Title: Stimulation of Proliferation of Rat Hepatic Stellate Cells by Galectin-1 and Galectin-3 through Different Intracellular Signaling Pathways
Abstract: We found that the expression of galectin-1 and galectin-3 was significantly up-regulated in hepatic stellate cells (HSCs) both in the course of their transdifferentiation into myofibroblasts, a process of "self-activation," and in the fibrosis of liver tissues. Recombinant galectin-1 and galectin-3 stimulated the proliferation of cultured HSCs via the MEK1/2-ERK1/2 signaling pathway. However, galectin-3 utilized protein kinases C and A to induce this process, whereas galectin-1 did not. We also found that thiodigalactoside, a potent inhibitor of β-galactoside binding, attenuated the effects of both galectins. In addition, galectin-1, but not galectin-3, promoted the migration of HSCs. Thus, it appears that galectin-1 and galectin-3, generated by activated HSCs, could participate in β-galactoside binding and induce different intracellular signaling pathways leading to the proliferation of HSCs. We found that the expression of galectin-1 and galectin-3 was significantly up-regulated in hepatic stellate cells (HSCs) both in the course of their transdifferentiation into myofibroblasts, a process of "self-activation," and in the fibrosis of liver tissues. Recombinant galectin-1 and galectin-3 stimulated the proliferation of cultured HSCs via the MEK1/2-ERK1/2 signaling pathway. However, galectin-3 utilized protein kinases C and A to induce this process, whereas galectin-1 did not. We also found that thiodigalactoside, a potent inhibitor of β-galactoside binding, attenuated the effects of both galectins. In addition, galectin-1, but not galectin-3, promoted the migration of HSCs. Thus, it appears that galectin-1 and galectin-3, generated by activated HSCs, could participate in β-galactoside binding and induce different intracellular signaling pathways leading to the proliferation of HSCs. Hepatic stellate cells (HSCs), 1The abbreviations used are: HSCs, hepatic stellate cells; ECMs, extracellular matrix materials; PDGF, platelet-derived growth factor; r-PDGF-BB, recombinant rat platelet-derived growth factor-BB; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-hydroxykinase; TAA, thioacetamide; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; r-galectin, recombinant human galectin; RT, reverse transcription; PKC, protein kinase C; PKA, protein kinase A; TDG, thiodigalactoside.1The abbreviations used are: HSCs, hepatic stellate cells; ECMs, extracellular matrix materials; PDGF, platelet-derived growth factor; r-PDGF-BB, recombinant rat platelet-derived growth factor-BB; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-hydroxykinase; TAA, thioacetamide; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; r-galectin, recombinant human galectin; RT, reverse transcription; PKC, protein kinase C; PKA, protein kinase A; TDG, thiodigalactoside. liver-specific pericytes, play a pivotal role in hepatic fibrogenesis (1Friedman S.L. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar). In liver injury, HSCs undergo proliferation and migration and generate a large amount of extracellular matrix materials (ECMs), including fibril-forming collagens, fibronectin, and proteoglycans, resulting in the formation of septa in chronically damaged liver (2Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1867) Google Scholar, 3Gressner A.M. Bachem M.G. Digestion. 1995; 56: 335-346Crossref PubMed Scopus (222) Google Scholar). Resident macrophages (Kupffer cells), infiltrating macrophages, platelets, and sinusoidal endothelial cells secrete growth factors at the inflammatory sites, such as transforming growth factor-β and platelet-derived growth factor (PDGF). These growth factors trigger the proliferation and the secretion of ECMs by HSCs. Recent studies have elucidated that MAPK and phosphatidylinositol 3-kinase (PI3K) are key signaling pathways involved in the growth factor-induced stimulation of HSCs (4Pinzani M. Marra F. Carloni V. Liver. 