary epithelial cells. Chronic obstruction of the bile duct causes massive activation of periductal myofibroblasts and ultimately results in biliary fibrosis/cirrhosis.23Xia JL Dai CS Michalopoulos GK Liu YH Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation.Am J Pathol. 2006; 168: 1500-1512Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 24Ezure T Sakamoto T Tsuji H Lunz JG Murase N Fung JJ Demetris AJ The development and compensation of biliary cirrhosis in interleukin-6-deficient mice.Am J Pathol. 2000; 156: 1627-1639Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar In this study, we evaluated the importance of S1P/S1P3 signaling in liver fibrogenesis through mediation of the homing of BM cells during cholestasis, which may represent a novel antifibrotic target. Enhanced green fluorescent protein (EGFP)-transgenic ICR mice (3 weeks old) were sacrificed by cervical dislocation at the time of BM harvest. EGFP-positive BM cells were extracted from the tibias and femurs by flushing with culture medium (Invitrogen, Grand Island, NY) using a 25-gauge needle. The cells were then passed through a 70-mm nylon mesh and were washed three times with PBS containing 2% fetal bovine serum (Biochrom, Berlin, Germany). All animal work was performed under the guidelines of the Ethics Committee of Capital Medical University. ICR mice aged 6 weeks received lethal irradiation (8 Gy) and immediately received transplantation by a tail vein injection of 1.5 × 107 whole BM cells, which were obtained from 3-week-old EGFP transgenic mice. Four weeks later, mice were subjected to the BDL-induced liver fibrosis as follows. After 2 weeks, mice were sacrificed, and tissue was harvested. To further demonstrate that homing of BM cells mediated by S1P is via the S1P3 receptor, we performed another series of experiments. After lethal irradiation and transplantation of EGFP-positive BM cells, mice underwent BDL in the absence or presence of suramin (Sigma-Aldrich, St. Louis, MO) administration. Suramin (20 mg/kg body weight, twice per week) in saline was injected i.p. All mice received 2 weeks of suramin or saline alone treatment, and liver tissue was harvested at next day after last injection. ICR mice were allocated randomly to two experimental groups, and either BDL or sham operations were performed as described previously by Uchinami et al25Uchinami H Seki E Brenner DA D'Armiento J Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis.Hepatology. 2006; 44: 420-429Crossref PubMed Scopus (151) Google Scholar In brief, mice were anesthetized to receive a midline laparotomy, and then the common bile duct was exposed and ligated three times. Two ligatures were placed in the proximal portion of the bile duct, and one ligature was located in the distal portion of the bile duct. The bile duct was then cut between the ligatures. The abdomen was closed in layers, and mice were allowed to recover on a heat pad. Two weeks later, mice were anesthetized to collect blood and liver samples. Liver samples were fixed in 4% paraformaldehyde and embedded in Tissue Tek OCT compound (Electron Microscopy Sciences, Hatfield, PA); 5-μm frozen sections were used for immunofluorescence. They were blocked with 3% bovine serum albumin for 1 hour and then incubated with anti-S1P3 receptor polyclonal antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) and Cy3-conjugated AffiniPure goat anti-rabbit IgG antibody (1:1000, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) as a secondary antibody. The sections were covered with Vectashield mounting medium containing 4,6-diamidino-2-phenylindole and observed under a confocal microscope (LSM510; Carl Zeiss MicroImaging GmbH, Jena, Germany). Immunohistochemical analysis was performed using anti-smooth muscle α-actin (α-SMA) (1:80, mouse monoclonal clone 1A4; Sigma-Aldrich) with a Mouse on Mouse kit (Vector Laboratories, Burlingame, CA). Detection of the primary antibody was performed by using a biotinylated antibody and DAB Peroxidase kit (Vector Laboratories). For negative controls, sections were processed as described earlier, except that incubation with the primary antibody was omitted. S1P concentrations in serum, liver tissue, and BM were determined as described previously by Min et al26Min JK Yoo HS Lee EY Lee WJ Lee YM Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase l.Anal Biochem. 2002; 303: 167-175Crossref PubMed Scopus (139) Google Scholar with minor modifications. Tissue samples were homogenized in 25 mmol/L HCl/1 mmol/L NaCl. BM was obtained by flushing the femur and tibia of donor mice with 25 mmol/L HCl/1 mol/L NaCl. The samples were ultrasonicated for 10 minutes in ice-cold water. Serum was used in measurements without any pretreatment. After transferring a 20-μl aliquot of the sample to a fresh tube for protein assay, 250 μl of methanol containing 0.6 μl of concentrated HCl were added and the samples were ultrasonicated for 10 minutes in ice-cold water. Lipids were extracted by addition of 500 μl of CHCl3/1 mol/L NaCl (1:1, v/v) and 25 μl of 3 N NaOH. The basic aqueous phase containing S1P was transferred to a siliconized glass tube. The residual S1P in the organic phase was extracted twice with 250 μl of methanol/1 mol/L NaCl (1:1, v/v) plus 15 μl of 3 N NaOH, and all of the aqueous fractions were then combined. The aqueous phase containing S1P was mixed with 150 μl of buffer (200 mmol/L Tris-HCl and 75 mmol/L MgCl2 in 2 mol/L glycine buffer, pH 9.0) and 50 units of alkaline phosphatase (bovine intestinal mucosa, type VII-T; Sigma-Aldrich). The mixture was incubated at 37°C for 45 minutes. To terminate the reaction, 15 μl of concentrated HCl was added and the dephosphorylated sphingosine was extracted twice with 500 μl of CHCl3. The pooled CHCl3 phase was washed three times with alkaline water and then dried under nitrogen in siliconized glass tubes. The dried lipid residue was resuspended in 200 μl of ethanol at 67°C for 30 minutes. The dissolved lipid solution was incubated with 30 μl of o-phthalaldehyde (Sigma Chemical) reagent for 1 hour at room temperature. Sample analysis was performed by using Agilent 1100 high-performance liquid chromatography equipment. The isocratic mobile phase was acetonitrile-deionized distilled water (90:10, v/v), and the flow rate was 1 ml/min. The derivatives were detected using a spectrofluorometer, with an excitation wavelength of 340 nm and an emission wavelength of 455 nm. Cell migration was determined in Boyden chambers as described previously.21Meriane M Duhamel S Lejeune L Galipeau J Annabi B Cooperation of matrix metalloproteinases with the RhoA/Rho kinase and mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase signaling pathways is required for the sphingosine-1-phosphate-induced mobilization of marrow-derived stromal cells.Stem Cells. 2006; 24: 2557-2565Crossref PubMed Scopus (67) Google Scholar In brief, serum-starved BM cells (4 × 104 cells) were seeded to the upper chamber. Cell migration was allowed to proceed for 6 hours at 37°C in 5% CO2 by adding S1P (1 nmol/L) to the lower chamber. Cells migrating to the lower surface of the filter were stained and quantified by cell counting. The migration index was defined as the number of cells in the lower chamber under the tested condition divided by the number of cells under control. The siRNA sequence targeting mouse S1P3 specifically was synthesized (L-040957-00; Dharmacon, Lafayette, CO). Forty to 50% confluent BM cells were prepared in six-well dishes. Transient transfection of siRNA (40 nmol/L) was performed by using Lipofectamine RNAiMAX as recommended by the manufacturer. Control cells were treated with 40 nmol/L RNAi negative control duplexes (scramble siRNA). After 48 hours cells were used to perform the migration assay. Total RNA was extracted from liver frozen specimens using an RNeasy kit (Qiagen, Hilden, Germany). Real-time RT-PCR was performed in an ABI Prism 7300 sequence detecting system (Applied Biosystems, Foster City, CA), as described previously.13Serriere-Lanneau V Teixeira-Clerc F Li LY Schippers M de Wries W Julien B Tran-Van-Nhieu J Manin S Poelstra K Chun J Carpentier S Levade T Mallat A Lotersztajn S The sphingosine 1-phosphate receptor S1P2 triggers hepatic wound healing.FASEB J. 2007; 21: 2005-2013Crossref PubMed Scopus (72) Google Scholar Primers (MWG Biotech, Ebersberg, Germany) used for real-time RT-PCR were as follows: mouse S1P1 receptor: sense, 5′-ACTTTGCGAGTGAGCTG-3′ and antisense, 5′-AGTGAGCCTTCAGTTACAGC-3′; S1P2 receptor: sense, 5′-TTCTGGAGGGTAACACAGTGGT-3′ and antisense, 5′-ACACCCTTTGTATCAAGTGGCA-3′; S1P3 receptor: sense, 5′-TGGTGTGCGGCTGTCTAGTCAA-3′ and antisense, 5′-CACAGCAAGCAGACCTCCAGA-3′; and 18S rRNA: sense, 5′-GTAACCCGTTGAACCCCATT-3′ and antisense, 5′-CCATCCAATCGGTAGTAGCG-3′. Probes (Applied Biosystems) used for real time RT-PCR were as follows: procollagen α1(I): MA00801666; procollagen α1(III): MA00802331; α-SMA: MA00725412; SphK1: MA00448841; S1P phosphatase: MA00473016; and S1P lyase: MA00486079. Liver tissues were fixed in PBS containing 4% paraformaldehyde for 24 hours and embedded in paraffin. Sections (5 μm) were stained with Sirius red for collagen visualization and H&E for analysis of necrotic area. The fibrotic area and necrotic area were assessed by computer-assisted image analysis with MetaMorph software (Universal Imaging Corporation, Downingtown, PA). The mean value of 15 randomly selected areas per sample was used as the expressed percentage of fibrosis or necrosis area. Hydroxyproline content of the liver was measured as described previously27Sakaida I Terai S Yamamoto N Aoyama K Ishikawa T Nishina H Okita K Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice.Hepatology. 2004; 40: 1304-1311Crossref PubMed Scopus (468) Google Scholar with minor modifications. In brief, three small fragments of each liver were pooled, homogenized in distilled water, and lyophilized, and 20 mg of the freeze-dried sample was hydrolyzed at 95°C for 20 minutes. After hydrolysis, the samples were neutralized at pH 6.0 to 6.8. The hydrolysates were then treated with activated charcoal. After centrifugation at 1000 × g for 10 minutes, aliquots of the hydrolysates were used to measure hydroxyproline content spectrophotometrically by reaction with Ehrlich’s reagent. Absorbance was measured at 560 nm. The hydroxyproline content of the liver was expressed as micrograms per gram of dry weight. Serum total bilirubin and serum aminotransferase were measured using commercial assay kits (Stanbio, Boerne, TX). ICR mice aged 6 weeks were given one-time suramin or saline alone administration (20 mg/kg b.wt. i.p.). After 24 hours, mice were anesthetized to receive midline laparotomy, and the common bile duct and duodenum were exposed. The bile duct was then cut in the proximal portion of the duodenal papilla. Bile flow was collected by drainage tube for 30 minutes. Western blot analysis of S1P3 receptor was performed with 50 μg of protein extract, obtained as described previously28Li LY Grenard P Van Nhieu JT Julien B Mallat A Habib A Lotersztajn S Heme oxygenase-1 is an antifibrogenic protein in human hepatic myofibroblasts.Gastroenterology. 2003; 125: 460-468Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar using rabbit polyclonal antibody to S1P3 (1:500; Santa Cruz Biotechnology) and peroxidase-conjugated goat anti-rabbit IgG antibody (1:10,000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) as a secondary antibody. Protein expression was visualized by using an enhanced chemiluminescence (ECL Plus) assay kit according to the manufacturer’s instructions (Amersham Biosciences, Arlington Heights, IL). Signals were normalized to the glyceraldehyde-3-phosphate dehydrogenase signals (rabbit monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase antibody, 1:1000; Sigma). Results are expressed as mean ± SEM. Statistical significance was assessed by using Student’s t-test or one-way analysis of variance for analysis of variance when appropriate. A value of P < 0.05 was considered to be statistically significant. We first examined S1P levels in cholestasis-induced liver fibrosis. High-performance liquid chromatography analysis of liver extracts from BDL mice revealed that S1P levels rose markedly compared with those in sham-operated liver (Figure 1A). Likewise we found that the serum S1P concentration in BDL mice was increased by approximate twofold higher than that for sham-operated mice (Figure 1B). We