ClC-2 mRNA Research Articles

ADIPOSE CLC-3 DEFICIENCY PROTECTS AGAINST DIET-INDUCED OBESITY AND INSULIN RESISTANCE BY REGULATING GLUT4 THROUGH THE PI3K/AKT PATHWAY

Obesity is a worldwide medical condition with excess body fat and has negative effects on personal health. Adipocyte hyperplasia or hypertrophy are considered to be accountable for excessive accumulation of adipose tissue. One of the main reasons that induce obesity is high-fat diet (HFD), HFD can also lead to a series of complications such as insulin resistance and type 2 diabetes. GLUT4 is an important glucose transporter regulating blood glucose, it is supervised by a complicated cascade known as PI3K/Akt pathway for sensing the high concentration of glucose in the blood. Upregulation of PI3K/Akt pathway can stimulate GLUT4 to be activated to transport glucose into adipose cells. Here, we identify voltage-gated chloride ion channel family protein ClC-3 has remarkable impact on the PI3K/Akt pathway to regulate glucose uptake mediated by GLUT4. Conditional knockout ClC-3 with CRISPR/Cas9-Cre/Lox method in adipocytes (ClC-3AKO) will decrease size of the adipocytes, which is mainly decreased in epididymal (eWAT), meanwhile, ClC-3AKO can upregulate the PI3K/Akt pathway phosphorylation level, and prompt glucose uptake by GLUT4. Our studies illustrate the important function of ClC-3 in regulating the HFD-induced obesity and provide new insight into treatment for the diabetic associated disease. 1. To generate ClC-3 systemic knockout mice, and measure the size of fat cells with HE staining; 2. To take human omentum tissue, detect ClC-3 protein and mRNA levels, and analyze its relationship with obesity indicators; 3. Use the CRISPR/Cas9-Cre/Lox method to construct ClC-3AKO mice, and measure their obesity, insulin resistance and changes in adipose tissue. And extract the epididymal fat protein and perform Western Blotting to detect the phosphorylation level of key protein molecules. 4. ClC-3 was overexpressed on the 3T3-L1 cell line, and immunofluorescence, glucose uptake experiments and Western Blotting were performed to determine whether the glucose uptake and signal pathways were restored. 1. ClC-3 systemic knockout mice have smaller fat cells, reduced fat tissue and improved obesity. 2. In human omentum fat, the expression of ClC-3 is positively correlated with obesity indicators. 3. We further found that specific fat knockout ClC-3 mice have reduced fat cells, mainly in eWAT, while ClC-3AKO mice can up-regulate the phosphorylation level of PI3K/Akt pathway. And promote GLUT4 uptake of glucose. 4. ClC-3 over-expression reversed the above effects. 1. ClC-3 knockout improves obesity and insulin resistance

Modulation of Chloride Currents in Human Lung Epithelial Cells Exposed to Exogenous Oxidative Stress.

Air pollution continues to be a major public health concern affecting 9 out of 10 individuals living in urban areas worldwide. Respiratory tract is the organ most exposed to gas pollution, and ozone has been shown to be one of the most noxious pollutants to which living organisms are exposed. In the present work, we have investigated the effects of 0.1 ppm of ozone on chloride currents in human lung epithelial cells (A549 line) and whether this effect could be modulated by vitamin E pre-treatment. Whole-cell patch clamp technique was applied to not excitable cells in order to obtain information about chloride currents behavior, important for epithelial lung cells homeostasis. Significant alteration of the I-V curve after ozone treatment was observed, with the appearance of a large outward rectifier component decreasing over time and returning to the basal state levels after 24 h. Statistical analysis indicated a modification of the amount of ions passing the membrane in the unit of time as a possible cause of this difference. RT-qPCR analysis showed an increase in ClC-2 and ORCC mRNA after ozone exposure. In addition, pre-treatment with vitamin E was able to suppress the outward rectifier component induced by ozone, bringing back the current values to the control level and preventing ozone induced chloride channels up regulation. Our data suggest that ozone exposure is able to modify chloride current density and the use of vitamin E can prevent the above-mentioned damage. J. Cell. Physiol. 232: 1817-1825, 2017. © 2016 Wiley Periodicals, Inc.

