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Functional and molecular characterization of the human neutral solute channel aquaporin-9
Abstract
In metabolically active cells, the coordinated transport of water and solutes is important for maintaining osmotic homeostasis. We recently identified a broad selective-neutral solute channel, AQP9, from rat liver that allows the passage of a wide variety of water and neutral solutes (H. Tsukaguchi, C. Shayakul, U. V. Berger, B. Mackenzie, S. Devidas, W. B. Guggino, A. N. van Hoek, and M. A. Hediger. J. Biol. Chem. 273: 24737-24743, 1998). A human homolog (hAQP9) with 76% amino acid sequence identity to rat AQP9 (rAQP9) was described, but its permeability was found to be restricted to water and urea (K. Ishibashi, M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki. Biochem. Biophys. Res. Commun. 244: 268-274, 1998). Here we report a reevaluation of the functional characteristics of hAQP9, its tissue distribution, the structure of its gene, and its chromosomal localization. When expressed in Xenopus oocytes, hAQP9 allowed passage of a wide variety of noncharged solutes, including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercurial-sensitive manner. These functional characteristics are similar to those of rAQP9. Based on Northern blot analysis, both rat and human AQP9 are abundantly expressed in liver, whereas, in contrast to rAQP9, hAQP9 is also expressed in peripheral leukocytes and in tissues that accumulate leukocytes, such as lung, spleen, and bone marrow. The human AQP9 gene is composed of 6 exons and 5 introns distributed over approximately approximately 25 kb. The gene organization is strikingly similar to that reported for human AQP3 and AQP7, suggesting their evolution from a common ancestral gene. The promoter region contains putative tonicity and glucocorticoid-responsive elements, suggesting that AQP9 may be regulated by osmolality and catabolism. Fluorescence in situ hybridization assigned its locus to chromosome 15 q22.1-22.2. Our data show that hAQP9 serves as a promiscuous solute channel expressed in both liver and peripheral leukocytes, where it is ideally suited to transport of metabolites and/or nutrients into and out of these cells
Supplementary resource (1)
Nucleotide Sequence
October 1998
... First cloned mammalian aquaglyceroporin was AQP3 which was able to f cilitate water and glycerol transportation [48], however in xenopus oocytes, AQP7, AQP and AQP10 transport water, glycerol, and urea [49]. Oocytes also allow the flow of a wid range of solutes through AQP9 [50]. Glycerol and urea are transported by aquagly eroporins, which are still poorly understood. ...
... First cloned mammalian aquaglyceroporin was AQP3 which was able to facilitate water and glycerol transportation [48], however in xenopus oocytes, AQP7, AQP9, and AQP10 transport water, glycerol, and urea [49]. Oocytes also allow the flow of a wide range of solutes through AQP9 [50]. Glycerol and urea are transported by aquaglyceroporins, which are still poorly understood. ...
Simple Summary
Aquaporins (AQPs) are transmembrane channel proteins that primarily transport water across the cellular membranes. AQPs have been found to be overexpressed in various human cancers, including prostate cancer. Clinical data suggest ideal prospects for AQPs as biomarkers. This review article mainly focuses on the opportunities for the development of AQPs as prognostic markers in prostate cancer.
Abstract
Prostate cancer is a complex heterogeneous disease that affects millions of males worldwide. Despite rapid advances in molecular biology and innovation in technology, few biomarkers have been forthcoming in prostate cancer. The currently available biomarkers for the prognosis of prostate cancer are inadequate and face challenges, thus having limited clinical utility. To date, there are a number of prognostic and predictive biomarkers identified for prostate cancer but lack specificity and sensitivity to guide clinical decision making. There is still tremendous scope for specific biomarkers to understand the natural history and complex biology of this heterogeneous disease, and to identify early treatment responses. Accumulative studies indicate that aquaporins (AQPs) a family of membrane water channels may serve as a prognostic biomarker for prostate cancer in monitoring disease advancement. In the present review, we discuss the existing prostate cancer biomarkers, their limitations, and aquaporins as a prospective biomarker of prognostic significance in prostate cancer.
... When combined with a related scorpion peptide KAaH2, migration was inhibited by 60%, despite the absence of anti-migratory effects of KAaH2 when administered alone [138]. The scorpion toxin chlorotoxin triggered internalization of a cell-surface complex containing MMP2 and the colocalized chloride channel ClC-3, also disrupting glioma cell volume regulation [131], and reduced invasiveness by 34% in acutely dissociated glioma cells from human biopsy tissue, and by 55% in cultured glioma cell lines [106,139]. Chlorotoxin preferentially binds to neuroectodermal tumors such as glioblastoma, with no effect on invasion in human melanoma, astrocyte or fibroblast cells in vitro [139], a property that is of interest for the development of glioblastoma therapeutics. Radio-iodinated TM-601, a synthetic form of chlorotoxin, reduced tumor growth with minimal toxic side effects in Phase I clinical trials [140]. ...
... The scorpion toxin chlorotoxin triggered internalization of a cell-surface complex containing MMP2 and the colocalized chloride channel ClC-3, also disrupting glioma cell volume regulation [131], and reduced invasiveness by 34% in acutely dissociated glioma cells from human biopsy tissue, and by 55% in cultured glioma cell lines [106,139]. Chlorotoxin preferentially binds to neuroectodermal tumors such as glioblastoma, with no effect on invasion in human melanoma, astrocyte or fibroblast cells in vitro [139], a property that is of interest for the development of glioblastoma therapeutics. Radio-iodinated TM-601, a synthetic form of chlorotoxin, reduced tumor growth with minimal toxic side effects in Phase I clinical trials [140]. ...
Comprising more than half of all brain tumors, glioblastoma multiforme (GBM) is a leading cause of brain cancer-related deaths worldwide. A major clinical challenge is presented by the capacity of glioma cells to rapidly infiltrate healthy brain parenchyma, allowing the cancer to escape control by localized surgical resections and radiotherapies, and promoting recurrence in other brain regions. We propose that therapies which target cellular motility pathways could be used to slow tumor dispersal, providing a longer time window for administration of frontline treatments needed to directly eradicate the primary tumors. An array of signal transduction pathways are known to be involved in controlling cellular motility. Aquaporins (AQPs) and voltage-gated ion channels are prime candidates as pharmacological targets to restrain cell migration in glioblastoma. Published work has demonstrated AQPs 1, 4 and 9, as well as voltage-gated potassium, sodium and calcium channels, chloride channels, and acid-sensing ion channels are expressed in GBM and can influence processes of cell volume change, extracellular matrix degradation, cytoskeletal reorganization, lamellipodial and filopodial extension, and turnover of cell-cell adhesions and focal assembly sites. The current gap in knowledge is the identification of optimal combinations of targets, inhibitory agents, and drug delivery systems that will allow effective intervention with minimal side effects in the complex environment of the brain, without disrupting finely tuned activities of neuro-glial networks. Based on published literature, we propose that co-treatments using AQP inhibitors in addition to other therapies could increase effectiveness, overcoming some limitations inherent in current strategies that are focused on single mechanisms. An emerging interest in nanobodies as drug delivery systems could be instrumental for achieving the selective delivery of combinations of agents aimed at multiple key targets, which could enhance success in vivo.
... Among the aquaglyceroporins, AQP9 is one of the most promiscuous AQPs. In addition to water, it is permeable to urea, nucleosides, lactate and glycerol [44,45]. When expressing AQP9 in oocytes the water permeability increased 30-fold [46]. ...
... In chronic inflammatory diseases, like Rheumatoid arthritis (RA) and psoriasis, the expression of AQP9 is increased and it has thus been proposed to be a potential marker of chronic inflammation [50]. The AQP9 gene holds a putative glucocorticoid binding motif [45] linking it to possible transcriptional responses in inflammatory reactions. The expression has also been shown to be affected by oestrogen [51]. ...
... In other words, aquaporins control the osmotic regulation of the schistosomes. Tsukaguchi et al., 1998Tsukaguchi et al., , 1999Braschi et al., 2006a,b;Gonen and Walz, 2006;Faghiri and Skelly, 2009;Faghiri et al., 2010 Tetraspanins They play an essential role in maintaining the plasma membrane structure of the schistosomes where they interact with one another. Also, interacts with many others, particularly associate proteins such as, integrins, MHC and co-stimulatory molecules to generate a huge signal transduction complexes known as tetraspanin-enriched microdomains (TEMs). ...
... Aquaporins act as channels to selectively control the influx and efflux of water molecules within cells. Certain aquaporins allow the diffusion of metabolites in and out of the cell (Tsukaguchi et al., 1998(Tsukaguchi et al., , 1999Gonen and Walz, 2006). ...
Schistosomiasis is one of the major parasitic diseases and second most prevalent among the group of neglected diseases. The prevalence of schistosomiasis may be due to environmental and socio-economic factors, as well as the unavailability of vaccines for schistosomiasis. To date, current treatment; mainly the drug praziquantel (PZQ), has not been effective in treating the early forms of schistosome species. The development of drug resistance has been documented in several regions globally, due to the overuse of PZQ, rate of parasitic mutation, poor treatment compliance, co-infection with different strains of schistosomes and the overall parasite load. Hence, exploring the schistosome tegument may be a potential focus for the design and development of targeted anti-schistosomal therapy, with higher bioavailability as molecular targets using nanotechnology. This review aims to provide a concise incursion on the use of various advance approaches to achieve targeted anti-schistosomal therapy, mainly through the use of nano-enabled drug delivery systems. It also assimilates the molecular structure and function of the schistosome tegument and highlights the potential molecular targets found on the tegument, for effective specific interaction with receptors for more efficacious anti-schistosomal therapy.
... AQP1, AQP3, AQP5, AQP8, AQP9, and, as recently reported [11], also AQP11 allow the transport of hydrogen peroxide, and, for this biophysical feature, are called peroxiporins [12][13][14][15]. Moreover, AQP7 and AQP9 have been reported to carry arsenite, AQP6 to transport nitrate/halide ions and AQP9 to facilitate monocarboxylate transport [16]. Some AQP channels have also been reported to facilitate the transmembrane exchange of physiologically important gases, such as CO 2 , NO, or O 2 [17][18][19]. ...
