CLIC4 Function in the Epithelial-Mesenchymal Transition of Epithelial Odontogenic Lesions.

BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.

The translocation of a chloride channel from the Golgi to the plasma membrane helps plants adapt to salt stress.

A key mechanism employed by plants to adapt to salinity stress involves maintaining ion homeostasis via the actions of ion transporters. While the function of cation transporters in maintaining ion homeostasis in plants has been extensively studied, little is known about the roles of their anion counterparts in this process. Here, we describe a mechanism of salt adaptation in plants. We characterized the chloride channel (CLC) gene AtCLCf, whose expression is regulated by WRKY transcription factor under salt stress in Arabidopsis thaliana. Loss-of-function atclcf seedlings show increased sensitivity to salt, whereas AtCLCf overexpression confers enhanced resistance to salt stress. Salt stress induces the translocation of GFP-AtCLCf fusion protein to the plasma membrane (PM). Blocking AtCLCf translocation using the exocytosis inhibitor brefeldin-A or mutating the small GTPase gene AtRABA1b/BEX5 (RAS GENES FROM RAT BRAINA1b homolog) increases salt sensitivity in plants. Electrophysiology and liposome-based assays confirm the Cl-/H+ antiport function of AtCLCf. Therefore, we have uncovered a mechanism of plant adaptation to salt stress involving the NaCl-induced translocation of AtCLCf to the PM, thus facilitating Cl- removal at the roots, and increasing the plant's salinity tolerance.

CLCA1 exacerbates lung inflammation via p38 MAPK pathway in acute respiratory distress syndrome.

Recent research has revealed that airway epithelial calcium-activated chloride channel-1 (CLCA1) is implicated in the inflammation of multiple human respiratory diseases, but the specific role in acute respiratory distress syndrome (ARDS) remains unknown. To investigate the role of CLCA1 in ARDS, 80 participants, including 26 ARDS patients, 26 patients with community-acquired pneumonia (CAP) and 28 control subjects, were enrolled in this study. As the result shows, the level of CLCA1 was significantly increased in ARDS patients and positively correlated with neutrophil infiltration and the poor prognosis of ARDS. Then, the level of CLCA1 also elevated in the LPS-induced ARDS mouse model, and the administration of CLCA1 significantly regulated the phenotypes of ARDS in mice, such as lung injury score, BALF protein concentration, neutrophils infiltration and the secretions of inflammatory factors. Furthermore, administration of CLCA1 substantially altered the phosphorylation of p38 in the ARDS mouse model, whereas repressing the expression of CLCA1 or inhibiting the activation of p38 both alleviated the inflammatory response of ARDS. In summary, CLCA1 was notably correlated with ARDS and exacerbated the ARDS phenotypes through the p38 MAPK pathway.

Acid-Sensitive Outwardly Rectifying Cl- Current in OV2944 Mouse Ovarian Cancer Cells.

BACKGROUND/AIMS: Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. METHODS: The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. RESULTS: OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3) but was slightly restored upon PS administration. CONCLUSION: These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Transport of CLCA2 to the nucleus by extracellular vesicles controls keratinocyte survival and migration.

Chloride channel accessory 2 (CLCA2) is a transmembrane protein, which promotes adhesion of keratinocytes and their survival in response to hyperosmotic stress. Here we show that CLCA2 is transported to the nucleus of keratinocytes via extracellular vesicles. The nuclear localization is functionally relevant, since wild-type CLCA2, but not a mutant lacking the nuclear localization signal, suppressed migration of keratinocytes and protected them from hyperosmotic stress-induced cell death. In the nucleus, CLCA2 bound to and activated ß-catenin, resulting in enhanced expression of Wnt target genes. Mass-spectrometry-based interaction screening and functional rescue studies identified RNA binding protein 3 as a key effector of nuclear CLCA2. This is of likely relevance in vivo because both proteins co-localize in the human epidermis. Together, these results identify an unexpected nuclear function of CLCA2 in keratinocytes under homeostatic and stress conditions and suggest a role of extracellular vesicles and their nuclear transport in the control of key cellular activities.

Renal antiporter ClC-5 regulates collagen I/IV through the ß-catenin pathway and lysosomal degradation.

