PPAR-γ/NF-kB/AQP3 axis in M2 macrophage orchestrates lung adenocarcinoma progression by upregulating IL-6.

Aquaporin 3 (AQP3), which is mostly expressed in pulmonary epithelial cells, was linked to lung adenocarcinoma (LUAD). However, the underlying functions and mechanisms of AQP3 in the tumor microenvironment (TME) of LUAD have not been elucidated. Single-cell RNA sequencing (scRNA-seq) was used to study the composition, lineage, and functional states of TME-infiltrating immune cells and discover AQP3-expressing subpopulations in five LUAD patients. Then the identifications of its function on TME were examined in vitro and in vivo. AQP3 was associated with TNM stages and lymph node metastasis of LUAD patients. We classified inter- and intra-tumor diversity of LUAD into twelve subpopulations using scRNA-seq analyses. The analysis showed AQP3 was mainly enriched in subpopulations of M2 macrophages. Importantly, mechanistic investigations indicated that AQP3 promoted M2 macrophage polarization by the PPAR-γ/NF-κB axis, which affected tumor growth and migration via modulating IL-6 production. Mixed subcutaneous transplanted tumor mice and Aqp3 knockout mice models were further utilized, and revealed that AQP3 played a critical role in mediating M2 macrophage polarization, modulating glucose metabolism in tumors, and regulating both upstream and downstream pathways. Overall, our study demonstrated that AQP3 could regulate the proliferation, migration, and glycometabolism of tumor cells by modulating M2 macrophages polarization through the PPAR-γ/NF-κB axis and IL-6/IL-6R signaling pathway, providing new insight into the early detection and potential therapeutic target of LUAD.

The presence of virulence factor genes downregulates uterine AQP3 and alters glutathione peroxidase activity and uterine histopathology in canine pyometra.

Present study was designed to evaluate the role of virulence factor genes (papG, cnf1 and hylA) in the pathogenesis of canine pyometra. Antimicrobial susceptibility test and detection of virulence genes were performed Escherichia coli (E. coli) detected in uterine swab samples. Animals were divided into two groups based on the presence (VF+, n:14) or absence (VF-, n:7) of the virulence factor genes papG, cnf1 and hylA. Blood and tissue glutathione peroxidase activity, uterine histopathologic analysis and AQP3, ESR1, PGR, OXTR gene expressions were determined in both groups. Statistical analyses were performed using Stata version 15.1. All E. coli isolates were susceptible to amikacin, whereas resistant to ampicillin, amoxicillin/clavulanic acid and lincomycin. None of the isolates were susceptible to cefotaxime. E. coli isolates had at least one virulence gene. The most prevalent gene was fimH (100%), followed by fyuA (95.8%), usp (83.3%), sfa (75%), cnf1 and hlyA (70.8%) genes. Blood GPx activity was greater in VF+ animals. On the other hand, uterine tissue GPx activity was lower in VF+ group compared to the control group. Expression levels of AQP3 were upregulated more than fivefold in VF-dogs compared to the control group. In addition, AQP3 expression levels were found approximately threefold higher in VF (-) than VF (+) group (p < .05). Varying degree of inflammation noted for all animals with pyometra, but the presence of bacteria noted only in VF+ animals. In conclusion, the presence of virulence factor genes does not play a role in the histopathological degree of inflammation, the presence of bacteria was found to vary. Serum GPx activity increased in VF+ animals. While the hormone receptor expressions were similar, AQP expression was upregulated in the absence of virulence factor genes.

[In silico analysis of molecular mimicry between human aquaporin 3, Aspergillus fumigatus aquaporin and aquaporins from allergic sources]. / Análisis en silico del mimetismo molecular entre acuaporina 3 humana y Aspergillus fumigatus: implicaciones para el potencial de reactividad cruzada.

OBJECTIVE: Conduct an in-silico assessment of potential molecular mimicry between human aquaporins, A. fumigatus, and diverse allergenic sources. METHODS: Amino acid sequences of human AQP3 and A. fumigatus aquaporin were compared through multiple alignments with 25 aquaporins from diverse allergenic sources. Phylogenetic analysis and homology-based modeling were executed, and the ElliPro server predicted conserved antigenic regions on 3D structures. RESULTS: Global identity among studied aquaporins was 32.6%, with a specific conserved local region at 71.4%. Five monophyletic clades (A-E) were formed, and Group B displayed the highest identity (95%), including 6 mammalian aquaporins, notably AQP3. A. fumigatus aquaporin exhibited the highest identity with Malassezia sympodialis (35%). Three linear and three discontinuous epitopes were identified in both human and A. fumigatus aquaporins. The Root Mean Square Deviation (RMSD) from overlapping aquaporin structures was 1.006. CONCLUSION: Identification of potential linear and conformational epitopes on human AQP3 suggests likely molecular mimicry with A. fumigatus aquaporins. High identity in a specific antigenic region indicates potential autoreactivity and a probable antigenic site involved in cross-reactivity. Validation through in vitro and in vivo studies is essential for further understanding and confirmation.

OBJETIVO: Realizar una evaluación in silico del posible mimetismo molecular entre las acuaporinas humanas, A. fumigatus y diversas fuentes alergénicas. MÉTODOS: Se compararon secuencias de aminoácidos de AQP3 humana y acuaporina de A. fumigatus mediante alineamientos múltiples con 25 acuaporinas de diversas fuentes alergénicas. Se ejecutaron análisis filogenéticos y modelos basados en homología, y el servidor ElliPro predijo regiones antigénicas preservadas en estructuras 3D. RESULTADOS: La identidad global entre las acuaporinas estudiadas fue del 32.6%, con una región local específica preservada en el 71.4%. Se formaron cinco clados monofiléticos (A-E), y el grupo B mostró la identidad más alta (95%), incluidas 6 acuaporinas de mamíferos, en particular AQP3. A. fumigatus aquaporin exhibió la mayor identidad con Malassezia sympodialis (35%). Se identificaron tres epítopos lineales y tres discontinuos en acuaporinas tanto humanas como de A. fumigatus. La desviación cuadrática media (RMSD) de las estructuras de acuaporinas superpuestas fue de 1,006. CONCLUSIÓN: La identificación de posibles epítopos lineales y conformacionales en AQP3 humano sugiere un probable mimetismo molecular con acuaporinas de A. fumigatus. La identidad alta en una región antigénica específica indica autorreactividad potencial y un sitio antigénico probable implicado en la reactividad cruzada. La validación mediante estudios in vitro e in vivo es desicivo para una mayor comprensión y confirmación.

Activation of ASIC3/ERK pathway by paeoniflorin improves intestinal fluid metabolism and visceral sensitivity in slow transit constipated rats.

Slow transit constipation (STC) is one of the most common gastrointestinal disorders in children and adults worldwide. Paeoniflorin (PF), a monoterpene glycoside compound extracted from the dried root of Paeonia lactiflora, has been found to alleviate STC, but the mechanisms of its effect remain unclear. The present study aimed to investigate the effects and mechanisms of PF on intestinal fluid metabolism and visceral sensitization in rats with compound diphenoxylate-induced STC. Based on the evaluation of the laxative effect, the abdominal withdrawal reflex test, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were used to detect the visceral sensitivity, fluid metabolism-related proteins, and acid-sensitive ion channel 3/extracellular signal-regulated kinase (ASIC3/ERK) pathway-related molecules. PF treatment not only attenuated compound diphenoxylate-induced constipation symptoms and colonic pathological damage in rats but also ameliorated colonic fluid metabolic disorders and visceral sensitization abnormalities, as manifested by increased colonic goblet cell counts and mucin2 protein expression, decreased aquaporin3 protein expression, improved abdominal withdrawal reflex scores, reduced visceral pain threshold, upregulated serum 5-hydroxytryptamine, and downregulated vasoactive intestinal peptide levels. Furthermore, PF activated the colonic ASIC3/ERK pathway in STC rats, and ASIC3 inhibition partially counteracted PF's modulatory effects on intestinal fluid and visceral sensation. In conclusion, PF alleviated impaired intestinal fluid metabolism and abnormal visceral sensitization in STC rats and thus relieved their symptoms through activation of the ASIC3/ERK pathway.

