J. Neurochem. (2012) 122, 702–713.
Although our recent report demonstrates the essential involvement of up-regulation of a regulator of intracellular Ca2+ concentration, type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs-1), mediated via dopamine D1-like receptor (D1DR) stimulation in the cocaine-induced psychological dependence, the exact mechanisms of regulation of IP3R-1 expression by D1DRs have not yet been clarified. This study attempted to clarify these mechanisms using mouse cerebral cortical neurons. An agonist for phosphatidylinositide-linked D1DRs, SKF83959, induced dose- and time-dependently IP3R-1 protein up-regulation following its mRNA increase without cAMP production. U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor), significantly inhibited the SKF83959-induced IP3R-1 up-regulation. Furthermore, immunohistochemical examinations showed that SKF83959 increased expression of both cFos and cJun in nucleus as well as enhanced translocation of both calcineurin and NFATc4 complex to nucleus from cytoplasm. In addition, SKF83959 directly recruited binding of both AP-1 and NFATc4 to IP3R-1 promoter region. These results indicate that D1DR activation induces IP3R-1 up-regulation via increased translocation of AP-1 as well as NFATc4 in Gαq protein-coupled calcium signaling transduction pathway.
dopamine D1 receptor
type 1 inositol 1,4,5-trisphosphate receptor
nuclear factor of activated T cells
Calcium is a ubiquitous intracellular signaling molecule required for initiating and regulating a wide variety of neuronal functions including neurotransmitter release, synaptic plasticity, neurite outgrowth, and neurodegeneration (Berridge 1998; Ciccolini et al. 2003). The changes of free intracellular Ca2+ concentration modifying physiological functions is a complex and multifaceted process regulated by various mechanisms. Changes of intracellular release of Ca2+ from intracellular stores via intracellular Ca2 + -releasing channels, inositol 1,4,5-trisphosphate receptors (IP3Rs), and ryanodine receptors (RyRs) on the endoplasmic reticulum, are supposed to be one of these mechanisms to alter intracellular Ca2 + concentration. Considering the importance of calcium signals for cellular functions, functional abnormalities in endoplasmic calcium channels could result in disturbance in cellular calcium homeostasis and, in turn, produce pathological conditions. For example, IP3Rs in brain have been hypothesized to contribute to opisthotonus in mice (Street et al. 1997). In addition, IP3R-knockdown mice showed antidepressant behavior, and type 1 IP3R (IP3R-1) deficient mice exhibited ataxia and epileptic seizures (Matsumoto et al. 1996; Galeotti et al. 2008). Furthermore, functional disturbance of IP3Rs as well as RyRs appears to be involved in neurodegenerative diseases such as Alzheimer’s disease (Mattson and Chan 2003).
There are three subtypes of IP3Rs [type 1 (IP3R-1), type 2 (IP3R-2), and type 3 IP3Rs (IP3R-3)], each of which has distinct physiological properties (Foskett et al. 2007; Mikoshiba 2007). IP3R-1 is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells and concentrated in neurons in cerebral cortex (Faure et al. 2001; Mikoshiba 2007). Although the most important regulators of IP3R channel functions are considered to be changes in intracellular Ca2+ concentration and phosphorylation of IP3Rs by numerous kinases and calcium modulators (Foskett et al. 2007), there are few available data to clarify how the expression of IP3Rs is regulated.
We have recently reported the functional correlation between animal behaviors with administration of several drugs of abuse and changes of intracellular Ca2+ dynamics, especially of expression of calcium channels such as L-type high voltage-gated calcium channels and ryanodine receptors. That is, up-regulation of L-type high voltage-gated calcium channels induced by methamphetamine, cocaine, and morphine participates in drug-dependent behavior (Shibasaki et al. 2010). Furthermore, up-regulation of IP3R-1 (Kurokawa et al. 2011c) as well as of RyRs (Kurokawa et al. 2010, 2011a,b) via the dopamine D1-like receptor (D1DR) activation participates in drug-dependent behaviors such as place preference induced by drugs of abuse. Thus, D1DRs play an important role in not only behavioral changes associated with drug dependence but also in regulating the expression of intracellular calcium channels to modify intracellular Ca2+ dynamics.
This study attempted to investigate mechanisms to regulate IP3R-1 expression, especially focused on Ca2+-related signal transduction pathways initiated by D1DR activation, using primary culture of mouse cerebral cortical neurons with functional D1DRs coupling to intracellular signal transduction system (Kurokawa et al. 2011a).
Materials and methods
Primary culture of mouse cerebral cortical neurons
Mouse cerebral cortical neurons for primary culture were isolated according to the method described previously (Ohkuma et al. 1986) with a minor modification. The neopallium free of the meninges was removed from 15-day-old fetus of ddY strain mouse (Japan SLC, Inc., Hamamatsu, Japan) deeply anesthetized with sodium pentobarbital under a stereoscopic microscope to exclude contaminating other brain regions. The dissected neopallium was minced, dispersed enzymatically, and centrifuged at 1 000 g at 4°C. The isolated cells were placed on a poly-l-lysine-coated culture dish with Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal bovine serum, cultured at 37°C in humidified 95% air/5% CO2 for 3 days, and then treated with 10 μM cytosine arabinoside in DMEM containing 10% horse serum (HS) for 24 h to suppress non-neuronal cell proliferation. The culture medium was thereafter exchanged to DMEM with 10% HS to continue further incubation under the same conditions mentioned above. The exchange of the culture medium to fresh DMEM with 10% HS was carried out every 3 days and the neurons were used for the following experiments on the 13th day in culture.
Immunohistochemical studies showed that more than 95% of cells were neurons (Ohkuma et al. 1986; Mohri et al. 2003). In addition, our previous study has confirmed the presence of D1DRs coupling to intracellular signal transduction system in the neurons used here (Kurokawa et al. 2011a).
All animal experiments carried out in this study were approved by the Animal Research Committee of Kawasaki Medical School and conducted according to the “Guide for Care and Use of Laboratory Animals” of Kawasaki Medical School that is based on the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.
cAMP was measured using a Cyclic AMP Complete EIA kit (Enzo Life Sciences Inc., Farmingdale, USA). Immediately after the neurons were exposed to vehicle, forskolin (3 μM), or SKF83959 (10 μM) for 1 h, they were treated with 0.1 N HCl for 20 min at 25°C, harvested, and centrifuged at 600 g for 10 min. Cyclic AMP concentration in the supernatant thus obtained was measured according to the protocol of the Cyclic AMP Complete EIA kit.
Treatment of the neurons with SKF83959 and other drugs
SKF83959, a selective agonist for D1DRs coupling to Gαq protein-phospholipase C (PLC) systems (Jin et al. 2003), was dissolved in DMSO and directly added in the culture medium. The added volume of DMSO containing SKF83959 was one hundredth of the volume of the culture medium.
To investigate effect of continuous exposure to SKF83959, the culture medium was exchanged to fresh one with SKF83959 (10 μM) on the 13th day of the culture and then the exposure continued for 6, 12, 24, and 48 h. In the case of the experiment to examine dose-dependent effect of SKF83959, the neurons were cultured with various concentrations of SKF83959 (0.3, 3, and 10 μM) for 24 h.
On the 13th day, the culture medium was exchanged to the fresh medium and SCH23390 (a selective D1DR antagonist; 1, 10, and 30 μM) dissolved in distilled water was added into the culture medium 1 h before the exposure to SKF83959. Thereafter, the neurons were incubated with both SKF83959 and SCH23390 for 24 h.
U73122 (a PLC inhibitor; 1, 10, and 30 μM), BAPTA-AM (an intracellular calcium chelating reagent; 10 μM), W7 (a calmodulin inhibitor; 30 μM), FK506 (a calcineurin inhibitor; 10 μM), and KN-93 (a calmodulin-dependent protein kinases inhibitor; 1, 3, and 10 μM) dissolved in DMSO or distilled water were exposed to the neurons 1 h before the SKF83959 exposure. Thereafter, the neurons were cultured with both SKF83959 and each of these agents for 24 h.
Treatment of the neurons with SKF82859 and KT5720
SKF82859 (30 μM), a selective agonist for D1DRs coupling to both Gαs protein-adenylate cyclase and Gαq protein-PLC systems, dissolved in distilled water was directly added in the culture medium on the 13th day in culture and the neurons were continued to culture for further 24 h. The volume of SKF82859 solution was one hundredth of the volume of the culture medium. KT5720 (1 μM), an inhibitor of protein kinase A (PKA), dissolved in DMSO, was added in the culture medium 1 h before the exposure to SKF82859 and then the neurons were cultured with both SKF82859 and KT5720 for 24 h.
Sample preparation for measuring expression of IP3Rs-1
After the treatment of the neurons with agents as described above, the neurons were washed twice with ice-cold phosphate buffer saline (PBS: pH 7.4), scraped off with ice-cold lysis buffer containing 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 0.5 mM EDTA, 10 mM NaF, 0.5% Triton X-100 with a protease inhibitor cocktail, and homogenized using a Potter-Elvehjem tissue grinder with a Teflon pestle. The homogenate was centrifuged at 1000 g for 10 min at 4°C. The resultant precipitate was resuspended with ice-cold nuclear extraction buffer containing 20 mM HEPES, 20% glycerol, 0.5 M NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 1 mM dithiothreitol, and a protease inhibitor cocktail, to use as the nuclear fraction. The supernatant was further centrifuged at 100 000 g for 60 min at 4°C and the pellets thus obtained were retained as the membrane fraction for the subsequent analysis.
