Calcium-activated Chloride Channel Family Research Articles

Abstract 7056: The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism

Abstract Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. In PCa the expression of anoctamin 7 (ANO7) has been shown to diminish as the cancer progresses. We have previously linked single-nucleotide polymorphisms in the ANO7 gene to the risk of aggressive prostate cancer and shown that in homozygous carriers of rs77559646, the variant leads to a total loss of ANO7 protein. ANO7 is a prostate-specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. To uncover the cellular functions of ANO7 has proven challenging because ANO7 is not expressed in commercially available cell lines. To study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and gene set enrichment analysis. Interestingly, we found enrichment and downregulation of mitochondrial genes participating in oxidative phosphorylation in ANO7 cells. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the basal and maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells. Furthermore, the glycolysis stress assay showed that glycolytic capacity is increased in ANO7 overexpressing cells. In addition, a targeted metabolite screening revealed that aspartate is decreased in ANO7 cells, which again points towards perturbed mitochondrial function as aspartate is produced from a tricarboxylic acid cycle intermediate. By inhibiting the uptake of extracellular aspartate, we measured slower proliferation of ANO7 expressing cells, while inhibiting aspartate had no effect on control cells. Interestingly, preliminary results also suggest ANO7's involvement in regulation of phospholipid acyl chain length and saturation, offering insights into altered signaling observed in ANO7 cells. Taken together, this study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism, which could explain why it is beneficial for cancer cells to lose ANO7 expression. Citation Format: Christoffer Löf, Nasrin Sultana, Neha Goel, Gudrun Wahlström, Johanna Schleutker. The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7056.

Experimental evaluation of the nonselective and selective TMEM16 family calcium-activated chloride channel blockers in the airways

Abstract Background The family of calcium-activated chloride channels, TMEM16, plays a significant role in contributing to the pathogenesis of airway inflammatory diseases. Targeting these ion channels and aiming to modulate them may provide an interesting new approach to the therapy of these potentially fatal diseases. Methods We tested this hypothesis in both healthy and ovalbumin (OVA)-sensitized male Dunkin-Hartley guinea pigs. The ion channel activity was modulated by TMEM16A-nonselective (benzbromarone) and TMEM16A-selective blockers (CaCCinh-A01). In vivo airway reactivity, induced by histamine and methacholine, was expressed as specific airway resistance (sRaw) values. The number of citric acid-induced coughs was counted using a double-chambered body plethysmograph, and the frequency of ciliary beating (CBF) was assessed in vitro by brushing method. For comparison, salbutamol and codeine were tested under the same conditions. Results The results showed significant differences in the responses of unsensitized and sensitized airways to both TMEM16 blockers administered. CaCCinh-A01 and benzbromarone significantly reduced the number of cough efforts in the group of OVA-sensitized guinea pigs. Significant improvement in sRaw values could be observed in OVA-sensitized TMEM16 blocker–treated animals compared to salbutamol when challenged with inhalational histamine, and the outcome was similar to salbutamol when challenged with methacholine. CBF was significantly inhibited in animals sensitized to OVA treated with selective inhibition of TMEM16A. Conclusions The results demonstrated that treatment with blockers of TMEM16 can reduce both cough effort and sRaw, and the difference between TMEM16A-selective and TMEM16A-nonselective blocking is only negligibly in favor of CaCCinh-A01. It is also worthwhile to note the impairment of CBF in OVA-sensitized animals treated with CaCCinh-A01.

Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window.

Many efforts have been made to understand excitotoxicity and develop neuroprotectants for the therapy of ischemic stroke. The narrow treatment time window is still to be solved. Given that the ischemic core expanded over days, treatment with an extended time window is anticipated. Bestrophin 1 (BEST1) belongs to a bestrophin family of calcium-activated chloride channels. We revealed an increase in neuronal BEST1 expression and function within the peri-infarct from 8 to 48h after ischemic stroke in mice. Interfering the protein expression or inhibiting the channel function of BEST1 by genetic manipulation displayed neuroprotective effects and improved motor functional deficits. Using electrophysiological recordings, we demonstrated that extrasynaptic glutamate release through BEST1 channel resulted in delayed excitotoxicity. Finally, we confirmed the therapeutic efficacy of pharmacological inhibition of BEST1 during 6-72h post-ischemia in rodents. This delayed treatment prevented the expansion of infarct volume and the exacerbation of neurological functions. Our study identifies the glutamate-releasing BEST1 channel as a potential therapeutic target against ischemic stroke with a wide time window.