Septic shock is the most severe complication of sepsis, characterized by a systemic inflammatory response following bacterial infection, leading to multiple organ failure and dramatically high mortality. AQP9 is an aquaglyceroporin/peroxiporin mainly expressed in hepatocytes and neutrophils and recently associated with inflammatory and infectious responses. Here, we evaluated whether AQP9 has a role in mouse systemic inflammation during endotoxic shock. Wild-type ( Aqp9+/+; WT) and Aqp9−/− knockout (KO) male mice were submitted to endotoxic shock by i.p. injection of LPS (40 mg/kg) and the related survival times were followed for 72 h. Nitric oxide (NO) and superoxide anion (O ²⁻ ) production, and the expression of inducible NO‐synthase (iNOS) and cyclooxigenase‐2 (COX‐2) were measured in liver, kidney, aorta, heart and lung of mice. LPS‐treated KO mice survived significantly longer than WT mice, and 25% of the KO mice fully recovered from LPS treatment. The LPS‐injected KO mice had lower inflammatory NO and O ²⁻ productions and reduced iNOS and COX‐2 through impaired NF‐κB p65 activation in liver, kidney, aorta, and heart as compared to LPS‐treated WT mice. Treatment of rodent hepatoma FaO cells with the AQP9 blocker HTS13286 prevented the LPS‐induced increase of NO and O ²⁻ . AQP9 seems to have a role in the early acute phase of LPS‐induced endotoxic shock likely involving the NF‐κB pathway, potentially through mediating uptake of extracellular H 2 O 2 , and PTP-dependent signaling.
Support from Italian Government to GC: “Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale 2017” PRIN2017 grant # 2017J92TM5 and “Fondo Integrativo Speciale per la Ricerca 2020”-FISR 2020 CoVAPin grant # FISR2020IP_04051); Support from University of Bari Aldo Moro to GC: grant “Horizon Europe Seeds 2022-2023” NextgenerationEU Programma MUR-Fondo Promozione e Sviluppo-DM 737/2021-Uniba Euroseeds ID #S10. Support from Regione Puglia to PG: “Research for Innovation (REFIN)”, POR Puglia 2014/2020—Asse X—Azione 10.4, UNIBA132-ID #091C54A8.
This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
... This is a novel observation not reported previously for any AQP, including other structurally similar NIPs or NOD26. AQP9 from humans and a rat were found to transport large molecules including polyols (glycerol, D-mannitol, D-sorbitol), purines (adenine), pyrimidines (uracil, 5-fluorouracil), and thiourea, but not cyclic saccharides (63,64). To define the saccharide specificity permeation by HvNIP2;1, an extensive panel of substrates was examined. ...
Aquaporins can facilitate the passive movement of water, small polar molecules, and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral (d-mannose, d-galactose, d-xylose, d-mannoheptaose) and charged (N-acetyl d-glucosamine, d-glucosamine, d-glucuronic acid) aldoses, disaccharides (cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols, were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.
... AQP6 is unique among other hAQPs since it is the only one hAQP permeable to a wide range of anions, presenting a high permeability to nitrate (Ikeda et al. 2002). AQP9 is an interesting case, since it is the first AQP reported as permeable to monocarboxylates, in particular lactic acid/lactate (Tsukaguchi et al. 1999). Finally, the transport of several reactive oxygen and nitrogen have been tested by molecular dynamic simulations in hAQP1, in particular H2O2, • OH, • NO2, and • NO showing that all these species could be transported through AQP1 pore (Yusupov et al. 2019), although experimental evidence is still lacking. ...
Peroxynitrite (ONOO−/ONOOH) is a strong oxidant formed in vivo. It is cytotoxic at high concentrations, participates in different diseases and the aging process, and contributes to immune cell response mechanisms to invading organisms. The mechanism of action of peroxynitrite in these processes depends on the oxidation of biomolecules either by direct reaction or by the reaction of derived secondary species. This review presents the biological chemistry of peroxynitrite and the derived radicals nitrogen dioxide, hydroxyl radical and carbonate radical, particularly discussing their diffusion properties across cellular membranes and in the context of their short biological half-lives. While in some cases simple diffusion is the main route, peroxynitrite can also use anion channels to traverse cellular membranes. Furthermore, the potential role of aquaporins in facilitating the diffusion of peroxynitrite and its neutral conjugate acid is discussed. The wide range of membrane permeabilities exhibited by precursors, peroxynitrite and its secondary radical species results in an unequal compartmentalization of peroxynitrite formation and reactions and decay, thus partially defining the biological sites of action of peroxynitrite.
... Aquaporins (AQPs) are a family of membrane channel proteins that transport water molecules across the cell membrane [15,16]. Some AQP subtypes also selectively transport neutral small molecules such as glycerol, urea and sugars, and were designated aquaglyceroporins [17][18][19]. ...
Hydrogen peroxide (H2O2) is a major form of reactive oxygen species that play an important role in the survival, proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). The regulatory mechanisms of H2O2 homeostasis in BMSCs are not fully understood. Here we demonstrate for the first time that aquaglyceroporin AQP7 is a functional peroxiporin expressed in BMSCs and remarkably upregulated upon adipodenic induction. The proliferation ability of BMSCs from AQP7−/− mice was significantly decreased, as indicated by fewer clonal formation and cell cycle arrest compared with wildtype BMSCs. AQP7 deficiency caused accumulation of intracellular generated H2O2 during BMSCs proliferation, leading to oxidative stress and inhibition of PI3K/AKT and STAT3 signaling pathways. After adipogenic induction, however, the AQP7−/− BMSCs exhibited greatly reduced adipogenic differentiation with fewer lipid droplets formation and lower cellular triglycerides content than wildtype BMSCs. In such case AQP7 deficiency was found to diminish import of extracellular H2O2 produced by plasma membrane NADPH Oxidases, resulting in altered AMPK and MAPK signaling pathways and reduced expression of lipogenic genes C/EBPα and PPARγ. Our data revealed a novel regulatory mechanism of BMSCs function through AQP7-mediated H2O2 transport across plasma membrane.
Graphical Abstract
AQP7 is a peroxiporin mediating H2O2 transport across the plasma membrane of BMSCs. During proliferation, AQP7 deficiency results in accumulation of intracellular generated H2O2 due to reduced export, which inhibited STAT3 and PI3K/AKT/insulin receptor signaling pathways and cell proliferation. During adipogenic differentiation, however, AQP7 deficiency blocked the uptake of extracelluar H2O2 generated through plasma membrane NOX enzymes. The reduced intracellular H2O2 level causes decreased expression of lipogenic genes C/EBPα and PPARγ due to altered AMPK and MAPK signaling pathways, leading to impaired adipogenic differentiation
... This is a novel observation not reported previously for any AQP, including other structurally similar NIPs or NOD26. AQP9 from humans and a rat were found to transport large molecules including polyols (glycerol, D-mannitol, D-sorbitol), purines (adenine), pyrimidines (uracil, 5-fluorouracil), and thiourea, but not cyclic saccharides (59,60). To define the saccharide specificity permeation by HvNIP2;1, an extensive panel of substrates was examined. ...
Aquaporins can facilitate the passive movement of water and small polar molecules and some ions. The barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus oocyte swelling assays was found to permeate water, boric and germanic acids, sucrose and L-arabinose but not D-glucose or D-fructose. Other saccharides, such as neutral (D-mannose, D-galactose, D-xylose, D-mannoheptaose) and charged (N-acetyl D-glucosamine, D-glucosamine, D-glucuronic acid) aldoses, disaccharides (lactose, cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols were permeated to a much lower extent. Apparent permeation of hydrated KCl and MgSO 4 ion pairs was observed, while CH 3 COONa and NaNO 3 permeated at significantly lower rates. Experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO 3 blocked the permeation of all solutes. Full-scale steered molecular dynamics simulations of HvNIP2;1 and spinach SoPIP2;1 revealed possible rectification for water, boric acid, and sucrose transport, and defined key residues interacting with permeants. In a biological context, the simulated sucrose rectification could mediate its apoplastic-to-intracellular transport but not the reverse, thus, constituting a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2,993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalised solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for sustained food production.
Significance Statement
Aquaporins are fundamental to water and solute movements in nearly all living organisms. Solute selectivity inspections of the HvNIP2;1 aquaporin revealed that it transported water, hydroxylated metalloids boric and germanic acids, sucrose, L-arabinose, KCl, and MgSO 4 ion pairs, but not D-glucose or D-fructose and to lesser extent urea, and acyclic polyols. This poly-selective transport by HvNIP2;1 classified in the NIP3 sub-clade aquaporins may afford nutritional and protective roles during plant development and in response to abiotic stresses. It is anticipated that the solute specificity definition of HvNIP2;1 inspires protein engineering and in silico mining to develop plants, which when exposed to suboptimal soil conditions of high soil metalloids, would overcome toxicity for sustained food production.
... AQP9 is an aquaglyceroporin and functions in the transport of water, urea and glycerol [116,117,237,238]. It was later found to also transport mannitol, purines and pyrimidines (Table 2) [137,177,222,[239][240][241][242]. ...
Astrocytes have distinctive morphological and functional characteristics, and are found throughout the central nervous system. Astrocytes are now known to be far more than just housekeeping cells in the brain. Their functions include contributing to the formation of the blood–brain barrier, physically and metabolically supporting and communicating with neurons, regulating the formation and functions of synapses, and maintaining water homeostasis and the microenvironment in the brain. Aquaporins (AQPs) are transmembrane proteins responsible for fast water movement across cell membranes. Various subtypes of AQPs (AQP1, AQP3, AQP4, AQP5, AQP8 and AQP9) have been reported to be expressed in astrocytes, and the expressions and subcellular localizations of AQPs in astrocytes are highly correlated with both their physiological and pathophysiological functions. This review describes and summarizes the recent advances in our understanding of astrocytes and AQPs in regard to controlling water homeostasis in the brain. Findings regarding the features of different AQP subtypes, such as their expression, subcellular localization, physiological functions, and the pathophysiological roles of astrocytes are presented, with brain edema and glioma serving as two representative AQP-associated pathological conditions. The aim is to provide a better insight into the elaborate “water distribution” system in cells, exemplified by astrocytes, under normal and pathological conditions.
... AQP9 was independently discovered in rats [51] and humans [42] where strong mRNA expression was found in liver and peripheral leukocytes in humans [42,52], as well as liver, Leydig cells, and immature spermatocytes in rats [51]. Since then, AQP9 immunoreactivity has been reported in many other tissues (Figure 2), including rat and human epididymis [53,54], various cell types of rodent and primate brain [53,[55][56][57][58], mouse spinal cord [59], human astrocytes [60], human chorioamnion and placenta [61,62], human and mouse inner ear [63,64], human and mouse small intestine [65], human and rat prostate [66,67], human skeletal muscle [68], urothelium [69], porcine and rat oviduct [70], human fallopian tube [71], human adipose tissue [72], human retina [73,74], and human and mouse skin [75]. ...
