Histidine triad nucleotide-binding protein 1 (HINT1) regulates Ca2+ signaling in mouse fibroblasts and neuronal cells via store-operated Ca2+ entry pathway

Cristina I. Linde, Bo Feng, Jia Bei Wang, Vera A. Golovina

Abstract

Recent findings indicate that histidine triad nucleotide-binding protein 1 (HINT1) is implicated in the pathophysiology of certain psychiatric disorders and also exhibits tumor suppressor properties. However, the authentic functions of HINT1 in cellular physiology and especially its role in Ca2+ signaling remain unclear. Here, we studied Ca2+ signaling in cultured embryonic fibroblasts derived from wild-type control and HINT1 knockout (KO) mice. The resting cytosolic Ca2+ level (measured with fura-2) was not altered in fibroblasts lacking HINT1. The stored Ca2+ evaluated by measuring peak amplitude of ATP (10 μM)-induced Ca2+ transients in Ca2+-free medium was significantly larger in HINT1 KO fibroblasts than in wild-type cells. Ca2+ influx after external Ca2+ restoration, likely via store- and receptor-operated channels (SOCs and ROCs, respectively), was greatly (by 2-fold) reduced in HINT1 KO fibroblasts. This correlated with a downregulated expression of Orai1 and stromal interacting molecule 1 (STIM1), essential components of store-operated Ca2+ entry pathway. Expression of canonical transient receptor potential (TRPC)3 and TRPC6, which function as ROCs, was not altered in HINT1 KO fibroblasts. Immunoblots also revealed that Orai1 was downregulated by twofold in brain lysates of HINT1 KO mice compared with the wild-type littermates. Importantly, silencer RNA knockdown of HINT1 in Neuro-2A cells markedly downregulated Orai1 and STIM1 protein expression and significantly (by 2.5-fold) reduced ATP-induced Ca2+ influx, while ATP-evoked Ca2+ release was not changed. Thus the study demonstrates a novel function of HINT1 that involves the regulation of SOC-mediated Ca2+ entry pathway (Orai1 and STIM1), essential for regulation of cellular Ca2+ homeostasis.

  • Orai1
  • STIM1
  • TRPC proteins
  • receptor-operated Ca2+ entry
  • HINT1 knockout mice

calcium signaling is essential for various processes in the central nervous system (CNS), including neuronal growth, differentiation, gene expression, secretion of neurotransmitters, and synaptic plasticity (5). Neuronal activation is associated with elevation of the cytosolic free Ca2+ concentration ([Ca2+]cyt) that triggers a large spectrum of physiological responses (5). The changes in [Ca2+]cyt generally consist of initial Ca2+ release from the intracellular Ca2+ stores, primarily in the endoplasmic reticulum (ER; Ref. 47), followed by sustained Ca2+ entry through plasma membrane Ca2+ channels (35).

Recent findings reveal that Ca2+ entry through store- and receptor-operated channels (SOCs and ROCs, respectively) plays an important role in shaping Ca2+ signals in neurons (6, 35). Substantial evidence indicates that two transmembrane proteins, Orai1 and stromal interacting molecule 1 (STIM1), are critical components of the store-operated Ca2+ entry pathway (12, 36, 48). Orai1 forms the Ca2+ selectivity filter of the Ca2+ release-activated Ca2+ (CRAC) channel (51), which may be another type of SOC. In addition to Orai1, two other subtypes, Orai2 and Orai3, were identified in the mammalian genome (15). Orai2 and Orai3 can constitute or contribute to SOCs when ectopically expressed but not in all tested cells (10). STIM1, the putative Ca2+ sensor in the ER, regulates SOCs and CRAC channels (19, 36). The canonical transient receptor potential (TRPC) proteins, mammalian homologs of the Drosophila transient receptor potential (trp) channel, have also been described as molecular components of SOCs and ROCs (18, 32). In particular, TRPC1, TRPC4, and TRPC5 may be part of the SOCs activated by ER Ca2+ store depletion (32), In contrast, TRPC3 and TRPC6, which are components of ROCs, can be activated by agonists via G protein-coupled receptors linked to phospholipase C and by diacylglicerols in a store depletion-independent manner (18).

Dysregulation of the neuronal Ca2+ signaling has been implicated in numerous neuropathological processes such as neurodegeneration, cognitive deficits, and schizophrenia (13, 24, 50). In view of the profound contribution of SOCs and ROCs in regulating neuronal Ca2+ signaling (6, 35), it is hardly surprising that these channels have been implicated in the neurological diseases such as Parkinson's, Alzheimer's, Huntington's, and bipolar disorder (13, 39, 49). For example, chlorpromazine, one of the phenothiazine neuroleptic drugs used to treat mental diseases including schizophrenia (40), inhibits SOC-mediated Ca2+ entry in PC12 cells (9).

Among the large number of proteins affected in psychiatric disorder patients is histidine triad nucleotide-binding protein 1 (HINT1; Refs. 11, 28, 43, 44), a small intracellular protein originally discovered as protein kinase C inhibitor-1 (PKCI-1; Ref. 33). The PKC inhibitory role is now viewed with skepticism although HINT1 can interact with PKC (23). HINT1 belongs to a histidine triad (HIT) superfamily, which contains a highly conserved His-X-His-X-His-XX motif (X is a hydrophobic amino acid) (23). HINT1 was initially isolated by Ca2+-dependent hydrophobic interaction chromatography and is widely expressed in different tissues, such as brain, liver and kidney (23, 26). The protein exhibits properties similar to those of calmodulin and related Ca2+ binding proteins (29). Nevertheless, a role of HINT1 in regulation of Ca2+ signaling remains unknown. Here, using fura-2 imaging, silencer RNA approach, and Western blot analysis, we demonstrate that HINT1 is involved in the regulation of SOC-mediated Ca2+ entry in mouse embryonic fibroblasts and Neuro-2A cells.

MATERIALS AND METHODS

Mouse embryonic fibroblasts and Neuro-2A cell lines.

Mouse embryonic fibroblast cultures were established from HINT1 knockout (KO) mice and wild-type (WT) control littermates (2). Briefly, mouse embryos of 12–13 days were collected from either HINT1 KO or WT mice. The head and all innards were removed from the embryos. The remaining embryo body was chopped into 1-mm cubes and the tissue was digested by incubation with trypsin at 37°C for 30 min. Then, the trypsin solution was aspirated and the tissue was resuspended into DMEM supplemented with 10% FBS, 100 U penicillin, and 100 μg streptomycin. The tissue was dispersed by trituration with a fire-polished Pasteur pipette. The dissociated cells were plated on 10-cm culture dishes and cultured to 100% confluence in a humidified atmosphere of 5% CO2-95% air at 37°C. Then, the cells were dissociated with trypsin and seeded to a new plate. The cells were replated every 3 days even if there was little or no growth. Growth of the cells was recovered after multiple rounds of passaging (about 20–25). The immortalized fibroblasts were then frozen using the DMEM media containing 20% FBS and 5% DMSO.

Neuro-2A mouse neuroblastoma cells were cultured in MEM GIBCO-BRL supplemented with 10% FBS and antibiotics. Mouse embryonic fibroblasts and Neuro-2A cells were split every 2–3 days and plated on either 25-mm coverslips for use in fluorescent microscopy experiments or on 10-cm culture dishes for Western blot analysis.

Small interfering RNA knockdown.

