Gamma tocopherol-enriched supplement reduces sputum eosinophilia and endotoxin-induced sputum neutrophilia in volunteers with asthma.

BACKGROUND: We and others have shown that the gamma tocopherol (γT) isoform of vitamin E has multiple anti-inflammatory and antioxidant actions and that γT supplementation reduces eosinophilic and endotoxin (LPS)-induced neutrophilic airway inflammation in animal models and healthy human volunteers. OBJECTIVE: We sought to determine whether γT supplementation reduces eosinophilic airway inflammation and acute neutrophilic response to inhaled LPS challenge in volunteers with asthma. METHODS: Participants with mild asthma were enrolled in a double-blinded, placebo-controlled crossover study to assess the effect of 1200 mg of γT daily for 14 days on sputum eosinophils, mucins, and cytokines. We also assessed the effect on acute inflammatory response to inhaled LPS challenge following γT treatment, focusing on changes in sputum neutrophilia, mucins, and cytokines. Mucociliary clearance was measured using gamma scintigraphy. RESULTS: Fifteen subjects with mild asthma completed both arms of the study. Compared with placebo, γT notably reduced pre-LPS challenge sputum eosinophils and mucins, including mucin 5AC and reduced LPS-induced airway neutrophil recruitment 6 and 24 hours after challenge. Mucociliary clearance was slowed 4 hours postchallenge in the placebo group but not in the γT treatment group. Total sputum mucins (but not mucin 5AC) were reduced at 24 hours postchallenge during γT treatment compared with placebo. CONCLUSIONS: When compared with placebo, γT supplementation for 14 days reduced inflammatory features of asthma, including sputum eosinophils and mucins, as well as acute airway response to inhaled LPS challenge. Larger scale clinical trials are needed to assess the efficacy of γT supplements as a complementary or steroid-sparing treatment for asthma.

A proof-of-concept clinical study examining the NRF2 activator sulforaphane against neutrophilic airway inflammation.

UNLABELLED: Sulforaphane (SFN), a naturally occurring isothiocyanate found in cruciferous vegetables, is implicated as a possible therapy for airway inflammation via induction of the transcription factor NF-E2-related factor 2 (NRF2). In this proof-of-concept clinical study, we show that supplementation of SFN with broccoli sprout homogenate in healthy human subjects did not induce expression of antioxidant genes or protect against neutrophilic airway inflammation in an ozone-exposure model. Therefore, dietary sulforaphane supplementation is not a promising candidate for larger scale clinical trials targeting airway inflammation. TRIAL REGISTRATION: NCT01625130 . Registered 19 June, 2012.

Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network.

The newly discovered Ca(2+)-activated Cl(-) channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca(2+)-activated Cl(-) currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca(2+)-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.

Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology.

RATIONALE: Ca2+ -activated Cl channels play pivotal roles in the cardiovascular system. They regulate vascular smooth muscle tone and participate in cardiac action potential repolarization in some species. Ca2+ -activated Cl channels were recently discovered to be encoded by members of the anoctamin (Ano, also called Tmem16) superfamily, but the mechanisms of Ano1 gating by Ca2+ remain enigmatic. OBJECTIVE: The objective was to identify regions of Ano1 involved in channel gating by Ca2+. METHODS AND RESULTS: The Ca2+ sensitivity of Ano1 was estimated from rates of current activation, and deactivation in excised patches rapidly switched between zero and high Ca2+ on the cytoplasmic side. Mutation of glutamates E702 and E705 dramatically altered Ca2+ sensitivity. E702 and E705 are predicted to be in an extracellular loop, but antigenic epitopes introduced into this loop are not accessible to extracellular antibodies, suggesting this loop is intracellular. Cytoplasmically applied membrane-impermeant sulfhydryl reagents alter the Ca2+ sensitivity of Ano1 E702C and E705C as expected if E702 and E705 are intracellular. Substituted cysteine accessibility mutagenesis of the putative re-entrant loop suggests that E702 and E705 are located adjacent to the Cl conduction pathway. CONCLUSIONS: We propose an alternative model of Ano1 topology based on mutagenesis, epitope accessibility, and cysteine-scanning accessibility. These data contradict the popular re-entrant loop model by showing that the putative fourth extracellular loop (ECL 4) is intracellular and may contain a Ca2+ binding site. These studies provide new perspectives on regulation of Ano1 by Ca2+.

Chloride channels: often enigmatic, rarely predictable.

Until recently, anion (Cl(-)) channels have received considerably less attention than cation channels. One reason for this may be that many Cl(-) channels perform functions that might be considered cell-biological, like fluid secretion and cell volume regulation, whereas cation channels have historically been associated with cellular excitability, which typically happens more rapidly. In this review, we discuss the recent explosion of interest in Cl(-) channels, with special emphasis on new and often surprising developments over the past five years. This is exemplified by the findings that more than half of the ClC family members are antiporters, and not channels, as was previously thought, and that bestrophins, previously prime candidates for Ca(2+)-activated Cl(-) channels, have been supplanted by the newly discovered anoctamins and now hold a tenuous position in the Cl(-) channel world.

Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization.