Western blot analysis
Protein samples (5 μg protein applied onto each lane) were separated by a 3–8% Tris–acetate gel (Invitrogen, Carlsbad, USA). The separated proteins were then transferred to a nitrocellulose membrane with a wet-type transblotter (90 V, 60 min). The nitrocellulose membranes were blocked with 5% non-fat milk and incubated with each of first antibodies for IP3R-1 (1 : 10 000 dilution), β actin (1 : 10 000), calmodulin-dependent protein kinase II (CaMKII) (1 : 5000 dilution), phosphorylated CaMKII (p-CaMKII) (1 : 5000 dilution), CaMKIV (1 : 5000 dilution), phosphorylated CaMKIV (1 : 5000 dilution), calcineurin (1 : 5000 dilution), and NFATc4 (1 : 5000 dilution) in PBS supplemented with 0.05% Tween 20 (T-PBS) at 4°C for 24 h, washed twice with T-PBS followed by the incubation with anti-rabbit IgG or anti-mouse IgG conjugated horseradish peroxidase (1 : 10 000 dilution) in T-PBS at 25°C for 1 h, and then washed twice with T-PBS. Finally the membrane was treated with SuperSignal West Femto Maximum Sensitivity Substrate, and separated proteins were detected with chemiluminescence. The data were analyzed using Image J (ver. 1.43u, National Institutes of Health, Bethesda, MD, USA).
To determine the quantity of IP3R-1 expression, the expression of β actin was used as internal standard. Under the conditions with the exposure to the drugs used in this study, β actin expression did not show any changes.
For examination of effects of SKF83959 and CaMKs on cFos and cJun expression in nucleus or of SKF83959 and calcineurin on translocation of NFATc4 from cytosol to nucleus, the neurons were treated with KN-93 or FK506 1 h before the exposure to SKF83959. Thereafter, the neurons were cultured with SKF83959 and KN-93 for additional 3 h or with SKF83959 and FK506 for additional 1 h. Immediately after the incubation, they were incubated with 4% paraformaldehyde at 25°C for 1 h and then blocked in the blocking solution containing 1% bovine serum albumin, 0.05% NaN3, and 0.3% Triton X-100 for 1 h. The fixed neurons were then incubated with each of antibodies for cFos (1 : 1000 dilution), cJun (1 : 1000 dilution), and NFATc4 (1 : 1000 dilution) in blocking solution overnight at 25°C, washed twice with ice-cold PBS, and finally incubated with Alexa-488 (for cJun) or Alexa-594 (for cFos and NFATc4) conjugated second antibodies diluted at 1 : 200 in blocking solution at 25°C for 2 h. Immunolabeling fluorescence was detected using OLYMPUS FV1000-D confocal laser scanning microscope.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay was carried out using a kit, ChIP-IT Express Enzymatic (Active Motif, Carlsbad, USA). After the treatment of the neurons with KN-93 (10 μM) or FK506 (10 μM) for 1 h, the neurons were further incubated with SKF83959 (10 μM) for 3 h. The neurons were cross-linked by formaldehyde (final concentration: 1%) with gentle shaking for 10 min at 25°C. Cross-linking was stopped by changing the medium to glycine stop-fix solution followed by further gentle shaking for 5 min at 25°C. Then soluble chromatin was digested and fragmented into ∼200–1500 bp using digestion buffer and enzymatic shearing cocktail (both are supplementary to the kit). The fragmented chromatin was immunoprecipitated with rabbit polyclonal antibodies for cFos, cJun, NFATc4, or a normal rabbit IgG non-immune antibody. One-tenth of lysate was stocked to quantify the amount of DNA before immunoprecipitation. Three microgram of DNA was used for immunoprecipitation using protein G magnetic beads according to the protocol supplied with the kit and other 0.03 μg of DNA was used as input. After immunoprecipitation, DNA-transcriptional factor complexes were uncross-linked, and then DNA fragment was subjected to quantitative real-time PCR with primers specific for AP-1 binding site in IP3R-1 promoter region (−2345–−2118) that was called as TPA responsive element (TRE) and for NFATc4 binding site in IP3R-1 promoter region (−1193–−958) previously reported (Graef et al. 1999). The sequence of the PCR primers used are as follows: for AP-1 binding site forward primer, 5′-TCTGAGCAGCTAAACGGCCCA-3′; reverse primer, 5′-TGGTCTTCCCTCCCCAGCCG-3′ (NC_000072.5); for NFATc4 forward primer, 5′-AGCGGTAGCAGACAGACCCCC-3′; reverse primer, 5′-AGGCTCCAGGCTGCAAAGTGC-3′ (NC_000072.5).
