CKII Control of Axonal Plasticity Is Mediated by Mitochondrial Ca2+ via Mitochondrial NCLX.

Mitochondrial Ca2+ efflux by NCLX is a critical rate-limiting step in mitochondria signaling. We previously showed that NCLX is phosphorylated at a putative Casein Kinase 2 (CKII) site, the serine 271 (S271). Here, we asked if NCLX is regulated by CKII and interrogated the physiological implications of this control. We found that CKII inhibitors down-regulated NCLX-dependent Ca2+ transport activity in SH-SY5Y neuronal cells and primary hippocampal neurons. Furthermore, we show that the CKII phosphomimetic mutants on NCLX inhibited (S271A) and constitutively activated (S271D) NCLX transport, respectively, rendering it insensitive to CKII inhibition. These phosphomimetic NCLX mutations also control the allosteric regulation of NCLX by mitochondrial membrane potential (ΔΨm). Since the omnipresent CKII is necessary for modulating the plasticity of the axon initial segment (AIS), we interrogated, in hippocampal neurons, if NCLX is required for this process. Similarly to WT neurons, NCLX-KO neurons can exhibit homeostatic plasticity following M-channel block. However, while WT neurons utilize a CKII-sensitive distal relocation of AIS Na+ and Kv7 channels to decrease their intrinsic excitability, we did not observe such translocation in NCLX-KO neurons. Thus, our results indicate that NCLX is regulated by CKII and is a crucial link between CKII signaling and fast neuronal plasticity.

ZnT1 is a neuronal Zn2+/Ca2+ exchanger.

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn2+ neurotoxicity. The mechanism mediating Zn2+ transport by ZnT1, however, has remained elusive. Here, we investigated ZnT1-dependent Zn2+ transport by measuring intracellular changes of this ion using the fluorescent indicator FluoZin-3. In primary mouse cortical neurons, which express ZnT1, transient addition of extracellular Zn2+ triggered a rise in cytosolic Zn2+, followed by its removal. Knockdown of ZnT1 by adeno associated viral (AAV)-short hairpin RNA (shZnT1) markedly increased rates of Zn2+ rise, and decreased rates of its removal, suggesting that ZnT1 is a primary route for Zn2+ efflux in neurons. Although Zn2+ transport by other members of the SLC30A family is dependent on pH gradients across cellular membranes, altered H+ gradients were not coupled to ZnT1-dependent transport. Removal of cytoplasmic Zn2+, against a large inward gradient during the initial loading phase, suggests that Zn2+ efflux requires a large driving force. We therefore asked if Ca2+ gradients across the membrane can facilitate Zn2+ efflux. Elimination of extracellular Ca2+ abolished Zn2+ efflux, while increased extracellular Ca2+ levels enhanced Zn2+ efflux. Intracellular Ca2+ rises, measured in GCaMP6 expressing neurons, closely paralleled cytoplasmic Zn2+ removal. Taken together, these results strongly suggest that ZnT1 functions as a Zn2+/Ca2+ exchanger, thereby regulating the transport of two ions of fundamental importance in neuronal signaling.

Zinc Signaling in the Mammary Gland: For Better and for Worse.

Zinc (Zn2+) plays an essential role in epithelial physiology. Among its many effects, most prominent is its action to accelerate cell proliferation, thereby modulating wound healing. It also mediates affects in the gastrointestinal system, in the testes, and in secretory organs, including the pancreas, salivary, and prostate glands. On the cellular level, Zn2+ is involved in protein folding, DNA, and RNA synthesis, and in the function of numerous enzymes. In the mammary gland, Zn2+ accumulation in maternal milk is essential for supporting infant growth during the neonatal period. Importantly, Zn2+ signaling also has direct roles in controlling mammary gland development or, alternatively, involution. During breast cancer progression, accumulation or redistribution of Zn2+ occurs in the mammary gland, with aberrant Zn2+ signaling observed in the malignant cells. Here, we review the current understanding of the role of in Zn2+ the mammary gland, and the proteins controlling cellular Zn2+ homeostasis and signaling, including Zn2+ transporters and the Gq-coupled Zn2+ sensing receptor, ZnR/GPR39. Significant advances in our understanding of Zn2+ signaling in the normal mammary gland as well as in the context of breast cancer provides new avenues for identification of specific targets for breast cancer therapy.

ZnR/GPR39 controls cell migration by orchestrating recruitment of KCC3 into protrusions, re-organization of actin and activation of MMP.

Actin re-organization and degradation of extracellular matrix by metalloproteases (MMPs) facilitate formation of cellular protrusions that are required for cell proliferation and migration. We find that Zn2+ activation of the Gq-coupled receptor ZnR/GPR39 controls these processes by regulating K+/Cl- co-transporter KCC3, which modulates cell volume. Silencing of KCC3 expression or activity reverses ZnR/GPR39 enhancement of cell proliferation, migration and invasion through Matrigel. Activation of ZnR/GPR39 recruits KCC3 into F-actin rich membrane protrusions, suggesting that it can locally control volume changes. Immunofluorescence analysis indicates that Zn2+ activation of ZnR/GPR39 and KCC3 are required to enhance formation of F-actin stress fibers and cellular protrusions. In addition, ZnR/GPR39 upregulation of KCC3-dependent transport increases the activity of matrix metalloproteases MMP2 and MMP9. Our study establishes a mechanism in which ZnR/GPR39 orchestrates localization and activation of KCC3, formation of F-actin rich cell protrusions and activation of MMPs, and thereby controls cell proliferation and migration.

ZnR/GPR39 upregulation of K+/Cl--cotransporter 3 in tamoxifen resistant breast cancer cells.

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca2+ signaling leading to tumor progression. The K+/Cl- co-transporters (KCC), activated by intracellular signaling, enhance breast cancer cell migration and invasion. We asked if ZnR/GPR39 enhances breast cancer cell malignancy by activating KCC. Activation of ZnR/GPR39 by Zn2+ upregulated K+/Cl- co-transport activity, measured using NH4+ as a surrogate to K+ while monitoring intracellular pH. Upregulation of NH4+ transport was monitored in tamoxifen resistant cells with functional ZnR/GPR39-dependent Ca2+ signaling but not in MCF-7 cells lacking this response. The NH4+ transport was Na+-independent, and we therefore focused on KCC family members. Silencing of KCC3, but not KCC4, expression abolished Zn2+-dependent K+/Cl- co-transport, suggesting that KCC3 is mediating upregulated NH4+ transport. The ZnR/GPR39-dependent KCC3 activation accelerated scratch closure rate, which was abolished by inhibiting KCC transport with [(DihydroIndenyl) Oxy] Alkanoic acid (DIOA). Importantly, silencing of either ZnR/GPR39 or KCC3 attenuated Zn2+-dependent scratch closure. Thus, a novel link between KCC3 and Zn2+, via ZnR/GPR39, promotes breast cancer cell migration and proliferation.

How cellular Zn2+ signaling drives physiological functions.

Zinc is an essential micronutrient affecting many aspects of human health. Cellular Zn2+ homeostasis is critical for cell function and survival. Zn2+, acting as a first or second messenger, triggers signaling pathways that mediate the physiological roles of Zn2+. Transient changes in Zn2+ concentrations within the cell or in the extracellular region occur following its release from Zn2+ binding metallothioneins, its transport across membranes by the ZnT or ZIP transporters, or release of vesicular Zn2+. These transients activate a distinct Zn2+ sensing receptor, ZnR/GPR39, or modulate numerous proteins and signaling pathways. Importantly, Zn2+ signaling regulates cellular physiological functions such as: proliferation, differentiation, ion transport and secretion. Indeed, novel therapeutic approaches aimed to maintain Zn2+ homeostasis and signaling are evolving. This review focuses on recent findings describing roles of Zn2+ and its transporters in regulating physiological or pathological processes.

Enhanced ZnR/GPR39 Activity in Breast Cancer, an Alternative Trigger of Signaling Leading to Cell Growth.

Acquired resistance to the estrogen receptor (ER) antagonist tamoxifen, is a major obstacle in treatment of breast cancer. Changes in Zn2+ accumulation and distribution are associated with tamoxifen-resistance and breast cancer progression. The Zn2+-sensing G-protein coupled receptor, ZnR/GPR39, triggers signaling leading to cell growth, but a role for this receptor in breast cancer in unknown. Using fluorescence imaging, we found Zn2+-dependent Ca2+ release, mediated by ZnR/GPR39 activity, in TAMR tamoxifen-resistant cells derived from MCF-7 cells, but not in ER-expressing MCF-7 or T47D cells. Furthermore, ZnR/GPR39 signaling was monitored in ER negative BT20, MDA-MB-453 and JIMT-1 cells. Expression of ZnR/GPR39 was increased in grade 3 human breast cancer biopsies compared to grade 2. Consistently, analysis of two breast cancer patient cohorts, GDS4057 and TCGA, indicated that in ER-negative tumors higher ZnR/GPR39 mRNA levels are associated with more aggressive tumors. Activation of ZnR/GPR39 in TAMR cells triggered MAPK, mTOR and PI3K signaling. Importantly, enhanced cell growth and invasiveness was observed in the ER negative breast cancer cells, TAMR, MDA-MB-453 and BT20 cells but not in the ER expressing MCF-7 cells. Thus, we suggest ZnR/GPR39 as a potential therapeutic target for combination treatment in breast cancer, particularly relevant in ER negative tumors.

The Zinc Sensing Receptor, ZnR/GPR39, in Health and Disease.

A distinct G-protein coupled receptor that senses changes in extracellular Zn2+, ZnR/GPR39, was found in cells from tissues in which Zn2+ plays a physiological role. Most prominently, ZnR/GPR39 activity was described in prostate cancer, skin keratinocytes, and colon epithelial cells, where zinc is essential for cell growth, wound closure, and barrier formation. ZnR/GPR39 activity was also described in neurons that are postsynaptic to vesicular Zn2+ release. Activation of ZnR/GPR39 triggers Gαq-dependent signaling and subsequent cellular pathways associated with cell growth and survival. Furthermore, ZnR/GPR39 was shown to regulate the activity of ion transport mechanisms that are essential for the physiological function of epithelial and neuronal cells. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner.

The zinc sensing receptor, ZnR/GPR39, in health and disease.

While zinc has had a well-established structural role for many years, it is only during the last two decades that its role as a signaling molecule has been recognized. Ionic zinc, Zn2+, that is endogenously released during physiological activity acts as a first messenger, triggering the activity of a distinct Zn2+-sensing-receptor, ZnR. The ZnR is a member of the Gq-coupled receptor family, and the molecular moiety mediating its activity is GPR39. In this review, we will discuss the role of the ZnR/GPR39 in mediating Zn2+-dependent signaling in epithelial tissues and in neurons, where Zn2+ homeostasis plays physiological as well as pathological roles. Importantly, ZnR/GPR39 activates signaling that regulates a remarkably wide range of cell functions, including proliferation, differentiation and survival, as well as modulation of ion transport, and thereby, regulation of Na+, H+ and Cl- homeostasis. Moreover, signaling activated by ZnR/GPR39 plays a key role in mediating effects of Zn2+ in health and disease. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner.

Mitochondria control store-operated Ca2+ entry through Na+ and redox signals.

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca2+ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca2+ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na+ that is critical in activating the mitochondrial Na+/Ca2+ exchanger (NCLX) causing enhanced mitochondrial Na+ uptake and Ca2+ efflux. Omission of extracellular Na+ prevents the cytosolic Na+ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na+ and Ca2+ signals to effect mitochondrial redox control over SOCE.

Amyloid ß attenuates metabotropic zinc sensing receptor, mZnR/GPR39, dependent Ca2+ , ERK1/2 and Clusterin signaling in neurons.

A hallmark of Alzheimer's disease is accumulation of amyloid beta (Aß) deposits, which are associated with neuronal dysfunction, spine loss, and impaired Ca2+ homeostasis. Amyloid beta (Aß) binds to and is aggregated by Zn2+ , a metal released from synaptic glutamatergic vesicles during neuronal activity. Synaptically released Zn2+ activates a metabotropic Gq-coupled Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling in post-synaptic neurons. We examined if Aß, as a Zn2+ binding protein, regulates neuronal Zn2+ -signaling mediated by mZnR/GPR39 using SHSY-5Y cells and cortical neurons from GPR39 wild-type and knockout mice. Following acute or chronic treatment with Aß neuronal Zn2+ -dependent Ca2+ release via mZnR/GPR39 is significantly reduced. This impairment is overcome when excess Zn2+ is applied, suggesting that impaired Ca2+ -signaling results from Aß binding of Zn2+ . The Zn2+ -dependent mZnR/GPR39 activation triggers phosphorylation of extracellular regulated kinase and up-regulates expression of the chaperone protein clusterin (Clu). Importantly, neuronal Zn2+ -dependent extracellular regulated kinase1/2 phosphorylation and up-regulation of Clu are attenuated by silencing mZnR/GPR39 as well as by Aß treatment. In contrast, Zn2+ -dependent AKT phosphorylation is not mediated by mZnR/GPR39 and is not attenuated by Aß treatment. Thus, Zn2+ signaling via mZnR/GPR39 is distinctively disrupted by a critical pathological component of Alzheimer's disease. Synaptically released Zn2+ activates a Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling, followed by ERK1/2 MAPK activation and up-regulation of clusterin. Amyloid beta (Aß) binds to Zn2+ thus forming oligomers that are a hallmark of Alzheimer's disease. We show that Aß attenuates Zn2+ -dependent Ca2+ -responses, abolishes ERK1/2 activation and down-regulates clusterin expression. Thus, Zn2+ signaling via mZnR/GPR39 is disrupted by Aß, a critical pathological component of Alzheimer's disease.

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons.

Mitochondrial Ca(2+) overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1) function, implicated in Parkinson disease, inhibits the mitochondrial Na(+)/Ca(2+) exchanger (NCLX), leading to impaired mitochondrial Ca(2+) extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA) pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLX(S258D)) prevents mitochondrial Ca(2+) overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca(2+) transport regulatory pathway that protects against mitochondrial Ca(2+) overload. Because mitochondrial Ca(2+) dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive decreases in pHe during acidosis. Hence, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain.

Mitotic slippage and expression of survivin are linked to differential sensitivity of human cancer cell-lines to the Kinesin-5 inhibitor monastrol.

The mitotic Kinesin-5 motor proteins crosslink and slide apart antiparallel spindle microtubules, thus performing essential functions in mitotic spindle dynamics. Specific inhibition of their function by monastrol-like small molecules has been examined in clinical trials as anticancer treatment, with only partial success. Thus, strategies that improve the efficiency of monastrol-like anticancer drugs are required. In the current study, we examined the link between sensitivity to monastrol and occurrence of mitotic slippage in several human cell-lines. We found that the rank of sensitivity to monastrol, from most sensitive to least sensitive, is: AGS > HepG2 > Lovo > Du145 ≥ HT29. We show correlation between the sensitivity of a particular cell-line to monastrol and the tendency of the same cell-line to undergo mitotic slippage. We also found that in the monastrol resistant HT29 cells, prolonged monastrol treatments increase mRNA and protein levels of the chromosomal passenger protein survivin. In contrast, survivin levels are not increased by this treatment in the monastrol-sensitive AGS cells. We further show that over-expression of survivin in the monastrol-sensitive AGS cells reduces mitotic slippage and increases resistance to monastrol. Finally, we show that during short exposure to monastrol, Si RNA silencing of survivin expression reduces cell viability in both AGS and HT29 cells. Our data suggest that the efficiency of anti-cancer treatment with specific kinesin-5 inhibitors may be improved by modulation of expression levels of survivin.

A crosstalk between Na⁺ channels, Na⁺/K⁺ pump and mitochondrial Na⁺ transporters controls glucose-dependent cytosolic and mitochondrial Na⁺ signals.

