Retinal pigment epithelium-specific CLIC4 mutant is a mouse model of dry age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Dry AMD has unclear etiology and no treatment. Lipid-rich drusen are the hallmark of dry AMD. An AMD mouse model and insights into drusenogenesis are keys to better understanding of this disease. Chloride intracellular channel 4 (CLIC4) is a pleomorphic protein regulating diverse biological functions. Here we show that retinal pigment epithelium (RPE)-specific Clic4 knockout mice exhibit a full spectrum of functional and pathological hallmarks of dry AMD. Multidisciplinary longitudinal studies of disease progression in these mice support a mechanistic model that links RPE cell-autonomous aberrant lipid metabolism and transport to drusen formation.

Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency.

The prevailing view is that the ClC-Ka chloride channel (mouse Clc-k1) functions in the thin ascending limb to control urine concentration, whereas the ClC-Kb channel (mouse Clc-k2) functions in the thick ascending limb (TAL) to control salt reabsorption. Mutations of ClC-Kb cause classic Bartter syndrome, characterized by renal salt wasting, with perinatal to adolescent onset. We studied the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2D and advanced 3D imaging of optically cleared kidneys. We show that Clc-k1 and Clc-k2 were broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and Clc-k2 revealed that both participated in NKCC2- and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 caused tubular injury and impaired renal medulla and TAL development. Inducible deletion of Clc-k2 beginning after medulla maturation produced mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and Clc-k2 contributed to salt reabsorption in TAL and distal convoluted tubule (DCT) in neonates, potentially explaining the less-severe phenotypes in classic Bartter syndrome. As opposed to the current understanding that salt wasting in adult patients with Bartter syndrome is due to Clc-k2 deficiency in adult TAL, our results suggest that it originates mainly from defects occurring in the medulla and TAL during development.

Transcriptome analysis of chloride intracellular channel knockdown in Drosophila identifies oxidation-reduction function as possible mechanism of altered sensitivity to ethanol sedation.

Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. Here, the sole Drosophila melanogaster ortholog, Clic, was targeted for RNAi knockdown to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 7% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, we evaluated the effect of hyperoxia on ethanol sedation in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under normoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90ß (Hsp90ß) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90ß-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

TMEM16C is involved in thermoregulation and protects rodent pups from febrile seizures.

Febrile seizures (FSs) are the most common convulsion in infancy and childhood. Considering the limitations of current treatments, it is important to examine the mechanistic cause of FSs. Prompted by a genome-wide association study identifying TMEM16C (also known as ANO3) as a risk factor of FSs, we showed previously that loss of TMEM16C function causes hippocampal neuronal hyperexcitability [Feenstra et al., Nat. Genet. 46, 1274-1282 (2014)]. Our previous study further revealed a reduction in the number of warm-sensitive neurons that increase their action potential firing rate with rising temperature of the brain region harboring these hypothalamic neurons. Whereas central neuronal hyperexcitability has been implicated in FSs, it is unclear whether the maximal temperature reached during fever or the rate of body temperature rise affects FSs. Here we report that mutant rodent pups with TMEM16C eliminated from all or a subset of their central neurons serve as FS models with deficient thermoregulation. Tmem16c knockout (KO) rat pups at postnatal day 10 (P10) are more susceptible to hyperthermia-induced seizures. Moreover, they display a more rapid rise of body temperature upon heat exposure. In addition, conditional knockout (cKO) mouse pups (P11) with TMEM16C deletion from the brain display greater susceptibility of hyperthermia-induced seizures as well as deficiency in thermoregulation. We also found similar phenotypes in P11 cKO mouse pups with TMEM16C deletion from Ptgds-expressing cells, including temperature-sensitive neurons in the preoptic area (POA) of the anterior hypothalamus, the brain region that controls body temperature. These findings suggest that homeostatic thermoregulation plays an important role in FSs.

ClC-2 inhibition prevents macrophage foam cell formation by suppressing Nlrp3 inflammasome activation.