Regulation of renal aquaporin water channels in acute pyelonephritis.

Acute pyelonephritis (APN) is most frequently caused by uropathogenic Escherichia coli (UPEC), which ascends from the bladder to the kidneys during a urinary tract infection. Patients with APN have been reported to have reduced renal concentration capacity under challenged conditions, polyuria, and increased aquaporin-2 (AQP2) excretion in the urine. We have recently shown increased AQP2 accumulation in the plasma membrane in cell cultures exposed to E. coli lysates and in the apical plasma membrane of inner medullary collecting ducts in a 5-day APN mouse model. This study aimed to investigate if AQP2 expression in host cells increases UPEC infection efficiency and to identify specific bacterial components that mediate AQP2 plasma membrane insertion. As the transepithelial water permeability in the collecting duct is codetermined by AQP3 and AQP4, we also investigated whether AQP3 and AQP4 localization is altered in the APN mouse model. We show that AQP2 expression does not increase UPEC infection efficiency and that AQP2 was targeted to the plasma membrane in AQP2-expressing cells in response to the two pathogen-associated molecular patterns (PAMPs), lipopolysaccharide and peptidoglycan. In contrast to AQP2, the subcellular localizations of AQP1, AQP3, and AQP4 were unaffected both in lysate-incubated cell cultures and in the APN mouse model. Our finding demonstrated that cellular exposure to lipopolysaccharide and peptidoglycan can trigger the insertion of AQP2 in the plasma membrane revealing a new regulatory pathway for AQP2 plasma membrane translocation, which may potentially be exploited in intervention strategies.NEW & NOTEWORTHY Acute pyelonephritis (APN) is associated with reduced renal concentration capacity and increased aquaporin-2 (AQP2) excretion. Uropathogenic Escherichia coli (UPEC) mediates changes in the subcellular localization of AQP2 and we show that in vitro, these changes could be elicited by two pathogen-associated molecular patterns (PAMPs), namely, lipopolysaccharide and peptidoglycan. UPEC infection was unaltered by AQP2 expression and the other renal AQPs (AQP1, AQP3, and AQP4) were unaltered in APN.

FTO attenuates TNF-α-induced damage of proximal tubular epithelial cells in acute pancreatitis-induced acute kidney injury via targeting AQP3 in an N6-methyladenosine-dependent manner.

BACKGROUND: Acute kidney injury (AKI) is a frequent complication of severe acute pancreatitis (SAP). Previous investigations have revealed the involvement of FTO alpha-ketoglutarate-dependent dioxygenase (FTO) and aquaporin 3 (AQP3) in AKI. Therefore, the aim of this study is to explore the association of FTO and AQP3 on proximal tubular epithelial cell damage in SAP-induced AKI. METHODS: An in-vitro AKI model was established in human proximal tubular epithelial cells (PTECs) HK-2 via tumor necrosis factor-α (TNF-α) induction (20 ng/mL), after which FTO and AQP3 expression was manipulated and quantified by quantitative real-time PCR and Western blotting. The viability and apoptosis of PTECs under various conditions, and reactive oxygen species (ROS), superoxide dismutase (SOD), and malonaldehyde (MDA) levels within these cells were measured using commercial assay kits and flow cytometry. Methylated RNA immunoprecipitation and mRNA stability assays were performed to elucidate the mechanism of FTO-mediated N6-methyladenosine (m6A) modification. Western blotting was performed to quantify ß-catenin protein levels in the PTECs. RESULTS: FTO overexpression attenuated the TNF-α-induced decrease in viability and SOD levels, elevated apoptosis, increased levels of ROS and MDA, and diminished TNF-α-induced AQP3 expression and reduced ß-catenin expression, but its silencing led to contradictory results. FTO negatively modulates AQP3 levels in RTECs in an m6A-depednent manner and compromises AQP3 stability. In addition, all FTO overexpression-induced effects in TNF-α-induced PTECs were neutralized following AQP3 upregulation. CONCLUSION: FTO alleviates TNF-α-induced damage to PTECs in vitro by targeting AQP3 in an m6A-dependent manner.