Abstract A020: Unraveling the role of ANO7 in prostate cancer metabolism

Abstract Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. We have previously linked single-nucleotide polymorphisms in the anoctamin 7 (ANO7) gene to the risk of aggressive prostate cancer and shown that carriers of a rs77559646 variant have improved treatment response to docetaxel. The expression of ANO7 is lost when prostate cancer progresses and is completely lacking in prostate cancer metastases. ANO7 is a prostate specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. Whether ANO7 has any of these capabilities is unclear. To uncover the cellular functions of ANO7 has proven challenging because the expression of ANO7 at protein level is either minimal or nonexistent in all commercially available cell lines. To overcome this and to study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. The characterization of the RWPE-1 cells overexpressing ANO7 showed that the viability and proliferation was increased both in 2D and 3D. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and pathway analysis. Interestingly, we found enrichment of mitochondrial genes participating in oxidative phosphorylation in cells overexpressing ANO7. The genes expressed from the mitochondrial genome were upregulated and the nuclear genes encoding for proteins involved in mitochondrial functions were mostly downregulated. This suggests that the mitochondrial metabolism has changed. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced compared to control cells. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells as there were no differences in maximal respiratory capacity when we treated the cells with the UK5099 inhibitor, which inhibits pyruvate import to mitochondria. Furthermore, preliminary results from the glycolysis stress assay indicates that glycolysis is increased in ANO7 overexpressing cells. In addition, electron microscopy study suggests that ANO7 overexpressing cells have less cristae compared to control. This study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism. Citation Format: Nasrin Sultana, Christoffer Lof, Neha Goel, Johanna Schleutker. Unraveling the role of ANO7 in prostate cancer metabolism [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A020.

CryoEM analysis of gating dynamics in mammalian bestrophins

The bestrophin family of calcium-activated chloride channels are integral to eye physiology. Normal function of the human Best1 channel (hBest1) is required for the maintenance of retinal function, and over 250 mutations in the BEST1 gene lead to a collection of related eye diseases broadly termed “bestrophinopathies.” The Best2 homolog is expressed in the basolateral membrane of the nonpigmented epithelial cells of the ciliary body, where it has been suggested to play a key role in aqueous humor production through its calcium-dependent activity.

Anoctamin 5 promotes osteosarcoma development by increasing degradation of Nel-like proteins 1 and 2.

Anoctamin 5 (ANO5) is a member of the Anoctamin (ANO) family of calcium-activated chloride channels. Although ANO5 expression is upregulated in various cancers, its role in osteosarcoma remains largely unknown. In this study, bioinformatics analysis, western blot, and immunohistochemical staining revealed that ANO5 was upregulated in osteosarcoma cell lines and osteosarcoma tissues, and ANO5 expression was positively associated with tumor size, tumor grade, and metastasis. Functional experiments demonstrated that inhibition of ANO5 decreased, while ANO5 overexpression increased, osteosarcoma cell proliferation and mobility in vitro. Immunoprecipitation, western blot, and confocal microscopy experiments showed that ANO5 bound to and promoted the degradation of Nel-like proteins 1 (NELL1) and 2 (NELL2). Moreover, a subcutaneous tumor transplantation model revealed that ANO5 knockdown reduced osteosarcoma cell proliferation and increased NELL1 and NELL2 expression in vivo. Finally, rescue experiments showed that knockdown of NELL1 or NELL2 reversed the inhibitory effects of ANO5 knockdown on osteosarcoma cell proliferation and migration. These results demonstrated that upregulation of ANO5 promoted osteosarcoma development by decreasing the stability of the NELL1 and NELL2 proteins and that ANO5 may be an effective target for the treatment of osteosarcoma.

