Down-regulation of endothelin receptors by transforming growth factor beta1 in hepatic stellate cells

BACKGROUND/AIMS: Hepatic endothelin-1 (ET-1) receptor density as well as the levels of both ET-1 and transforming growth factor beta1 (TGF-beta1) increase in liver cirrhosis. Considering their potent contractile (ET-1) and fibrogenic (TGF-beta1) actions on myofibroblastic stellate cells found in the fibrotic/cirrhotic liver, we aimed to investigate the effects of TGF-beta1 on ET-1 receptors and ET-1 synthesis in these cells. METHODS: Stellate cells isolated from rat liver by enzymatic digestion were cultured and subjected to TGF-beta1 treatment. Cellular ET-1 receptors and ET-1 released in the medium were determined. RESULTS: TGF-beta1 treatment produced time- and dose-dependent decrease in ET-1 binding sites, but did not affect the affinity of the receptors for ET-1. TGF-beta1 also stimulated the release of ET-1 from stellate cells. The extent of TGF-beta1-induced inhibition of [125I]ET-1 binding was much greater for ETB subtype (73+/-18% inhibition), which comprised a major portion (78+/-12%) of the total ET-1 receptors, than for ETA subtype (35+/-11% inhibition). The mRNA expression of the ET-1 receptors also was reduced by TGF-beta1 treatment. TGF-beta1-induced reduction in ET-1 receptor density was coupled to the inhibition of ET-1-stimulated release of [3Hlarachidonic acid from the prelabeled cells. The effects of TGF-beta1 were inhibited by a TGF-beta1 neutralizing monoclonal antibody. CONCLUSIONS: These results suggest that the TGF-beta1-induced decrease in ET-1 receptor density may be an important mechanism in limiting the pathologic actions of ET-1 on stellate cells in chronic liver disease.     

转化生长因子β1对大鼠肝星状细胞表达β-连环蛋白的影响

目的研究转化生长因子β1（TGF—β1）对大鼠肝星状细胞（HSC）表达β-连环蛋白（β-catenin）的影响。方法采用逆转录聚合酶链反应（RT—PCR）比较经不同浓度的TGF-β1刺激不同时间后HSC表达β-catenin、smad3和α-SMA mRNA的变化,并比较三者之间的关系。结果1ng／ml TGF—β1刺激2h后,HSC表达β—catenin mRNA、smad3 mRNA和α—SMA mRNA量最大,β—catenin mRNA表达与两者均具有明显的相关关系（r=0．947,P〈0．01;r=0．950,P〈0．01）。结论β—catenin在TGF—β1活化HSC的过程中发挥重要作用。     

DNase I-hypersensitive sites enhance alpha1(I) collagen gene expression in hepatic stellate cells

Liver fibrosis is characterized by a dramatic increase in the expression of type I collagen. Several deoxyribonuclease (DNase) I-hypersensitive sites (HS) have been located in the distal 5'-flanking region of the alpha1(I) collagen gene that are specific to collagen-producing cells. To assess the role of the DNase I-HS in regulating alpha1(I) collagen gene expression in hepatic stellate cells (HSCs), 3 transgenic mouse lines expressing collagen-alpha1(I) reporter genes were used (Krempen et al. Gene Expr 1999;8:151-163). The pCol9GFP transgene contains the collagen gene promoter (-3122 to +111) linked to the green fluorescent protein (GFP) reporter gene. The pCol9GFP-HS4,5 transgene contains HS4,5 and pColGFP-HS8,9 contains HS8,9 positioned upstream of the collagen promoter in pCol9GFP. HSCs isolated from transgenic mice containing pCol9GFPHS4,5 and pColGFP-HS8,9 showed earlier and higher GFP expression patterns than HSCs isolated from pCol9GFP mice. HSCs from pCol9GFP-HS4,5 showed the highest levels of GFP expression and culture-induced expression correlated with induction of the endogenous alpha1(I) collagen gene. After CCl(4) administration, pCol9GFP-HS4,5 mice showed increased GFP expression compared with pCol9GFP mice in both whole liver extracts and isolated HSCs. Several sites for DNA-protein interactions in both HS4 and HS5 were identified that included a binding site for activator protein 1. In conclusion, DNase I-HS4,5 enhance expression of the alpha1(I) collagen gene promoter in HSCs both in vitro and in vivo after a fibrogenic stimulus. The collagen-GFP transgenic mice provide a convenient and reliable model system to investigate the molecular mechanisms controlling increased collagen expression during fibrosis.     