Neuro2A cells were transfected with the small interfering RNAs (siRNAs) ON-Target plus SMARTpool (20 μM) designed against HINT1 or siCONTROL (Dharmacon, Lafayette, CO). The sequences of the HINT1/siRNA duplexes were as follows: 5′-UGAAAGUCUUCUAGGACAU-3′, 5′-ACGAGAUUGCCAAGGCUCA-3′, 5′-CGGCAAGAUCAUCCGCAAA-3′, and 5′-GGUAAAUGGCACACGUAGU-3′. The sequences of nontargeted siRNA (siCONTROL) duplexes were as follows: 5′-UAGCGACUAAACACAUCAA-3′ and 5′-UAAGGCUAUGAAGAGAUAC-3′. Twenty-four hours before treatment, Neuro2A cells were placed in the culture medium without antibiotics and further transfected with siRNA using Lipofectamine 2000 reagent in Opti-MEM (Invitrogen). After a 24-h incubation, the medium was aspirated and replaced with DMEM (10% FBS) without siRNA for 72 h before Ca2+ measurements or Western blot analysis was performed.

Calcium imaging.

[Ca2+]cyt was measured with fura-2 using digital imaging (25). Mouse embryonic fibroblasts or Neuro 2A cells were loaded with fura-2 by incubation for 35 min in culture medium containing 3.3 μM fura-2 AM (20–22°C, 5% CO2-95% air). After the dye loading, coverslips were transferred to a tissue chamber mounted on a microscope stage, where the cells were superfused for 15–20 min (35–36°C) with standard physiological salt solution (PSS) to wash away extracellular dye. The PSS contained the following (in mM): 140 NaCl, 5.9 KCl, 1.2 NaH2PO4, 5 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES (pH 7.4). In Ca2+-free PSS, CaCl2 was omitted and 50 μM EGTA were added to chelate residual Ca2+. Cells were studied for 40–60 min during continuous superfusion with PSS (35°C).

The imaging system included a Zeiss Axiovert 100 microscope (Carl Zeiss, Thornwood, NY). The fura-2-loaded cells were illuminated with a diffraction grating-based system (Polychrome V; TILL Photonics, Germany). Fluorescent images were recorded with a CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ). Image acquisition and analysis were performed with a MetaFluor/MetaMorph Imaging System (Molecular Devices, Downingtown, PA). [Ca2+]cyt was calculated by determining the ratio of fura-2 fluorescence emission (510 nm) excited at 380 and 360 nm, as previously described (25). Intracellular fura-2 was calibrated in situ separately in cultured mouse embryonic fibroblasts and Neuro 2A cells (25).

Immunoblot analysis.

Membrane proteins were solubilized in SDS buffer containing 5% 2-mercaptoethanol and were separated by SDS-PAGE as previously described (25). The following antibodies were used: rabbit polyclonal anti-HINT1 (26); rabbit polyclonal anti-Orai1 (ProSci, Poway, CA); rabbit polyclonal anti-STIM1 (a gift from Dr. J. Roos, Torrey Pines Therapeutics, La Jolla, CA). Gel loading was controlled with monoclonal anti-β-actin antibodies (Sigma-Aldrich, St. Louis, MO). After being washed, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h at room temperature. The immune complexes on the membranes were detected by enhanced chemiluminescence-plus (Amersham BioSciences, Piscataway, NJ) and exposure to X-ray film (Eastman Kodak, Rochester, NY). Quantitative analysis of immunoblots was performed by using a Kodak DC120 digital camera and 1D Image Analysis Software (Eastman Kodak).

Materials.

Neuro-2A cells were purchased from American Type Culture Collection, (Manassas, VA). FBS was obtained from Atlanta Biologicals (Lawrenceville, GA). DMEM was purchased from Cellgro (Manassas, VA). All other tissue culture reagents were obtained from GIBCO-BRL (Grand Island, NY). Fura-2 AM was obtained from Molecular Probes (Invitrogen Detection Technologies, Eugene, OR). All other reagents were analytic grade or the highest purity available.

Statistical analysis.

The numerical data presented in results are means ± SE from n single cells (one value per cell). Immunoblots were repeated at least three to six times for each protein. Data from three transfections were obtained for siRNA protocols. Statistical significance was determined using Student's paired or unpaired t-test or two-way ANOVA, as appropriate. P < 0.05 was considered significant.

RESULTS

ATP-induced Ca2+ responses are altered in cultured embryonic fibroblasts derived from HINT1 KO mice.