Cell migration is essential for many biological and pathological processes. Establishing cell polarity with a trailing edge and forming a single lamellipodium at the leading edge of the cell is crucial for efficient directional cell migration and is a hallmark of mesenchymal cell motility. Lamellipodia formation is regulated by spatial-temporal activation of the small GTPases Rac and Cdc42 at the front edge, and RhoA at the rear end. At a molecular level, partitioning-defective (Par) protein complex comprising Par3, Par6, and atypical Protein Kinase (aPKC isoforms ζ and λ/ι) regulates front-rear axis polarization. At the front edge, integrin clustering activates Cdc42, prompting the formation of Par3/Par6/aPKC complexes to modulate MTOC positioning and microtubule stabilization. Consequently, the Par3/Par6/aPKC complex recruits Rac1-GEF Tiam to activate Rac1, leading to lamellipodium formation. At the rear end, RhoA-ROCK phosphorylates Par3 disrupting its interaction with Tiam and inactivating Rac1. RhoA activity at the rear end allows the formation of focal adhesions and stress fibers necessary to generate the traction forces that allow cell movement. Nox1-based NADPH oxidase is necessary for PDGF-induced migration in vitro and in vivo for many cell types, including fibroblasts and smooth muscle cells. Here, we report that Nox1-deficient cells failed to acquire a normal front-to-rear polarity, polarize MTOC, and form a single lamellipodium. Instead, these cells form multiple protrusions that accumulate Par3 and active Tiam. The exogenous addition of H2O2 rescues this phenotype and is associated with the hyperactivation of Par3, Tiam, and Rac1. Mechanistically, Nox1 deficiency induces the inactivation of PP2A phosphatase, leading to increased activation of aPKC. These results were validated in Nox1y/- primary mouse aortic smooth muscle cells (MASMCs), which also showed PP2A inactivation after PDGF-BB stimulation consistent with exacerbated activation of aPKC. Moreover, we evaluated the physiological relevance of this signaling pathway using a femoral artery wire injury model to generate neointimal hyperplasia. Nox1y/- mice showed increased staining for the inactive form of PP2A and increased signal for active aPKC, suggesting that PP2A and aPKC activities might contribute to reducing neointima formation observed in the arteries of Nox1y/- mice.

ANOs 3-7 in the anoctamin/Tmem16 Cl- channel family are intracellular proteins.

Ca(2+)-activated Cl(-) channels (CaCCs) participate in numerous physiological functions such as neuronal excitability, sensory transduction, and transepithelial fluid transport. Recently, it was shown that heterologously expressed anoctamins ANO1 and ANO2 generate currents that resemble native CaCCs. The anoctamin family (also called Tmem16) consists of 10 members, but it is not known whether all members of the family are CaCCs. Expression of ANOs 3-7 in HEK293 cells did not generate Cl(-) currents activated by intracellular Ca(2+), as determined by whole cell patch clamp electrophysiology. With the use of confocal imaging, only ANO1 and ANO2 traffic to the plasma membrane when expressed heterologously. Furthermore, endogenously expressed ANO7 in the human prostate is predominantly intracellular. We took a chimeric approach to identify regions critical for channel trafficking and function. However, none of the chimeras of ANO1 and ANO5/7 that we made trafficked to the plasma membrane. Our results suggest that intracellular anoctamins may be endoplasmic reticulum proteins, although it remains unknown whether these family members are CaCCs. Determining the role of anoctamin family members in ion transport will be critical to understanding their functions in physiology and disease.

Physiological roles and diseases of Tmem16/Anoctamin proteins: are they all chloride channels?

The Tmem16 gene family was first identified by bioinformatic analysis in 2004. In 2008, it was shown independently by 3 laboratories that the first two members (Tmem16A and Tmem16B) of this 10-gene family are Ca(2+)-activated Cl(-) channels. Because these proteins are thought to have 8 transmembrane domains and be anion-selective channels, the alternative name, Anoctamin (anion and octa=8), has been proposed. However, it remains unclear whether all members of this family are, in fact, anion channels or have the same 8-transmembrane domain topology. Since 2008, there have been nearly 100 papers published on this gene family. The excitement about Tmem16 proteins has been enhanced by the finding that Ano1 has been linked to cancer, mutations in Ano5 are linked to several forms of muscular dystrophy (LGMDL2 and MMD-3), mutations in Ano10 are linked to autosomal recessive spinocerebellar ataxia, and mutations in Ano6 are linked to Scott syndrome, a rare bleeding disorder. Here we review some of the recent developments in understanding the physiology and structure-function of the Tmem16 gene family.

The role of Nox-mediated oxidation in the regulation of cytoskeletal dynamics.

Nox generated ROS, particularly those derived from Nox1, Nox2 and Nox4, have emerged as important regulators of the actin cytoskeleton and cytoskeleton-supported cell functions, such as migration and adhesion. The effects of Nox-derived ROS on cytoskeletal remodeling may be largely attributed to the ability of ROS to directly modify proteins that constitute or are associated with the cytoskeleton. Additionally, Nox-derived ROS may participate in signaling pathways governing cytoskeletal remodeling. In addition to these more extensively studied signaling pathways involving Nox-derived ROS, there also exist redox sensitive pathways for which the source of ROS is unclear. ROS from as of yet undetermined sources play a role in modifying, and thus regulating, the activity of several proteins critical for remodeling of the actin cytoskeleton. In this review we discuss ROS sensitive targets that are likely to affect cytoskeletal dynamics, as well as the potential involvement of Nox proteins.

Drosophila bestrophin-1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.

Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca(2+). The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca(2+)/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.

A comprehensive strategy to identify stoichiometric membrane protein interactomes.

There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies.