CREB-1在TGF-β3抗肝纤维化中的作用
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摘要
目的：通过观察外源性转化生长因子β3（TGF-β3）对大鼠肝星状细胞（HSC）中TGF-β3启动子活性和磷酸化cAMP反应元件结合蛋白-1（CREB-1）的影响，研究TGF-β3自反馈调节信号通路中正向调节因子。
方法：（1）构建并检测TGF-β3启动子和CRE位点突变的TGF-β3启动子荧光素酶报告基因质粒（PGL3-TGFβ3-P和PGL3-TGFβ3-MP），并判断脂质体转染法对细胞形态的影响；（2）用外源性TGF-β3诱导大鼠HSC细胞，2h后提取总RNA，通过实时荧光定量PCR法测定对照组和实验组中TGF-β3mRNA的表达情况；（3）用外源性TGF-β3诱导转染了PGL3-TGFβ3-P的大鼠HSC细胞，于不同时间点，用荧光素酶报告基因分析法检测TGF-β3启动子活性；（4）将TGF-β3启动子上的CRE结合位点基因突变，并将该突变的报告质粒与正常质粒一起转染与大鼠HSC细胞，加/不加TGF-β3进行诱导，通过荧光素酶报告基因分析法检测启动子的活性；（5）用外源性TGF-β3诱导大鼠HSC细胞，于不同时间点提取核蛋白，通过电泳迁移率分析法（EMSA）检测各时间点磷酸化CREB-1的DNA结合活性，同时采用western-blot方法检测磷酸化CREB-1各时间点的变化情况；（6）用外源性TGF-β3诱导大鼠HSC细胞，于不同时间点分别提取细胞总RNA和核蛋白，用western-bloting法检测CREB-1的表达情况以及实时荧光定量PCR检测CREB-1mRNA的表达情况。
结果（1）电泳结果显示成功构建正常和突变的TGF-β3启动子报告基因质粒，用Lipofectamine2000脂质体转染法对大鼠HSC细胞进行转染后无明显毒性；（2）大鼠HSC细胞被外源性TGFβ3诱导2h后，TGFβ3mRNA的表达明显上升（上升3.7倍，P=0.003）；（3）外源性TGF-β3处理HSC后，TGF-β3启动子活性表现出时间依赖性增高，并于诱导后24h达峰值（2.2倍于对照组，P＜0.05）；（4）CRE位点的缺失显示出TGF-β3启动子对外源性TGF-β3诱导HSC时的不应答，TGF-β3启动子的基础活性下降85%（P＜0.05）；（5）EMSA结果显示，DNA探针有很好的特异性，外源性TGF-β3可诱导磷酸化CREB-1的DNA结合活性持续增强，同时western-blot结果显示外源性TGF-β3可以引起磷酸化CREB-1表达增加；（6）外源性TGF-β3对大鼠HSC中CREB-1mRNA和CREB-1蛋白的表达无影响。
结论（1）外源性TGF-β3可促进TGF-β3启动子活性增强，且CRE位点在外源性TGF-β3介导的TGF-β3启动子活性中发挥重要作用；（2）外源性TGF-β3可促进CRE结合蛋白磷酸化CREB-1的DNA活性及表达，但不能促进CREB-1蛋白及mRNA的表达。本研究显示TGF-β3可自身活化，CREB-1的磷酸化在该信号通路中发挥重要作用。
目的：通过研究外源性TGF-β3对HSC中TGF-β1/smad信号通路的影响，探讨TGF-β3抗肝纤维化机制。
方法：（1）用外源性TGF-β3（10ng/ml）处理/不处理HSC细胞，2h后提取细胞总RNA，通过实时荧光定量PCR测定TGF-β1/smad信号通路中各因子的mRNA表达情况；（2）用外源性TGF-β3（10ng/ml）处理HSC细胞，于不同时间点收集细胞总RNA和总蛋白，通过实时荧光定量PCR和western-blot测定smad7的表达情况；（3）将具有最佳抑制率的smad3siRNA干扰片段和空白质粒通过Lipofectamine2000脂质转染法导入HSC细胞中，24h后，加/不加外源性TGF-β3（10ng/ml）处理HSC细胞，2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况；（4）将具有最佳抑制率的CREB-1shRNA干扰质粒、pSRV-CREB-1表达质粒和空白质粒通过Lipofectamine2000脂质转染法导入HSC细胞中，24h后，加/不加外源性TGF-β3（10ng/ml）处理HSC细胞，2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况；（5）将CREB-1shRNA干扰质粒、pSRV-CREB-1表达质粒和空白质粒导入HSC细胞中，24h后，加/不加外源性TGF-β1（10ng/ml）处理HSC细胞，2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况；（6）将HSC细胞用ERK抑制剂（20mM)、JNK抑制剂（20mM）、p38抑制剂（20mM）和PKA抑制剂（5mM）预处理30分钟，然后加/不加外源性TGF-β3（10ng/ml）,2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况。
结果：（1）在HSC中，外源性TGF-β3可诱导smad6低表达（1.5倍于对照组）和smad7高表达（3.6倍于对照组），其差异具有统计学意义（P≤0.001）,但外源性TGF-β3对TGF-β/smad信号通路中的其他因子均无影响；（2）外源性TGF-β3可在短时间诱导HSC中smad7的大量表达，1hmRNA达到顶峰(4.1倍于对照组），2h蛋白达到最高，随后这一诱导作用逐渐下降，但24h其差异具有统计学意义（P<0.05）；（3）在HSC中， smad3的降低可显著抑制基础状态（非外源性TGF-β3诱导）smad7的表达，同时也可阻断外源性TGF-β3对smad7的诱导作用，与空白质粒对照组相比，其下降50%，有显著统计学意义（P<0.05）；（4）在HSC中，降低或过表达CREB-1蛋白均不能影响正常状态下smad mRNA的表达，然而降低CREB-1可显著抑制外源性TGF-β3对smad7的诱导作用，与空白质粒对照组相比，其下降42%，有显著统计学意义（P<0.05），过表达的CREB-1也可加强外源性TGF-β3对smad7的诱导作用，与空白质粒对照组相比其差异有统计学意义（P<0.05）；（5）经过激酶抑制剂的预处理后，外源性TGF-β3对smad7的诱导作用明显受到p38抑制剂的影响，与阳性对照组相比下降40%（2.64±0.37），其差异有统计学意义（P<0.05），然而各激酶抑制剂不影响基础状态下smad7mRNA的表达，ERK抑制剂、JNK抑制剂和PKA抑制剂均不能影响外源性TGF-β3对smad7的诱导。（6）在HSC中，外源性TGF-β1可诱导smad7mRNA的表达（1.5倍于对照组，P<0.05），然而抑制或过表达CREB-1均不能影响外源性TGF-β1对smad7的诱导作用，与阳性对照组相比，其差异没有统计学意义（P>0.05）。
结论：(1) TGF-β3促进HSC中smad7的表达；（2）smad3是调控smad7的关键转录蛋白；（3）TGF-β3通过p38活化CREB-1，后者入核参与调节smad7的表达。因此，TGF-β3可能通过活化CREB-1辅助smad3稳定调节HSC中smad7的表达，提示CREB-1可能是一个十分重要的抗肝纤维化蛋白。
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 (10.68_0.57,2.2-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.
方法：（1）构建并检测TGF-β3启动子和CRE位点突变的TGF-β3启动子荧光素酶报告基因质粒（PGL3-TGFβ3-P和PGL3-TGFβ3-MP），并判断脂质体转染法对细胞形态的影响；（2）用外源性TGF-β3诱导大鼠HSC细胞，2h后提取总RNA，通过实时荧光定量PCR法测定对照组和实验组中TGF-β3mRNA的表达情况；（3）用外源性TGF-β3诱导转染了PGL3-TGFβ3-P的大鼠HSC细胞，于不同时间点，用荧光素酶报告基因分析法检测TGF-β3启动子活性；（4）将TGF-β3启动子上的CRE结合位点基因突变，并将该突变的报告质粒与正常质粒一起转染与大鼠HSC细胞，加/不加TGF-β3进行诱导，通过荧光素酶报告基因分析法检测启动子的活性；（5）用外源性TGF-β3诱导大鼠HSC细胞，于不同时间点提取核蛋白，通过电泳迁移率分析法（EMSA）检测各时间点磷酸化CREB-1的DNA结合活性，同时采用western-blot方法检测磷酸化CREB-1各时间点的变化情况；（6）用外源性TGF-β3诱导大鼠HSC细胞，于不同时间点分别提取细胞总RNA和核蛋白，用western-bloting法检测CREB-1的表达情况以及实时荧光定量PCR检测CREB-1mRNA的表达情况。
结果（1）电泳结果显示成功构建正常和突变的TGF-β3启动子报告基因质粒，用Lipofectamine2000脂质体转染法对大鼠HSC细胞进行转染后无明显毒性；（2）大鼠HSC细胞被外源性TGFβ3诱导2h后，TGFβ3mRNA的表达明显上升（上升3.7倍，P=0.003）；（3）外源性TGF-β3处理HSC后，TGF-β3启动子活性表现出时间依赖性增高，并于诱导后24h达峰值（2.2倍于对照组，P＜0.05）；（4）CRE位点的缺失显示出TGF-β3启动子对外源性TGF-β3诱导HSC时的不应答，TGF-β3启动子的基础活性下降85%（P＜0.05）；（5）EMSA结果显示，DNA探针有很好的特异性，外源性TGF-β3可诱导磷酸化CREB-1的DNA结合活性持续增强，同时western-blot结果显示外源性TGF-β3可以引起磷酸化CREB-1表达增加；（6）外源性TGF-β3对大鼠HSC中CREB-1mRNA和CREB-1蛋白的表达无影响。
