The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, th...The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, the regulatory effect of HSS on EGF-receptor(EGF-R) and the receptor phosphorylation at molecular level was studied. HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated. The results showed that preincubation of hepatocytes with HSS could lead to an increase in [125I]-EGF binding to its receptors and inhibit EGFinduced receptor down-regulation. Furthermore, the overexpression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation. Additionally, it was demonstrated with human hepatoma sMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/timedependency after HSS treatment. These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transduction.展开更多
IM To partially isolate and identify hepatic stimulator substance mRNA from human fetal liver tissues.METHODS The poly (A)mRNA was extracted from human fetal liver tissues of 4-5 month gestation, fractionated by s...IM To partially isolate and identify hepatic stimulator substance mRNA from human fetal liver tissues.METHODS The poly (A)mRNA was extracted from human fetal liver tissues of 4-5 month gestation, fractionated by size on sucrose gradient centrifugation, translated into protein from each fraction in vitro and then its products were tested for HSS activity.RESULTS Twentytwo 500μg total RNA was obtained from human fetal liver tissues and pooled. mRNA of 420μg was yielded, processed by oligo(dT)cellulose column chromatography, then was sizefractionated by ultracentrifution on a continuous sucrose density gradient (5%-25%), and separated into 18 fractions. Translated products of mRNA in fraction 8 and 9 could produce a twofold increase in the incorporation of 3HTdR into DNA of SMMC7721 hepatoma cells and in a heatresistant and organspecific way.CONCLUSION The partially purified HSS mRNA was obtained and this would facilitate the cloning of HSS using expression vectors.展开更多
Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS)...Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growthpromoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream RasMAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 μM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely.Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059.Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.展开更多
To optimize the experimental conditions of MTT colorimetric assay for HSS bioactivity in vitro,we studied the optimal combination of the major conditions of the MTT assay by orthogonal test and other experiments,and c...To optimize the experimental conditions of MTT colorimetric assay for HSS bioactivity in vitro,we studied the optimal combination of the major conditions of the MTT assay by orthogonal test and other experiments,and compared HSS bioactivity in vitro measured by the improved MTT protocol and published MTT assay at serial protein doses.Results showed that the absorbance value(A value)of the MTT assay directly correlated with the number of human hepatoma cell lines SMMC7721.The result of orthogonal test was the number of 5×104 SMMC7721 cells/ml,culture period 6 h before adding HSS,concentration of HSS 100μg/ml,incubation time with HSS 36 h.Additionally,several experiments demonstrated the optimal combination of other conditions was 50μg MTT,incubation time for MTT 6 h,DMSO was used to dissolve the MTT formazan crystals and measured with ELISA scanner at 570 nm.The result of determining HSS bio-activity in vitro by optimized MTT protocol showed that sHSS bio-activity increased with the growth of protein dose,but decreased when it beyond a certain dose.The optimized MTT protocol was a sensitive,convenient and stable quantitative method to evaluate HSS bio-activity.展开更多
文摘The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, the regulatory effect of HSS on EGF-receptor(EGF-R) and the receptor phosphorylation at molecular level was studied. HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated. The results showed that preincubation of hepatocytes with HSS could lead to an increase in [125I]-EGF binding to its receptors and inhibit EGFinduced receptor down-regulation. Furthermore, the overexpression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation. Additionally, it was demonstrated with human hepatoma sMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/timedependency after HSS treatment. These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transduction.
文摘IM To partially isolate and identify hepatic stimulator substance mRNA from human fetal liver tissues.METHODS The poly (A)mRNA was extracted from human fetal liver tissues of 4-5 month gestation, fractionated by size on sucrose gradient centrifugation, translated into protein from each fraction in vitro and then its products were tested for HSS activity.RESULTS Twentytwo 500μg total RNA was obtained from human fetal liver tissues and pooled. mRNA of 420μg was yielded, processed by oligo(dT)cellulose column chromatography, then was sizefractionated by ultracentrifution on a continuous sucrose density gradient (5%-25%), and separated into 18 fractions. Translated products of mRNA in fraction 8 and 9 could produce a twofold increase in the incorporation of 3HTdR into DNA of SMMC7721 hepatoma cells and in a heatresistant and organspecific way.CONCLUSION The partially purified HSS mRNA was obtained and this would facilitate the cloning of HSS using expression vectors.
基金This work was supported by the National Natural Science Foundation of China(No.39870285,No.0070342).
文摘Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growthpromoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream RasMAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 μM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely.Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059.Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.
文摘To optimize the experimental conditions of MTT colorimetric assay for HSS bioactivity in vitro,we studied the optimal combination of the major conditions of the MTT assay by orthogonal test and other experiments,and compared HSS bioactivity in vitro measured by the improved MTT protocol and published MTT assay at serial protein doses.Results showed that the absorbance value(A value)of the MTT assay directly correlated with the number of human hepatoma cell lines SMMC7721.The result of orthogonal test was the number of 5×104 SMMC7721 cells/ml,culture period 6 h before adding HSS,concentration of HSS 100μg/ml,incubation time with HSS 36 h.Additionally,several experiments demonstrated the optimal combination of other conditions was 50μg MTT,incubation time for MTT 6 h,DMSO was used to dissolve the MTT formazan crystals and measured with ELISA scanner at 570 nm.The result of determining HSS bio-activity in vitro by optimized MTT protocol showed that sHSS bio-activity increased with the growth of protein dose,but decreased when it beyond a certain dose.The optimized MTT protocol was a sensitive,convenient and stable quantitative method to evaluate HSS bio-activity.
