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TMEM16A knockdown abrogates two different Ca(2+)-activated Cl (-) currents and contractility of smooth muscle in rat mesenteric small arteries.

ABSTRACT: The presence of Ca(2+)-activated Cl(-) channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca(2+)-activated Cl(-) currents (I Cl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent I Cl(Ca) depends on bestrophin expression, while the "classical" I Cl(Ca) is not. Downregulation of bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca(2+)]i, and SMC membrane potential were measured in isolated arterial segments. I Cl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent I Cl(Ca) and the "classical" I Cl(Ca) in SMCs. TMEM16A downregulation also reduced expression of bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K(+)-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca(2+)]i. Surprisingly, K(+)-induced depolarization was unchanged but Ca(2+) entry was reduced. We suggested that this is due to reduced expression of the L-type Ca(2+) channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs I Cl(Ca) and vascular function and suggests an interaction between TMEM16A and L-type Ca(2+) channels.

SUBMITTER: Dam VS

PROVIDER: S-EPMC4062836 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTransmembrane protein 16A (TMEM16A) Ca2+-activated Cl- channels have diverse physiological functions, such as epithelial secretion of Cl- and fluid and sensation of pain. Recent studies have demonstrated that TMEM16A contributes to the pathogenesis of infectious and non-infectious inflammatory diseases. However, the role of TMEM16A in inflammation has not been clearly elucidated.Aim of reviewIn this review, we aimed to provide comprehensive information regarding the roles of TMEM16A in inflammation by summarizing the mechanisms underlying TMEM16A expression and activation under inflammatory conditions, in addition to exploring the diverse inflammatory signaling pathways activated by TMEM16A. This review attempts to develop the idea that TMEM16A plays a diverse role in inflammatory processes and contributes to inflammatory diseases in a cellular environment-dependent manner.Key scientific concepts of reviewMultiple inflammatory mediators, including cytokines (e.g., interleukin (IL)-4, IL-13, IL-6), histamine, bradykinin, and ATP/UTP, as well as bacterial and viral infections, promote TMEM16A expression and/or activity under inflammatory conditions. In addition, TMEM16A activates diverse inflammatory signaling pathways, including the IP3R-mediated Ca2+ signaling pathway, the NF-κB signaling pathway, and the ERK signaling pathway, and contributes to the pathogenesis of many inflammatory diseases. These diseases include airway inflammatory diseases, lipopolysaccharide-induced intestinal epithelial barrier dysfunction, acute pancreatitis, and steatohepatitis. TMEM16A also plays multiple roles in inflammatory processes by increasing vascular permeability and leukocyte adhesion, promoting inflammatory cytokine release, and sensing inflammation-induced pain. Furthermore, TMEM16A plays its diverse pathological roles in different inflammatory diseases depending on the disease severity, proliferating status of the cells, and its interacting partners. We herein propose cellular environment-dependent mechanisms that explain the diverse roles of TMEM16A in inflammation.

Project description:IntroductionTransmembrane protein 16A (TMEM16A) is a Ca2+-activated chloride channel that plays a role in cancer cell proliferation, migration, invasion, and metastasis. However, whether TMEM16A contributes to breast cancer metastasis remains unknown.ObjectiveIn this study, we investigated whether TMEM16A channel activation by ROCK1/moesin promotes breast cancer metastasis.MethodsWound healing assays and transwell migration and invasion assays were performed to study the migration and invasion of MCF-7 and T47D breast cancer cells. Western blotting was performed to evaluate the protein expression, and whole-cell patch clamp recordings were used to record TMEM16A Cl- currents. A mouse model of breast cancer lung metastasis was generated by injecting MCF-7 cells via the tail vein. Metastatic nodules in the lung were assessed by hematoxylin and eosin staining. Lymph node metastasis, overall survival, and metastasis-free survival of breast cancer patients were assessed using immunohistochemistry and The Cancer Genome Atlas dataset.ResultsTMEM16A activation promoted breast cancer cell migration and invasion in vitro as well as breast cancer metastasis in mice. Patients with breast cancer who had higher TMEM16A levels showed greater lymph node metastasis and shorter survival. Mechanistically, TMEM16A promoted migration and invasion by activating EGFR/STAT3/ROCK1 signaling, and the role of the TMEM16A channel activity was important in this respect. ROCK1 activation by RhoA enhanced the TMEM16A channel activity via the phosphorylation of moesin at T558. The cooperative action of TMEM16A and ROCK1 was supported through clinical findings indicating that breast cancer patients with high levels of TMEM16A/ROCK1 expression showed greater lymph node metastasis and poor survival.ConclusionOur findings revealed a novel mechanism underlying TMEM16A-mediated breast cancer metastasis, in which ROCK1 increased TMEM16A channel activity via moesin phosphorylation and the increase in TMEM16A channel activities promoted cell migration and invasion. TMEM16A inhibition may be a novel strategy for treating breast cancer metastasis.

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TMEM16A knockdown abrogates two different Ca(2+)-activated Cl (-) currents and contractility of smooth muscle in rat mesenteric small arteries.

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