Protein measurement of sample
The protein content in the samples prepared from the neurons was determined using a Micro BCA Protein Assay Kit (Thermo; Rockford, USA).
Each of the data were represented as the mean ± SEM. The statistical significance was assessed by the method described in each figure legend after the application of one-way anova.
The antibody for IP3Rs-1 (ab5804) and ChIP grade antibodies for cFos (sc-52X), cJun (ab31419), and NFATc4 (sc-1153 X) were obtained from Abcam Inc. (Cambridge, UK) and Santa Cruz Biotechnology (Santa Cruz, USA), respectively. The antibody for calcineurinB (SA-212) was purchased from BIOMOL Research Laboratories Inc. (Plymouth Meeting, USA). The antibodies for β actin (sc-47778), CaMKII (sc-5306), p-CaMKII (sc-12806), p-CaMKIV (sc-28443-R), and a rabbit IgG non-immune antibody (sc-2027) were the products of Santa Cruz Biotechnology. The antibody for CaMKIV (#4032) was purchased from Cell Signaling Technology Inc (Boston, USA). SKF82859, SKF83959, SCH23390, U73122, BAPTA-AM, and W7 were the products of Sigma-Aldrich Co. (St. Louis, USA). Protease inhibitor cocktail and Super Signal West Femto Maximum Sensitivity Substrate were obtained from Roche Diagnostics (Indianapolis, USA) and Thermo Fisher Scientific (Rockford, USA), respectively. Other reagents used were commercially available and of analytical grade.
Effect of SKF83959 on expression of IP3R-1 in the cerebral cortical neurons
For determining the optimum conditions of continuous SKF83959 (an agonist for D1DRs coupled to Gαq protein) exposure of the neurons to change IP3R-1 expression, the neurons were continuously treated with various concentrations (0.3, 3, and 10 μM) of SKF83959 and with 10 μM SKF83959 for various durations of incubation (for IP3R-1 protein; 6, 12, 24, and 48 h, for its mRNA; 1, 3, 6, 12, and 24 h). As shown in Fig. 1a, SKF83959 dose-dependently increased IP3R-1 protein expression and showed its maximal effect at 3 μM. SKF83959 (10 μM) up-regulated IP3R-1 protein dependent on the incubation duration and the increase of the expression attained its plateau 24 and 48 h after the initiation of SKF83959 exposure (Fig. 1b). On the other hand, SKF83959 (10 μM) induced two peaks of the increased expression of IP3R-1 mRNA after the initiation of SKF83959 exposure, that is, significant increases of IP3R-1 mRNA expression were found 3 and 24 h after the initiation of the exposure, though the expression 6 and 12 h after the exposure was almost same as that before the exposure to SKF83959 (Fig. 1c).
Therefore, we treated the neurons with 10 μM SKF83959 for 24 h as the experimental conditions to up-regulate IP3Rs-1 in the following experiments.
To determine the quantity of IP3R-1 expression, the expression of β actin was used as internal standard. Under the conditions with the exposure to the drugs used in this study, β actin exression did not show any changes.
IP3R-1 up-regulation by D1DR stimulation was not mediated via PKA-linked signal transduction pathway
We examined effect of SCH23390, a selective D1DRs antagonist, on the IP3R-1 up-regulation by SKF83959 to confirm whether SKF83959 increased IP3R-1 expression via direct D1DR activation or not. Figure 1d shows that SCH23390 suppressed the up-regulation of IP3R-1 protein induced by SKF83959 in a dose-dependent manner, though SCH23390 alone has no potential to modify IP3R-1 expression. To determine the quantity of IP3R-1 expression, the expression of β actin was used as internal standard. Similarly, the increase of mRNA expression by SKF83959 was completely abolished by the treatment with both SKF83959 and SCH23390 at 3 h and 24 h after the exposure to these agents (Fig. 1e).
To check whether or not the SKF83959-induced IP3R-1 up-regulation was mediated via PKA-linked signal transduction pathway, we examined the activity of SKF83959 to produce cAMP. SKF83959 showed no effects on cAMP production, whereas forskolin significantly increased cAMP in the neurons used here (Fig. 1f). These results are in good agreement with the previous data demonstrating that the activation of D1DRs by SKF83959 has not been coupled to cAMP generation (Jin et al. 2003). Furthermore, as shown in Fig. 1g, SKF82859, a selective agonist for D1DRs functionally coupled to both Gαs- and Gαq-linked signal transduction pathways, induced significant IP3R-1 up-regulation, which was not affected at all by a PKA inhibitor, KT5720. These results therefore suggest that SKF82859 up-regulates IP3R-1 expression via other pathways rather than the pathway linked to cAMP.
