Protein kinase C overactivity impairs prefrontal cortical regulation of working memory.

The prefrontal cortex is a higher brain region that regulates thought, behavior, and emotion using representational knowledge, operations often referred to as working memory. We tested the influence of protein kinase C (PKC) intracellular signaling on prefrontal cortical cognitive function and showed that high levels of PKC activity in prefrontal cortex, as seen for example during stress exposure, markedly impair behavioral and electrophysiological measures of working memory. These data suggest that excessive PKC activation can disrupt prefrontal cortical regulation of behavior and thought, possibly contributing to signs of prefrontal cortical dysfunction such as distractibility, impaired judgment, impulsivity, and thought disorder.

Glucocorticoid responsiveness of the rat phenylethanolamine N-methyltransferase gene.

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 microM). The type II glucocorticoid receptor antagonist RU38486 (10 microM) fully inhibited dexamethasone (1 microM) activation of the PNMT promoter, consistent with classical glucocorticoid receptors mediating corticosteroid-stimulated transcriptional activity. Relative IC(50) 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.

Phenylethanolamine N-methyltransferase gene expression. Sp1 and MAZ potential for tissue-specific expression.

Phenylethanolamine N-methyltransferase (PNMT) promoter-luciferase reporter gene constructs (pGL3RP863, pGL3RP444, and pGL3RP392) transfected into COS1, RS1, PC12, NIH/3T3, or Neuro2A cells showed the highest basal luciferase activity in the Neuro2A cells. DNase I footprinting with Neuro2A cell nuclear extract identified protected PNMT promoter regions spanning the -168/-165 and -48/-45 base pair Sp1/Egr-1 binding sites. Gel mobility shift assays and transient transfection assays using site-directed mutant PNMT promoter-luciferase reporter gene constructs indicated that the elevated basal luciferase activity in the Neuro2A cells was mediated by Sp-1. Furthermore, activation of the PNMT promoter by Sp1 depends on both its binding affinity for its cognate target sequences and its intracellular concentrations. When Sp1 levels were increased through an expression plasmid, luciferase reporter gene expression rose well beyond basal wild-type levels, even with either Sp1 binding element mutated. Finally, another transcription factor expressed in the Neuro2A cells competes with Sp1 by interacting with DNA sequences 3' to the -48 base pair Sp1 site to prevent Sp1 binding and induction of the PNMT promoter. The DNA consensus sequence, Southwestern analysis, and gel mobility shift assays with antibodies identify MAZ as the competitive factor. These findings suggest that Sp1 may potentially contribute to the tissue-specific expression of the PNMT gene, with the competition between Sp1 and MAZ conferring additional tissue-specific control.