Botulinum toxin type a blocks aquaporin 5 trafficking by decreasing synaptosomal-associated protein 23 in submandibular acinar cells.

The trafficking of aquaporin 5 (AQP5) is critical for salivary secretion. Synaptosomal-associated protein 23 (SNAP23) is an important regulator in the process of membrane fusion. However, the role of SNAP23 on AQP5 trafficking has not been explored. Botulinum toxin type A (BoNT/A) is a bacterial toxin that effectively treats sialorrhea. We previously reported that BoNT/A induced AQP5 redistribution in cultured acinar cells, but the mechanism remained unclear. In this study, SNAP23 was predominantly localized to the plasma membrane of acinar cells in the rat submandibular gland (SMG) and colocalized with AQP5 at the apical membrane of acinar cells. In stable GFP-AQP5-transfected SMG-C6 cells, the acetylcholine receptor agonist carbachol (CCh) induced trafficking of AQP5 from intracellular vesicles to the apical membrane. Furthermore, SNAP23 knockdown by siRNA significantly inhibited CCh-induced AQP5 trafficking, whereas this inhibitory effect was reversed by SNAP23 re-expression, indicating that SNAP23 was essential in AQP5 trafficking. More importantly, BoNT/A inhibited salivary secretion from SMGs, and the underlying mechanism involved that BoNT/A blocked CCh-triggered AQP5 trafficking by decreasing SNAP23 in acinar cells. Taken together, these results identified a crucial role for SNAP23 in AQP5 trafficking and provided new insights into the mechanism of BoNT/A in treating sialorrhea and thereby a theoretical basis for clinical applications.

Involvement of aquaporin 5 and Na-K-2Cl cotransporter 1 in the pathogenesis of primary focal hyperhidrosis: evidence from the primary sweat gland cell culture.

People with primary focal hyperhidrosis (PFH) usually have an overactive sympathetic nervous system, which can activate the sweat glands through the chemical messenger of acetylcholine. The role of aquaporin 5 (AQP5) and Na-K-2Cl cotransporter 1 (NKCC1) in PFH is still unknown. The relative mRNA and protein levels of AQP5 and NKCC1 in the sweat gland tissues of three subtypes of patients with PFH (primary palmar hyperhidrosis, PPH; primary axillary hyperhidrosis, PAH; and primary craniofacial hyperhidrosis, PCH) were detected with real-time PCR (qPCR) and Western blot. Primary sweat gland cells from healthy controls (NPFH-SG) were incubated with different concentrations of acetylcholine, and the relative mRNA and protein expression of AQP5 and NKCC1 were also detected. NPFH-SG cells were also transfected with si-AQP5 or shNKCC1, and acetylcholine stimulation-induced calcium transients were assayed with Fluo-3 AM calcium assay. Upregulated AQP5 and NKCC1 expression were observed in sweat gland tissues, and AQP5 demonstrated a positive Pearson correlation with NKCC1 in patients with PPH (r = 0.66, P < 0.001), patients with PAH (r = 0.71, P < 0.001), and patients with PCH (r = 0.62, P < 0.001). Upregulated AQP5 and NKCC1 expression were also detected in primary sweat gland cells derived from three subtypes of patients with PFH when compared with primary sweat gland cells derived from healthy control. Acetylcholine stimulation could induce the upregulated AQP5 and NKCC1 expression in NPFH-SG cells, and AQP5 or NKCC1 inhibitions attenuated the calcium transients induced by acetylcholine stimulation in NPFH-SG cells. The dependence of ACh-stimulated calcium transients on AQP5 and NKCC1 expression may be involved in the development of PFH.NEW & NOTEWORTHY The dependence of ACh-stimulated calcium transients on AQP5 and Na-K-2Cl cotransporter 1 (NKCC1) expression may be involved in the development of primary focal hyperhidrosis (PFH).

Loss of aquaporin 5 contributes to the corneal epithelial pathogenesis via Wnt/ß-catenin pathway.

