In the previous project period we have found that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds cause a reduction in epidermal growth factor (EGF) binding to its receptor with a concurrent rise in activities of several protein kinases, particularly protein tyrosine kinases in the hepatic plasma membrane of several experimental species. The major objective of this project is to ask what the toxicological consequences of elevated protein tyrosine kinase activities (Project 1-3) are and why TCDD causes such an effect on EGF receptor (Project 4 and 5). In more specific terms we plan to study (1) the consequence of TCDD-caused down-regulation of EGF receptor (EGF-R) in skin cells to test the possibility that TCDD-caused activation of EGF-R is the major cause for their subsequent proliferation and differentiation response; (2) TCDD-caused EGF-R and other protein tyrosine kinase changes in thymus, particularly with regard to their implication on thymocyte maturation and thymic involution; (3) effects of activation of EGF-R insulin-R and other tyrosine kinases and protein kinase C on pancreas, especially changes in several specific protein substrates known to regulate insulin secretion; (4) the possibility that TCDD's action is mediated through c-erbA, a member of a family of genes known to mediate steroid and thyroid hormones; and (5) to test the hypothesis that c-erbA eventually activates c-erbB, a family of genes producing protein tyrosine kinases including EGF-R. The rationale of such an approach is that TCDD given in vivo causes marked increases in protein tyrosine kinase activities at an early stage of poisoning (24 to 48 hrs) and that in the past few years a tremendous advance has been made in the understanding of the vital roles of some of the protein tyrosine kinases. We will, therefore, examine several highly TCDD-sensitive tissues where certain protein tyrosine kinases have been already shown to play key roles. We plan to test our hypothesis that TCDD first stimulates the expression of one of c-erbA genes (as in the case of steroid/thyroid hormones) which in turn signals a rise in expression of the c-erbB family of genes, endcoding proteins with tyrosine kinase activities. For this purpose several gene transfection/deletion experiments on otherwise TCDD nonresponsive cells will be carried out using c-erbA and c-erbB (EGF-R) to analyze each step of TCDD-triggered cascade of events leading to elevation of the level of target receptors such as EGF-R and other protein tyrosine kinases.
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