Vol. 61, Issue 6, 1385-1392, June 2002

Glucocorticoid Responsiveness of the Rat Phenylethanolamine N-Methyltransferase Gene

T. C. Tai, R. Claycomb, S. Her, A. K. Bloom, and Dona L. Wong

Department of Psychiatry, Harvard Medical School, Boston, Massachusetts; and Laboratory of Molecular and Developmental Neurobiology, McLean Hospital, Belmont, Massachusetts

Two newly identified, overlapping (1 bp) glucocorticoid response elements (GREs) at 759 and 773 bp in the promoter of the rat phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) gene are primarily responsible for its glucocorticoid sensitivity, rather than the originally identified 533-bp GRE. A dose-dependent increase in PNMT promoter activity was observed in RS1 cells transfected with a wild-type PNMT promoter-luciferase reporter gene construct and treated with dexamethasone (maximum activation at 0.1 µM). The type II glucocorticoid receptor antagonist RU38486 (10 µM) fully inhibited dexamethasone (1 µM) activation of the PNMT promoter, consistent with classical glucocorticoid receptors mediating corticosteroid-stimulated transcriptional activity. Relative IC₅₀ values from gel mobility shift competition assays showed that the 759-bp GRE has a 2-fold greater affinity for the glucocorticoid receptor than the 773-bp GRE. Site-directed mutation of the 533-, 759-, and 773-bp GREs alone or in tandem demonstrated that the 759-bp GRE was also functionally more important, but both the 759- and 773-bp GREs are required for maximum glucocorticoid responses. Moreover, the 533-bp GRE, rather than increasing glucocorticoid sensitivity of the promoter, may limit corticosteroid responsiveness mediated via the 759- and 773-bp GREs. Finally, the glucocorticoid receptor bound to the 759- and 773-bp GREs interacts cooperatively with Egr-1 and/or AP-2 to stimulate PNMT promoter activity in RS1 cells treated with dexamethasone. In contrast, glucocorticoid receptors bound to the 533-bp GRE only seem to participate in synergistic activation of the PNMT promoter through interaction with activator protein 2.