Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Intracellular Sphingosine 1-Phosphate Contributes to Collagen Expression of Hepatic Myofibroblasts in Human Liver Fibrosis Independent of Its Receptors

2014; Elsevier BV; Volume: 185; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2014.09.023

ISSN

1525-2191

Autores

Lei Xiu, Na Chang, Le Yang, Xin Liu, Lin Yang, Jingjing Ge, Liying Li,

Tópico(s)

Fibroblast Growth Factor Research

Resumo

Sphingosine 1-phosphate (S1P) is involved in multiple pathological processes, including fibrogenesis. S1P participates in mouse liver fibrogenesis via a paracrine manner. Herein, we investigated the involvement of S1P in human liver fibrosis. Human fibrotic samples were obtained from livers of patients undergoing liver transplantation. Expression of sphingosine kinase (SphK1), collagen (Col) α1(I), Col α1(III), α-smooth muscle actin, and p-Smad2/3 was characterized by immunofluorescence, real-time RT-PCR, high-content analysis, or Western blot analysis in the fibrotic liver, human bone marrow–derived mesenchymal stem cells, and human hepatogenic profibrotic cells. The effect of SphK1 was assessed using siSphK1 or SphK-specific inhibitor. SphK1, which was expressed in human fibrotic liver myofibroblasts, could be detected in human bone marrow–derived mesenchymal stem cells or human hepatogenic profibrotic cells activated by transforming growth factor β1 (TGF-β1). TGF-β1 evoked the activation of SphK1, increased intracellular S1P, and up-regulated expression of SphK1, Col α1(I), and Col α1(III) in a TGF-β receptor–dependent manner. TGF-β1 induced expression of Col α1(I) and Col α1(III) via SphK1, which was mediated by intracellular S1P, independent of S1P receptors. TGF-β1 evoked nuclear translocation of p-Smad2 and p-Smad3 in TGF-β receptor–dependent, but SphK1-independent, manner. In conclusion, intracellular S1P plays a crucial role in the TGF-β1–induced expression of Col α1(I) and Col α1(III), which is required for human fibrosis development. S1P exerts its effects in S1P receptor–independent manner. Sphingosine 1-phosphate (S1P) is involved in multiple pathological processes, including fibrogenesis. S1P participates in mouse liver fibrogenesis via a paracrine manner. Herein, we investigated the involvement of S1P in human liver fibrosis. Human fibrotic samples were obtained from livers of patients undergoing liver transplantation. Expression of sphingosine kinase (SphK1), collagen (Col) α1(I), Col α1(III), α-smooth muscle actin, and p-Smad2/3 was characterized by immunofluorescence, real-time RT-PCR, high-content analysis, or Western blot analysis in the fibrotic liver, human bone marrow–derived mesenchymal stem cells, and human hepatogenic profibrotic cells. The effect of SphK1 was assessed using siSphK1 or SphK-specific inhibitor. SphK1, which was expressed in human fibrotic liver myofibroblasts, could be detected in human bone marrow–derived mesenchymal stem cells or human hepatogenic profibrotic cells activated by transforming growth factor β1 (TGF-β1). TGF-β1 evoked the activation of SphK1, increased intracellular S1P, and up-regulated expression of SphK1, Col α1(I), and Col α1(III) in a TGF-β receptor–dependent manner. TGF-β1 induced expression of Col α1(I) and Col α1(III) via SphK1, which was mediated by intracellular S1P, independent of S1P receptors. TGF-β1 evoked nuclear translocation of p-Smad2 and p-Smad3 in TGF-β receptor–dependent, but SphK1-independent, manner. In conclusion, intracellular S1P plays a crucial role in the TGF-β1–induced expression of Col α1(I) and Col α1(III), which is required for human fibrosis development. S1P exerts its effects in S1P receptor–independent manner. Sphingosine 1-phosphate (S1P) is a bioactive lipid that regulates vital physiological and pathological actions.1Hla T. Physiological and pathological actions of sphingosine 1-phosphate.Semin Cell Dev Biol. 2004; 15: 513-520Crossref PubMed Scopus (347) Google Scholar There is much evidence demonstrating that S1P is a pivotal regulator of fibrosis diseases, including cardiac fibrosis, pulmonary fibrosis, renal fibrosis, systemic sclerosis, and liver fibrosis.2Gellings 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, 3Ren S. Babelova A. Moreth K. Xin C. Eberhardt W. Doller A. Pavenstadt H. Schaefer L. Pfeilschifter J. Huwiler A. Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression.Kidney Int. 2009; 76: 857-867Crossref PubMed Scopus (66) Google Scholar, 4Vogler R. Sauer B. Kim D.S. Schafer-Korting M. Kleuser B. Sphingosine-1-phosphate and its potentially paradoxical effects on critical parameters of cutaneous wound healing.J Invest Dermatol. 2003; 120: 693-700Crossref PubMed Scopus (105) Google Scholar, 5Kono 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, 6Davaille 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: 37323-37330Crossref PubMed Scopus (90) Google Scholar It has been recently reported that S1P participates in mouse hepatic myofibroblast activation and rat hepatocyte apoptosis, which contribute to liver fibrosis.7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Karimian G. Buist-Homan M. Schmidt M. Tietge U.J. de Boer J.F. Klappe K. Kok J.W. Combettes L. Tordjmann T. Faber K.N. Moshage H. Sphingosine kinase-1 inhibition protects primary rat hepatocytes against bile salt-induced apoptosis.Biochim Biophys Acta. 2013; 1832: 1922-1929Crossref PubMed Scopus (25) Google Scholar Under normal conditions, S1P is produced by the phosphorylation of sphingosine, which is catalyzed by sphingosine kinase (SphK). S1P can function as an intracellular second messenger. It has been demonstrated that S1P binds and alters the functions of several intracellular proteins, such as histone deacetylases, tumor necrosis factor (TNF) receptor–associated factor 2, and ceramide synthase 2, which influence diverse biological actions.9Hait N.C. Allegood J. Maceyka M. Strub G.M. Harikumar K.B. Singh S.K. Luo C. Marmorstein R. Kordula T. Milstien S. Spiegel S. Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate.Science. 2009; 325: 1254-1257Crossref PubMed Scopus (746) Google Scholar, 10Alvarez S.E. Harikumar K.B. Hait N.C. Allegood J. Strub G.M. Kim E.Y. Maceyka M. Jiang H. Luo C. Kordula T. Milstien S. Spiegel S. Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2.Nature. 2010; 465: 1084-1088Crossref PubMed Scopus (592) Google Scholar, 11Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate.J Biol Chem. 2008; 283: 5677-5684Crossref PubMed Scopus (363) Google Scholar S1P can also be secreted into the extracellular space, providing access to S1P receptors (S1PRs) on the cell surface, thereby allowing paracrine signaling of S1P.12Takabe K. Paugh S.W. Milstien S. Spiegel S. "Inside-out" signaling of sphingosine-1-phosphate: therapeutic targets.Pharmacol Rev. 2008; 60: 181-195Crossref PubMed Scopus (586) Google Scholar After binding to S1PRs, S1P activates diverse downstream signaling pathways and induces diverse cellular responses.7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13Spiegel S. Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid.Nat Rev Mol Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1756) Google Scholar Liver fibrosis is characterized by the excessive deposition of collagen (Col), especially Col α1(I) and Col α1(III).14Bataller R. Brenner D.A. Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4135) Google Scholar, 15Friedman S.L. Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2183) Google Scholar Myofibroblasts are the crucial collagen-producing cells during liver fibrosis.16Hinz B. Gabbiani G. Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives.F1000 Biol Rep. 2010; 2: 78Crossref PubMed Scopus (137) Google Scholar Comprehensive sources contribute to myofibroblasts in liver, including hepatic stellate cells (HSCs), portal fibroblasts, bone marrow–derived mesenchymal stem cells (BMSCs), and epithelial mesenchymal transition from hepatocytes and cholangiocytes.15Friedman S.L. Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2183) Google Scholar HSCs were separated from fat-storing cells from outgrowth of liver explants.17Casini A. Pinzani M. Milani S. Grappone C. Galli G. Jezequel A.M. Schuppan D. Rotella C.M. Surrenti C. Regulation of extracellular matrix synthesis by transforming growth factor beta 1 in human fat-storing cells.Gastroenterology. 