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The Antioxidant (–)-Epigallocatechin-3-gallate Inhibits Rat Hepatic Stellate Cell Proliferation in Vitro by Blocking the Tyrosine Phosphorylation and Reducing the Gene Expression of Platelet-derived Growth Factor-β Receptor

2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês

10.1074/jbc.m212042200

1083-351X

Anping Chen, Li Zhang,

Liver Disease Diagnosis and Treatment

During hepatic fibrogenesis, quiescent hepatic stellate cells (HSC) become active and trans-differentiate into myofibroblast-like cells. This process coincides with an increase in cell proliferation, loss of stored vitamin A droplets, and excessive production and deposition of extracellular matrix components. HSC activation is coupled with the sequential expression of cytokine receptors, including platelet-derived growth factor-β receptor (PDGF-βR). Although the underlying mechanisms remain incompletely understood, it is widely accepted that oxidative stress plays critical roles in activation of HSC during hepatic fibrogenesis. We have recently demonstrated that the antioxidant (–)-epigallocatechin gallate (EGCG), a major component in green tea extracts, significantly inhibited the proliferation of passaged HSC. The aim of the present study is to elucidate the underlying mechanisms. Since PDGF is a potent mitogen for HSC and mediates the early proliferative response, it was hypothesized that EGCG might inhibit HSC proliferation by interfering with the PDGF signal transduction. In this report, we demonstrated that EGCG, in two steps, significantly and effectively inhibited the proliferation of primary and passaged HSC. The polyphenolic compound initiated its inhibitory action by rapidly blocking the phosphorylation of tyrosines in PDGF-βR elicited by PDGF in serum. This action was short lived, persisting for a few hours. In addition, this antioxidant inhibited the gene expression of PDGF-βR by blocking the activation of transcription factors activator protein-1 and NF-κB, which were required for the gene transcription. The latter action remained effective for no less than 48 hours. These results provided a novel insight into the mechanisms by which EGCG inhibits HSC growth. The inhibitory effect of the natural antioxidant, its long history of beverage consumption without adverse health effects, and higher potent antioxidant capability make it a good candidate for therapeutic treatment and prevention of hepatic fibrosis. During hepatic fibrogenesis, quiescent hepatic stellate cells (HSC) become active and trans-differentiate into myofibroblast-like cells. This process coincides with an increase in cell proliferation, loss of stored vitamin A droplets, and excessive production and deposition of extracellular matrix components. HSC activation is coupled with the sequential expression of cytokine receptors, including platelet-derived growth factor-β receptor (PDGF-βR). Although the underlying mechanisms remain incompletely understood, it is widely accepted that oxidative stress plays critical roles in activation of HSC during hepatic fibrogenesis. We have recently demonstrated that the antioxidant (–)-epigallocatechin gallate (EGCG), a major component in green tea extracts, significantly inhibited the proliferation of passaged HSC. The aim of the present study is to elucidate the underlying mechanisms. Since PDGF is a potent mitogen for HSC and mediates the early proliferative response, it was hypothesized that EGCG might inhibit HSC proliferation by interfering with the PDGF signal transduction. In this report, we demonstrated that EGCG, in two steps, significantly and effectively inhibited the proliferation of primary and passaged HSC. The polyphenolic compound initiated its inhibitory action by rapidly blocking the phosphorylation of tyrosines in PDGF-βR elicited by PDGF in serum. This action was short lived, persisting for a few hours. In addition, this antioxidant inhibited the gene expression of PDGF-βR by blocking the activation of transcription factors activator protein-1 and NF-κB, which were required for the gene transcription. The latter action remained effective for no less than 48 hours. These results provided a novel insight into the mechanisms by which EGCG inhibits HSC growth. The inhibitory effect of the natural antioxidant, its long history of beverage consumption without adverse health effects, and higher potent antioxidant capability make it a good candidate for therapeutic treatment and prevention of hepatic fibrosis. Hepatic fibrosis occurs as a wound-healing process after many forms of chronic liver injury, including virus infection, autoimmune liver diseases, and sustained alcohol abuse (1Bissell D.M. J. Gastroenterol. 1998; 33: 295-302Google Scholar). Hepatic stellate cells (HSC) 1The abbreviations used are: HSC, hepatic stellate cell(s); AP-1, activator protein-1; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; EGCG, (–)-epigallocatechin-3-gallate; JNK, Jun N-terminal kinase; RPA, RNase protection assay; PDGF, platelet-derived growth factor; PDGF-βR, platelet-derived growth factor-β receptor; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: HSC, hepatic stellate cell(s); AP-1, activator protein-1; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; EGCG, (–)-epigallocatechin-3-gallate; JNK, Jun N-terminal kinase; RPA, RNase protection assay; PDGF, platelet-derived growth factor; PDGF-βR, platelet-derived growth factor-β receptor; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. are the most relevant cell type responsible for the excessive production and deposition of extracellular matrix during the development of hepatic fibrosis. HSC are normally low mitotic ("quiescent") and mainly responsible for the uptake, storage, and delivery of retinoids. Upon liver injury, these cells become active and trans-differentiate into α-smooth muscle actin-positive myofibroblast-like cells. Several key phenotypic changes accompany the process, including an increase in cell proliferation, loss of stored vitamin A droplets, and excessive production and deposition of extracellular matrix components. Culturing quiescent HSC on plastic plates causes spontaneous activation, leading to a myofibroblast-like phenotype, mimicking the process seen in vivo. This provides a simple model for studying mechanisms underlying the activation and trans-differentiation of these cells. Platelet-derived growth factor (PDGF) is a potent mitogen for HSC proliferation. The actions of PDGF and its receptors have been recently reviewed (2Heldin C.H. Westermark B. Physiol. Rev. 1999; 79: 1283-1316Google Scholar). PDGF is composed of two partly homologous chains, A and B, forming three isoforms (AA, AB, and BB). Both the A- and B-chains of PDGF bind to PDGF-α receptor, whereas only the B-chain binds to platelet-derived growth factor-β receptor (PDGF-βR). PDGF-AA, however, is much less potent to HSC than are PDGF-BB and PDGF-AB, suggesting a predominant expression of PDGF-βR (3Pinzani M. Gentilini A. Caligiuri A. De Franco R. Pellegrini G. Milani S. Marra F. Gentilini P. Hepatology. 