1998; 18: 2-13Crossref PubMed Scopus (231) Google Scholar, 5Marra F. Gentilini A. Pinzani M. Choudhury G.G. Parola M. Herbst H. Dianzani M.U. Laffi G. Abboud H.E. Gentilini P. Gastroenterology. 1997; 112: 1297-1306Abstract Full Text PDF PubMed Scopus (185) Google Scholar). Galectins form a group of β-galactoside-binding animal lectins (6Sharon N. Lis H. Science. 1989; 246: 227-234Crossref PubMed Scopus (1061) Google Scholar, 7Barondes S.H. Castronovo V. Cooper D.N. Cummings R.D. Drickamer K. Feizi T. Gitt M.A. Hirabayashi J. Hughes C. Kasai K.-i. Leffler H. Liu F. Lotan R. Mercurio A.M. Monsigni M. Pillai S. Poirer F. Raz A. Rigby P.W.J. Rini J.M. Wang J.L. Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1074) Google Scholar, 8Kasai K.-i. Hirabayashi J. J. Biochem. 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Pathol. 2001; 32: 302-310Crossref PubMed Scopus (75) Google Scholar). A study on concanavalin A-induced acute liver injury showed that galectin-1 administration protects mice from liver injury by selectively eliminating activated T cells and preventing the synthesis of proinflammatory Th1-derived cytokines (34Santucci L. Fiorucci S. Cammilleri F. Servillo G. Federici B. Morelli A. Hepatology. 2000; 31: 399-406Crossref PubMed Scopus (138) Google Scholar). Our previous proteome analysis of rat HSC proteins revealed that the amount of galectin-1 is greatly increased in activated HSCs compared with quiescent HSCs; the secretion of galectin-1 is also much enhanced by activated HSCs (32Kristensen D.B. Kawada N. Imamura K. Miyamoto Y. Tateno C. Seki S. Kuroki T. Yoshizato K. Hepatology. 2000; 32: 268-277Crossref PubMed Scopus (200) Google Scholar). Although a detailed analysis has not yet been performed, these results suggest that an enhanced level of galectin-1 modifies the function of HSCs themselves as well as other hepatic constituent cells in chronically injured liver. In this study, we investigated in detail the expression pattern of galectin-1 and galectin-3 in HSCs and in fibrotic liver tissues. We further demonstrated that both types of galectins activate MAPK pathways presumably by cross-linking with target molecules through their β-galactoside-containing glycoconjugates, leading to the proliferation of HSCs. Materials—Collagenase, thioacetamide (TAA), isopropyl-β-d-thiogalactopyranoside, Me2SO, and 3,3′-diaminobenzidine tetrahydrochloride) were purchased from Wako Pure Chemical Co. (Osaka, Japan). Pronase E was from Merck (Darmstadt, Germany). Rat PDGF-BB was from R&D Systems (Minneapolis, MN). Rat hepatocyte growth factor was from Toyobo Co. (Japan). Polyclonal antibodies against ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), MEK1/2, phospho-MEK1/2 (Ser217/Ser221), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), Akt, and phospho-Akt (Ser473) were from Cell Signaling Technology, Inc. (Beverly, MA) and those against PDGF receptor-β were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against smooth muscle α-actin were purchased from Dako A/S (Glostrup, Denmark), and those against galectin-3 purchased from Affinity Bioreagents, Inc. (Golden, CO). Polyclonal antibodies against galectin-1 were generated in our laboratory as described previously (35Hirabayashi J. Ayaki H. Soma G. Kasai K.-i. FEBS Lett. 1989; 250: 161-165Crossref PubMed Scopus (50) Google Scholar, 36Oda Y. Leffler H. Sakakura Y. Kasai K.-i. Barondes S.H. Gene (Amst.). 1991; 99: 279-283Crossref PubMed Scopus (70) Google Scholar, 37Hirabayashi J. Kasai K.-i. Biochem. Biophys. Res. Commun. 