BEST4+ cells in the intestinal epithelium.

The recent development of single-cell transcriptomics highlighted the existence of a new lineage of mature absorptive cells in the human intestinal epithelium. This subpopulation is characterized by the specific expression of Bestrophin 4 (BEST4) and of other marker genes including OTOP2, CA7, GUCA2A, GUCA2B, and SPIB. BEST4+ cells appear early in development and are present in all regions of the small and large intestine at a low abundance (<5% of all epithelial cells). Location-specific gene expression profiles in BEST4+ cells suggest their functional specialization in each gut region, as exemplified by the small intestine-specific expression of the ion channel CFTR. The putative roles of BEST4+ cells include sensing and regulation of luminal pH, tuning of guanylyl cyclase-C signaling, transport of electrolytes, hydration of mucus, and secretion of antimicrobial peptides. However, most of these hypotheses lack functional validation, notably because BEST4+ cells are absent in mice. The presence of BEST4+ cells in human intestinal organoids indicates that this in vitro model should be suitable to study their role. Recent studies showed that BEST4+ cells are also present in the intestinal epithelium of macaque, pig, and zebrafish and, here, we report their presence in rabbits, which suggests that these species could be appropriate animal models to study BEST4+ cells during the development of diseases and their interactions with environmental factors such as diet or the microbiota. In this review, we summarize the existing literature regarding BEST4+ cells and emphasize the description of their predicted roles in the intestinal epithelium in health and disease.NEW & NOTEWORTHY BEST4+ cells are a novel subtype of mature absorptive cells in the human intestinal epithelium highlighted by single-cell transcriptomics. The gene expression profile of BEST4+ cells suggests their role in pH regulation, electrolyte secretion, mucus hydration, and innate immune defense. The absence of BEST4+ cells in mice requires the use of alternative animal models or organoids to decipher the role of this novel type of intestinal epithelial cells.

BEST1 Positive Monocytes in Circulation: Visualize Intratumoral Crosstalk between Cancer Cells and Monocytes.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by an abundance of monocytes and macrophages recruited from the peripheral blood. However, it has not been determined whether these infiltrated cells can be released back into circulation with a tumor-associated neobiosignature. This study reports that Bestrophin1 (BEST1), a component protein of Ca2+ -activated Cl- channels (CaCCs), is highly expressed on classical monocytes in the peripheral blood of HNSCC patients. This is due to monocyte education by tumor cells, in which tumoral VEGF-A upregulates BEST1 expression on monocytes through the MEK-ERK-ELK1 pathway. This leads to improved secretion of IL-6 and IL-8, which promotes tumor cell proliferation. This work also finds that BEST1 facilitates the motility of monocytes, contributing to the migration of these cells back into circulation. These results suggest that the expression of BEST1 on peripheral monocytes may be a potential tool for monitoring tumor progression, and opens up the possibility of searching for cancer biomarkers on monocytes rather than on the tumor or its products.

Bestrophin-2 and glutamine synthetase form a complex for glutamate release.

Bestrophin-2 (BEST2) is a member of the bestrophin family of calcium-activated anion channels that has a critical role in ocular physiology1-4. Here we uncover a directional permeability of BEST2 to glutamate that heavily favours glutamate exit, identify glutamine synthetase (GS) as a binding partner of BEST2 in the ciliary body of the eye, and solve the structure of the BEST2-GS complex. BEST2 reduces cytosolic GS activity by tethering GS to the cell membrane. GS extends the ion conducting pathway of BEST2 through its central cavity and inhibits BEST2 channel function in the absence of intracellular glutamate, but sensitizes BEST2 to intracellular glutamate, which promotes the opening of BEST2 and thus relieves the inhibitory effect of GS. We demonstrate the physiological role of BEST2 in conducting chloride and glutamate and the influence of GS in non-pigmented ciliary epithelial cells. Together, our results reveal a novel mechanism of glutamate release through BEST2-GS.

Low expression of bestrophin-2 is associated with poor prognosis in colon cancer.

