In this manuscript, we showed that following a fibrogenic stimulus of leptin, hepatic stellate cells (HSCs) underwent a complex activation process characterized by increased proliferation and excessive deposition of type I collagen. Studies with special chemical inhibitors demonstrated that this process involved Janus protein tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT), mitogen-activated protein kinases (MAPK), and phosphatidylinositol 3-linase (PI3K)/Protein kinase B (AKT) signal pathways. Leflunomide pretreatment significantly inhibited the deposition of type I collagen in HSCs and the proliferation of primary HSC by interrupting the three proliferative signal transduction pathways in vitro, which was indicated by [3H]thymidine incorporation and cell cycle analysis. Furthermore, leptin-induced cyclin D1 protein expression, which correlates well with HSC proliferation, was also significantly inhibited by leflunomide. On the other hand, leflunomide also prevented leptin-induced Kupffer cell (KC) activation and HSC collagen synthesis induced by KC-conditioned medium (KCCM). Collectively, these results provided a novel insight into the mechanisms by which leflunomide may exert in liver fibrosis.
Get full access to this article
View all access options for this article.
References
1.
Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. Hepatology13:1193–1202, 1991.
2.
Schmitt-Graff A, Kruger S, Bochard F, Gabbiani G, Denk H. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am J Pathol138:1233–1242, 1991.
3.
Honda H, Ikejima K, Hirose M, Yoshikawa M, Lang T, Enomoto N, Kitamura T, Takei Y, Sato N. Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver. Hepatology36: 12–21, 2002.
4.
Henriksen JH, Holst JJ, Moller S, Brinch K, Bendtsen F. Increased circulating leptin in alcoholic cirrhosis: relation to release and disposal. Hepatology29:1818–1824, 1999.
5.
Uygun A, Kadayifci A, Yesilova Z, Erdil A, Yaman H, Saka M, Deveci S, bagci S, Gulsen M, Karaeren N, Dagalp K. Serum leptin levels in patients with nonalcoholic steatohepatitis. Am J Gastroenterol95: 3584–3589, 2000.
6.
Jiang YC, Liu J, Waalkes M, Kang Y J. Changes in the gene expression associated with carbon tetrachloride-induced liver fibrosis persist after cessation of dosing in mice. Toxicol Sci79:404–410, 2004.
Leclercq IA, Farrell GC, Schriemer R, Robertson GR. Leptin is essential for the hepatic response to chronic liver injury. J Hepatol37: 206–213, 2002.
9.
White DW, Kuropatwinski KK, Devos R, Baumann H, Tartaglia LA. Leptin receptor (OB-R) signaling. Cytoplasmic domain mutational analysis and evidence for receptor homooligomerization. J Biol Chem272:4065–4071, 1997.
10.
Takahashi Y, Okimura Y, Mizuno I, Iida K, Takahashi T, Kaji H, Abe H, Chihara K. Leptin induces mitogen activated protein kinase-dependent proliferation of C3H10T1/2 cells. J Biol Chem272:12897–12900, 1997.
11.
Harvey J, McKay NG, Walker KS, Van der Kaay J, Downes CP, Ashford MLJ. Essential role of phosphoinositide 3-kinase in leptin-induced KATP channel activation in the rat CRI-G1 insulinoma cell line. J Biol Chem275:4660–4669, 2000.
12.
Martin-Romero C, Sanchez-Margalet V. Human leptin activates PI3K and MAPK pathways in human peripheral blood mononuclear cells. Possible role of Sam68. Cell Immunol212:83–91, 2001.
13.
Zhou H, Xin-Ming L, Meinkoth J, Pittman RN. AKT regulates cell survival and apoptosis at a postmitochondrial level. J Cell Biol151: 483–494, 2000.
14.
Reif S, Lang A, Jeffery N, Lindquist, Yata Y, Gäbele E, Scanga A, David A. Brenner, Richard A, Rippe. The role of focal adhesion kinase-phosphatidylinositol 3-kinase-AKT signaling in hepatic stellate cell proliferation and type I collagen expression. J Biol Chem278:8083–8090, 2003.
15.
Shiratori Y, Geerts A, Ichida T, Kawase T, Wisse E. Kupffer cells from CCl4-induced fibrotic livers stimulate proliferation of fat-storing cells. J Hepatol3:294–303, 1986.
16.
Ikejima K, Honda H, Yoshikawa M, Hirose M, Kitamura T, Takei Y, Sato N. Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals. Hepatology34: 288–297, 2001.
17.
Santos-Alvarez J, Goberna R, Sanchez-Margalet V. Human leptin stimulates proliferation and activation of human circulating monocytes. Cell Immunol194:6–11, 1999.
18.
Herrmann ML, Schleyerbach R, Kirschbaum BJ. Leflunomide: an immunomodulatory drug for the treatment of rheumatoid arthritis and other autoimmune diseases. Immunopharmacology47:273–289, 2000.
19.
Li WD, Ran GX, Teng HL, Lin ZB. Dynamic effects of leflunomide on IL-1, IL-6, and TNF-α activity produced from peritoneal macrophages in adjuvant arthritis rats. Acta Pharmacol Sin23:752–756, 2002.
20.
Manna SK, Mukhopadhyay A, Aggarwal BB. Leflunomide suppresses TNF-α induced cellular responses: effects on NF-κB, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis. J Immunol165: 5962–5969, 2000.
21.
Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydrogenase. Biochemistry35:1270–1273, 1996.
