This research proposal will investigate the underlying mechanisms and neuronal specificity of glucocorticoid regulation of calcitonin/CGRP gene expression. The foundation for this investigation is the observation that glucocorticoids can either increase or decrease calcitonin/CGRP gene transcription in different cultured cell lines and possibly in the rat nervous system. To establish that neuron-specific glucocorticoid regulation of calcitonin/CGRP gene expression occurs in vivo, we will compare the effect of glucocorticoids on CGRP mRNA levels in the hypothalamus, brainstem, and sensory ganglia of adrenalectomized and steroid replaced rats. To address the mechanisms modulating the glucocorticoid regulation, the first step will be to identify the glucocorticoid response element(s) (GREs) responsible for positive and negative regulation of the transcription rate. Chimeric fusion genes containing fragments of the calcitonin/CGRP promoter and intragenic regions will be linked to the chloramphenicol acetyltransferase reporter gene and transfected into the CA77 and 44-2C medullary thyroid carcinoma cell lines. We have shown that the endogenous calcitonin/CGRP transcription rate is stimulated in CA77 and repressed in 44-2C cells by glucocorticoids. The presence of glucocorticoid receptor binding sites within the functional response elements will be demonstrated and localized using an in vitro receptor-DNA binding assay with purified glucocorticoid receptor. The significance of these in vitro binding sites will be functionally tested by transfection assays into the CA77 and 44-2C cells using reporter fusion genes. The final experiments will attempt to identify accessory factors required for cell-specific positive and negative glucocorticoid regulation. The approach will be to examine both hormone inducible factors and cell-specific DNA binding proteins that recognize sequences near the GRE(s). The overall expectation is that these studies will provide evidence for a cell-specific factor that modulates glucocorticoid regulation of calcitonin/CGRP gene expression. Identification of this factor may be pertinent towards defining the network of regulatory mechanisms that control other steroid regulated genes. Furthermore, an understanding of glucocorticoid regulation of calcitonin/CGRP gene expression may potentially provide additional insight into deficient calcitonin expression in Williams Syndrome and the myriad of developmental, homeostatic, and pathophysiological manifestations of glucocorticoids in the neuroendocrine system.
