GLUCOCORTICOID ACTIVATION OF CHROMOGRANIN-A GENE-EXPRESSION - IDENTIFICATION AND CHARACTERIZATION OF A NOVEL GLUCOCORTICOID RESPONSE ELEMENT

Glucocorticoids regulate catecholamine biosynthesis and storage at sev eral sites. Chromogranin A, an abundant protein complexed with catecho lamines in secretory vesicles of chromaffin cells and sympathetic axon s, is also augmented by glucocorticoids. This study reports isolation of the rat chromogranin A promoter to elucidate transcriptional regula tion of chromogranin A biosynthesis by glucocorticoids in neuroendocri ne cells. Endogenous chromogranin A gene expression was activated up t o 3.5-fold in chromaffin cells by glucocorticoid, in time-dependent fa shion. Inhibition of new protein synthesis by cycloheximide did not al ter the rise in chromogranin A mRNA, suggesting that glucocorticoids d irectly activate the chromogranin A promoter; nuclear runoff assays co nfirmed a 3.3-fold increased rate of initiation of new chromogranin A transcripts after glucocorticoid. Transfected rat chromogranin A promo ter/luciferase reporter constructs were activated 2.6-3.1-fold by gluc ocorticoid, and selective agonist/antagonist studies determined that d examethasone effects were mediated by glucocorticoid receptors. Both r at and mouse chromogranin A promoter/luciferase reporter constructs we re activated by glucocorticoid. A series of promoter deletions narrowe d the region of glucocorticoid action to a 93-bp section of the promot er, from position -526 to -619 bp upstream of the cap site. A 15-bp se quence ([-583 bp] 5'-ACATGAGTGTGTCCT-3' [-597 bp]) within this region showed partial homology to a glucocorticoid response element (GRE; hal f-site in italics) consensus sequence, and several lines of experiment al evidence confirmed its function as a GRE: (a) site-directed mutatio n of this GRE prevented glucocorticoid activation of a chromogranin A promoter/reporter; (b) transfer of this GRE to a heterologous (thymidi ne kinase) promoter/reporter conferred activation by glucocorticoid, i n copy number-dependent and orientation-independent fashion; and (c) e lectrophoretic gel mobility shifts demonstrated binding of this GRE by ligand-activated glucocorticoid receptor, though at 2.75-fold lower a ffinity than the glucocorticoid receptor interaction with a consensus GRE. The rat chromogranin A GRE showed functional and structural simil arities to GREs in other genes proportionally regulated by glucocortic oids. We conclude that a discrete domain of the chromogranin A promote r is both necessary and sufficient to confer glucocorticoid regulation onto the gene, and that the activity of this region also explains the degree of activation of the endogenous gene by glucocorticoid.