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Dopamine D2 receptor (D2R) has known to activate Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin by increasing in the intracellular Ca(2+). We previously showed that D2LR (long isoform) and D2SR (short isoform) enhanced SRE and NF-kappaB, and conversely suppressed CRE transcriptional activities in NG108-15 cells stably expressed with these receptors (NGD2LR and NGD2SR). In this study, to investigate whether activation of calcineurin is involved in the transcriptional regulations through D2R, we evaluated effect of cyclosporin A, a selective calcineurin inhibitor, on them in NGD2LR and NGD2SR cells using luciferase reporter gene assay. We first confirmed that D2LR activates calcineurin in NG108-15 cells by measurement of dephosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein Mr 32 000 (DARPP-32) at threonin 34 by immunoblot analysis with its phospho-specific antibody. Cyclosporin A treatment showed no change in suppression of forskolin-induced CRE activation or activation of SRE but significantly attenuated NF-kappaB activation by D2LR stimulation in NGD2LR cells. Interestingly, D2SR-induced NF-kappaB activation, which was weaker than that by D2LR stimulation, was not affected by cyclosporin A treatment in NGD2SR cells. Furthermore, D2SR stimulation did not cause dephosphorylation of DARPP-32 at threonin 34. Taken together, D2SR and D2LR may employ different signaling pathway on intracellular Ca(2+) mobilization, thereby showing different NF-kappaB activation in the calcineurin-dependent manner.

We previously showed that dopamine receptors D1R and D2R expressed in NG108-15 cells activated protein kinase A and extracellular signal-regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)-kappaB activity. To investigate whether other dopamine receptor subtypes regulate NF-kappaB, we established NG108-15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF-kappaB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF-kappaB-Luci activity, although forskolin-induced cyclic AMP responsive element-Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element-luciferase reporter not observed following stimulation with quinpirole in D3R- or D4R-expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF-kappaB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF-kappaB activity. By contrast, risperidone treatment enhanced NF-kappaB activity. Taken together, D2R and D1R/D5R had opposite effects on NF-kappaB activity in NG108-15 cells. Risperidone up-regulated and haloperidol down-regulated NF-kappaB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone.

To investigate transcriptional regulation by dopamine receptors, we established NG108-15 cells stably expressing D1R, D2LR and D2SR (NGD1R, NGD2LR and NGD2SR) and evaluated the effects of these receptors on NF-kappaB, SRE and CRE activity using luciferase reporter constructs. Stimulation with quinpirole, a selective D2R agonist, increased NF-kappaB and SRE activity but decreased CRE activity in both NGD2R cell lines. By contrast, stimulation with SKF 38393, a selective D1R agonist, decreased NF-kappaB and SRE activity but increased CRE activity in NGD1R cells. Stimulation with forskolin and overexpression of constitutively active PKA suppressed NF-kappaB activity, likely due to D1R stimulation. D2R stimulation activated ERK, and treatment with U1026, a selective MEK inhibitor, eliminated D2R-induced NF-kappaB activation. D2R stimulation also activated the neural cell adhesion molecule (NCAM) promoter, which includes a potential NF-kappaB site. Furthermore, by transfecting constitutively active CaM KII and MEKK, and dominant negative p38 MAPK, we show that the NCAM promoter is positively regulated by CaM KII but negatively regulated by p38 MAPK. These results indicate that D2R-induced NF-kappaB activation through ERK may be involved in activation of the NCAM promoter, and additionally that other protein kinases such as CaM KII and p38 MAPK also regulate NCAM expression.

Dopamine D2 receptor (D2R) has known to activate Ca2+/calmodulin-dependent protein phosphatase, calcineurin by increasing in the intracellular Ca2+. We previously showed that D2LR (long isoform) and D2SR (short isoform) enhanced SRE and NF-κB, and conversely suppressed CRE transcriptional activities in NG108-15 cells stably expressed with these receptors (NGD2LR and NGD2SR). In this study, to investigate whether activation of calcineurin is involved in the transcriptional regulations through D2R, we evaluated effect of cyclosporin A, a selective calcineurin inhibitor, on them in NGD2LR and NGD2SR cells using luciferase reporter gene assay. We first confirmed that D2LR activates calcineurin in NG108-15 cells by measurement of dephosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein Mr 32 000 (DARPP-32) at threonin 34 by immunoblot analysis with its phospho-specific antibody. Cyclosporin A treatment showed no change in suppression of forskolin-induced CRE activation or activation of SRE but significantly attenuated NF-κB activation by D2LR stimulation in NGD2LR cells. Interestingly, D2SR-induced NF-κB activation, which was weaker than that by D2LR stimulation, was not affected by cyclosporin A treatment in NGD2SR cells. Furthermore, D2SR stimulation did not cause dephosphorylation of DARPP-32 at threonin 34. Taken together, D2SR and D2LR may employ different signaling pathway on intracellular Ca2+ mobilization, thereby showing different NF-κB activation in the calcineurin-dependent manner. [ABSTRACT FROM AUTHOR]

We previously showed that dopamine receptors D1R and D2R expressed in NG108-15 cells activated protein kinase A and extracellular signal-regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)-κB activity. To investigate whether other dopamine receptor subtypes regulate NF-κB, we established NG108-15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF-κB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF-κB-Luci activity, although forskolin-induced cyclic AMP responsive element-Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element- luciferase reporter not observed following stimulation with quinpirole in D3R- or D4R-expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF-κB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF-κB activity. By contrast, risperidone treatment enhanced NF-κB activity. Taken together, D2R and D1R/D5R had opposite effects on NF-κB activity in NG108-15 cells. Risperidone up-regulated and haloperidol down-regulated NF-κB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone. [ABSTRACT FROM AUTHOR]