AQP5 plays a crucial role in maintaining corneal transparency and the barrier function of the cornea. Here, we found that in the corneas of Aqp5-/- mice at older than 6 months, loss of AQP5 significantly increased corneal neovascularization, inflammatory cell infiltration, and corneal haze. The results of immunofluorescence staining showed that upregulation of K1, K10, and K14, and downregulation of K12 and Pax6 were detected in Aqp5-/- cornea and primary corneal epithelial cells. Loss of AQP5 aggravated wound-induced corneal neovascularization, inflammation, and haze. mRNA sequencing, western blotting, and qRT-PCR showed that Wnt2 and Wnt6 were significantly decreased in Aqp5-/- corneas and primary corneal epithelial cells, accompanied by decreased aggregation in the cytoplasm and nucleus of ß-catenin. IIIC3 significantly suppressed corneal neovascularization, inflammation, haze, and maintained corneal transparent epithelial in Aqp5-/- corneas. We also found that pre-stimulated Aqp5-/- primary corneal epithelial cells with IIIC3 caused the decreased expression of K1, K10, and K14, the increased expression of K12, Pax6, and increased aggregation in the cytoplasm and nucleus of ß-catenin. These findings revealed that AQP5 may regulate corneal epithelial homeostasis and function through the Wnt/ß-catenin signaling pathway. Together, we uncovered a possible role of AQP5 in determining corneal epithelial cell fate and providing a potential therapeutic target for corneal epithelial dysfunction.

Goji Berry Juice Prevents Tumor Necrosis Factor Alpha-Induced Xerostomia in Human Salivary Gland Cells.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by oral dryness that is primarily attributed to tumor necrosis factor alpha (TNF-α)-mediated reduction in saliva production. In traditional Chinese medicine, goji berries are recognized for their hydrating effect and are considered suitable to address oral dryness associated with Yin deficiency. In the present study, we used goji berry juice (GBJ) to investigate the potential preventive effect of goji berries on oral dryness caused by SS. Pretreatment of human salivary gland cells with GBJ effectively prevented the decrease in aquaporin-5 (AQP-5) mRNA and protein levels induced by TNF-α. GBJ also inhibited histone H4 deacetylation and suppressed the generation of intracellular reactive oxygen species (ROS). Furthermore, GBJ pretreatment reserved mitochondrial membrane potential and suppressed the upregulation of Bax and caspase-3, indicating that GBJ exerted an antiapoptotic effect. These findings suggest that GBJ provides protection against TNF-α in human salivary gland cells and prevents the reduction of AQP-5 expression on the cell membrane. Altogether, these results highlight the potential role of GBJ in preventing oral dryness caused by SS.

Methazolamide Reduces the AQP5 mRNA Expression and Immune Cell Migration-A New Potential Drug in Sepsis Therapy?

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 (Aqp5) knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide (10-5 M) and furosemide (10-6 M) reduced AQP5 mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-α compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced AQP5 expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce AQP5 expression and migration of immune cells. However, after LPS administration, the reduction in AQP5 expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing AQP5 expression and has the potential to be used in sepsis prophylaxis.

The cell polarity protein Scribble is downregulated by the water channel aquaporin-5 in breast cancer cells.

Breast carcinomas originate from cells in the terminal duct-lobular unit. Carcinomas are associated with increased cell proliferation and migration, altered cellular adhesion, as well as loss of epithelial polarity. In breast cancer, aberrant and high levels of aquaporin-5 (AQP5) are associated with increased metastasis, poor prognosis, and cancer recurrence. AQP5 increases the proliferation and migration of cancer cells, and ectopic expression of AQP5 in normal epithelial cells reduces cell-cell adhesion and increases cell detachment and dissemination from migrating cell sheets, the latter via AQP5-mediated activation of the Ras pathway. Here, we investigated if AQP5 also affects cellular polarity by examining the relationship between the essential polarity protein Scribble and AQP5. In tissue samples from invasive lobular and ductal carcinomas, the majority of cells with high AQP5 expression displayed low Scribble levels, indicating an inverse relationship. Probing for interactions via a Glutathione S-transferase pull-down experiment revealed that AQP5 and Scribble interacted. Moreover, overexpression of AQP5 in the breast cancer cell line MCF7 reduced both size and circularity of three-dimensional (3-D) spheroids and induced cell detachment and dissemination from migrating cell sheets. In addition, Scribble levels were reduced. An AQP5 mutant cell line, which cannot activate Ras (AQP5S156A) signaling, displayed unchanged spheroid size and circularity and an intermediate level of Scribble, indicating that the effect of AQP5 on Scribble is, at least in part, dependent on AQP5-mediated activation of Ras. Thus, our results suggest that high AQP5 expression negatively regulates the essential polarity protein Scribble and thus, can affect cellular polarity in breast cancer.

TGF-ß1 Triggers Salivary Hypofunction via Attenuating Protein Secretion and AQP5 Expression in Human Submandibular Gland Cells.

Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.

Aquaporin 5 maintains lens transparency by regulating the lysosomal pathway using circRNA.

The lens is transparent, non-vascular, elastic and wrapped in a transparent capsule. The lens oppacity of AQP5-/- mice was increased more than that of wild-type (AQP5+/+ ) mice. In this study, we explored the potential functional role of circular RNAs (circRNAs) and transcription factor HSF4 in lens opacity in aquaporin 5 (AQP5) knockout (AQP5-/- ) mice. Autophagy was impaired in the lens tissues of AQP5-/- mice. Autophagic lysosomes in lens epithelial cells of AQP5-/- mice were increased compared with AQP5+/+ mice, based on analysis by transmission electron microscopy. The genetic information of the mice lens was obtained by high-throughput sequencing, and then the downstream genes were analysed. A circRNA-miRNA-mRNA network related to lysosomal pathway was constructed by the bioinformatics analysis of the differentially expressed circRNAs. Based on the prediction of the TargetScan website and the validation by dual luciferase reporter assay and RNA immunoprecipitation-qPCR, we found that circRNA (Chr16: 33421321-33468183+) inhibited the function of HSF4 by sponging microRNA (miR-149-5p), and it downregulated the normal expression of lysosome-related mRNAs. The accumulation of autophagic lysosome may be one of the reasons for the abnormal development of the lens in AQP5-/- mice.

Ablation of AQP5 gene in mice leads to olfactory dysfunction caused by hyposecretion of Bowman's gland.

Smell detection depends on nasal airflow, which can make absorption of odors to the olfactory epithelium by diffusion through the mucus layer. The odors then act on the chemo-sensitive epithelium of olfactory sensory neurons (OSNs). Therefore, any pathological changes in the olfactory area, for instance, dry nose caused by Sjögren's Syndrome (SS) may interfere with olfactory function. SS is an autoimmune disease in which aquaporin (AQP) 5 autoantibodies have been detected in the serum. However, the expression of AQP5 in olfactory mucosa and its function in olfaction is still unknown. Based on the study of the expression characteristics of AQP5 protein in the nasal mucosa, the olfaction dysfunction in AQP5 knockout (KO) mice was found by olfactory behavior analysis, which was accompanied by reduced secretion volume of Bowman's gland by using in vitro secretion measure system, and the change of acid mucin in nasal mucus layer was identified. By excluding the possibility that olfactory disturbance was caused by changes in OSNs, the result indicated that AQP5 contributes to olfactory functions by regulating the volume and composition of OE mucus layer, which is the medium for the dissolution of odor molecules. Our results indicate that AQP5 can affect the olfactory functions by regulating the water supply of BGs and the mucus layer upper the OE that can explain the olfactory loss in the patients of SS, and AQP5 KO mice might be used as an ideal model to study the olfactory dysfunction.

Aquaporin-5 Dynamic Regulation.

