ISO调控颌下腺细胞ADPR环化酶基因表达的信号转导通路研究

As a 42-45 kDa transmembrane glycoprotein, adenosine diphosphate ribosyl cyclase(ADPRC) is ubiquitously expressed in various cells and tissues including hematopoietic cells and organs such as spleen, liver, thymus, salivary gland, pancreas, heart, thyroid gland, ileum, colon, jejunum, brain, and eye et al. As a receptor on the plasma membrane, ADPRC has been shown to be an important surface immunoregulatory molecule with a multitude of diverse functions. As one of adhesion molecule (CD38), ADPRC also has adhesion properties and has been suggested to mediate selectin-type adhesion between different blood populations and vascular endothelial cells. As an enzyme, ADPRC has a catalytic domain in its long extracellular region that produces not only cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from NAD+ but also NAADP from NADP+. It should be noted that all of these products could enhance intracellular Ca2+ concentration in different ways. cADPR has been reported to mobilize Ca2+ from endoplasmic reticulum (ER) by affecting the ryanodine receptor on ER membrane. NAADP also induces Ca2+ release from the intracellular Ca2+ store, but not, it is thought, from ER. In the mouse submandibular glands, In pre-experiments, we found that ADPRC expression were significantly increased by isoproterenol(ISO,10-7mol/L), a β-adrenoceptors agonist, and the fuction of saliva secretion was enhanced. Despite a number of evidences of physiological function of molecules produced by ADPRC and itself in many cells and tissues, the signal pathway of ADPRC expression is still unclear. In pre-experiments,we found that the increased ADPRC expression was inhibited by actinomycin D, an transcriptional activity inhibitor of AP-1 and transfection of plasmid uncontain ras. Overexpression of RasGRP1 resulted in activation of Ras and JNK，as well as phosphatization of c-Jun. So, we hypothesize that after RasGRP1 is activated by ISO，the activated RasGRP1 regulated promoter of ADPRC by promoting the bind of Ras to GTP and activating Ras，which activate JNK and AP-1 subsequently. In this study, we will detect and analyse the effects of ISO on expression of ADPRC and elucidate the role of ISO in regulation of ADPRC promoter activation. And confirm the hypothesis of that the enhanced activity of ADPRC promoter by ISO vias promoting the bind of AP-1 and DNA reactive sequence. And make certain the role of activation of RasGRP1 and downstream of Ras/JNK pathway by ISO in the regulation of AP-1 DNA binding activity and ADPRC promoter activation.

腺苷二磷酸核糖环化酶（ADPRC）调控cADPR、NAADP等信息分子合成，调节细胞功能，又具有多种表面免疫调节和促分裂增生作用。异丙肾上腺素（ISO）促进颌下腺等组织表达ADPRC，但其表达调控信号转导机制尚不清楚。预实验发现，ISO增加ADPRC表达的作用可被AP-1抑制剂和转染Ras阴性质粒所抑制，RasGRP1过表达促进Ras激活，活化JNK，磷酸化c-Jun。设想：ISO激活RasGRP1后，通过促进Ras与GTP结合活化Ras，后者再激活JNK和AP-1，调控ADPRC启动子活性。本研究将检测ISO对颌下腺ADPRC表达的影响，明确ISO对ADPRC基因启动子活性的调控作用；证实ISO通过促进AP-1与相应DNA反应序列结合增强ADPRC启动子活性，确定ISO激活的RasGRP1及其下游Ras/JNK通路在调控AP-1 DNA结合活性及ADPRC启动子活性中的作用。

本项目通过整体动物模型和培养的颌下腺细胞探讨异丙肾上腺素（ISO）调控颌下腺细胞腺苷二磷酸核糖环化酶（ADPRC）基因表达的信号转导通路。整体水平的实验研究发现，ISO促进大鼠颌下腺的分泌功能，使颌下腺体积增大、重量增加，组织及细胞的ADPRC表达增多，发生细胞内转位，证实了ISO增强颌下腺细胞ADPRC表达进而介导功能增强的作用。这一结果拓展了ISO作为功能性受体的配体在涎腺特别是颌下腺分泌功能机制研究上的新靶点，可以为临床上涎腺分泌功能障碍等的治疗提供依据。通过转染重组质粒pGL3-basic-ADPRC promoter至颌下腺细胞证实了ISO激活ADPRC基因启动子、调控其活性的作用。用AP-1抑制剂curcumin或对照溶剂进一步探讨核转录因子作用的实验研究发现，ISO明显上调了磷酸化c-Fos及c-Jun的表达，促进了二聚体的形成，使AP-1合成增多。用生物素标记的AP-1双链探针通过凝胶迁移实验发现ISO刺激下增多的AP-1其DNA结合活性明显升高，进而通过转染含ADPRC启动子序列的荧光素酶报告基因质粒证实了ISO使AP-1结合到ADPRC基因启动子区的AP-1位点而产生正性调控作用；AP-1结合位点点突变实验研究从相反的方面证实了ISO/AP-1的这一作用。RNA干扰实验研究和免疫沉淀法检测的结果显示，ISO通过上调RasGRP1磷酸化激活Ras；Ras活化对下游激酶JNK影响的实验研究发现，ISO通过磷酸化RasGRP1活化Ras进而激活JNK使c-Jun和c-Fos磷酸化上调AP-1，证实了JNK处于ISO调控ADPRC基因表达信号转导通路的中心位置。进一步的实验研究发现，转染含ADPRC启动子序列荧光素酶报告基因质粒的细胞在ISO作用下，随着RasGRP1/Ras/JNK的顺序激活，荧光素酶活性明显升高，即ADPRC基因启动子活性明显上调，这一作用被进一步转染RasGRP1 siRNA、dominant-negative Ras质粒或与JNK抑制剂SP600125预孵育而消除，证实了RasGRP1/Ras/JNK的顺序激活上调了转录因子AP-1并增强其DNA结合活性介导了ISO上调ADPRC基因启动子活性的作用，明确了ISO调控ADPRC基因表达的信号转导通路，为这些信号分子功能研究提供了基础，也为临床相关疾病防治研究提供了参考。