Blockage of aquaporin-3 peroxiporin activity by organogold compounds affects melanoma cell adhesion, proliferation and migration.

Aquaporin-3 (AQP3) is a membrane channel with dual aquaglyceroporin/peroxiporin activity, facilitating the diffusion of water, glycerol and H2O2 across cell membranes. AQP3 shows aberrant expression in melanoma and its role in cell adhesion, migration and proliferation is well described. Gold compounds were shown to modulate AQP3 activity with reduced associated toxicity, making them promising molecules for cancer therapy. In this study, we validated the phenotype resulting from AQP3-silencing of two melanoma cell lines, MNT-1 and A375, which resulted in decreased H2O2 permeability. Subsequently, the AQP3 inhibitory effect of a new series of organogold compounds derived from Auphen, a potent AQP3 inhibitor, was first evaluated in red blood cells (RBCs) that highly express AQP3, and then in HEK-293T cells with AQP3 overexpression to ascertain the compounds' specificity. The first screening in RBCs unveiled two organogold compounds as promising blockers of AQP3 permeability. Moderate reduction of glycerol permeability but drastic inhibition of H2O2 permeability was detected for some of the gold derivatives in both AQP3-overexpressing cells and human melanoma cell lines. Additionally, all compounds were effective in impairing cell adhesion, proliferation and migration, although in a cell type-dependent manner. In conclusion, our data show that AQP3 peroxiporin activity is crucial for melanoma progression and highlight organogold compounds as promising AQP3 inhibitors with implications in melanoma cell adhesion, proliferation and migration, unveiling their potential as anticancer drugs against AQP3-overexpressing tumours. KEY POINTS: AQP3 affects cellular redox balance. Gold compounds inhibit AQP3 permeability in melanoma cells. AQP3 is involved in cell adhesion, proliferation and migration of melanoma. Blockage of AQP3 peroxiporin activity impairs melanoma cell migration. Gold compounds are potential anticancer drug leads for AQP3-overexpressing cancers.

Cellular Distribution of Aquaporin 3, 7 and 9 in the Male Reproductive System: A Lesson from Bovine Study (Bos taurus).

The increasing incidence of male infertility in humans and animals creates the need to search for new factors that significantly affect the course of reproductive processes. Therefore, the aim of this study was to determine the temporospatial expression of aquaglyceroporins (AQP3, AQP7 and AQP9) in the bovine (Bos taurus) reproductive system using immunohistochemistry and Western blotting. The study also included morphological analysis and identification of GATA-4. In brief, in immature individuals, AQP3 and AQP7 were found in gonocytes. In reproductive bulls, AQP3 was observed in spermatocytes and spermatogonia, while AQP7 was visible in all germ cells and the Sertoli cells. AQP7 and AQP9 were detected in the Leydig cells. Along the entire epididymis of reproductive bulls, aquaglyceroporins were visible, among others, in basal cells (AQP3 and AQP7), in epididymal sperm (AQP7) and in the stereocilia of the principal cells (AQP9). In males of all ages, aquaglyceroporins were identified in the principal and basal cells of the vas deferens. An increase in the expression of AQP3 in the testis and cauda epididymis and a decrease in the abundance of AQP7 in the vas deferens with age were found. In conclusion, age-related changes in the expression and/or distribution patterns of AQP3, AQP7 and AQP9 indicate the involvement of these proteins in the normal development and course of male reproductive processes in cattle.

Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens.

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

The Aquaporin 3 Polymorphism (rs17553719) Is Associated with Sepsis Survival and Correlated with IL-33 Secretion.

