This application is designed to study the regulatory interactions between the calcitropic hormones and Chromogranin A (CgA). The general hypothesis is that CgA and its processed peptides interact at the level of gene expression, post-translational processing, and secretion with the calcitropic hormones, namely: (a) the peptides encoded by the calcitonin (CT) gene(s), especially CT itself and CGRP (calcitonin-gene-relate-peptide); (b) parathyroid hormone (PTH); and (c) the PTH-related protein (PTHrP) associated with the hypercalcemia of malignancy. This hypothesis is derived from the studies with cell lines from calcitropic hormone-producing endocrine tissues that also produce and secrete CgA. Previous work has demonstrated common regulatory pathways for CgA and the calcitropic hormones in studies of mRNA expression, biosynthesis, and secretion with these cell lines. It has also been demonstrated by pulse-chase studies and Western blotting, that CgA is processed to different peptides in a tissue-specific manner by calcitropic hormone-producing cells. The experimental plan now proposes to study the molecular mechanisms of these regulatory interactions. For studies of CgA processing, pulse-chase and Western studies will be continued with antibodies to CgA-peptides based on the processing of the protein at its dibasic residues. These studies, along with analytical biochemical procedures, are intended to lead to the identification of tissue-specific CgA-derived peptides for the different cell lines. In vivo and in vitro secretory studies of these peptides will be conducted to further evaluate their biological relevance. For studies of gene expression, efforts will identify the regulatory sequences of the CgA gene by genomic cloning, restriction mapping, and DNA sequencing. Further studies will include investigation of the promoter and enhancer functions of these regulatory elements with reporter genes such as in the CAT assay. Such CAT assays will be performed in different cell types to evaluate the tissue specificity of CgA regulation. The hormone- and second messenger-responsiveness of these regulatory elements will be evaluated by studying the effects of the calcitropic hormones themselves, and relevant second messengers such as cAMP and calcium. These transcription units can be compared to known hormone-specific transcription units. It may thus be possible to discern those structural features of CgA DNA regulatory sequences that are unique and those that they share with the calcitropic proteins and peptides, and thus elucidate such mechanisms of regulatory interactions for CgA and the calcitropic hormones.
