REGULATION OF MIS TYPE II RECEPTOR AND TARGET GENES

MIS II 型受体和靶基因的调控

基本信息

批准号：
2722990
负责人：
JOSE M. TEIXEIRA
金额：
$ 11.97万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-04-01 至 2003-03-31
项目状态：
已结题

项目摘要

Mullerian Inhibiting Substance, a member of the TGF-beta superfamily of growth and differentiation factors, is produced by Sertoli cells during embryonal development and is required for normal reproductive development in male embryos. The signal activity of MIS is the regression of the Mullerian duct, the precursor of the uterus, fallopian tubes and upper vagina. MIS is also produced both in the adult testis by Sertoli cells and in the ovary by granulosa cells, where its exact role remains to be fully explored. Based on in vitro and in vivo evidence, it is our hypothesis that signal transduction by MIS, via its heteromeric serine/threonine kinase receptor, is required to maintain reproductive competence of the gonad and to prevent hyperplastic growth. The goal of this proposal is to (I) understand the developmental, cell- specific and sexually dimorphic molecular mechanisms regulating the expression of the MIS type II receptor (MISrII) in Leydig cells, Sertoli cells, and granulosa cells during different stages of the development and also in the mesenchymal cells surrounding the Mullerian duct during embryonal development and (II) to uncover target genes whose expression is regulated by MIS signal transduction. Since we have cloned the MISrII gene with its TATA-less promoter and have identified cell lines expressing endogenous MISrII, we now have the tools to study expression of MISrII and its downstream target genes. To analyze the MISrII promoter, we will express chimeric promoter/ reporter constructs in MIS type II receptor expressing cells, then perform DNase I footprinting and gel shift analysis, with nuclear extracts prepared from those cells, to determine the cis-acting DNA elements necessary and sufficient for transcription and to examine the role of TFII-I in assembly of the pre-initiation complex. Sequences identified will be used for oligoaffinity purification of trans-acting factors which bind to those sequences. We will also immortalize the Mullerian duct mesenchymal cells which undergo apoptosis and regression in response to MIS to provide cell lines in which to identify downstream, transcriptionally regulated target genes of MIS participating in this important process. We will approach this by studying candidate genes that we might expect to be regulated. We will also perform subtractive hybridization with both R2C cells which respond to MIS, express the receptor, and from which we recently constructed a cDNA library. Information uncovered by these studies will contribute to our understanding of how MIS initiates apoptosis and causes G1 arrest and that these molecular mechanisms can be harnessed for the control of tumors known to respond to MIS, such as human ovarian cancer.

苗勒管抑制物质，TGF-β超家族的成员生长和分化因子，由支持细胞在生长过程中产生胚胎发育，是正常生殖所必需的男性胚胎的发育。 MIS 的信号活动是苗勒氏管（子宫、输卵管的前身）退化管和上阴道。 MIS 也在成人睾丸中产生由支持细胞和卵巢中的颗粒细胞组成，其确切的位置的作用还有待充分探讨。基于体外和体内证据，我们的假设是 MIS 的信号转导通过其异聚丝氨酸/苏氨酸激酶受体，需要维持性腺的生殖能力并防止增生性生长。该提案的目标是（I）了解发育、细胞- 调节特定和性别二态性的分子机制 MIS II 型受体 (MISrII) 在间质细胞、支持细胞中的表达不同发育阶段的细胞和颗粒细胞以及苗勒氏管周围的间充质细胞胚胎发育和（II）发现其表达的靶基因受 MIS 信号转导调节。由于我们已经克隆了 MISrII 基因及其无 TATA 启动子，并且已经鉴定出表达内源性 MISrII 的细胞系，我们现在拥有研究 MISrII 及其下游靶基因表达的工具。为了分析 MISrII 启动子，我们将表达嵌合启动子/ 在 MIS II 型受体表达细胞中构建报告基因，然后使用核进行 DNase I 足迹分析和凝胶位移分析从这些细胞中制备提取物，以确定顺式作用 DNA 转录和检查所必需和充分的元素 TFII-I 在预起始复合物组装中的作用。序列鉴定将用于反式作用的寡亲和纯化与这些序列结合的因子。我们也将永垂不朽苗勒氏管间充质细胞发生凋亡和退化响应 MIS，提供可识别的细胞系 MIS 的下游转录调控靶基因参与这一重要进程。我们将通过以下方式解决这个问题研究我们可能期望受到调控的候选基因。我们将还与响应的 R2C 细胞进行消减杂交到 MIS，表达受体，我们最近从中构建了一个 cDNA文库。这些研究发现的信息将有助于帮助我们了解 MIS 如何启动细胞凋亡并导致 G1 期停滞并且可以利用这些分子机制来控制已知对 MIS 有反应的肿瘤，例如人类卵巢癌。