DIFFERENTIAL GENE EXPRESSION IN SCLERODERMA FIBROBLASTS

系统性硬化症的潜在基础是硬皮病，目前尚不清楚。硬皮病患者培养的真皮成纤维细胞过度表达细胞外基质成分，从而在培养模型中保留了硬皮病皮肤的特征。我们使用差异显示和对大量表达序列标签的杂交来比较硬皮病和健康成纤维细胞中的基因表达。我们最近发表的数据显示，调节基质代谢的蛋白--蛋白酶Nexin 1在硬皮病皮肤中表达，但在健康人的皮肤中不表达。由于已知蛋白酶Nexin 1可以抑制胶原酶的激活，而且我们已经证明了蛋白酶Nexin 1可以诱导胶原转录，因此我们在巨细胞病毒转录单元中创建了含有人蛋白酶Nexin 1基因的转基因小鼠。当前提议的一部分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差异更大的基因。与大量表达序列标签的杂交表明，热休克蛋白90(HSP90)在硬皮病成纤维细胞中过表达。Northern分析表明，HSP90在硬皮病成纤维细胞中高表达，而在健康成纤维细胞中未检测到。健康细胞的过度表达或热休克本身会导致内源性胶原信息的显著增加。HSP90的过表达还导致胶原酶启动子活性(MMP1)降低3.6倍。此外，热休克蛋白90的特异性抑制剂格尔达那霉素可以阻断转化生长因子β诱导的胶原转录。HSP90被认为是一种分子伴侣。HSP90的伴侣活性对激素受体的正常功能是必不可少的。TGFbeta激活一个受体系统，进而导致一种名为Smad的细胞质蛋白的磷酸化。Smad的磷酸化导致其运输到细胞核，在那里它与特定的转录调控序列结合。我们最近的新发现是，HSP90是Smad信号复合体的一个组成部分。因此，这项建议的第二个目的是更确切地了解硬皮病皮肤中HSP90的过度表达。这项建议的最终目的是确定Smad、HSP90和Smad信号复合体中相关蛋白质之间相互作用的性质，从而了解HSP90如何在有关转化生长因子-β信号转导中发挥作用。

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.