结论（1）外源性TGF-β3可促进TGF-β3启动子活性增强，且CRE位点在外源性TGF-β3介导的TGF-β3启动子活性中发挥重要作用；（2）外源性TGF-β3可促进CRE结合蛋白磷酸化CREB-1的DNA活性及表达，但不能促进CREB-1蛋白及mRNA的表达。本研究显示TGF-β3可自身活化，CREB-1的磷酸化在该信号通路中发挥重要作用。
目的：通过研究外源性TGF-β3对HSC中TGF-β1/smad信号通路的影响，探讨TGF-β3抗肝纤维化机制。
方法：（1）用外源性TGF-β3（10ng/ml）处理/不处理HSC细胞，2h后提取细胞总RNA，通过实时荧光定量PCR测定TGF-β1/smad信号通路中各因子的mRNA表达情况；（2）用外源性TGF-β3（10ng/ml）处理HSC细胞，于不同时间点收集细胞总RNA和总蛋白，通过实时荧光定量PCR和western-blot测定smad7的表达情况；（3）将具有最佳抑制率的smad3siRNA干扰片段和空白质粒通过Lipofectamine2000脂质转染法导入HSC细胞中，24h后，加/不加外源性TGF-β3（10ng/ml）处理HSC细胞，2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况；（4）将具有最佳抑制率的CREB-1shRNA干扰质粒、pSRV-CREB-1表达质粒和空白质粒通过Lipofectamine2000脂质转染法导入HSC细胞中，24h后，加/不加外源性TGF-β3（10ng/ml）处理HSC细胞，2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况；（5）将CREB-1shRNA干扰质粒、pSRV-CREB-1表达质粒和空白质粒导入HSC细胞中，24h后，加/不加外源性TGF-β1（10ng/ml）处理HSC细胞，2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况；（6）将HSC细胞用ERK抑制剂（20mM)、JNK抑制剂（20mM）、p38抑制剂（20mM）和PKA抑制剂（5mM）预处理30分钟，然后加/不加外源性TGF-β3（10ng/ml）,2h后收集细胞总RNA，通过实时荧光定量PCR测定smad7mRNA的表达情况。
结果：（1）在HSC中，外源性TGF-β3可诱导smad6低表达（1.5倍于对照组）和smad7高表达（3.6倍于对照组），其差异具有统计学意义（P≤0.001）,但外源性TGF-β3对TGF-β/smad信号通路中的其他因子均无影响；（2）外源性TGF-β3可在短时间诱导HSC中smad7的大量表达，1hmRNA达到顶峰(4.1倍于对照组），2h蛋白达到最高，随后这一诱导作用逐渐下降，但24h其差异具有统计学意义（P<0.05）；（3）在HSC中， smad3的降低可显著抑制基础状态（非外源性TGF-β3诱导）smad7的表达，同时也可阻断外源性TGF-β3对smad7的诱导作用，与空白质粒对照组相比，其下降50%，有显著统计学意义（P<0.05）；（4）在HSC中，降低或过表达CREB-1蛋白均不能影响正常状态下smad mRNA的表达，然而降低CREB-1可显著抑制外源性TGF-β3对smad7的诱导作用，与空白质粒对照组相比，其下降42%，有显著统计学意义（P<0.05），过表达的CREB-1也可加强外源性TGF-β3对smad7的诱导作用，与空白质粒对照组相比其差异有统计学意义（P<0.05）；（5）经过激酶抑制剂的预处理后，外源性TGF-β3对smad7的诱导作用明显受到p38抑制剂的影响，与阳性对照组相比下降40%（2.64±0.37），其差异有统计学意义（P<0.05），然而各激酶抑制剂不影响基础状态下smad7mRNA的表达，ERK抑制剂、JNK抑制剂和PKA抑制剂均不能影响外源性TGF-β3对smad7的诱导。（6）在HSC中，外源性TGF-β1可诱导smad7mRNA的表达（1.5倍于对照组，P<0.05），然而抑制或过表达CREB-1均不能影响外源性TGF-β1对smad7的诱导作用，与阳性对照组相比，其差异没有统计学意义（P>0.05）。
结论：(1) TGF-β3促进HSC中smad7的表达；（2）smad3是调控smad7的关键转录蛋白；（3）TGF-β3通过p38活化CREB-1，后者入核参与调节smad7的表达。因此，TGF-β3可能通过活化CREB-1辅助smad3稳定调节HSC中smad7的表达，提示CREB-1可能是一个十分重要的抗肝纤维化蛋白。
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 (10.68_0.57,2.2-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.
引文
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