IP3R-1 up-regulation mediated by SKF83959 via Gαq-linked calcium signaling pathway
As the SKF83959-incuced IP3R-1 up-regulation was not mediated by PKA transduction pathway as described above, we examined how another signal transduction pathway coupling to D1DRs, Gαq signal transduction pathway, affected on the SKF83959-incuced IP3R-1 up-regulation. A PLC inhibitor, U73122, dose-dependently inhibited the SKF83959-induced IP3R-1 up-regulation with complete abolishment of the up-regulation at 30 μM, indicating that the SKF83959-induced IP3R-1 up-regulation could be mediated via Gαq-PLC signal transduction pathway coupling to D1DRs (Fig. 2a). We further examined how intracellular Ca2+ and a Ca2+ modulator, calmodulin, worked in the SKF83959-induced IP3R-1 up-regulation. Although 10 μM BAPTA-AM, a membrane-permeable Ca2+ chelating agent, alone showed no effects on IP3R-1 expression without SKF83959, BAPTA-AM (10 μM) significantly inhibited the SKF83959-induced IP3R-1 up-regulation. In addition, W7 (30 μM), an inhibitor of calmodulin, also significantly suppressed the SKF83959-induced IP3R-1 up-regulation, though W7 alone did not show any effects on IP3R-1 protein expression (Fig. 2b and c). These results clearly indicate that intracellular Ca2+ and calmodulin functionally promoted by PLC activation after D1DRs are essential to the up-regulation of IP3R-1 protein induced by SKF83959.
Change of calmodulin-dependent protein kinase (CaMK) activation after SKF83959 treatment
Changes of expression of phosphorylated CaMKII and CaMKIV as parameters of their activity were examined. Western blot analysis showed that the ratio of pCaMKIV to CaMKIV time-dependently increased (Fig. 2d) with time-dependent reduction of the ratio of pCaMKII to CaMKII (Fig. 2e). Furthermore, KN-93, an inhibitor non-selective to CaMKs, dose-dependently suppressed the SKF83959-induced IP3R-1 up-regulation, and completely abolished the up-regulation at 10 μM (Fig. 2f), whereas 10 μM KN-93 itself did not have any potential to modify IP3R-1 expression. These data indicate that activated CaMKIV is, at least in part, involved in the SKF83959-induced IP3R-1 up-regulation.
Facilitation of IP3R-1 gene transcription by AP-1 binding to mouse IP3R-1 gene promoter region
As mentioned above (see Fig. 1c and e), the SKF83959-induced IP3R-1 up-regulation was associated with its mRNA increase, which suggests that this up-regulation may be because of increased transcription of IP3R-1 gene. IP3R-1 promoter region has a sequence of TRE that is considered to bind to AP-1 (Furutama et al. 1996), which leads us to examine whether SKF83959 promotes AP-1 binding to TRE region. Figure 3b shows that SKF83959 recruits both cFos and cJun to AP-1 binding promoter region of IP3R-1 gene and that the inhibition of CaMKs by KN-93 significantly suppresses this stimulatory effect of SKF83959. The quantitative data obtained from chromatin immunoprecipitation with cFos and cJun demonstrated that D1DR activation by SKF83959 significantly increased the binding of AP-1, (dimmer of cFos and cJun) to IP3R-1 promoter regions including TRE, to which AP-1 is considered to bind, and that KN-93 completely abolished the interaction of both cFos and cJun with IP3R-1 promoter region, though KN-93 alone showed no effects on the chromatin immunoprecipitation (Fig. 3c).
Localization of cFos and cJun in nucleus after treatment with SKF83959 in the mouse cerebral cortical neurons
To check whether KN-93 indeed decreased the expression of both cFos and cJun in nucleus because IP3R-1 expression is mediated by both cFos and cJun in the transcriptional level as mentioned above, we further examined changes of the expression of cFos and cJun in nucleus of the neurons under the conditions with or without SKF83959 using immunohistochemical analysis. SKF83959 (10 μM) increased the expression of both cFos and cJun immunoreactivity in nucleus and this stimulatory effect of SKF83959 was clearly suppressed by the pretreatment with 10 μM KN-93 (Fig. 3d). Based on these data, it is considered to be reasonable that the increased expression of both cFos and cJun in nucleus occurs in the SKF83959-induced IP3R-1 up-regulation.