1995; 21: 232-239Google Scholar, 4Heldin P. Pertoft H. Nordlinder H. Heldin C.H. Laurent T.C. Exp. Cell Res. 1991; 193: 364-369Google Scholar). Dimerization of PDGF-βR by PDGF-BB results in the phosphorylation of tyrosines in the intracellular domain of the receptor, which creates binding sites for association of tyrosine-dependent proteins, such as Shc, Grb2, Src, etc. (5Heldin C.H. Ostman A. Ronnstrand L. Biochim. Biophys. Acta. 1998; 1378: 79-113Google Scholar), followed by activation of signal transduction pathways (6Claesson-Welsh L. J. Biol. Chem. 1994; 269: 32023-32026Google Scholar). The tyrosine phosphorylation is, therefore, essential for activation of a series of intracellular signaling pathways that mediate changes in gene expression, cell migration, and cell proliferation (6Claesson-Welsh L. J. Biol. Chem. 1994; 269: 32023-32026Google Scholar). Studies have indicated that during liver injury, the early proliferative response in HSC was mainly mediated by PDGF (7Kinnman N. Goria O. Wendum D. Gendron M.C. Rey C. Poupon R. Housset C. Lab. Invest. 2001; 81: 1709-1716Google Scholar). The onset of HSC proliferation coincides with the induction of the expression of PDGF-βR, whereas PDGF-α receptor is not changed (8Wong L. Yamasaki G. Johnson R.J. Friedman S.L. J. Clin. Invest. 1994; 94: 1563-1569Google Scholar). Although the underlying mechanisms remain incompletely understood, accumulating evidence has indicated that oxidative stress plays critical roles in the activation of HSC and hepatic fibrogenesis (9Lee K.S. Buck M. Houglum K. Chojkier M. J. Clin. Invest. 1995; 96: 2461-2468Google Scholar, 10Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Google Scholar, 11Fernandez-Checa J.C. Kaplowitz N. Garcia-Ruiz C. Colell A. Semin. Liver Dis. 1998; 18: 389-401Google Scholar). Oxidative stress is formed by excessive production of reactive oxygen species, including hydrogen peroxide (H2O2), which are generated endogenously by all aerobic cells as by-products of a number of metabolic reactions (12Fridovich I. Science. 1978; 201: 875-880Google Scholar). Oxidative stress has been implicated in stimulating HSC entry into S-phase, NF-κB activation, and gene expression (9Lee K.S. Buck M. Houglum K. Chojkier M. J. Clin. Invest. 1995; 96: 2461-2468Google Scholar). The antioxidant vitamin E shows its role in inhibiting the activation of HSC (9Lee K.S. Buck M. Houglum K. Chojkier M. J. Clin. Invest. 1995; 96: 2461-2468Google Scholar) as well as in preventing iron-induced hepatic fibrogenesis (13Pietrangelo A. Gualdi R. Casalgrandi G. Montosi G. Ventura E. J. Clin. Invest. 1995; 95: 1824-1831Google Scholar). However, the therapeutic efficacy of current well known antioxidants, including superoxide dismutase, vitamin E, and ascorbic acid, in treatment of human hepatic fibrosis is generally unimpressive (14Halliwell B. Drugs. 1991; 42: 569-605Google Scholar). Natural antioxidants, such as polyphenols from green tea extracts, have recently attracted considerable attention for prevention of oxidative stress-related diseases including cancers, cardiovascular diseases, and degenerative diseases (15Yang C.S. Landau J.M. J. Nutr. 2000; 130: 2409-2412Google Scholar, 16Trevisanato S.I. Kim Y.I. Nutr. Rev. 2000; 58: 1-10Google Scholar). Green tea is the most consumed beverage in the world (17Mukhtar H. Ahmad N. Am. J. Clin. Nutr. 2000; 71 (discussion 1703S–1694S): 1698S-1702SGoogle Scholar, 18Katiyar S.K. Mukhtar H. World Rev. Nutr. Diet. 1996; 79: 154-184Google Scholar). Of the polyphenols purified from green tea, (–)-epigallocatechin gallate (EGCG) is a major constituent and the most potent antioxidant (19Ahmad N. Mukhtar H. Nutr Rev. 1999; 57: 78-83Google Scholar). The antioxidant potential of EGCG is far greater than that of vitamin E and/or C (20Rice-Evans C. Proc. Soc. Exp. Biol. Med. 1999; 220: 262-266Google Scholar). Accumulating evidence has demonstrated that EGCG results in a dose-based differential inhibition of tumor necrosis factor-α- and lipopolysaccharide-mediated activation of NF-κB in cancer cells but not in normal cells (21Ahmad N. Gupta S. Mukhtar H. Arch. Biochem. Biophys. 