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1.
Sci Rep ; 14(1): 11595, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773164

RESUMO

Despite growing evidence implicating the calcium-activated chloride channel anoctamin1 (ANO1) in cancer metastasis, its direct impact on the metastatic potential of prostate cancer and the possible significance of epigenetic alteration in this process are not fully understood. Here, we show that ANO1 is minimally expressed in LNCap and DU145 prostate cancer cell lines with low metastatic potential but overexpressed in high metastatic PC3 prostate cancer cell line. The treatment of LNCap and DU145 cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) potentiates ANO1 expression, suggesting that DNA methylation is one of the mechanisms controlling ANO1 expression. Consistent with this notion, hypermethylation was detected at the CpG island of ANO1 promoter region in LNCap and DU145 cells, and 5-Aza-CdR treatment resulted in a drastic demethylation at promoter CpG methylation sites. Upon 5-Aza-CdR treatment, metastatic indexes, such as cell motility, invasion, and metastasis-related gene expression, were significantly altered in LNCap and DU145 cells. These 5-Aza-CdR-induced metastatic hallmarks were, however, almost completely ablated by stable knockdown of ANO1. These in vitro discoveries were further supported by our in vivo observation that ANO1 expression in xenograft mouse models enhances the metastatic dissemination of prostate cancer cells into tibial bone and the development of osteolytic lesions. Collectively, our results help elucidate the critical role of ANO1 expression in prostate cancer bone metastases, which is epigenetically modulated by promoter CpG methylation.


Assuntos
Anoctamina-1 , Neoplasias Ósseas , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias , Regiões Promotoras Genéticas , Neoplasias da Próstata , Masculino , Anoctamina-1/metabolismo , Anoctamina-1/genética , Humanos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Ósseas/secundário , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Camundongos , Ilhas de CpG , Decitabina/farmacologia , Movimento Celular/genética , Epigênese Genética , Azacitidina/farmacologia
2.
Am J Physiol Cell Physiol ; 326(4): C1237-C1247, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38581667

RESUMO

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3ß levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3ß signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.


Assuntos
Diabetes Mellitus , Insulinas , Animais , Camundongos , Anoctamina-1/metabolismo , Artérias/metabolismo , Diabetes Mellitus/metabolismo , Músculo Liso Vascular/metabolismo , Receptor de Insulina/metabolismo
3.
J Cell Mol Med ; 28(9): e18320, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38685684

RESUMO

Liver diseases include all types of viral hepatitis, alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), cirrhosis, liver failure (LF) and hepatocellular carcinoma (HCC). Liver disease is now one of the leading causes of disease and death worldwide, which compels us to better understand the mechanisms involved in the development of liver diseases. Anoctamin 1 (ANO1), a calcium-activated chloride channel (CaCC), plays an important role in epithelial cell secretion, proliferation and migration. ANO1 plays a key role in transcriptional regulation as well as in many signalling pathways. It is involved in the genesis, development, progression and/or metastasis of several tumours and other diseases including liver diseases. This paper reviews the role and molecular mechanisms of ANO1 in the development of various liver diseases, aiming to provide a reference for further research on the role of ANO1 in liver diseases and to contribute to the improvement of therapeutic strategies for liver diseases by regulating ANO1.


Assuntos
Anoctamina-1 , Hepatopatias , Humanos , Anoctamina-1/metabolismo , Anoctamina-1/genética , Hepatopatias/metabolismo , Hepatopatias/patologia , Hepatopatias/genética , Animais , Transdução de Sinais , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Regulação da Expressão Gênica
4.
Cell Calcium ; 120: 102885, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38642428