Macrophage foam cell formation and inflammation are a pathological hallmark of atherosclerosis. ClC-2 has been implicated in various pathological processes, including inflammation and lipid metabolic disorder. However, the functional role of ClC-2 in macrophage foam cell formation and inflammation is unclear. Here, we found that ClC-2 was dominantly expressed in macrophages of atherosclerotic plaque and increased in atherogenesis. Knockdown of ClC-2 inhibited ox-LDL -induced lipid uptake and deposition in macrophages. The increase in CD36 expression and the decrease in ABCA1 expression induced by ox-LDL were alleviated by ClC-2 downregulation. Further, ClC-2 lacking limited the ox-LDL-induced secretion of inflammatory cytokines and chemokine, and suppressed Nlrp3 inflammasome activation. Restoration of Nlrp3 expression reversed the effect of ClC-2 downregulation on macrophage lipid accumulation and inflammation. Collectively, our study demonstrates that ClC-2 knockdown ameliorates ox-LDL-induced macrophage foam cell formation and inflammation by inhibiting Nlrp3 inflammasome activation.

The chloride intracellular channel protein CLIC4 inhibits filopodium formation induced by constitutively active mutants of formin mDia2.

Chloride intracellular channel 4 (CLIC4) functions in diverse actin-dependent processes. Upon Rho activation, CLIC4 reversibly translocates from the cytosol to the plasma membrane to regulate cell adhesion and migration. At the plasma membrane, CLIC4 counters the formation of filopodia, which requires actin assembly by the formin mammalian Diaphanous (mDia)2. To this end, mDia2 must be activated through conversion from the closed to the open conformation. Thus, CLIC4 could harness the activation or the open conformation of mDia2 to inhibit filopodium formation. Here, we find that CLIC4 silencing enhances the filopodia induced by two constitutively active mDia2 mutants. Furthermore, we report that CLIC4 binds the actin-regulatory region of mDia2 in vitro. These results suggest that CLIC4 modulates the activity of the open conformation of mDia2, shedding new light into how cells may control filopodia.

Chloride intracellular channel 1 cooperates with potassium channel EAG2 to promote medulloblastoma growth.

Ion channels represent a large class of drug targets, but their role in brain cancer is underexplored. Here, we identify that chloride intracellular channel 1 (CLIC1) is overexpressed in human central nervous system malignancies, including medulloblastoma, a common pediatric brain cancer. While global knockout does not overtly affect mouse development, genetic deletion of CLIC1 suppresses medulloblastoma growth in xenograft and genetically engineered mouse models. Mechanistically, CLIC1 enriches to the plasma membrane during mitosis and cooperates with potassium channel EAG2 at lipid rafts to regulate cell volume homeostasis. CLIC1 deficiency is associated with elevation of cell/nuclear volume ratio, uncoupling between RNA biosynthesis and cell size increase, and activation of the p38 MAPK pathway that suppresses proliferation. Concurrent knockdown of CLIC1/EAG2 and their evolutionarily conserved channels synergistically suppressed the growth of human medulloblastoma cells and Drosophila melanogaster brain tumors, respectively. These findings establish CLIC1 as a molecular dependency in rapidly dividing medulloblastoma cells, provide insights into the mechanism by which CLIC1 regulates tumorigenesis, and reveal that targeting CLIC1 and its functionally cooperative potassium channel is a disease-intervention strategy.

ClC-3 Deficiency Impairs the Neovascularization Capacity of Early Endothelial Progenitor Cells by Decreasing CXCR4/JAK-2 Signalling.