The absence of HINT1 expression in fibroblasts from HINT1 KO mice was confirmed at the protein level by Western blotting (Fig. 1A). The average resting [Ca2+]cyt level was not significantly altered in fibroblasts lacking HINT1 (135 ± 3 vs. 139 ± 3 nM in WT fibroblasts). Under physiological conditions, Ca2+ mobilization from the ER stores is mediated by a variety of agonists, which are known to trigger inositol trisphosphate (IP3) synthesis (4). To activate the phosphoinositide/Ca2+ signaling cascade, fibroblasts were stimulated with the purinergic receptor agonist ATP (Fig. 1B). The application of 10 μM ATP, in physiological media, induced altered Ca2+ signals in the HINT1 KO fibroblasts (Fig. 1, B and C). The peak initial response, believed to be the result of IP3-mediated ER Ca2+ release, was significantly augmented in the HINT1 KO fibroblasts (924 ± 30 vs. 743 ± 29 nM in WT fibroblasts, P < 0.001; Fig. 1, B and C). The later plateau, likely mediated by Ca2+ entry through SOCs and/or ROCs, was, however, greatly reduced in HINT1 KO fibroblasts (Fig. 1B). Superimposed records of the ATP-induced Ca2+ response show that the integral of the rise of [Ca2+]cyt (area under the [Ca2+]cyt curve) in HINT1 KO fibroblasts was decreased to 56 ± 4% of the area in WT fibroblasts (n = 26 WT cells; n = 26 HINT1 KO fibroblasts; P < 0.001). In subsequent experiments we investigated the contribution of ER Ca2+ release and extracellular Ca2+ entry through SOCs and ROCs to the altered Ca2+ homeostasis in HINT1 KO fibroblasts.

Fig. 1.

Altered ATP-induced Ca2+ responses in embryonic fibroblasts derived from histidine triad nucleotide-binding protein 1 (HINT1) knockout (KO) mice. A: Western blot showing the absence of HINT1 expression in fibroblasts derived from HINT1 KO mice. Membrane proteins (20 μg/lane) from wild-type (WT) and HINT1 KO fibroblasts were loaded and probed with specific anti-HINT1 antibodies. Blots were later incubated with anti-β-actin antibodies to verify uniform protein loading. Representative Western blot is shown; comparable results were obtained in 3 immunoblots. B: ATP-induced Ca2+ responses in WT (blue line) and HINT1 KO fibroblasts (red line). Representative records show the time course of cytosolic free Ca2+ concentration ([Ca2+]cyt) changes induced by 10 μM ATP. C: summarized data showing the resting [Ca2+]cyt and the peak amplitude of ATP-induced Ca2+ transients (n = 105 WT cells and n = 78 HINT1 KO cells). Data are means ± SE. **P < 0.001 vs. WT fibroblasts.

ER Ca2+ stores and SOC/ROC-mediated Ca2+ entry in HINT1 KO fibroblasts.

Figure 2A illustrates the protocol: it shows the time course of ATP-induced changes in [Ca2+]cyt in the absence and presence of extracellular Ca2+ in control WT and HINT1 KO fibroblasts. The ATP-induced ER Ca2+ release (indicated by the initial rise in [Ca2+]cyt in Ca2+-free medium) was larger in HINT1 KO than WT fibroblasts (1,032 ± 42 vs. 838 ± 35 nM in WT fibroblasts; P < 0.001; Fig. 2, A and B). Ca2+-free media were used to avoid complications from Ca2+ entry. Subsequent restoration of external Ca2+ evoked a secondary rise in [Ca2+]cyt, associated with Ca2+ entry from the extracellular fluid via both SOCs and ROCs. To eliminate the contribution of voltage-gated Ca2+ channels to ATP-induced Ca2+ entry, all solutions in the experiments contained 10 μM nifedipine (25). This rise in [Ca2+]cyt was significantly smaller in HINT1 KO fibroblasts (100 ± 9 vs. 202 ± 10 nM in WT fibroblasts; P < 0.001; Fig. 2, A and B). The implication is that SOC/ROC activity is reduced in HINT1 KO fibroblasts. Whether this was simply due to decreased entry through an unchanged number of channels or to a decrease in the number of channels available was tested by immunoblot analysis. The results revealed that expression of Orai1 (a protein component of SOC) in HINT1 KO cells was twofold smaller than in WT fibroblasts (Fig. 3, A and B). Moreover, expression of STIM1, essential component of the store-operated Ca2+ entry pathway, was also significantly downregulated in HINT1 KO fibroblasts (Fig. 3, C and D). The expression of TRPC3 and TRPC6, which belong to the TRPC3/6/7 subfamily of diacylglycerol-activated ROCs, was not significantly altered in HINT1 KO cells (Fig. 3, EH).