22.
Xu X, Williams JW, Bremer EG, Finnegan A, Chong ASF. Inhibition of protein tyrosin phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. J Biol Chem270:12398–12403, 1995.
23.
Cherwinski HM, Cohn RG, Cheung P, Webster DJ, Xu YZ, Caulfield JP, Young JM, Nakano G, Ransom JT. The immunosuppressant leflunomide inhibits lymphocyte proliferation by inhibiting pyrimidine biosynthesis. J Pharmacol Exp Ther275:1043–1049, 1995.
24.
Elder RT, Xu X, Williams JW, Gong H, Finnegan A, Chong AS. The immunosuppressive metabolite of leflunomide, A771726, affects murine T cells through two biochemical mechanisms. J Immunol159:22–27, 1997.
25.
Carloni V, Pinzani M, Giusti S, Romanelli RG, Parola M, Bellomo G, Failli P, Hamilton AD, Sebti SM, Laffi G, Gentilini P. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent in ras in human stellate cells. Hepatology31:131–140, 2000.
26.
Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endotheliul cells by density gradient centrifugation with stractan. Anal Biochem161:207–218, 1987.
27.
Takase S, Leo MA, Nouchi T, Lieber CS. Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat. J Hepatol6:267–276, 1988.
28.
Lam S, Verhagen NA, Strutz F, van der Pijl JW, Daha MR, van Kooten C. Glucose-induced fibronectin and collagen type III expression in renal fibroblasts can occur independent of TGF-β. Kidney Int63:878–888, 2003.
29.
Andrew NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res19:2499, 1991.
30.
Wagner BJ, Hayes TE, Hoban CJ, Cochran BH. The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter. EMBO J9:4477–4484, 1990.
31.
Friedman, SL. molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem275:2247–2250, 2000.
32.
Kawada N, Ikeda K, Seki S, Kuroki T. Expression of cyclins D1, D2 and E correlates with proliferation of rat stellate cells in culture. J Hepatol30:1057–1064, 1999.
33.
Kim YB, Uotani S, Pierroz DD, Flier JS, and Khan BB. In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin. Endocrinology141:2328–2339, 2000.
34.
Rebbaa A, Hurh J, Yamamoto H, Donna S, Kersey, Bremer EG. Ganglioside GM3 inhibition of EGF receptor mediated signal transduction. Glycobiology6:399–406, 1996.
35.
Rivera CA, Bradford BU, Hunt KJ, Adachi Y, Schrum LW, Koop DR, Burchardt ER, Rippe RA, Thurman RG. Attenuation of CCl4-induced hepatic fibrosis by GdCl3 treatment or dietary glycine. Am J Physiol Gastrointest Liver Physiol281:200–207, 2001.
36.
Friedman SL, Wei S, Blaner WS. Retinol release by activated rat hepatic lipocytes: regulation by Kupffer cell-conditioned medium and PDGF. Am J Physiol Gastrointest Liver Physiol264:947–952, 1993.
37.
Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest84:1780–1785, 1989.
38.
Saxena NK, Titus MA, Ding X, Floyd J, Srinivasan S, Sitaraman SV, Frank A. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and AKT phosphorylation. Faseb J18:1612–164, 2004.
39.
Ikejima K, Takei Y, Honda H, Hirose M, Yoshikawa M, Zhang YJ, Lang T, Fukuda T, Yamashina S, Kitamura T, Sato N. Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat. Gastroenterology122:1399–1410, 2002.
40.
Saxena NK, Ikeda K, Rockey DC, Friedman SL, Anania FA. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice. Hepatology35:762–771, 2002.
41.
Dai K, Qi JY, Tian DY. Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice. World J Gastroenterol11:4822–4826, 2005.
42.
Arthur MJP. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am J Physiol Gastrointest Liver Physiol279:245–249, 2000.
43.
Chog AS, Rezai K, Gebel HM, Finnegan A, Foster P, Xu X, Williams JW. Effects of leflunomide and other immunosuppressive agents on T cell proliferation in vitro. Transplantation61:140–145, 1996.
44.
Banks AS, Davis SM, Bates SH, Myers MG.. Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 275:14563–14572, 2000.
45.
Xu X, Williams JW, Gong H, Finnegan A, Chong AS. Two activities of immunosuppressive metabolite of leflunomide, A771726. Inhibition of pyrimidine nucleotide synthesis and protein tyrosine phosphorylation. Biochem Pharmacol52:527–534, 1996.
46.
Matter T, Kochhar K, Bartlett R, Bremer EG, Finnegan A. Inhibition of the epidermal growth factor receptor tyrosine kinase activity by leflunomide. FEBS Lett334:161–164, 1993.
47.
Manna SK, Aggarwal BB. Immunosuppressive leflunomide metabolite (A771726) blocks TNF-dependent nuclear factor-κB activation and gene expression. J Immunol162:2095–2102, 1999.
48.
Matsuoka M, Tsukamoto H. Stimulation of hepatic collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenic role in alcoholic liver fibrogenesis. Hepatology11: 599–605, 1990.
49.
Arend WP, Dayer JM. Inhibition of the production and effects of interleukin-1 and tumor necrosis factor alpha in rheumatoid arthritis. Arthritis Rheum38:151–160, 1995.
50.
Yao HW, Li J, Chen JQ, Xu SY. Leflunomide attenuates hepatocyte injury by inhibiting kupffer cells. World J Gastroenterol10:1608–1611, 2004.