BACKGROUND: Endothelial progenitor cell (EPC) therapy has been suggested as a major breakthrough in the treatment of ischemic diseases. However, the molecular mechanism that underlies EPC functional regulation is still unclear. METHODS: We examined the angiogenic capacity of EPCs in a hindlimb ischemia model of wild-type and ClC-3 knockout mice. RESULTS: Mice lacking of ClC-3 exhibited reduced blood flow recovery and neovascularization in ischemic muscles 7 and 14 days after hind limb ischemia. Moreover, compared with wild-type EPCs, the hindlimb blood reperfusion in mice receiving ClC-3 knockout EPCs was significantly impaired, accompanied by reduced EPC homing and retention. In vitro, EPCs derived from ClC-3 knockout mice displayed impaired migratory, adhesive, and angiogenic activity. CXC chemokine receptor 4 (CXCR4) expression was significantly reduced in EPC from ClC-3 knockout mice compared with wild-type. Moreover, the expression and phosphorylation of Janus kinase 2 (JAK-2), a downstream signalling of CXCR4, was also reduced in ClC-3 knockout EPC, indicating that CXCR4/JAK-2 signalling is dysregulated by ClC-3 deficiency. Consistent with this assumption, the migratory capacity of wild-type EPCs was attenuated by either CXCR4 antagonist AMD3100 or JAK-2 inhibitor AG490. More importantly, the impaired migratory capacity of ClC-3 knockout EPCs was rescued by overexpression of CXCR4. CONCLUSIONS: ClC-3 plays a critical role in the angiogenic capacity of EPCs and EPC-mediated neovascularization of ischemic tissues. Disturbance of CXCR4/JAK-2 signalling may contribute to the functional impairment of ClC-3 deficient EPCs. Thus, ClC-3 may be a potential therapeutic target for modulating neovascularization in ischemic diseases.

Knockdown of CLC-3 in the hippocampal CA1 impairs contextual fear memory.

Previous studies support a critical role of hippocampus in contextual fear memory. Structural and functional alterations of hippocampus occur frequently in posttraumatic stress disorders (PTSD). Recent reports reveal that knockout of CLC-3, a member of the CLC family of anion channels and transporters, leads to neuronal degeneration and loss of hippocampus. However, the role of CLC-3 in contextual fear memory remains unknown. Using adenovirus and adeno-associated virus gene transfer to knockdown CLC-3 in hippocampal CA1, we investigate the role of CLC-3 in contextual fear memory. CLC-3 expression is increased in hippocampal CA1 after formation of long-term contextual fear memory. Knockdown of CLC-3 by adenovirus infusion in hippocampal CA1 significantly attenuates the contextual fear memory, reduces spine density, induces defects of excitatory synaptic ultrastructure showed by the decreased PSD length, PSD thickness and active zone length, and impairs L-LTP induction and maintenance. Knockdown of CLC-3 also induces the synaptic NMDAR subunit composition to an increased GluN2A/GluN2B ratio pattern and reduces the activity of CaMKII-α. Furthermore, selectively knockdown of CLC-3 in excitatory neurons by adeno-associated virus driven from CaMKII-α promoter is sufficient to impair long-term contextual fear memory. These findings highlight that CLC-3 in hippocampal CA1 is necessary for contextual fear memory.

The apoptotic effect of Zoledronic acid on the nasopharyngeal carcinoma cells via ROS mediated chloride channel activation.

Zoledronic acid (ZA), a third-generation bisphosphonate, has been applied for treatment of bone metastases caused by malignant tumors. Recent studies have found its anti-cancer effects on various tumor cells. One of the mechanisms of anti-cancer effects of ZA is induction of apoptosis. However, the mechanisms of ZA-induced apoptosis in tumor cells have not been clarified clearly. In this study, we investigated the roles of chloride channels in ZA-induced apoptosis in nasopharyngeal carcinoma CNE-2Z cells. Apoptosis and chloride current were induced by ZA and suppressed by chloride channel blockers. After the knockdown of ClC-3 expression by ClC-3 siRNA, ZA-induced chloride current and apoptosis were significantly suppressed, indicating that the chloride channel participated in ZA-induced apoptosis may be ClC-3. When reactive oxygen species (ROS) generation was inhibited by the antioxidant N-acetyl-L-cysteine (L-NAC), ZA-induced apoptosis and chloride current were blocked accordingly, suggesting that ZA induces apoptosis through promoting ROS production and subsequently activating chloride channel.