Change of translocation of calcineurin and NFATc4 after treatment with SKF83959
Previous investigations have demonstrated that calcineurin binds to dephosphorylate NFAT family followed by facilitation of translocation to nucleus (Clipstone and Crabtree 1992; Jain et al. 1993; Hogan et al. 2003) and that IP3R-1 mRNA expression is regulated by NFATc4 (Graef et al. 1999). We therefore investigated whether facilitated translocation of both NFATc4 and calcineurin to nucleus from cytoplasm occurred after the activation of D1DRs coupling to Gαq protein by SKF83959. Western blot analysis shows that translocation of both calcineurin and NFATc4 significantly increased in the nuclear fractions prepared from the neurons after SKF83959 exposure for 1 h (Fig. 4a and b). Furthermore, the SKF83959-induced IP3R-1 up-regulation was completely suppressed by the pretreatment with 10 μM FK506, an inhibitor for calcineurin (Fig. 4c).
Facilitation of IP3R-1 gene transcription by NFATc4 binding to mouse IP3R-1 gene promoter region
On the basis of the data that D1DR stimulation facilitated the translocation of calcineurin and NFATc4 into neuronal nucleus from cytosol, we examined to confirm the recruitment of NFATc4 to NFAT-binding promoter region of IP3R-1 gene using chromatin immunoprecipitation analysis. Figure 4e and f showed that SKF83959 clearly recruited NFATc4 to NFAT-binding promoter region of IP3R-1 gene and that FK506 significantly suppressed this SKF83959 effect. The quantitative data for chromatin immunoprecipitation with NFATc4 demonstrated significant increase of NFATc4 binding to IP3R-1 promoter regions, to which NFAT family has been considered to bind, by D1DR activation with SKF83959 (Fig. 4f). In addition, FK506 completely abolished the increase of interaction of NFATc4 with IP3R-1 promoter region, though FK506 alone shows no effects on NFATc4 binding with IP3R-1 promoter region (Fig. 4f).
Localization of NFATc4 in nucleus after treatment with SKF83959 in the mouse cerebral cortical neurons
To check whether FK506 indeed decreased translocation of NFATc4 into nucleus of the neurons, we examined translocation of NFATc4 to nucleus with or without SKF83959 by immunohistochemical analysis. SKF83959 (10 μM) enhanced translocation of NFATc4 to neuronal nucleus, which was clearly suppressed by the pretreatment with 10 μM FK506 (Fig. 4g). On the basis of the results shown in Fig. 4, it is considered that the increased translocation of NFATc4 to nucleus of the neurons is involved in the SKF83959-induced IP3R-1 up-regulation.
A variety of functional proteins and mediators involved in neuronal signal transduction systems, such as inositol 1,4,5-triphosphate (IP3), Ca2+, ATP, and various kinases including cAMP-dependent protein kinase, PKA, cGMP-dependent protein kinase, calmodulin-dependent protein kinase II (CaMKII), protein kinase C, and protein tyrosine kinases, have been reported to modify IP3R channel activity (Foskett et al. 2007). Among these factors, Ca2+ signal transduction pathway has been reported to regulate IP3R-1 expression (Genazzani et al. 1999; Graef et al. 1999), though exact mechanisms to regulate IP3R-1 expression via D1DR are not fully clarified. Our previous reports have demonstrated that drugs of abuse, such as psychostimulants and morphine, up-regulate several calcium channel subunits consisting of L-type voltage-gated calcium channels, such as α1C, α1D, and α2/δ1 subunits (Shibasaki et al. 2010), and ryanodine receptors (RyRs) (Kurokawa et al. 2010, 2011a,b). Furthermore, these changes are mediated via D1DR activation (Kurokawa et al. 2010, 2011a,b) and are involved in animal behaviors produced by drug abuse (Kurokawa et al. 2010, 2011b; Shibasaki et al. 2010). Among these changes of channels to regulate intracellular Ca2+ concentration, RyR up-regulation was induced by increased transcription of its gene information via cyclic AMP-responsive element-binding protein (CREB) following PKA activation after D1DR activation (Kurokawa et al. 2011a) as well as stimulation of nicotinic acetylcholine receptors (Ziviani et al. 2011). In addition, as far regulation of IP3R-1 expression, we previously reported that IP3R-1 up-regulation in association with increased place preference by cocaine was also mediated via D1DR activation (Kurokawa et al. 2011c). These results suggest the possibility that IP3R-1 expression may be also regulated via signal transduction pathway coupled to D1DRs, which promotes us to carry out this study.
We considered that the primary culture of mouse cerebral cortical neurons was suitable for the purpose of this study because these neurons had D1DRs functionally coupling with intracellular signal transduction system (Kurokawa et al. 2011a).