干扰素α对Ⅰ型胶原及转化生长因子β1基因表达的影响

目的 观察干扰素α(IFN α)对血小板衍生生长因子-BB(PDGF-BB)刺激的大鼠肝星状细胞(HSC)分泌Ⅰ型胶原及转化生长因子β1(TGF β1)基因表达的影响,探讨IFN α抗肝纤维化的可能机制.方法 体外培养大鼠HSC系rHSC-99,分别用0、0.0125、0.0250、0.0500,0.1000、0.2000,0.4000 ng/ml IFN α,PDGF-BB干预和两者共同干预,用四甲基偶氮唑盐实验观察各组对HSC细胞活力的影响,采用逆转录聚合酶链反应方法测定各组对HSC细胞Ⅰ型胶原mRNA和TGF β1 mRNA表达的影响.结果 (1)HSC细胞活力(A值)PDGF-BB干预组为1.35±0.22,空白对照组为0.89±0.12,两组比较,F=16.311,P＜0.05,差异有统计学意义,说明PDGF-BB可提高HSC细胞活力.0.025,0.050、0.100、0.2000,0.400ng/ml IFN α加PDGF-BB共干预组,A值分别为0.84±0.18.0.45±0.15、0.26±0.01、0.33±0.07,0.30±0.06,较空白对照组明显降低,F=7.430,P＜0.05,差异有统计学意义,说明IFN α与PDGF-BB共同作用可抑制HSC细胞活力,且在0.025-0.100 ng/ml范围内随着IFN α浓度的增加其抑制作用越明显.(2)0.050、0.100、0.200ng/ml IFN α加PDGF-BB共干预各组Ⅰ型胶原mRNA相对表达值分别为0.94±0.19、0.61±0.12,0.52±0.02,空白对照组为1.41±0.01,共干预各组比空白对照组均明显降低,F=127.921,P＜0.05,差异有统计学意义.0.050、0.100,0.200ng/mlIFN α加PDGF-BB共干预组各组TGFβ1 mRNA相对表达值分别为1.18±0.06、1.15±0.10、1.39±0.04,空白对照组为1.62±0.12,共干预各组比空白对照组均明显降低,F=82.115,P＜0.05,差异有统计学意义,说明IFN α与PDGF-BB共同作用对HSC细胞Ⅰ型胶原、TGFβ1基因的表达有抑制作用,且随着浓度的增加其抑制作用越明显.结论 IFN α对PDGF-BB诱导的HSC细胞活力及Ⅰ型胶原、TGF β1基因的表达有抑制作用,且随着浓度的增加其抑制作用越明显.这可能是IFN α发挥抗肝纤维化作用的途径之一.     

肝素对大鼠肝星状细胞中转化生长因子β1启动活性的影响

在影响转化生长因子(TGF β1)合成的化学物质和药物中,葡萄糖和低分子量肝素可以在肾脏系膜细胞中促进或降低TGF β1 mRNA和蛋白的合成,而且这些作用是通过影响TGF β1基因的启动子活性而发挥的。拟通过检测报告基因活性探讨葡萄糖和肝素对TGF β1启动子的影响。     

大鼠肝星状细胞转化生长因子β3/β1 mRNA比值与Ⅰ型胶原合成的关系

转化生长因子β1(TGF β 1)是激活肝星状细胞(HSC)并促进其胶原合成的最重要因子.TGF β 3被认为具有抗组织纤维化的功能.Carrington等[1]研究提示:TGF β 1与TGF β 3的比值是纤维化发生程度的关键因素.本实验研究大鼠HSC中TGF β 3/TGF β 1 mRNA比值的变化及与Ⅰ型胶原合成的关系,阐明TGF β 3是拮抗TGF β 1的重要因子,为TGF β 3对肝纤维化的防治作用提供实验依据.     

PD98059 inhibits expression of pERK1 protein and collagen alpha1(I) mRNA in rat pancreatic stellate cells activated by platelet-derived growth factor.

OBJECTIVE: Pancreatic stellate cells (PSC) are considered as the principal effector cells in pancreatic fibrosis. We studied the role of platelet-derived growth factor (PDGF) in the activation of PSC. METHODS: Cultured rat PSC were co-incubated with PDGF-BB (25 ng/mL) and different doses (0-40 ng/mL) of PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK). Expressions of p ERK1 protein and of collagen a1(I) mRNA were measured. RESULTS: Expression of p ERK1 protein was up-regulated by PDGF-BB, and was down-regulated in a dose-dependent manner by PD98059. Expression of collagen a1(I) mRNA also showed an increase with PDGF-BB and non-dose-dependent inhibition by PD98059. CONCLUSION: Our findings suggest that PSC activation is mediated by PDGF signal pathway, and ERK1 protein plays an important role in this activation.     

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.     

