Chronic olanzapine activates the Stat3 signal transduction pathway and alters expression of components of the 5-HT2A receptor signaling system in rat frontal cortex

Abstract

The mechanisms underlying desensitization of serotonin 2A (5-HT(2A)) receptor signaling by antagonists are unclear but may involve changes in gene expression mediated via signal transduction pathways. In cells in culture, olanzapine causes desensitization of 5-HT(2A) receptor signaling and increases the levels of regulators of G protein signaling (RGS) 7 protein dependent on phosphorylation/activation of the Janus kinase 2 (Jak2)/signal transducers and activators of transcription 3 (Stat3) signaling pathway. In the current study, the 5-HT(2A) receptor signaling system in rat frontal cortex was examined following 7 days of daily treatment with 0.5, 2.0 or 10.0 mg/kg i.p. olanzapine. Olanzapine increased phosphorylation of Stat3 in rats treated daily with 10 mg/kg olanzapine and caused a dose-dependent desensitization of 5-HT(2A) receptor-mediated phospholipase C activity. There were dose-dependent increases in the levels of membrane-associated 5-HT(2A) receptor, G(alpha11) and G(alphaq) protein levels but no changes in the G(beta) protein levels. With olanzapine treatment, RGS4 protein levels increase in the membrane-fraction and decrease in the cytosolic fraction by similar amounts suggesting a redistribution of RGS4 protein within neurons. RGS7 protein levels increase in both the membrane and cytosolic fractions in rats treated daily with 10mg/kg olanzapine. The olanzapine-induced increase in Stat3 activity could underlie the increase in RGS7 protein expression in vivo as previously demonstrated in cultured cells. Furthermore, the increases in membrane-associated RGS proteins could play a role in desensitization of signaling by terminating the activated G(alphaq/11) proteins more rapidly.

Figure 1

5-HT_2A receptor agonists cause activation of G_αq/11 proteins which in turn activate the second messenger enzyme PLC. Chronic treatment with olanzapine causes desensitization of this pathway as measured by either production of inositol phosphate in cells or PLC activity in brain tissue. We hypothesize that 5-HT_2A receptor antagonism (induced by olanzapine) activates the Jak2/Stat3 pathway causing phosphorylated Stat3 to dimerize and translocate to the nucleus, stimulate immediate early genes and subsequently increase RGS7 transcription and RGS7 protein levels in the membrane. The increased membrane-associated RGS7 protein can then increase hydrolysis of activated G_αq/11 and result in desensitization of 5-HT_2A receptor signaling.

Figure 2

Rat body weight is not altered by 0.5, 2, or 10 mg/kg olanzapine injected daily for 1, 3, 5, or 7 days.

Figure 3

(A) 5-HT-stimulated PLC activity in the frontal cortex was significantly attenuated by daily treatments with olanzapine for 7 days. Each dose of olanzapine (Olan), 0.5, 2.0 and 10.0 mg/kg, significantly reduced PLC activity compared to saline-treated controls (** indicates significantly different from saline-treated controls rats at p< 0.01). (B) GTPγS-stimulated-PLC activity was not altered by the olanzapine treatments.

Figure 4

(A) Olanzapine (Olan) treatment increased the membrane-associated levels of the 5-HT_2A receptor protein in rat frontal cortex measured on western blots. (B) There were no changes in the levels of G_β1-4 in the membrane fraction with olanzapine treatment. Actin was used to verify equal loading of lanes in the SDS PAGE gel. * indicates significantly different from saline-treated controls at p < 0.05.

Figure 5

Chronic olanzapine differentially altered G_α protein levels in the cytosolic and membrane fraction of the frontal cortex as measured by western blotting. The levels of G_αq (A) and G_α11 (B) in the membrane fraction were significantly increased in the rats treated with 10 mg/kg olanzapine. In contrast, the levels of G_αq (C) and G_α11 (D) proteins in the cytosol fraction of rat frontal cortex were not significantly altered by chronic olanzapine treatments. * indicates significantly different from saline-treated control rats at p< 0.05.

Figure 6

Chronic olanzapine differentially altered RGS4 and RGS7 protein levels in the frontal cortex. RGS4 levels in the membrane fraction (A) were increased by daily treatment with 2.0 and 10.0 mg/kg olanzapine. The levels of RGS4 in the cytosol (B) were reduced by approximately half with all doses of olanzapine. In contrast, RGS7 protein levels in the membrane (C) and cytosol (D) were significantly increased by daily injections of 10 mg/kg olanzapine. * indicates significantly different from saline-treated control rats and # indicates significantly different compared to 0.5 mg/kg olanzapine at p< 0.05.

Figure 7

Chronic olanzapine increased Stat3 activity in the frontal cortex. The levels of phosphorylated Stat 3 were significantly increased by daily treatments of 10 mg/kg olanzapine (Olan 10). Stat protein levels were not altered by olanzapine. Actin protein levels were used to verify equal loading of lanes on the SDS-PAGE gel. * indicates significantly different from saline-treated control rats at p< 0.05.