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In this study, we investigated the role of two inducible repressor proteins, inducible cAMP early repressor (ICER) and Fos-related antigen 2 (Fra-2) in the adrenergic induction of MAPK phosphatase-1 (MKP-1) as compared with their roles in the induction of arylalkylamine-N-acetyltransferase (AA-NAT) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA and protein levels of MKP-1 and AA-NAT, as well as in the AA-NAT activity and melatonin production. NE stimulation also caused a simultaneous increase in the mRNA and protein levels of ICER and Fra-2. Transient knockdown of icer using adenovirus expressing small interfering RNA (siRNA) abolished the NE induction of icer expression but had little effect on the NE induction of mkp-1 or aa-nat expression. In contrast, pretreatment with adenovirus overexpressing icer was effective in reducing the NE induction of mkp-1 and aa-nat. The inhibitory effect of overexpressing icer was reversed by cotreatment with siRNA against icer. siRNA against fra-2 also abolished the NE-stimulated expression of fra-2 but had little effect on the NE induction of mkp-1 and aa-nat expression. Proteasomal inhibition, which reduced the NE-stimulated induction of aa-nat, caused a reduction of ICER and Fra-2. Together, these results indicate that whereas overexpression of ICER can suppress the NE induction of aa-nat and mkp-1, the amount of the repressors, ICER and Fra-2, present during NE induction appears insufficient to exert a significant effect in controlling the expression of these genes.

The norepinephrine-driven increase in mitogen-activated protein kinase (MAPK) activity is part of the mechanism that regulates arylalkylamine N-acetyltransferase (AA-NAT) activity in the rat pineal gland. We now report a marked nocturnal increase in the expression of a MAPK phosphatase, MAP kinase phosphatase-1 (MKP-1), that was blocked by maintaining animals in constant light or treatment with propranolol. MKP-1 expression was regulated by norepinephrine acting through both a-and b-adrenergic receptors. These results establish a nocturnal increase in pineal MKP-1 expression that is under the control of a photoneural system. Because substrates of MKP-1 can influence AA-NAT activity, our findings suggest the involvement of MKP-1 in the regulation of the nocturnal AA-NAT signal.

In this study, we investigated the effect of proteasomal inhibition on the induction of arylalkylamine-N-acetyltransferase (AA-NAT) enzyme in cultured rat pinealocytes, using two proteasome inhibitors, MG132 and clastolactacystin beta-lactone (c-lact). Addition of c-lact or MG132 3 h after norepinephrine (NE) stimulation produced a significant increase in AA-NAT protein level and enzyme activity. However, when the proteasome inhibitors were added before or together with NE, significant reductions of the NE-induced aa-nat mRNA, protein, and enzyme activity were observed. A similar inhibitory effect of MG132 on aa-nat transcription was observed when cells were stimulated by dibutyryl cAMP, indicating an effect distal to a post-cAMP step. The inhibitory effect of MG132 on adrenergic-induced aa-nat transcription was long lasting because it remained effective after 14 h of washout and appeared specific for aa-nat because the induction of another adrenergic-regulated gene, MAPK phosphatase-1, by NE was not affected. Time-profile studies revealed that the inhibitory effect of MG132 on NE-stimulated aa-nat induction was detected after 1 h, suggesting accumulation of a protein repressor as a possible mechanism of action. This possibility was also supported by the finding that the inhibitory effect of c-lact on NE-induced aa-nat induction was markedly reduced by cycloheximide, a protein synthesis inhibitor. Together, these results support an important role of proteasomal proteolysis in the adrenergic-mediated induction of aa-nat transcription through its effect on a protein repressor.

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