The CYP1A1 protein contributes to the phase 1 metabolism of a variety of xenobiotics and carcinogens, and has been implicated as a factor in the development of environmentally-based human disease. Induction of the CYP1A1 gene by ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent upon the cell cycle stage and proliferative/ differentiation status of the target tissue, and is suppressed in some cell types by exposure to mitogens and phorbol esters and constitutive expression of activated p21-ras. In each of these cases suppression of Cyp1a-1 induction correlates with an activation of components of the MEK/MAP kinase cascade, and an inability of the aryl hydrocarbon receptor (AhR) complex to bind to DNA. The hypothesis to be tested in this proposal is that members of MEK/MAP kinase cascade, and the signal transduction pathways that activate them, modulate CYP1A1 induciblity as a consequence of their altering the function of the AhR complex. This hypothesis will be tested by analyzing Cyp1a-1 inducibility and AhR function in cell lines in which constitutive activation of the MEK/MAP kinase cascade is achieved by transfection of v-Ha-ras, v-raf, or mutated MEK genes. Analyses of Cyp1a-1 induction will also be performed in keratinocyte preparations isolated from transgenic mice have constitutively activated MEK/MAP kinases as a consequence of targeted expression of a Ha-ras oncogene in their skins. A cell-free in vitro reconstitution system will be used to determine if cellular extracts containing activated MEK/MAP kinases suppress the TCDD-dependent transformation of the AhR and its binding to dioxin response elements (DREs) in DNA, and whether this suppression can be eliminated by prior immunodepletion of the MEK and MAP kinases. If warranted, the investigators will determine if DNA-AhR complex binding can be suppressed by incubation of components of the AhR complex with purified MEK/MAP kinases in vitro. If so, they will determine which component of the AhR complex is phosphorylated. The relationship between DRE binding and the phosphorylation of specific sites on either the AhR or ARNT proteins will be assessed by site- directed mutagenesis. Sucrose gradient analyses, immune coprecipitation protocols, and in vivo footprinting will be used to determine whether loss of DNA binding in gel retardation assays reflects inhibition of AhR and ARNT heterodimer formation, or an inability of the AhR-ARNT hetrodimer to bind to DNA. Lastly, since MAP kinases are activated in G2/M, late G1, and shortly after serum stimulation of Go arrested cells, one would predict that Cyp1a-1 inducibility and AhR function should be suppressed at various stages of the cell cycle. This prediction will be tested by analyzing Cyp1a-1 inducibility and AhR function in cell cycle stage specific cell populations. The proposed research examines an innovative hypothesis that provides a unified mechanism for the regulation of Cyp1a-1 induction and AhR function by multiple and diverse signal transduction pathways.
Showing the most recent 10 out of 47 publications
