| Part one. Effects of phospho-CREB-1on transforming growth factor-β3auto-regulation in hepatic stellate cellsAims:Previous studies have demonstrated that transforming growth factor-β3(TGF-β3) protected liver against fibrosis in vivo and vitro, but its regulation is poorly understood. In addition, the cAMP-responsive element (CRE) in TGF-β3promoter is recognized as an important regulatory site for TGF-b3auto-regulation. Thus, we hypothesize that transcription factor CRE-binding protein-1(CREB-1) regulates the auto-induction of TGF-β3in rat hepatic stellate cells (HSC).Methods:1) Construct TGF-β3promoter and CRE-mutant TGF-β3promoter luciferase reporter plasmid PGL3-TGFβ3-P and PGL3-TGFβ3-MP), and confirm the correction of these plasmids. Additionally, check the cytotoxisity of Lipofectamine2000.2) HSC were treated with or without exogenous TGF-β3for2h, the total RNA were extracted and Real-time PCR was performed to detect the mRNA expression of TGF-β3.3) HSC were transinfected with PGL3-TGFβ3-P, then treated with or without exogenous TGF-β3for series time, and the promoter activity of TGF-β3were tested by luciferase reporter assay.4) HSC were transinfected with PGL3-TGFβ3-MP, then treated with or without exogenous TGF-β3for24h, and the promoter activity of TGF-β3were tested by luciferase reporter assay.5) HSC were treated with or without exogenous TGF-β3for series time, then nuclear proteins were extracted, phospho-CREB-1expression were examined by western blot and the DNA-binding activity of phospho-CREB-1were detected by electrophoretic mobility assay (EMSA).6) HSC were treated with or without exogenous TGF-β3for series time, total RNA and nuclear proteins were extracted, western blot and real-time PCR were performed to detect the mRNA and protein expression of CREB-1.Results:1) Agarose gel electrophoresis indicated that the construction of PGL3-TGFβ3-P and PGL3-TGFβ3-MP were correct, and Lipofectamine2000had no significant effect on HSC status.2) Exogenous TGF-P3significantly increased TGF-β3mRNA expression in HSC after, there is3.7-fold up-regulation compared with control.3) Exogenous TGF-β3induced the activity of PGL3-TGF-β3-P at6h, peaked at24h (10.680.57,2.2-fold higher than control group, P<0.05), and decreased at48h.4) the promoter activity of PGL3-TGF-β3-MP containing the mutational CRE site was completely blocked in the presence of exogenous TGF-β3, and there was no significantly statistical difference between treatment group and control group (P>0.05). Additionally, TGF-β3promoter activity was decreased by85%compared with PGL3-TGF-β3-P, when the CRE site was mutated.5) EMSA indicated that CRE probe showed a good specificity in binding with phospho-CREB-1, and exogenous TGF-β3dramatically increased the complex formation at CRE site in a time-dependent manner, with a peak level occurring at1h (2.4-fold higher than control group, P<0.05) after TGF-β3treatment, and maintained for the next11h.6) Exogenous TGF-β3had no effect on mRNA and protein expression of CREB-1in HSC.Conclusion:TGF-β3can trigger its auto-regulation, the phosphorylation of CREB-1plays a important role in this signaling by binding to the CRE site in TGF-β3promoter. Part two. The effect of CREB-1on TGF-p3-induced smad7expression in rat hepatic satellite cellsAims and Background:Test the effect of transforming growth factor-β3(TGF-β3) on TGF-β/smad signaling pathway in rat hepatic satellite cells (HSC), due to find out the mechanism which contributes to TGF-β3-resisted liver fibrosis. cAMP-responsive element binding protein-1(CREB-1) is an important transcription factor in TGF-β3auto-regulation signaling pathway.Methods:1) HSC were treated with or without exogenous TGF-β3(10ng/ml) for2hours, then total RNA were extracted and the factors in TGF-β/smad signaling pathway were detected by Real-time PCR.2) HSC were treated with exogenous TGF-β3in series time, then total RNA and total protein were collected, Real-time PCR and western-blot were performed to examine the expression of smad7.3) The most efficiency smad3siRNA was chosen, control plasmid and siRNA-smad3were trans-infected into HSC by following Lipofectamine2000protocol, after24h culture, cells were treated with or without exogenous TGF-β3for2hours, then total RNA were collected, smad3and smad7expression was detected by Real-time PCR.4) According to the Lipofectamine2000protocol, control plasmid, shRNA-CREB-1and pSRV-CREB-1were trans-infected into HSC, after culturing for24h, cells were exposed with or without exogenous TGF-β3for2hours, then total RNA were collected, CREB-1and smad7expression was detected by Real-time PCR.5) HSC were pretreated with ERK inhibitor (20mM), JNK inhibitor (20mM), p38inhibitor (20mM) and PKA inhibitor (5mM) for30min, then cells were presented with or without exogenous TGF-β3for2hours, total RNA were collected and smad7expression was detected by Real-time PCR.6) Similarly to method4, HSC were trans-infected with control plasmid, shRNA-CREB-1and pSRV-CREB-1, after24h culture, cells treated with or without exogenous TGF-β1(lOng/ml) for2hours, then smad7mRNA expression was tested by Real-time PCR.Results:1) Exogenous TGF-β3significantly increased the expression of smad6and smad7in HSC, the induction is1.5-fold and3.6-fold higher than that in control (P<0.001),but Exogenous TGF-β3had no effect on the expression of smad3, smad4, TGF-β type1receptor, TGF-β type2receptor, smurfl and smurf2(P>0.05).2) Exogenous TGF-p3increased smad7expression rapidly, peak at1h after the stimulation (4.1-fold higher compared to control), but the induction of protein was decreased after2hours stimulation, all of the inductions had statistic significance within12hours (P<0.05).3) In HSC, smad3deficiency markedly reduced the smad7mRNA expression in the basal condition (50%reduction), which was trans-infected with control plasmid without exogenous TGF-β3treatment (P<0.05). Also, smad3deficiency obviously inhibited exogenous TGF-β3-induced smad7expression, that is an approximated a half reduction compared to the positive control (P<0.05).4) The inhibition or over-expression of CREB-1could not influence the expression of smad7in HSC (P>0.05), but CREB-1deficiency significantly inhibited exogenous TGF-β3-induced smad7expression (42%reduction, P<0.05), while the over-expression of CREB-1enhanced the induction of smad7mediated by exogenous TGF-P3(P<0.05).5)After the pretreatment of inhibitors, there were no changes of smad7in basal condition, but p38inhibitor obviously blocked the induction of smad7by exogenous TGF-β3, that is a40percent decreasing (P<0.05), while other inhibitors (ERK inhibitor, JNK inhibitor and PKA inhibitor) had no effect on the induction of smad7by exogenous TGF-β3stimulation (P>0.05).6) In basal condition, exogenous TGF-β1also increased smad7mRNA expression in HSC (1.5-fold higher than control, P<0.05), but this induction is lower than it by exogenous TGF-03. Additionally, the inhibition and over-expression of CREB-1had no effect on exogenous TGF-β1-induced smad7expression in HSC (P>0.05).Conclusion:1) TGF-β3increases smad7expression in HSC.2) smad3is an important transcriptional regulator for smad7.3) CREB-1is critical for TGF-β3-induced samd7in HSC.4) TGF-β3activates CREB-1by p38in HSC. Taken together, TGF-β3might activate both smad3and CREB-1, and CREB-1is an important co-transcriptional factor which enhances the binding of smad3with DNA, caused a continuous induction of smad7in HSC, and CREB-1might contribute to resist liver fibrosis. |
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