RESUMO

When activated by increase in intracellular Ca2+, anoctamins (TMEM16 proteins) operate as phospholipid scramblases and as ion channels. Anoctamin 1 (ANO1) is the Ca2+-activated epithelial anion-selective channel that is coexpressed together with the abundant scramblase ANO6 and additional intracellular anoctamins. In salivary and pancreatic glands, ANO1 is tightly packed in the apical membrane and secretes Cl-. Epithelia of airways and gut use cystic fibrosis transmembrane conductance regulator (CFTR) as an apical Cl- exit pathway while ANO1 supports Cl- secretion mainly by facilitating activation of luminal CFTR and basolateral K+ channels. Under healthy conditions ANO1 modulates intracellular Ca2+ signals by tethering the endoplasmic reticulum, and except of glands its direct secretory contribution as Cl- channel might be small, compared to CFTR. In the kidneys ANO1 supports proximal tubular acid secretion and protein reabsorption and probably helps to excrete HCO3-in the collecting duct epithelium. However, under pathological conditions as in polycystic kidney disease, ANO1 is strongly upregulated and may cause enhanced proliferation and cyst growth. Under pathological condition, ANO1 and ANO6 are upregulated and operate as secretory channel/phospholipid scramblases, partly by supporting Ca2+-dependent processes. Much less is known about the role of other epithelial anoctamins whose potential functions are discussed in this review.


Assuntos
Anoctaminas , Humanos , Animais , Anoctaminas/metabolismo , Cálcio/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Anoctamina-1/metabolismo
5.
Cell Calcium ; 120: 102889, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38677213

RESUMO

Chemical senses allow animals to detect and discriminate a vast array of molecules. The olfactory system is responsible of the detection of small volatile molecules, while water dissolved molecules are detected by taste buds in the oral cavity. Moreover, many animals respond to signaling molecules such as pheromones and other semiochemicals through the vomeronasal organ. The peripheral organs dedicated to chemical detection convert chemical signals into perceivable information through the employment of diverse receptor types and the activation of multiple ion channels. Two ion channels, TMEM16B, also known as anoctamin2 (ANO2) and TMEM16A, or anoctamin1 (ANO1), encoding for Ca2+-activated Cl¯ channels, have been recently described playing critical roles in various cell types. This review aims to discuss the main properties of TMEM16A and TMEM16B-mediated currents and their physiological roles in chemical senses. In olfactory sensory neurons, TMEM16B contributes to amplify the odorant response, to modulate firing, response kinetics and adaptation. TMEM16A and TMEM16B shape the pattern of action potentials in vomeronasal sensory neurons increasing the interspike interval. In type I taste bud cells, TMEM16A is activated during paracrine signaling mediated by ATP. This review aims to shed light on the regulation of diverse signaling mechanisms and neuronal excitability mediated by Ca-activated Cl¯ channels, hinting at potential new roles for TMEM16A and TMEM16B in the chemical senses.


Assuntos
Anoctaminas , Animais , Humanos , Anoctaminas/metabolismo , Anoctamina-1/metabolismo
6.
Discov Med ; 36(183): 753-764, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38665024

RESUMO

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.


Assuntos
Cálcio , Suplementos Nutricionais , Fluorose Dentária , Animais , Masculino , Camundongos , Fator 6 Ativador da Transcrição/metabolismo , Adenina/análogos & derivados , Ameloblastos/metabolismo , Ameloblastos/patologia , Ameloblastos/efeitos dos fármacos , Anoctamina-1/metabolismo , Anoctamina-1/antagonistas & inibidores , Anoctamina-1/genética , Cálcio/metabolismo , Modelos Animais de Doenças , eIF-2 Quinase/metabolismo , eIF-2 Quinase/genética , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Fluoretos/toxicidade , Fluoretos/efeitos adversos , Fluorose Dentária/patologia , Fluorose Dentária/metabolismo , Fluorose Dentária/etiologia , Indóis , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores
7.
Brain Res ; 1834: 148915, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38582414

RESUMO

Bestrophin-1 and anoctamin-1 are members of the calcium-activated chloride channels (CaCCs) family and are involved in inflammatory and neuropathic pain. However, their role in pain hypersensitivity induced by REM sleep deprivation (REMSD) has not been studied. This study aimed to determine if anoctamin-1 and bestrophin-1 are involved in the pain hypersensitivity induced by REMSD. We used the multiple-platform method to induce REMSD. REM sleep deprivation for 48 h induced tactile allodynia and a transient increase in corticosterone concentration at the beginning of the protocol (12 h) in female and male rats. REMSD enhanced c-Fos and α2δ-1 protein expression but did not change activating transcription factor 3 (ATF3) and KCC2 expression in dorsal root ganglia and dorsal spinal cord. Intrathecal injection of CaCCinh-A01, a non-selective bestrophin-1 blocker, and T16Ainh-A01, a specific anoctamin-1 blocker, reverted REMSD-induced tactile allodynia. However, T16Ainh-A01 had a higher antiallodynic effect in male than female rats. In addition, REMSD increased bestrophin-1 protein expression in DRG but not in DSC in male and female rats. In marked contrast, REMSD decreased anoctamin-1 protein expression in DSC but not in DRG, only in female rats. Bestrophin-1 and anoctamin-1 promote pain and maintain tactile allodynia induced by REM sleep deprivation in both male and female rats, but their expression patterns differ between the sexes.