[Bartter syndrome, severe rare orphan kidney disease: a step towards therapy through pharmacogenetic and epidemiological studies].

Bartter syndromes (BS) types 1-5 are rare salt-losing tubulopathies presenting with overlapping clinical phenotypes including marked salt wasting and hypokalemia leading to polyuria, polydipsia, volume contraction, muscle weakness and growth retardation. These diseases are due to an impairment of sodium, potassium, chloride reabsorption caused by mutations in genes encoding for ion channel or transporters expressed in specific nephron tubule segments. Particularly, BS type 3 is a clinically heterogeneous form caused by mutations in CLCNKB gene which encodes the ClC-Kb chloride channel involved in NaCl reabsorption in the renal tubule. Specific therapy for BS is lacking and the only pharmacotherapy up today available is purely symptomatic and characterized by limiting side effects. The improvement of our understanding of the phenotype/genotype correlation and of the precise pathogenic mechanisms associated with BS type 3 as well as the pharmacological characterization of ClC-K chloride channels are fundamental to design therapies tailored upon patients' mutation. This mini review focused on recent studies representing relevant forward steps in the field as well as noteworthy examples of how basic and clinical research can cooperate to gain insight into the pathophysiology of this renal channelopathy, paving the way for a personalized therapy.

Soluble mucus component CLCA1 modulates expression of leukotactic cytokines and BPIFA1 in murine alveolar macrophages but not in bone marrow-derived macrophages.

The secreted airway mucus cell protein chloride channel regulator, calcium-activated 1, CLCA1, plays a role in inflammatory respiratory diseases via as yet unidentified pathways. For example, deficiency of CLCA1 in a mouse model of acute pneumonia resulted in reduced cytokine expression with less leukocyte recruitment and the human CLCA1 was shown to be capable of activating macrophages in vitro. Translation of experimental data between human and mouse models has proven problematic due to several CLCA species-specific differences. We therefore characterized activation of macrophages by CLCA1 in detail in solely murine ex vivo and in vitro models. Only alveolar but not bone marrow-derived macrophages freshly isolated from C57BL6/J mice increased their expression levels of several pro-inflammatory and leukotactic cytokines upon CLCA1 stimulation. Among the most strongly regulated genes, we identified the host-protective and immunomodulatory airway mucus component BPIFA1, previously unknown to be expressed by airway macrophages. Furthermore, evidence from an in vivo Staphylococcus aureus pneumonia mouse model suggests that CLCA1 may also modify BPIFA1 expression in airway epithelial cells. Our data underscore and specify the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the Clca1-deficient S. aureus pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense.

Bone marrow transplantation improves proximal tubule dysfunction in a mouse model of Dent disease.

Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow-derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5Y/- proximal tubule cells with bone marrow-derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow-derived cells and proximal tubule cells were observed in vivo and in vitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow-derived cells and diseased tubular cells play a key role in the rescue mechanism.

Differential effects of anoctamins on intracellular calcium signals.

The Ca2+-activated Cl- channel TMEM16A [anoctamin (ANO)1] is homologous to yeast Ist2 and has been shown to tether the cortical endoplasmic reticulum (ER) to the plasma membrane. We therefore examined whether ANO1 and other members of the ANO family affect intracellular Ca2+ ([Ca2+]i) signals. It is shown that expression of ANO1 augments Ca2+ store release upon stimulation of GPCRs, whereas knockdown of ANO1, or lack of Ano1 expression in Ano1-/- animals, as shown in an earlier report, inhibits Ca2+ release. ANO6, and -10 show similar effects, whereas expression of ANO4, -8, and -9 attenuate filling of the ER store. The impact of ANO1 and -4 were examined in more detail. ANO1 colocalized and interacted with IP3R, whereas ANO4 colocalized with SERCA Ca2+ pumps and interacted with ORAI-1 channels, respectively. ANO1 Cl currents were rapidly activated exclusively through Ca2+ store release, and remained untouched by influx of extracellular Ca2+ In contrast expression of ANO4 caused a delayed activation of membrane-localized ANO6 channels, solely through store-operated Ca2+ entry via ORAI. Ca2+ signals were inhibited by knocking down expression of endogenous ANO1, -5, -6, and -10 and were also reduced in epithelial cells from Ano10-/- mice. The data suggest that ANOs affect compartmentalized [Ca2+]i signals, which may explain some of the cellular defects related to ANO mutations.-Cabrita, I., Benedetto, R., Fonseca, A., Wanitchakool, P., Sirianant, L., Skryabin, B. V., Schenk, L. K., Pavenstädt, H., Schreiber, R., Kunzelmann, K. Differential effects of anoctamins on intracellular calcium signals.