1984; 122: 938-944Crossref PubMed Scopus (85) Google Scholar). [3H]Thymidine, [α-32P]dCTP, Hybond-N+, the Rediprime DNA labeling system, and ECL detection reagent were purchased from Amersham Biosciences (Buckinghamshire, UK). PD98059, SB203580, LY294002, and GF109203X were purchased from Calbiochem. U0126, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were purchased from Sigma. Williams' Medium E was from Invitrogen. H-89 was from Seikagaku Corp. (Tokyo, Japan). Type I collagen-coated dishes were products of Iwaki (Tokyo). The GeneAmp RNA PCR core kit was obtained from PerkinElmer Life Sciences. Isogen and agarose S were from Nippon Gene (Tokyo). Immobilon P membranes were from Millipore Corp. (Bedford, MA). pET21a was from Novagen (Madison, WI). Kodak XAR-5 film was from Eastman Kodak Co. Avidin-biotin-peroxidase complexes were from Vector Laboratories, Inc. (Burlingame, CA). Cell culture insert was from BD Biosciences. All other reagents were purchased from Sigma unless indicated otherwise. PD98059, U0126, SB203580, LY294002, GF109203X, and H-89 were dissolved in Me2SO. The final concentration of Me2SO in the culture medium was always below 0.1%, which did not affect the function of cultured HSCs. Animals—Pathogen-free male Wistar rats were obtained from Japan SLC, Inc. (Shizuoka, Japan). Animals were housed at a constant temperature and supplied with laboratory chow and water ad libitum. Production of Recombinant Galectin-1 and Galectin-3—Recombinant human galectin-1 (r-galectin-1) and r-galectin-3 were generated in our laboratory as described previously (35Hirabayashi J. Ayaki H. Soma G. Kasai K.-i. FEBS Lett. 1989; 250: 161-165Crossref PubMed Scopus (50) Google Scholar). Briefly, DNA fragments encoding either human galectin-1 or galectin-3 were amplified by PCR using cloned cDNA as a template. The amplified fragments were ligated to pET21a. Generated prokaryotic expression vectors were used to transform Escherichia coli BL21(DE3) cells. Recombinant proteins were induced in the cells by 1 mm isopropyl-β-d-thiogalactopyranoside. They were purified by affinity chromatography on asialofetuin-Sepharose 4B, which was prepared according to Oda et al. (36Oda Y. Leffler H. Sakakura Y. Kasai K.-i. Barondes S.H. Gene (Amst.). 1991; 99: 279-283Crossref PubMed Scopus (70) Google Scholar) and Hirabayashi and Kasai (37Hirabayashi J. Kasai K.-i. Biochem. Biophys. Res. Commun. 1984; 122: 938-944Crossref PubMed Scopus (85) Google Scholar). Liver Fibrosis Models—Liver fibrosis was induced in rats either by intraperitoneal injection of TAA (50 mg/body) twice a week for 8 weeks (38Okuyama H. Shimahara Y. Kawada N. Seki S. Kristensen D.B. Yoshizato K. Uyama N. Yamaoka Y. J. Biol. Chem. 2001; 276: 28274-28280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) or by ligation of the common bile duct for 2 weeks. After fibrosis developed, the peritoneal cavities of the rats were opened under ether anesthesia. The liver was perfused with phosphate-buffered saline via the portal vein to remove the blood completely and subsequently removed. Part of the liver was fixed in 4% paraformaldehyde and used for histological evaluation. The remaining parts were quickly frozen in liquid nitrogen and stored at —80 °C until used. Preparation of Hepatic Constituent Cells—Hepatic constituent cells were isolated from rat livers as previously described in detail (39Kawada N. Kristensen D.B. Asahina K. Nakatani K. Minamiyama Y. Seki S. Yoshizato K. J. Biol. Chem. 2001; 276: 25318-25323Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Kupffer cells and sinusoidal endothelial cells were used immediately after isolation. HSCs were plated on plastic dishes in DMEM supplemented with 10% FBS (FBS/DMEM). Hepatocytes were cultured on type I collagen-coated dishes in Williams' Medium E supplemented with 10% FBS (FBS/Williams' Medium E). The culture media were changed every day. HSCs isolated from normal and fibrotic livers are referred to as quiescent HSCs and in vivo activated HSCs, respectively, in this study (40Kawada N. Seki S. Inoue M. Kuroki T. Hepatology. 1998; 27: 1265-1274Crossref PubMed Scopus (379) Google Scholar). Quiescent HSCs cultured for 7 days are referred to as in vitro activated HSCs (41Kawada N. Ikeda K. Seki S. Kuroki T. J. Hepatol. 