Antipsychotic Drugs Inhibit the Human Corticotropin-Releasing-Hormone Gene Promoter Activity in Neuro-2A Cells—an Involvement of Protein Kinases

Abstract

Antipsychotic drugs can regulate transcription of some genes, including those involved in regulation of hypothalamic-pituitary-adrenal (HPA) axis, whose activity is frequently disturbed in schizophrenic patients. However, molecular mechanism of antipsychotic drug action on the corticotropin-releasing hormone (CRH) gene activity has not been investigated so far. This study was undertaken to examine the influence of conventional and atypical antipsychotic drugs on the CRH gene promoter activity in differentiated Neuro-2A cell cultures stably transfected with a human CRH promoter fragment linked to the chloramphenicol acetyltransferase (CAT) reporter gene. It has been found that chlorpromazine (0.1-5.0 mM), haloperidol (0.5-5.0 mM), clozapine (1.0-5.0 mM), thioridazine (1.0-5.0 mM), promazine (5.0 and 10 mM), risperidone (5.0 and 10.0 mM), and raclopride (only at the highest used concentrations, ie 30 and 100 mM) present in culture medium for 5 days inhibited the CRH-CAT activity. Sulpiride and remoxipride had no effect. Since CRH gene activity is most potently enhanced by cAMP/protein kinase A pathway, the effect of antipsychotics on the forskolin-induced CRH-CAT activity was determined. Chlorpromazine (1.0-5.0 mM), haloperidol (1.0-5.0 mM), clozapine (1.0-5.0 mM), thioridazine (3.0 and 5.0 mM), and raclopride (30 and 100 mM), but not promazine, sulpiride, risperidone, and remoxipride, inhibited the forskolin-stimulated CRH gene promoter activity. A possible involvement of protein kinases in chlorpromazine and clozapine inhibitory action on CRH activity was also investigated. It was found that wortmannin (0.01 and 0.02 mM), an inhibitor of phosphatidylinositol 3-kinase (PI3-K), significantly attenuated the inhibitory effect of chlorpromazine and clozapine on CRH gene promoter activity. In line with these results, a Western blot study showed that these drugs increased phospho-Ser-473 Akt level, had no effect on total Akt, and decreased glycogen synthase kinase-3b level. Additionally, we found that clozapine decreased protein kinase C (PKC) level and that its action on CRH activity was attenuated by PKC activator (TPA, 0.1 mM). The obtained results indicate that inhibition of CRH gene promoter activity by some antipsychotic drugs may be a molecular mechanism responsible for their inhibitory action on HPA axis activity. Clozapine and chlorpromazine action on CRH activity operates mainly through activation of the PI3-K/Akt pathway. Moreover, PKC-mediated pathway seems to be involved in clozapine action on CRH gene activity.

Neuroleptics Affect Neuropeptide S and NPSR mRNA Levels in the Rat Brain

“…Inhibition of HPA axis by neuroleptics may be mediated by their ability to inhibit CRH gene promoter activity. Clozapine and chlorpromazine modulate CRH activity mainly via activation of the PI3K/Akt pathway, but PKC-related pathway may be also involved (Basta-Kaim et al 2006). Interestingly, olanzapine increased calcitonin gene-related peptide (CGRP) immunoreactivity in the striatum, frontal cortex, and hypothalamus (Angelucci et al 2008), whereas the pronounced adrenocorticotropic hormone (ACTH) and cortisol lowering performed by atypical neuroleptics is possibly related to the downregulation of serotonergic, adrenergic, and histaminergic receptors (Cohrs et al 2006).…”

A Role for the PKC Signaling System in the Pathophysiology and Treatment of Mood Disorders: Involvement of a Functional Imbalance?

“…Finally, even atypical antipsychotics, used for the treatment of acute mania, seem to modulate the PKC pathway. Clozapine for instance, decreased PKC activity after 5 days of exposure in mouse neuroblastoma cells [39].…”

Antipsychotic drug mechanisms: links between therapeutic effects, metabolic side effects and the insulin signaling pathway

“…86 A similar analysis in mouse neuroblastoma cells demonstrated that 5 days of both clozapine and chlorpromazine increased phospho-Ser473-Akt without increasing total Akt levels. 87 Olanzapine has been similarly shown to increase levels of phosphoSer473-Akt in PC12 cells although total Akt remained the same. 88 This effect was dependent on PI3K, suggests an additional mechanism of antipsychotic effects on Akt and may be related to differences in cell line.…”

“…86 Similar results were observed in another study of mouse neuroblastoma cells exposed to 5 days of antipsychotic; active, nonphosphorylated GSK-3b decreased after exposure to both chlorpromazine and clozapine. 87 In mice that had been treated with clozapine for 68 days it was observed that although insulin receptor activity decreased and phospho-Ser21-GSK-3a and phospho-Ser9-GSK-3b remained the same, total GSK-3a and -b levels, and thus activity, were decreased. 74 Opposite effects were noted in glioblastoma cells in which clozapine had the effect of dephosphorylating GSK-3b at Ser9, 92 possibly related to differences in cell line.…”