Title: Bone Marrow-Derived Mesenchymal Stem Cells Differentiate to Hepatic Myofibroblasts by Transforming Growth Factor-β1 via Sphingosine Kinase/Sphingosine 1-Phosphate (S1P)/S1P Receptor Axis
Abstract: Sphingosine kinase (SphK) is involved in numerous biological processes, including cell growth, proliferation, and differentiation. However, whether SphK participates in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to myofibroblasts has been unknown. In a carbon tetrachloride-treated mouse model, SphK1 was expressed in BMSCs in damaged liver. Furthermore, mRNA expression of both SphK1 and transforming growth factor β1 (TGF-β1) was significantly increased after liver injury, with a positive correlation between them. The SphK inhibitor SKI significantly blocked BMSC differentiation to myofibroblasts during liver injury (the proportion of BMSC-derived myofibroblasts decreased markedly, compared with no SKI treatment) and attenuated the extent of liver fibrosis. Using primary mouse BMSCs, we demonstrated that TGF-β1 induced BMSC differentiation to myofibroblasts, accompanied by the up-regulation of SphK1 and modulation of sphingosine 1-phosphate (S1P) receptor (S1PR) expression. Notably, pharmacological or siRNA-mediated inhibition of SphK1 abrogated the prodifferentiating effect of TGF-β1. Moreover, using either S1PR subtype-specific antagonists or specific siRNAs, we found that the prodifferentiating effect of TGF-β1 was mediated by S1PR1 and S1PR3. These data suggest that SphK1 activation by TGF-β1 leads to differentiation of BMSCs to myofibroblasts mediated by S1PR1 and S1PR3 up-regulation, thus providing new information on the mechanisms by which TGF-β1 gives rise to fibrosis and opening new perspectives for pharmacological treatment of liver fibrosis. Sphingosine kinase (SphK) is involved in numerous biological processes, including cell growth, proliferation, and differentiation. However, whether SphK participates in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to myofibroblasts has been unknown. In a carbon tetrachloride-treated mouse model, SphK1 was expressed in BMSCs in damaged liver. Furthermore, mRNA expression of both SphK1 and transforming growth factor β1 (TGF-β1) was significantly increased after liver injury, with a positive correlation between them. The SphK inhibitor SKI significantly blocked BMSC differentiation to myofibroblasts during liver injury (the proportion of BMSC-derived myofibroblasts decreased markedly, compared with no SKI treatment) and attenuated the extent of liver fibrosis. Using primary mouse BMSCs, we demonstrated that TGF-β1 induced BMSC differentiation to myofibroblasts, accompanied by the up-regulation of SphK1 and modulation of sphingosine 1-phosphate (S1P) receptor (S1PR) expression. Notably, pharmacological or siRNA-mediated inhibition of SphK1 abrogated the prodifferentiating effect of TGF-β1. Moreover, using either S1PR subtype-specific antagonists or specific siRNAs, we found that the prodifferentiating effect of TGF-β1 was mediated by S1PR1 and S1PR3. These data suggest that SphK1 activation by TGF-β1 leads to differentiation of BMSCs to myofibroblasts mediated by S1PR1 and S1PR3 up-regulation, thus providing new information on the mechanisms by which TGF-β1 gives rise to fibrosis and opening new perspectives for pharmacological treatment of liver fibrosis. Mesenchymal stem cells (MSCs) are a heterogeneous population of cells with the potential for multilineage differentiation. They have been identified and isolated from various tissues, including blood, adipose tissue, trabecular bone, muscle, and dermis.1Caplan A.I. Mesenchymal stem cells.J Orthop Res. 1991; 9: 641-650Crossref PubMed Scopus (3492) Google Scholar Under appropriate culture conditions, bone marrow-derived MSCs (BMSCs) are capable of differentiating to a variety of mesenchymal tissues, including bone, cartilage, muscle, ligament, tendon, adipose tissue, and stroma.2Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17988) Google Scholar, 3Watabe T. Miyazono K. Roles of TGF-beta family