The role of MAP kinases in the regulation of steroid hormone synthesis will be studied in a insect model system, the prothoracic gland of the tobacco hornworm Manduca sexta. Episodic increases in ecdysteroid synthesis by the gland coordinate gene expression during development. Ecdysteroid synthesis is acutely regulated by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH), in a system with considerable homology to vertebrate systems such as the ACT'H-adrenal cortex axis. These studies will expand upon previous work on signal transduction and steroidogenesis, with the ultimate goal of understanding the extrinsic and intrinsic controls of ecdysteroid synthesis. Preliminary experiments revealed that FTFH evokes a rapid increase in the phosphorylation of the MAP kinases known as ERKs (extracellular signal-regulated kinases). Inhibitors of ERK phosphorylation caused at least a partial inhibition of PTTH-stimulated ecdysteroid synthesis. Biochemical, physiological, pharmacological and molecular techniques will be used to rigorously determine the degree to which PTTH-dependent ecdysteroid and protein synthesis are dependent on activated ERKs. A second goal of the project is to determine as far as possible the upstream effectors of ERK activation and the downstream targets of ERKs. Among the candidate ERK targets, modulators of general and specific protein synthesis are of special interest because PTTH-stimulated ecdysteroid synthesis requires de novo translation. Upstream of ERKs, determining the possible involvement of cyclic AMP-dependent protein kinase (PKA) is of prime concern, since PKA activity is required for maximal PTTH-stimulated ecdysteroid synthesis but new data suggest that tyrosine kinases may also be involved. Feedback of ecdysteroids on the prothoracic gland down-regulates the ability of the gland to further synthesize ecdysteroids. The third goal of the project will address the possible ecdysteroid regulation of ERK levels as one way by which this negative feedback is accomplished. These studies will shed light on how intracellular regulatory pathways interact and should be of special interest to the endocrine field because prothoracic glands are not proliferative. They thus provide a system in which the acute stimulation of steroid synthesis is uncoupled from cell division.
