Genetic mechanisms for adrenergic control during stress.

Cortisol and epinephrine released in response to stress are replenished via activation of the hypothalamic-pituitary-adrenal (HPA or stress) axis. Immobilization (IMMO) stress in rats stimulates epinephrine production in part via the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT mRNA rose up to 7.0-fold with acute or chronic stress. Two transcription factors mediating stress induction of the PNMT gene are the glucocorticoid receptor (GR) and Egr-1, which interact with -533, -759, and -773 bp, and -165 bp binding sites in the rat PNMT promoter, respectively. To identify molecular mechanisms involved, effects of hypoxic stress on PNMT promoter activity were examined in PC12 cells transfected with the PNMT promoter-luciferase reporter gene construct pGL3RP893. Oxygen reduction to 5% increased PNMT promoter-driven luciferase expression, with maximum activity at 6 h. Pretreatment of the cells with protein kinase A (PKA) and protein kinase C (PKC) inhibitors, H-89 and GF109203X, respectively, attenuated the rise in luciferase. Similarly, PKA-deficient PC12 cells transfected with pGL3RP893 and exposed to hypoxia also showed attenuated PNMT promoter-driven luciferase expression. Mutation of the Egr-1 binding site completely prevented PNMT promoter activation, indicating that Egr-1 is essential to the stress response. Consistent with this result, hypoxia increased Egr-1 protein. Hypoxia also increased endogenous PNMT mRNA. However, a shift to intron-retaining mRNA from which truncated, nonfunctional protein is produced, occurred, suggesting that posttranscriptional regulation may be an important genetic mechanism controlling adrenergic expression and hence, epinephrine, during stress.