Aquaporin-5 (AQP5), belonging to the aquaporins (AQPs) family of transmembrane water channels, facilitates osmotically driven water flux across biological membranes and the movement of hydrogen peroxide and CO2. Various mechanisms have been shown to dynamically regulate AQP5 expression, trafficking, and function. Besides fulfilling its primary water permeability function, AQP5 has been shown to regulate downstream effectors playing roles in various cellular processes. This review provides a comprehensive overview of the current knowledge of the upstream and downstream effectors of AQP5 to gain an in-depth understanding of the physiological and pathophysiological processes involving AQP5.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

CHRNA1 promotes the pathogenesis of primary focal hyperhidrosis.

The aim of the study was to elucidate the involvement of cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) in the pathogenesis of primary focal hyperhidrosis (PFH). The hyperhidrosis mouse model was constructed using pilocarpine injection. The expression levels of CHRNA1 in sweat gland tissues of PFH patients and hyperhidrosis mice were compared using Western blots and quantitative real-time PCR (qRT-PCR) analyses. Sweat secretion in hyperhidrosis mice treated with small-interfering RNA (siRNA) targeting CHRNA1 (si-CHRNA1) or non-specific siRNA were compared. Sweat secretory granules in the sweat gland cells of hyperhidrosis mice were examined using transmission electron microscopy. The serum level of acetylcholine was measured using enzyme-linked immunosorbent assay, while markers associated with PFH, including Aquaporin 5 (AQP5) and Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), were assessed using immunohistochemical assay and Western blots. Brain-derived neurotrophic factor (BDNF) and Neuregulin 1 (NRG-1) in sympathetic ganglia axons of hyperhidrosis mice were quantified using Western blots. CHRNA1 up-regulation is a characteristic of the sweat glands of PFH patients and Hyperhidrosis mice. Silencing CHRNA1 decreased sweat secretion and the number of sweat secretory granules of hyperhidrosis mice. Serum acetylcholine, as well as AQP5 and CACNA1C expression in the sweat glands, was reduced by siCHRNA1. BDNF1 and NRG-1 levels in the sympathetic ganglia axons were also attenuated by siCHRNA1 treatment. CHRNA1 up-regulation is a potential biomarker of PFH and downregulating CHRNA1 could alleviate the symptoms of PFH through inactivating the sympathetic system.

Physiological Cooperation between Aquaporin 5 and TRPV4.

Aquaporins-among them, AQP5-are responsible for transporting water across biological membranes, which is an important process in all living organisms. The transient receptor potential channel 4 (TRPV4) is a cation channel that is mostly calcium-permeable and can also be activated by osmotic stimuli. It plays a role in a number of different functions in the body, e.g., the development of bones and cartilage, and it is involved in the body's osmoregulation, the generation of certain types of sensation (pain), and apoptosis. Our earlier studies on the uterus and the literature data aroused our interest in the physiological role of the cooperation of AQP5 and TRPV4. In this review, we focus on the co-expression and cooperation of AQP5 and TRPV4 in the lung, salivary glands, uterus, adipose tissues, and lens. Understanding the cooperation between AQP5 and TRPV4 may contribute to the development of new drug candidates and the therapy of several disorders (e.g., preterm birth, cataract, ischemia/reperfusion-induced edema, exercise- or cold-induced asthma).

AQP5-1364A/C Polymorphism Affects AQP5 Promoter Methylation.