next examined whether the enzymes involved in determining S1P abundance (SphK, S1P phosphatase, and S1P lyase) in liver tissue were affected by BDL. As expected, 2 weeks after BDL, expression of SphK1 mRNA was significantly increased compared with that in sham-operated mice (Figure 1C), although expression of SphK2 mRNA was unchanged (data not shown). Moreover, expression of S1P phosphatase and lyase mRNAs was unchanged after BDL (Figure 1, D and E). These findings indicated that the up-regulation of SphK1 expression resulted in increased S1P concentration in the liver during cholestasis. In addition, we tested S1P levels in BM and found that there was no difference in S1P concentration of BM between the BDL-operated and sham-operated mice (Figure 1F). Likewise, these enzymes (SphK, S1P phosphatase, and S1P lyase) in BM were unaffected after BDL (data not shown), indicating that the hepatic S1P system was exclusively activated after BDL-induced liver injury. We further determined expression of S1PRs in sham- and BDL-operated mice liver. Real time RT-PCR analysis showed that expression of S1P3 receptor, but not that of S1P1 or S1P2, was markedly up-regulated in BDL-operated liver (Figure 2A). Meanwhile, we evaluated the protein levels of S1P3 receptor in the BDL liver tissue. As shown in Figure 2B, Western blot analysis also revealed a pronounced increase in S1P3 receptor expression after BDL. Taken together, these data reveal that SphK1/S1P and related receptors (here, in particular, S1P3) are significantly increased in BDL mice and may play a role in the pathogenesis of cholestatic liver injury. In addition, immunohistochemical staining for α-SMA was performed to evaluate the BDL-induced fibrosis model (Figure 3, A and B). Collagen deposition around the portal tracts with the formation of bridging fibrosis was seen in the livers of the BDL mice. S1P has been shown to serve as a very good candidate for the induction of cell mobilization.29Spiegel S English D Milstien S Sphingosine 1-phosphate signaling: providing cells with a direction.Trends Cell Biol. 2002; 12: 236-242Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar There is, in fact, a growing body of evidence to indicate that S1P strongly stimulates BM stem cells migration in vitro.20Annabi B Thibeault S Lee YT Bousquet-Gagnon N Eliopoulos N Barrette S Galipeau J Beliveau R Matrix metalloproteinase regulation of sphingosine-1-phosphate-induced angiogenic properties of bone marrow stromal cells.Exp Hematol. 2003; 31: 640-649Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Meriane M Duhamel S Lejeune L Galipeau J Annabi B Cooperation of matrix metalloproteinases with the RhoA/Rho kinase and mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase signaling pathways is required for the sphingosine-1-phosphate-induced mobilization of marrow-derived stromal cells.Stem Cells. 2006; 24: 2557-2565Crossref PubMed Scopus (67) Google Scholar, 22Jaganathan BG Ruester B Dressel L Stein S Grez M Seifried E Henschler R Rho inhibition induces migration of mesenchymal stromal cells.Stem Cells. 2007; 25: 1966-1974Crossref PubMed Scopus (80) Google Scholar Recently, several studies have shown that the BM contributes significantly to liver fibrosis. Thus, it was of interest to examine whether S1P mediated migration of BM cells to the damaged liver during cholestasis. We first transplanted BM cells from EGFP transgenic mice into recipients. Four weeks later, hematological reconstitution was complete.16Russo FP Alison MR Bigger BW Amofah E Florou A Amin F Bou-Gharios G Jeffery R Iredale JP Forbes SJ The bone marrow functionally contributes to liver fibrosis.Gastroenterology. 2006; 130: 1807-1821Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar We then sought to examine BM-derived cells in mouse fibrotic liver induced by BDL. Two weeks after BDL, a confocal microscopy image of livers showed that significant numbers of EGFP-positive cells (BM origin) were found in the fibrotic foci (Figure 3C). Thus, we confirm in our BM transplant model that BM cells can migrate to the damaged liver. Next,
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