REGULATION OF EXTRACELLULAR MATRIX BIOSYNTHESIS

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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.