It is known that several hormonal primary messengers are capable of regulating developmentally significant biological responses in embryonic palatal tissue by modulating intracellular levels of cAMP. The effects of cAMP on metabolic pathways in eukaryotes are mediated by cAMP- dependent protein kinases (cAMP-dPK) and we have demonstrated spatial and temporal alterations in these enzymes in tissue of the developing mammalian secondary palate. Our studies characterizing cAMP-dPK in tissue derived from the developing mammalian palate has allowed consideration of cAMP-dPK as a key regulatory enzyme, capable of transducing hormonally elevated intracellular levels of cAMP into metabolic responses during orofacial ontogenesis. Our overall objectives in this application include: 1) Determination of whether the temporal alterations in soluble cAMP-dPK that we have defined are the result of transcriptional regulation of cAMP-dPK gene expression; 2) Utilizing a variety of """"""""blocking"""""""" strategies (introduction of A) antisense RNA or b) a recombinant expression vector encoding cAMP-dPK inhibitor protein or c) a membrane permeable specific competetive inhibitor of cAMP-dPK types I and II) to precisely define a functional role for cAMP-dPK in palate medial edge epithelial cell differentiation, mesenchymal cell proliferation and/or GAG synthesis; 3) Analysis of the regulation of cAMP-dPK regulatory subunit gene expression by transfection of cAMP-dPK regulatory and catalytic subunits into palate mesenchymal cells; 4) Determination of the presence and distribution of a cyclic AMP response element-binding protein (CREBP) in developing embryonic palatal tissue. These studies will directly test hypotheses regarding the functional role that cAMP-dPK plays in various aspects of palatal tissue differentiation and define the means by which cellular regulatory signals affect gene expression during embryonic palate development.
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