FUNCTION OF COFACTORS MODIFYING THYROID HORMONE RECEPTOR FUNCTION

1.皮质醇腺瘤p53和ACTH受体基因突变的分析在良性皮质醇腺瘤中筛选p53和ACTH受体基因可能的突变。在11例CPA手术标本中，p53和ACTH-R基因均未检测到突变。因此，这些基因的突变是CPA的罕见原因。促肾上腺皮质激素受体（ACTH-R）在皮质醇分泌腺瘤（CPA）中表达的分析我们和其他人证明ACTH-R基因的表达受其自身配体ACTH的上调，如果同样的调节作用也适用于CPA，则ACTH-R的表达应受到抑制，因为内源性ACTH分泌的缺乏。然而，临床证据表明，对库欣综合征患者给予ACTH导致皮质醇产生进一步增加，提示功能性ACTH-R的表达。Northem印迹分析表明CPA表达ACTH-R mRNA比邻近的非肿瘤组织更丰富。 ...更多信息 stic肾上腺。提示ACTH-R基因表达的调控与正常肾上腺不同。为了更深入地了解ACTH-R的表达是如何调控的，我们克隆了ACTH-R基因的启动子区。为了评估启动子的功能，通过将启动子区或其缺失突变体置于萤火虫荧光素酶基因的上游来构建报告基因。通过构建报告基因，证明SF-1负责肾上腺特异性表达。此外，AP-1和SF-1均参与ACTH依赖性的受体基因上调。皮质醇生成腺瘤（CPA）特异性表达基因的鉴定为了深入了解CPA的发病机制，从10个CPA样品和邻近的萎缩正常组织中分离mRNA。在合成相应的双链cDNA后，用Dpn II限制它们并连接接头，从而构建代表CPA或正常cDNA的每个扩增子。PCR差减分析显示，有几个克隆在CPA中优先表达，其中谷胱甘肽-S-转移酶A-1（GST-A1）在所有CPA组织中的表达均明显高于相应正常组织。抑制这种酶的依他克林酸引起严重减少的细胞系的增殖来自肾上腺皮质癌。提示GST-A1在CPA的发生发展中起重要作用。少

1. ANALYSIS OP MUTATIONS IN p53 AND ACTH RECEPTOR (ACTH-R) GENES IN CORTISOL-PRODUCING ADENOMAS (CPA)Possible mutations in p53 and ACTH-R genes were screened in benign adrenocortical adenomas producing cortisol. In eleven CPA samples obtained at surgery, no mutation was detected either in p53 and ACTH-R genes. It was thus suggested that mutations in these genes are rare cause of CPA.2. ANALYSIS OF ACTH RECEPTOR (ACTH-R) EXPRESSION IN CORTISOL-PRODUCING ADENOMAS (CPA)We and others demonstrated that expression of ACTH-R gene is upregulated by its own ligand ACTH.If the same regulation applies to CPA, expression of ACTH-R should be suppressed because of the lack of endogenous ACTH secretion. However, clinical evidence that ACTH administration to the patients with Cushing's syndrome resulted in further increase in cortisol production, suggesting the expression of functional ACTH-R.Northem blot analysis demonstrated that CPA expresses ACTH-R mRNA more abundantly than the adjacent non-neopla … More stic adrenal gland. It was thus suggested that regulation of ACTH-R gene expression is different from that of normal adrenal gland. To gain more insight how ACTH-R expression is regulated, we cloned the promoter region of ACTH-R gene. To assess the function of the promoter, reporter genes were constructed by placing the promoter region or its deletion mutants to the upstream of the firefly luciferase gene. By transfecting the reporter gene constructs, it was demonstrated that SF-1 is responsible for adrenal specific expression. Furthermore, both AP-1 and SF-1 are shown to be involved in ACTH-dependent upregulation of its receptor gene.3. SEARCH FOR GENES SPECIFICALLY EXPRESSED IN CORTISOL-PRODUCING ADENOMAS (CPA)To gain insight for the pathogenesis of CPA, mRNAs were separated from 10 CPA samples and adjacent atrophic normal tissues. After the synthesis of corresponding double stranded cDNA, they were restricted with DpnII and adaptors were ligated, resulting in the construction of each amplicon representing CPA or normal cDNAs. PCR-based subtraction revealed that several clones were identified to be preferentially expressed in CPA.Among them, glutathione-S-transferase A-1 (GST-A1) was marked upregulated in all the CPA samples when the expression was compared with that in corresponding normal tissues. Inhibition of this enzyme by ethacrinic acid caused severe reduction of the proliferation of the cell line derived from adrenocortical cancer. It was thus suggested GST-A1 plays an important role for the tumorigenesis of CPA. Less