Down-regulation of transforming growth factor β1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

AIM: To investigate the effect of herbal compound 861(Cpd861) on the transforming growth factor-131 (TGFβ1)/activin receptor-like kinase 1 (ALK1, type I receptor)signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells.METHODS: LX-2 cells were treated with TGFβ1(5 ng/mL) Cpd861 (0.1 mg/mL), TGFβ1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA (α-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smadl,and immunocytochemical analysis for the expression of α-SMA.RESULTS: In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathwayrelated expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h.CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.     

Effects of herbal compound 861 on human hepatic stellate cell proliferation and activation

AIM:To investigate the effects of herbal compound 861(Cpd 861)on cell proliferation in human hepatic stellatecells(LX-2)and human hepatocellular liver carcinoma cells(HepG2),and expression of α-smooth muscle actin(α-SMA)in LX-2 cells.METHODS:LX-2 and HepG2 cells were incubated withvarious concentrations of Cpd 861(0.1-0.003 mg/mL)for 1,2,3,5 and 7 d.Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Effects of Cpd861on the expression of α-SMA mRNA in LX-2 cells weremeasured by real-time quantitative PCR method using SYBRGreen I technology.RESULTS:Cpd 861,at 0.1 mg/mL,significantly inhibitedLX-2 cell proliferation(15% decrease relative to control,P<0.05)after 3 d of incubation.The inhibitory effects seemedto increase with the treatment time(25% decrease after5 d of incubation and 35% decrease after 7 d of incubation,P<0.01).However,Cpd 861 did not affect HepG2 cellproliferation at the same concentration used for LX-2 cells.The expression levels of α-SMA mRNA decreased significantlywhen LX-2 cells were exposed to Cpd 861 for 48 h(59?crease relative to control,P<0.05)or 72 h(60% decreaserelative to control,P<0.01).CONCLUSION:Cpd 861 can significantly inhibit LX-2 cellproliferation in a dose-dependant manner,and reduce theexpression levels of α-SMA mRNA in LX-2 cells.Since hepaticcell proliferation and high level of α-SMA are associatedwith liver fibrosis,the results suggest that Clod 861 may beuseful in the treatment of this disease.     

Effects of herbal compound 861 on human hepatic stellate cell proliferation and activation

AIM: To investigate the effects of herbal compound 861 (Cpd 861) on cell proliferation in human hepatic stellate cells (LX-2) and human hepatocellular liver carcinoma cells (HepG2), and expression of α-smooth muscle actin (α-SMA) in LX-2 cells.METHODS: LX-2 and HepG2 cells were incubated with various concentrations of Cpd 861 (0.1-0.003 mg/mL)for 1, 2, 3, 5 and 7 d. Cell proliferation was analyzed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects of Cpd861on the expression of cc-SMA mRNA in LX-2 cells weremeasured by real-time quantitative PCR method using SYBRGreen I technology.RESULTS: Cpd 861, at 0.1 mg/mL, significantly inhibitedLX-2 cell proliferation (15% decrease relative to control,P&lt;0.05) after 3 d of incubation. The inhibitory effects seemedto increase with the treatment time (25% decrease after5 d of incubation and 35% decrease after 7 d of incubation,P&lt;0.01). However, Cpd 861 did not affect HepG2 cellproliferation at the same concentration used for I_X-2 cells.The expression levels of ~-SMA mRNA decreased significantlywhen LX-2 cells were exposed to Cpd 861 for 48 h (59%decrease relative to control, P&lt;0.05) or 72 h (60% decreaserelative to control, P&lt;0.01).CONCLUSION: Cpd 861 can significantly inhibit LX-2 cellproliferation in a dose-dependant manner, and reduce theexpression levels of o~-SMA mRNA in LX-2 cells. Since hepaticcell proliferation and high level of ~-SMA are associatedwith liver fibrosis, the results suggest that Cpd 861 may beuseful in the treatment of this disease.     

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM： To investigate the role of insulin-like growth factor binding protein-7 （IGFBP-7） in the activation and transdifferentiation of hepatic stellate cells （HSC） in vitro.
METHODS： Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor （TGF）-β1, IGFBP-7 or anti- IGFBP-7 antibody for 24 h. The supernatant or a cytoplasm suspension was obtained from cultured HSC, followed by transfer of cells to form cell-coated dishes. Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin, collagen Ⅰ and α-smooth muscle actin （SMA）. The pro-apoptotic effect of anti- IGFBP-7 antibody was determined by flow cytometry.
RESULTS： Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group. In addition, fibronectin, collagen Ⅰ and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent. Moreover, flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC, which is responsible for the development of liver fibrosis, and may represent a novel pathway and target for therapeutic intervention.
CONCLUSION： IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC. Anti- IGFBP-7 antibody may ameliorate liver fibrogenesis.     

