The ZIP3 Zinc Transporter Is Localized to Mossy Fiber Terminals and Is Required for Kainate-Induced Degeneration of CA3 Neurons.

Tight regulation of neuronal Zn2+ is critical for physiological function. Multiple Zn2+ transporters are expressed in the brain, yet their spatial distribution and distinct roles are largely unknown. Here, we show developmental regulation of the expression of Zn2+ transporters ZIP1 and ZIP3 in mouse hippocampal neurons, corresponding to previously described increase in neuronal vesicular Zn2+ during the first postnatal month. Rates of Zn2+ uptake in cultured mouse hippocampal neurons, monitored using FluoZin-3 fluorescence, were higher in mature neurons, which express higher levels of ZIP1 and ZIP3. Zn2+ uptake was attenuated by ∼50% following silencing of either ZIP1 or ZIP3. Expression of both ZIP1 and ZIP3 was ubiquitous on somas and most neuronal processes in the cultured neurons. In contrast, we observed distinct localization of the transporters in adult mouse hippocampal brain, with ZIP1 predominantly expressed in the CA3 stratum pyramidale, and ZIP3 primarily localized to the stratum lucidum. Consistent with their localization, silencing of ZIP1 expression in vivo reduced Zn2+ uptake in CA3 neurons while ZIP3 silencing reduced Zn2+ influx into dentate gyrus (DG) granule cells in acute hippocampal slices. Strikingly, in vivo silencing of ZIP3, but not ZIP1, protected CA3 neurons from neurodegeneration following kainate-induced seizures. Our results indicate that distinct Zn2+ transporters control Zn2+ accumulation and toxicity in different neuronal populations in the hippocampus and suggest that selective regulation of Zn2+ transporters can prevent seizure induced brain damage.SIGNIFICANCE STATEMENT Zinc plays a major role in neuronal function and its dysregulation is associated with neurodegeneration. Multiple zinc transporters are expressed in neurons, yet little is known on their distinct roles. Here, we show that the plasma membrane ZIP1 and ZIP3 zinc transporters are expressed on distinct neuronal populations in the CA3 region of the hippocampus. We show that ZIP1 mediates zinc influx into postsynaptic cells, while ZIP3 is responsible for zinc re-uptake from this synapse into dentate granule cells. We further show that silencing of ZIP3, but not ZIP1, can rescue the postsynaptic cells from kainate-induced neurodegeneration. This suggests that neuronal zinc toxicity and degeneration can be modulated by regulation of specific zinc transporters function.

SNAP23 regulates KCC2 membrane insertion and activity following mZnR/GPR39 activation in hippocampalneurons.

Modulation of the neuronal K+/Cl- cotransporter 2 (KCC2) activity, which mediates Cl- export, is critical to neuronal function. Here, we demonstrate that KCC2 interacts with the SNARE protein synaptosome-associated protein 23, SNAP23, an essential component of membrane insertion machinery. Using KCC2 truncated mutants, we show that KCC2 C-terminal domain is essential for membrane targeting and SNAP23-dependent upregulation of KCC2 activity triggered by activation of the Zn2+-sensitive receptor mZnR/GPR39 in HEK293 cells. Expression of SNAP23 phosphorylation-insensitive mutants or inhibition of its upstream activator IκB kinase (IKK) prevents mZnR/GPR39 upregulation of KCC2 activity in mouse hippocampal neurons. We further find that SNAP23 interacts with Syntaxin 1A and KCC2, and that all three proteins exhibit increased membrane insertion following mZnR/GPR39 activation in neurons. Our results elucidate a G-protein-coupled receptor-dependent pathway for regulation of KCC activity, mediated via interaction with SNARE proteins.

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.

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.

Synaptic zinc inhibition of NMDA receptors depends on the association of GluN2A with the zinc transporter ZnT1.

The NMDA receptor (NMDAR) is inhibited by synaptically released zinc. This inhibition is thought to be the result of zinc diffusion across the synaptic cleft and subsequent binding to the extracellular domain of the NMDAR. However, this model fails to incorporate the observed association of the highly zinc-sensitive NMDAR subunit GluN2A with the postsynaptic zinc transporter ZnT1, which moves intracellular zinc to the extracellular space. Here, we report that disruption of ZnT1-GluN2A association by a cell-permeant peptide strongly reduced NMDAR inhibition by synaptic zinc in mouse dorsal cochlear nucleus synapses. Moreover, synaptic zinc inhibition of NMDARs required postsynaptic intracellular zinc, suggesting that cytoplasmic zinc is transported by ZnT1 to the extracellular space in close proximity to the NMDAR. These results challenge a decades-old dogma on how zinc inhibits synaptic NMDARs and demonstrate that presynaptic release and a postsynaptic transporter organize zinc into distinct microdomains to modulate NMDAR neurotransmission.

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.

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 Zn2+-sensing receptor, ZnR/GPR39, upregulates colonocytic Cl- absorption, via basolateral KCC1, and reduces fluid loss.