转化生长因子β1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

目的 观察不同活化状态肝星状细胞(HSC)对外源性转化生长因子-β_1(TGF-β_1)旁分泌刺激的生物学效应作用。方法 原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500 pmol/L TGF-β_1温育细胞24h,~3H—TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原蛋白表达沉积,~3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量。100pmol/L TGF-β_1温育细胞15～90min,northern blot法检测细胞Ⅰ型前胶原mRNA的表达水平。结果 TGF-β_1浓度依赖性抑制培养1d HSC的细胞增殖,10～500 pmol/L TGF-β_1浓度组细胞内~3H—TdR掺入率分别为对照组的52.8％～16.8％,与对照组比较,q值为5.44～10.37,P<0.01。但TGF-β_1对培养4d与7d的细胞增殖无影响。随细胞活化,HSC基础性α-SMA、Ⅰ型胶原蛋白与mRNA水平明显增加,而TGF-β_1刺激各培养时间HSC以上蛋白与基因的表达。培养1、4、7d HSC基础水平与TGF-β_1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1 200±708)dpm/孔;(2 966±1 701)dpm/孔与(6 160±1 123)dpm/孔;(2 580±767)dpm/孔与(4 583±1 467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05。以培养4d HSC     

蛋白激酶C及转化生长因子β1信号通路对肝星状细胞激活的影响

目的探讨蛋白激酶C（PKC）活性改变对HSC表达TGF β1的影响及在HSC激活中的作用。方法将肝星状细胞系rHSC-99分为3组：对照组（A组），PKC激动剂佛波酯0．5μmol／L组（B组），PKC抑制剂Calphostin C 100nmol／L组（C组）。加药后0、3、6、12h和24h分别检测各组细胞PKC活性的变化；作用24h后，采用Western blot和RT—PCR方法检测各组细胞TGF β1，Smad 4，Ⅰ、Ⅲ型胶原和α-平滑肌肌动蛋白的表达；采用MTT法检测细胞的增殖情况。结果 佛波酯作用后PKC的活性显著增强，而Calphostin C则抑制PKC的活性。PKC活性增强后，与对照组相比TGF β1及其下游信号分子Smad 4的表达分别升高了4．8倍和13．1倍（P〈0．01）；HSC的Ⅰ、Ⅲ型胶原和α-平滑肌肌动蛋白的表达分别升高了2．4倍、1．8倍和1．3倍（P〈0．01），并促进HSC的增殖；PKC活性被抑制后则能抑制以上作用。结论PKC活性的改变能调控HSC中TGF β1的表达，在HSC的激活中发挥调节作用。     

The dependence of transforming growth factor-beta-induced collagen production on autocrine factor activin A in hepatic stellate cells

The present study was conducted to examine the role of activin A in the activation of cultured rat hepatic stellate cells (HSC). HSC expressed mRNA for the beta(A)-subunit of activin and the type I and II activin receptors. TGF-beta increased the mRNA expression of the beta(A)-subunit of activin as well as the release of the beta(A) dimer, activin A. Exogenous activin A activated HSC and increased the expression of alpha-smooth muscle actin and collagen. Exogenous follistatin, an antagonist of activin A, blocked not only the effect of activin A but also the effect of TGF-beta on the expression of type I collagen. Similarly, follistatin inhibited TGF-beta-induced secretion of collagen from HSC. Additionally, the effect of TGF-beta was markedly reduced in HSC overexpressing the dominant-negative type II activin receptor. In contrast, the effect of activin A on the collagen production was not affected in HSC overexpressing the dominant-negative type II TGF-beta receptor. In conclusion, an autocrine factor activin A mediates part of the action of TGF-beta on the production of collagen in HSC. The results also suggest that follistatin may be useful for the treatment of hepatic fibrosis.     

Modulation of transforming growth factor beta function in hepatocytes and hepatic stellate cells in rat liver injury

BACKGROUND: Transforming growth factor beta (TGF-beta) regulates hepatocyte proliferation and biosynthesis of the extracellular matrix. AIMS: This study investigated alternations in sensitivity to TGF-beta1 and binding properties for ligand in hepatocytes and hepatic stellate cells (HSC) after CCl(4) administration. METHODS: Plasma TGF-beta1 levels in rats after CCl(4) administration were determined using ELISA. Effects of TGF-beta1 were examined by DNA synthesis in hepatocytes and by measurement of fibronectin production in HSC after CCl(4) administration. Binding of (125)I TGF-beta1 was tested in these cells. RESULTS: Plasma TGF-beta1 levels were increased as early as 24 hours and were maximal by 48 hours. The antiproliferative response to TGF-beta1 decreased in hepatocytes at 48 hours and normalised at 72 hours. Fibronectin production of both normal and injured HSC was affected by TGF-beta1 treatment. Cross linked ligand/receptor complexes were detected in normal hepatocytes and HSC. However, these levels decreased specifically in hepatocytes at 48 hours and normalised by 72 hours. CONCLUSIONS: Downregulation of TGF-beta receptor occurred in hepatocytes after chemical insult and TGF-beta1 could not transduce its antiproliferative signal. Recovery of TGF-beta receptor expression causes the signal to transduce to the nucleus at 72 hours. In HSC, whenever TGF-beta1 is increased, TGF-beta1 can transduce its signal for fibronectin production via its receptor because signalling receptors are expressed constantly.     