Sepsis is a common life-threatening disease caused by dysregulated immune response and metabolic acidosis which lead to organ failure. An abnormal expression of aquaporins plays an important role in organ failure. Additionally, genetic variants in aquaporins impact on the outcome in sepsis. Thus, we investigated the polymorphism (rs17553719) and expression of aquaporin-3 (AQP3) and correlated these measurements with the survival of sepsis patients. Accordingly, we collected blood samples on several days (plus clinical data) from 265 sepsis patients who stayed in different ICUs in Germany. Serum plasma, DNA, and RNA were then separated to detect the promotor genotypes of AQP3 mRNA expression of AQP3 and several cytokines. The results showed that the homozygote CC genotype exhibited a significant decrease in 30-day survival (38.9%) compared to the CT (66.15%) and TT genotypes (76.3%) (p = 0.003). Moreover, AQP3 mRNA expression was significantly higher and nearly doubled in the CC compared to the CT (p = 0.0044) and TT genotypes (p = 0.018) on the day of study inclusion. This was accompanied by an increased IL-33 concentration in the CC genotype (day 0: p = 0.0026 and day 3: p = 0.008). In summary, the C allele of the AQP3 polymorphism (rs17553719) shows an association with increased AQP3 expression and IL-33 concentration accompanied by decreased survival in patients with sepsis.

AQP3 and AQP9-Contrary Players in Sepsis?

Sepsis involves an immunological systemic response to a microbial pathogenic insult, leading to a cascade of interconnected biochemical, cellular, and organ-organ interaction networks. Potential drug targets can depict aquaporins, as they are involved in immunological processes. In immune cells, AQP3 and AQP9 are of special interest. In this study, we tested the hypothesis that these aquaporins are expressed in the blood cells of septic patients and impact sepsis survival. Clinical data, routine laboratory parameters, and blood samples from septic patients were analyzed on day 1 and day 8 after sepsis diagnosis. AQP expression and cytokine serum concentrations were measured. AQP3 mRNA expression increased over the duration of sepsis and was correlated with lymphocyte count. High AQP3 expression was associated with increased survival. In contrast, AQP9 expression was not altered during sepsis and was correlated with neutrophil count, and low levels of AQP9 were associated with increased survival. Furthermore, AQP9 expression was an independent risk factor for sepsis lethality. In conclusion, AQP3 and AQP9 may play contrary roles in the pathophysiology of sepsis, and these results suggest that AQP9 may be a novel drug target in sepsis and, concurrently, a valuable biomarker of the disease.

Red ginseng polysaccharide promotes ferroptosis in gastric cancer cells by inhibiting PI3K/Akt pathway through down-regulation of AQP3.

OBJECTIVE: This study aims to investigate the effect of red ginseng polysaccharide (RGP) on gastric cancer (GC) development and explore its mechanism. METHODS: GC cell lines AGS were treated with varying concentrations of RGP (50, 100, and 200 µg/mL). AGS cells treated with 200 µg/mL RGP were transfected with aquaporin 3 (AQP3) overexpression vector. Cell proliferation, viability, and apoptosis were evaluated by MTT, colony formation assay, and flow cytometry, respectively. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression of AQP3. The levels of Fe2+, malondialdehyde, and lactate dehydrogenase were measured using their respective detection kits, and the reactive oxygen species levels was determined by probe 2',7'-dichlorodihydrofluorescein diacetate. The expression of ferroptosis-related protein and PI3K/Akt pathway-related protein were assessed by western blot. In vivo experiments in nude mice were performed and the mice were divided into four groups (n = 5/group) which gavage administrated with 150 mg/kg normal saline, and 75, 150, 300 mg/kg RGP, respectively. Their tumor weight and volume were recorded. RESULTS: RGP treatment effectively inhibited the proliferation and viability of AGS cells in a dosage-dependent manner and induced apoptosis. It induced ferroptosis in AGS cells, as well as inhibiting the expression of PI3K/Akt-related proteins. AQP3 overexpression could reversed the effect of RGP treatment on ferroptosis. Confirmatory in vivo experiments showed that RGP could reduce the growth of implanted tumor, with increased RGP concentration resulting in greater tumor inhibitory effects. CONCLUSION: RGP might have therapeutic potential against GC, effectively inhibiting the proliferation and viability of AGS cells.

The novel mechanism of human norovirus induced diarrhea: Activation of PKD2 caused by HuNoVs destroyed AQP3 expression through AP2γ in intestinal epithelial cells.