The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin (AQP5)-1364A/C polymorphism was shown to be associated with decreased AQP5 expression, which was shown to be relevant in this context leading towards improved outcomes in sepsis. To date, the underlying mechanism of the C-allele-leading to lower AQP5 expression-has been unknown. Knowing the detailed mechanism depicts a crucial step with a target to further interventions. Genotype-dependent regulation of AQP5 expression might be mediated by the epigenetic mechanism of promoter methylation and treatment with epigenetic-drugs could maybe provide benefit. Hence, we tested the hypothesis that AQP5 promoter methylation differs between genotypes in specific types of immune cells.: AQP5 promoter methylation was quantified in cells of septic patients and controls by methylation-specific polymerase chain reaction and quantified by a standard curve. In cell-line models, AQP5 expression was analyzed after demethylation to determine the impact of promoter methylation on AQP5 expression. C-allele of AQP5-1364 A/C promoter polymorphism is associated with a five-fold increased promoter methylation in neutrophils (p = 0.0055) and a four-fold increase in monocytes (p = 0.0005) and lymphocytes (p = 0.0184) in septic patients and healthy controls as well. In addition, a decreased AQP5 promoter methylation was accompanied by an increased AQP5 expression in HL-60 (p = 0.0102) and REH cells (p = 0.0102). The C-allele which is associated with lower gene expression in sepsis is accompanied by a higher methylation level of the AQP5 promoter. Hence, AQP5 promoter methylation could depict a key mechanism in genotype-dependent expression.

Deficiency of CARMA3 attenuates the development of bleomycin induced pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) has attracted more and more attention due to its irreversibility and high mortality rate. Currently, there is no effective treatment option is available to reverse the disease. Caspase recruitment domain-containing membrane-associated guanylate kinase protein (CARMA3) has been recognized as a proinflammatory molecule involved in many lung diseases, such as Allergic airway inflammation and lung cancer. Bleomycin (Bleo), as an alkaline sugar peptide antibiotics, is often used as a first-line anti-tumor agent. Its toxic effect is to induce pulmonary fibrosis (PF) and its clinical symptoms, so it has been widely used in the construction of pulmonary fibrosis model. METHODS: Wild type mice (WT, n = 20) and CARMA3 knockout mice (CARMA3-KO, n = 20) were generated and injected with bleomycin or saline via trachea. The severity of fibrosis was evaluated by fibrosis markers and lung histological morphology. Furthermore, the amount of alveolar epithelial cells and inflammation in lung tissue were examined. Finally, epithelial-mesenchymal transition was further investigated. RESULTS: We found CARMA3 expression in the mice alveolar epithelial cells. And compared with WT mice, CARMA3-KO mice showed reduced deposition of collagen fibers, inflammation and destruction of alveolar epithelial cells in lung tissue. In addition, after bleomycin induction, the expressions of proinflammatory factors and collagen-related factors in CARMA3-KO mice were much lower than those in WT mice. The epithelial-mesenchymal transformation phenotype was also improved in CARMA3-KO mice compared to WT mice. CONCLUSION: Our Results shows that CARMA3 plays an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. CARMA3 could alleviate the fibrosis by improving inflammation, deposition of collagen and damage of alveolar epithelial cells, which revealed that CARMA3 may be a potential target for pulmonary fibrosis.

Uterine kisspeptin receptor critically regulates epithelial estrogen receptor α transcriptional activity at the time of embryo implantation in a mouse model.

Embryo implantation failure is a major cause of infertility in women of reproductive age and a better understanding of uterine factors that regulate implantation is required for developing effective treatments for female infertility. This study investigated the role of the uterine kisspeptin receptor (KISS1R) in the molecular regulation of implantation in a mouse model. To conduct this study, a conditional uterine knockout (KO) of Kiss1r was created using the Pgr-Cre (progesterone receptor-CRE recombinase) driver. Reproductive profiling revealed that while KO females exhibited normal ovarian function and mated successfully to stud males, they exhibited significantly fewer implantation sites, reduced litter size and increased neonatal mortality demonstrating that uterine KISS1R is required for embryo implantation and a healthy pregnancy. Strikingly, in the uterus of Kiss1r KO mice on day 4 (D4) of pregnancy, the day of embryo implantation, KO females exhibited aberrantly elevated epithelial ERα (estrogen receptor α) transcriptional activity. This led to the temporal misexpression of several epithelial genes [Cftr (Cystic fibrosis transmembrane conductance regulator), Aqp5 (aquaporin 5), Aqp8 (aquaporin 8) and Cldn7 (claudin 7)] that mediate luminal fluid secretion and luminal opening. As a result, on D4 of pregnancy, the lumen remained open disrupting the final acquisition of endometrial receptivity and likely accounting for the reduction in implantation events. Our data clearly show that uterine KISS1R negatively regulates ERα signaling at the time of implantation, in part by inhibiting ERα overexpression and preventing detrimentally high ERα activity. To date, there are no reports on the regulation of ERα by KISS1R; therefore, this study has uncovered an important and powerful regulator of uterine ERα during early pregnancy.

