| Aims: Previous studies have demonstrated that transforming growth factor-β3（TGF-β3） protected liver against fibrosis in vivo and vitro, but itsregulation is poorly understood. In addition, the cAMP-responsiveelement （CRE） in TGF-β3promoter is recognized as an importantregulatory site for TGF-b3auto-regulation. Thus, we hypothesize thattranscription factor CRE-binding protein-1（CREB-1） regulates theauto-induction of TGF-β3in rat hepatic stellate cells （HSC）.Methods:1) Construct TGF-β3promoter and CRE-mutant TGF-β3promoterluciferase reporter plasmid PGL3-TGFβ3-P and PGL3-TGFβ3-MP),and confirm the correction of these plasmids. Additionally, check thecytotoxisity of Lipofectamine2000.2) HSC were treated with or without exogenous TGF-β3for2h, the total RNA were extracted andReal-time PCR was performed to detect the mRNA expression ofTGF-β3.3) HSC were transinfected with PGL3-TGFβ3-P, then treatedwith or without exogenous TGF-β3for series time, and the promoteractivity of TGF-β3were tested by luciferase reporter assay.4) HSCwere transinfected with PGL3-TGFβ3-MP, then treated with orwithout exogenous TGF-β3for24h, and the promoter activity ofTGF-β3were tested by luciferase reporter assay.5) HSC were treatedwith or without exogenous TGF-β3for series time, then nuclearproteins were extracted, phospho-CREB-1expression were examinedby western blot and the DNA-binding activity of phospho-CREB-1were detected by electrophoretic mobility assay （EMSA）.6) HSC weretreated with or without exogenous TGF-β3for series time, total RNAand nuclear proteins were extracted, western blot and real-time PCRwere performed to detect the mRNA and protein expression ofCREB-1.Results:1) Agarose gel electrophoresis indicated that the construction ofPGL3-TGFβ3-P and PGL3-TGFβ3-MP were correct, andLipofectamine2000had no significant effect on HSC status.2)Exogenous TGF-β3significantly 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 ofPGL3-TGF-β3-MP containing the mutational CRE site was completelyblocked in the presence of exogenous TGF-β3, and there was nosignificantly statistical difference between treatment group and controlgroup （P>0.05）. Additionally, TGF-β3promoter activity was decreasedby85%compared with PGL3-TGF-β3-P, when the CRE site wasmutated.5) EMSA indicated that CRE probe showed a good specificityin binding with phospho-CREB-1, and exogenous TGF-β3dramatically increased the complex formation at CRE site in atime-dependent manner, with a peak level occurring at1h （2.4-foldhigher than control group, P<0.05） after TGF-β3treatment, andmaintained for the next11h.6) Exogenous TGF-β3had no effect onmRNA and protein expression of CREB-1in HSC.Conclusion: TGF-β3can trigger its auto-regulation, the phosphorylation ofCREB-1plays a important role in this signaling by binding to the CREsite in TGF-β3promoter. Aims and Background: Test the effect of transforming growth factor-β3（TGF-β3） on TGF-β/smad signaling pathway in rat hepatic satellitecells （HSC）, due to find out the mechanism which contributes toTGF-β3-resisted liver fibrosis. cAMP-responsive element bindingprotein-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) HSCwere treated with exogenous TGF-β3in series time, then total RNA andtotal protein were collected, Real-time PCR and western-blot wereperformed to examine the expression of smad7.3) The most efficiencysmad3siRNA was chosen, control plasmid and siRNA-smad3weretrans-infected into HSC by following Lipofectamine2000protocol, after24h culture, cells were treated with or without exogenous TGF-β3for2hours, then total RNA were collected, smad3and smad7expression wasdetected by Real-time PCR.4) According to the Lipofectamine2000 protocol, control plasmid, shRNA-CREB-1and pSRV-CREB-1weretrans-infected into HSC, after culturing for24h, cells were exposed withor without exogenous TGF-β3for2hours, then total RNA werecollected, CREB-1and smad7expression was detected by Real-timePCR.5) HSC were pretreated with ERK inhibitor （20mM）, JNKinhibitor （20mM）, p38inhibitor （20mM） and PKA inhibitor （5mM） for30min, then cells were presented with or without exogenous TGF-β3for2hours, total RNA were collected and smad7expression wasdetected by Real-time PCR.6) Similarly to method4, HSC weretrans-infected with control plasmid, shRNA-CREB-1and pSRV-CREB-1,after24h culture, cells treated with or without exogenous TGF-β1（10ng/ml） for2hours, then smad7mRNA expression was tested byReal-time PCR.Results:1) Exogenous TGF-β3significantly increased the expression ofsmad6and smad7in HSC, the induction is1.5-fold and3.6-fold higherthan that in control （P≤0.001）,but Exogenous TGF-β3had no effect onthe expression of smad3, smad4, TGF-β type1receptor, TGF-β type2receptor, smurf1and smurf2（P>0.05）.2) Exogenous TGF-β3increasedsmad7expression rapidly, peak at1h after the stimulation （4.1-foldhigher compared to control）, but the induction of protein was decreased after2hours stimulation, all of the inductions had statistic significancewithin12hours （P<0.05）.3) In HSC, smad3deficiency markedlyreduced the smad7mRNA expression in the basal condition （50%reduction）, which was trans-infected with control plasmid withoutexogenous TGF-β3treatment （P<0.05）. Also, smad3deficiencyobviously inhibited exogenous TGF-β3-induced smad7expression, thatis an approximated a half reduction compared to the positive control（P<0.05）.4) The inhibition or over-expression of CREB-1could notinfluence 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 ofCREB-1enhanced the induction of smad7mediated by exogenousTGF-β3（P<0.05）.5)After the pretreatment of inhibitors, there were nochanges of smad7in basal condition, but p38inhibitor obviouslyblocked the induction of smad7by exogenous TGF-β3, that is a40percent decreasing （P<0.05）, while other inhibitors （ERK inhibitor, JNKinhibitor 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-β3. Additionally, the inhibition and over-expressionof CREB-1had no effect on exogenous TGF-β1-induced smad7expression in HSC （P>0.05）.Conclusion:1) TGF-β3increases smad7expression in HSC.2) smad3is animportant transcriptional regulator for smad7.3) CREB-1is critical forTGF-β3-induced samd7in HSC.4) TGF-β3activates CREB-1by p38inHSC. Taken together, TGF-β3might activate both smad3and CREB-1,and CREB-1is an important co-transcriptional factor which enhancesthe binding of smad3with DNA, caused a continuous induction ofsmad7in HSC, and CREB-1might contribute to resist liver fibrosis. |
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