signaling in stem cell renewal and differentiation.Cell Res. 2009; 19: 103-115Crossref PubMed Scopus (344) Google Scholar Their multipotency, easy isolation, and ready availability make MSCs particularly suited for tissue engineering and clinical applications. Thus, it is desirable to identify at the molecular level the regulatory mechanisms underlying the differentiation of MSCs. Myofibroblasts play a central role in the pathogenesis of liver fibrosis. De novo formation of differentiated myofibroblasts is thought to be primarily responsible for excessive extracellular matrix production; thus, elucidating the mechanisms of myofibroblast activation seems indispensable for designing rational therapeutic strategies to inhibit the fibrogenic process the leads to cirrhosis.4Friedman S.L. Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2183) Google Scholar, 5Desmoulière A. Darby I.A. Gabbiani G. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis.Lab Invest. 2003; 83: 1689-1707Crossref PubMed Scopus (299) Google Scholar A high proportion of myofibroblasts in the fibrotic liver are of BMSC origin.6Forbes S.J. Russo F.P. Rey V. Burra P. Rugge M. Wright N.A. Alison M.R. 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 (395) Google Scholar, 7Kallis Y.N. Alison M.R. Forbes S.J. Bone marrow stem cells and liver disease.Gut. 2007; 56: 716-724Crossref PubMed Scopus (126) Google Scholar, 8Russo F.P. Alison M.R. Bigger B.W. Amofah E. Florou A. Amin F. Bou-Gharios G. Jeffery R. Iredale J.P. Forbes S.J. The bone marrow functionally contributes to liver fibrosis.Gastroenterology. 2006; 130: 1807-1821Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar In a previous study, we demonstrated that BMSCs can migrate to the damaged liver, mediated by endogenous sphingosine 1-phosphate (S1P), which can produce a concentration gradient between liver and bone marrow after liver injury, and that BMSCs differentiate to myofibroblast-like cells expressing α-smooth muscle actin (α-SMA) in mouse fibrotic liver.9Li C. Kong Y. Wang H. Wang S. Yu H. Liu X. Yang L. Jiang X. Li L. Li L. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis [Erratum appeared in J Hepatol 2009, 51:973].J Hepatol. 2009; 50: 1174-1183Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar However, which factor mediates differentiation of BMSCs to myofibroblasts and the molecular mechanisms governing this process are not fully understood. Sphingosine kinase (SphK) catalyzes the phosphorylation of sphingosine to generate the bioactive lipid S1P.10Watterson K.R. Lanning D.A. Diegelmann R.F. Spiegel S. Regulation of fibroblast functions by lysophospholipid mediators: potential roles in wound healing.Wound Repair Regen. 2007; 15: 607-616Crossref PubMed Scopus (91) Google Scholar The two distinct isoforms of mammalian SphK (SphK1 and SphK2) differ in sequence, catalytic properties, localization, and function.11Maceyka M. Sankala H. Hait N.C. Le Stunff H. Liu H. Toman R. Collier C. Zhang M. Satin L.S. Merrill A.J. Milstien S. Spiegel S. SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism.J Biol Chem. 2005; 280: 37118-37129Crossref PubMed Scopus (488) Google Scholar SphK1 is activated by various factors, including ganglioside GM1 (GM1), G-protein coupled receptors, small GTPases, tyrosine kinase receptors, proinflammatory cytokines, immunoglobulin receptors, and calcium and protein kinase activators, and is involved in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, motility, and cytoskeletal rearrangement.12Taha T.A. Hannun Y.A. Obeid L.M. Sphingosine kinase: biochemical and cellular regulation and role in disease.J Biochem Mol Biol. 2006; 39: 113-131Crossref PubMed Google Scholar, 13Donati C. Cencetti F. Nincheri P. Bernacchioni C. Brunelli S. Clementi E. Cossu G. Bruni P. Sphingosine 1-phosphate mediates proliferation and survival of mesoangioblasts.Stem Cells. 2007; 25: 1713-1719Crossref PubMed Scopus (66) Google Scholar, 14Meacci E. Nuti F. Donati C. Cencetti F. Farnararo M. Bruni P. Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts.J Cell Physiol. 2008; 214: 210-220Crossref PubMed Scopus (56) Google Scholar, 15Karliner J.S. Honbo N. Summers K. Gray M.O. Goetzl E.J. The lysophospholipids sphingosine-1-phosphate and lysophosphatidic acid enhance survival during hypoxia in neonatal rat cardiac myocytes.J Mol Cell Cardiol. 2001; 33: 1713-1717Abstract Full Text PDF PubMed Scopus (158) Google Scholar SphK1 activation translocates from the cytosol to the plasma membrane, leading to S1P formation, which then activates a set of five G-protein-coupled receptors referred to as S1P receptor (S1PR) types 1 to 5 (S1PR1-5), leading to regulation of numerous downstream signaling components.16Rosen H. Goetzl E.J. Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network.Nat Rev Immunol. 2005; 5: 560-570Crossref PubMed Scopus (620) Google Scholar It is becoming increasingly clear that the expression pattern of S1PRs is critical for the final effect elicited by the bioactive lipid in some cell types, given that individual receptor subtypes are coupled to different signaling cascades and are consequently implicated in the regulation of a variety of biological responses.17Alvarez S.E. 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, 18Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat Rev Mol Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2452) Google Scholar Transforming growth factor-β1 (TGF-β1) is a member of a large family of growth factors involved in numerous biological processes, including cell proliferation, differentiation, embryonic development, carcinogenesis, immune function, inflammation, and wound healing.19Massagué J. TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3985) Google Scholar, 20Blobe G.C. Schiemann W.P. Lodish H.F. Role of transforming growth factor beta in human disease.N Engl J Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2185) Google Scholar, 21Jeon E.S. Moon H.J. Lee M.J. Song H.Y. Kim Y.M. Bae Y.C. Jung J.S. Kim J.H. Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-beta-dependent mechanism.J Cell Sci. 2006; 119: 4994-5005Crossref PubMed Scopus (150) Google Scholar, 22Song H.Y. Kim M.Y. Kim K.H. Lee I.H. Shin S.H. Lee J.S. Kim J.H. Synovial fluid of patients with rheumatoid arthritis induces alpha-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells through a TGF-beta1-dependent mechanism.Exp Mol Med. 2010; 42: 565-573Crossref PubMed Scopus (21) Google Scholar Some of the effects elicited by TGF-β1 are transmitted by the pathway initiated by enhancement of SphK. The SphK/S1P/S1PR pathway is critically implicated in the mechanism by which TGF-β1 elicits invasive behavior of esophageal cancer cells,23Miller A.V. Alvarez S.E. Spiegel S. Lebman D.A. Sphingosine kinases and sphingosine-1-phosphate are critical for transforming growth factor beta-induced extracellular signal-regulated kinase 1 and 2 activation and promotion of migration and invasion of esophageal cancer cells.Mol Cell Biol. 2008; 28: 4142-4151Crossref PubMed Scopus (72) Google Scholar antiapoptotic action in mesoangioblasts,24Donati C. Cencetti F. De Palma C. Rapizzi E. Brunelli S. Cossu G. Clementi E. Bruni P. TGFbeta protects mesoangioblasts from apoptosis via sphingosine kinase-1 regulation.Cell Signal. 2009; 21: 228-236Crossref PubMed Scopus (27) Google Scholar and prodifferentiating effect in various types of fibroblasts25Yamanaka M. Shegogue D. Pei H. Bu S. Bielawska A. Bielawski J. Pettus B. Hannun Y.A. Obeid L. Trojanowska M. Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor-beta and mediates TIMP-1 up-regulation.J Biol Chem. 2004; 279: 53994-54001Crossref PubMed Scopus (120) Google Scholar, 26Kono 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-beta1.Am J Respir Cell Mol Biol. 2007; 37: 395-404Crossref PubMed Scopus (126) Google Scholar, 27Gellings Lowe N. Swaney J.S. Moreno K.M. Sabbadini R.A. Sphingosine-1-phosphate and sphingosine kinase are critical for transforming growth factor-beta-stimulated collagen production by cardiac fibroblasts.Cardiovasc Res. 2009; 82: 303-312Crossref PubMed Scopus (119) Google Scholar and myoblasts.28Cencetti F. Bernacchioni C. Nincheri P. Donati C. Bruni P. Transforming growth factor-beta1 induces transdifferentiation of myoblasts into myofibroblasts via up-regulation of sphingosine kinase-1/S1P3 axis.Mol Biol Cell. 2010; 21: 1111-1124Crossref PubMed Scopus (125) Google Scholar In particular, up-regulation of SphK1 has been found to be critical for TGF-β1–induced transcriptional regulation of the tissue inhibitor of metalloproteinases 1 (TIMP-1), which inhibits degradation of extracellular matrix in fibroblasts.25Yamanaka M. Shegogue D. Pei H. Bu S. Bielawska A. Bielawski J. Pettus B. Hannun Y.A. Obeid L. Trojanowska M. Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor-beta and mediates TIMP-1 up-regulation.J Biol Chem. 2004; 279: 53994-54001Crossref PubMed Scopus (120) Google Scholar Moreover, recent reports have shown that SphK1 and S1PRs play a role in TGF-β1–dependent extracellular matrix remodeling and myofibroblast differentiation of lung fibroblasts and myoblasts and in collagen production by cardiac fibroblasts.26Kono 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-beta1.Am J Respir Cell Mol Biol. 2007; 37: 395-404Crossref PubMed Scopus (126) Google Scholar, 27Gellings Lowe N. Swaney J.S. Moreno K.M. Sabbadini R.A. Sphingosine-1-phosphate and sphingosine kinase are critical for transforming growth factor-beta-stimulated collagen production by cardiac fibroblasts.Cardiovasc Res. 2009; 82: 303-312Crossref PubMed Scopus (119) Google Scholar, 28Cencetti F. Bernacchioni C. Nincheri P. Donati C. Bruni P. Transforming growth factor-beta1 induces transdifferentiation of myoblasts into myofibroblasts via up-regulation of sphingosine kinase-1/S1P3 axis.Mol Biol Cell. 2010; 21: 1111-1124Crossref PubMed Scopus (125) Google Scholar The well-established prodifferentiating effect of TGF-β1, together with the emerging role of the SphK/S1P/S1PR axis as the TGF-β1–regulated signaling pathway, spurred us to investigate whether TGF-β1 could induce differentiation of BMSCs to myofibroblasts and whether SphK1 and S1PRs participate in its biological action. With the present study, we demonstrate for the first time that TGF-β1 induces differentiation of BMSCs to myofibroblasts via up-regulation of SphK1 and modification of S1PR expression. Importantly, S1PR1 and S1PR3, which are up-regulated on TGF-β1 challenge, seem to be critically implicated in the differentiation of BMSCs to myofibroblasts. In vivo administration of SphK inhibitor significantly blocked differentiation of BMSCs to myofibroblasts during liver injury, and thus attenuated the extent of liver fibrosis. These results represent the first experimental evidence that induction of the SphK/S1P/S1PR axis is exploited by TGF-β1 to induce BMSC differentiation. These results may open new perspectives for pharmacological treatment of liver fibrosis. Antibodies against α-SMA and SphK1 were from Sigma-Aldrich (St. Louis, MO) and ECM Biosciences (Versailles, KY), respectively. Anti-CD105 and anti-CD166 antibodies used for flow cytometry analysis were from AbD Serotec (Kidlington, UK) and BD Biosciences (San Jose, CA), respectively. TGF-β1 was from PeproTech (London, UK), and GM1 was from Sigma-Aldrich. The sphingosine kinase inhibitor SKI [2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole] and N,N-dimethylsphingosine (DMS) were from Calbiochem (Bad Soden, Germany) and Biomol (Hamburg, Germany), respectively. The specific S1PR1/S1PR3 antagonist VPC23019 and the specific S1PR1 antagonist W146 were from Avanti Polar Lipids (Alabaster, AL). The specific S1PR2 antagonist JTE-013 and the S1PR1 agonist SEW2871 were from Cayman Chemical (Ann Arbor, MI). PCR reagents were from Applied Biosystems (Life Technologies, Foster City, CA). Alkaline phosphatase type VII-T, o-phthalaldehyde, and other common reagents were from Sigma-Aldrich. ICR mice aged 3 weeks (Laboratory Animal Center, Capital Medical University) were sacrificed by cervical dislocation. Whole bone marrow cells were extracted from the tibias and femurs of mice by flushing with Gibco culture medium (Life Technologies, Grand Island, NY) using a 25-gauge needle. The cells were then passed through 70-mm nylon mesh and were washed three times with PBS containing 2% fetal bovine serum (Biochrom, Berlin, Germany). BMSCs were isolated from bone marrow of enhanced green fluorescent protein (EGFP) transgenic ICR mice or wild-type mice, and were cultured as described previously.9Li C. Kong Y. Wang H. Wang S. Yu H. Liu X. Yang L. Jiang X. Li L. Li L. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis [Erratum appeared in J Hepatol 2009, 51:973].J Hepatol. 2009; 50: 1174-1183Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar Characterization of BMSCs was performed by flow cytometry analysis.9Li C. Kong Y. Wang H. Wang S. Yu H. Liu X. Yang L. Jiang X. Li L. Li L. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis [Erratum appeared in J Hepatol 2009, 51:973].J Hepatol. 2009; 50: 1174-1183Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar All animal experiments were performed according to guidelines of the Ethics Committee of Capital Medical University. ICR mice aged 6 weeks received intraperitoneal injections of 1 μL/g body weight of a carbon tetrachloride/olive oil (CCl4/OO) mixture, 1:9 v/v, twice per week. The mice were sacrificed at 3 days or at 2, 4, 6, or 8 weeks of CCl4 treatment (n = 7 per group), always on the day after the last injection. Liver tissue was collected for real-time RT-PCR and high-performance liquid chromatography (HPLC) analysis. Another group of mice received lethal irradiation (8 Gy), and then immediately received transplantation by a tail-vein injection of 1.2 × 106 enriched EGFP-positive BMSCs and 1.08 × 107 EGFP-negative whole bone marrow cells. Four weeks later, mice received intraperitoneal injections of CCl4 twice per week for 4 weeks. SKI (10 mg/kg body weight) or saline was administered the day before CCl4 or OO treatment (n = 7 per group). Liver tissue was collected for real-time RT-PCR, for H&E and Sirius Red staining, and for immunofluorescence analysis. Liver samples were fixed in 4% paraformaldehyde and embedded in Tissue Tek optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA). Frozen sections (7 μm thick) were used for immunofluorescence staining. Sections were blocked with 2% bovine serum albumin in PBS for 1 hour and then incubated with anti-SphK1 polyclonal antibody (1:100) diluted in PBS. Cy3-conjugated AffiniPure goat anti-rabbit IgG antibody (1:500) was used as a secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). The sections were covered with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing DAPI and were observed under a confocal microscope (Leica TCS SP5 with LAS AF version 2.5 software; Leica Microsystems, Wetzlar, Germany). For negative controls, sections were processed the same way, except that incubation with the primary antibody was omitted. Immunofluorescent detection of α-SMA in liver tissue was performed using a Vector M.O.M. (mouse-on-mouse) immunodetection kit (Vector Laboratories) and a 1:1000 dilution of a monoclonal antibody to α-SMA. The proportion of BMSC-derived myofibroblasts was calculated as the number of both EGFP-positive and α-SMA–positive cells divided by number of α-SMA–positive cells, using Image-Pro Plus version 6.0 software (Media Cybernetics, Silver Spring, MD). Cultured BMSCs with or without treatments were fixed in 4% paraformaldehyde in PBS for 30 minutes. Then cells were washed twice with PBS, permeabilized in 0.5% Triton X-100 in PBS for 15 minutes, blocked with 2% BSA for 1 hour, and then incubated with anti-α-SMA monoclonal antibody (1:2000), followed by secondary antibody conjugated with Cy3 (1:1000; Jackson ImmunoResearch Laboratories). Total RNA was extracted from frozen liver specimens or cultured BMSCs with or without treatments, using an RNeasy kit (Qiagen, Hilden, Germany). Real-time RT-PCR was performed with an ABI Prism 7300 sequence-detecting system (Life Technologies, Foster City, CA), as described previously.29Li C. Zheng S. You H. Liu X. Lin M. Yang L. Li L. Sphingosine 1-phosphate (S1P)/S1P receptors are involved in human liver fibrosis by action on hepatic myofibroblasts motility [Erratum appeared in J Hepatol 2012, 56:749].J Hepatol. 