SKF83959 exposure of the neurons for 24 h induced a significant increase in IP3R-1 protein expression with the enhancement of its mRNA expression. Both types of changes were completely suppressed by D1DR blockade with a selective antagonist to D1DRs, SCH23390. Such involvement of D1DRs in increased IP3R-1 expression is in good agreement with that observed in the brain of mouse treated with cocaine (Kurokawa et al. 2011c). In contrast to the time course of change in IP3R-1 protein expression, that of mRNA expression showed different pattern of increases, that is, there were two peaks of increase in mRNA expression during continuous exposure to SKF83959. Previous investigations reported that dopamine D2 receptor-coupled signal transduction works with two different time schedules, early and late phases, of changes in intracellular signaling processes. The early phase was mediated via G proteins, and β-arrestin mediated the late phase of signal transduction (Beaulieu et al. 2004, 2005). Therefore, the late phase of change of IP3R-1 mRNA after D1DR activation by SKF83959 presented in this study may be mediated via other pathways different from G protein-linked pathway. When considering the time course of changes of both IP3R-1 protein and its mRNA, it is reasonable to consider that the early phase of IP3R-1 mRNA increase is mediated via G protein-participating signal transduction pathway as described below. However, the exact physiological and/or pathophysiological roles and significance of increase of IP3R-1 mRNA in possible late phase found in this study remain to be elucidated.
Previous investigations have confirmed that D1DRs functionally couple to both Gαs and Gαq proteins (Jin et al. 2001; Rashid et al. 2007). As D1DRs stimulate adenylate cyclase to facilitate cAMP formation, we examined whether IP3R-1 expression was mediated via PKA-coupling transduction pathway activated by cAMP. SKF83959, a selective D1DR agonist, has been reported to couple to Gαq protein to facilitate selectively phosphatidylinositide turnover, but not to couple to Gαs protein, and has no activity to form cAMP (Jin et al. 2003). The latter pharmacological property of SKF83959 was also found in this study. On the other hand, another full agonist selective to D1DRs, SKF82859, coupling to both Gαs and Gαq proteins, up-regulated IP3R-1 and this SKF82859-induced up-regulation of IP3R-1 expression was not affected by KT5720, a PKA inhibitor. These results obtained from the experiments using two D1DR antagonists coupling to different intracellular signal transduction systems indicate that IP3R-1 up-regulation by D1DR activation is mediated via Gαq-linked signal transduction pathway, but not via Gαs-linked one.
D1DRs coupling to Gαq protein transduce their signaling through PLC activation to produce inositol-1, 4, 5-triphosphate (IP3) and diacylgricerol. As previous studies have reported that Ca2+ signal transduction pathway may regulate IP3R-1 expression (Genazzani et al. 1999; Graef et al. 1999), we focused on the roles of Ca2+ signal transduction pathway for clarifying the exact regulatory mechanisms of IP3R-1 expression by D1DR activation. For this purpose, we examined effects of U73122, a PLC inhibitor, BAPTA, an intracellular calcium chelating agent, and W7, a calmodulin inhibitor, on the SKF83959-induced IP3R-1 up-regulation. As demonstrated here, the inhibition of the SKF83959-induced increase of IP3R-1 expression by U73122, BAPTA, and W7 is considered to suggest that the SKF83959-induced increase of IP3R-1 expression is mediated via Ca2+ signaling pathway. Taken together with these results presented here and the previous reports suggesting that IP3R-1 expression appears to be regulated by Ca2+ influx through L-type voltage-gated calcium channels and N-methyl-d-aspartate receptors (Genazzani et al. 1999; Graef et al. 1999), it is reasonable to conclude that calcium was an important key factor for IP3R-1 up-regulation by D1DR stimulation.
Both CaMKs and calcineurin are well known as metabolic components appearing downstream of calcium and calmodulin signal transduction pathways (Klee 1991; Solàet al. 1999, 2001; Nakanishi and Okazawa 2006). Previous reports showed that activated CaMKs phosphorylated cyclic AMP-responsive element-binding protein and phosphorylated-CREB (p-CREB), in turn, increased cFos production (Sato et al. 2006). In addition, activated CaMKs were also shown to mediate cJun activation (Enslen et al. 1996; Størling et al. 2005). Thus Ca2+-dependent CaMKs pathway promotes cFos expression and cJun activity to enhance possibly activator protein-1 (AP-1) activity. Among various types of CaMK isoforms, this study suggests that CaMKIV showing its activation as increased p-CaMKIV may, at least in part, play an important role in the SKF83959-induced IP3R-1 up-regulation, which is supported by the results showing complete abolishment of the SKF83959-induced IP3R-1 up-regulation by KN-93, a non-selective inhibitor of CaMKs. On the other hand, p-CAMKII decreased by SKF83959 in a time-dependent manner. However, the pathophysiological significance of these changes of p-CAMKII in the SKF83959-induced IP3R-1 up-regulation is not clear at present.