Assuntos
Bestrofinas , Gânglios Espinais , Hiperalgesia , Privação do Sono , Medula Espinal , Animais , Privação do Sono/metabolismo , Privação do Sono/complicações , Hiperalgesia/metabolismo , Masculino , Feminino , Ratos , Gânglios Espinais/metabolismo , Medula Espinal/metabolismo , Bestrofinas/metabolismo , Canais de Cloreto/metabolismo , Sono REM/fisiologia , Ratos Wistar , Anoctamina-1 , Canais de Cálcio Tipo L
8.
Molecules ; 29(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474548

RESUMO

Transient receptor potential vanilloid 4 (TRPV4) is a widely expressed cation channel that plays an important role in many physiological and pathological processes. However, most TRPV4 drugs carry a risk of side effects. Moreover, existing screening methods are not suitable for the high-throughput screening (HTS) of drugs. In this study, a cell model and HTS method for targeting TRPV4 channel drugs were established based on a calcium-activated chloride channel protein 1 Anoctamin 1 (ANO1) and a double mutant (YFP-H148Q/I152L) of the yellow fluorescent protein (YFP). Patch-clamp experiments and fluorescence quenching kinetic experiments were used to verify that the model could sensitively detect changes in intracellular Ca2+ concentration. The functionality of the TRPV4 cell model was examined through temperature variations and different concentrations of TRPV4 modulators, and the performance of the model in HTS was also evaluated. The model was able to sensitively detect changes in the intracellular Ca2+ concentration and also excelled at screening TRPV4 drugs, and the model was more suitable for HTS. We successfully constructed a drug cell screening model targeting the TRPV4 channel, which provides a tool to study the pathophysiological functions of TRPV4 in vitro.


Assuntos
Ensaios de Triagem em Larga Escala , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Anoctamina-1 , Cálcio/metabolismo
9.
Front Immunol ; 15: 1341209, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38352864

RESUMO

Background: Aminooctylamine (ANO1) plays an oncogenic role in various cancers. However. its role in pancreatic cancer (PC) has rarely been studied. This study investigated the prognostic value of ANO1 and its correlation with the tumor microenvironment (TME) in PC. Methods: Consecutive patients with PC (n = 119) were enrolled. The expression of ANO1 in cancer cells, the expression of fibroblast activation protein (FAP) and alpha smooth muscle actin in cancer-associated fibroblasts (CAFs), and the numbers of CD8- and FOXP3-positive tumor-infiltrating lymphocytes (TILs) were evaluated using immunohistochemistry. The prognostic value of ANO1 and its correlation with CAF subgroups and TILs were analyzed. The possible mechanism of ANO1 in the TME of PC was predicted using the the Cancer Genome Atlas (TCGA) dataset. Results: The expression of AN01 was correlated with overall survival (OS) and disease-free survival. Multi-factor analysis showed that high ANO1 expression was an independent adverse prognostic factor for OS (hazard ratio, 4.137; P = 0.001). ANO1 expression was positively correlated with the expression of FAP in CAFs (P < 0.001) and negatively correlated with the number of CD8-positive TILs (P = 0.005), which was also validated by bioinformatics analysis in the TCGA dataset. Moreover, bioinformatic analysis of the TCGA dataset revealed that ANO1 may induce an immunosuppressive tumor microenvironment in pancreatic cancer in a paracrine manner. Conclusion: ANO1 is a prognostic factor in patients with PC after radical resection. ANO1 may induce an immunosuppressive tumor microenvironment in PC in a paracrine manner, suggesting that ANO1 may be a novel therapeutic target.


