FUNCTION AND REGULATION OF TRESPIN, A NOVEL SERPIN

本提案的目的是阐明一种新的丝氨酸蛋白酶抑制剂的生物学功能和作用机制，该丝氨酸蛋白酶抑制剂名为Trespin（TGF-β-抑制性丝氨酸蛋白酶抑制剂），我们已经通过差异显示RT-PCR在NRP-152细胞中鉴定出该丝氨酸蛋白酶抑制剂。我们还将研究TGF-β和其他凋亡诱导剂下调Trespin转录的机制，以及Trespin致癌的潜在作用。我们认为，由于以下原因，Trespin作为由TGF-β和许多其他凋亡诱导剂诱导的凋亡的负调节剂发挥作用。Trespin的缺失与许多药物诱导的细胞凋亡密切相关，重组Trespin阻断HL 60和Jurkat细胞胞质中caspase 3的活化以及NRP-152细胞胞质中ICE样caspase的活化。此外，由TGF-β引起的侵入蛋白表达的丧失先于细胞凋亡的诱导，并且通过转录机制发生。我们计划通过检查用含有正义或反义Trespin cDNA的表达载体转染或逆转录病毒感染的细胞中Trespin的体内生物学功能来检验Trespin是细胞凋亡调节因子的假设。我们计划研究的调控Trespin表达的启动子元件和转录因子的特点负责其负调控的肿瘤诱导剂。为此，我们将分离的侵入启动子，研究转录调控的侵入启动子融合到荧光素酶报告构建体的各种元素，并确定与这些因素和迁移率变化分析与核提取物的表达构建体共转染所涉及的转录因子。我们认为，从这些后者的研究中获得的数据将有助于识别一种新的转录因子和/或一个已知的转录因子在细胞凋亡的调控及其作用机制的作用。最后，我们将研究Trespin在癌症发生中的作用。这将通过将多种恶性和癌前细胞和组织中的Trespin表达与其恶性表型相关联来完成。这也将通过确定在用有义或反义侵入蛋白转染/感染的细胞中侵入蛋白cDNA的过表达或低表达是否会改变其致瘤表型来完成。

The goal of this proposal is to elucidate the biological function, and mechanism of action of a novel serpin named Trespin (for TGF-beta- repressible serpin) that we have identified in NRP-152 cells by differential display RT-PCR. We will also study the mechanism of transcriptional down-regulation of Trespin by TGF-beta and other apoptosis inducers, and the potential role of Trespin carcinogenesis. We believe that Trespin plays a role as a negative-regulator apoptosis induced by TGF-beta and many other apoptosis inducing agents for the following reasons. Loss of Trespin correlates well with the induction of apoptosis by many agents, and recombinant Trespin blocks the activation of caspase 3 in HL60 and Jurkat cell cytosols and an ICE-like caspase in NRP-152 cell cytosol. Moreover, loss of Trespin expression by TGF-beta precedes the induction of apoptosis and occurs through a transcriptional mechanism. We plan to test the hypothesis that Trespin is a regulator of apoptosis by examining the biological function of Trespin in vivo in cells transfected or retrovirally infected with expression vectors containing sense or anti-sense Trespin cDNAs. We plan to study the regulation of Trespin expression by characterizing the promoter elements and transcription factors responsible for its negative regulation by apoptosis-inducing agents. For this we will isolate the Trespin promoter, study transcriptional regulation of various elements of the Trespin promoter fused to a luciferase reporter construct and identify the transcription factors involved by co-transfection with expression constructs for such factors and mobility shift assays with nuclear extracts. We feel that the data obtained from these latter studies will help the identification of a novel transcriptional factor and/or the role of a known transcription factor in the regulation of apoptosis and its mechanism of action. Lastly, we will study the role of Trespin in carcinogenesis. This will be done by correlating expression Trespin in a variety of malignant and pre-malignant cells and tissues with their malignant phenotype. This will also be done by determining whether over- expression or under-expression of Trespin cDNA in cells transfected/infected with sense or anti-sense Trespin will alter their tumorigenic phenotype.