ClC-7 Deficiency Impairs Tooth Development and Eruption.

CLCN7 gene encodes the voltage gated chloride channel 7 (ClC-7) in humans. The mutations in CLCN7 have been associated with osteopetrosis in connection to the abnormal osteoclasts functions. Previously, we found that some osteopetrosis patients with CLCN7 mutations suffered from impacted teeth and root dysplasia. Here we set up two in vivo models under a normal or an osteoclast-poor environment to investigate how ClC-7 affects tooth development and tooth eruption. Firstly, chitosan-Clcn7-siRNA nanoparticles were injected around the first maxillary molar germ of newborn mice and caused the delay of tooth eruption and deformed tooth with root dysplasia. Secondly, E13.5 molar germs infected with Clcn7 shRNA lentivirus were transplanted under the kidney capsule and presented the abnormal changes in dentin structure, periodontal tissue and cementum. All these teeth changes have been reported in the patients with CLCN7 mutation. In vitro studies of ameloblasts, odontoblasts and dental follicle cells (DFCs) were conducted to explore the involved mechanism. We found that Clcn7 deficiency affect the differentiation of these cells, as well as the interaction between DFCs and osteoclasts through RANKL/OPG pathway. We conclude that ClC-7 may affect tooth development by directly targeting tooth cells, and regulate tooth eruption through DFC mediated osteoclast pathway.

Null mutation of chloride channel 7 (Clcn7) impairs dental root formation but does not affect enamel mineralization.

ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl(-) by the ruffled border of osteoclasts enables H(+) secretion by v-H(+)-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl(-) as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7(-/-) mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7(-/-) mice. Craniofacial bones in Clcn7(-/-) mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7(-/-) mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization.

ClC-3 deficiency prevents atherosclerotic lesion development in ApoE-/- mice.

BACKGROUND: Recent evidence suggested that ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, plays a critical role in regulation of a variety of physiological functions. However, remarkably little is known about whether ClC-3 is involved in atherosclerosis. This study aims to establish the involvement and direct role of ClC-3 in atherogenesis and underlying mechanisms by using ClC-3 and ApoE double null mice. METHODS AND RESULTS: After a 16-week western-type high-fat diet, the ClC-3(+/+)ApoE(-/-) mice developed widespread atherosclerotic lesions in aorta. However, the lesion size was significantly reduced in aorta of ClC-3(-/-)ApoE(-/-) mice. Compared with the ClC-3(+/+) controls, there was significantly decreased ox-LDL binding and uptake in isolated peritoneal macrophages from ClC-3(-/-) mice. Moreover, the expression of scavenger receptor SR-A, but not CD36, was significantly decreased in both ClC-3(-/-) peritoneal macrophages and aortic lesions from ClC-3(-/-)ApoE(-/-) mice. These findings were further confirmed in ox-LDL-treated RAW264.7 macrophages, which showed that silence of ClC-3 inhibited SR-A expression, ox-LDL accumulation and foam cell formation, whereas overexpression of ClC-3 produced the opposite effects. In addition, ClC-3 siRNA significantly inhibited, whereas ClC-3 overexpression increased, the phosphorylation of JNK/p38 MAPK in ox-LDL-treated RAW264.7 foam cells. Pretreatment with JNK or p38 inhibitor abolished ClC-3-induced increase in SR-A expression and ox-LDL uptake. Finally, the increased JNK/p38 phosphorylation and SR-A expression induced by ClC-3 could be mimicked by reduction of [Cl(-)]i by low Cl(-) solution. CONCLUSIONS: Our findings demonstrated that ClC-3 deficiency inhibits atherosclerotic lesion development, possibly via suppression of JNK/p38 MAPK dependent SR-A expression and foam cell formation.