1993; 105: 245-253Abstract PubMed Google Scholar, 18Marra F. Pinzani M. DeFranco R. Laffi G. Gentilini P. Involvement of phosphatidylinositol 3-kinase in the activation of extracellular signal-regulated kinase by PDGF in hepatic stellate cells.FEBS Lett. 1995; 376: 141-145Abstract Full Text PDF PubMed Scopus (80) Google Scholar But, in general, these cells are a mixture of HSCs, portal myofibroblasts, and fibroblasts. We named these cells as hepatogenic profibrotic cells (HPCs). HPCs and BMSCs are thought to be the main sources of hepatic myofibroblasts.19Lee U.E. Friedman S.L. Mechanisms of hepatic fibrogenesis.Best Pract Res Clin Gastroenterol. 2011; 25: 195-206Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 20Forbes 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, 21Mederacke I. Hsu C.C. Troeger J.S. Huebener P. Mu X. Dapito D.H. Pradere J.P. Schwabe R.F. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology.Nat Commun. 2013; 4: 2823Crossref PubMed Scopus (825) Google Scholar Thus, it is interesting to understand the regulatory mechanisms underlying the collagen production in BMSCs and HPCs. It has been proved that the synthesis of collagen is highly regulated by different factors and cytokines at various levels,22Ghosh A.K. Factors involved in the regulation of type I collagen gene expression: implication in fibrosis.Exp Biol Med (Maywood). 2002; 227: 301-314Crossref PubMed Scopus (214) Google Scholar and transforming growth factor (TGF)-β1 is a central mediator of collagen production.23Prime S.S. Pring M. Davies M. Paterson I.C. TGF-beta signal transduction in oro-facial health and non-malignant disease (part I).Crit Rev Oral Biol Med. 2004; 15: 324-336Crossref PubMed Scopus (53) Google Scholar, 24Wahl S.M. Transforming growth factor-beta: innately bipolar.Curr Opin Immunol. 2007; 19: 55-62Crossref PubMed Scopus (147) Google Scholar, 25Inagaki Y. Okazaki I. Emerging insights into transforming growth factor beta Smad signal in hepatic fibrogenesis.Gut. 2007; 56: 284-292Crossref PubMed Scopus (386) Google Scholar, 26Breitkopf K. Godoy P. Ciuclan L. Singer M.V. Dooley S. TGF-beta/Smad signaling in the injured liver.Z Gastroenterol. 2006; 44: 57-66Crossref PubMed Scopus (152) Google Scholar TGF-β1 usually exerts its functions by canonical TGF-β receptor (TGFβR)/Smad pathway.27Shi Y. Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4815) Google Scholar TGF-β binds to its receptor TGFβR, finally resulting in the phosphorylation of Smad2/3, which then associate with Smad4 and translocate into the nucleus to control transcriptional gene expression in chondrocytes, fibroblasts, or hepatocytes, resulting in extracellular matrix production or experimental liver fibrosis.28Finnson K.W. Parker W.L. Chi Y. Hoemann C.D. Goldring M.B. Antoniou J. Philip A. Endoglin differentially regulates TGF-beta-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes.Osteoarthritis Cartilage. 2010; 18: 1518-1527Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 29Piek E. Ju W.J. Heyer J. Escalante-Alcalde D. Stewart C.L. Weinstein M. Deng C. Kucherlapati R. Bottinger E.P. Roberts A.B. Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.J Biol Chem. 2001; 276: 19945-19953Crossref PubMed Scopus (356) Google Scholar, 30Ferguson C.M. Schwarz E.M. Reynolds P.R. Puzas J.E. Rosier R.N. O'Keefe R.J. Smad2 and 3 mediate transforming growth factor-beta1-induced inhibition of chondrocyte maturation.Endocrinology. 2000; 141: 4728-4735Crossref PubMed Scopus (115) Google Scholar, 31Gressner O.A. Lahme B. Siluschek M. Rehbein K. Weiskirchen R. Gressner A.M. Connective tissue growth factor is a Smad2 regulated amplifier of transforming growth factor beta actions in hepatocytes–but without modulating bone morphogenetic protein 7 signaling.Hepatology. 2009; 49: 2021-2030Crossref PubMed Scopus (49) Google Scholar Previously, we have shown that S1P is crucial in liver fibrosis,7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Li C. Kong Y. Wang H. Wang S. Yu H. Liu X. Yang L. Jiang X. Li L. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis.J Hepatol. 2009; 50: 1174-1183Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 33Li 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, 34Li 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.J Hepatol. 2011; 54: 1205-1213Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 35Liu 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, 36Yang L. Yue S. Liu X. Han Z. Zhang Y. Li L. Sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis is involved in liver fibrosis-associated angiogenesis.J Hepatol. 2013; 59: 114-123Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar and the mode of action of S1P is diverse because of the differences in various species. It has been reported that S1P participates in the differentiation of mouse BMSCs to myofibroblasts via S1PR1 and S1PR3 up-regulation.7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar But, in human BMSCs (hMSCs), collagen expression is regulated negatively by S1PR1 and S1PR3.37Chang N. Xiu L. Li L. Sphingosine 1-phosphate receptors negatively regulate collagen type I/III expression in human bone marrow-derived mesenchymal stem cell.J Cell Biochem. 2014; 115: 359-367Crossref PubMed Scopus (16) Google Scholar These results elucidate the disparity between human and animal experiments, and strongly suggest the necessity of verifying the experiments performed in animal models in a human context. Previously, we found that TGF-β1 induces activation of mouse BMSCs via SphK1 up-regulation, and this process is S1PR dependent.7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar But, the operating mechanisms in human cells are still unclear. To explore the molecular mechanisms of human liver fibrosis, it is necessary to study the mode of action of S1P in the expression of collagen in primary human HPCs (hHPCs) and hMSCs. Herein, we demonstrate, for the first time, that the production of Col α1(I) and Col α1(III) is increased by both activation and up-regulation of SphK1, which elevates intracellular S1P in hMSCs and hHPCs after TGF-β1 treatment. In addition, TGF-β1–induced expression of SphK1 and collagen is TGFβR dependent. Moreover, phosphorylations of Smad2 or Smad3 are SphK1 independent. More important, these results represent the first experimental evidence that S1P participates in the production of collagens in an S1PR-independent manner, and unravel a novel mode of action of intracellular S1P in the process of human liver fibrogenesis. Snap-frozen surgical liver resections from 21 patients (13 men and 8 women; mean age, 56 years; age range, 42 to 69 years) were studied retrospectively. Normal liver samples were collected from five patients undergoing hepatic resection for colorectal metastasis. Fibrotic samples (fibrosis stage: F2 to F4) were obtained from 16 livers of patients undergoing liver transplantation. Fibrosis was consecutive to chronic hepatitis C virus (n = 4) or hepatitis B virus (n = 10) infections, and alcohol-induced liver disease (n = 2). Normal serum samples were collected from 16 healthy volunteers (10 men and 6 women; mean age, 46 years; age range, 36 to 58 years), and fibrotic serum samples were obtained from 17 patients (11 men and 6 women; mean age, 49 years; age range, 40 to 65 years) with liver fibrosis. All tissues were obtained with donor consent and the approval of the Capital Medical University (Beijing, China) Ethics Committee (2011SY08). hMSCs (Cyagen, Sunnyvale, CA) were cultured as previously described.37Chang N. Xiu L. Li L. Sphingosine 1-phosphate receptors negatively regulate collagen type I/III expression in human bone marrow-derived mesenchymal stem cell.J Cell Biochem. 2014; 115: 359-367Crossref PubMed Scopus (16) Google Scholar hMSCs of passage 6 to passage 10 were used in the experiments per the user manual recommendations. hHPCs were obtained and cultured by outgrowth of explants prepared from surgical specimens of normal livers, as previously described.38Li L. Grenard P. Nhieu J.T. Julien B. Mallat A. Habib A. Lotersztajn S. Heme oxygenase-1 is an antifibrogenic protein in human hepatic myofibroblasts.Gastroenterology. 