Glucose-dependent cytosolic Na(+) influx in pancreatic islet ß cells is mediated by TTX-sensitive Na(+) channels and is propagated into the mitochondria through the mitochondrial Na(+)/Ca(2+) exchanger, NCLX. Mitochondrial Na(+) transients are also controlled by the mitochondrial Na(+)/H(+) exchanger, NHE, while cytosolic Na(+) changes are governed by Na(+)/K(+) ATPase pump. The functional interaction between the Na(+) channels, Na(+)/K(+) ATPase pump and mitochondrial Na(+) transporters, NCLX and NHE, in mediating Na(+) signaling is poorly understood. Here, we combine fluorescent Na(+) imaging, pharmacological inhibition by TTX, ouabain and EIPA, with molecular control of NCLX expression, so as to investigate the crosstalk between Na(+) transporters on both the plasma membrane and the mitochondria. According to our results, glucose-dependent cytosolic Na(+) response was enhanced by ouabain and was followed by a rise in mitochondrial Na(+) signal. Silencing of NCLX expression using siNCLX, did not affect the glucose- or ouabain-dependent cytosolic rise in Na(+). In contrast, the ouabain-dependent rise in mitochondrial Na(+) was strongly suppressed by siNCLX. Furthermore, mitochondrial Na(+) influx rates were accelerated in cells treated with the Na(+)/H(+) exchanger inhibitor, EIPA or by combination of EIPA and ouabain. Similarly, TTX blocked the cytosolic and mitochondrial Na(+) responses, which were enhanced by ouabain or EIPA, respectively. Our results suggest that Na(+)/K(+) ATPase pump controls cytosolic glucose-dependent Na(+) rise, in a manner that is mediated by TTX-sensitive Na(+) channels and subsequent mitochondrial Na(+) uptake via NCLX. Furthermore, these results indicate that mitochondrial Na(+) influx via NCLX is antagonized by Na(+) efflux, which is mediated by the mitochondrial NHE; thus, the duration of mitochondrial Na(+) transients is set by the interplay between these pivotal transporters.

Pancreatic ß-cell Na+ channels control global Ca2+ signaling and oxidative metabolism by inducing Na+ and Ca2+ responses that are propagated into mitochondria.

Communication between the plasma membrane and mitochondria is essential for initiating the Ca(2+) and metabolic signals required for secretion in ß cells. Although voltage-dependent Na(+) channels are abundantly expressed in ß cells and activated by glucose, their role in communicating with mitochondria is unresolved. Here, we combined fluorescent Na(+), Ca(2+), and ATP imaging, electrophysiological analysis with tetrodotoxin (TTX)-dependent block of the Na(+) channel, and molecular manipulation of mitochondrial Ca(2+) transporters to study the communication between Na(+) channels and mitochondria. We show that TTX inhibits glucose-dependent depolarization and blocks cytosolic Na(+) and Ca(2+) responses and their propagation into mitochondria. TTX-sensitive mitochondrial Ca(2+) influx was largely blocked by knockdown of the mitochondrial Ca(2+) uniporter (MCU) expression. Knockdown of the mitochondrial Na(+)/Ca(2+) exchanger (NCLX) and Na(+) dose response analysis demonstrated that NCLX mediates the mitochondrial Na(+) influx and is tuned to sense the TTX-sensitive cytosolic Na(+) responses. Finally, TTX blocked glucose-dependent mitochondrial Ca(2+) rise, mitochondrial metabolic activity, and ATP production. Our results show that communication of the Na(+) channels with mitochondria shape both global Ca(2+) and metabolism signals linked to insulin secretion in ß cells.- Nita, I. I., Hershfinkel, M., Kantor, C., Rutter, G. A., Lewis, E. C., Sekler, I. Pancreatic ß-cell Na(+) channels control global Ca(2+) signaling and oxidative metabolism by inducing Na(+) and Ca(2+) responses that are propagated into mitochondria.

Impact of S100A8/A9 expression on prostate cancer progression in vitro and in vivo.

The proinflammatory S100A8/A9 proteins, which are expressed in myeloid cells under physiological conditions, are strongly expressed in human prostate cancer epithelial cells. Their role in the tumor cells and in tumor progression is largely unclear. We established a prostate cancer epithelial cell line (PC-3 TO-A8/A9) expressing S100A8 and S100A9 simultaneously under doxycycline control, to study the role of S100A8/A9 on tumor growth and infiltration of immune cells in subcutaneous xenografts in male NMRI nu/nu mice. Colonization of distant organs was studied after intracardial injection of the tumor cells in male NOD/SCID mice. PC-3 TO-A8/A9 cells grown in vitro and subcutaneous xenografts in mice not treated with doxycycline expressed high levels of S100A8/A9 mRNA and protein, whereas doxycycline treatment suppressed S100A8/A9 expression. S100A8/A9 expression did not significantly alter growth rate and invasion of the subcutaneous tumors into surrounding tissues. However, S100A8/A9 expression caused increased infiltration of immune cells, especially neutrophils. In intracardially injected mice sporadic tumor settlement was observed in muscle and lymph nodes. Colonies of tumor cells and micro-metastases were observed in the lung of 64.3% (9 out of 14) of mice not treated with doxycycline and in 33.3% (5 out of 15) of mice treated with doxycycline. Our data demonstrate for the first time that S100A8/A9 expression in epithelial cancer cells causes enhanced infiltration of immune cells, especially neutrophils, and stimulates settlement of the cancer cells in the lung.