2000; 376: 338-346Google Scholar, 22Lin Y.L. Lin J.K. Mol. Pharmacol. 1997; 52: 465-472Google Scholar). NF-κB inhibition by EGCG results in cell cycle deregulation and apoptosis (21Ahmad N. Gupta S. Mukhtar H. Arch. Biochem. Biophys. 2000; 376: 338-346Google Scholar, 22Lin Y.L. Lin J.K. Mol. Pharmacol. 1997; 52: 465-472Google Scholar). Effects of the antioxidant EGCG on HSC growth and activation have, however, not been well studied. We recently observed that this phytoantioxidant significantly inhibited cell growth of activated HSC in vitro by inducing cell cycle arrest and apoptosis (23Chen A. Zhang L. Xu J. Tang J. Biochem. J. 2002; 368: 695-704Google Scholar). The present studies were mainly aimed at elucidating molecular mechanisms by which EGCG inhibited HSC proliferation. We examined the effect of EGCG on activated HSC proliferation in vitro, evaluated the roles of EGCG in blocking the PDGF-elicited tyrosine phosphorylation in PDGF-βR and in inhibiting the PDGF-βR gene expression, and elucidated molecular mechanisms underlying regulation of the gene expression. Results in this paper supported our initial hypothesis that EGCG might inhibit HSC proliferation by interfering with PDGF signaling. These actions, together with others, contribute to the effect of EGCG on inhibition of HSC growth. Stellate Cell Isolation and Culture—HSC were isolated from male Sprague-Dawley rats (less than 300 g) as previously described (24Chen A. Davis B.H. J. Biol. Chem. 1999; 274: 158-164Google Scholar, 25Chen A. Davis B.H. Mol. Cell. Biol. 2000; 20: 2818-2826Google Scholar). Primary cells were incubated in Dulbecco's modified eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Passaged cells were maintained in DMEM supplemented with 10% FBS. Without a specific indication, activated HSC were used at the age of passage 4–8 in experiments. For some experiments, cells were serum-starved for 48 h in DMEM with 0.4% FBS prior to the addition of 10% FBS and/or EGCG (99.5% purity; Sigma) for the indicated times. Pyrrolidine dithiocarbamate, an inhibitor of NF-κB (26Tsai S.H. Liang Y.C. Chen L. Ho F.M. Hsieh M.S. Lin J.K. J. Cell. Biochem. 2002; 84: 750-758Google Scholar), was purchased from Sigma. The process of serum starvation of HSC is generally considered to render them "quiescent" (27Galli A. Crabb D. Price D. Ceni E. Salzano R. Surrenti C. Casini A. Hepatology. 2000; 31: 101-108Google Scholar, 28Svegliati-Baroni G. Ridolfi F. Di Sario A. Saccomanno S. Bendia E. Benedetti A. Greenwel P. Hepatology. 2001; 33: 1130-1140Google Scholar). Determination of Cell Growth—Quiescent HSC were isolated from normal rats and incubated in DMEM containing 20% FBS. After a 24-h recovery, cells were treated with or without EGCG at the indicated concentrations for an additional 6 days. Cell growth was determined by attached cell numbers counted by a cell counter (Coulter Corp., Miami, FL). Cell proliferation was analyzed by [3H]thymidine incorporation assays, as described previously (23Chen A. Zhang L. Xu J. Tang J. Biochem. J. 2002; 368: 695-704Google Scholar). 125I-PDGF-BB Binding Assay—After a 24-h recovery, freshly isolated quiescent HSC were treated with or without EGCG at the indicated concentrations for an additional 6 days. Cells were washed once with binding buffer (25 mm HEPES, pH 7.4, 125 mm NaCl, 5 mm KCl, 5 mm MgSO4, and 1 mm CaCl2). Cells (104) were subsequently incubated with 125I-PDGF-BB (Amersham Biosciences) (1–50 ng/ml) in the binding buffer containing 0.2% bovine serum albumin at 4 °C for 4 h in the absence or presence of a 50-fold excess of unlabeled PDGF-BB (Calbiochem) to measure total or nonspecific binding, respectively. Cells were then washed at least three times with the binding buffer and lysed with 1% Triton