2003; 125: 460-469Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar This procedure was performed in accordance with ethical regulations imposed by the Chinese legislation. Cells were used between the fourth and ninth passage for experiments, without any noticeable differences in results observed with cells obtained from various passages or from various livers. Unless otherwise indicated, cells prepared for experiments were cultured in medium without serum for 24 hours and treated with TGF-β1 (PeproTech, London, UK), TGFβR1 inhibitor (LY364947; Calbiochem, La Jolla, CA), N,N-dimethylsphingosine (DMS; Biomol, Hamburg, Germany), S1P and dihydro-S1P (H2S1P; Biomol, Tebu, France), W146 (Avanti Polar Lipids, Alabaster, AL), and JTE-013 and CAY10444 (Cayman Chemical, Ann Arbor, MI). Immunofluorescence of cultured hMSCs or hHPCs was performed as described.36Yang L. Yue S. Liu X. Han Z. Zhang Y. Li L. Sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis is involved in liver fibrosis-associated angiogenesis.J Hepatol. 2013; 59: 114-123Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Cells were incubated with antibodies of anti-Col α1(I), Col α1(III), SphK1 (dilution 1:50; Santa Cruz Biotechnology, Santa Cruz, CA), p-Smad2 (dilution 1:50; Cell Signaling, Beverly, MA), p-Smad3 (dilution 1:50; Santa Cruz Biotechnology), or α-smooth muscle actin (α-SMA; dilution 1:1000; Sigma, St. Louis, MO), followed by secondary antibodies conjugated with Cy3 or fluorescein isothiocyanate (diluted 1:100; Jackson Immunoresearch, West Grove, PA). Double staining of SphK1 and α-SMA was performed in human liver sections. Immunofluorescence was performed as described.7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Sections were first incubated with anti-SphK1 antibody described above, and incubated with Cy3-conjugated AffiniPure donkey anti-goat IgG antibody (diluted 1:100; Jackson Immunoresearch, West Grove, PA) as a secondary antibody. Next, sections were incubated with anti–α-SMA antibody described above, and incubated with fluorescein isothiocyanate–conjugated AffiniPure goat anti-mouse IgG antibody (diluted 1:100) as a secondary antibody. Sections or cells were incubated without primary antibodies as negative control. hMSCs (n = 5000) and hHPCs (n = 6000) were plated in the wells of 96-well plates (Corning, NY), attached overnight, and then treated with chemicals. Immunofluorescence staining for Col α1(I), Col α1(III), p-Smad2, or p-Smad3 was performed as described above. The plates were imaged on Thermo Scientific CellInsight personal cell imaging platform (Cellomics, Inc., Thermo Fisher Scientific Inc., Waltham, MA). High content analysis was performed as described previously.37Chang N. Xiu L. Li L. Sphingosine 1-phosphate receptors negatively regulate collagen type I/III expression in human bone marrow-derived mesenchymal stem cell.J Cell Biochem. 2014; 115: 359-367Crossref PubMed Scopus (16) Google Scholar Extraction of total RNA and real-time RT-PCR was performed as described previously.32Li C. Kong Y. Wang H. Wang S. Yu H. Liu X. Yang L. Jiang X. Li L. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis.J Hepatol. 2009; 50: 1174-1183Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar Primers were as follows: 18S rRNA, 5′-GTAACCCGTTGAACCCCATT-3′ (sense) and 5′-CCATCCAATCGGTAGTAGCG-3′ (anti-sense); Col α1(I), 5′-AGGTCCCCCTGGAAAGAA-3′ (sense) and 5′-AATCCTCGAGCACCCTGA-3′ (anti-sense); Col α1(III), 5′-AGCTGGAAAGAGTGGTGACAG-3′ (sense) and 5′-CCTTGAGGACCAGGAGCAC-3′ (anti-sense); S1PR1, 5′-TCTGCGGGAAGGGAGTATGT-3′ (sense) and 5′-CGATGGCGAGGAGACTGAA-3′ (anti-sense); S1PR2, 5′-TGCCCGCCTTCAGCAT-3′ (sense) and 5′-AAAAAGTAGTGGGCTTTGTAGAGGAT-3′ (anti-sense); and S1PR3, 5′-TCTCAGCCTTCATCCATTAACTCTAC-3′ (sense) and 5′-AGGGAGCCTTATGTCATACCACAA-3′ (anti-sense). Probe (Life Technologies, Foster City, CA) used for real-time RT-PCR was as follows: SphK1: Hs00184211_m1. Western blot analysis was performed as described.7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar The blots were incubated with primary antibodies against SphK1 or p-SphK1 (dilution 1:1000; ECM Biosciences, Versailles, KY) or Col α1(I) or Col α1(III) (dilution 1:500; Santa Cruz Biotechnology). Results were normalized relative to glyceraldehyde-3-phosphate dehydrogenase (rabbit anti–glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody, dilution 1:1000; Sigma) or tubulin (rabbit monoclonal anti-tubulin antibody, dilution 1:1000; Epitomics, Burlingame, CA) expression to correct for variations in protein loading and transfer. The siRNA sequences specifically targeting human SphK1, S1PR1, S1PR2, S1PR3, or negative control siRNA were synthesized (S16957, S4448, S17766, S4455, or 4390843, respectively; Ambion, Austin, TX). RNA interferences of hMSCs or hHPCs were performed as described.7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar TGF-β1 concentrations in human serum were determined using commercially specific enzyme-linked immunosorbent assay and were processed according to the manufacturer's specifications (Promega, Charbonnie`res, France). hMSCs and hHPCs, with or without stimulation of 10 ng/mL TGF-β1, were collected. The preparation and analysis of sample were performed as described.35Liu 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 Sample analysis was performed using Agilent 1200 high-performance liquid chromatographic equipment (Agilent Technologies, Wilmington, DE). All results were confirmed in at least three independent experiments. Data were expressed as means ± SEM. Statistical analyses were performed by Student's t-test or analysis of variance when appropriate. Statistical significance was defined as P < 0.05. It is well established that myofibroblasts are the principal effector cells in fibrotic liver.15Friedman S.L. Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2183) Google Scholar In liver tissue sections of patients with liver fibrosis, there was strong immunoreactivity for α-SMA, a marker for myofibroblasts, in the fibrotic areas (Figure 1B). Double-immunofluorescence staining for SphK1 and α-SMA showed that the numbers of α-SMA–positive cells in the fibrotic areas were significantly positive for SphK1 (Figure 1B). We also detected the basal expression of SphK1 and α-SMA in normal human liver tissue sections (Figure 1A); there was basal expression of SphK1 and α-SMA in the portal area, in the vascular endothelial cells (SphK1), and in smooth muscle cells (α-SMA), respectively. TGF-β1 is a well-known profibrotic mediator that plays an important role in activation of myofibroblasts.15Friedman S.L. Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2183) Google Scholar In serum of patients with liver fibrosis, the level of TGF-β1 was increased by approximately 1.6-fold (Figure 1C). Hence, we performed immunofluorescence of α-SMA and SphK1 in TGF-β1–stimulated hMSCs or hHPCs, the main sources of human myofibroblasts, in vitro. There was strong expression of both α-SMA and SphK1 in these activated myofibroblasts (Figure 1D). These results showed that SphK1 probably participated in activation of myofibroblasts. TGF-β1 has been shown to elevate expression of SphK1 in many cell types.2Gellings 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, 5Kono 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, 7Yang L. Chang N. Liu X. Han Z. Zhu T. Li C. Li L. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis.Am J Pathol. 2012; 181: 85-97Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 39Cencetti 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 explore the compliance of SphK1 to TGF-β1 in hMSCs and hHPCs, mRNA and protein levels of SphK1 were evaluated by real-time RT-PCR and Western blot analysis. Cells were treated with increasing concentrations of TGF-β1 (1, 2, 5, 7.5, or 10 ng/mL), which induced a dose-dependent increase in the mRNA expression of SphK1 (Figure 2A). SphK1 mRNA expression increased in a time-dependent manner at 10 ng/mL TGF-β1, with a maximal increase at 24 hours in both hMSCs and hHPCs (Figure 2B). On the basis of these experiments, stimulation by 10

Referência(s)