The ZnR/GPR39 interacts with the CaSR to enhance signaling in prostate and salivary epithelia.

Zinc signaling is mediated by the zinc sensing receptor, ZnR, recently suggested to be the same receptor as G-protein coupled receptor 39, GPR39. However, it is unknown if GPR39 is mediating Zn(2+) -dependent signaling in prostate and salivary tissue where changes in zinc concentrations are frequent and of physiological significance. Here, we show that GPR39 is mediating Zn(2+) -dependent Ca(2+) responses and is regulating activity of MAP and PI3 pathways in prostate cancer cells, PC3, and ductal salivary gland cells, HSY. We next ask whether ZnR/GPR39 interacts with other GPCR family members. We find that endogenous ZnR/GPR39 activity is regulated by the expression and activity of another cation sensing GPCR, the Ca(2+) -sensing receptor (CaSR). Although CaSR is not activated by Zn(2+), co-expression of CaSR and ZnR/GPR39 synergistically enhances Ca(2+) responses in PC3 and HSY cells. Silencing of the CaSR using siRNA or a dominant negative construct reduces the Zn(2+) -dependent signaling. Importantly, overexpression of GPR39 in HEK293 cells is sufficient to trigger Zn(2+) -dependent responses. Nevertheless, application of the CaSR agonist spermine, at concentration below its threshold, enhanced Zn(2+) -dependent Ca(2+) response. Our results suggest that the CaSR interacts with ZnR/GPR39 and thereby regulates its activity. Finally, we show that in PC3 cells ZnR/GPR39 is required for mediating the Zn(2+) -dependent activation of MAPK and PI3K, pathways leading to enhanced cell growth. Importantly, Zn(2+) -dependent activation of ZnR/GPR39 also enhances the expression of the Ca(2+) -binding protein S100A4 that is linked to invasion of prostate cancer cells.

The mitochondrial Na+/Ca2+ exchanger upregulates glucose dependent Ca2+ signalling linked to insulin secretion.

Mitochondria mediate dual metabolic and Ca(2+) shuttling activities. While the former is required for Ca(2+) signalling linked to insulin secretion, the role of the latter in ß cell function has not been well understood, primarily because the molecular identity of the mitochondrial Ca(2+) transporters were elusive and the selectivity of their inhibitors was questionable. This study focuses on NCLX, the recently discovered mitochondrial Na(+)/Ca(2+) exchanger that is linked to Ca(2+) signalling in MIN6 and primary ß cells. Suppression either of NCLX expression, using a siRNA construct (siNCLX) or of its activity, by a dominant negative construct (dnNCLX), enhanced mitochondrial Ca(2+) influx and blocked efflux induced by glucose or by cell depolarization. In addition, NCLX regulated basal, but not glucose-dependent changes, in metabolic rate, mitochondrial membrane potential and mitochondrial resting Ca(2+). Importantly, NCLX controlled the rate and amplitude of cytosolic Ca(2+) changes induced by depolarization or high glucose, indicating that NCLX is a critical and rate limiting component in the cross talk between mitochondrial and plasma membrane Ca(2+) signalling. Finally, knockdown of NCLX expression was followed by a delay in glucose-dependent insulin secretion. These findings suggest that the mitochondrial Na(+)/Ca(2+) exchanger, NCLX, shapes glucose-dependent mitochondrial and cytosolic Ca(2+) signals thereby regulating the temporal pattern of insulin secretion in ß cells.

Extracellular pH regulates zinc signaling via an Asp residue of the zinc-sensing receptor (ZnR/GPR39).

Zinc activates a specific Zn(2+)-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca(2+) responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na(+)/H(+) exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn(2+) binding site, His(17) or His(19), or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp(313) with alanine resulted in similar Ca(2+) responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na(+)/H(+) exchange at pH 7.4 and pH 6.5. Substitution of Asp(313) to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp(313), which was shown to modulate Zn(2+) binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.