Our previous work has demonstrated protein kinase D2 (PKD2) played a critical influence in experimental colitis in animal. However, the role of PKD2 in human norovirus (HuNoVs)-induced diarrhea remained unknown. Aquaporin 3 (AQP3) expression, a critical protein mediating diarrhea, was assessed by western blot, qRT-PCR in intestinal epithelial cells (IECs). Luciferase, IF, IP and ChIP assay were used to explore the mechanism through which HuNoVs regulated AQP3. Herein, we found that AQP3 expression was drastically decreased in IECs in response to VP1 transfection, the major capsid protein of HuNoVs, or HuNoVs infection. Mechanistically, HuNoVs triggered phosphorylation of PKD2 through TLR2/MyD88/IRAK4, which further inhibited AP2γ activation and nuclear translocation, leading to suppress AQP3 transactivation in IECs. Most importantly, PKD2 interacted with MyD88/IRAK4, and VP1 overexpression enhanced this complex form, which, in turn, to increase PKD2 phosphorylation. In addition, endogenous PKD2 interacted with AP2γ, and this interaction was enhanced in response to HuNoVs treatment, and subsequently resulting in AP2γ phosphorylation inhibition. Moreover, inhibition of PKD2 activation could reverse the inhibitory effect of HuNoVs on AQP3 expression. In summary, we established a novel mechanism that HuNoV inhibited AQP3 expression through TLR2/MyD88/IRAK4/PKD2 signaling pathway, targeting PKD2 activity could be a promising strategy for prevention of HuNoVs-induced gastroenteritis.

Targeting Aquaporin-3 Attenuates Skin Inflammation in Rosacea.

Rosacea is a common inflammatory skin disorder mediated by the dysregulation of both keratinocytes and T cells. Here, we report that aquaporin 3 (AQP3), a channel protein that mediates the transport of water/glycerol, was highly expressed in the epidermis and CD4+ T cells of both rosacea patients and experimental mice. Specifically, AQP3 deletion blocked the development of rosacea-like skin inflammation in model mice with LL37-induced rosacea-like disease. We also present mechanistic evidence showing that AQP3 was essential to the activation of NF-κB signaling and subsequent production of disease-characteristic chemokines in keratinocytes. Moreover, we show that AQP3 was upregulated during T cell differentiation and promotes helper T (Th) 17 differentiation possibly via the activation of STAT3 signaling. Our findings reveal that AQP3-mediated activation of NF-κB in keratinocytes and activation of STAT3 in CD4+ T cells acted synergistically and contributed to the inflammation in rosacea.

Salinity effects on expression and localization of aquaporin 3 in gills of the euryhaline milkfish (Chanos chanos).

Milkfish (Chanos chanos) are important euryhaline fish in Southeast Asian countries that can tolerate a wide range of salinity changes. Previous studies have revealed that milkfish have strong ion regulation and survival abilities under osmotic stress. In addition to ion regulation, water homeostasis in euryhaline teleosts is important during environmental salinity shifts. Aquaporins (AQP) are vital water channels in fish, and different AQPs can transport water influx or outflux from the body. AQP3 is one of the AQP channels, and the function of AQP3 in the gills of euryhaline milkfish is still unknown. The aim of this study was to investigate the expression and localization of AQP3 in the gills of euryhaline milkfish to contribute to our understanding of the physiological role and localization of AQP3 in fish. The AQP3 sequence was found in the milkfish next-generation sequencing (NGS) database and is mainly distributed in the gills of freshwater (FW)-acclimated milkfish. Under hypoosmotic and hyperosmotic stress, the osmolality of milkfish immediately shifted, similar to the aqp3 gene expression. Moreover, the abundance of AQP3 protein significantly decreased 3 h after transferring milkfish from FW to seawater (SW). However, there was no change within 7 days when the milkfish experienced hypoosmotic stress. Moreover, double immunofluorescence staining of milkfish gills showed that AQP3 colocalized with Na+ /K+ ATPase at the basolateral membrane of ionocytes. These results combined indicate that milkfish have a strong osmoregulation ability under acute osmotic stress because of the quick shift in the gene and protein expression of AQP3 in their gills.

Population genomics analysis to identify ion and water transporter genes involved in the adaptation of Tibetan naked carps to brackish water.

Understanding how evolutionary processes shape the genetic variations and influence the response of species to environmental alterations is critical for biodiversity conservation and molecular breeding. Gymnocypris przewalskii przewalskii is the only known cyprinid fish that dwells in the brackish water of Lake Qinghai on the Qinghai-Tibetan Plateau. To reveal the genetic basis of its adaptation to high salinity and alkalinity, whole-genome sequencing was performed in G. p. przewalskii and its freshwater relatives Gymnocypris eckloni and Gymnocypris przewalskii ganzihonensis. Compared with freshwater species, lower genetic diversity and higher linkage disequilibrium were observed in G. p. przewalskii. Selective sweep analysis identified 424 core-selective genes enriched in transport activities. Transfection analysis showed that genetic changes in the positively selected gene aquaporin 3 (AQP3) improved cell viability after salt treatment, suggesting its involvement in brackish water adaptation. Our analysis indicates that ion and water transporter genes experienced intensive selection, which might have contributed to the maintenance of high osmolality and ion content in G. p. przewalskii. The current study identified key molecules involved in the adaptation of fish to brackish water, providing valuable genomic resources for the molecular breeding of salt-tolerant fish.

E-cadherin and aquaporin-3 are downregulated in wound edges of human chronic wounds.

Chronic wounds are defined as wounds that fail to proceed through the normal phases of wound healing; a complex process involving different dynamic events including migration of keratinocytes in the epidermis. Chronic wounds are estimated to affect 1-2% of the human population worldwide and are a major socioeconomic burden. The prevalence of chronic wounds is expected to increase with the rising number of elderly and patients with diabetes and obesity, who are at high risk of developing chronic wounds. Since E-cadherin and the water channel aquaporin-3 are important for both skin function and cell migration, and aquaporin-3 is furthermore involved in wound healing of the skin demonstrated by impaired wound healing in aquaporin-3-null mice, we hypothesized that E-cadherin and aquaporin-3 expression may be dysregulated in chronic wounds. Therefore, we investigated the expression of E-cadherin and aquaporin-3 in biopsies from the edges of chronic wounds from human patients. This was accomplished by immunohistochemical stainings of E-cadherin and aquaporin-3 on serial sections followed by qualitative evaluation of staining patterns, which revealed low expression of both E-cadherin and aquaporin-3 at the wound edge. Future studies are needed to reveal if this downregulation is associated with the pathophysiology of chronic wounds.

AQP3-Dependent PI3K/Akt Modulation in Breast Cancer Cells.

Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated H2O2 uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2-related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.

LncRNA PVT1 delays skin photoaging by sequestering miR-551b-3p to release AQP3 expression via ceRNA mechanism.

Understanding human skin photoaging requires in-depth knowledge of the molecular and functional mechanisms. Human dermal fibroblasts (HDFs) gradually lose their ability to produce collagen and renew intercellular matrix with aging. Therefore, our study aims to reveal the mechanistic actions of a novel ceRNA network in the skin photoaging by regulating HDF activities. Photoaging-related genes were obtained in silico, followed by GO and KEGG enrichment analyses. Differentially expressed lncRNAs and miRNAs were screened from the GEO database to construct the ceRNA co-expression network. In skin photoaging samples, PVT1 and AQP3 were poorly expressed, while miR-551b-3p was highly expressed. The relationships among the lncRNA, miRNA and mRNA were explored through the ENCORI database and dual luciferase reporter assay. Mechanistically, PVT1 could sequester miR-551b-3p to upregulate the expression of AQP3, which further inactivated the ERK/p38 MAPK signaling pathway. HDFs were selected to construct an in vitro cell skin photoaging model, where the senescence, cell cycle distribution and viability of young and senescent HDFs were detected by SA-ß-gal staining, flow cytometry and CCK-8 assay. In vitro cell experiments confirmed that overexpression of PVT1 or AQP3 enhanced viability of young and senescent HDFs and inhibited HDF senescence, while miR-551b-3p upregulation counteracted the effect of PVT1. In conclusion, PVT1-driven suppression of miR-551b-3p induces AQP3 expression to inactivate the ERK/p38 MAPK signaling pathway, thereby inhibiting HDF senescence and ultimately delaying the skin photoaging.