Dataset Information

Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells.

ABSTRACT: Regulation of the Ca2+-activated Cl- channel TMEM16A by Ca2+/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca2+/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca2+/CAM-regulated K+ channel KCNN4, 1-EBIO, activated TMEM16A in overexpressing cells, but not in cells with endogenous expression of TMEM16A. Evidence is provided that CAM-interaction with TMEM16A modulates the Ca2+ sensitivity of the Cl- channel. Enhanced Ca2+ sensitivity of overexpressed TMEM16A explains its activity at basal (non-elevated) intracellular Ca2+ levels. The present results correspond well to a recent report that demonstrates a Ca2+-unbound form of CAM (apo-CAM) that is pre-associated with TMEM16A and mediates a Ca2+-dependent sensitization of activation (and inactivation). However, when using activators or inhibitors for holo-CAM, CAMKII, or calcineurin, we were unable to detect a significant impact of CAM, and limit evidence for regulation by CAM-dependent regulatory proteins on receptor-mediated activation of endogenous TMEM16A in airway or colonic epithelial cells. We propose that regulatory properties of TMEM16A and and other members of the TMEM16 family as detected in overexpression studies, should be validated for endogenous TMEM16A and physiological stimuli such as activation of phospholipase C (PLC)-coupled receptors.

SUBMITTER: Talbi K

PROVIDER: S-EPMC8471323 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells.

Talbi Khaoula K Ousingsawat Jiraporn J Centeio Raquel R Schreiber Rainer R Kunzelmann Karl K

Membranes 20210921 9

Regulation of the Ca2+-activated Cl- channel TMEM16A by Ca2+/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca2+/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca2+/CAM-regulated K+ channel K ...[more]

PMID: 34564540

Similar Datasets

Project description:BackgroundAirway inflammation is the core pathological process of asthma, with the key inflammatory regulators incompletely defined. Recently, fibroblast growth factor 2 (FGF2) has been reported to be an inflammatory regulator; however, its role in asthma remains elusive. This study aimed to investigate the immunomodulatory role of FGF2 in asthma.MethodsFirst, FGF2 expression was characterised in clinical asthma samples and the house dust mite (HDM)-induced mouse chronic asthma model. Second, recombinant mouse FGF2 (rm-FGF2) protein was intranasally delivered to determine the effect of FGF2 on airway inflammatory cell infiltration. Third, human airway epithelium-derived A549 cells were stimulated with either HDM or recombinant human interleukin-1β (IL-1β) protein combined with or without recombinant human FGF2. IL-1β-induced IL-6 or IL-8 release levels were determined using enzyme-linked immunosorbent assay, and the involved signalling transduction was explored via Western blotting.ResultsCompared with the control groups, the FGF2 protein levels were significantly upregulated in the bronchial epithelium and alveolar areas of clinical asthma samples (6.70 ± 1.79 vs. 16.32 ± 2.40, P = 0.0184; 11.20 ± 2.11 vs. 21.00 ± 3.00, P = 0.033, respectively) and HDM-induced asthmatic mouse lung lysates (1.00 ± 0.15 vs. 5.14 ± 0.42, P < 0.001). Moreover, FGF2 protein abundance was positively correlated with serum total and anti-HDM IgE levels in the HDM-induced chronic asthma model (R2 = 0.857 and 0.783, P = 0.0008 and 0.0043, respectively). Elevated FGF2 protein was mainly expressed in asthmatic bronchial epithelium and alveolar areas and partly co-localised with infiltrated inflammatory cell populations in HDM-induced asthmatic mice. More importantly, intranasal instillation of rm-FGF2 aggravated airway inflammatory cell infiltration (2.45 ± 0.09 vs. 2.88 ± 0.14, P = 0.0288) and recruited more subepithelial neutrophils after HDM challenge [(110.20 ± 29.43) cells/mm2 vs. (238.10 ± 42.77) cells/mm2, P = 0.0392] without affecting serum IgE levels and Th2 cytokine transcription. In A549 cells, FGF2 was upregulated through HDM stimulation and promoted IL-1β-induced IL-6 or IL-8 release levels (up to 1.41 ± 0.12- or 1.44 ± 0.14-fold change vs. IL-1β alone groups, P = 0.001 or 0.0344, respectively). The pro-inflammatory effect of FGF2 is likely mediated through the fibroblast growth factor receptor (FGFR)/mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) pathway.ConclusionOur findings suggest that FGF2 is a potential inflammatory modulator in asthma, which can be induced by HDM and acts through the FGFR/MAPK/NF-κB pathway in the airway epithelial cells.

Project description:Niclosamide and benzbromarone have been described as inhibitors of the calcium activated chloride channel, TMEM16A, and on this basis have been considered and tested as clinical candidates for the treatment of airway diseases. However, both compounds have previously demonstrated activity on a range of additional biological targets and it is unclear from the literature to what extent any activity on TMEM16A may contribute to efficacy in these models of airway disease. The aim of the present study was therefore to examine the pharmacology and selectivity of these clinical candidates together with a structurally unrelated TMEM16A blocker, Ani9, in a range of functional assays to better appreciate the putative role of TMEM16A in the regulation of both epithelial ion transport and the development of an airway epithelial mucus secretory phenoptype. Benzbromarone and Ani9 both attenuated recombinant TMEM16A activity in patch clamp studies, whereas in contrast, niclosamide induced a paradoxical potentiation of the TMEM16A-mediated current. Niclosamide and benzbromarone were also demonstrated to attenuate receptor-dependent increases in intracellular Ca2+ levels ([Ca2+]i) which likely contributed to their concomitant attenuation of the Ca2+-stimulated short-circuit current responses of FRT-TMEM16A and primary human bronchial epithelial (HBE) cells. In contrast, Ani9 attenuated the Ca2+-stimulated short-circuit current responses of both cell systems without influencing [Ca2+]i which supports a true channel blocking mechanism for this compound. Additional studies using HBE cells revealed effects of both niclosamide and benzbromarone on global ion transport processes (absorptive and secretory) as well as signs of toxicity (elevated LDH levels, loss of transepithelial resistance) that were not shared by Ani9. Ani9 also failed to influence the IL-13 induced differentiation of HBE towards a goblet cell rich, mucus hypersecreting epithelium, whereas niclosamide and benzbromarone attenuated numbers of both goblet and multiciliated cells, that would be consistent with cellular toxicity. Together these data challenge the description of niclosamide as a TMEM16A blocker and illustrate a range of off-target effects of both niclosamide and benzbromarone which may contribute to the reported activity in models of airway function.

Dataset Information

Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells.

Publications

Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets