REGULATION OF EXTRACELLULAR MATRIX BIOSYNTHESIS

这项建议的总体目标是阐明 人细胞中纤维连接蛋白生物合成的调控。此外， 将被调查的问题可能会发现新的 可应用于配位合成的调节机制 其他细胞外基质成分。这些研究也很重要 因为富含纤维连接蛋白的细胞外基质在 致癌转化是导致肿瘤许多特性的原因 恶性细胞。 纤维连接蛋白在所有类型的贴壁细胞中都有表达，但不表达 来自循环中的血细胞。转基因实验将用于 鉴定和鉴定纤维连接蛋白中的“沉默”元件 启动子，并研究它在这个不寻常的组织中的可能作用- 专一性。 我们的实验室已经获得了证据表明稳定性的变化 都参与了纤维连接蛋白的诱导 地塞米松及其对ras癌基因的抑制作用。纤维粘连蛋白 微基因和几个嵌合基因将用于转基因 检测5‘和3’端对信使核糖核酸稳定性作用的实验 纤维连接蛋白基因的非翻译区和第一外显子。 在确定了参与稳定性控制的RNA区域后， 与这些纤维连接蛋白mRNA位点特异结合的蛋白质将是 特色化的。 最后，各种癌基因对纤维连接蛋白的影响 将对生物合成进行分析。细胞由不同的 癌基因，包括ras、mos和src，降低了 纤维连接蛋白，与纤维连接蛋白的缺失之间有很强的相关性 纤维连接蛋白与肿瘤发生和转移表型的获得 在活体内。然而，癌基因作用的直接靶点和机制 人们对此仍然知之甚少。重金属诱导的癌基因将是 构造并用来表征不同的 TE-85细胞纤维连接蛋白生物合成的癌基因片段 骨肉瘤细胞。转录效应将与 癌基因的转录后效应及其机制 将被描述为。

The overall goal of this proposal is to elucidate various aspects of the regulation of biosynthesis of fibronectin in human cells. Furthermore, the questions that will be investigated are likely to uncover new regulatory mechanisms that may apply to the coordinate synthesis of other extracellular matrix components. These studies are also important because the disappearance of fibronectin-rich extracellular matrix upon oncogenic transformation is responsible for many of the properties of malignant cells. Fibronectin is expressed in all types of adherent cells but is absent from circulating blood cells. Transfection experiments will be used to identify and characterize a "silencer" element in the fibronectin promoter, and to investigate its possible role in this unusual tissue- specificity. Our laboratory has obtained evidence that alterations in the stability of fibronectin mRNA are involved in both the induction of fibronectin by dexamethasone and its repression by the ras oncogene. A fibronectin minigene and several chimeric genes will be used in transfection experiments to examine the role in mRNA stability of the 5' and 3' untranslated regions and of the first exon of the fibronectin gene. After identifying RNA regions involved in the control of stability, the proteins binding specifically to these fibronectin mRNA sites will be characterized. Finally, the effects of the various classes of oncogenes on fibronectin biosynthesis will be analyzed. Cells transformed by different oncogenes, including ras, mos and src, have decreased levels of fibronectin, and there is a strong correlation between loss of fibronectin and acquisition of the tumorigenic and metastatic phenotypes in vivo. However, the direct targets and mechanisms of oncogene action remain poorly understood. Heavy metal-inducible oncogenes will be constructed and used to chracterize the direct effects of different oncogene clases on fibronectin biosynthesis biosynthesis in TE-85 osteosarcoma cells. Transcriptional effects will be distinguished from posttranscriptional effects of oncogenes, and the mechanisms involved will be characterized.