Lacrimal gland homeostasis is maintained by the AQP5 pathway by attenuating endoplasmic reticulum stress inflammation in the lacrimal gland of AQP5 knockout mice.

Purpose: AQP5-/- mice spontaneously exhibit dry eye symptoms. The purpose of this study was to assess the endoplasmic reticulum (ER) stress-mediated inflammation generated by a deficiency of aquaporin 5 (AQP5) in the lacrimal gland. Methods: Hematoxylin and eosin (H&E) staining, Oil Red O staining, and transmission electron microscopy (TEM) analysis were performed to identify structural changes in lacrimal gland epithelial cells because of AQP5 deficiency. Corneal epithelial defects were assessed with sodium fluorescein staining. The expression profiles of mRNA and proteins were determined by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot. Mice in the quercetin group were injected intraperitoneally with 40 mg/kg of quercetin, and the control group was injected with an equal volume of dimethyl sulfoxide (DMSO) for 4 weeks. Results: Aqueous tear secretion fell at about 50% in 1- and 6-month-old AQP5-/- mice compared with that of AQP5+/+ mice. TEM showed that the ER structure was damaged. ER stress was significantly increased in the lacrimal gland of AQP5-/- mice. Lipid droplets accumulated in the matrix and acinar cells, and changes occurred in the lipid metabolism and gene expression levels for PPARα, CPT1α, and CPT2 in the AQP5-/- mice. Immune cell infiltration and increases in the gene expression levels of the chemokines CXCL1, CXCL2, and CCL5 were found in the lacrimal gland of AQP5-/- mice. Quercetin partially reversed ER stress levels, inflammation, and lipid accumulation, and it inhibited tear secretion. Conclusions: The study data indicated that a deficiency of AQP5 induced pathophysiological changes and functional decompensation of the lacrimal gland. Quercetin may improve the inflammation in the lacrimal glands of AQP5-/- mice by regulating the ER stress levels.

Exosomes co-expressing AQP5-targeting miRNAs and IL-4 receptor-binding peptide inhibit the migration of human breast cancer cells.

Aquaporin-5 (AQP5) plays a role in breast cancer cell migration. This study aimed to identify AQP5-targeting miRNAs and examine their effects on breast cancer cell migration through exosome-mediated delivery. Bioinformatic analyses identified miR-1226-3p, miR-19a-3p, and miR-19b-3p as putative regulators of AQP5 mRNA. Immunoblotting revealed a decrease of AQP5 protein abundance when each of these miRNAs was transfected into human breast cancer MDA-MB-231 cells. Quantitative real-time PCR demonstrated the reduction of AQP5 mRNA expression by the transfection of miR-1226-3p and a luciferase reporter assay revealed the reduction of AQP5 translation after the transfection of miR-19b-3p in MDA-MB-231 cells. Consistently, the transfection of each miRNA impeded cell migration. Pathway enrichment analyses showed that these three miRNAs regulate target genes, which were predominantly enriched in the gap junction pathway. For the efficient delivery of AQP5-targeting miRNAs to breast cancer cells, exosomes expressing both miRNAs and a peptide targeting interleukin-4 receptor, which is highly expressed in breast cancer cells, were bioengineered and their inhibitory effects on AQP5 protein expression and cell migration were demonstrated in MDA-MB-231 cells. Taken together, AQP5-regulating miRNAs are identified, which could be exploited for the inhibition of breast cancer cell migration via the exosome-mediated delivery.

