Activation of the JAK-STAT pathway is necessary for desensitization of 5-HT2A receptor-stimulated phospholipase C signalling by olanzapine, clozapine and MDL 100907

Abstract

We have previously demonstrated that olanzapine-induced desensitization of 5-HT2A receptor-stimulated phospholipase C (PLC) activity is associated with increases in RGS7 protein levels both in vivo and in cells in culture, and the increase in RGS7 is dependent on activation of the JAK-STAT pathway in cells in culture. In the present study, we found that desensitization of 5-HT2A receptor-stimulated PLC activity induced by olanzapine is dependent on activation of the JAK-STAT pathway. Similar to olanzapine, clozapine-induced desensitization of 5-HT2A receptor signalling is accompanied by increases in RGS7 and activation of JAK2. Treatment with the selective 5-HT2A receptor antagonist MDL 100907 also increased RGS7 protein levels and JAK2 activation. Using a JAK2 inhibitor AG490, we found that clozapine and MDL 100907-induced increases in RGS7 are dependent on activation of the JAK-STAT pathway. Olanzapine, clozapine, and MDL 100907 treatment increased mRNA levels of RGS7. Using a chromatin immunoprecipitation assay we found STAT3 binding to the putative RGS7 promoter region. Taken together, olanzapine-induced activation of the JAK-STAT pathway, and STAT3 binding to the RGS7 gene could underlie the increase in RGS7 mRNA which could subsequently increase protein expression. Furthermore, the increase in RGS7 protein could play a role in the desensitization of 5-HT2A receptor signalling by terminating the activated Galphaq/11 proteins more rapidly. Overall, our data suggest that the complete desensitization of 5-HT2A receptor-stimulated PLC activity by olanzapine, clozapine and MDL 100907 requires activation of the JAK-STAT pathway, which in turn increases RGS7 expression probably by direct transcriptional activity of STAT3.

Figure 1

Olanzapine or clozapine decreases DOI-stimulated inositol phosphate (IP) accumulation. A1A1v cells were treated with either vehicle or with various concentrations of (A) olanzapine or (B) clozapine for 24 h, and incubated with [3H]-myoinositol for same 24 h period. Then cells were stimulated with 10⁻⁴ M DOI. The bar graph represents IP accumulation normalized to DOI-stimulated IP accumulation in vehicle-treated cells from at least three independent experiments. Inositol phosphate accumulation was significantly decreased in olanzapine or clozapine-treated cells compared to vehicle-treated cells. * indicates significantly different from vehicle-treated cells at p < 0.05, ** indicates significantly different from vehicle-treated cells at p < 0.01 # indicates significantly different from 5µM clozapine at p < 0.05 as analyzed by one-way ANOVA and Newman-Keuls post-hocs tests.

Figure 2

Clozapine and MDL100907 stimulate JAK2 phosphorylation and pretreatment with AG490 reverses this effect. (A) A1A1v cells were treated either with vehicle (DMSO) or 20µM clozapine or 1µM MDL100907 for 24 h. Membrane fractions of cells were analyzed by western blot with anti-phosphoJAK2 antibody, stripped and reprobed with anti-JAK2 and anti-actin antibodies. The bar graph represents quantification of phosphoJAK2 protein levels divided by JAK2 protein levels from five independent experiments. Phosphorylation of JAK2 was significantly increased (p<0.05) with both clozapine and MDL100907 treatment compared with vehicle-treated cells. * indicates significantly different from vehicle-treated cells at p < 0.05. (B) Cells were pretreated with AG490 (30 µM) for 1 h before treatment with either MDL100907 or clozapine for 24 h. Membrane fractions of cells were analyzed as described above. The bar graph represents quantification of phosphoJAK2 protein levels divided by JAK2 protein levels from three independent experiments. # indicates significantly different from vehicle-pretreated and the same drug treated cells at p < 0.01, * indicates significantly different from vehicle-pretreated and vehicle-treated cells at p < 0.01 as analyzed by one-way ANOVA and Newman-Keuls post-hocs tests.

Figure 3

Pretreatment with AG490 prevented the clozapine- and MDL100907-induced increase in RGS7 protein levels. (A) A1A1v cells were treated either with vehicle (DMSO) or 20µM clozapine or 1µM MDL100907 for 24 h. Membrane fractions of cells were analyzed by western blot with an anti-RGS7 antibody, stripped and reprobed with an anti-actin antibody as a loading control. Bar graph represents quantification of RGS7 protein levels divided by actin protein levels from four independent experiments. RGS7 protein levels were significantly increased (p<0.05) with clozapine and MDL100907 compared to vehicle-treated cells. * indicates significantly different from vehicle-treated cells at p < 0.05. (B) Cells were pretreated with AG490 (30 µM) for 1 h before treatment with either MDL100907 or clozapine for 24 h. Membrane fractions of cells were analyzed as described above. Bar graph represents quantification of RGS7 protein levels divided by actin protein levels from three independent experiments. # and ## indicates significantly different from vehicle-pretreated and the same drug treated cells at p < 0.05 and p < 0.01 respectively, * indicates significantly different from vehicle-pretreated and vehicle treated cells at p < 0.05 as analyzed by one-way ANOVA and Newman-Keuls post-hocs tests.

Figure 4

Time course studies show JAK2 phosphorylation preceded the increase in RGS7 protein in response to olanzapine. (A) cells were treated with olanzapine (300 nM) or vehicle (20% acetic acid) for 3, 6, 12 or 24 hours. Membrane fractions of cells were analyzed by western blot with anti-phospho-JAK2, then stripped and reprobed with anti-JAK2 antibody. The bar graph represents quantification of phosphoJAK2 protein levels divided by JAK2 protein levels from three independent experiments. * indicates significantly different from vehicle-treated cells at the same time point at p < 0.01. # and ## indicate significantly different from 3 h olanzapine-treated cells at p < 0.05 and p < 0.01 respectively,+ indicates significantly different from 6 h olanzapine treated cells at p < 0.01. (B)The same membrane was stripped and reprobed with anti-RGS7 and anti-actin antibody. Bar graph represents quantification of RGS7 protein levels divided by actin protein levels from three independent experiments. * indicates significantly different from vehicle treated cells at the same time point at p < 0.01. # indicates significantly different from 3 h olanzapine treated cells at p < 0.01 as analyzed by one-way ANOVA and Newman-Keuls post-hocs tests.

Figure 5

A JAK inhibitor partly attenuated the olanzapine-induced decrease in PLC activity. A1A1v cells were pretreated for 1h with the 30µM AG490 prior to treating with either vehicle (20% acetic acid) or 300nM olanzapine for 24 h. (A) 5-HT-stimulated PLC activity was significantly (p<0.01) reduced by olanzapine treatment compared to vehicle-treated control (**indicates significantly different at p < 0.01). AG490 alone did not have any effect on PLC activity as analyzed by two-way ANOVA and Newman-Keuls post-hocs tests. However, in cells pretreated with AG490, the olanzapine-induced decrease in PLC activity was significantly attenuated (* indicates significantly different at p<0.05). (B) GTPγS-stimulated-PLC activity was not altered either by the olanzapine or AG490 treatments. (C). Olanzapine treatment had no effect on bradykinin-stimulated PLC activity, whereas 5-HT-stimulated PLC activity was significantly reduced suggesting olanzapine treatment selectively affect 5-HT_2A receptor mediated-PLC activity as analyzed by two-way ANOVA and Newman-Keuls post-hocs tests. Experiments were performed three independent times.

Figure 6

Olanzapine, clozapine and MDL100907 increase RGS7 mRNA levels. A1A1v cells were treated either with vehicle (20% acetic acid), 300nM olanzapine, vehicle (DMSO), 20µM clozapine or 1µM MDL100907 for 24 h. Total RNA was isolated from vehicle-treated control or drug-treated cells, and equal amounts of cDNA were reverse-transcribed. Bar graph represents quantification of RGS7 mRNA levels normalized to GAPDH levels from five independent experiments and represents the fold change over control. RGS7 mRNA levels were significantly increased in olanzapine, clozapine and MDL100907-treated compared to vehicle-treated cells (p<0.05) as analyzed by two-way ANOVA and Newman-Keuls post-hocs tests. * indicates significantly different from vehicle-treated cells at p<0.05.

Figure 7

STAT3 binds to the putative RGS7 promoter region at site 2. ChIP assays were performed as described in “Materials and Methods” with an anti-STAT3 antibody, non-immune IgG, or with beads alone. Primers that amplify the STAT3 binding site previously identified in the hepcidin gene were used as a positive control. Non-precipitated genomic DNA (input) was used as a positive control. Amplification was not observed in lanes loaded with samples immunoprecipitated with beads alone, i.e., without the STAT3 antibody, and non-specific IgG in place of the STAT3 antibody confirming specificity of the reaction with the STAT3 antibody.