UNASSIGNED: Paneth cells play a central role in intestinal innate immune response. These cells are localized at the base of small intestinal crypts of Lieberkuhn. The calcium-activated chloride channel TMEM16A and the phospholipid scramblase TMEM16F control intracellular Ca2+ signaling and exocytosis. We analyzed the role of TMEM16A and TMEM16F for Paneth cells secretion.
UNASSIGNED: Mice with intestinal epithelial knockout of Tmem16a (Tmem16a-/-) and Tmem16f (Tmem16f-/-) were generated. Tissue structures and Paneth cells were analyzed, and Paneth cell exocytosis was examined in small intestinal organoids in vitro. Intracellular Ca2+ signals were measured and were compared between wild-type and Tmem16 knockout mice. Bacterial colonization and intestinal apoptosis were analyzed.
UNASSIGNED: Paneth cells in the crypts of Lieberkuhn from Tmem16a-/- and Tmem16f-/- mice demonstrated accumulation of lysozyme. Tmem16a and Tmem16f were localized in wild-type Paneth cells but were absent in cells from knockout animals. Paneth cell number and size were enhanced in the crypt base and mucus accumulated in intestinal goblet cells of knockout animals. Granule fusion and exocytosis on cholinergic and purinergic stimulation were examined online. Both were strongly compromised in the absence of Tmem16a or Tmem16f and were also blocked by inhibition of Tmem16a/f. Purinergic Ca2+ signaling was largely inhibited in Tmem16a knockout mice. Jejunal bacterial content was enhanced in knockout mice, whereas cellular apoptosis was inhibited.
UNASSIGNED: The present data demonstrate the role of Tmem16 for exocytosis in Paneth cells. Inhibition or activation of Tmem16a/f is likely to affect microbial content and immune functions present in the small intestine.

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.

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.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

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.

Enhancer-gene communication is dependent on topologically associating domains (TADs) and boundaries enforced by the CCCTC-binding factor (CTCF) insulator, but the underlying structures and mechanisms remain controversial. Here, we investigate a boundary that typically insulates fibroblast growth factor (FGF) oncogenes but is disrupted by DNA hypermethylation in gastrointestinal stromal tumors (GISTs). The boundary contains an array of CTCF sites that enforce adjacent TADs, one containing FGF genes and the other containing ANO1 and its putative enhancers, which are specifically active in GIST and its likely cell of origin. We show that coordinate disruption of four CTCF motifs in the boundary fuses the adjacent TADs, allows the ANO1 enhancer to contact FGF3, and causes its robust induction. High-resolution micro-C maps reveal specific contact between transcription initiation sites in the ANO1 enhancer and FGF3 promoter that quantitatively scales with FGF3 induction such that modest changes in contact frequency result in strong changes in expression, consistent with a causal relationship.

Oviductal smooth muscle exhibits spontaneous rhythmic contraction (SRC) and controls the passage of the ova at the exact time, but its mechanistic regulation remains to be determined. In this study, female mice with Ano1SMKO (smooth muscle-specific deletion of Ano1) had reduced fertility. Deficiency of Ano1 in mice resulted in impaired oviductal SRC function and reduced calcium signaling in individual smooth muscle cells in the oviduct. The Ano1 antagonist T16Ainh-A01 dose-dependently inhibited SRCs and [Ca2+]i in the oviducts of humans and mice. A similar inhibitory effect of SRCs and [Ca2+]i was observed after treatment with nifedipine. In our study, ANO1 acted primarily as an activator or amplifier in [Ca2+]i and contraction of tubal smooth muscle cells. We found that tubal SRC was markedly attenuated in patients with ectopic pregnancy. Then, our study was designed to determine whether chloride channel Ano1-mediated smooth muscle motility is associated with tubal SRC. Our findings reveal a new mechanism for the regulation of tubal motility that may be associated with abnormal pregnancies such as ectopic pregnancies.

The aim of this study was to explore the function and mechanism of circular RNA (circRNA) matrix metallopeptidase 1 (circMMP1) in the progression of esophageal squamous cell carcinoma (ESCC).
CircMMP1 expression was detected by quantitative real-time PCR (qRT-PCR), and its relationship with the prognosis of ESCC patients was evaluated by Kaplan-Meier analysis. Cells were transfected using corresponding plasmids, and the cell proliferation activity, migration and invasion capabilities in vitro were assessed. The protein level in tissues and cells was analyzed using western blotting. RNA pulldown, dual-luciferase reporter assay and RNA immunoprecipitation assay were performed in ESCC cells to detect the interaction between circMMP1 and miR-671-5p, or the correlation between miR-671-5p and ANO1. Xenograft tumor experiment was carried out to uncover the function of circMMP1 in vivo.
The high level of circMMP1 in tumor tissues was associated with poor prognoses of ESCC patients. Knockdown of circMMP1 suppressed ESCC cell proliferation, migration and invasion in vitro. MiR-671-5p was the target of circMMP1 and mediated the inhibition effect of circMMP1 on ESCC cells. CircMMP1 targeted miR-671-5p to regulate ANO1 expression, which was downstream of miR-671-5p. Overexpression of ANO1 weakened tumor-repressive function of circMMP1 knockdown in ESCC cells. Moreover, silencing of circMMP1 impeded ESCC tumor growth in vivo.
Our study provided novel evidence that circMMP1 accelerated ESCC progression by acting as a miR-671-5p sponge to enhance ANO1 expression.

The TMEM16A calcium-activated chloride channel is a promising therapeutic target for various diseases. Niclosamide, an anthelmintic medication, has been considered as a TMEM16A inhibitor for treating asthma and chronic obstructive pulmonary disease, but was recently found to possess broad-spectrum off-target effects. Here we show that, under physiological conditions, niclosamide acutely potentiates TMEM16A without having any inhibitory effect. Our computational and functional characterizations pinpoint a putative niclosamide binding site on the extracellular side of TMEM16A. Mutations in this site attenuate the potentiation. Moreover, niclosamide potentiates endogenous TMEM16A in vascular smooth muscle cells, triggers intracellular calcium increase, and constricts the murine mesenteric artery. Our findings advise caution when considering niclosamide as a TMEM16A inhibitor to treat diseases such as asthma, COPD, and hypertension. The identification of the putative niclosamide binding site provides insights into the mechanism of TMEM16A pharmacological modulation, shining light on developing specific TMEM16A modulators to treat human diseases.

The application of immunotherapy in gastrointestinal (GI) cancers remains challenging because of the limited response rate and emerging therapeutic resistance. Combining clinical cohorts, multi-omics study, and functional/molecular experiments, it is found that ANO1 amplification or high-expression predicts poor outcomes and resistance to immunotherapy for GI cancer patients. Knocking-down or inhibiting ANO1 suppresses the growth/metastasis/invasion of multiple GI cancer cell lines, cell-derived xenograft, and patient-derived xenograft models. ANO1 contributes to an immune-suppressive tumor microenvironment and induces acquired resistance to anti-PD-1 immunotherapy, while ANO1 knockdown or inhibition enhances immunotherapeutic effectiveness and overcomes resistance to immunotherapy. Mechanistically, through inhibiting cancer ferroptosis in a PI3K-Akt signaling-dependent manner, ANO1 enhances tumor progression and facilitates cancer-associated fibroblast recruitment by promoting TGF-β release, thus crippling CD8+ T cell-mediated anti-tumor immunity and generating resistance to immunotherapy. This work highlights ANO1\'s role in mediating tumor immune microenvironment remodeling and immunotherapeutic resistance, and introduces ANO1 as a promising target for GI cancers\' precision treatment.