Aquaporins (AQPs) are transmembrane channels essential for water, energy, and redox homeostasis, with proven involvement in a variety of pathophysiological conditions such as edema, glaucoma, nephrogenic diabetes insipidus, oxidative stress, sepsis, cancer, and metabolic dysfunctions. The 13 AQPs present in humans are widely distributed in all body districts, drawing cell lineage-specific expression patterns closely related to cell native functions. Compelling evidence indicates that AQPs are proteins with great potential as biomarkers and targets for therapeutic intervention. Aquaporin-9 (AQP9) is the most expressed in the liver, with implications in general metabolic and redox balance due to its aquaglyceroporin and peroxiporin activities, facilitating glycerol and hydrogen peroxide (H2O2) diffusion across membranes. AQP9 is also expressed in other tissues, and their altered expression is described in several human diseases, such as liver injury, inflammation, cancer, infertility, and immune disorders. The present review compiles the current knowledge of AQP9 implication in diseases and highlights its potential as a new biomarker for diagnosis and prognosis in clinical medicine.
... AQP1, AQP3, AQP5, AQP8, AQP9, and, as recently reported [22], also AQP11, allow the movement of hydrogen peroxide; hence, they are also counted as peroxiporins [23,24]. Moreover, AQP7 and AQP9 have been reported to mediate the transmembrane transport of arsenite, AQP6 to transport nitrate/halide ions, and AQP9 to facilitate monocarboxylate transport [25]. Some AQP channels have also been reported to mediate the transmembrane exchange of physiologically important gases, such as CO 2 , NO, or O 2 [26][27][28], although these gases can freely diffuse through the phospholipid bilayer. ...
The skin is the largest organ of the human body, serving as an effective mechanical barrier between the internal milieu and the external environment. The skin is widely considered the first-line defence of the body, with an essential function in rejecting pathogens and preventing mechanical, chemical, and physical damages. Keratinocytes are the predominant cells of the outer skin layer, the epidermis, which acts as a mechanical and water-permeability barrier. The epidermis is a permanently renewed tissue where undifferentiated keratinocytes located at the basal layer proliferate and migrate to the overlying layers. During this migration process, keratinocytes undertake a differentiation program known as keratinization process. Dysregulation of this differentiation process can result in a series of skin disorders. In this context, aquaporins (AQPs), a family of membrane channel proteins allowing the movement of water and small neutral solutes, are emerging as important players in skin physiology and skin diseases. Here, we review the role of AQPs in skin keratinization, hydration, keratinocytes proliferation, water retention, barrier repair, wound healing, and immune response activation. We also discuss the dysregulated involvement of AQPs in some common inflammatory dermatological diseases characterised by skin barrier disruption.
... [50] VNN2 (vascular noninflammatory molecule 2) participates in hematopoietic cell trafficking and oxidative stress while [51] AQP9 stimulates urea transport and osmotic water permeability. [52] IL1R1 (interleukin-1 alpha receptor alfa-1) an important mediator involved in many cytokine-induced immune and inflammatory responses, binds to the agonist ligands IL-1 and is inhibited by the antagonist IL-1Ra. Natural resistance-associated macrophage protein 1, encoded by the SLC11A1 gene, regulates macrophage activation and has been associated with infectious, autoimmune diseases and tuberculosis susceptibility. ...
Background: Yoga is a multifaceted spiritual tool that helps in maintaining health, peace of mind, and positive thoughts. In the context of asana, yoga is similar to physical exercise. This study aims to construct a molecular network to find hub genes that play important roles in physical exercise and yoga.
Methodology: We combined differentially expressed genes (DEGs) in yoga and exercise using computational bioinformatics from publicly available gene expression omnibus (GEO) datasets and identified the co-differentially expressed mRNAs with GEO2R. The co‑DEGs were divided into four different groups and each group was subjected to protein–protein interaction (PPI) network, pathways analysis, and gene ontology.
Results: Our study identified immunological modulation as a dominant target of differential expression in yoga and exercise. Yoga predominantly modulated genes affecting the Th1 and NK cells, whereas Cytokines, Macrophage activation, and oxidative stress were affected by exercise. We also observed that while yoga regulated genes for two main physiological functions of the body, namely Circadian Rhythm (BHLHE40) and immunity (LBP, T‑box transcription factor 21, CEACAM1), exercise‑regulated genes involved in apoptosis (BAG3, protein kinase C alpha), angiogenesis, and cellular adhesion (EPH receptor A1).
Conclusion: The dissimilarity in the genetic expression patterns in Yoga and exercise highlights the discrete effect of each in biological systems. The integration and convergences of multi‑omics signals can provide deeper and comprehensive insights into the various biological mechanisms through which yoga and exercise exert their beneficial effects and opens up potential newer research areas.
... In addition, AQP3 expression has been reported in macrophages (Zhu et al. 2011;Ikezoe et al. 2016;Marchini et al. 2003), lymphocytes (Hara-Chikuma et al. 2012Moon et al. 2004), and dendritic cells (Song et al. 2011;Moon et al. 2004;Marchini et al. 2003) in both mice and humans. Similarly, AQP9 expression has been reported in rodent (Elkjaer et al. 2000) and human (Tsukaguchi et al. 1999;Jelen et al. 2011;Matsushima et al. 2014;Ishibashi et al. 1998) polymorphonuclear leukocytes (granulocytes) where a role in neutrophil motility has been suggested (Karlsson et al. 2011;Loitto et al. 2002). AQP9 expression has also been reported in murine activated T-lymphocytes (Cui et al. 2015) and dendritic cells (De Santis et al. 2018). ...
The glycerol channel AQP7 facilitates glycerol efflux from adipose tissue (AT), and AQP7 deficiency has been suggested to promote obesity. However, the release of glycerol from AT is not fully blocked in AQP7-deficient mice, which suggests that either alternative glycerol channels are present in AT or significant simple diffusion of glycerol occurs. Previous investigations of the expression of other aquaglyceroporins (AQP3, AQP9, AQP10) than AQP7 in AT are contradictory. Therefore, we here aim at determining the cellular localization of AQP3 and AQP9 in addition to AQP7 in human and mouse AT using well-characterized antibodies for immunohistochemistry (IHC) and immunoblotting as well as available single-cell transcriptomic data from human and mouse AT. We confirm that AQP7 is expressed in endothelial cells and adipocytes in human AT and find ex vivo evidence for interaction between AQP7 and perilipin-1 in adipocytes. In addition, labeling for AQP7 in human AT also includes CD68-positive cells. No labeling for AQP3 or AQP9 was identified in endothelial cells or adipocytes in human or mouse AT using IHC. Instead, in human AT, AQP3 was predominantly found in erythrocytes, whereas AQP9 expression was observed in a small number of CD15-positive cells. The transcriptomic data revealed that AQP3 mRNA was found in a low number of cells in most of the identified cell clusters, whereas AQP9 mRNA was found in myeloid cell clusters as well as in clusters likely representing mesothelial progenitor cells. No AQP10 mRNA was identified in human AT. In conclusion, the presented results do not suggest a functional overlap between AQP3/AQP9/AQP10 and AQP7 in human or mouse white AT.
... The observed increase in glycerol release in ex vivo lipolysis experiments and the reduction in adipocyte size, could be explained by AQP7 upregulation for its crucial role in adipocyte glycerol permeability and control of triglyceride accumulation (Boque et al., 2013;Fiorentini et al., 2015). Phloretin, a chalcone abundant in apples, was identified as a non-specific AQP9 inhibitor that can reduce the size of bone-resorbing cells, the osteoclasts, with inhibition of osteoclast differentiation, a biological process mediated by AQP9 (Tsukaguchi et al., 1999;Aharon and Bar-Shavit, 2006). Recently, silybin, the major bioactive component of milk thistle, was found to upregulate AQP9 mRNA and protein expression in hepatic steatosis with increase in glycerol uptake, preventing triglycerides accumulation and liver dysfunction with decreased fat-induced autophagy. ...
Aquaglyceroporins, a sub-class of aquaporins that facilitate the diffusion of water, glycerol and other small uncharged solutes across cell membranes, have been recognized for their important role in human physiology and their involvement in multiple disorders, mostly related to disturbed energy homeostasis. Aquaglyceroporins dysfunction in a variety of pathological conditions highlighted their targeting as novel therapeutic strategies, boosting the search for potent and selective modulators with pharmacological properties. The identification of selective inhibitors with potential clinical applications has been challenging, relying on accurate assays to measure membrane glycerol permeability and validate effective functional blockers. Additionally, biologicals such as hormones and natural compounds have been revealed as alternative strategies to modulate aquaglyceroporins via their gene and protein expression. This review summarizes the current knowledge of aquaglyceroporins’ involvement in several pathologies and the experimental approaches used to evaluate glycerol permeability and aquaglyceroporin modulation. In addition, we provide an update on aquaglyceroporins modulators reported to impact disease, unveiling aquaglyceroporin pharmacological targeting as a promising approach for innovative therapeutics.
... These H 2 O 2 -permeable AQPs, now called peroxiporins, are proposed as key players to regulate redox signaling in various organisms [9,12]. In mammals, AQP9 is an aquaglyceroporin localized in the plasma membrane of hepatocytes facing the sinusoids [13,14] that transports H 2 O, glycerol and some other neutral solutes [15]. AQP9 is also a peroxiporin that efficiently transports H 2 O 2 [16]. ...
Aquaporin-9 (AQP9) is an aquaglyceroporin strongly expressed in the basolateral membrane of hepatocytes facing the sinusoids. AQP9 is permeable to hydrogen peroxide (H2O2) and glycerol as well as to water. Here, we report impaired liver regeneration in AQP9−/− mice which involves altered steady-state H2O2 concentration and glucose metabolism in hepatocytes. AQP9−/− mice showed remarkably delayed liver regeneration and increased mortality following 70% or 90% partial hepatectomy. Compared to AQP9+/+ littermates, AQP9−/− mice showed significantly greater hepatic H2O2 concentration and more severe liver injury. Fluorescence measurements indicated impaired H2O2 transport across plasma membrane of primary cultured hepatocytes from AQP9−/− mice, supporting the hypothesis that AQP9 deficiency results in H2O2 accumulation and oxidative injury in regenerating liver because of reduced export of intracellular H2O2 from hepatocytes. The H2O2 overload in AQP9−/− hepatocytes reduced PI3K-Akt and insulin signaling, inhibited autophagy and promoted apoptosis, resulting in impaired proliferation and increased cell death. In addition, hepatocytes from AQP9−/− mice had low liver glycerol and high blood glycerol levels, suggesting decreased glycerol uptake and gluconeogenesis in AQP9−/− hepatocytes. Adeno-associated virus (AAV)-mediated expression of hepatic expression of aquaglyceroporins AQP9 and AQP3 in AQP9−/− mice, but not water-selective channel AQP4, fully rescued the impaired liver regeneration phenotype as well as the oxidative injury and abnormal glucose metabolism. Our data revealed a pivotal role of AQP9 in liver regeneration by regulating hepatocyte H2O2 homeostasis and glucose metabolism, suggesting AQP9 as a novel target to enhance liver regeneration following injury, surgical resection or transplantation.
... In the rodent CNS, AQP9 is expressed in astrocytes, catecholaminergic neurons and endothelial cells of pial vessels [134,135], although CNS expression may be more restricted in humans [136]. AQP9 is permeable to glycerol and lactic acid [137], and thus it has been hypothesized that it has a role in brain energy metabolism in addition to water homeostasis [138]. In murine primary hepatocyte culture, AQP9 knockout reduced the glucose output from the liver when using glycerol as a substrate, suggesting that AQP9 facilitates glycerol uptake for gluconeogenesis [139,140]. ...
The aquaporins (AQPs) form a family of integral membrane proteins that facilitate the movement of water across biological membrane by osmosis, as well as facilitating the diffusion of small polar solutes. AQPs have been recognised as drug targets for a variety of disorders associated with disrupted water or solute transport, including brain oedema following stroke or trauma, epilepsy, cancer cell migration and tumour angiogenesis, metabolic disorders, and inflammation. Despite this, drug discovery for AQPs has made little progress due to a lack of reproducible high-throughput assays and difficulties with the druggability of AQP proteins. However, recent studies have suggested that targetting the trafficking of AQP proteins to the plasma membrane is a viable alternative drug target to direct inhibition of the water-conducting pore. Here we review the literature on the trafficking of mammalian AQPs with a view to highlighting potential new drug targets for a variety of conditions associated with disrupted water and solute homeostasis.
... AQP1, AQP3, AQP5, AQP8, AQP9, and AQP11 also allow the passage of hydrogen peroxide, and are therefore called peroxiporins [8e11]. Moreover, AQP7 and AQP9 have been reported to move arsenite [12], AQP6 to be permeable to nitrate and halide ions [13,14], AQP1 to be a gated ion channel [15], and AQP9 to facilitate monocarboxylate passage [16]. Some AQP channels have also been reported to facilitate transmembrane movement of gases of biological relevance such as NO, CO 2 or O 2 [17e19]. ...
Aquaglyceroporins are a group of the aquaporin (AQP) family of transmembrane water channels. While AQPs facilitate the passage of water, small solutes, and gases across biological membranes, aquaglyceroporins allow passage of water, glycerol, urea and some other solutes. Thanks to their glycerol permeability, aquaglyceroporins are involved in energy homeostasis. This review provides an overview of what is currently known concerning the functional implication and control of aquaglyceroporins in tissues involved in energy metabolism, i.e. liver, adipose tissue and endocrine pancreas. The expression, role and (dys)regulation of aquaglyceroporins in disorders affecting energy metabolism, and the potential relevance of aquaglyceroporins as drug targets to treat the alterations of the energy balance is also addressed.
... AQP1, AQP3, AQP5, AQP8, AQP9, and, as recently reported [11], also AQP11 allow the transport of hydrogen peroxide, and, for this biophysical feature, are called peroxiporins [12][13][14][15]. Moreover, AQP7 and AQP9 have been reported to carry arsenite, AQP6 to transport nitrate/halide ions and AQP9 to facilitate monocarboxylate transport [16]. Some AQP channels have also been reported to facilitate the transmembrane exchange of physiologically important gases, such as CO2, NO, or O2 [17][18][19]. ...
Septic shock is the most severe complication of sepsis, being characterized by a systemic inflammatory response following bacterial infection, leading to multiple organ failure and dramatically high mortality. Aquaporin-9 (AQP9), a membrane channel protein mainly expressed in hepatocytes and leukocytes, has been recently associated with inflammatory and infectious responses , thus triggering strong interest as a potential target for reducing septic shock-dependent mortality. Here, we evaluated whether AQP9 contributes to murine systemic inflammation during endotoxic shock. Wild type (Aqp9 +/+ ; WT) and Aqp9 gene knockout (Aqp9 −/− ; KO) male mice were submitted to endotoxic shock by i.p. injection of lipopolysaccharide (LPS; 40 mg/kg) and the related survival times were followed during 72 h. The electronic paramagnetic resonance and confocal microscopy were employed to analyze the nitric oxide (NO) and superoxide anion (O2 −) production , and the expression of inducible NO-synthase (iNOS) and cyclooxigenase-2 (COX-2), respectively , in the liver, kidney, aorta, heart and lung of the mouse specimens. LPS-treated KO mice survived significantly longer than corresponding WT mice, and 25% of the KO mice fully recovered from the endotoxin treatment. The LPS-injected KO mice showed lower inflammatory NO and O2 − productions and reduced iNOS and COX-2 levels through impaired NF-κB p65 activation in the liver, kidney, aorta, and heart as compared to the LPS-treated WT mice. Consistent with these results, the treatment of FaO cells, a rodent hepatoma cell line, with the AQP9 blocker HTS13268 prevented the LPS-induced increase of inflammatory NO and O2 −. A role for AQP9 is suggested in the early acute phase of LPS-induced endotoxic shock involving NF-κB signaling. The modulation of AQP9 expression/function may reveal to be useful in developing novel endotoxemia therapeutics.
... AQP9 is supposed to be a glycerol channel in liver cells. Transcriptional activities of AQP7 and 9 are both negatively regulated by insulin (Kishida, et al., 2000;Kishida et al., 2001;Tsukaguchi et al., 1999). ...
Thirteen members of aquaporin (AQP), a water channel, are expressed in mammals. In this review, we briefly overview these mammalian AQPs, then focus on AQP5, an exocrine gland-type AQP. Namely, we discuss: (1) the mechanism for coupling of AQP5 dynamics with the secretion and restoration cycle of amylase after isoproterenol (IPR) in the parotid gland (PG); (2) roles of parasympathetic nerve for maintaining AQP5 level in the submandibular gland (SMG), and; (3) AQP5 down-regulation in an experimental pathological model by LPS administration in the PG. We then move to the effects of single nucleotide mutation (SNP) found in rats and humans and its affected phenotypes. That is, G308A point mutation found in rat AQP5 cDNA resulted in amino acid substitutions of Gly103 for Asp103, and causes diminished expression of its protein product. In humans, several SNPs in AQP5 are found in European and Chinese families and cause autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma.
... AQP9'un üre transportu hariç su ve solut geçirgenliği HgCl2 ile inhibe edilebilir (50). Deneysel Diabetes mellitusta belirgin olarak düşen karaciğer AQP9 m-RNA miktarı, insülin uygulamasından sonra normal sevilerine ulaşır (49). ...
... AQP9'un üre transportu hariç su ve solut geçirgenliği HgCl2 ile inhibe edilebilir (50). Deneysel Diabetes mellitusta belirgin olarak düşen karaciğer AQP9 m-RNA miktarı, insülin uygulamasından sonra normal sevilerine ulaşır (49). ...
... Therefore, the osmotic regulation of AQP1 may be mediated by JNK pathways, which are induced by hypertonicity [12]. Chaoyang Zhang et al. found that erythropoietin maintained the integrity of the outer blood-retinal barrier and retinal edema by downregulating HIF-1α and JNK signals, thus upregulating the expression of ZO-1 and occludin in the RPE cells of early diabetic rats [15]. This nding indicates that JNK may also affect the permeability of blood vessels, thus changing the osmotic pressure inside and outside the vessels. ...
Background: The development and recovery(REC) of myopia is associated with changing of choroidal thickness(CT) in model of guinea pigs. In this process, Aquaporin-1 (AQP-1) is related to changes in choroidal thickness during the recovery from myopia, but the corresponding signaling pathway has not been clarified. The aim of this study was to investigate the effect of JNK1 on CT/AQP-1 and the recovery from myopia.
Methods: According to the different intravitreal injections in eyes that underwent form deprivation for 21 days, guinea pigs were divided into four groups: the REC group, the REC+anisomycin (agonist for JNK1, REC-AN) group, the REC+SP600125 (inhibitor for JNK1, REC-SP) group, and the REC+DMSO (REC-DM) group. Each group was divided into three subgroups according to the time of removal of the deprivation: 3 days (d), 7 d and 10 d. All animals underwent biometric measurements (refractive error, axial length (AL), and CT), and the protein expression of AQP-1 and p-JNK1 in the choroid was also measured.
Results: In the REC and REC-DM groups, significant differences in CT/ refractive error /AL/p-JNK1 or AQP-1 were only found in the 3d group compared with the normal control (NC) group (all p<0.05). In the REC-AN group, CT/p-JNK1 or AQP-1 in the 3d group was significantly higher than that in the other 3d’ groups (all p<0.05), but no significant difference in refractive error or AL was found compared with that in the NC group (all p>0.05). In the REC-SP group, a significant difference in refractive error /CT/p-JNK1 or AQP1 was found in the 3d/7d group compared with the NC group (all p<0.05), but AL was only found in the 3d groups (both p=0.001).
Conclusions: Changes in JNK1 phosphorylation can regulate AQP-1 and CT during the recovery from myopia and the recovery time. Thus, JNK1 may be a potential therapeutic target for preventing/treating myopia.
... This finding indicates that JNK may also affect the permeability of blood vessels, thus changing the osmotic pressure inside and outside the vessels. Second, AQP1, -4, and − 9 have an AP1 element in their promoter region, allowing JNK-dependent transcriptional regulation [16]. Furthermore, AQP1 may lead to increased vascular permeability and interstitial fluid effusion, thus causing choroid thickening. ...
Background: The development and recovery(REC) of myopia is associated with changing of choroidal thickness(CT) in modelof guinea pigs.In this process, Aquaporin-1 (AQP-1) is related to changes in choroidal thickness during the recovery from myopia, but the corresponding signaling pathway has not been clarified. The aim of this study was to investigate the effect of JNK1 on CT/AQP-1 and the recovery from myopia.
Methods: According to the different intravitreal injections in eyes that underwent form deprivation for 21 days, guinea pigs were divided into four groups: the REC group, the REC+anisomycin (agonist for JNK1, REC-AN) group, the REC+SP600125 (inhibitor for JNK1, REC-SP) group, and the REC+DMSO (REC-DM) group. Each group was divided into three subgroups according to the time of removal of the deprivation : 3 days (d), 7 d and 10 d. All animals underwent biometric measurements (refractive error, axial length (AL), and CT), and the protein expression of AQP-1 and p-JNK1 in the choroid was also measured.
Results: In the REC and REC-DM groups, significant differences in CT/ refractive error /AL/p-JNK1 or AQP-1 were only found in the 3d group compared with the normal control ( NC ) group (all p<0.05). In the REC-AN group, CT/p-JNK1 or AQP-1 in the 3d group was significantly higher than that in the other 3d’ groups (all p<0.05), but no significant difference in refractive error or AL was found compared with that in the NC group (all p>0.05). In the REC-SP group, a significant difference in refractive error /CT/p-JNK1 or AQP1 was found in the 3d/7d group compared with the NC group (all p<0.05), but AL was only found in the 3d groups (both p=0.001).
Conclusions: Changes in JNK1 phosphorylation can regulate AQP-1 and CT during the recovery from myopia and the recovery time. Thus, JNK1 may be a potential therapeutic target for preventing/treating myopia.
... OxSR, oxidative stress response; TonSR, tonicity stress response; UPR, unfolded protein response; HypSR, hypoxia stress response. (Yasui et al., 1999), or monocarboxylates by AQP9 (Tsukaguchi, Weremowicz, Morton, & Hediger, 1999), made this classification somehow obsolete. Moreover, H 2 O 2 can be transported by AQP3, AQP8, AQP9, and possibly AQP11 (Nordzieke & Medraño-Fernandez, 2018) and ammonia by AQP1, AQP3, AQP4, AQP6, AQP7, AQP8, and AQP9 (Assentoft et al., 2016;Geyer, Musa-Aziz, Qin, & Boron, 2013;Jahn et al., 2004). ...
Life entails an unpredictable succession of physical, chemical, mechanical and biological challenges, which perturb homeostasis and may cause damage. Hence, from the simplest unicellular beings to complex mammals, the capacity to promptly respond to different stresses is a fundamental pillar of life and one of the strongest elements in driving evolution. Reactive oxygen species (ROS) are archetypes of the friend/foe, eu/caco stress paradigm nicely introduced by Helmut Sies, the editor of this book. This chapter deals with H2O2, the most important ROS in human pathobiology. Capable of tuning the intensity and duration of tyrosine kinase signalling circuits, H2O2 is an essential second messenger in physiological conditions. At the same time, it becomes toxic for cells when at high concentrations. Its transport across membranes and diffusion must be therefore precisely regulated. Here we summarize recent findings that highlight how cells manage to target a most reactive species across membranes and within molecularly crowded compartments and tune its activity to integrate different stress circuits and optimize adaptive responses.
... In contrast, As 5+ utilizes phosphate transporters for cellular uptake (Csanaky and Gregus 2001;Kwong 2004). AQPs are almost present in every organ like the lung, liver, spleen, adipose tissues, and kidney (Tsukaguchi et al. 1999). Uptake of organic methylated MMAIII form is done by the AQP9 channel transporter. ...
Arsenic (As) is a toxic and ubiquitously present element. It is present in some places of the world in excessively high concentrations in water and soil that threaten public health. North America constitutes one of the hotspots of As contamination. In Canada and the USA, a number of reports show the contamination of As in a number of food items including rice, rice-based products, fruits, beverages, animal food items, etc. Further, As contamination of water sources is also known to occur in different states of both Canada and the USA. Thus, the problem of As contamination is widespread and needs attention. This chapter provides an overview of As contamination of water and food sources of North America.
... These authors showed also that deficiency of AQP9 decreased H 2 O 2 -induced cytotoxicity, suggesting an involvement of AQP9 in redox signaling. The potential phosphorylation sites located at Ser28, Ser11 and Ser222, probably representing PKC targets, have been suggested for the human isoform [94,95], supporting the hypothesis of AQP9 involvement in the redox signaling processes. ...
The transport of H2O2 across membranes by specific aquaporins (AQPs) has been considered the last milestone in the timeline of hydrogen peroxide discoveries in biochemistry. According to its concentration and localization, H2O2 can be dangerous or acts as a signaling molecule in various cellular processes as either a paracrine (intercellular) and/or an autocrine (intracellular) signal. In this review, we investigate and critically examine the available information on AQP isoforms able to facilitate H2O2 across biological membranes (“peroxiporins”), focusing in particular on their role in cancer. Moreover, the ability of natural compounds to modulate expression and/or activity of peroxiporins is schematically reported and discussed.
... Polyols are widely present in diverse organisms (Lewis and Smith, 1967;Pfyffer and Rast, 1980;Honegger et al., 1993;Gustavs et al., 2011), and they function to remove reactive oxygen species (Parida and Das, 2005), to adjust the osmotic pressure of compatible solutes (Wegmann, 1986;Reed et al., 1987), and to store the reducing power provided by NADH or NADPH (Jennings, 1985;Williamson et al., 2002). Polyol transporters have been reported in bacteria, such as D-arabinitol and ribitol transporters from Klebsiella pneumoniae (Heuel et al., 1997), in red alga, such as sugar and mannitol transporters from Galdieria sulphuraria (Schilling and Oesterhelt, 2007), in plants, such as the first reported mannitol transporter from celery (Noiraud et al., 2001), and in humans, such as glycerol, mannitol, sorbitol and the other neutral solute channels (Tsukaguchi et al., 1999). The orthologs or paralogs of these proteins have also been characterized as polyol transporters. ...
In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.
... The promoter region of AQP9 contains putative tonicity and glucocorticoid-responsive elements, suggesting that AQP9 may be regulated by osmolality and catabolism (42), involving osmoregulation of small molecules (43). This mechanism is important to the function of intestinal epithelial barrier (44). ...
The present study aimed to investigate the expression and function of aquaporin (AQP)9 in the intestinal tract of acute liver injury rat models. A total of 20 Sprague Dawley rats were randomly divided into four groups: Normal control (NC) group and acute liver injury groups (24, 48 and 72 h). Acute liver injury rat models were established using D‑amino galactose, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Tbil) and albumin were determined using an automatic biochemical analyzer. Proteins levels of myosin light chain kinase (MLCK) in rat intestinal mucosa were investigated via immunohistochemistry. Pathological features were observed using hematoxylin and eosin (H&E) staining. MLCK, AQP9 and claudin‑1 protein expression levels were detected via western blotting. Levels of ALT and AST in acute liver injury rats were revealed to steadily increase between 24 and 48 h time intervals, reaching a peak level at 48 h. Furthermore, TBil levels increased significantly until 72 h. Levels of ALT were revealed to significantly increase until the 48 h time interval, and then steadily decreased until the 72 h time interval. The acute liver injury 72 h group exhibited the greatest levels of MLCK expression among the three acute liver injury groups; however, all three acute liver injury groups exhibited enhanced levels of MLCK expression compared with the NC group. Protein levels of AQP9 and claudin‑1 were enhanced in the NC group compared with the three acute liver injury groups. H&E staining demonstrated that terminal ileum mucosal layer tissues obtained from the acute liver injury rats exhibited visible neutrophil infiltration. Furthermore, the results revealed that levels of tumor necrosis factor‑α, interleukin (IL)‑6 and IL‑10 serum cytokines were significantly increased in the acute liver injury groups. In addition, AQP9 protein expression was suppressed in acute liver injury rats, which induced pathological alterations in terminal ileum tissues may be associated with changes of claudin‑1 and MLCK protein levels.
... Permeability studies by stopped-flow light scattering also demonstrated that mSCs permeability to glycerol is inhibited by phloretin in 55% in relation to non-treated cells. Since phloretin is known as a general inhibitor of aquaglyceroporins [22,37,38] and that only two isoforms able to transport glycerol are expressed in mSCs, it is clear that AQP3 and AQP9 are pivotal for the transport of glycerol in the mSCs. Glycerol movement across membranes also occur through the phospholipid bilayer, by "simple diffusion", a thermodynamically disadvantaged pathway, of less importance and, above all, not controllable with respect to the "facilitated" pathway offered by aquaglyceroporins [20,21]. ...
High 17β-Estradiol (E2) levels are known to cause alterations of spermatogenesis and environments throughout the male reproductive tract. Sertoli cells (SCs) ensure an adequate environment inside the seminiferous tubule. Glycerol stands as essential for the maintenance of blood–testis barrier created by SCs, however, the role of E2 in this process is not known. Herein, we hypothesized that the effect of E2 on glycerol permeability in mouse SCs (mSCs) could be mediated by aquaglyceroporins. The expression of aquaglyceroporins was assessed by RT-PCR and qRT-PCR. Glycerol permeability was evaluated by stopped-flow light scattering. We were able to identify the expression of AQP3 and AQP9 in mSCs where AQP9 is more abundant than AQP3. Our results show that high E2 levels decrease AQP9 mRNA abundance with no influence on AQP3 in mSCs. Interestingly, high E2 levels decreased mSCs’ permeability to glycerol, while downregulating AQP9 expression, thus suggesting a novel mechanism by which E2 modulates fluid secretion in the testis. In conclusion, E2 is an important regulator of mSCs physiology and secretion through changes in AQP9 expression and function. Thus, alterations in glycerol permeability induced by E2 may be the cause for male infertility in cases associated with the presence of high E2 levels.
... Phloretin, an apple chalcone, has anti-oxidative and anti-inflammatory actions (Aliomrani et al., 2016). Phloretin is widely used as an inhibitor of AQP-mediated glycerol transport (Calamita et al., , 2012Rodríguez et al., 2014;Tsukaguchi, Weremowicz, Morton, & Hediger, 1999). Notably, glycerol is a key substrate of gluconeogenesis and synthesis of triacylglycerols (TAG) (Calamita et al., 2012). ...
A series of plant-derived bioactive compounds belonging to the class of polyphenols, terpenes, and capsaicinoids, interact with important pathophysiological pathways at a molecular, cellular and systemic level. Mechanisms of action include cell growth and differentiation, apoptosis, autophagy, inflammation, redox balance and metabolic and energy homeostasis. These effects might also involve the expression and function of Aquaporins (AQPs), a family of membrane channel proteins, involved in several body functions. The ultimate translational beneficial effect of such phytocompounds on AQPs in health and disease is a matter of intensive research. Results might provide novel therapeutic approaches to a number of human diseases. Here, we give an updated overview of this fast growing and promising field, discussing a number of phytocompounds and their action on AQPs and related potential clinical achievements.
... certain AQPs are also known as aquaglyceroporins since they participate in the transport of other small molecules, such as glycerol, urea or ammonia. AQP9, which was first identified in leukocytes (14), is an aquaglyceroporin that is expressed in numerous organs, with high expression levels detected in the liver, epididymis, testis, spleen and brain (15). There have been several studies on the functions of AQPs in certain organs. ...
Aquaporins (AQPs) are small integral membrane proteins that are essential for water transport across membranes. AQP9, one of the 13 mammalian AQPs (including AQP0 to 12), has been reported to be highly expressed in hydrarthrosis and synovitis patients. Given that several studies have identified signal transduction as an additional function of AQPs, it is hypothesized that AQP9 may modulate inflammatory signal transduction in chondrocytes. Therefore, the present study used a model of interleukin (IL)‑1β‑induced inflammation to determine the mechanisms associated with AQP9 functions in chondrocytes. Osteoarthritis (OA) and normal cartilage samples were subjected to immunohistological analysis. In addition, matrix metalloproteinase (MMP)3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‑5) mRNA and protein analysis was conducted in normal human articular chondrocytes from the knee (NHAC‑Kn) stimulated with IL‑1β by reverse transcription‑polymerase chain reaction (RT‑qPCR) and western blotting, respectively. AQP9 knockdown was also performed by transfection of AQP9‑specific small interfering RNA using Lipofectamine. AQP1, 3, 7, 9 and 11 mRNA expression levels were detected in OA human chondrocytes and in IL‑1β‑treated normal human chondrocytes. The levels of AQP9, MMP‑3, MMP‑13 and ADAMTS‑5 mRNA were increased by treatment with 10 ng/ml IL‑1β in a time‑dependent manner, while knockdown of AQP9 expression significantly decreased the mRNA levels of the MMP3, MMP13 and ADAMTS‑5 genes, as well as the phosphorylation of IκB kinase (IKK). Treatment with a specific IKK inhibitor also significantly decreased the expression levels of MMP‑3, MMP‑13 and ADAMTS‑5 in response to IL‑1β stimulation. Furthermore, immunohistochemical analysis demonstrated that AQP9 and inflammatory markers were highly expressed in OA cartilage. In addition, the downregulation of AQP9 in cultured chondrocytes decreased the catabolic gene expression in response to IL‑1β stimulation through nuclear factor‑κB signaling. Therefore, AQP9 may be a promising target for the treatment of OA.
... Phloretin, an abundant chalcone in apples, is a protective agent with anti-oxidative stress and anti-inflammatory actions (Aliomrani et al., 2016). Phloretin is also known for gating the aquaglyceroporins by inhibiting the AQP-mediated transport of glycerol and urea (Tsukaguchi et al., 1999;Calamita et al., 2008Calamita et al., , 2012Rodriguez et al., 2014), a metabolic intermediate substrate of gluconeogenesis and triacylglycerols (TAG) synthesis (Calamita et al., 2012), and a variety of urea transporters and urea-conducting AQPs such as AQP3, AQP7, and AQP9 (Shayakul et al., 2001;Fenton et al., 2004). AQP9-facilitated urea extrusion out of liver was evoked to explain the loss of urea and diuresis that characterizes mice submitted to high protein diet. ...
Plant-derived bioactive compounds have protective role for plants but may also modulate several physiological processes of plant consumers. In the last years, a wide spectrum of phytochemicals have been found to be beneficial to health interacting with molecular signaling pathways underlying critical functions such as cell growth and differentiation, apoptosis, autophagy, inflammation, redox balance, cell volume regulation, metabolic homeostasis, and energy balance. Hence, a large number of biologically active phytocompounds of foods have been isolated, characterized, and eventually modified representing a natural source of novel molecules to prevent, delay or cure several human diseases. Aquaporins (AQPs), a family of membrane channel proteins involved in many body functions, are emerging among the targets of bioactive phytochemicals in imparting their beneficial actions. Here, we provide a comprehensive review of this fast growing topic focusing especially on what it is known on the modulatory effects played by several edible plant and herbal compounds on AQPs, both in health and disease. Phytochemical modulation of AQP expression may provide new medical treatment options to improve the prognosis of several diseases.
... At the subcellular level, AQP9 is localized in the plasma membrane as well as in the inner mitochondrial membrane [23,26]. AQP9 belongs to the aquaglyceroporin subfamily of water channels and is permeable to a broad range of substrates including glycerol and urea [27][28][29], monocarboxylates (lactate and β-hydroxybutyrate) [28], purines [28], ammonia [30] and arsenite [31]. ...
More than 90% of the cases of Parkinson’s disease have unknown etiology. Gradual loss of dopaminergic neurons of substantia nigra is the main cause of morbidity in this disease. External factors such as environmental toxins are believed to play a role in the cell loss, although the cause of the selective vulnerability of dopaminergic neurons remains unknown. We have previously shown that aquaglyceroporin AQP9 is expressed in dopaminergic neurons and astrocytes of rodent brain. AQP9 is permeable to a broad spectrum of substrates including purines, pyrimidines, and lactate, in addition to water and glycerol. Here we test our hypothesis that AQP9 serves as an influx route for exogenous toxins and, hence, may contribute to the selective vulnerability of nigral dopaminergic (tyrosine hydroxylase-positive) neurons. Using Xenopus oocytes injected with Aqp9 cRNA, we show that AQP9 is permeable to the parkinsonogenic toxin 1-methyl-4-phenylpyridinium (MPP⁺). Stable expression of AQP9 in HEK cells increases their vulnerability to MPP+ and to arsenite—another parkinsonogenic toxin. Conversely, targeted deletion of Aqp9 in mice protects nigral dopaminergic neurons against MPP⁺ toxicity. A protective effect of Aqp9 deletion was demonstrated in organotypic slice cultures of mouse midbrain exposed to MPP⁺in vitro and in mice subjected to intrastriatal injections of MPP⁺in vivo. Seven days after intrastriatal MPP⁺ injections, the population of tyrosine hydroxylase-positive cells in substantia nigra is reduced by 48% in Aqp9 knockout mice compared with 67% in WT littermates. Our results show that AQP9 –selectively expressed in catecholaminergic neurons—is permeable to MPP⁺ and suggest that this aquaglyceroporin contributes to the selective vulnerability of nigral dopaminergic neurons by providing an entry route for parkinsonogenic toxins. To our knowledge this is the first evidence implicating a toxin permeable membrane channel in the pathophysiology of Parkinson’s disease.
Fatty acid (FA) metabolism dysfunction of white adipose tissue (WAT) underlies obesity and insulin resistance in response to high calorie intake and/or endocrine-disrupting chemicals (EDCs), among other factors. Arsenic is an EDC that has been associated with metabolic syndrome and diabetes. However, the combined effect of a high-fat diet (HFD) and arsenic exposure on WAT FA metabolism has been little studied. FA metabolism was evaluated in visceral (epididymal and retroperitoneal) and subcutaneous WAT of C57BL/6 male mice fed control or HFD (12 and 40% kcal fat, respectively) for 16 weeks together with an environmentally relevant chronic arsenic exposure through drinking water (100 μg/l) during the second half of the study. In mice fed HFD, arsenic potentiated the increase of serum markers of selective insulin resistance in WAT and fatty acid re-esterification and the decrease in the lipolysis index. Retroperitoneal was the WAT most affected, where the combination of arsenic and HFD in contrast to HFD, generated higher weight, larger adipocytes, increased triglyceride content, and decreased fasting stimulated lipolysis evidenced by lower phosphorylation of HSL and perilipin. At the transcriptional level, arsenic in mice fed either diet downregulated genes involved in fatty acid uptake (LPL, CD36), oxidation (PPARα, CPT1), lipolysis (ADRß3) and glycerol transport (AQP7 and AQP9). Additionally, arsenic potentiated hyperinsulinemia induced by HFD, despite a slight increase in weight gain and food efficiency. Thus, the second hit of arsenic in sensitized mice by HFD worsens fatty acid metabolism impairment in WAT, mainly retroperitoneal, along with an exacerbated insulin resistance phenotype.
Aquaporins (AQPs) are a family of membrane water channels that basically function as regulators of intracellular and intercellular water flow. To date, 13 AQPs, distributed widely in specific cell types in various organs and tissues, have been characterized in humans. A pair of NPA boxes forming a pore is highly conserved among all aquaporins and is also key residues for the classification of AQP superfamily into four groups according to primary sequences. AQPs may also be classified based on their transport properties. So far, chromosome localization and gene structure of 13 human AQPs have been identified, which is definitely helpful for studying phenotypes and potential targets in naturally occurring and synthetic mutations in human or cells.
Aquaporins (AQPs) are a family of small transmembrane proteins that selectively transport water and other small molecules and ions following an osmotic gradient across cell plasma membranes. This enables them to regulate numerous functions including water homeostasis, fat metabolism, proliferation, migration, and adhesion. Previous structural and functional studies highlight a strong biological relationship between AQP protein expression, localization, and key biological functions in normal and cancer tissues, where aberrant AQP expression correlates with tumorigenesis and metastasis. In this review, we discuss the roles of AQP1, AQP3, AQP4, AQP5, and AQP7 in breast cancer progression and metastasis, including the role of AQPs in the tumor microenvironment, to highlight potential contributions of stromal-derived to epithelial-derived AQPs to breast cancer. Emerging evidence identifies AQPs as predictors of response to cancer therapy and as targets for increasing their sensitivity to treatment. However, these studies have not evaluated the requirements for protein structure on AQP function within the context of breast cancer. We also examine how AQPs contribute to a patient’s response to cancer treatment, existing AQP inhibitors and how AQPs could serve as novel predictive biomarkers of therapy response in breast cancer. Future studies also should evaluate AQP redundancy and compensation as mechanisms used to overcome aberrant AQP function. This review highlights the need for additional research into how AQPs contribute molecularly to therapeutic resistance and by altering the tumor microenvironment.
The aquaporins (AQPs) are a family of small integral membrane proteins that facilitate the bidirectional transport of water across biological membranes in response to osmotic pressure gradients as well as enable the transmembrane diffusion of small neutral solutes (such as urea, glycerol, and hydrogen peroxide) and ions. AQPs are expressed throughout the human body. Here, we review their key roles in fluid homeostasis, glandular secretions, signal transduction and sensation, barrier function, immunity and inflammation, cell migration, and angiogenesis. Evidence from a wide variety of studies now supports a view of the functions of AQPs being much more complex than simply mediating the passive flow of water across biological membranes. The discovery and development of small-molecule AQP inhibitors for research use and therapeutic development will lead to new insights into the basic biology of and novel treatments for the wide range of AQP-associated disorders.
Background & aims:
Patient-derived human induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes (hiPSC-Heps) have facilitated the study of rare genetic liver diseases. Here, we aimed to establish an in vitro liver disease model of the urea cycle disorder ornithine transcarbamylase deficiency (OTCD) using patient-derived hiPSC-Heps.
Approach & results:
Before modeling OTCD, we addressed the question of why hiPSC-Heps generally secrete less urea than adult primary human hepatocytes (PHHs). Since hiPSC-Heps are not completely differentiated and maintain some characteristics of fetal PHHs, we compared gene expression levels in human fetal and adult liver tissue to identify genes responsible for reduced urea secretion in hiPSC-Heps. We found lack of aquaporin 9 (AQP9) expression in fetal liver tissue as well as in hiPSC-Heps, and showed that forced expression of AQP9 in hiPSC-Heps restores urea secretion and normalizes the response to ammonia challenge by increasing ureagenesis. Furthermore, we proved functional ureagenesis by challenging AQP9-expressing hiPSC-Heps with ammonium chloride labeled with the stable isotope [15 N] (15 NH4 Cl) and by assessing enrichment of [15 N]-labeled urea. Finally, using hiPSC-Heps derived from patients with OTCD, we generated a liver disease model that recapitulates the hepatic manifestation of the human disease. Restoring OTC expression-together with AQP9-was effective in fully correcting OTC activity and normalizing ureagenesis as assessed by 15 NH4 Cl stable-isotope challenge.
Conclusion:
Our results identify a critical role for AQP9 in functional urea metabolism and establish the feasibility of in vitro modeling of OTCD with hiPSC-Heps. By facilitating studies of OTCD genotype/phenotype correlation and drug screens, our model has potential for improving the therapy of OTCD.
Introduction
This study aimed to examine maternal serum aquaporin 9 levels in pregnant women with gestational diabetes mellitus and to compare them with non-diabetic pregnant women.
Methods
Forty-one pregnant women between 37 and 39 weeks of gestation complicated with gestational diabetes mellitus and 39 non-diabetic pregnant women at similar gestational weeks without additional obstetric complications were included in this cross-sectional study. Maternal serum aquaporin 9 levels and leptin levels of the cases were measured.
Results
Maternal serum leptin and aquaporin 9 levels in pregnant women with GDM were found to be significantly higher than in the control group (p < .001). In the study group, first-minute Apgar scores were significantly lower and birth weight significantly higher (p = .001 and .005, respectively). A weak but significant positive correlation between aquaporin 9 levels and maternal body mass index (r = 0.279, p = .012), birth weight (r = 0.433, p < .001), and hemoglobin A1c (r = 0.354, p = .001) levels was detected. A significant positive correlation was detected between maternal serum aquaporin 9 levels and leptin levels (r = 0.331, p = .003).
Conclusion
The increased aquaporin 9 levels detected in cases with gestational diabetes mellitus might be a marker of the poor maternal metabolic environment specific to diabetes and might contribute to the pathophysiology of gestational diabetes.
The spectrum of putative and experimentally shown permeants of cellular water and solute channels of the ubiquitous aquaporin family is still increasing. Virtually all AQP substrates, e.g. water, glycerol, urea, hydrogen peroxide, or carbon dioxide, are permanently neutral small molecule compounds. Several reports, however, describe aquaporins that exhibit lactate permeability. Lactate in aqueous solution undergoes a pH-dependent protonation equilibrium with neutral lactic acid, which likely represents the actual substrate form passing the aquaporin channel. Certain aquaporins, however, appear to be better geared for lactate/lactic acid permeability even at low proton availability. Here, we discuss the structural properties of such aquaporins and compare them to the microbial protein family of the formate-nitrite (lactate) transporters that assume the aquaporin fold despite unrelated protein sequences.
Aquaporins are membrane channels in the broad family of major intrinsic proteins (MIPs), with 13 classes showing tissue-specific distributions in humans. As key physiological modulators of water and solute homeostasis, mutations, and dysfunctions involving aquaporins have been associated with pathologies in all major organs. Increases in aquaporin expression are associated with greater severity of many cancers, particularly in augmenting motility and invasiveness for example in colon cancers and glioblastoma. However, potential roles of altered aquaporin (AQP) function in reproductive cancers have been understudied to date. Published work reviewed here shows distinct classes aquaporin have differential roles in mediating cancer metastasis, angiogenesis, and resistance to apoptosis. Known mechanisms of action of AQPs in other tissues are proving relevant to understanding reproductive cancers. Emerging patterns show AQPs 1, 3, and 5 in particular are highly expressed in breast, endometrial, and ovarian cancers, consistent with their gene regulation by estrogen response elements, and AQPs 3 and 9 in particular are linked with prostate cancer. Continuing work is defining avenues for pharmacological targeting of aquaporins as potential therapies to reduce female and male reproductive cancer cell growth and invasiveness.
Purpose
The development and recovery(REC) of myopia is associated with changing of choroidal thickness(CT) in model of guinea pigs. Aquaporin-1 (AQP-1) is related to the changes of CT during the recovery from myopia, but the corresponding signaling pathway has not been clarified. This study aimed to investigate the effect of JNK1 on CT/AQP-1 and the recovery from myopia.
Materials and Methods
According to the different single intravitreal injections in eyes that underwent form deprivation for 21 days, guinea pigs were divided into four groups: the REC group, the REC+anisomycin (REC-AN, agonist for JNK1, 0.2 nmol) group, the REC+SP600125 (REC-SP, inhibitor for JNK1, 0.2 nmol) group, and the REC+dimethyl sulfoxide (REC-DM) group. Each group was divided into three subgroups based on the duration of the form deprivation: 3 days (d), 7 d and 10 d. All animals underwent biometric measurements (refractive error, axial length (AL), and CT), and the protein expression of AQP-1 and p-JNK1 in the choroid was also measured.
Results
In REC and REC-DM groups, significant differences in CT/refractive error/AL/p-JNK1 or AQP-1 were only found in the 3d group compared with normal control (NC) group (all p < .05). In REC-AN group, CT/p-JNK1 or AQP-1 in 3d group was significantly higher than that in other 3d groups (all p < .05), but no significant difference in refractive error or AL was found compared with NC group at three time points (all p > .05). In REC-SP group, a significant difference in refractive error/CT/p-JNK1 or AQP1 was found in 3d/7d group compared with NC group (all p < .05), but AL was only found in 3d groups (p = .001).
Conclusions
Changes in JNK1 phosphorylation can regulate AQP-1 and CT during the recovery from myopia and the recovery time. Thus, JNK1 may be a potential therapeutic target for preventing/treating myopia.
Water can diffuse freely through the lipid bilayer at a limited rate or by water channel proteins in a rapid speed. Aquaporins (AQPs) are a family of membrane water channel proteins that mainly function as regulators of intracellular and intercellular water flow. They are distributed wildly in specific cell types in multiple organs and tissues. Since the first AQP was identified, 13 AQPs have been characterized in mammals. Structural analysis shows that AQPs are homotetramers with each AQP monomer containing six transmembrane α-helices, two half helices and five connecting loops with two conserved asparagine-proline-alanine (NPA) motifs embedding into the plasma membrane. AQPs are demonstrated to selectively transport water but also some other small molecules. The cellular functions of aquaporins are regulated mainly by posttranslational modifications, e.g., phosphorylation, ubiquitination, glycosylation, subcellular distribution, degradation, and protein interactions. Aquaporins, in particular, AQP2 plays an important role in some disease conditions such as water loss and gain. Insights into the molecular mechanisms regulating AQPs and its clinical significance are proving to be fundamental for development of novel therapeutic targets or reliable diagnostic and prognostic biomarkers. In this chapter, we summarize the molecular aspects of aquaporins which include the isoforms, crystal structure and the molecular mechanisms underlying the regulation of AQPs, with most focus on arginine vasopressin-regulated AQP2.
10‐Hydroxy‐trans‐2‐decenoic acid (10H2DA) is a unique lipid component of royal jelly produced by worker honeybees that exerts insulin‐like effects. We herein investigated the effects of 10H2DA on the gene expression of aquaporin 9 (AQP9), which functions as a glycerol transporter in the liver, to clarify whether 10H2DA modulates energy metabolism. 10H2DA suppressed AQP9 gene expression in HepG2 cells by promoting the phosphorylation of Akt and AMP‐activated protein kinase (AMPK). This suppression was partially recovered by the treatment of cells with inhibitors for Akt and AMPK. Based on the result showing that leptomycin B partially recovered the suppression of AQP9 gene expression, 10H2DA inhibited the expression of Foxa2, a transcription factor for the AQP9 gene, and also induced its nuclear exclusion. Although 10H2DA up‐regulated phosphoenolpyruvate carboxykinase and glucose‐6‐phosphatase gene expression, this was suppressed through the modulation of Foxa2 by insulin. These results suggest that 10H2DA suppresses AQP9 gene expression through the phosphorylation of Akt and AMPK and down‐regulation of Foxa2 expression. 10H2DA suppressed AQP9 gene expression in HepG2 cells by promoting the phosphorylation of Akt and AMP‐activated protein kinase (AMPK). 10H2DA inhibited the expression of Foxa2, a transcription factor for the AQP9 gene, a transcription factor for the AQP9 gene, and also induced its nuclear exclusion. 10H2DA suppresses AQP9 gene expression through the phosphorylation of Akt and AMPK and down‐regulated expression of Foxa2.
Arsenic, a ubiquitous metalloid, is naturally present in the lithosphere (Earth’s crusts, soil, rock, etc.), hydrosphere (surface water, aquifers, deep wells, etc.), atmosphere, and biosphere. In South Asia, Some studies reported that GSH contamination in groundwater in the Ganga- Brahmaputra fluvial plains in India and Padma-Meghna fluvial plains in Bangladesh has been found to have a huge impact on human health, and its consequences have been reported as the world’s biggest natural groundwater calamities. After entering into the body, it is distributed in a large number of organs including the lungs, liver, kidney, and skin. The clinical manifestations of arsenic poisoning are myriad, and the correct diagnosis depends largely on awareness of the problem. It is very difficult to diagnose early symptoms of arsenicosis because such non-specific symptoms may also be present in many other diseases. Medicine used for remedy of arsenicosis has been found to be unsatisfactory by repeated application and experience. The number of chelating drugs has been used for the treatment of arsenic poisoning. Some of them have been found to be effective during acute poisoning; however, there is still no safe and effective antidote available for treating chronic arsenicosis. Few natural and herbal extracts too have been attempted with limited success. This chapter reviews some of the fundamentals of arsenic toxicity and also summarizes currently available remedial measures.