Mutations in Cl-/H+ antiporter ClC-5 cause Dent's disease type 1 (DD1), a rare tubulopathy that progresses to renal fibrosis and kidney failure. Here, we have used DD1 human cellular models and renal tissue from DD1 mice to unravel the role of ClC-5 in renal fibrosis. Our results in cell systems have shown that ClC-5 deletion causes an increase in collagen I (Col I) and IV (Col IV) intracellular levels by promoting their transcription through the ß-catenin pathway and impairing their lysosomal-mediated degradation. Increased production of Col I/IV in ClC-5-depleted cells ends up in higher release to the extracellular medium, which may lead to renal fibrosis. Furthermore, our data have revealed that 3-mo-old mice lacking ClC-5 (Clcn5 +/- and Clcn5 -/- ) present higher renal collagen deposition and fibrosis than WT mice. Altogether, we describe a new regulatory mechanism for collagens' production and release by ClC-5, which is altered in DD1 and provides a better understanding of disease progression to renal fibrosis.

Chloride ions in health and disease.

Chloride is a key anion involved in cellular physiology by regulating its homeostasis and rheostatic processes. Changes in cellular Cl- concentration result in differential regulation of cellular functions such as transcription and translation, post-translation modifications, cell cycle and proliferation, cell volume, and pH levels. In intracellular compartments, Cl- modulates the function of lysosomes, mitochondria, endosomes, phagosomes, the nucleus, and the endoplasmic reticulum. In extracellular fluid (ECF), Cl- is present in blood/plasma and interstitial fluid compartments. A reduction in Cl- levels in ECF can result in cell volume contraction. Cl- is the key physiological anion and is a principal compensatory ion for the movement of the major cations such as Na+, K+, and Ca2+. Over the past 25 years, we have increased our understanding of cellular signaling mediated by Cl-, which has helped in understanding the molecular and metabolic changes observed in pathologies with altered Cl- levels. Here, we review the concentration of Cl- in various organs and cellular compartments, ion channels responsible for its transportation, and recent information on its physiological roles.

Spinal bestrophin-1 and anoctamin-1 channels have a pronociceptive role in the tactile allodynia induced by REM sleep deprivation in rats.

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.

Genotype-phenotype correlation in CLCN4-related developmental and epileptic encephalopathy.

CLCN4-related disorder is a rare X-linked neurodevelopmental condition with a pathogenic mechanism yet to be elucidated. CLCN4 encodes the vesicular 2Cl-/H+ exchanger ClC-4, and CLCN4 pathogenic variants frequently result in altered ClC-4 transport activity. The precise cellular and molecular function of ClC-4 remains unknown; however, together with ClC-3, ClC-4 is thought to have a role in the ion homeostasis of endosomes and intracellular trafficking. We reviewed our research database for patients with CLCN4 variants and epilepsy, and performed thorough phenotyping. We examined the functional properties of the variants in mammalian cells using patch-clamp electrophysiology, protein biochemistry, and confocal fluorescence microscopy. Three male patients with developmental and epileptic encephalopathy were identified, with differing phenotypes. Patients #1 and #2 had normal growth parameters and normal-appearing brains on MRI, while patient #3 had microcephaly, microsomia, complete agenesis of the corpus callosum and cerebellar and brainstem hypoplasia. The p.(Gly342Arg) variant of patient #1 significantly impaired ClC-4's heterodimerization capability with ClC-3 and suppressed anion currents. The p.(Ile549Leu) variant of patient #2 and p.(Asp89Asn) variant of patient #3 both shift the voltage dependency of transport activation by 20 mV to more hyperpolarizing potentials, relative to the wild-type, with p.(Asp89Asn) favouring higher transport activity. We concluded that p.(Gly342Arg) carried by patient #1 and the p.(Ile549Leu) expressed by patient #2 impair ClC-4 transport function, while the p.(Asp89Asn) variant results in a gain-of-transport function; all three variants result in epilepsy and global developmental impairment, but with differences in epilepsy presentation, growth parameters, and presence or absence of brain malformations.

Chloride intracellular channel (CLIC) proteins function as fusogens.

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

Fluoride Ion Binding and Translocation in the CLCF Fluoride/Proton Antiporter: Molecular Insights from Combined Quantum-Mechanical/Molecular-Mechanical Modeling.

CLCF fluoride/proton antiporters move fluoride ions out of bacterial cells, leading to fluoride resistance in these bacteria. However, many details about their operating mechanisms remain unclear. Here, we report a combined quantum-mechanical/molecular-mechanical (QM/MM) study of a CLCF homologue from Enterococci casseliflavus (Eca), in accord with the previously proposed windmill mechanism. Our multiscale modeling sheds light on two critical steps in the transport cycle: (i) the external gating residue E118 pushing a fluoride in the external binding site into the extracellular vestibule and (ii) an incoming fluoride reconquering the external binding site by forcing out E118. Both steps feature competitions for the external binding site between the negatively charged carboxylate of E118 and the fluoride. Remarkably, the displaced E118 by fluoride accepts a proton from the nearby R117, initiating the next transport cycle. We also demonstrate the importance of accurate quantum descriptions of fluoride solvation. Our results provide clues to the mysterious E318 residue near the central binding site, suggesting that the transport activities are unlikely to be disrupted by the glutamate interacting with a well-solvated fluoride at the central binding site. This differs significantly from the structurally similar CLC chloride/proton antiporters, where a fluoride trapped deep in the hydrophobic pore causes the transporter to be locked down. A free-energy barrier of 10-15 kcal/mol was estimated via umbrella sampling for a fluoride ion traveling through the pore to repopulate the external binding site.

TP63 truncating mutation causes increased cell apoptosis and premature ovarian insufficiency by enhanced transcriptional activation of CLCA2.

BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear. METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI. RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression. CONCLUSION: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Astrocytic chloride regulates brain function in health and disease.

Chloride ions (Cl-) play a pivotal role in synaptic inhibition in the central nervous system, primarily mediated through ionotropic mechanisms. A recent breakthrough emphathizes the significant influence of astrocytic intracellular chloride concentration ([Cl-]i) regulation, a field still in its early stages of exploration. Typically, the [Cl-]i in most animal cells is maintained at lower levels than the extracellular chloride [Cl-]o, a critical balance to prevent cell swelling due to osmotic pressure. Various Cl- transporters are expressed differently across cell types, fine-tuning the [Cl-]i, while Cl- gradients are utilised by several families of Cl- channels. Although the passive distribution of ions within cells is governed by basic biophysical principles, astrocytes actively expend energy to sustain [Cl-]i at much higher levels than those achieved passively, and much higher than neuronal [Cl-]i. Beyond the role in volume regulation, astrocytic [Cl-]i is dynamically linked to brain states and influences neuronal signalling in actively behaving animals. As a vital component of brain function, astrocytic [Cl-]i also plays a role in the development of disorders where inhibitory transmission is disrupted. This review synthesises the latest insights into astrocytic [Cl-]i, elucidating its role in modulating brain function and its implications in various pathophysiological conditions.

A Pharmacological Investigation of the TMEM16A Currents in Murine Skeletal Myogenic Precursor Cells.

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.

CLCA1 is Poorly Expressed in Stomach Adenocarcinoma and Correlates with Immune Infiltration / CLCA1 se Expresa Deficientemente en el Adenocarcinoma de Estómago y se Correlaciona con la Infiltración Inmune

SUMMARY: Calcium-activated chloride channel regulator 1 (CLCA1) is associated with cancer progression. The expression and immunologic function of CLCA1 in stomach adenocarcinoma (STAD) remain unclear. In this investigation, the expression of CLCA1 in STAD tissues and its involvement in the progression and immune response of STAD were examined using databases such as cBioPortal, TISIDB, and UALCAN. In order to validate the expression level of CLCA1 protein in gastric adenocarcinoma, thirty clinical tissue specimens were gathered for immunohistochemical staining. The findings indicated a downregulation of CLCA1 in STAD patients, which was correlated with race, age, cancer grade, Helicobacter pylori infection, and molecular subtype. Through the examination of survival analysis, it was identified that diminished levels of CLCA1 within gastric cancer cases were linked to decreased periods of post-progression survival (PPS), overall survival (OS), and first progression (FP) (P<0.05). The CLCA1 mutation rate was lower in STAD, but the survival rate was higher in the variant group. The correlation between the expression level of CLCA1 and the levels of immune infiltrating cells in STAD, as well as the immune activating molecules, immunosuppressive molecules, MHC molecules, chemokines, and their receptor molecules, was observed. Gene enrichment analysis revealed that CLCA1 may be involved in STAD progression through systemic lupus erythematosus (SLE), proteasome, cell cycle, pancreatic secretion, and PPAR signaling pathways. In summary, CLCA1 is anticipated to function as a prognostic marker for patients with STAD and is linked to the immunization of STAD.

El regulador 1 del canal de cloruro activado por calcio (CLCA1) está asociado con la progresión del cáncer. La expresión y la función inmunológica de CLCA1 en el adenocarcinoma de estómago (STAD) aún no están claras. En esta investigación, se examinó la expresión de CLCA1 en tejidos STAD y su participación en la progresión y respuesta inmune de STAD utilizando bases de datos como cBioPortal, TISIDB y UALCAN. Para validar el nivel de expresión de la proteína CLCA1 en el adenocarcinoma gástrico, se recolectaron treinta muestras de tejido clínico para tinción inmunohistoquímica. Los hallazgos indicaron una regulación negativa de CLCA1 en pacientes con STAD, que se correlacionó con la raza, la edad, el grado del cáncer, la infección por Helicobacter pylori y el subtipo molecular. Mediante el examen del análisis de supervivencia, se identificó que los niveles reducidos de CLCA1 en los casos de cáncer gástrico estaban relacionados con períodos reducidos de supervivencia posterior a la progresión (PPS), supervivencia general (OS) y primera progresión (FP) (P <0,05). La tasa de mutación CLCA1 fue menor en STAD, pero la tasa de supervivencia fue mayor en el grupo variante. Se observó la correlación entre el nivel de expresión de CLCA1 y los niveles de células inmunes infiltrantes en STAD, así como las moléculas activadoras inmunes, moléculas inmunosupresoras, moléculas MHC, quimiocinas y sus moléculas receptoras. El análisis de enriquecimiento genético reveló que CLCA1 puede estar involucrado en la progresión de STAD a través del lupus eritematoso sistémico (LES), el proteasoma, el ciclo celular, la secreción pancreática y las vías de señalización de PPAR. En resumen, se prevé que CLCA1 funcione como un marcador de pronóstico para pacientes con STAD y está vinculado a la inmunización de STAD.

Predicting Mutational Effects on Ca2+-Activated Chloride Conduction of TMEM16A Based on a Simulation Study.

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.

Ano1 regulates embryo transport by modulating intracellular calcium levels in oviduct smooth muscle.

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.

Galectin-8 modulates human osteoclast activity partly through isoform-specific interactions.

In overactive human osteoclasts, we previously identified an alternative splicing event in LGALS8, encoding galectin-8, resulting in decreased expression of the long isoform. Galectin-8, which modulates cell-matrix interactions and functions intracellularly as a danger recognition receptor, has never been associated with osteoclast biology. In human osteoclasts, inhibition of galectin-8 expression revealed its roles in bone resorption, osteoclast nuclearity, and mTORC1 signaling regulation. Galectin-8 isoform-specific inhibition asserted a predominant role for the short isoform in bone resorption. Moreover, a liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis of galectin-8 isoforms performed in HEK293T cells identified 22 proteins shared by both isoforms. Meanwhile, nine interacting partners were specific for the short isoform, and none were unique to the long isoform. Interactors specific for the galectin-8 short isoform included cell adhesion proteins and lysosomal proteins. We confirmed the interactions of galectin-8 with CLCN3, CLCN7, LAMP1, and LAMP2, all known to localize to secretory vesicles, in human osteoclasts. Altogether, our study reveals direct roles of galectin-8 in osteoclast activity, mostly attributable to the short isoform.

TIMP1/CHI3L1 facilitates glioma progression and immunosuppression via NF-κB activation.

Gliomas are highly heterogeneous brain tumours that are resistant to therapies. The molecular signatures of gliomas play a high-ranking role in tumour prognosis and treatment. In addition, patients with gliomas with a mesenchymal phenotype manifest overpowering immunosuppression and sophisticated resistance to treatment. Thus, studies on gene/protein coexpression networks and hub genes in gliomas holds promise in determining effective treatment strategies. Therefore, in this study, we aimed to. Using average linkage hierarchical clustering, 13 modules and 224 hub genes were described. Top ten hub genes (CLIC1, EMP3, TIMP1, CCDC109B, CASP4, MSN, ANXA2P2, CHI3L1, TAGLN2, S100A11), selected from the most meaningful module, were associated with poor prognosis. String analysis, co-immunoprecipitation and immunofluorescence revealed a significant correlation between TIMP1 and CHI3L1. Furthermore, we found, both in vivo and in vitro, that TIMP1 promoted gliomagenesis via CHI3L1 overexpression as well as NF-κB activation. TIMP1 expression correlated with tumour immune infiltration and immune checkpoint-related gene expression. In addition, TIMP1 resulted in immunosuppressive macrophage polarization. In summary, TIMP1/CHI3L1 might be perceived as a diagnostic marker and an immunotherapy target for gliomas.