Activated ClC-2 Inhibits p-Akt to Repress Myelination in GDM Newborn Rats

This study aims to investigate the effect and mechanism of type 2 voltage-gated chloride channel (ClC-2) on myelin development of newborn rats' cerebral white matter with gestational diabetes mellitus (GDM). In this study, GDM model was induced in late pregnant rat model. The alteration of ClC-2 expression in various developmental stages of cerebral white matter with/without being exposed to high glucose was analyzed using RT-PCR, active oxygen detection, TUNEL staining, Western Blot as well as immuno-histochemical staining. Our results showed that ClC-2 mRNA and protein expressions in GDM group were significantly increased in white matter of fetal rats after E18 stage, and elevated the level of TNF-α and iNOS in white matter at P0 and P3 stage of newborn rats. Meanwhile, In GDM group, reactive oxygen species (ROS) levels of the white matter at E18, P0, and P3 stage were significantly higher than control group. Furthermore, the expression level of myelin transcription factor Olig2 at P0 stage and CNPase at P3 stage were strikingly lower than that of the control group. In GDM group, ClC-2 expression in the corpus callosum (CC) and cingulate gyrus (CG) regains, and TUNEL positive cell number were increased at P0 and P3 stage. However, PDGFα positive cell number at P0 stage and CNPase expression at P3 stage were significantly decreased. Caspase-3 was also increased in those white matter regions in GDM group, but p-Akt expression was inhibited. While DIDS (a chloride channel blocker) can reverse these changes. In conclusion, ClC-2 and caspase-3 were induced by GDM, which resulted in apoptosis and myelination inhibition. The effect was caused by repressing PI3K-Akt signaling pathway. Application of ClC-2 inhibitor DIDS showed protective effects on cerebral white matter damage stimulated by high glucose concentration.

The CLC-2 Chloride Channel Modulates ECM Synthesis, Differentiation, and Migration of Human Conjunctival Fibroblasts via the PI3K/Akt Signaling Pathway.

Recent evidence suggests that chloride channels are critical for cell proliferation, migration, and differentiation. We examined the effects of transforming growth factor (TGF)-β1 on chloride channel expression and associations with human conjunctival fibroblast (HConF) biology. To investigate the potential role of chloride channel (CLC)-2 in migration, transition to myofibroblasts and extracellular matrix (ECM) synthesis of HconF, a small interfering RNA (siRNA) approach was applied. TGF-β1-induced migration and transition of fibroblasts to myofibroblasts characterized by α-smooth muscle actin (α-SMA) expression, supported by increased endogenous expression of CLC-2 protein and mRNA transcripts. ECM (collagen I and fibronectin) synthesis in HConF was enhanced by TGF-β1. CLC-2 siRNA treatment reduced TGF-β1-induced cell migration, transition of fibroblasts to myofibroblasts, and ECM synthesis of HConF. CLC-2 siRNA treatment in the presence of TGF-β1 inhibited phosphorylation of PI3K and Akt in HConF. These findings demonstrate that CLC-2 chloride channels are important for TGF-β1-induced migration, differentiation, and ECM synthesis via PI3K/Akt signaling in HConF.

Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel.