2011; 54: 1205-1213Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Primers (MWG Biotech, Ebersberg, Germany) used for real-time RT-PCR were as follows: 18S rRNA, sense, 5′-GTAACCCGTTGAACCCCATT-3′, and antisense, 5′-CCATCCAATCGGTAGTAGCG-3′; α-SMA, sense, 5′-ATGCTCCCAGGGCTGTTTT-3′, and anti-sense, 5′-TTCCAACCATTACTCCCTGATGT-3′; SphK1, sense, 5′-TGTCACCCATGAACCTGCTGTCCCTGCACA-3′, and anti-sense, 5′-AGAAGGCACTGGCTCCAGAGGAACAAG-3′; SphK2, sense, 5′-ACAGAACCATGCCCGTGAG-3′, and anti-sense, 5′-AGGTCAACACCGACAACCTG-3′; procollagen α1(I) [Colα1(I)], sense, 5′-AGGGCGAGTGCTGTGCTT T-3′, and anti-sense, 5′-CCCTCGACTCCTACATCTTCTGA-3′; procollagen α1(III) [Colα1(III)], sense, 5′-TGAAACCCCAGCAAAACAAAA-3′, and anti-sense, 5′-TCACTTGCACTGGTTGATAAGATTAA-3′; S1PR1, sense, 5′-ACTTTGCGAGTGAGCTG-3′, and anti-sense, 5′-AGTGAGCCTTCAGTTACAGC-3′; S1PR2, sense, 5′-TTCTGGAGGGTAACACAGTGGT-3′, and anti-sense, 5′-ACACCCTTTGTATCAAGTGGCA-3′; S1PR3, sense, 5′-TGGTGTGCGGCTGTCTAGTCAA-3′, and anti-sense, 5′-CACAGCAAGCAGACCTCCAGA-3′; and TGF-β1, sense, 5′-TGCGCTTGCAGAGATTAAAA-3′, and anti-sense, 5′-TCACTGGAGTTGTACGGCAG-3′. Liver tissues were fixed in PBS containing 4% paraformaldehyde for 24 hours and then were embedded in paraffin. Sections (6 μm thick) were stained with Sirius Red for collagen visualization and with H&E for liver histology, as described previously.30Li C. Jiang X. Yang L. Liu X. Yue S. Li L. Involvement of sphingosine 1-phosphate (SIP)/S1P3 signaling in cholestasis-induced liver fibrosis.Am J Pathol. 2009; 175: 1464-1472Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar The fibrotic area was assessed by means of computer-assisted image analysis with Leica Qwin V3 software. The expressed percentage of fibrotic or inflammatory area was calculated based on the mean value of 15 randomly selected areas per sample. Western blot analysis of α-SMA and SphK1 was performed with 20 μg of protein extract, obtained as described previously,31Manton K.J. Leong D.F. Cool S.M. Nurcombe V. Disruption of heparan and chondroitin sulfate signaling enhances mesenchymal stem cell-derived osteogenic differentiation via bone morphogenetic protein signaling pathways.Stem Cells. 2007; 25: 2845-2854Crossref PubMed Scopus (91) Google Scholar using mouse monoclonal antibody and rabbit polyclonal antibody to α-SMA (1:1000) and SphK1 (1:1000), respectively. Peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1:5000; Jackson ImmunoResearch Laboratories) was used as a secondary antibody. Protein expression was visualized by using an enhanced chemiluminescence assay kit according to the manufacturer's instructions (Amersham ECL Plus; GE Healthcare Life Sciences, Arlington Heights, IL). The bands were quantified using GeneSnap and GeneTools software from PerkinElmer (Waltham, MA), and results were normalized relative to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (rabbit monoclonal anti-GAPDH antibody, 1:1000; Sigma-Aldrich) or tubulin (rabbit monoclonal anti-tubulin antibody, 1:1000, Epitomics, Burlingame, CA) expression to correct for variations in protein loading and transfer. The siRNA sequences specifically targeting mouse SphK1, S1PR1, S1PR2, or S1PR3 were synthesized by Dharmacon: L-040671-00, L-051684-00, L-063765-00, and L-040959-00, respectively (Thermo Scientific, Lafayette, CO). At 40% to 50% confluency, BMSCs were prepared in 60-mm dishes. Transient transfection of siRNA (40 nmol/L) was performed by using Invitrogen Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA), as recommended by the manufacturer. Control cells were treated with 40 nmol/L RNAi negative control duplexes (scrambled siRNA). After 48 hours, cells were used to perform the differentiation assay. After 24 hours stimulation of TGF-β1 or GM1, both BMSCs and their supernatants were collected. Extraction of S1P and sample analysis were performed as described previously.32Liu X. Yue S. Li C. Yang L. You H. Li L. Essential roles of sphingosine 1-phosphate receptor types 1 and 3 in human hepatic stellate cells motility and activation.J Cell Physiol. 