X-100, 0.1% bovine serum albumin, and 0.1 m NaOH. The radioactivity of the aliquots of soluble fraction was measured by a γ-counter. The difference in radioactivity between cells incubated with (i.e. nonspecific binding) and without (i.e. total binding) excessive unlabeled PDGF-BB was considered as specific binding. Scatchard analysis of 125-I-PDGF-BB-specific binding data was used to evaluate the effect of EGCG on the affinity of receptors to PDGF-BB (29Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-671Google Scholar). Transient Transfection—Sixty-eighty percent confluent HSC in six-well plastic plates were transiently transfected using the LipofectAMINE reagent (Invitrogen). Each treatment or sample (3 μg of DNA/well) was run in triplicate for every experiment. Experiments were repeated at least three times (n ≥ 3). Luciferase assays were performed as previously described (25Chen A. Davis B.H. Mol. Cell. Biol. 2000; 20: 2818-2826Google Scholar, 30Chen A. Beno D.W.A. Davis B.H. J. Biol. Chem. 1996; 271: 25994-25998Google Scholar). Transfection efficiency was determined by co-transfection of a β-galactosidase reporter, pSV-β-gal (0.5 μg of DNA/well) (Promega). Plasmid Constructs—The PDGF-βR reporter plasmid pβ12 was a gift from Dr. Keiko Funa (Ludwig Institute for Cancer Research, Uppsala, Sweden). In pβ12, a fragment, containing 1366 base pair nucleotides upstream from the start codon of the mouse PDGF-βR gene, was subcloned into a luciferase expression vector pGL2 (basic) (31Ballagi A.E. Ishizaki A. Nehlin J.O. Funa K. Biochem. Biophys. Res. Commun. 1995; 210: 165-173Google Scholar). Other PDGF reporter plasmids with various sizes of the gene promoter region were derived from the parental pβ12 generated by restriction enzyme digestion as indicated or by PCR. The expression plasmids of dominant negative JNK, the dominant negative c-Jun, and the AP-1 reporter plasmid were previously described (24Chen A. Davis B.H. J. Biol. Chem. 1999; 274: 158-164Google Scholar, 25Chen A. Davis B.H. Mol. Cell. Biol. 2000; 20: 2818-2826Google Scholar). Plasmids with site-directed mutations in either the AP-1 site or the NF-κB site or in both sites were derived from the plasmid pβ12/T+E, by using the GeneEditor™ in vitro site-directed mutagenesis system (Promega). The two bases CA at –693 and –692 in the AP-1 binding site were mutated to TG, whereas the other two bases GG at –751 and –750 in the NF-κB binding site were mutated to AA. Site-directed mutations were confirmed by DNA sequencing. RNA Isolation and RNase Protection Assay (RPA)—Total RNA was isolated by the TRI-Reagent, following the protocol recommended by the manufacturer (Sigma). PDGF-βR cDNA was a 606-bp PstI fragment of the extracellular domain, kindly provided by Dr. Dan Bowen-Pope (University of Washington). The 115 bp of 28 S rRNA probe was used as an internal control (Ambion, Austin, TX). The antisense probes were synthesized and 32P-labeled by MAXIscript™ (Ambion). RPA was carried out with RPA II™ kits (Ambion) following the protocol provided by the manufacturer. The radioactivity in each band was measured by a PhosphorImager (Amersham Biosciences), as described previously (24Chen A. Davis B.H. J. Biol. Chem. 1999; 274: 158-164Google Scholar, 25Chen A. Davis B.H. Mol. Cell. Biol. 2000; 20: 2818-2826Google Scholar). Real Time PCR—Real-time PCR was performed as previously described (23Chen A. Zhang L. Xu J. Tang J. Biochem. J. 2002; 368: 695-704Google Scholar). No genomic DNA contamination or pseudogenes were detected by PCR without the reverse transcription step in the total RNA used. GAPDH was used as an internal control. Primers used in the real time PCR were as follows: PDGF-βR (F), 5′-CTG CCA CAG CAT GAT GAG GAT TGA T-3′ (bp 1783–1806); PDGF-βR (R), 5′-GCC AGG ATG GCT GAG ATC ACC AC (bp 1733–1755) (32Herren B. Weyer K.A. Rouge M. Lotscher P. Pech M. Biochim. Biophys. Acta. 1993; 1173: 294-302Google Scholar); GAPDH (F), 5′-GGC AAA TTC AAC GGC ACA GT-3′; GAPDH (R), 5′-AGA TGG TGA TGG GCT TCC C-3′. Electrophoretic Mobility Shift Assay (EMSA)—EMSA was performed as previously described (24Chen A. Davis B.H. J. Biol. Chem. 1999; 274: 158-164Google Scholar). The integrity of nuclear extracts was tested by EMSA with a 32P-labeled SP-1 consensus probe, resulting in distinct SP-1 shifts from all extracts (data not shown). The following double-stranded oligonucleotides, containing binding sites interested in the PDGF-βR promoter, were synthesized by Invitrogen and used as probes in EMSA: 1) AP-1 probe (–706 to –684), 5′-ATA AAA GTG ACTCAG TGG CTG G-3′;2)NF-κB probe (–759 to –736), 5′-TGG TAA AGGGAG GCT CCA TTT ACA-3′. Immunoprecipitation—Passaged HSC, treated with or without EGCG at the indicated concentrations, were lysed in radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris, 0.1% SDS, 1% Nonidet P-40, and 0.5% sodium deoxycholate, 1 mm iodoacetamide (freshly prepared), 1 mm phenylmethylsulfonyl fluoride, 0.2 units of aprotinin). Protein concentration was determined by Bio-Rad protein assays. The same amount of proteins (50 μg) in each sample was incubated at 4 °C with Protein G-Sepharose beads (Amersham Biosciences) (one-tenth volume) for 1 h to eliminate nonspecific binding. The protein samples were incubated with a 1:100 dilution of rabbit anti-PDGF-βR polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight. Protein G-Sepharose (one-tenth volume) was then added and incubated for 1 h at 4 °C. After five washes with a cold buffer (20 mm Tris-HCl, pH 7.4, 500 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.2% SDS), the beads were resuspended in 20 μl of 2× SDS sample buffer containing dithiothreitol. Samples were boiled for 3 min and ready for Western blotting analyses. Western Blotting Analyses—Using standard techniques, SDS-PAGE with 10% resolving gel was used to separate proteins, which were subsequently detected by using anti-PDGF-βR or anti-phosphotyrosine antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-phosphorylated JNK1/2 (Promega), or anti-JNK1/2 antibodies (Santa Cruz Biotechnology). The first antibodies were recognized by horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology). Protein bands were visualized by utilizing chemiluminescence reagent (Kirkegaard & Perry Laboratories). Statistical Analysis—Differences between means were evaluated using an unpaired two-sided Student's t test (p < 0.05 considered as significant). Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by analysis of variance with the Dunnett's test for post hoc analysis. EGCG Inhibits Primary HSC Proliferation in Vitro—We have previously demonstrated that EGCG significantly inhibited the proliferation of passaged HSC (23Chen A. Zhang L. Xu J. Tang J. Biochem. J. 2002; 368: 695-704Google Scholar). To further evaluate the effect of EGCG on primary HSC growth, HSC were isolated from normal rat livers. After recovery in DMEM with 20% FBS for 24 h, the same number of cells were incubated in the medium with or without EGCG at the indicated concentrations for an additional 6 days. Cell growth was determined by attached cell numbers. As shown in Fig. 1A, compared with the control, EGCG at 20, 50, and 100 μm caused a dose-dependent reduction in cell numbers by 16.6 ± 3.6, 65.1 ± 6.9, and 75 ± 9.8%, respectively (Fig. 1A). Further studies demonstrated that EGCG, in a dose-dependent manner, reduced the incorporation of [3H]thymidine into primary HSC chromosomal DNA by 11.4 ± 5.2, 67.1 ± 8.6, or 71.4 ± 6.2% at 20, 50, or 100 μm, respectively (Fig. 1B), suggesting the EGCG inhibitory effect on primary HSC proliferation. PDGF is a major and potent mitogen for HSC proliferation in vivo and in vitro. Further experiments were performed to determine the effect of PDGF in serum on stimulating cell proliferation. As shown in Fig. 1B, compared with the control, the addition of neutralizing anti-PDGF antibodies to the medium resulted in a significant reduction in the incorporation of [3H]thymidine into primary HSC DNA, indicating a critical role of PDGF in the 20% FBS medium in primary HSC proliferation. Taken together, our current and previous studies demonstrated that EGCG inhibited both primary and passaged HSC proliferation, in which PDGF in serum played a critical role. These results prompted us to hypothesize that EGCG might block PDGF roles by interfering with PDGF signaling in culture-activated HSC. EGCG Rapidly Blocks the PDGF-initiated Phosphorylation of Tyrosines in PDGF-βR—To test the hypothesis, studies were first focused on evaluating EGCG effects on phosphorylation of tyrosines in PDGF-βR elicited by PDGF in serum. To reduce phosphorylated tyrosines in PDGF-βR, passaged HSC were serum-starved in serum-free DMEM for 3 h. Some of the cells were stimulated with exogenous PDGF-BB (20 ng/ml) for1has a positive control. The others were stimulated with 10% FBS, with or without neutralizing anti-PDGF antibodies, in the presence or absence of EGCG either at different concentrations for a fixed time (1 h) (Fig. 2A) or at a fixed dose (50 μm) for different times (Fig. 2B). PDGF-βR was subsequently immunoprecipitated from cell lysates and then analyzed for phosphorylated tyrosines or for associated Src by Western blotting analyses (Fig. 2). As shown in Fig. 2A, serum (lane 2) as well as PDGF-BB (lane 6), elicited the phosphorylation of tyrosines in PDGF-βR, which was blocked by EGCG in a dose-dependent pattern (lanes 3–5). Depleting PDGF from the medium by neutralizing anti-PDGF antibodies (30 μg/ml) led to a marked reduction in the tyrosine phosphorylation (lane 1), suggesting that PDGF in serum was the major factor eliciting the tyrosine phosphorylation. Experiments of co-immunoprecipitation demonstrated that blocking the tyrosine phosphorylation by EGCG resulted in disassociation of tyrosine-dependent proteins, such as Src, with PDGF-βR in activated HSC (Fig. 2A). Additional studies indicated that EGCG at 50 μm rapidly exerted its effect on inhibition of the tyrosine phosphorylation in less than 0.5 h (Fig. 2B, lane 2). This inhibitory effect gradually diminished in 5 h (lanes 2–5), suggesting that the inhibitory effect of EGCG on the tyrosine phosphorylation in PDGF-βR was instant and short lived. Cells treated with 10% FBS (serum) (lane 1) or PDGF at 20 ng/ml (lane 6) for 1 h was used as a positive control. These results collectively indicated that EGCG effectively and rapidly blocked the phosphorylation of tyrosines in PDGF-βR elicited by PDGF in serum and instantly interfered with PDGF signaling in culture-activated HSC. EGCG Inhibits the Expression of PDGF-βR in Both Primary and Passaged HSC—As demonstrated earlier, the effect of EGCG on blocking the phosphorylation of tyrosines in PDGF-βR was only sustained for less than 5 h (Fig. 2B), whereas the effect of EGCG on inhibiting cell proliferation lasted for days in primary HSC (Fig. 1A) and in passaged HSC (23Chen A. Zhang L. Xu J. Tang J. Biochem. J. 2002; 368: 695-704Google Scholar). It was therefore assumed that in addition to the rapid, short life effect, EGCG should have additional, long life effects on inhibiting HSC proliferation, including reducing the gene expression of PDGF-βR. Primary HSC were treated with or without EGCG at the indicated concentrations starting at