ClC-3 is a volume-sensitive chloride channel that is responsible for cell volume adjustment and regulatory cell volume decrease (RVD). In order to evaluate the effects of fluid shear stress (FSS) stimulation on the osteoblast ClC-3 chloride channel, MC3T3-E1 cells were stimulated by FSS in the experimental group. Fluorescence quantitative polymerase chain reaction was used to detect changes in ClC-3 mRNA expression, the chloride ion fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used to detect the chloride channel activity, and whole-cell patch clamping was used to monitor the changes in the volume-sensitive chloride current activated by a hypotonic environment following mechanical stimulation. The results show that the expression of the osteoblast chloride channel ClC-3 was significantly higher in the FSS group compared with the control group. MQAE fluorescence intensity was significantly reduced in the FSS group compared to the control group, suggesting that mechanical stimulation increased chloride channel activity and increased the efflux of intracellular chloride ions. Image analysis of osteoblast volume changes showed that osteoblast RVD was enhanced by mechanical stimulation. Whole-cell patch clamping showed that the osteoblast volume-sensitive chloride current was larger in the stimulated group compared to the control group, suggesting that elevated ClC-3 chloride channel expression results in an increased volume-sensitive chloride current. In conclusion, FSS stimulation enhances the RVD of osteoblast cell by increasing the expression of the ClC-3 and enhancing the chloride channel activity.

High glucose inhibits ClC-2 chloride channels and attenuates cell migration of rat keratinocytes.

BackgroundAccumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25 mM) on ClC-2 chloride channels and cell migration of keratinocytes.MethodsNewborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies.ResultsWe found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25 mM) medium, accompanied by the decline of volume-activated Cl− current (ICl,vol), migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including ICl,vol and migration keratinocytes were inhibited.ConclusionOur study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling.

Swelling-activated Cl− currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells

Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl currents (ICl,swell) are upregulated in PASMC proliferation and that nonspecific Cl current blockers inhibit proliferation. However, the specific role of ICl,swell in PASMC proliferation and its molecular underpinning remain unknown. In the present study, we found that the specific ICl,swell blocker, DCPIB (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC50 = 2.7 μmol/l) ICl,swell and inhibited (IC50 = 6.9 μmol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl channel genes underlying ICl,swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to ICl,swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by >45%) compared with control-shRNA, it did not alter ICl,swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for ICl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution. Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie ICl,swell in these cells.

Asthma Severity and Expression Of CLC3 On Human Peripheral Blood and Nasal Lavage Eosinophils

RationaleWe have reported the role of CLC3 in TGF-β-induced migration and activation of eosinophils, and increased expression of CLC3b and CLC3e transcript variants on TGF-β and eotaxin-treated eosinophils. Severity of asthma and corticosteroid treatment may determine the CLC3 expression on eosinophils.MethodsEosinophils were isolated from human venous blood (>99%pure; >98%viable) with negative selection from healthy (n=10), mild-to-moderate (n=10) and moderate-to-severe (n=10) allergic asthmatics. Nasal washings were taken from respective groups. Q-PCR, Western blot, and fluorescence microscopy were used to obtain outcome measures.ResultsmRNA transcripts of CLC3 variants varied among groups based on the severity of asthma and corticosteroid treatment. Eosinophils from mild-to-moderate asthmatics had 5-6-fold increase in CLC3 mRNA transcripts compared to healthy volunteers. However, moderate-to-severe asthmatics on inhaled corticosteroid had 2-3-fold increase in CLC3 transcripts. The CLC3b and CLC3e transcripts were increased 5-7 and 6-8-fold in mild-to-moderate asthmatics, and in moderate-severe asthmatics increase by 4-5 and 3-4 fold, respectively. Protein expression of CLC3 also followed the same trend. Asthmatics nasal lavage contained multiple eosinophils expressing different levels of CLC3 with increased expression in mild-to-moderate than moderate-to-severe asthmatic and no inflammatory cells in healthy subjects.ConclusionsThe CLC3 expression on eosinophils correlates with asthma severity. Reduction in CLC3 expression may be an additional mechanism of corticosteroid effects in allergic airway inflammation. Differential CLC3 expression on nasal eosinophils could be a biomarker of asthma severity and treatment with corticosteroids. RationaleWe have reported the role of CLC3 in TGF-β-induced migration and activation of eosinophils, and increased expression of CLC3b and CLC3e transcript variants on TGF-β and eotaxin-treated eosinophils. Severity of asthma and corticosteroid treatment may determine the CLC3 expression on eosinophils. We have reported the role of CLC3 in TGF-β-induced migration and activation of eosinophils, and increased expression of CLC3b and CLC3e transcript variants on TGF-β and eotaxin-treated eosinophils. Severity of asthma and corticosteroid treatment may determine the CLC3 expression on eosinophils. MethodsEosinophils were isolated from human venous blood (>99%pure; >98%viable) with negative selection from healthy (n=10), mild-to-moderate (n=10) and moderate-to-severe (n=10) allergic asthmatics. Nasal washings were taken from respective groups. Q-PCR, Western blot, and fluorescence microscopy were used to obtain outcome measures. Eosinophils were isolated from human venous blood (>99%pure; >98%viable) with negative selection from healthy (n=10), mild-to-moderate (n=10) and moderate-to-severe (n=10) allergic asthmatics. Nasal washings were taken from respective groups. Q-PCR, Western blot, and fluorescence microscopy were used to obtain outcome measures. ResultsmRNA transcripts of CLC3 variants varied among groups based on the severity of asthma and corticosteroid treatment. Eosinophils from mild-to-moderate asthmatics had 5-6-fold increase in CLC3 mRNA transcripts compared to healthy volunteers. However, moderate-to-severe asthmatics on inhaled corticosteroid had 2-3-fold increase in CLC3 transcripts. The CLC3b and CLC3e transcripts were increased 5-7 and 6-8-fold in mild-to-moderate asthmatics, and in moderate-severe asthmatics increase by 4-5 and 3-4 fold, respectively. Protein expression of CLC3 also followed the same trend. Asthmatics nasal lavage contained multiple eosinophils expressing different levels of CLC3 with increased expression in mild-to-moderate than moderate-to-severe asthmatic and no inflammatory cells in healthy subjects. mRNA transcripts of CLC3 variants varied among groups based on the severity of asthma and corticosteroid treatment. Eosinophils from mild-to-moderate asthmatics had 5-6-fold increase in CLC3 mRNA transcripts compared to healthy volunteers. However, moderate-to-severe asthmatics on inhaled corticosteroid had 2-3-fold increase in CLC3 transcripts. The CLC3b and CLC3e transcripts were increased 5-7 and 6-8-fold in mild-to-moderate asthmatics, and in moderate-severe asthmatics increase by 4-5 and 3-4 fold, respectively. Protein expression of CLC3 also followed the same trend. Asthmatics nasal lavage contained multiple eosinophils expressing different levels of CLC3 with increased expression in mild-to-moderate than moderate-to-severe asthmatic and no inflammatory cells in healthy subjects. ConclusionsThe CLC3 expression on eosinophils correlates with asthma severity. Reduction in CLC3 expression may be an additional mechanism of corticosteroid effects in allergic airway inflammation. Differential CLC3 expression on nasal eosinophils could be a biomarker of asthma severity and treatment with corticosteroids. The CLC3 expression on eosinophils correlates with asthma severity. Reduction in CLC3 expression may be an additional mechanism of corticosteroid effects in allergic airway inflammation. Differential CLC3 expression on nasal eosinophils could be a biomarker of asthma severity and treatment with corticosteroids.

Pathological impact of hyperpolarization-activated chloride current peculiar to rat pulmonary vein cardiomyocytes

Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs+ resistant, and was not affected by removal of external Na+ or K+. The current was inhibited with Cd2+, and the reversal potential was sensitive to changes in [Cl−] on either side of the membrane in a manner consistent with a Cl− selective channel. Cl− channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl− current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl− current in rat PVs was shifted to negative potentials as [Cl−]i increased, (ii) the Cl− current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl− channel, and that the channel may play a functional role in norepinephrine-induced automaticity.

Effect of CLC-2 on the cytoskeleton in human trabecular meshwork cells

The chloride channel protein 2 (CLC‑2) is important in maintaining the volume of trabecular meshwork cells by adjusting the outflow of aqueous solutions and maintaining the fluid balance. However, little is known concerning the functions of CLC‑2 in the cytoskeleton, specifically in human trabecular meshwork (HTM) cells. In the present study, two CLC-2 specific siRNAs (siRNA1 and siRNA2) that target CLC-2 mRNA were designed. The siRNAs were transfected into the HTM cells and the results showed that siRNA1 in particular decreased the expression of CLC‑2 by ~45%. Furthermore, an siRNA1‑mediated CLC-2 knockdown significantly reconstructed the actin cytoskeleton and formed cross‑linked actin networks. In addition, the downregulation of the expression of CLC‑2 was associated with increased TGF‑β and Smad2 activities in the HTM cells following 24 h of transfection. In conclusion, these results suggest that CLC‑2 knockdown promotes trabecular meshwork cytoskeletal disorders and may activate the TGF‑β/Smad signaling pathway. Thus, CLC‑2 may be a promising and potential novel therapeutic strategy for combating primary open‑angle glaucoma.

The ClC-3 chloride channel and osmoregulation in the European Sea Bass, Dicentrarchus labrax

Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na(+)/K(+)-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10min to 30days. In gills, NKA-α1 subunit expression transiently increased from 10min and reached a stabilized intermediate expression level after 24h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50%) 1h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30% higher 7days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-α1 subunit expression was followed by significantly higher stable levels from 24h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.

Lubiprostone Activates Cl− Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84

Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl(-) transport in T84 cells by activating ClC-2 channels. To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl(-) transport. Cl(-) transport was assessed either as I(sc) across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP](i) was measured by enzyme immunoassay, and [Ca(2+)](i) by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR. Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of I(sc) in T84 cells. The I(sc) effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP](i). H89, bumetanide, or CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation. Lubiprostone activates Cl(-) secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.

Chloride channel ClC‐3 promotion of osteogenic differentiation through Runx2

ClC-3 chloride channel has been speculated to contribute to the acidification of synaptic vesicles and endosomes. However, the biological function of ClC-3 in osteogenesis remains to be determined. In this study, we first analyzed ClC-3 expression in MC3T3-E1 cells and primary mouse osteoblasts and then performed the osteoinductive procedure to determine the effects on gene expression. Subsequently, we transiently transfected ClC-3 cDNA or ClC-3-siRNA into MC3T3-E1 cells to determine the changed phenotype and gene expression. Lastly, we assessed the underlying mechanism responsible for ClC-3-induced osteodifferentiation. We found that ClC-3 mRNA was expressed in primary mouse osteoblasts and MC3T3-E1 cells and induced by using an osteoinductive procedure. We also found that overexpression of ClC-3 contributed to osteodifferentiation, such as increase in the expression of osteogenic markers [alkaline phosphatase (Alp), osteocalcin (Oc), bone sialoprotein (Bsp), osterix (Osx), and runt-related transcription factor 2 (Runx2)], morphological changes, and mineralized nodules in MC3T3-E1 cells. ClC-3 gene silencing suppressed gene expression of these osteogenic markers. Moreover, overexpressed ClC-3 protein co-localized with TGF-beta1 in intracellular organelles, inhibited TGF-beta1 protein expression and induced endosomal acidification. Nevertheless, knockdown of Runx2 expression antagonized the effects of ClC-3 in osteodifferentiation and expression of osteogenic markers. The data from the current study suggest that the function of ClC-3 in osteodifferentiation may be through the Runx2 pathway.

Expression patterns of ClC-3 mRNA and protein in aortic smooth muscle, kidney and brain in diabetic rats

ClC-3, a member of the ClC family of voltage-gated chloride channels, regulates cell proliferation of cultured rat aortic vascular smooth muscle cells, pathogenesis of allergic rhinitis and tumor cell migration. However, its role in diabetic animals is still unknown. To address this issue, we investigated the expression patterns of ClC-3 in diabetic rats. Five-week-old Sprague-Dawley rats were divided into two groups, 50 non-diabetic control rats (non-DM) and 50 diabetic model rats (DM). ClC-3 mRNA and protein expression in aortic smooth muscle, kidney and brain tissues were examined by fluorimeter-based quantitive RT-PCR assay and Western blot analysis, respectively. ClC-3 mRNA and protein were endogenously expressed in aortic smooth muscle, kidney (cortex and medulla) and brain tissues of both control and streptozotocin-induced diabetic rats. ClC-3 mRNA and protein expression levels were significantly higher in aortic smooth muscle and brain tissues of diabetic rats, but significantly decreased in kidney medulla tissue, relative to non-DM controls. There were no significant differences in ClC-3 mRNA and protein expression in kidney cortex between non-diabetic control and diabetic rats. Furthermore, the altered ClC-3 expression patterns in diabetic rat aortic smooth muscle, brain, and kidney medulla tissues all correlated with the changes in blood glucose levels (p < 0.05). In conclusion, our data show for the first time that diabetes alters both the gene and protein expression of ClC-3 channels. These changes may contribute to the impaired vascular, brain and kidney functions observed in diabetes.

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.

Involvement of chloride channels in TGF-β1-induced apoptosis of human bronchial epithelial cells

Widespread damage of airway epithelium and defective epithelial repair are hallmarks of chronic asthma. Growth factors and cytokines spatially and temporally regulate epithelial shedding and repair. Within this context, a key function is exerted by transforming growth factor (TGF)-beta. Recent growing evidence suggests that chloride (Cl(-)) channels are critical to cell apoptosis. We examined the effects of TGF-beta1 on Cl(-) channel expression and activity and its relationship with apoptosis in human bronchial epithelial cells (HBECs). The small interfering RNA (siRNA) approach was used to investigate the potential role of CLC-3, a member of the volume-regulated Cl(-) channel family, in apoptosis of HBECs. TGF-beta1 significantly induced HBEC apoptosis, which paralleled to a significant decrease in the endogenous expression of CLC-3 protein and mRNA transcripts. Outward rectifying and voltage-dependent CLC-3-like Cl(-) currents in HBECs were diminished by TGF-beta1. siRNA for CLC-3 abolished Cl(-) current and enhanced TGF-beta1-induced cell apoptosis. Overexpression of CLC-3 in HBECs inhibited TGF-beta1-induced cell apoptosis. Bcl-2 was also downregulated after TGF-beta stimulation. TGF-beta1-induced cell apoptosis was suppressed in Bcl-2-transfected HBECs. Our data demonstrate that CLC-3-like voltage-gated chloride channels play a critical role in TGF-beta-induced apoptosis of human airway epithelial cells.

Hypertonic saline solution stimulated CLC-3 production in primary cultured human nasal epithelial cells

Objective To determine the molecular mechanisms of hypertonic saline on nasal disorders. Methods Primary human nasal epithelial cells (HNECs) were treated with 0.9 and 3% saline for 12 h. CLC-3 was detected in HNECs by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Results Positive expression of CLC-3 was detected in HNECs in all subjects by immunohistochemistry. The relative density of CLC-3 mRNA was 0.22 ± 0.09 and 0.12 ± 0.05 in HNECs treated with 3 and 0.9% saline, respectively. The effect of 3% saline on the expression of CLC-3 in HNECs was observed in a time-dependent manner. Conclusions Hypertonic saline decreases the nasal edema and influences the mucocilliary function through the fluid secretion in vitro and may be associated with the upregulation of CLC-3.

Possible Role of Transforming Growth Factor Beta and Interleukin-4 in the Up-Regulation of CLC-2 and CLC-3 in Chronic Rhinosinusitis

Chronic rhinosinusitis (CRS) may cover different disease entities, and the pathogenic mechanism remains unclear. The aim of this study was to evaluate the expression of chloride channel protein CLC-2 and CLC-3 in CRS without nasal polyps (CRSsNP) and evaluate the roles of interleukin (IL)-4 and transforming growth factor (TGF) beta in the up-regulation of CLC-2 and CLC-3. We detected expression of CLC-2 and CLC-3 in 17 patients with CRSsNP by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), and we examined the concentration of TGF-beta, IL-4, IL-5, and interferon (IFN) gamma in ethmoid sinus mucosa by enzyme-linked immunosorbent assay (ELISA). We found that CLC-2 and CLC-3 is up-regulated in CRSnNP and located in submucosal glands and epithelium of the ethmoid sinus. CLC-2 and CLC-3 mRNA correlated with IL-4 in CRSsNP (r = 0.57 and 0.65; p < 0.05). CLC-2 and CLC-3 mRNA correlated negatively with mucosal TGF-beta in CRSsNP (r = -0.49 and -0.54; p < 0.05). We concluded that CLC-2 and CLC-3 is up-regulated in ethmoid mucosa and may affect the development of CRSsNP. TGF-beta and IL-4 may modulate the expression of CLC-2 and CLC-3 in CRSsNP.

Involvement of chloride channels in IGF-I-induced proliferation of porcine arterial smooth muscle cells

The existence of Cl- channels in vascular smooth muscle cells (VSMCs) has been increasingly investigated, but the biological functions are not yet clear. Insulin-like growth factor (IGF)-I affects proliferation and migration of VSMCs, and dysregulation of this axis may be involved in atherogenesis and intimal hyperplasia. We examined the effects of Cl- channel blockers on IGF-I-induced proliferation in porcine VSMCs. The siRNA approach was used to support the role of ClC-2, a member of the volume-regulated Cl- channel family, in cell proliferation of VSMCs. The IGF-I-induced VSMC proliferation was significantly suppressed by the Cl- channel blockers NPPB and IAA94 but not by DIDS. IGF-I-induced cell proliferation parallels a significant increase in the endogenous expression of ClC-2 mRNA and protein. Inhibitors of PI3-kinase, LY294002 and wortmannin, significantly attenuated the IGF-I-upregulated ClC-2 expression and cell proliferation. We observed ClC-2-like Cl- current, and this current was augmented by IGF-I. SiRNA specifically targeted to ClC-2 abolished IGF-I-induced cell proliferation. Our data demonstrate that ClC-2 plays a role in IGF-1-induced regulation of VSMC proliferation in cardiovascular diseases.

Interaction between Cl- channels and CRAC-related Ca2+ signaling during T lymphocyte activation and proliferation1

To test the hypothesis that Cl channel blockers affect T cell proliferation through Ca2+-release-activated Ca2+ (CRAC) signaling and examine the effects of the combination of a CRAC channel blocker and a Cl channel blocker on concanavalin A (ConA; 5 mg/mL)-induced Ca2+ signaling, gene expression and cellular proliferation in human peripheral T lymphocytes. [3H]Thymidine incorporation, Fura-2 fluorescent probe, RNase protection assay, and reverse transcription-polymerase chain reaction were used. The Cl channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ConA-induced Ca2+ influx, interleukin-2 mRNA expression and T lymphocyte proliferation in a concentration-dependent manner, and also enhanced the inhibitory effects of 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imidazole (SK&F96365) on the above key events during T cell activation. A combination of DIDS (1 micromol/L) and SK&F96365 (1 micromol/L) significantly diminished ConA-induced ClC-3 mRNA expression by 64%, whereas DIDS(1 micromol/L) or SK&F96365 (1 micromol/L) alone decreased ConA-induced ClC-3 mRNA expression by only 16% and 9%, respectively. These results suggest that there is an interaction between CRAC-mediated Ca2+ signaling and DIDS-sensitive Cl channels during ConA-induced T cell activation and proliferation. Moreover, the DIDS-sensitive Cl channels may be related to the ClC-3 Cl channels.