OBJECTIVES: The purpose of this research was to confirm the prognostic value of bestrophin-2 (BEST2), one of the hub genes in colon cancer, via bioinformatics analysis and validation in public databases and immunohistochemistry detection. METHODS: The GEO2R online tool and Venn diagram software were utilized to identify differentially expressed genes (DEGs) from expression profiles, including GSE20916, GSE44861 and GSE74602, from the Gene Expression Omnibus (GEO). The overall survival (OS) and disease-free survival (DFS) of colon cancer patients from The Cancer Genome Atlas (TCGA) were analyzed through Kaplan-Meier survival curves. Verification of the significance of BEST2 in colon cancer was based on TCGA, Genotype Tissue Expression (GTEx) and 10 datasets from GEO. BEST2 expression was detected with immunohistochemistry (IHC) in 330 colon tissue samples on microarrays including 165 colon cancerand 165 adjacent normal tissues. For further validation, comprehensive analysis from tissue microarrays and multiple datasets was performed by the summarizing of receiver operating characteristic (SROC) curves and the standard mean differences (SMDs). BEST2 expression in various kinds of colon cancer tissues and cell lines in the context of pancancer was obtained from the Expression Atlas database. The CBioPortal database was queried to identify BEST2 gene alterations and mutation status in colon cancer. Correlated genes (CEGs) with BEST2 and DEGs from public database data were assembled for functional and pathway enrichment analysis. RESULTS: We identified 85 DEGs from the three datasets and screened out BEST2 as a prognostic predictor via the TCGA database. Colon cancer patients with high expression of BEST2 had better survival than patients with low BEST2 (HR = 0.5, P = 0.006) as shown in Kaplan-Meier survival curves in GEPIA. In all, 1463 colon cancer tissues and 1023 colon normal tissues were gathered via public databases as well as in-house tissue microarrays. The comprehensiveexpression analysis suggested low-expression of BEST2 in colon cancer (SMD = -2.48, 95% CI [-3.15- -1.80]) and the notable efficacy of BEST2 expression in differentiating colon cancer from noncancer samples (AUC = 0.97). Gene alteration status of BEST2 occurred in 5% of colon cancer cases, mostly missense mutations and deep deletions. Genes positively correlated with BEST2 and DEGs primarily aggregated in pathways such as anion absorption, digestive juice secretion, cAMP signaling and so on (P < 0.05). CONCLUSION: Ampleevidencesupportsthe role of BEST2 in distinguishing colon cancer from normal tissues in this research. Low expression of BEST2 is correlated with a shorter OS, which implies that BEST2 can be employed as a potential biomarker and therapeutictarget in colon cancer.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells.

OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

The PKC-ζ pseudosubstrate peptide induces glutamate release from retinal pigment epithelium cells through kinase- independent activation of Best1.

AIMS: The retinal pigment epithelium (RPE) is a highly specialized cell monolayer, that plays a key role in the maintenance of photoreceptor function and the blood-retina barrier (BRB). In this study, we found that a myristoylated pseudosubstrate of PKC-ζ (PKCζ PS), considered as a PKC-ζ inhibitor, plays a distinct role in RPE. MAIN METHODS: We demonstrated that PKCζ PS stimulates the release of Glutamate (Glu) using in vitro3H-Glutamate release experiments. By western blot, kinase assays, and Fluoresence Ca+2 Concentration Measurements, we determined the cellular mechanisms involved in such release. KEY FINDINGS: Surprisingly, PKCζ PS has no effect on either phosphorylation of T560, essential for catalytic activity, nor it has an effect on kinase activity. It induces the dose-dependent release of Glu by increasing intracellular Ca+2 levels. Interestingly, this release was not observed upon stimulation by other non-competitive PKC-ζ inhibitors. We here demonstrated that the PKCζ PS stimulates the release of Glutamate from RPE by activating the Ca2+-dependent Cl channel Bestrophin 1 (Best1). SIGNIFICANCE: These results question PKCζ PS specificity as an inhibitor of this enzyme. Furthermore, the present results underline the relevance of clarifying the molecular mechanisms involved in glutamate release from the retina under conditions derived from excitotoxic stimuli.

Nanomedicine-based Curcumin Approach Improved ROS Damage in Best Dystrophy-specific Induced Pluripotent Stem Cells.