RGDS肽对TGFβ刺激肝星状细胞分泌细胞外基质的影响

探讨RGDS肽体外对TGFβ1刺激肝星状细胞分泌细胞外基质的影响。从正常大鼠肝脏中分离肝星状细胞原代培养,分6组,单纯对照组,TGFβ1对照组,RGDS组,RGDS＋TGFβ1组、RGES＋TGFβ1组及INFα-2b TGFβ1组。应用酶联免疫吸附测定方法检测各组培养上清中的Ⅲ型胶原。RGDS＋TGFβ1组和IFN＋TGFβ1组Ⅲ型胶原水平明显低于TGFβ1对照组RGES对照组（P＜0．05）。RGDS可抑制TGFβ1促进肝星状细胞合成Ⅲ型胶原的作用。     

重组转化生长因子β3基因对大鼠肝星状细胞Ⅰ型胶原表达的影响

目的观察转化生长因子D3基因（TGFβ3）对大鼠肝星状细胞株（HSC—T6）Ⅰ型胶原合成的影响。方法TGFβ3表达质粒[pcDNA3．1（＋）-TGFβ31和TGFβ1表达质粒[pcDNA3．1（＋）-TGFD11的构建。通过脂质体介导方法，将pcDNA3．1（＋）-TGFβ1、pcDNA3．1（＋）-TGFβ3分别及共同转染体外培养的HSC—T6细胞，荧光定量PCR法及Westernblot法分别检测转染后TGFβ1、TGFD3、Ⅰ型胶原mRNA及蛋白质的表达。将pcDNA3．1（＋）-TGFD1转染HSC—T6细胞，经G418筛选建立高表达TGFD1的HSC～T6细胞克隆，pcDNA3．1（＋）-TGFD3转染克隆细胞，荧光定量PCR法检测转染后TGFβ3、TGFβ1及Ⅰ型胶原mRNA的表达，Westernblot法检测TGFβ1、Ⅰ型胶原蛋白的表达情况。结果构建的pcDNA3．1（＋）TGFD3、pcDNA3．1（＋）-TGFD1质粒可转染HSC—T6细胞，转染率28．2％。pcDNA3．1（＋）TGF侈3转染细胞后，Ⅰ型胶原mRNA及蛋白的表达较空白组及对照组增加，以72h增高最为明显（P〈0．05）；共转染组Ⅰ型胶原mRNA及蛋白质的表达较pcDNA3．1（＋）-TGFβ1转染组明显降低（P〈0．05）。TGF侈3转染克隆细胞后，TGFD1mRNA表达较克隆组无明显改变（P〉0．05），而蛋白质表达明显下降（P〈0．05），Ⅰ型胶原mRNA及蛋白质表达均较克隆组明显降低（P〈0．05）。结论TGFD3基因转染正常培养的HSC—T6细胞，增加Ⅰ型胶原的表达；转染高表达TGFβ1的克隆组HSC—T6细胞，Ⅰ型胶原表达明显降低，提示TGFβ3对肝纤维化的发生有抑制作用。     

肝素对大鼠肝星状细胞中转化生长因子β1和Ⅰ型胶原的影响

目的:研究肝素与大鼠肝星状细胞(hepatic stellate cells,HSC)作用后转化生长因子β1 (transforming growth factorβ1,TGF-β1)和Ⅰ型胶原表达的变化及意义.方法:大鼠肝星状细胞以1×10~8/L浓度接种于96孔培养板,每孔100μL.实验分组为肝素Ⅰ组、肝素Ⅱ组、肝素Ⅲ组,加入肝素使各组培养液中肝素浓度分别是10,100,1000 mg/L,加生理盐水为对照组(每组6孔重复3次)培养48 h.培养终止后吸取上清液-20℃冰冻保存,ELISA法检测其上清液TGF-β1和Ⅰ型胶原水平,MTT法观察细胞增殖情况.结果:肝素Ⅱ组和Ⅲ组HSC培养上清液TGF-β1水平均显著低于对照组(4.59±1.27 ng/L,3.34±1.13 ng/L vs 5.95±1.72 ng/L,P均<0.01),肝素各组Ⅰ型胶原水平均低于对照组(87.20±9.30 ng/L,73.17±12.04 ng/L vs 95.61±12.55 ng/L,63.31±10.93 ng/L vs 95.61±12.55 ng/L,P均<0.05),肝素Ⅲ组平均吸光度低于肝素Ⅰ组和对照组(0.29±0.07 vs 0.42±0.12,0.46±0.17,P均<0.05).结论:大鼠肝星状细胞在肝素作用下TGF-β1和Ⅰ型胶原分泌受抑制,其增殖减少.