Calcium-Activated Chloride Channel ANO1/TMEM16A: Regulation of Expression and Signaling.

Anoctamin-1 (ANO1), also known as TMEM16A, is the most studied member of anoctamin family of calcium-activated chloride channels with diverse cellular functions. ANO1 controls multiple cell functions including cell proliferation, survival, migration, contraction, secretion, and neuronal excitation. This review summarizes the current knowledge of the cellular mechanisms governing the regulation of ANO1 expression and of ANO1-mediated intracellular signaling. This includes the stimuli and mechanisms controlling ANO1 expression, agonists and processes that activate ANO1, and signal transduction mediated by ANO1. The major conclusion is that this field is poorly understood, remains highly controversial, and requires extensive and rigorous further investigation.

Mathematical Models of Protein Induced Membrane Deformation

I will describe recent advances we have made in using fast, continuum elasticity theory to describe membrane deformations around proteins. I will show that our calculations match the deformations predicted from all atom molecular dynamics simulations for two proteins: gramicidin (a small antibiotic ion channel) and nhTMEM16 (a member of the calcium activated chloride channel family). Our calculations reveal that nhTMEM16 produces large distortions in the membrane potentially related to its ability to scramble lipids from one leaflet to the other. This hypothesis is supported by atomistic simulations in which we observe lipids flipping from one leaflet to the other. Experiments to test the lipid flipping mechanism in a mammalian TMEM16 family member will also be discussed.

Platelet Phosphatidylserine Exposure, Survival and Blood Coagulation in Mice Lacking TMEM16F

Scott Syndrome is a rare, moderately severe bleeding disorder caused by a defect in platelet, red cell, and lymphocyte phosphatidylserine exposure. The syndrome has been linked to mutations in TMEM16F, a Ca++-activated ion channel. A moderately severe bleeding disorder in German Shephard dogs, characterized by decreased platelet phosphatidylserine exposure, has also linked to mutations in TMEM16F.TMEM16F, is a member of a recently-identified family of calcium-activated chloride channels that are also called anoctamins. Members of the family apparently serve both as ion channels and phospholipid scrambling channels. A crystal structure of one member of the TMEM16 family shows a homo-dimeric structure of an integral membrane protein with each unit containing 10 transmembrane helices. A transmembrane hydrophilic cavity lies at the interface, and contains a cation binding site, and a slot with dimensions that could accommodate acyl chains.Tmem16f-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding. We have developed an independent gene-targeted (gt) Tmem16f allele generated in C57BL/6 ES cells and find results that both confirm prior reports and contrast with them.JM8 ES cells were obtained from EUCOMM, and Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f+/gt intercross progeny identified no surviving Tmem16fgt/gt mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryos. Blinded pathological evaluation of E17.5 gt/gt pups indicated reduction of ossification and angular limb changes, consistent with 2 prior reports. Thus, our results confirm previously reported bone changes, but in contrast with prior reports, indicate that Tmem16f deficiency is lethal in the C57BL/6 genetic background.An F2 intercross of +/gt mice outcrossed one generation to 129x16vJ resulted in gt/gt mice surviving to weaning, though at only 30% of the expected Mendelian frequency. Progeny analysis points to a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3. Further efforts to localize the responsible gene(s) are underway.Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time.Our data suggest the existence of a viability-determining genetic modifier of the TMEM16F deficiency phenotype in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover a novel regulator of hemostatic function. The observation that heterozygous deficiency leads to a PS exposure and hemostatic phenotype also suggests the possibility that heterozygous TMEM16F mutations may influence hemostasis or thrombosis in humans. DisclosuresNo relevant conflicts of interest to declare.

Stoichiometry and specific assembly of Best ion channels.