1999; 30: 1057-1064Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). RT-PCR—Total RNA was extracted from hepatic constituent cells and liver tissues using Isogen. mRNA expression was determined by RT-PCR using the GeneAmp RNA PCR core kit. The following primers were used: galectin-1, 5′-ATGGCCTGTGGTCTGGTCGC-3′ (forward) and 3′-AATTCACACACCGGAAACTC-5′ (reverse); galectin-3, 5′-ATGGCAGACGGCTTCTCACT-3′ (forward) and 3′-CGCGAAGGGTGCGGTACTAG-5′ (reverse); collagen α2(I), 5′-ATGCTCAGCTTTGTGGAT-3′ (forward) and 3′-CCCTTGAAACGACGAGTC-5′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 3′-TCCACCACCCTGTTGCTGTA-5′ (reverse). Northern Blotting—Total RNA was extracted from cultured HSCs and liver tissues using Isogen. Total RNA (10 μg) was separated on a 1% agarose gel and transferred onto a nylon membrane. After prehybridization, the membrane was incubated in buffer supplemented with PCR-amplified double-stranded cDNAs for galectin-1, galectin-3, and glyceraldehyde-3-phosphate dehydrogenase, which were labeled with [α-32P]dCTP using the Rediprime DNA labeling system; this was followed by autoradiography on Kodak XAR-5 x-ray film. Immunoblotting—HSCs were cultured in the presence or absence of test agents and then homogenized in buffer consisting of 62.5 mm Tris, 0.1% glycerol, 2% SDS, and 5% 2-mercaptoethanol (pH 6.8). After the samples (10 μg of protein) were heat-denatured, they were analyzed by 7.5–15% SDS-PAGE and then transferred onto Immobilon P membranes. After blocking, the membranes were treated for 2 h at room temperature with individual antibodies. After washing, they were incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized on Kodak XAR-5 film using ECL detection reagent. Immunohistochemistry—Immunohistochemical detection of galectin-1 and galectin-3 in rat liver tissues was performed according to the method described in detail by Nakatani et al. (42Nakatani K. Seki S. Kawada N. Kitada T. Yamada T. Sakaguchi H. Kadoya H. Ikeda K. Kaneda K. Virchows Arch. 2002; 441: 466-474Crossref PubMed Scopus (48) Google Scholar). Immunoprecipitates were visualized using 0.025% 3,3′-diaminobenzidine tetrahydrochloride and 0.003% H2O2. Specimens were observed under an Olympus IX70 microscope. Cell Growth Assays—Isolated HSCs were cultured on plastic dishes for 3 days in FBS/DMEM and then maintained for 24 h in serum-free DMEM. Isolated hepatocytes were cultured on type I collagen-coated dishes for 24 h in FBS/Williams' Medium E and then maintained for 24 h in serum-free Williams' Medium E. The cells were successively stimulated with test agents for 24 h and pulse-labeled with 1.0 μCi/ml [3H]thymidine during the last 6 h. The incorporated radioactivity was subjected by liquid scintillation counting as previously described (38Okuyama H. Shimahara Y. Kawada N. Seki S. Kristensen D.B. Yoshizato K. Uyama N. Yamaoka Y. J. Biol. Chem. 2001; 276: 28274-28280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In another experiment, isolated HSCs (1 × 105 cells/well) were plated in six-well culture plates, incubated for 2 days in FBS/DMEM, and maintained for 24 h in serum-free DMEM. The cells were successively stimulated with either r-galectin-1 or r-galectin-3 for 24 or 48 h. They were successively fixed in 100% methanol and stained with a Giemsa solution. The number of HSCs was counted in a microscopic field (0.84 mm2) at a magnification of ×100. Five microscopic fields were randomly chosen for each specimen. Migration Assay—The migration activity of HSCs was assayed using cell culture insert as previously described (43Ikeda K. Wakahara T. Wang Y.Q. Kadoya H. Kawada N. Kaneda K. Hepatology. 1999; 29: 1760-1767Crossref PubMed Scopus (151) Google Scholar). HSCs (1 × 105 cells) cultured for 2 days were detached from the plates using trypsin, suspended in 400 μl of FBS/DMEM, introduced into the insert, and allowed to adhere to the upper surface of the membrane. HSCs were then maintained in serum-free DMEM for 24 h. Recombinant rat PDGF-BB (r-PDGF-BB; 20 ng/ml) or r-galectin-1 or r-galectin-3 (10 μg/ml) was successively introduced into the upper chamber. After 48 h of incubation, the culture medium was removed. Cells adhering to the membrane were fixed in 100% methanol and stained with a Giemsa solution. The number of HSCs on the upper surface of the membrane was counted at a magnification of ×400, and that on the lower surface was counted similarly by changing the focus. Five microscopic fields were randomly