Interaction between insulin-like growth factor binding protein-related protein 1 and trans-forming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND:We previously showed that insulin-like growth factor binding protein-related protein 1(IGFBPrP1) is a novel mediator in liver fibrosis.Transforming growth factor beta 1(TGFβ1) is known as the strongest effector of liver fibrosis.Therefore,we aimed to investigate the detailed interaction between IGFBPrP1 and TGFβ1 in primary hepatic stellate cells(HSCs).METHODS:We overexpressed TGFβ1 or IGFBPrP1 and inhibited TGFβ1 expression in primary HSCs for 6,12,24,48,72,and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin(α-SMA),collagen I,fibronectin,and phosphorylated-mothers against decapentaplegic homolog 2/3(p-Smad2/3).RESULTS:We found that the adenovirus vector encoding the TGFβ1 gene(Ad TGFβ1) induced IGFBPrP1 expression while that of α-SMA,collagen I,fibronectin,and TGFβ1 increased gradually.Concomitantly,Ad IGFBPrP1 upregulated TGFβ1,α-SMA,collagen I,fibronectin,and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours.Inhibition of TGFβ1 expression reduced the IGFBPrP1-stimulated expression of α-SMA,collagen I,fibronectin,and p-Smad2/3.CONCLUSIONS:These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFβ1,which likely accelerates liver fibrosis progression.Furthermore,IGFBPrP1 likely participates in liver fibrosis in a TGFβ1-depedent manner,and may act as an upstream regulatory factor of TGFβ1 in the Smad pathway.     

Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

AIM： To investigate the effects of transforming growth factor β1 （TGF-β1） on the differentiation of colonic lamina propria fibroblasts （CLPF） into myofibroblasts in vitro.
METHODS： Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of m-smooth muscle actin （α-SMA）, fibronectin （FN） and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase （FAK） in CLPF were analyzed after induction of migration.did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SIVlA production. Short term incubation （6 h） with TGF-β1 enhanced CLPF migration, while long term treatment （6 d） of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 （2 d） in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.
CONCLUSION： Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact （6 h） of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β(TGF-β) signaling in vitro. METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/ DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1, 2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-α-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲ procollagen (PCⅢ) in supernatants were determined by radioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively. RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%, 75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RT-PCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P<0.01), and both were significantly correlated with α-SMA protein expression (r= -0.938, P<0.01; r = 0.938,P<0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well. CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

Effect of hepatocyte growth factor and transforming growth factor-β1 on atrial fibroblasts fibrosis

Objective To investigate the effect of hepatocyte growth factor (HGF) and transforming growth factor-β1 (TGFβ1) on the expression of α-smooth muscle actin(α-SMA) and collagen I in human atrial fibroblast in vitro, and to explore the possible molecular mechanism of atrial fibrosis in patients     

Id1 is a critical mediator in TGF-beta-induced transdifferentiation of rat hepatic stellate cells

Transforming growth factor (TGF)-beta is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-beta antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and alpha-smooth muscle actin (alpha-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-beta pathways that control the transdifferentiation process. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with alpha-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-beta1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-beta/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).     

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。     

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。     

复方861对HSC-T6细胞组织基质金属蛋白酶抑制因子1基因表达的影响

目的 观察复方861对HSC-T6组织基质金属蛋白酶抑制因子1(TIMP1)基因表达的影响。方法复方861 0.25、0.5、1.0mg/ml等浓度作用于HSC-T6细胞48h,以逆转录定量PCR方法测定其对HSC-T6细胞TIMP1基因表达的影响。结果 复方861 0.25、0.5、1.0mg/ml等不同浓度作用后,HSC-T6细胞基因表达水平依次为2.50±0.71、0.50±0.01、0.11±0.03,与空白对照组门(3.78±0.67)比较,可明显抑制TIMP1基因表达水平(P＜0.05或P＜0.01)。结论 复方861抑制HSC-T6细胞TIMP1基因表达水平,从而促进胶原降解,可能是抗肝纤维化的作用机制之一。     

TGF beta-induced Smad signaling remains intact in primary human ovarian cancer cells

Disruptions in TGF beta signaling have been implicated in various human cancers, including ovarian cancer. Our goal was to determine whether ovarian cancer cells isolated from patient ascites fluid were growth inhibited by TGF beta 1 treatment and further characterize the expression and activity profile of TGF beta/Smad signaling components in human ovarian cancer cells. We found that 9 of 10 primary cultures of ovarian cancer cells (OC2-10) were growth inhibited by 16 pM TGF beta 1. One primary ovarian cancer sample (OC1) and the established ovarian cancer cell lines CaOV3 and SkOV3 continued to grow in the presence of TGF beta 1. All cells expressed components of the TGF beta/Smad signaling pathway including TGF beta 1, T beta RI, T beta RII, Smad2, -3, -4, and Smad anchor for receptor activation. Although OC1, CaOV3, and SkOV3 are not growth inhibited by TGF beta 1, they can transmit the TGF beta 1 signal to turn on a transfected TGF beta/Smad reporter gene, p3TP.lux. In addition, all cells up-regulate the endogenous TGF beta target genes Smad7 and PAI-1. p15(Ink4B) mRNA is also up-regulated with TGF beta 1 treatment in OC2-9, whereas the p15(Ink4B) gene has been deleted in OC1, CaOV3, and SkOV3 cells. Homozygous deletion of p15(Ink4B) may account for TGF beta resistance in some populations of ovarian cancer cells. Our data demonstrate that the TGF beta/Smad signaling pathway remains functional in human ovarian cancer cells and suggest that if abnormalities exist in the cellular response of TGF beta signals, they must lie downstream of the Smad proteins.