Human Bestrophin 1 (hBest1) is a calcium-activated chloride channel that regulates neuronal excitability, synaptic activity, and retinal homeostasis. Mutations in hBest1 cause the autosomal-dominant Best macular dystrophy (BMD). Because hBest1 mutations cause BMD, but a knockout does not, we wondered if hBest1 mutants exert a dominant negative effect through interaction with other calcium-activated chloride channels, such as hBest2, 3, or 4, or transmembrane member 16A (TMEM16A), a member of another channel family. The subunit architecture of Best channels is debated, and their ability to form heteromeric channel assemblies is unclear. Using single-molecule subunit analysis, we find that each of hBest1, 2, 3, and 4 forms a homotetrameric channel. Despite considerable conservation among hBests, hBest1 has little or no interaction with other hBests or mTMEM16A. We identify the domain responsible for assembly specificity. This domain also plays a role in channel function. Our results indicate that Best channels preferentially self-assemble into homotetramers.

Murine calcium-activated chloride channel family member 3 induces asthmatic airway inflammation independently of allergen exposure

Background Expression of murine calcium-activated chloride channel family member 3 (mCLCA3) has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin (OVA). However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified. Methods mCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid (BALF) were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC (MUC5AC) were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 (IL-13) were determined quantitatively. Results mCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-γ, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group (P &lt;0.05). The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALF. The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue. Conclusion These findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.

Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle

Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.

CLCA2, a novel RUNX1 partner gene in a therapy-related leukemia with t(1;21)(p22;q22)

The RUNX1 gene is frequently rearranged in de novo and therapy-related leukemia. In the present study, we identified the CLCA2 gene as a novel fusion partner of RUNX1 in a case of therapy-related acute myeloid leukemia associated with t(1;21)(p22;q22). Reverse transcriptase–polymerase chain reaction analysis and sequencing revealed that the t(1;21) results in out-of-frame RUNX1-CLCA2 fusions. Alternative splicing generates at least six fusion transcripts, including a major transcript fusing RUNX1 exon 6 with CLCA2 exon 2. These out-of-frame fusions produce putative truncated RUNX1 isoforms retaining the DNA binding Runt domain but not the transcriptional regulatory domain of RUNX1. No mutations were found in the exons encoding the Runt and C-terminal domains of the nonrearranged RUNX1 gene. Similar to truncated RUNX1 isoforms previously described, these shortened products could act as dominant negative inhibitors of RUNX1-dependent transactivation. CLCA2 is a breast tumor suppressor gene that encodes a member of the calcium-activated chloride channel family and is involved for the first time in a chromosomal translocation. The RUNX1-CLCA2 fusion is another example of out-of-frame fusion generating truncated RUNX1 isoforms that represent a recurrent molecular mechanism in RUNX1-related leukemias.

Bestrophin 2: An anion channel associated with neurogenesis in chemosensory systems

The chemosensory neuroepithelia of the vertebrate olfactory system share a life-long ability to regenerate. Novel neurons proliferate from basal stem cells that continuously replace old or damaged sensory neurons. The sensory neurons of the mouse and rat olfactory system specifically express bestrophin 2, a member of the bestrophin family of calcium-activated chloride channels. This channel was recently proposed to operate as a transduction channel in olfactory sensory cilia. We raised a polyclonal antibody against bestrophin 2 and characterized the expression pattern of this protein in the mouse main olfactory epithelium, septal organ of Masera, and vomeronasal organ. Comparison with the maturation markers growth-associated protein 43 and olfactory marker protein revealed that bestrophin 2 was expressed in developing sensory neurons of all chemosensory neuroepithelia, but was restricted to proximal cilia in mature sensory neurons. Our results suggest that bestrophin 2 plays a critical role during differentiation and growth of axons and cilia. In mature olfactory receptor neurons, it appears to support growth and function of sensory cilia.

The Role of CLCA Proteins in Inflammatory Airway Disease

Inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) exhibit stereotyped traits that are variably expressed in each person. In experimental mouse models of chronic lung disease, these individual disease traits can be genetically segregated and thereby linked to distinct determinants. Functional genomic analysis indicates that at least one of these traits, mucous cell metaplasia, depends on members of the calcium-activated chloride channel (CLCA) gene family. Here we review advances in the biochemistry of the CLCA family and the evidence of a role for CLCA family members in the development of mucous cell metaplasia and possibly airway hyperreactivity in experimental models and in humans. On the basis of this information, we develop the model that CLCA proteins are not integral membrane proteins with ion channel function but instead are secreted signaling molecules that specifically regulate airway target cells in healthy and disease conditions.

Expression analysis of the CLCA gene family in mouse and human with emphasis on the nervous system

BackgroundMembers of the calcium-activated chloride channel (CLCA) gene family have been suggested to possess a variety of functions including cell adhesion and tumor suppression. Expression of CLCA family members has mostly been analyzed in non-neural tissues. Here we describe the expression of mouse and human CLCA genes in the nervous system.ResultsWe show that from the six mouse CLCA family members only Clca1, Clca2 and Clca4 mRNAs are expressed in the adult brain, predominantly in olfactory ensheathing cells. During mouse nervous system development Clca1/2 is more widely expressed, particularly in cranial nerves, the diencephalon and in the cerebral cortex. While human CLCA2 and CLCA4 genes are widely expressed in brain, and at particularly high levels in the optic nerve, human CLCA3, the closest homologue of mouse Clca1, Clca2 and Clca4, is not expressed in the brain. Furthermore, we characterize the expression pattern of mouse Clca1/2 genes during embryonic development by in situ hybridization.ConclusionThe data published in this article indicate that within the nervous system mouse Clca1/2 genes are highly expressed in the cells ensheathing cranial nerves. Human CLCA2 and CLCA4 mRNAs are expressed at high level in optic nerve. High level expression of CLCA family members in mouse and human glial cells ensheathing nerves suggests a specific role for CLCA proteins in the development and homeostasis of these cells.

LPS-induced mucin expression in human sinus mucosa can be attenuated by hCLCA inhibitors

hCLCA1 is a member of the calcium-activated chloride channel family and is associated with disease-inducible mucus expression. Niflumic acid (NFA) and a closely related chemical structure are reported inhibitors of calcium-activated chloride channels and endotoxin-inducible mucus expression in the mouse. Therefore, we tested the hypothesis that hCLCA1 may be involved in lipopolysaccharide (LPS) induced mucin up-regulation in human airways. We also investigated the effect of NFA and MSI-2216 on LPS-induced mucin up-regulation. Explanted human airways and the muco-epidermoid cell line Calu-3 were stimulated with LPS. Different concentrations of NFA or MSI-2216 were added to LPS-stimulated airway mucosa and Calu-3 cells. Expression of hCLCA1 and MUC5AC mRNA and protein was quantified in human airways using real-time PCR and PAS staining. In addition, immunohistochemistry was performed for quantification of inflammatory cells (lymphocytes, monocytes, eosinophils, and neutrophils) in the submucosa of the airways. Expression of hCLCA1 protein in Calu-3 cells was analysed by FACS. LPS significantly induced hCLCA1 and MUC5AC mRNA and protein expression in human airway mucosa (P < 0.05). NFA and MSI-2216 significantly decreased LPS-induced mucus expression in explanted airway mucosa in a dose-dependent manner (P < 0.05). In Calu-3 cells, LPS significantly increased hCLCA1 surface expression whereas intracellular expression was significantly decreased (P < 0.05). In Calu-3 cells, NFA and MSI-2216 also significantly reduced MUC5AC mRNA expression (P < 0.05). These data suggest that hCLCA1 may play a role in LPS-induced mucin expression in human airway mucosa. Calcium-activated chloride channel inhibitors significantly decreased LPS-induced mucus expression both ex vivo and in vitro . Therefore, blocking of hCLCA1 may offer a therapeutic approach to reduce bacterial-induced mucus hypersecretion.