Pharmaceutical Activation or Genetic Absence of ClC-2 Alters Tight Junctions During Experimental Colitis.

BACKGROUND: We have previously reported that the ClC-2 chloride channel has an important role in regulation of tight junction barrier function during experimental colitis, and the pharmaceutical ClC-2 activator lubiprostone initiates intestinal barrier repair in ischemic-injured intestine. Thus, we hypothesized that pharmaceutical ClC-2 activation would have a protective and therapeutic effect in murine models of colitis, which would be absent in ClC-2 mice. METHODS: We administered lubiprostone to wild-type or ClC-2 mice with dextran sulfate sodium (DSS) or 2, 4, 5-trinitrobenzene sulfonic acid-induced colitis. We determined the severity of colitis and assessed intestinal permeability. Selected tight junction proteins were analyzed by Western blotting and immunofluorescence/confocal microscopy, whereas proliferative and differentiated cells were examined with special staining and immunohistochemistry. RESULTS: Oral preventive or therapeutic administration of lubiprostone significantly reduced the severity of colitis and reduced intestinal permeability in both DSS and trinitrobenzene sulfonic acid-induced colitis. Preventive treatment with lubiprostone induced significant recovery of the expression and distribution of selected sealing tight junction proteins in mice with DSS-induced colitis. In addition, lubiprostone reduced crypt proliferation and increased the number of differentiated epithelial cells. Alternatively, when lubiprostone was administered to ClC-2 mice, the protective effect against DSS colitis was limited. CONCLUSIONS: This study suggests a central role for ClC-2 in restoration of barrier function and tight junction architecture in experimental murine colitis, which can be therapeutically targeted with lubiprostone.

Development of the Olfactory Epithelium and Nasal Glands in TMEM16A-/- and TMEM16A+/+ Mice.

TMEM16A/ANO1 is a calcium-activated chloride channel expressed in several types of epithelia and involved in various physiological processes, including proliferation and development. During mouse embryonic development, the expression of TMEM16A in the olfactory epithelium is dynamic. TMEM16A is expressed at the apical surface of the entire olfactory epithelium at embryonic day E12.5 while from E16.5 its expression is restricted to a region near the transition zone with the respiratory epithelium. To investigate whether TMEM16A plays a role in the development of the mouse olfactory epithelium, we obtained the first immunohistochemistry study comparing the morphological properties of the olfactory epithelium and nasal glands in TMEM16A-/- and TMEM16A+/+ littermate mice. A comparison between the expression of the olfactory marker protein and adenylyl cyclase III shows that genetic ablation of TMEM16A did not seem to affect the maturation of olfactory sensory neurons and their ciliary layer. As TMEM16A is expressed at the apical part of supporting cells and in their microvilli, we used ezrin and cytokeratin 8 as markers of microvilli and cell body of supporting cells, respectively, and found that morphology and development of supporting cells were similar in TMEM16A-/- and TMEM16A+/+ littermate mice. The average number of supporting cells, olfactory sensory neurons, horizontal and globose basal cells were not significantly different in the two types of mice. Moreover, we also observed that the morphology of Bowman's glands, nasal septal glands and lateral nasal glands did not change in the absence of TMEM16A. Our results indicate that the development of mouse olfactory epithelium and nasal glands does not seem to be affected by the genetic ablation of TMEM16A.