Best dystrophy (BD), also termed best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and can cause central visual loss. Unfortunately, there is no clear definite therapy for BD or improving the visual function on this progressive disease. The human induced pluripotent stem cell (iPSC) system has been recently applied as an effective tool for genetic consultation and chemical drug screening. In this study, we developed patient-specific induced pluripotent stem cells (BD-iPSCs) from BD patient-derived dental pulp stromal cells and then differentiated BD-iPSCs into retinal pigment epithelial cells (BD-RPEs). BD-RPEs were used as an expandable platform for in vitro candidate drug screening. Compared with unaffected sibling-derived iPSC-derived RPE cells (Ctrl-RPEs), BD-RPEs exhibited typical RPE-specific markers with a lower expression of the tight junction protein ZO-1 and Bestrophin-1 (BEST1), as well as reduced phagocytic capabilities. Notably, among all candidate drugs, curcumin was the most effective for upregulating both the BEST1 and ZO-1 genes in BD-RPEs. Using the iPSC-based drug-screening platform, we further found that curcumin can significantly improve the mRNA expression levels of Best gene in BD-iPSC-derived RPEs. Importantly, we demonstrated that curcumin-loaded PLGA nanoparticles (Cur-NPs) were efficiently internalized by BD-RPEs. The Cur-NPs-based controlled release formulation further increased the expression of ZO-1 and Bestrophin-1, and promoted the function of phagocytosis and voltage-dependent calcium channels in BD-iPSC-derived RPEs. We further demonstrated that Cur-NPs enhanced the expression of antioxidant enzymes with a decrease in intracellular ROS production and hydrogen peroxide-induced oxidative stress. Collectively, these data supported that Cur-NPs provide a potential cytoprotective effect by regulating the anti-oxidative abilities of degenerated RPEs. In addition, the application of patient-specific iPSCs provides an effective platform for drug screening and personalized medicine in incurable diseases.

Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.

Vitamin C Prevents Hydrocortisone-Induced Injury in HMEC-1 through Promoting Bestrophin-3 Expression.

OBJECTIVE: To investigate the protective effects and underlying mechanisms of Vitamin C (VC) on hydrocortisone (HC)-induced cell injury in human microvascular endothelial cells (HMEC). METHODS: Cell viability was measured by CCK-8 assay and the expression of Best-3 was detected by Western blotting assay. The experiment was divided into normal control, HC injury group, VC treatment groups, HC + Best-3 siRNA group, HC + VC + Best-3 siRNA group, HC + pcDNA3.1 Best-3 group, and HC + VC + pcDNA3.1 Best-3 group. RESULTS: HC inhibited HMEC-1 cell viability was balanced with lower expression of Best-3 in a dose-dependent manner. Conversely, VC promoted HMEC-1 cell viability was paralleled to higher expression of Best-3 in a dose-dependent manner. Silencing Best-3 with Best-3 siRNA inhibited HMEC-1 cell viability, however, over-expression of Best-3 with pcDNA3.1 Best-3 promoted HMEC-1 cell viability. Moreover, VC and over-expression of Best-3 prevented HC-induced HMEC-1 cell apoptosis; however, silencing Best-3 further enhanced HC-induced HMEC-1 cell apoptosis. HC reduced Best-3 expression, which was alleviated by VC treatment. HC treatment decreased Bcl-2 expression, facilitated Bax expression. Both of VC and over-expression of Best-3 promoted Bcl-2 expression and decreased Bax expression. Additionally, VC and Best-3 expression have a synergistic effect. CONCLUSIONS: VC can efficiently attenuate HC-induced HMEC-1 cell injury, which may be related to promote Best-3 expression.

Clinical Course of Autosomal Recessive Bestrophinopathy Complicated by Choroidal Neovascularization.

The authors report the clinical course of two cases of autosomal recessive bestrophinopathy (ARB) complicated by choroidal neovascularization (CNV). One patient presenting with a novel BEST1 mutation (c.658 C>T, p.Gln220*) underwent anti-vascular endothelial growth factor therapy. Response to treatment was documented on optical coherence tomography angiography (OCTA). Despite initial response to treatment, recurrent CNV exudation with progressive subretinal fibrosis was observed. In the second patient, the CNV was not treated and spontaneous regression was observed. This report indicates that the clinical course of CNV in ARB may vary considerably, ranging from spontaneous regression to progressive subretinal fibrosis despite intervention. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:888-892.].

ATP activates bestrophin ion channels through direct interaction.

Human Bestrophin1 (hBest1) is a Ca2+-activated Cl- channel in retinal pigment epithelium (RPE) essential for retina physiology, and its mutation results in retinal degenerative diseases that have no available treatments. Here, we discover that hBest1's channel activity in human RPE is significantly enhanced by adenosine triphosphate (ATP) in a dose-dependent manner. We further demonstrate a direct interaction between ATP and bestrophins, and map the ATP-binding motif on hBest1 to an intracellular loop adjacent to the channel activation gate. Importantly, a disease-causing mutation of hBest1 located within the ATP-binding motif, p.I201T, diminishes ATP-dependent activation of the channel in patient-derived RPE, while the corresponding mutants in bestrophin homologs display defective ATP binding and a conformational change in the ATP-binding motif. Taken together, our results identify ATP as a critical activator of bestrophins, and reveal the molecular mechanism of an hBest1 patient-specific mutation.

Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl- secretion in murine colon by downregulating TMEM16A.

Attenuated Ca2+-activated Cl- secretion has previously been observed in the model of dextran sulfate sodium (DSS)-induced colitis. Prior studies have implicated dysfunctional muscarinic signaling from basolateral membranes as the potential perpetrator leading to decreased Ca2+-activated Cl- secretion. However, in our chronic model of DSS-colitis, cholinergic receptor muscarinic 3 ( Chrm3) transcript (1.028 ± 0.12 vs. 1.029 ± 0.27, P > 0.05) and CHRM3 protein expression (1.021 ± 0.24 vs. 0.928 ± 0.09, P > 0.05) were unchanged. Therefore, we hypothesized that decreased carbachol (CCH)-stimulated Cl- secretion in DSS-induced colitis could be attributed to a loss of Ca2+-activated Cl- channels (CaCC) in apical membranes of colonic epithelium. To establish this chemically-induced colitis, Balb/C mice were exposed to 4% DSS for five alternating weeks to stimulate a more moderate, chronic colitis. Upon completion of the protocol, whole thickness sections of colon were mounted in an Ussing chamber under voltage-clamp conditions. DSS-induced colitis demonstrated a complete inhibition of basolateral administration of CCH-stimulated Cl- secretion that actually displayed a reversal in polarity (15.40 ± 2.22 µA/cm2 vs. -2.47 ± 0.25 µA/cm2). Western blotting of potential CaCCs, quantified by densitometric analysis, demonstrated no change in bestrophin-2 and cystic fibrosis transmembrane regulator, whereas anoctamin-1 [ANO1, transmembrane protein 16A (TMEM16A)] was significantly downregulated (1.001 ± 0.13 vs. 0.510 ± 0.12, P < 0.05). Our findings indicate that decreased expression of TMEM16A in DSS-induced colitis contributes to the decreased Ca2+-activated Cl- secretion in murine colon.

Direct interaction with 14-3-3γ promotes surface expression of Best1 channel in astrocyte.

BACKGROUND: Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer's disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells. METHODS: The protein-protein interaction between Best1 and 14-3-3γ was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3γ on Best1-mediated current was measured by whole-cell patch clamp technique. RESULTS: We identified 14-3-3γ as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3γ was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3γ. We also revealed that interaction between Best1 and 14-3-3γ was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3γ without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3γ-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3γ-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3γ. CONCLUSIONS: 14-3-3γ promotes surface expression of Best1 channel in astrocytes through direct interaction.

The Ca2+-activated chloride channel anoctamin-2 mediates spike-frequency adaptation and regulates sensory transmission in thalamocortical neurons.

Neuronal firing patterns, which are crucial for determining the nature of encoded information, have been widely studied; however, the molecular identity and cellular mechanisms of spike-frequency adaptation are still not fully understood. Here we show that spike-frequency adaptation in thalamocortical (TC) neurons is mediated by the Ca2+-activated Cl- channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in TC neurons results in significantly reduced spike-frequency adaptation along with increased tonic spiking. Moreover, thalamus-specific knockdown of ANO2 increases visceral pain responses. These results indicate that ANO2 contributes to reductions in spike generation in highly activated TC neurons and thereby restricts persistent information transmission.

Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model.

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl- channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl- ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl- conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1.