DIFFERENTIAL GENE EXPRESSION IN SCLERODERMA FIBROBLASTS

硬皮病成纤维细胞中的差异基因表达

• 批准号: 6375363
• 负责人: DAVID R STREHLOW
• 金额: $ 23.72万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375363
• 关键词: collagenase; differential display technique; enzyme activity; fibroblasts; gel mobility shift assay; gene expression; genetically modified animals; heat shock proteins; immunoprecipitation; laboratory mouse; molecular chaperones; protease inhibitor; protein binding; protein protein interaction; scleroderma; serine proteinases; tissue /cell culture; transcription factor

The underlying basis of systemic sclerosis, scleroderma, is unknown. Cultured dermal fibroblasts from scleroderma patients overexpress extracellular matrix components, thus retaining a feature of scleroderma skin in the culture model. We used differential display and hybridization to large arrays of expressed sequence tags to compare gene expression in scleroderma and healthy fibroblasts. Our recently published data show that protease nexin 1, a protein that regulates matrix metabolism, is expressed in scleroderma skin but not in skin from healthy individuals. Because protease nexin 1 is known to inhibit the activation of collagenase, and because we have shown that protease nexin 1 induces collagen transcription, we created transgenic mice containing the human protease nexin 1 cDNA in a cytomegalovirus transcription unit. Part of the current proposal is to examine these mice as a potential model of fibrotic disease. We recently found another gene with more dramatic differential expression. Hybridization to large arrays of expressed sequence tags demonstrated that heat shock protein 90 (hsp90) is overexpressed in scleroderma fibroblasts. Northern analysis showed that hsp90 is highly expressed in scleroderma fibroblasts and not detected in healthy fibroblasts. Overexpression in healthy cells or heat shock itself caused a significant increase in endogenous collagen message. Overexpression of hsp90 also causes a 3.6-fold reduction in collagenase promoter activity (MMP1). Furthermore, a specific inhibitor of hsp90, geldanamycin, obliterates TGFbeta-induced collagen transcription. Hsp90 is known as a molecular chaperone. The chaperone activity of hsp90 is essential to the normal function of the hormone receptor. TGFbeta activates a receptor system, which in turn causes phosphorylation of a cytoplasmic protein called Smad. Phosphorylation of Smad causes its transport to the nucleus where it binds to a specific transcriptional regulatory sequence. Our recent novel finding is that hsp90 is a component of the Smad signaling complex. The second aim of this proposal is therefore to more firmly understand the overexpression of hsp90 in scleroderma skin. The final aim in this proposal is to define the nature of the interactions between Smad, hsp90 and related proteins in the Smad signaling complex and thus to understand how hsp90 functions in regarding TGF-beta signaling.

系统性硬化症，硬皮病的基础尚不清楚。 来自硬皮病患者的培养的真皮成纤维细胞过表达细胞外基质组分，从而在培养模型中保留硬皮病皮肤的特征。 我们使用差异显示和杂交表达序列标签的大阵列比较硬皮病和健康成纤维细胞的基因表达。我们最近发表的数据表明，蛋白酶nexin 1，一种调节基质代谢的蛋白质，在硬皮病皮肤中表达，但在健康个体的皮肤中不表达。 因为已知蛋白酶连接蛋白1抑制胶原酶的活化，并且因为我们已经表明蛋白酶连接蛋白1诱导胶原转录，所以我们创建了在巨细胞病毒转录单位中含有人蛋白酶连接蛋白1 cDNA的转基因小鼠。目前的部分建议是将这些小鼠作为纤维化疾病的潜在模型进行研究。我们最近发现了另一个具有更显著差异表达的基因。 杂交到大阵列的表达序列标签表明，热休克蛋白90（hsp 90）在硬皮病成纤维细胞过表达。 北方分析表明，热休克蛋白90在硬皮病成纤维细胞中高度表达，而在健康成纤维细胞中未检测到。 在健康细胞中的过表达或热休克本身引起内源性胶原蛋白信息的显著增加。hsp 90的过表达还导致胶原酶启动子活性（MMP 1）降低3.6倍。 此外，热休克蛋白90的特异性抑制剂格尔德霉素可消除TGF β诱导的胶原蛋白转录。Hsp 90被称为分子伴侣。 hsp 90的伴侣活性对激素受体的正常功能是必不可少的。 TGF β激活受体系统，这反过来又导致称为Smad的细胞质蛋白的磷酸化。Smad的磷酸化导致其转运至细胞核，在细胞核中其与特异性转录调控序列结合。 我们最近的新发现是，热休克蛋白90是一个组成部分的Smad信号复合物。因此，该建议的第二个目的是更坚定地理解硬皮病皮肤中hsp 90的过表达。 本提案的最终目的是确定Smad，hsp 90和Smad信号复合物中相关蛋白之间相互作用的性质，从而了解hsp 90如何在TGF-β信号传导中发挥作用。