2011; 226: 2370-2377Crossref PubMed Scopus (51) Google Scholar Data are expressed as means ± SEM and were analyzed by Student's t-test or analysis of variance when appropriate. P < 0.05 was considered significant. We first reconstituted bone marrow in the irradiated mice by transplantation of the genetic EGFP-labeled BMSCs. Liver fibrosis was then induced by the administration of CCl4 for 4 weeks, and expression of α-SMA, a marker for myofibroblasts, was examined in the damaged liver by immunofluorescence analysis. In OO-treated liver, α-SMA was expressed only in the vascular area, and there were only a few, scattered EGFP-positive cells (BMSCs) in the liver. No colocalization was observed between α-SMA–positive and EGFP-positive cells (Figure 1A). After CCl4 treatment, however, there was strong immunoreactivity for α-SMA in the fibrotic areas, with distribution similar to that of EGFP-positive cells (Figure 1B). Confocal microscopy images showed that these EGFP-positive cells (BMSCs) had a myofibroblast phenotype (Figure 1C). We then determined the proportion of BMSC-derived myofibroblasts and found that a large proportion (69%) of α-SMA–positive cells in the fibrotic areas were also positive for EGFP, suggesting that BMSCs could differentiate to myofibroblasts during liver fibrosis (Figure 1E). We also quantitated the numbers of myofibroblasts of non-bone marrow origin (including hepatic stellate cells); the percentage was only 31%. Given the significant contribution of BMSCs to the myofibroblast lineage, we focused on the myofibroblasts derived from BMSC differentiation. To prove fully that BMSCs actually leave the bone marrow and then engraft the liver, we used flow cytometry analysis to explore whether the EGFP-positive cells in the blood of chimeric mice were MSCs. We first characterized these EGFP-positive cells and found that more EGFP-positive cells were detected in the blood of chimeric animals treated with CCl4, compared with the OO-treated control (see Supplemental Figure S1A at http://ajp.amjpathol.org). Moreover, we used the typical MSC marker proteins, CD166 and CD105, to confirm the phenotype of MSCs in mice treated with OO or CCl4. Some of the EGFP-positive cells circulating in the blood of chimeric animals were also positive for CD166 (see Supplemental Figure S1B at http://ajp.amjpathol.org) or CD105 (see Supplemental Figure S1C at http://ajp.amjpathol.org). These results suggest that, after liver injury, these MSCs actually leave the bone marrow, enter the circulation, and then traffic to the liver (see Supplemental Figure S1 at http://ajp.amjpathol.org). SphK is reported to play a pivotal role in the differentiation of many types of cells.25Yamanaka M. Shegogue D. Pei H. Bu S. Bielawska A. Bielawski J. Pettus B. Hannun Y.A. Obeid L. Trojanowska M. Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor-beta and mediates TIMP-1 up-regulation.J Biol Chem. 2004; 279: 53994-54001Crossref PubMed Scopus (120) Google Scholar, 26Kono 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-beta1.Am J Respir Cell Mol Biol. 2007; 37: 395-404Crossref PubMed Scopus (126) Google Scholar, 27Gellings Lowe N. Swaney J.S. Moreno K.M. Sabbadini R.A. Sphingosine-1-phosphate and sphingosine kinase are critical for transforming growth factor-beta-stimulated collagen production by cardiac fibroblasts.Cardiovasc Res. 2009; 82: 303-312Crossref PubMed Scopus (119) Google Scholar, 28Cencetti F. Bernacchioni C. Nincheri P. Donati C. Bruni P. Transforming growth factor-beta1 induces transdifferentiation of myoblasts into myofibroblasts via up-regulation of sphingosine kinase-1/S1P3 axis.Mol Biol Cell. 2010; 21: 1111-1124Crossref PubMed Scopus (125) Google Scholar To investigate whether SphK/S1P/S1PR signaling is involved in BMSC differentiation, we first examined the mRNA expression of SphK1 in CCl4-induced liver fibrosis models. SphK1 mRNA level incr