Immunofluorescent staining demonstrates the increase of both cFos and cJun expression in nucleus after D1DR stimulation with SKF83959 and significant suppression of this increased expression of cFos and cJun in nucleus of the neurons by KN-93. Western blot analysis showed that KN-93 dose-dependently suppressed the SKF83959-induced IP3R-1 up-regulation. Furthermore, SKF83959 time-dependently activated CaMKIV, but not CaMKII. Chromatin immunoprecipitation also showed enhanced binding of both cFos and cJun to IP3R-1 promotor region following D1DR stimulation with SKF83959, which was significantly suppressed by KN-93. These findings therefore indicate that CaMKIV activated by D1DR stimulation promotes the binding of AP-1, including both cFos and cJun, to IP3R-1 promoter region to accelerate IP3R-1 mRNA transcription via Gαq protein-coupled intracellular calcium signaling system. Previous studies reported that CaMKIV plays an essential role in facilitating CREB phosphorylation (Lonze and Ginty 2002; West et al. 2002; Deisseroth et al. 2003) and that CaMKIV-CREB pathway is required for protein synthesis-dependent long-term potentiation (Ho et al. 2000; Kang et al. 2001). CaMKIV signal transduction pathway also mediated protein synthesis (Li et al. 2010; Chen et al. 2011). Our investigation suggests that CaMKIV may be one of key factors, which regulates IP3R-1 expression via D1DR stimulation. However, the pathophysiological role of decreased phosphorylated CAMK II in the SKF83959-induced IP3R-1 up-regulation remains to be elucidated.
NFAT activity appears to be downstream of calcium signaling pathways (Clipstone and Crabtree 1992; Jain et al. 1993; Hogan et al. 2003). Among members of NFAT family, NFATc4 is predominantly localized in the spinal cord and brain and is also abundantly present in the olfactory bulb, cerebellum, and certain regions of the cortex (Bradley et al. 2005; Seybold et al. 2006; Groth et al. 2007), and also binds to calcineurin followed by translocation of NFAT-calcineurin complex into nucleus (Groth and Mermelstein 2003). The immunofluorescent staining and western blot analysis in this study showed that D1DR stimulation by SKF83959 facilitated the translocation of both calcineurin and NFATc4 to nucleus from cytosol and that the translocated NFATc4 to nucleus directly bound to IP3R-1 promoter region. Similar findings have been reported in the previous investigation (Graef et al. 1999). Furthermore, as demonstrated here, FK506 significantly reduced the translocation of NFATc4 to nucleus from cytosol and, in turn, suppressed the binding of NFATc4 to IP3R-1 promoter region in nucleus. Therefore, the findings presented in this study used the cortical neurons confirm that calcineurin activated by D1DR stimulation promotes the binding of NFATc4 to IP3R-1 promoter region to accelerate IP3R-1 mRNA transcription via Gαq protein-coupled intracellular calcium mobilizing system as previously reported (Graef et al. 1999). Previous reports have shown that NFAT family mediates several kinds of gene expression, such as BDNF, neurotrophin, and growth hormone gene, in the central nervous system (Groth and Mermelstein 2003; Asai et al. 2004; Groth et al. 2007). These findings suggested that the regulation of NFAT-mediated expression of genes including IP3R-1 may also occur in the central nervous system function.
This study is considered to confirm the first direct evidence that the expression of IP3R-1 in the neurons is regulated by phosphatidylinositide-linked D1DRs via calcium signaling transduction pathway including AP-1 with activated CAMKIV as well as via calcineurin-linked NTATc4 transcription promoting processes. Our recent investigation reports that cocaine induces IP3R-1 up-regulation through D1DR activation in the brains of mice showing cocaine-conditioned place preference and suggests that this up-regulation may be functionally related with addictive behaviors (Kurokawa et al. 2011c). In addition, the data that amphetamine induced IP3R sensitization in vivo (Ahn et al. 2010) and that a calcineurin inhibitor FK506 suppressed nicotine-mediated motor activity (Addy et al. 2007) were also reported. Taken together, these data suggest that up-regulation of IP3Rs-1 by drugs of abuse such as psychostimulants is regulated by D1DRs coupling to Gαq-PLC signaling system following not only facilitation of calcineurin-NFATc4 translocation into nucleus but also increase of AP-1 expression including increased moving of both cFos and cJun into nucleus and accelerating transcription of IP3R-1 gene information. Therefore, inhibitors on these activated components of the pathway described above, especially the inhibitors of IP3R-1, inhibitors of CaMKIV, and calcineurin inhibitors, may become candidates as possible therapeutics to treat and/or prevent psychological dependence on drugs of abuse, such as cocaine and methamphetamine.
The authors would like to thank to Junko Katayama and Noriko Ohtsuki for their excellent technical assistance. This work was supported in part by Grants-in-Aid for ministry of Health, Labour and Welfare, and by a Research Project Grant from Kawasaki Medical University (23-A46).
All authors declare that they have no conflicts of interest in this study.