Activation of Anoctamin-1 Limits Pulmonary Endothelial Cell Proliferation via p38–Mitogen-activated Protein Kinase–Dependent Apoptosis

Abstract

Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.

TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels

“…In our study, we investigated the mechanism of action of Eact, a putative activator of the TMEM16A chloride channel. Eact, which was initially discovered by its high efficacy in FRT cells expressing TMEM16A (Namkung et al 2011b), has been used in many studies to demonstrate the role of TMEM16A in transepithelial ion transport, smooth muscle contraction, endocrine function, mucus release and cell proliferation and migration (Namkung et al 2011b;Berglund et al 2014;Sun et al 2014;Allawzi et al 2018;Song et al 2018;Benedetto et al 2019). In contrast with Eact being a direct activator of TMEM16A, we found little effect of this compound in bronchial epithelial cells under conditions of high TMEM16A expression, i.e.…”

Transmembrane member 16A participates in hydrogen peroxide‐induced apoptosis by facilitating mitochondria‐dependent pathway in vascular smooth muscle cells

“…For example, the protein level and the activity of TMEM16A negatively correlate with VSMC proliferation (Wang et al ., ; Zhang et al ., ), but these correlations were found to be positive in epithelial cells (Ruffin et al ., ; Buchholz et al ., ; Cha et al ., ) and tumour cells (Britschgi et al ., ; Deng et al ., ). Unlike the role of TMEM16A in cell proliferation, which has been fully explored in various cell types, the effect of TMEM16A on cell apoptosis has only been reported for tumour cells (Britschgi et al ., ; Berglund et al ., ; Seo et al ., ; Deng et al ., ) and ECs (Allawzi et al ., ). In this study, we provided evidence that TMEM16A contributes to VSMC apoptosis, and the results were also opposite to those of tumour cells.…”

“…In this study, we used immunoblotting and immunogold staining to prove the presence of endogenous TMEM16A expression in mitochondria. This result is consistent with a previous study in endothelial cells (Allawzi et al ., ). Moreover, we explored the intrinsic mechanism of TMEM16A on mitochondrial function.…”

“…Our previous studies demonstrated that TMEM16A inhibits proliferation in basilar artery smooth muscle cells (BASMCs) and ameliorates hypertension‐induced cerebrovascular remodelling (Wang et al ., ). Recent findings revealed that TMEM16A promotes apoptosis of pulmonary endothelial cells (ECs) (Allawzi et al ., ). Therefore, we assumed that TMEM16A had a facilitatory effect on apoptosis in BASMCs, unlike its effect observed in cancer cells.…”

Recent advances in TMEM16A: Structure, function, and disease

“…It has been reported that TMEM16A is expressed in various smooth muscle cells, such as hypertensive rat basilar smooth muscle cells, pulmonary artery smooth muscle cells (Allawzi et al, ; Davis et al, ; M. Wang et al, ). Indeed, TMEM16A expression and activity were confirmed in smooth muscle cells from various arteries and veins (U. Oh & Jung, ).…”