Fig. 2.

Comparison of ATP-induced Ca2+ release and Ca2+ entry in WT and HINT1 KO fibroblasts. A: representative records showing the time course of [Ca2+]cyt changes in control WT (blue line) and HINT1 KO fibroblasts (red line). ATP (10 μM) was applied in Ca2+-free and Ca2+-containing solutions. Nifedipine (10 μM) was applied 10 min before the records shown and was maintained throughout the experiment. B: summarized data showing ATP-induced Ca2+ release and Ca2+ influx in WT (blue bars) and HINT1 KO fibroblasts (red bars). Peak amplitudes of the ATP-induced Ca2+ responses in the absence and presence of external Ca2+ were studied in 105 WT and 75 HINT1 KO cells. The data are shown as quantitative difference of ATP-induced transient Ca2+ peaks relative to the baseline [Ca2+]cyt levels (before any treatment). **P < 0.001 vs. WT cells.

Fig. 3.

Expression of Orai1, stromal interacting molecule 1 (STIM1), canonical transient receptor potential (TRPC)3, and TRPC6 in WT and HINT1 KO fibroblasts. AH: Western blot analysis of ORAI1 (A), STIM1 (C), TRPC 3 (E), and TRPC 6 (G) protein expression in WT and HINT1 KO fibroblasts. A, C, E, and G: representative blots. All lanes were loaded with 30 μg of membrane protein. Summary data (B, D, F, and H) are normalized to the amount of β-actin and are expressed as means ± SE from 6 (B), 6 (D), 3 (F), and 3 (H) immunoblots. *P < 0.05 and **P < 0.001 vs. WT fibroblasts.

Knockdown of HINT1 gene in Neuro-2A cells downregulates expression of Orai1 and STIM1 and decreases ATP-induced Ca2+ influx.

To study the acute effects of HINT1 knockdown and to determine whether HINT1 is involved in regulation of Ca2+ signaling in neuronal cells, we used siRNA-mediated silencing of HINT1 in Neuro-2A cells. Transfection with HINT1/siRNA resulted in 82 ± 2% knockdown of HINT1 protein in cultured Neuro-2A cells (Fig. 4, A and B). Neuro-2A cells are a mouse neuroblastoma cell line that endogenously expresses HINT1. Importantly, knockdown of HINT1 greatly reduced the expression of Orai1 (Fig. 4, C and D) and STIM1 (Fig. 4, E and F) in Neuro-2A cells. Moreover, the absence of HINT1 expression in the brain of HINT1 KO mice (Fig. 4A) is also associated with a significant (by 2-fold) downregulation of Orai1 protein (Fig. 4, C and D).

Fig. 4.

Knockdown or absence of HINT1 gene downregulates expression of ORAI1 and STIM1 proteins. AF: Western blot analysis of HINT1 (A and B), Orai1 (C and D), and STIM1 (E and F) protein expression in Neuro-2A cells treated with HINT1 small interfering (si)RNA. Contr, cells treated with nontargeting siRNA. All lanes were loaded with 20 μg of membrane protein. Blots were later incubated with anti-β-actin antibodies to verify uniform protein loading. Brain whole cell lysate (20 μg/lane) from WT and HINT1 KO mice were used as a control. B, D, and F: data are normalized to the amount of β-actin and expressed as means ± SE from 3 (B), 5 (D; Neuro-2A cells), 3 (D; brain), and 3 (F) immunoblots. **P < 0.001 vs. control Neuro2A cells (B, D, and F) and vs. WT brain (D).

Selective inhibition of HINT1 expression in Neuro-2A cells significantly attenuated the ATP-induced Ca2+ entry (26 ± 2 vs. 63 ± 3 nM in cells treated with siControl RNA; P < 0.001; Fig. 5, A, B, and E). The average resting [Ca2+]cyt level was also significantly reduced in Neuro-2A cells transfected with HINT1/siRNA (65 ± 1 vs. 70 ± 1 nM in control cells, P < 0.05; Fig. 5C), while the ATP-induced Ca2+ release was not altered in the cells (Fig. 5 A, B, and D).

Fig. 5.

Knockdown of the HINT1 gene markedly reduces ATP-induced Ca2+ entry in Neuro-2A cells. Representative records showing the time course of [Ca2+]cyt changes in Neuro-2A cells treated with siControl (A) and HINT1/siRNA (B). ATP (10 μM) was applied in Ca2+-free and Ca2+-containing solutions. Nifedipine (10 μM) was applied 10 min before the records shown and was maintained throughout the experiment. CE: summarized data showing the resting [Ca2+]cyt (C), the ATP-induced transient Ca2+ peak in the absence of extracellular Ca2+ (D), and the secondary rise in [Ca2+]cyt when Ca2+ was added back (E). Data in D and E are shown as quantitative differences of ATP-induced transient Ca2+ peaks relative to the baseline [Ca2+]cyt levels (before any treatment). Data are means ± SE (n = 187 cells transfected with nontargeted siRNA and n = 141 cells transfected with HINT1/siRNA). *P < 0.05 and **P < 0.001 vs. control cells.

DISCUSSION

The results described in this report show the effects of knockdown of HINT1 in vivo and in vitro on the expression of components of SOC-mediated Ca2+ entry pathway (Orai1 and STIM1), essential for regulation of cellular Ca2+ homeostasis. This is the first study demonstrating a functional role of HINT1 in the regulation of Ca2+ signaling in mouse fibroblasts and neuronal cells.

Ca2+ homeostasis is altered in cultured embryonic fibroblasts derived from HINT1 KO mice and in Neuro-2A cells transfected with HINT1/siRNA.

The functional role of HINT1 in the regulation of cell Ca2+ signaling was largely unknown. Therefore, we first studied Ca2+ homeostasis in cultured fibroblasts from mice lacking HINT1. A key observation in this study is that the HINT1 KO fibroblasts exhibit Ca2+ dysregulation: augmented ATP-evoked ER Ca2+ release and decreased ATP-induced Ca2+ entry (Fig. 2, A and B), which is reflected in much shorter plateau of ATP-induced Ca2+ response (Fig. 1B) compared with that in WT fibroblasts. Immunoblots revealed that the decreased ATP-induced Ca2+ entry is likely a consequence of downregulated expression of Orai1 and STIM1 proteins (Fig. 3, AD), critical components of store-operated Ca2+ entry in different cell types including fibroblasts (1, 15, 19, 36). It is noteworthy that the absence of HINT1 expression in the brain of HINT1 KO mice is also associated with downregulation of Orai1 (Fig. 4, C and D). In contrast, expression of TRPC3 and TRPC6, components of ROCs, was not altered in HINT1 KO fibroblasts (Fig. 3, EH), indicating that the effects of HINT1 gene KO are specific only for the expression of SOCs. The augmentation of ATP-mediated ER Ca2+ release in HINT1 KO fibroblasts can be explained by the increased ER Ca2+ store content resulting, apparently, from upregulation of SERCA2 and by increased expression of IP3 receptors, which should enhance mobilization of the larger Ca2+ store.

Importantly, the effects of genetic ablation of the HINT1 gene in vivo could be replicated in vitro in Neuro2A cells transfected with HINT1/siRNA. Indeed, inhibition of HINT1 expression in the cells with HINT1/siRNA also greatly downregulated Orai1 and STIM1 (Fig. 4), although the mechanisms by which this occur are not yet understood and require further investigation. This is reflected, functionally, by the significantly attenuated extracellular Ca2+ influx triggered by ER Ca2+ store depletion with ATP (Fig. 5). Thus the results provide strong experimental support for a new functional role of HINT1 in the regulation of SOC-mediated Ca2+ entry not only in fibroblasts but also in neuronal cells.

HINT1 deficiency may negatively modulate Ca2+ signaling via PKC-regulated activity of SOCs and ROCs.

The [Ca2+]cyt in cells with suppressed HINT1 expression can be modulated not only by downregulated expression of Orai1 and STIM1 but also suppressed activity of SOCs and, apparently, ROCs via PKC-mediated phosphorylation of the channels. HINT1 is known as a PKC interacting protein (23). A yeast two-hybrid screen identified a putative PKC interaction with HINT1, although it was not confirmed at the cellular level (23, 42). Recent findings revealed that protein expression of PKCγ, which belongs to the conventional PKC subfamily (α, βI, βII, and γ; Ref. 30), is elevated in brain lysates from HINT1 KO mice compared with WT mouse brain samples (42). It is noteworthy that the PKCγ isoform is exclusively found in neurons of the CNS and is expressed mostly after postnatal development (16). Furthermore, PKC enzymatic activity was higher in HINT1 KO than in WT mice (42). Of critical relevance, PKC has been shown to regulate SOC activation in different cell types (17, 31, 34). Recently, Kawasaki et al. (21) demonstrated that PKC activation in HEK293 cells suppresses SOC activity by phosphorylation of Orai1 at NH2-terminal serine residues Ser-27 and Ser-30 and, thereby, downregulates store-operated Ca2+ entry. Note that Ser-27 and Ser-30 are unique to Orai1 and are not conserved in Orai2 and Orai3. Therefore, phosphorylation of Orai2 and Orai3 upon PKC activation was negligible (21). Conversely, PKC inhibitors or knockdown of PKCβ with the short hairpin RNA resulted in increased Ca2+ influx (21). Thus, the data indicate that PKC negatively regulates store-operated Ca2+ entry by direct phosphorylation of Orai1, although exactly how the phosphorylation of Ser-27 and Ser-30 interferes with channel activation remains unclear.

TRPC3 and TRPC6, which function as ROCs, are also negatively regulated by PKC-mediated phospholylation at COOH-terminal serine residues Ser-712 and Ser-714, respectively (41, 46). For instance, protein kinase C activation by phorbol 12-myristate 13-acetate induced TRPC3 phosphorylation and completely inhibited TRPC3 channel activity (41). Replacement of Ser-712 by alanine not only impaired PKC-mediated TRPC3 phosphorylation but also channel inhibition, while WT TRPC activity was completely inhibited (41). Thus our new findings demonstrating suppressed ATP-induced Ca2+ influx in cells from HINT1 KO mice (Fig. 2), which are characterized by upregulated PKC expression and activity (42), are in good agreement with the published results (21, 41). We do not exclude, however, that Ca2+ signaling can be modulated by HINT1 deficiency also via PKC-mediated phosphorylation of other Ca2+ transporters and receptors, as well as via other protein kinase signaling pathways, such as Src signaling. Indeed, recent data revealed that HINT1 might be a negative regulator of expression of Src (7), which can phosphorylate/regulate TRPC channels (20, 45).

HINT1, Ca2+ signaling and their implications in psychiatric disorders.