Activation of Aquaporin 5 by carcinogenic Helicobacter pylori infection promotes epithelial-mesenchymal transition via the MEK/ERK pathway.

BACKGROUND: Helicobacter pylori (H. pylori) is a major risk factor for gastric cancer. The water channel protein Aquaporin 5 (AQP5) is involved in the tumorigenesis and progression of various cancers. In this study, we aimed to explore the role of AQP5 in H. pylori-induced gastric carcinogenesis. MATERIALS AND METHODS: We collected 160 samples which inculded CNAG, IM, Dys and gastric cancer from patients who underwent endoscopy and detected the expression of AQP5. In vivo and vitro H. pylori infection models, we explored the relationship between AQP5 and H. pylori. Plasmid, siRNA and inhibitors were used to investigated the relationship between AQP5 and EMT and the role of AQP5 in H. pylori-induced gastric carcinogenesis. RESULT: AQP5 expression was gradually increased in human gastric tissues with the progression of chronic nonatrophic gastritis to gastric cancer and associated with the H. pylori infection status. In vivo and in vitro studies showed that H. pylori infection induced AQP5 expression in gastric epithelial cells in a CagA-dependent manner. Knockdown of AQP5 reversed H. pylori-induced cell proliferation and invasion, and -suppressed cell apoptosis. Additionally, knockdown of AQP5 suppressed H. pylori-induced Epithelial-mesenchymal transition (EMT) phenotypes by regulating transcriptional factors, mesenchymal markers, and epithelial markers. CONCLUSIONS: We explored the underlying mechanism and our results indicated that knockdown of AQP5 significantly suppressed H. pylori infection-induced phosphorylation of ERK1/2, MEK and the expression levels of downstream genes. Treatment with an ERK inhibitor suppressed the EMT induced by H. pylori infection. Taken together, this study suggest that pathogenic H. pylori infection promotes AQP5 expression to induce the EMT via the MEK/ERK signaling pathway.

Pannexin 1 Mediates Gastric Cancer Cell Epithelial-Mesenchymal Transition via Aquaporin 5.

Pannexin 1 (PANX1) has been implicated in cancer emergence and progression. However, its roles in gastric cancer remain unclear. In the present study, the function and molecular mechanisms of PANX1 in gastric cancer were investigated in vitro. Two gastric cancer cell lines exhibiting low and high PANX1 expression (SNU-16 and HCG-27, respectively) were transfected using a PANX1-containing plasmid or PANX1 transcript-targeting short hairpin (sh)RNA. In addition, HCG-27 cells and PANX1-overexpressing SNU-16 cells were subjected to short interfering (si)RNA-mediated aquaporin 5 (AQP5) knockdown. In vitro cell migration (scratch) and transwell invasion assays were performed to evaluate the cell migratory and invasive abilities. Real-time fluorescence quantitative PCR was used to detect transcripts encoding epithelial-mesenchymal transition markers. Immunofluorescence and Western blotting were conducted to quantify corresponding proteins. In SNU-16 cells, PANX1 overexpression induced conversion from round (cobblestone-like) to elongated (spindle-like) morphologies and enhanced the cell migratory and invasive abilities. PANX1 knockdown had the opposite effect in HGC-27 cells. In PANX1-overexpressing SNU-16 cells, expression of SLUG, vimentin, and AQP5 was significantly upregulated, whereas expression of E-cadherin was downregulated. In HGC-27 cells, PANX1 knockdown showed the opposite effect. In both PANX1-overexpressing SNU-16 cells and untransfected HGC-27 cells, silencing of AQP5 expression significantly inhibited PANX1-induced upregulation of SLUG and vimentin expression, as well as downregulation of E-cadherin expression and enhanced migratory and invasive abilities. In summary, elevated PANX1 expression induces gastric cancer cell epithelial-mesenchymal transition and the associated promotion of migratory and invasive abilities by inducing expression of AQP5, which facilitates SLUG-mediated regulation of vimentin and E-cadherin expression.