Assuntos
Neoplasias Pancreáticas , Microambiente Tumoral , Humanos , Prognóstico , Neoplasias Pancreáticas/patologia , Linfócitos do Interstício Tumoral/metabolismo , Modelos de Riscos Proporcionais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
10.
BMC Cancer ; 24(1): 233, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38373988

RESUMO

Head and neck squamous cell carcinoma (HNSCC) constitutes one of the most common types of human cancers and often metastasizes to lymph nodes. Platinum-based chemotherapeutic drugs are commonly used for treatment of a wide range of cancers, including HNSCC. Its mode of action relies on its ability to impede DNA repair mechanisms, inducing apoptosis in cancer cells. However, due to acquired resistance and toxic side-effects, researchers have been focusing on developing novel combinational therapeutic strategies to overcome cisplatin resistance. In the current study, we identified p90RSK, an ERK1/2 downstream target, as a key mediator and a targetable signaling node against cisplatin resistance. Our results strongly support the role of p90RSK in cisplatin resistance and identify the combination of p90RSK inhibitor, BI-D1870, with cisplatin as a novel therapeutic strategy to overcome cisplatin resistance. In addition, we have identified TMEM16A expression as a potential upstream regulator of p90RSK through the ERK pathway and a biomarker of response to p90RSK targeted therapy in the context of cisplatin resistance.


Assuntos
Antineoplásicos , Neoplasias de Cabeça e Pescoço , Proteínas Quinases S6 Ribossômicas 90-kDa , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Anoctamina-1/genética , Anoctamina-1/metabolismo
11.
Commun Biol ; 7(1): 151, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38317010

RESUMO

Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl- channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence.


Assuntos
Canal Anal , Anoctamina-1 , Contração Muscular , Animais , Camundongos , Canal Anal/inervação , Canal Anal/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Miócitos de Músculo Liso , Anoctamina-1/fisiologia
12.
J Am Chem Soc ; 146(7): 4665-4679, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38319142

RESUMO

The dysfunction and defects of ion channels are associated with many human diseases, especially for loss-of-function mutations in ion channels such as cystic fibrosis transmembrane conductance regulator mutations in cystic fibrosis. Understanding ion channels is of great current importance for both medical and fundamental purposes. Such an understanding should include the ability to predict mutational effects and describe functional and mechanistic effects. In this work, we introduce an approach to predict mutational effects based on kinetic information (including reaction barriers and transition state locations) obtained by studying the working mechanism of target proteins. Specifically, we take the Ca2+-activated chloride channel TMEM16A as an example and utilize the computational biology model to predict the mutational effects of key residues. Encouragingly, we verified our predictions through electrophysiological experiments, demonstrating a 94% prediction accuracy regarding mutational directions. The mutational strength assessed by Pearson's correlation coefficient is -0.80 between our calculations and the experimental results. These findings suggest that the proposed methodology is reliable and can provide valuable guidance for revealing functional mechanisms and identifying key residues of the TMEM16A channel. The proposed approach can be extended to a broad scope of biophysical systems.


Assuntos
Canais de Cloreto , Cloretos , Humanos , Cloretos/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Mutação , Transdução de Sinais , Cálcio/metabolismo
13.
Biophys Chem ; 308: 107194, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38401241

RESUMO

The TMEM16/Anoctamin protein family (TMEM16x) is composed of members with different functions; some members form Ca2+-activated chloride channels, while others are lipid scramblases or combine the two functions. TMEM16x proteins are typically activated in response to agonist-induced rises of intracellular Ca2+; thus, they couple Ca2+-signalling with cell electrical activity or plasmalemmal lipid homeostasis. The structural domains underlying these functions are not fully defined. We used a Naïve Bayes classifier to gain insights into these domains. The method enabled identification of regions involved in either ion or lipid transport, and suggested domains for possible pharmacological exploitation. The method allowed the prediction of the transport property of any given TMEM16x. We envisage this strategy could be exploited to illuminate the structure-function relationship of any protein family composed of members playing different molecular roles.


Assuntos
Anoctaminas , Lipídeos , Anoctaminas/metabolismo , Teorema de Bayes , Anoctamina-1/metabolismo , Transporte de Íons , Cálcio/metabolismo
14.
Int J Mol Sci ; 25(4)2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38396901

RESUMO

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.


Assuntos
Anoctamina-1 , Canais de Cloreto , Células Musculares , Animais , Camundongos , Anoctamina-1/metabolismo , Anoctamina-1/fisiologia , Transporte Biológico , Cálcio/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Canais Iônicos/metabolismo , Mamíferos/metabolismo , Células Musculares/metabolismo
15.
Sci Rep ; 14(1): 246, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-38168913

RESUMO

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death worldwide. Cigarette smoke which has approximately 2-3 µg of Cadmium (Cd) per cigarette contributes to the environmental exposure and development and severity of COPD. With the lack of a cadmium elimination mechanism in humans, the contribution of cadmium induced stress to lung epithelial cells remains unclear. Studies on cadmium responsive miRNAs suggest regulation of target genes with an emphasis on the critical role of miRNA-mRNA interaction for cellular homeostasis. Mir-381, the target miRNA in this study is negatively regulated by cadmium in airway epithelial cells. miR-381 is reported to also regulate ANO1 (Anoctamin 1) expression negatively and in this study low dose cadmium exposure to airway epithelial cells was observed to upregulate ANO1 mRNA expression via mir-381 inhibition. ANO1 which is a Ca2+-activated chloride channel has multiple effects on cellular functions such as proliferation, mucus hypersecretion and fibroblast differentiation in inflamed airways in chronic respiratory diseases. In vitro studies with cadmium at a high concentration range of 100-500 µM is reported to activate chloride channel, ANO1. The secretory epithelial cells are regulated by chloride channels like CFTR, ANO1 and SLC26A9. We examined "ever" smokers with COPD (n = 13) lung tissue sections compared to "never" smoker without COPD (n = 9). We found that "ever" smokers with COPD had higher ANO1 expression. Using mir-381 mimic to inhibit ANO1, we demonstrate here that ANO1 expression is significantly (p < 0.001) downregulated in COPD derived airway epithelial cells exposed to cadmium. Exposure to environmental cadmium contributes significantly to ANO1 expression.


Assuntos
MicroRNAs , Doença Pulmonar Obstrutiva Crônica , Humanos , Cádmio/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Células Epiteliais/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/genética , Proteínas de Neoplasias/metabolismo , Transportadores de Sulfato/metabolismo , Antiporters/metabolismo
16.
Sci Rep ; 14(1): 1464, 2024 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233410

RESUMO

The Ca2+ activated Cl- channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.


Assuntos
Asma , Fibrose Cística , Humanos , Camundongos , Ratos , Animais , Niclosamida/farmacologia , Benzobromarona/farmacologia , Benzobromarona/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/uso terapêutico , Fibrose Cística/tratamento farmacológico , Anoctamina-1 , Muco , Intestinos
17.
Am J Physiol Lung Cell Mol Physiol ; 326(1): L111-L123, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-38084409

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.


Assuntos
Fibrose Pulmonar Idiopática , Miofibroblastos , Humanos , Anoctamina-1/metabolismo , Diferenciação Celular , Cloretos/metabolismo , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Miofibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fatores de Crescimento Transformadores/metabolismo , Fatores de Crescimento Transformadores/farmacologia
18.
Pflugers Arch ; 476(2): 211-227, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37979051

RESUMO

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.


Assuntos
Asma , COVID-19 , Camundongos , Animais , Anoctamina-1/metabolismo , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Anoctaminas/metabolismo , Pulmão/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Cálcio/metabolismo , Inflamação/tratamento farmacológico , Anti-Inflamatórios , Canais de Cloreto/metabolismo
20.
Sci Signal ; 16(811): eadk5661, 2023 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-37963193

RESUMO

The TMEM16A channel represents a key depolarizing mechanism in arterial smooth muscle and contractile pericytes, where it is activated by several endogenous contractile agonists. In this issue of Science Signaling, Mata-Daboin et al. demonstrate a previously unidentified role for TMEM16A in endothelial cells for acetylcholine-mediated vasorelaxation. Collectively, TMEM16A serves as a transducer of vasoactive stimuli to enable fine modulation of vessel tone.


Assuntos
Canais de Cloreto , Células Endoteliais , Canais de Cloreto/genética , Músculo Liso , Artérias , Ânions , Anoctamina-1/genética
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