The Putative Chloride Channel hCLCA2 Has a Single C-terminal Transmembrane Segment

Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl(-) channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680. Earlier investigations of transmembrane geometry suggested five membrane passes. However, analysis by the more recently derived simple modular architecture research tool algorithm predicts that a C-terminal 22-amino-acid hydrophobic segment comprises the only transmembrane pass. To resolve this question, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. Cell surface biotinylation and endoglycosidase H analysis revealed a 128-kDa precursor confined to the endoplasmic reticulum and a maturely glycosylated 141-kDa precursor at the cell surface by 48 h post-transfection. By 72 h, 109-kDa N-terminal and 35-kDa C-terminal cleavage products were detected at the cell surface but not in the endoplasmic reticulum. Surprisingly, however, the 109-kDa product was spontaneously shed into the medium or removed by acid washes, whereas the precursor and 35-kDa product were retained by the membrane. Two other CLCA family members, bCLCA2 and hCLCA1, also demonstrated preferential release of the N-terminal product. Transfer of the hCLCA2 C-terminal hydrophobic segment to a secreted form of green fluorescent protein was sufficient to target that protein to the plasma membrane. Together, these data indicate that hCLCA2 is mostly extracellular with only a single transmembrane segment followed by a short cytoplasmic tail and is itself unlikely to form a channel.

Gob-5 Contributes to Goblet Cell Hyperplasia and Modulates Pulmonary Tissue Inflammation

Gob-5 is a member of the calcium-activated chloride channel family and has been associated with allergic response in mouse models of pulmonary inflammation. Gene expression of Gob-5 has been shown to be induced in allergic airways and has been strongly associated with mucin gene regulation and goblet cell hyperplasia. We investigated the physiologic role of Gob-5 in murine models of pulmonary inflammation using mice deficient in Gob-5. After sensitization and aerosol challenge with ovalbumin (OVA), Gob-5 knockout mice exhibit significantly increased bronchoalveolar lavage (BAL) inflammation as compared with wild-type controls. The augmented inflammation in BAL consisted predominantly of neutrophils. Examination of perivascular inflammation revealed that tissue inflammation was decreased in OVA-challenged Gob-5-/- mice. OVA-challenged Gob-5 knockout mice also had decreased goblet cell hyperplasia as well as decreased mucus production. These mice also had decreased airway hypersensitivity after cholinergic provocation with methacholine. Gob-5 knockout mice were also challenged via intranasal LPS, a TLR-4 agonist. Gob-5-/- mice responded with increased neutrophilic BAL inflammation and decreased perivascular tissue inflammation as compared with wild-type controls. There was little effect on goblet cell hyperplasia and mucus production after LPS challenge. These observations reinforce findings that associate Gob-5 with goblet cell hyperplasia and mucus production in the allergic immune response, but also implicate Gob-5 in the regulation of tissue inflammation in the innate immune response.

Genetic segregation of airway disease traits despite redundancy of calcium-activated chloride channel family members

Complex airway diseases such as asthma and chronic obstructive pulmonary disease exhibit stereotyped traits (especially airway hyperreactivity and mucous cell metaplasia) that are variably expressed in each patient. Here, we used a mouse model for virus-induced long-term expression of these traits to determine whether individual traits can be genetically segregated and thereby linked to separate determinants. We showed that an F2 intercross population derived from susceptible and nonsusceptible mouse strains can manifest individual phenotypic extremes that exhibit one or the other disease trait. Functional genomic analysis of these extremes further indicated that a member of the calcium-activated chloride channel (CLCA) gene family designated mClca3 was inducible with mucous cell metaplasia but not airway hyperreactivity. In confirmation of this finding, we found that mClca3 gene transfer to mouse airway epithelium was sufficient to induce mucous cell metaplasia but not airway hyperreactivity. However, newly developed mClca3(-/-) mice exhibited the same degree of mucous cell metaplasia and airway hyperreactivity as wild-type mice. Bioinformatic analysis of the Clca locus led to the identification of mClca5, and gene transfer indicated that mClca5 also selectively drives mucous cell metaplasia. Thus, in addition to the capacity of CLCA family members to exhibit diverse functional activities, there is also preserved function so that more than one family member mediates mucous cell metaplasia. Nonetheless, Clca expression appears to be a selective determinant of mucous cell metaplasia so that shared homologies between CLCA family members may still represent a useful target for focused therapeutic intervention in hypersecretory airway disease.