chosen for each specimen. The proportion of migrated cells (termed migration index) was calculated as follows: migration index (%) = (number of cells on lower surface of membrane)/(number on upper and lower surfaces of membrane) × 100. Statistical Analysis—Data presented as bar graphs are means ± S.D. of three independent experimental series. Statistical analysis was performed by Student's t test at a significance level of p < 0.05. Expression of Galectin-1 and Galectin-3 in Rat Hepatic Constituent Cells and Liver Tissues—HSCs, hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated from normal rat livers. The expression of galectin-1 and galectin-3 mRNAs was determined by RT-PCR (Fig. 1A). Galectin-1 mRNA was not detectable in any of the cells, whereas galectin-3 mRNA was detectable in Kupffer cells. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells underwent apoptosis after a few days of culture as reported previously (44Ni R. Tomita Y. Matsuda K. Ichihara A. Ishimura K. Ogasawara J. Nagata S. Exp. Cell Res. 1994; 215: 332-337Crossref PubMed Scopus (183) Google Scholar, 45Takei Y. Kawano S. Nishimura Y. Goto M. Nagai H. Chen S.S. Omae A. Fusamoto H. Kamada T. Ikeda K. Kawada N. Kaneda K. J. Gastroenterol. Hepatol. 1995; 10: S65-S67Crossref PubMed Scopus (26) Google Scholar). In contrast, HSCs increased in number and morphologically transdifferentiated into myofibroblasts after 4 days of culture. The expression of both galectin-1 and galectin-3 mRNAs was increased in HSCs in a time-dependent manner as revealed by RT-PCR and Northern blot analysis (Fig. 1B). Western blotting revealed that the protein levels of galectin-1 and galectin-3 increased in a time course similar to that for the expression of smooth muscle α-actin and PDGF receptor-β, well characterized markers for activated HSCs. In accordance with these observations on in vitro activated HSCs, in vivo activated HSCs isolated from TAA-induced fibrotic livers expressed mRNAs and proteins for galectin-1 and galectin-3, both of which were undetectable in quiescent HSCs isolated from normal livers (Fig. 1C). Immunohistochemistry showed that galectin-1 was negligible in intact liver tissues (Fig. 1D, panel a), whereas in the septa of TAA-induced fibrotic livers, where activated HSCs were localized, it was prevalent (panel b). Galectin-3 immunoreactivity, which was sporadically positive in Kupffer cells in normal livers (panel c), was augmented around periportal areas and septa in fibrotic livers (panel d). These observations support the results obtained with HSCs and Kupffer cells isolated from normal and fibrotic livers described above. RT-PCR showed that the mRNA levels of both galectin-1 and galectin-3 were up-regulated in two models of liver fibrosis, the model obtained by TAA administration and the model obtained by common bile duct ligation (Fig. 1E). These results conclusively demonstrate that the expression of galectin-1 and galectin-3 is augmented in activated HSCs. Effects of Galectin-1 and Galectin-3 on HSC Proliferation— To clarify the role of galectin-1 and galectin-3 in the process of liver fibrosis, we examined their effects on the proliferation of HSCs and hepatocytes. As depicted in Fig. 2A, both galectin-1 and galectin-3 were found to stimulate DNA synthesis in HSCs in a dose-dependent manner, with the stimulation by the former higher than that by the latter. Their mitogenic activity at 10 μg/ml was less than that at 20 ng/ml PDGF-BB, the most potent mitogen for HSCs (4Pinzani M. Marra F. Carloni V. Liver. 1998; 18: 2-13Crossref PubMed Scopus (231) Google Scholar, 5Marra F. Gentilini A. Pinzani M. Choudhury G.G. Parola M. Herbst H. Dianzani M.U. Laffi G. Abboud H.E. Gentilini P. Gastroenterology. 