Administration of zinc, as a complement to oral rehydration solutions, effectively diminishes duration and severity of diarrhea, but it is not known whether it merely fulfills a nutritional deficiency, or if zinc has a direct role of regulating solute absorption. We show that Zn2+ acts via a specific receptor, ZnR/GPR39, to reduce fluid loss. Intestinal fluid secretion triggered by cholera toxin (CTx) was lower in WT mice compared to ZnR/GPR39 KO. In the absence of dietary Zn2+ we observed similar fluid accumulation in WT and ZnR/GPR39 KO mice, indicating that Zn2+ and ZnR/GPR39 are both required for a beneficial effect of Zn2+ in diarrhea. In primary colonocytes and in Caco-2 colonocytic cells, activation of ZnR/GPR39 enhanced Cl- transport, a critical factor in diarrhea, by upregulating K+/Cl- cotransporter (KCC1) activity. Importantly, we show basolateral expression of KCC1 in mouse and human colonocytes, thus identifying a novel Cl- absorption pathway. Finally, inhibition of KCC-dependent Cl- transport enhanced CTx-induced fluid loss. Altogether, our data indicate that Zn2+ acting via ZnR/GPR39 has a direct role in controlling Cl- absorption via upregulation of basolateral KCC1 in the colon. Moreover, colonocytic ZnR/GPR39 and KCC1 reduce water loss during diarrhea and may therefore serve as effective drug targets.

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.

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.

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs.

Single-chain (SC) gonadotropins have been genetically engineered to increase the repertoire of analogs for potential use in humans and domestic animals. The major aim of the current study was to examine the steroidogenic related activity of SC FSH analogs carrying structural differences. To address this issue, we designed and expressed three SC bovine FSH analogs in CHO cells: (i) FSHßα in which the tethered subunit domains are linked in tandem; (ii) FSHßCTPα that contains the carboxy terminal peptide (CTP) of the human choriogonadotropin (hCG) ß subunit as a spacer, and (iii) FSHßboCTPα in which the linker is derived from a CTP-like sequence (boCTP) decoded from the bovine LHß DNA. The data suggested that the secretion efficiency of these variants from the transfected cells was unaffected by the presence or absence of the CTP linker, N-glycans were attached to the analogs and the hCGß-CTP domain in the FSHßCTPα variant was O-glycosylated. In a rat immortalized granulosa cell bioassay the potency of the three variants towards progesterone secretion varied. In immature mice, the analogs increased the ovary weight and induced StAR, Cyp11a (P450scc), Cyp17 (P450c17) and Cyp19 (P450aromatase) transcripts. However, the dose dependence and amplitude of these transcript levels differed in response to FSHßα, FSHßboCTPα and FSHßCTPα. Collectively, these data suggest that the design of the FSH analog can modulate the bioactivity in vitro and in vivo. A systematic analysis of receptor activation with ligands carrying structural differences may identify new regulatory factor/s involved in the pleiotropic FSH activity.

Nitric oxide signaling modulates synaptic inhibition in the superior paraolivary nucleus (SPN) via cGMP-dependent suppression of KCC2.

Glycinergic inhibition plays a central role in the auditory brainstem circuitries involved in sound localization and in the encoding of temporal action potential firing patterns. Modulation of this inhibition has the potential to fine-tune information processing in these networks. Here we show that nitric oxide (NO) signaling in the auditory brainstem (where activity-dependent generation of NO is documented) modulates the strength of inhibition by changing the chloride equilibrium potential. Recent evidence demonstrates that large inhibitory postsynaptic currents (IPSCs) in neurons of the superior paraolivary nucleus (SPN) are enhanced by a very low intracellular chloride concentration, generated by the neuronal potassium chloride co-transporter (KCC2) expressed in the postsynaptic neurons. Our data show that modulation by NO caused a 15 mV depolarizing shift of the IPSC reversal potential, reducing the strength of inhibition in SPN neurons, without changing the threshold for action potential firing. Regulating inhibitory strength, through cGMP-dependent changes in the efficacy of KCC2 in the target neuron provides a postsynaptic mechanism for rapidly controlling the inhibitory drive, without altering the timing or pattern of the afferent spike train. Therefore, this NO-mediated suppression of KCC2 can modulate inhibition in one target nucleus (SPN), without influencing inhibitory strength of other target nuclei (MSO, LSO) even though they are each receiving collaterals from the same afferent nucleus (a projection from the medial nucleus of the trapezoid body, MNTB).

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.

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.

Modulation of hepatitis C virus release by the interferon-induced protein BST-2/tetherin.

Hepatitis C virus is a leading cause of chronic hepatitis and liver cancer. Little information exists on the interplay between innate defense mechanisms and viral antagonists that promote viral egress. Herein, the effects of Tetherin/BST-2 on HCV release were investigated. In Huh-7.5 hepatocytes, low expression levels of BST-2 were detected. Treatment of Huh-7.5 cells with IFNα, elevated BST-2 expression levels. However, HCV could not alter the expression of IFNα-induced BST-2, nor of stably over-expressed BST-2. Significantly, over expressed BST-2 moderately blocked HCV production and release from Huh-7.5 cells. Functional analysis of BST-2, confirmed its ability to inhibit the release of HIV delta-Vpu from Huh-7.5-BST-2 cells. HIV-Vpu antagonized BST-2 activity and rescued HIV delta-Vpu release from Huh-7.5-BST-2 cells. However, vpu slightly rescued HCV release and production from Huh-7.5-BST-2. We conclude that BST-2 moderately restricts HCV production and release from Huh-7.5 hepatocytes, while the virus lacks mechanisms to counteract this restriction.