环磷酸腺苷信号通路参与调控生长抑素受体5激活对垂体催乳素腺瘤激素分泌的抑制作用

Inhibitory effect of somatostatin receptor 5 activation on hormone secretion in pituitary prolactinoma through cyclic adenosine monophosphate pathway

目的探究环磷酸腺苷(cAMP)信号通路在生长抑素受体5(SSTR5)激活对垂体催乳素腺瘤激素分泌抑制作用中的调控机制.方法使用构建的SSTR5过表达GH3细胞系作为垂体催乳素腺瘤的体外细胞模型,采用不同浓度SSTR5激动剂BIM23052联合cAMP激动剂Forskolin(5μmol/L)或百日咳毒素(10 nmol/L)进行细胞刺激,或联合转染过表达环磷腺苷效应元件结合蛋白(CREB)作为干预.通过荧光素酶报告基因系统检测不同刺激组间PRL-luc、环磷酸腺苷反应元件(CRE)-Luc、Pit1-Luc、AP1-Luc和雌激素反应元件(ERE)-Luc启动子活性,Renilla荧光素酶基因用作内参以标准化转染效率.组间统计分析采用Student's t检验和单向方差分析.结果为验证GH3细胞中SSTR5过表达的有效性,使用cAMP响应元件报告载体进行监测,GH3SSTR5细胞中CRE报告基因活性显著低于GH3mock组[(74.87±6.66)%比(100.00±5.21)%,t=5.15,P<0.05],提示SSTR5过表达显著抑制CRE转录活性.不同浓度BIM23052(0、0.1、1.0、10.0、100.0nmol/L)联合Forskolin(5μmol/L)预处理GH3SSTR5细胞,CRE报告基因活性分别为(100.00±2.23)%、(17.79±2.30)%、(6.25±0.37)%、(25.27±2.99)%、(44.60±3.25)%,提示BIM23052降低了Forskolin诱导的CRE转录活性,呈现U型剂量响应曲线,在1.0 nmol/L时抑制效果最强(t=71.68,P<0.01).此外,Pit1启动子包含CRE元件,与未添加BIM23052和Forskolin组[CRE报告基因活性(100.00±4.24)%]比较,不同浓度BIM23052(0、0.1、1.0、10.0、100.0 nmol/L)联合Forskolin(5μmol/L)预处理GH3SSTR5细胞,Pit1报告基因活性分别为(317.91±9.03)%、(278.11±4.94)%、(250.72±14.33)%、(176.21±8.88)%、(162.34±7.54)%,表明BIM23052处理同样降低了GH3SSTR5细胞中Forskolin诱导的Pit1启动子活性,且这种降低具有剂量依赖性(P<0.01).然而,BIM23052和百日咳毒素处理对Prl启动子上的其他响应元件,如AP1或ERE(雌激素反应元件),并未表现出明显抑制作用(P>0.05).过表达CREB后,Prl启动子活性从空白转染组[Ctrl组(100.00±12.01)%比BIM组(63.02±15.35)%,t=5.10,P<0.01]到CREB过表达组[Ctrl组(102.17±11.80)%比BIM组(106.27±14.62)%,t=0.58,P>0.05].结论在垂体催乳素腺瘤中,SSTR5通过抑制cAMP-CREB途径来调控催乳素合成.

Objective To explore the involvement of the cyclic adenosine monophosphate(cAMP)signaling pathway in the regulation of hormone secretion inhibition by somatostatin receptor 5(SSTR5)acti-vation in pituitary prolactinoma through in vitro experiments.Methods We utilized our developed SSTR5-overexpressing GH3 cell line as an in vitro model for pituitary prolactinoma.Cells were stimulated using the SSTR5 agonist BIM23052 combined with the cAMP activator Forskolin(5μmol/L)or pertussis toxin(10 nmol/L),or with cAMP response element binding protein(CREB)overexpression.The activity of PRL-luc,cAMP response element(CRE)-Luc,Pit1-Luc,AP1-Luc,and estrogen response element(ERE)-Luc promoters was assessed using a luciferase reporter gene assay.The Renilla luciferase gene was used as an internal reference to standardize transfection efficiency.Statistical analysis between groups was performed using Student's t-test and one-way ANOVA.Results To verify the effectiveness of SSTR5 overexpression in GH3 cells,we monitored CRE reporter activity.It was found that CRE reporter gene ac-tivity in GH3SSTR5 cells was significantly reduced[(74.87±6.66)%]compared to GH3mock cells[(100.00±5.21)%,t=5.15,P<0.05],indicating that SSTR5 overexpression inhibits CRE transcrip-tional activity.Pretreatment of GH3SSTR5 cells with various concentrations of BIM23052(0,0.1,1.0,10.0,100.0 nmol/L)combined with Forskolin(5μmol/L)resulted in CRE reporter gene activities of(100.00±2.23)%,(17.79±2.30)%,(6.25±0.37)%,(25.27±2.99)%,and(44.60±3.25)%,respectively.This suggests that BIM23052 reduces Forskolin-induced CRE transcriptional activity,exhibi-ting a U-shaped dose-response curve,with the maximum inhibitory effect observed at 1.0 nmol/L(t=71.68,P<0.01).Additionally,since the Pit1 promoter contains CRE elements,BIM23052 also reduced Forskolin-induced Pit1 promoter activity in a dose-dependent manner[(317.91±9.03)%,(278.11±4.94)%,(250.72±14.33)%,(176.21±8.88)%,(162.34±7.54)%,P<0.01].However,BIM23052 and pertussis toxin treatment did not significantly inhibit other response elements on the Prl pro-moter,such as AP1 or ERE(P>0.05).Overexpression of CREB showed that Prl promoter activity changed from[Ctrl group(100.00±12.01)%vs.BIM group(63.02±15.35)%,t=5.10,P<0.01]in the blank transfection group to[Ctrl group(102.17±11.80)%vs.BIM group(106.27±14.62)%,t=0.58,P>0.05]in the CREB overexpression group,indicating that BIM23052 treatment does not affect Prl promoter activity in CREB-overexpressing GH3 cells.This further confirms that inhibition of CREB ac-tivity is necessary for the SSTR5 agonist to inhibit PRL synthesis.Conclusion In pituitary prolactinoma,SSTR5 regulates prolactin synthesis by inhibiting the cAMP-CREB pathway.