day 2 until day 7. Cells (104) were subsequently incubated with 125I-PDGF-BB (1–50 ng/ml) at 4 °C for 4 h in the absence or presence of a 50-fold excess of unlabeled PDGF-BB to measure total or nonspecific binding, respectively. Specific binding curves were obtained by subtraction of nonspecific binding from total binding (Fig. 3A). The results indicated that EGCG caused a dose-dependent reduction in specific binding of 125I-PDGF-BB to primary HSC. Scatchard analyses of these data indicated that EGCG did not alter the binding affinity of receptor(s) to PDGF-BB (data not shown). These results collectively suggested that EGCG might reduce the level of membrane PDGF-βR in primary cells. RPA were used for further analyses of EGCG effects on PDGF-βR mRNA in primary HSC (Fig. 3B). After recovery for 24 h, freshly isolated primary HSC were incubated for another 3 days. A part of the cells were then harvested (day 4). The rest of the cells started the treatment with or without EGCG at the indicated concentrations for an additional 3 days (day 7). As shown in Fig. 3B by RPA, compared with cells aged at day 4 (Day 4), primary HSC aged at day 7 (Day 7) acquired a higher level of PDGF-βR mRNA, which was inhibited by EGCG in a dose-dependent manner. 28 S rRNA was used as an internal control for equal loading. Similar results were obtained from serum-starved passaged HSC (Fig. 4). The process of serum starvation of HSC is generally considered to render them "quiescent" (27Galli A. Crabb D. Price D. Ceni E. Salzano R. Surrenti C. Casini A. Hepatology. 2000; 31: 101-108Google Scholar, 28Svegliati-Baroni G. Ridolfi F. Di Sario A. Saccomanno S. Bendia E. Benedetti A. Greenwel P. Hepatology. 2001; 33: 1130-1140Google Scholar). Serum stimulation significantly increased the abundance of steady state levels of PDGF-βR mRNA demonstrated by real time PCR (Fig. 4, A and B). EGCG, in dose-dependent (Fig. 4A) and time-dependent manners (Fig. 4B), reduced the levels of PDGF-βR mRNA in passaged HSC. Further immunoprecipitation and immunoblotting analyses confirmed the EGCG-inhibitory effect on the protein expression of PDGF-βR in activated passaged HSC (Fig. 4C). Taken together, these results demonstrated that EGCG significantly inhibited the serum-induced expression of PDGF-βR in both primary and passaged HSC in dose- and time-dependent manners, which lasted for no less than 48 h.Fig. 4EGCG inhibits the expression of PDGF-βR in passaged HSC. Passaged HSC were incubated in serum-depleted medium (0.4% FBS) for 48 h prior to the addition of 10% FBS in the presence or absence of EGCG at the indicated doses for the indicated times. Total RNA was isolated from these cells for real time PCR. The mRNA -fold change was calculated by using GAPDH as an internal control. Values were means ± S.D. (n = 6). †, p < 0.05 versus cells without serum (–), without EGCG (–); *, p < 0.05, versus cells with serum (+), without EGCG (–). A, real time PCR of PDGF-βR mRNA in cells treated with EGCG at different concentrations for a fixed time (24 h); B, real time PCR of PDGF-βR mRNA in cells in the presence or absence of EGCG at a fixed concentration (50 μm) for different times; C, passaged HSC grown in medium with 10% FBS were treated with or without EGCG at 50 μm for the indicated times. A representative of three independent immunoprecipitations and Western blots of PDGF-βR protein is shown here.View Large Image Figure ViewerDownload (PPT) EGCG Reduces the mRNA Stability and the Gene Promoter Activity of PDGF-βR—To begin exploring the mechanisms by which EGCG inhibited the expression of PDGF-βR in HSC, we evaluated the effect of EGCG on PDGF-βR mRNA stability and on the gene promoter activity of the receptor. Serum-starved HSC were stimulated with 10%