Bile acid-induced colon cancer cell proliferation

描述（由申请方提供）：本提案中待检验的中心假设是胆汁酸通过与M3毒蕈碱受体（M3 R）相互作用诱导结肠癌细胞增殖，从而引起表皮生长因子受体（EGFR）的反式激活。为了阐明导致胆汁酸诱导的结肠癌细胞增殖的受体后信号传导并确定EGFR反式激活的需要和机制，提出了以下具体目的：1.进一步阐明受体后信号转导途径介导胆碱能激动剂诱导的EGFR反式激活并刺激结肠癌细胞增殖。1a.为了用于拟定的研究，除了天然表达M3 R和EGFR的细胞外，还将用M3 R和/或EGFR的cDNA克隆转染结肠癌细胞。1b.胆碱能激动剂诱导的细胞增殖的机制将使用免疫印迹来探测p44/42 MARK、p38 MARK和JNK通路中的活化蛋白，并通过检查磷脂酶C和蛋白激酶C活化和Ca 2+动员的作用来研究。将通过检查EGFR磷酸化和使用EGFR抑制剂、反义寡核苷酸和显性阴性突变体来证实胆碱能激动剂诱导的EGFR反式激活的需要。确定M3 R和EGFR共表达的必要性，并阐明胆汁酸调节结肠癌细胞增殖的分子机制。2a.将使用放射性配体结合试验、活化p44/42 MARK、p38 MARK和JNK级联蛋白的免疫印迹以及EGFR抑制剂、反义寡核苷酸和显性阴性突变体，确定胆汁酸诱导的受体后信号传导以及M3 R和EGFR共表达的要求。2b.将阐明胆汁酸诱导的与结肠癌增殖相关的转录因子（p90 RSK、p38 MARK和JNK）和基因（CREB、NF-κ B、c-Fos和c-Jun）的活化和表达。3.探讨胆汁酸诱导人结肠癌细胞EGFR反式激活的分子机制。3a）EGFR配体（包括HB-EGF）的表达和释放将使用免疫印迹、北方印迹和RT-PCR来确定，并且胆汁酸诱导的EGFR反式激活对EGFR配体释放的依赖性将使用EGFR抗体、金属蛋白酶抑制剂和EGFR的特异性抗体和抑制剂来确定。3b）通过免疫印迹和原位杂交鉴定结肠癌细胞金属蛋白酶，并通过使用金属蛋白酶抑制剂和抗血清以及通过敲低金属蛋白酶表达来确定这些酶在介导胆汁酸诱导的HB-EGF释放中的作用。3c）胆汁酸激活金属蛋白酶的机制将通过探索PKC、Ca 2+和Src在胆汁酸诱导的HB-EGF释放中的作用来确定。

DESCRIPTION (provided by applicant): The central hypothesis to be tested in this proposal is that bile acids induce colon cancer cell proliferation by interaction with M3 muscarinic receptors (M3R), thereby causing transactivation of epidermal growth factor receptors (EGFR). To elucidate post-receptor signaling that results in bile acid-induced colon cancer cell proliferation and to determine the requirement for, and mechanism of, transactivation of EGFR the following Specific Aims are proposed: 1. To elucidate further post-receptor signal transduction pathways which mediate cholinergic agonist-induced transactivation of EGFR and stimulate colon cancer cell proliferation. 1a. For use in the proposed studies, in addition to cells that naturally express M3R and EGFR, colon cancer cells will be transfected with cDNA clones for M3R and/or EGFR. 1b. The mechanism of cholinergic agonist-induced cell proliferation will be studied using immunoblotting to probe for activated proteins in the p44/42 MARK, p38 MARK and JNK pathways, and by examining the roles of phospholipase C and protein kinase C activation and Ca2+ mobilization.1c. The requirement for cholinergic agonist-induced transactivation of EGFR will be confirmed by examining EGFR phosphorylation and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants.2. To determine the requirement for co-expression of M3R and EGFR and delineate the molecular mechanisms whereby bile acids regulate colon cancer cell proliferation. 2a. Bile acid-induced post-receptor signaling and the requirement for co-expression of M3R and EGFR will be determined using radioligand binding assays, immunoblotting for activated p44/42 MARK, p38 MARK and JNK cascade proteins, and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants. 2b. Bile acid-induced activation and expression of transcription factors (p90RSK, p38 MARK and JNK) and genes (CREB, NF-kappaB, c-Fos and c-Jun) related to colon cancer proliferation will be elucidated. 3. To determine the molecular mechanism in human colon cancer cells of bile acid-induced transactivation of EGFR. 3a) Expression and release of EGFR ligands, including HB-EGF, will be determined using immunoblots, northern blots, and RT-PCR, and the dependence of bile acid-induced EGFR transactivation on release of EGFR ligands will be determined using EGFR antibodies, metalloproteinase inhibitors, and specific antibodies and inhibitors for EGFR. 3b) Colon cancer cell metalloproteases will be identified by immunoblotting and in situ hybridization and the role of these enzymes in mediating bile acid-induced HB-EGF release will be determined by using metalloprotease inhibitors and antisera, and by knocking down metalloprotease expression. 3c) The mechanism whereby bile acids activate metalloproteases will be determined by exploring the roles of PKC, Ca2+, and Src in bile acid-induced HB-EGF release.