Human Choriogonadotropin Signaling in Cell Proliferation

描述(申请人提供)：人绒毛膜促性腺激素在人类生殖过程中起关键作用，在怀孕前三个月产生。它也是由多种肿瘤引起的，包括绒毛膜癌、葡萄胎和睾丸的胚胎癌。人绒毛膜促性腺激素在结构上与肿瘤生长因子-β、神经生长因子和血小板衍生生长因子-β在某些方面相似。它们都含有半胱氨酸结基序，与多种肿瘤的发生发展密切相关。人绒毛膜促性腺激素能刺激细胞生长和分化，因此，其功能很可能是由三个成员的MAK信号通路调节的，即有丝分裂原激活的蛋白激酶-激酶(MAPKKK)--MAPKK--细胞外信号调节激酶(ERK)。本研究旨在探讨MAPK通路在hCG诱导的细胞生长和分化中的作用。利用特定途径的DNA微阵列技术比较hCG和EGF信号在大鼠SIGC-hCG/；促性腺激素受体(LHR)和SIGC-表皮生长因子受体(EGFR)中与各自受体相互作用时的基因表达谱。这应该会给我们提供一些关于两个信号通路共同的基因表达的信息。ERK下游和MAPKKK上游的信号蛋白将被研究。最后，将寻找一种支架蛋白，在其上可能组织这些激酶。这些研究将在大鼠SIGC上进行。SIGC具有上皮形态，在无黄体化的培养中生长。初步研究的具体短期目标将包括：1)分别将hCG/LH和EGF受体cDNA导入大鼠SIG细胞，筛选稳定的转染体SIGC-hCG/LHR和SIGC-EGFR；2)SIGC-hCG/LHR和SIGC-EGFR的初步鉴定，包括a)用125IhCG和125IEGF结合分析hCG/LH和EGF受体的存在和浓度；b)研究hCG对腺苷酸环化酶、磷脂酶C和EGF对受体酪氨酸激酶活性的影响；3)研究hCG和EGF对Raf、MEK和ERK活性的影响；4)用DNA芯片比较hCG和EGF在SIGC-hCG/LHR和SIGC-EGFR中的信号基因表达谱；5)利用特异性多克隆抗体结合或免疫沉淀技术分别检测ERK和Raf激酶的下游和上游信号蛋白；6)寻找MAP激酶模块的支架蛋白。

DESCRIPTION (provided by applicant): hCG plays a key role in human reproduction and is produced during the first trimester of pregnancy. It is also produced by a variety of tumors including choriocarcinoma, hydatidiform mole, and embryonic carcinoma of the testis. Structurally, hCG is similar in some respects to tumor growth factor (TGF)-Beta, nerve growth factor (NGF) and platelet-derived growth factor (PDGF)-Beta. All of them contain cystine knot motif and have been implicated in the development of a variety of tumors. hCG stimulates cell growth and differentiation and, therefore, it is highly likely that its function is regulated by the three member MAK kinase cascade, mitogen-activated protein kinase-kinase-kinase (MAPKKK)--MAPKK-extracellular signal-regulated kinase (ERK). The objectives of the proposed studies are to investigate the involvement of the MAP kinase pathway in the cell growth and differentiation by hCG. Pathway-specific DNA microarray technology will be employed to compare the gene expression profiles of hCG and epidermal growth factor (EGF) signaling when they interact with their respective receptors in signaling in a rat SIGC-hCG/; lutropin receptor (LHR) and SIGC-epidermal growth factor receptor (EGFR). This should give us some information on the expression of genes that are common to both signaling pathways. The signaling proteins downstream of ERK and upstream of MAPKKK will be investigated. Lastly, search for a scaffold protein on which these kinases might be organized will be made. The studies will be carried out with the rat SIGC. SIGC has an epithelial morphology and grows in culture without luteinization. Specific short-term goals of the initial studies will include: 1) Transfection of rat SIG cells with hCG/LH and EGF receptor cDNAs separately and selection of stable transfectants, SIGC-hCG/LHR and SIGC-EGFR; 2) Preliminary characterization of SIGC-hCG/LHR and SIGC-EGFR, which will involve a) determination of the presence and concentration of hCG/LH and EGF receptors by binding assays using 125IhCG and 125IEGF and b) study of the effect of hCG on the activation of adenylyl cyclase and phospholipase C and of EGF on the activation of receptor tyrosine kinase; 3) Study the effect of hCG and EGF on the activities of Raf, MEK and ERK; 4) Comparison of the gene expression profiles of signaling by hCG and EGF in SIGC-hCG/LHR and SIGC-EGFR by DNA microarrays; 5) Determination of the downstream and upstream signaling proteins of ERK and Raf kinases respectively by association or immunoprecipitation techniques using specific polyclonaI antibodies; and 6) Search for a scaffold protein for MAP kinase module will be made.