1997; 112: 1297-1306Abstract Full Text PDF PubMed Scopus (185) Google Scholar). Neither galectin-1 nor galectin-3 affected DNA synthesis in hepatocytes (Fig. 2B). Additionally, the cell number of HSCs treated with either galectin-1 or galectin-3 (10 μg/ml) significantly increased at 2 or 4 days after stimulation compared with that of unstimulated control cells (Fig. 2C, panel a). In fact, microscopic observation showed that the cell density of HSCs increased at 4 days after stimulation with either galectin-1 or galectin-3 (10 μg/ml) compared with the unstimulated control culture of HSCs (panels b–e). The mitogenic activity of both galectins was again less than that of PDGF-BB (20 ng/ml). Intracellular Signaling Pathways in Galectin-stimulated HSCs—To reveal the molecular mechanism underlying the mitogenic activity of galectin-1 and galectin-3 for HSCs, we investigated their effects on the activity of MAPK cascades in HSCs. Both galectin-1 and galectin-3 induced the phosphorylation of ERK1/2 in a time- and dose-dependent manner (Fig. 3A, panel a). No such activation was observed with p38 MAPK, SAPK/JNK, and Akt (Fig. 3B). ERK1/2 was phosphorylated rather gradually, with a maximum intensity at 120 min of galectin (10 μg/ml) treatment. It was interesting that this time course of ERK1/2 phosphorylation was sharply different from that of PDGF-BB-induced phosphorylation, where the initial and maximum level of ERK1/2 phosphorylation was observed as early as 6 min after treatment. Phosphorylation of MEK1/2, an upstream signal of ERK1/2, also occurred later in galectinstimulated HSCs than in PDGF-BB-stimulated HSCs (Fig. 3A, panel b). Effects of MEK1/2 Inhibitors on ERK1/2 Phosphorylation and DNA Synthesis in Galectin-stimulated HSCs—We examined the effects of PD98059 and U0126, MEK1/2 inhibitors, on the galectin-induced appearance of phospho-ERK1/2 in HSCs (Fig. 4A). Both inhibitors attenuated the galectin-induced appearance of phospho-ERK1/2 in a dose-dependent manner. On the other hand, neither SB203580, a p38 MAPK inhibitor, nor LY294002, a PI3K inhibitor, affected this process (Fig. 4B). Thus, it appears that galectin-1 and galectin-3 stimulate HSCs through the phosphorylation of MEK1/2 and ERK1/2. In fact, galectin-induced DNA synthesis in HSCs was abolished by PD98059 or U0126 in a dose-dependent manner (Fig. 4C). Effects of Inhibitors of PKC and PKA on ERK1/2 Phosphorylation in Galectin-stimulated HSCs—To obtain further insight into the mechanism of the galectin-induced activation of MEK1/2 and ERK1/2 in HSCs, the phosphorylation of ERK1/2 in galectin-stimulated HSCs was investigated in the presence of GF109203X, a specific PKC inhibitor, and H-89, a specific PKA inhibitor. The PDGF-BB-induced phosphorylation of ERK1/2 was suppressed by GF109203X, but not by H-89 (Fig. 5A), implying that this activation was PKC (but not PKA)-dependent, as previously reported (4Pinzani M. Marra F. Carloni V. Liver. 1998; 18: 2-13Crossref PubMed Scopus (231) Google Scholar). Interestingly, the phosphorylation of ERK1/2 induced by galectin-1 was not affected by GF109203X or by H-89, whereas that induced by galectin-3 was. These results indicate that galectin-1 and galectin-3 activate different signaling pathways leading to the phosphorylation of ERK1/2. Inhibition of ERK1/2 Phosphorylation and DNA Synthesis by Thiodigalactoside—Thiodigalactoside (TDG) is one of the potent hapten inhibitors of β-galactoside binding (46Bianchet M.A. Ahmed H. Vasta G.R. Amzel L.M. Proteins. 2000; 40: 378-388Crossref PubMed Scopus (75) Google Scholar, 47Levy Y. Arbel-Goren R. Hadari Y.R. Eshhar S. Ronen D. Elhanany E. Geiger B. Zick Y. J. Biol. Chem. 2001; 276: 31285-31295Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Preliminary studies were performed to ensure that TDG has no stimulatory effect on both ERK1/2 activation and DNA synthesis in HSCs. Fig. 6A shows the results of such experiments. In fact, TDG at 10 and 20 mM did not affect either process at all, whereas PDGF-BB at 20 ng/ml stimulated them as expected. Next, galectin-1 and galectin-3 (10 μg/ml each) were pretreated with TDG at 20 mM, and HSCs were successively challenged with the pretreated galectin-1 or galectin-3 in the presence of excess TDG and examined for the phosphorylation of ERK1/2 (Fig. 6B). TDG significantly decreased the potential of galectin-1 or galectin-3 to activate the phosphorylation of ERK1/2 in HSCs. TDG (20 mM) also