REGULATION OF EXTRACELLULAR MATRIX BIOSYNTHESIS

本提案的总体目标是阐明 调节人细胞中纤连蛋白的生物合成。 此外，委员会认为， 将被调查的问题可能会发现新的 可能适用于协调合成的调节机制 其他细胞外基质成分。 这些研究也很重要 因为富含纤连蛋白的细胞外基质的消失， 致癌转化是负责的许多性质， 恶性细胞 纤维连接蛋白在所有类型的贴壁细胞中表达，但不表达 从循环的血细胞。 转染实验将用于 鉴定和表征纤连蛋白中的"沉默物"元件 启动子，并研究其在这种不寻常的组织中的可能作用- 的特异性 我们的实验室已经获得证据表明， 纤维连接蛋白mRNA的表达参与纤维连接蛋白的诱导， 地塞米松及其被ras癌基因抑制。 纤连蛋白 在转染中将使用小基因和几种嵌合基因 实验以检查5 '和3'末端的mRNA稳定性中的作用。 非翻译区和纤连蛋白基因的第一个外显子。 在鉴定了参与稳定性控制的RNA区域后， 与这些纤连蛋白mRNA位点特异性结合的蛋白质将被 表征了 最后，各种类型的癌基因对纤维连接蛋白的影响 生物合成将被分析。 细胞转化不同 癌基因，包括ras，mos和src， 纤维连接蛋白，并且在纤维连接蛋白的丢失之间存在很强的相关性。 纤维连接蛋白与肿瘤发生和转移表型的获得 in vivo. 然而，癌基因作用的直接靶点和机制 仍然知之甚少。 重金属诱导的癌基因将被 建造并用于描述不同的直接影响 TE-85细胞中纤维连接蛋白生物合成的癌基因分类 骨肉瘤细胞 转录效应将与 癌基因的转录后效应及其机制 将被定性。

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.