These studies explore molecular mechanisms by which epidermal growth factor and glucocorticoids modulate synthesis of bioactive lipids. They develop our new findings that: (1) treatment of confluent primary cultures of mouse embryo palate mesenchyme (MEPM) cells with epidermal growth factor (EGF) induces gene expression of the newly discovered, arachidonyl-specific phospholipase A2c(PLA2c), and (2) dexamethasone, a cleft palate-inducing glucocorticoid, inhibits EGF-induced accumulation of PLA2c mRNA. These data demonstrate for the first time coordinate regulation of transcription and translation of the PLA2c gene and its inhibition by glucocorticoids. Furthermore, they reveal a common molecular pathway by which EGF and glucocorticoids may regulate biochemical differentiation of embryonic palatal cells. This project aims to determine the molecular mechanisms underlying regulation of gene expression of PLA2c. Cis-acting promoter/enhancer sequences in a PLA2c genomic clone will be sequenced and defined by transient transfection assay. The cis-acting control elements involved in glucocorticoid-inhibition of EGF-induced gene expression of PLA2c will be sequenced to determine the presence of glucocorticoid-responsive elements and analyzed by transfection assay to determine those elements necessary for glucocorticoid-inhibition of EGF-induced gene expression of PLA2c. Cis-acting control elements which interact with glucocorticoid-induced trans-acting proteins will be identified by band- shift and photo-affinity labeling techniques. Lastly, the possibility that genetic differences in glucocorticoid-sensitivity to induction of cleft palate might reside in genetic differences in regulation of gene expression of PLA2c will be determined (1) by structural analysis of cis- regulatory sequences from the glucocorticoid-sensitive A/J strain and the -less sensitive C57BL/6J strain embryo palatal cells, and (2) by assay of the effects of dexamethasone on PLA2c gene expression in A/J and C57BL/6J strain MEPM cells transfected with homologous and heterologous cis-acting elements. In completing these aims we will define a molecular mechanism by which EGF and glucocorticoids induce and alter differentiation of embryonic cells at the biochemical level. Understanding such molecular mechanisms is essential to the long term goal of devising strategies to prevent birth defect.