Widespread expression of HINT1 in the CNS (26) implies that HINT1 can play an essential role in brain function under normal as well as pathological disease conditions. We previously demonstrated that HINT1 is involved in modulation of the μ-opioid receptor signaling (14). It specifically interacts with the μ-opioid receptor and, thereby, attenuates PKC-mediated phosphorylation and receptor desensitization. HINT1 KO mice exhibit enhanced basal and morphine-induced analgesia and increased morphine tolerance compared with control WT mice (14). HINT1 deficiency also leads to higher locomotion in response to the psychostimulant d-amphetamine and the dopamine-receptor agonist apomorphine, apparently through dysregulation of postsynaptic dopamine transmission (3), which may contribute to the pathophysiology of schizophrenia (38). Although schizophrenia is likely to have a multifactorial origin (37), one of the unifying elements of the great majority of molecular changes reported in the disease is their association with potential modifications in Ca2+ signaling (24). For instance, alterations in Ca2+ homeostasis are featured as the main links between glutamatergic and dopaminergic abnormalities frequently ascribed to schizophrenia (50).

Therefore, our new findings that HINT1 is involved in the regulation of neuronal Ca2+ signaling, along with its role in central dopaminergic and μ-opioid mechanisms, provide support for a potential role of HINT1 in the pathophysiology of schizophrenia and certain other mental disorders (11, 43, 44). Indeed, human genomic studies showed downregulation of HINT1 mRNA in the prefrontal cortex of schizophrenic and bipolar disorder patients compared with healthy controls (11, 43, 44). Linkage studies also revealed that HINT1 gene variants are associated with schizophrenia (8). Moreover, the levels of HINT1 are increased in brains from patients with major depressive disorder (28). A potential role of HINT1 in depression and anxiety disorders was also described in studies of HINT1 KO mice (2, 3, 42). Recently, using a combination of linkage analysis and next generation sequencing, Zimon et al. (52) demonstrated that loss-of-function mutations in HINT1 cause axonal neuropathy with neuromyotonia. Finally, evidence of tumor suppressor properties of HINT1 was recently uncovered (27) indicating its role in diverse cellular functions.

Despite the documented links between HINT1 and the pathophysiology of the various aforementioned disorders, the authentic functions of HINT1 in cellular physiology and especially Ca2+ signaling remained elusive. The data described in this report provide the first evidence that HINT1 is a novel endogenous regulator of cellular Ca2+ signaling. Our results show that loss of HINT1 leads to an inappropriate downregulation of components of store-operated Ca2+ entry pathway (Orai1 and STIM1) and disturbed Ca2+ homeostasis, critical for cellular function. Thus the remarkable ability of HINT1 to regulate Ca2+ signaling provides an additional support for its potential role in the pathophysiology of certain disorders.

GRANTS

This work was supported by National Institute of Mental Health MH092940 (to J. B. Wang) and by funds from the University of Maryland, School of Medicine (to V. A. Golovina).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: C.I.L., J.B.W., and V.A.G. conception and design of research; C.I.L. and B.F. performed experiments; C.I.L. and V.A.G. analyzed data; C.I.L., J.B.W., and V.A.G. interpreted results of experiments; C.I.L. and V.A.G. prepared figures; C.I.L., J.B.W., and V.A.G. edited and revised manuscript; C.I.L., J.B.W., and V.A.G. approved final version of manuscript; V.A.G. drafted manuscript.

ACKNOWLEDGMENTS

We thank Dr. J. Roos, (Torrey Pines Therapeutics, La Jolla, CA) for generous gift of anti-STIM1 antibodies.

REFERENCES

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
  22. 22.
  23. 23.
  24. 24.
  25. 25.
  26. 26.
  27. 27.
  28. 28.
  29. 29.
  30. 30.
  31. 31.
  32. 32.
  33. 33.
  34. 34.
  35. 35.
  36. 36.
  37. 37.
  38. 38.
  39. 39.
  40. 40.
  41. 41.
  42. 42.
  43. 43.
  44. 44.
  45. 45.
  46. 46.
  47. 47.
  48. 48.
  49. 49.
  50. 50.
  51. 51.
  52. 52.
View Abstract