Dendritic cells (DCs) are the most potent antigen-presenting cells able to trigger the adaptive immune response to specific antigens. When non-self-antigens are captured, DCs switch from an “immature” to a “mature” state to fulfill their function. Among the several surface proteins involved in DCs maturation, the role of aquaporins (AQPs) is still poorly understood. Here we investigated the expression profile of Aqps in murine bone marrow derived dendritic cells (BMDCs). Among the Aqps analyzed, Aqp9 was the most expressed by DCs. Its expression level was significantly upregulated 6 h following LPS exposure. Chemical inhibition of Aqp9 led to a decreased inflammatory cytokines secretion. BMDCs from AQP9-KO mice release lower amount of inflammatory cytokines and chemokines and increased release of IL-10. Despite the reduced release of inflammatory cytokines, Aqp9-KO mice were not protected from DSS induced colitis. All together, our data indicate that AQP9 blockade can be an efficient strategy to reduce DCs inflammatory response but it is not sufficient to protect from acute inflammatory insults such as DSS induced colitis.
Aquaporins (AQPs) constitute an ancient and diverse protein family present in all living organisms, indicating a common ancient ancestor. However, during evolution, these organisms appear and evolve differently, leading to different cell organizations and physiological processes. Amongst the eukaryotes, an important distinction between plants and animals is evident, the most conspicuous difference being that plants are sessile organisms facing ever-changing environmental conditions. In addition, plants are mostly autotrophic, being able to synthesize carbohydrates molecules from the carbon dioxide in the air during the process of photosynthesis, using sunlight as an energy source. It is therefore interesting to analyze how, in these different contexts specific to both kingdoms of life, AQP function and regulation evolved. This review aims at highlighting similarities and differences between plant and mammal AQPs. Emphasis is given to the comparison of isoform numbers, their substrate selectivity, the regulation of the subcellular localization, and the channel activity.
The purpose of this research was to explore the behavior of aquaporins (AQPs) in an in vitro model of Parkinson's disease that is a recurrent neurodegenerative disorder caused by the gradual, progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Because of postmortem studies have provided evidences for oxidative damage and alteration of water flow and energy metabolism, we carried out an investigation about AQP4 and 9, demonstrated in the brain to maintain water and energy homeostasis. As an appropriate in vitro cell model, we used SH-SY5Y cultures and induced their differentiation into a mature dopaminergic neuron phenotype with retinoic acid (RA) alone or in association with phorbol-12-myristate-13-acetate (MPA). The association RA plus MPA provided the most complete and mature neuron phenotype, as demonstrated by high levels of β-Tubulin III, MAP-2, and tyrosine hydroxylase. After validation of cell differentiation, the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and H2O2 were applied to reproduce a Parkinson's-like stress. The results confirmed RA/MPA differentiated SH-SY5Y as a useful in vitro system for studying neurotoxicity and for using in a MPTP and H2O2-induced Parkinson's disease cell model. Moreover, the data demonstrated that neuronal differentiation, neurotoxicity, neuroinflammation, and oxidative stress are strongly correlated with dynamic changes of AQP4 and 9 transcription and transduction. New in vitro and in vivo experiments are needed to confirm these innovative outcomes.
The cDNA for the fifth mammalian aquaporin (AQP5) was isolated from rat, and expression was demonstrated in rat salivary and lacrimal glands, cornea, and lung (Raina, S.,
Preston, G. M., Guggino, W. B., and Agre, P.(1995) J. Biol. Chem. 270, 1908-1912). Here we report the isolation and characterization of the human AQP5 cDNA and gene. The AQP5 cDNA from a human submaxillary gland library contains a 795-base pair open reading frame encoding a 265-amino acid protein.
The deduced amino acid sequences of human and rat AQP5 are 91% identical with 6 substitutions in the 22-amino acid COOH-terminal domain. Expression of human AQP5 in Xenopus oocytes conferred mercurial-sensitive osmotic water permeability (P) equivalent to other aquaporins. The human AQP5 structural gene resides within a 7.4-kilobase SalI-EcoRI fragment with four exons corresponding to amino acids 1-121, 122-176, 177-204, and 205-265 separated by introns of 1.2,
0.5, and 0.9 kilobases. A transcription initiation site was identified 518 base pairs upstream of the initiating methionine.
Genomic Southern analysis indicated that AQP5 is a single copy gene which localized to human chromosome 12q13; this coincides with the chromosomal locations of the homologous
human genes MIP and AQP2, thus confirming 12q13 as the site of an aquaporin gene cluster. The mouse gene localized to distal chromosome 15. This information
may permit molecular characterization of AQP5 expression during normal development and in clinical disorders.
This report describes the identification and purification of a nuclear protein from rat liver that binds selectively to DNA sequences associated with several animal virus enhancers. The binding activity was tracked by direct DNase I footprinting through four steps of biochemical fractionation. These procedures led to the identification of a polypeptide species exhibiting an apparent molecular weight of 20 kD that accounts for enhancer binding activity. DNase I and dimethyl sulfate footprinting assays were used to examine the manner in which the purified protein binds to enhancer elements associated with SV40, murine sarcoma virus, and polyoma virus. The results of these assays indicate that the initial interaction established between the 20-kD protein and each viral enhancer occurs via a common DNA sequence known as the enhancer core homology.
Deletion experiments have defined two stretches of DNA (genetic elements), lying close to the promoter for a human gene for metallothionein, that separately mediate the induction of the gene by heavy metal ions, particularly cadmium, and by glucocorticoid hormones. The element responsible for induction by cadmium is duplicated, yet a single copy is fully functional; the element responsible for induction by glucocorticoid hormones is coincident with the DNA-binding site for the glucocorticoid hormone receptor.
The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.
Splice junction sequences from a large number of nuclear and viral genes encoding protein have been collected. The sequence
ACAG/GTGAAGT was found to be a consensus of 139 exon-intron boundaries (or donor sequences) and (CT)nNTCAG/G was found to be a consensus of 130 intron-exon boundaries (or acceptor sequences). The possible role of splice junction
sequences as signals for processing is discussed.
Water transport in highly water-permeable membranes is conducted by water-selective pores--namely, water channels. The recent cloning of water channels revealed the water-selective characteristics of these proteins when expressed in Xenopus oocytes or reconstituted in liposomes. Currently, it is assumed that the function of water channels is to transport only water. We now report the cloning of a member of the water channel that also transports nonionic small molecules such as urea and glycerol. We named this channel aquaporin 3 (AQP3) for its predominant water permeability. AQP3 has amino acid sequence identity with major intrinsic protein (MIP) family proteins including AQP-channel-forming integral membrane protein, AQP-collecting duct, MIP, AQP-gamma tonoplast intrinsic protein, nodulin 26, and glycerol facilitator (33-42%). Thus, AQP3 is an additional member of the MIP family. Osmotic water permeability of Xenopus oocytes measured by videomicroscopy was 10-fold higher in oocytes injected with AQP3 transcript than with water-injected oocytes. The increase in osmotic water permeability was inhibited by HgCl2, and this effect was reversed by a reducing agent, 2-mercaptoethanol. Although to a smaller degree, AQP3 also facilitated the transport of nonionic small solutes such as urea and glycerol, while the previously cloned water channels are permeable only to water when expressed in Xenopus oocytes. AQP3 mRNA was expressed abundantly in kidney medulla and colon. In kidney, it was exclusively immunolocalized at the basolateral membrane of collecting duct cells. AQP3 may function as a water and urea exit mechanism in antidiuresis in collecting duct cells.
The human gene encoding aquaporin-CD (AQP-CD) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the human genome and comprises four exons distributing over 5 kilobases. The size range of exons is 81-761 base pairs, and that for introns is approximately 3000 to approximately 250 base pairs. The exon-intron boundaries of human AQP-CD gene are identified at identical positions in other related genes, the human AQP-CHIP gene and the human major intrinsic protein gene. The major transcription initiation sites were identified to positions 93 and 94 base pairs upstream of the ATG initiation codon by primer extension and ribonuclease protection assay. The 5'-flanking region of the hAQP-CD gene was characterized by a TATA box, two GATA consensus sequences, an AP-1 site, an AP-2 site, three E-boxes, and a cyclic AMP-responsive element. These structural features will lead to a better understanding of the mechanisms of tissue-specific expression and the regulation by dehydration in AQP-CD gene and will also be of help in search for possible genetic disorders in human AQP-CD gene.
The terminal part of the inner medullary collecting duct exhibits a high degree of water permeability that is independent of increased intracellular cAMP and not accounted for by the activity of the known renal epithelial water channels CHIP28 (28-kDa channel-forming integral protein) and WCH-CD (collecting duct water channel protein). Starting with rat kidney papilla mRNA, reverse transcription PCR was performed with degenerate primers assuming that the putative channel would be a member of the major intrinsic protein (MIP) family of proteins. A cDNA fragment was identified and used to screen a rat kidney cDNA library. A 1.9-kb cDNA clone was isolated. The open reading frame of 876 bp coded for a protein of 292 amino acids (M(r), 31,431). Aquaporin 3 (AQP3; 31.4-kDa water channel protein) is a newly discovered member of the MIP family. Northern blot analysis showed a single transcript for AQP3 of approximately 1.9 kb present in the renal medulla, predominantly in the inner medulla. With in situ hybridization, abundant message was found in the cells of the medullary collecting ducts. Injection of the complementary RNA of AQP3 into Xenopus oocytes markedly increased the osmotic water permeability. This permeability had an energy of activation of 3.0 kcal/mol (1 cal = 4.184 J), it was fully blocked by 1 mM p-chloromercuriphenylsulfonate, and this inhibition was reversed by 5 mM dithiothreitol. cAMP did not increase this water permeability. AQP3 did not permit passage of monovalent ions (Na, K, Cl); however, it is slightly permeable to urea. The present study demonstrates the existence of an additional water channel, AQP3, in epithelial cells of the medullary collecting duct.
Human aquaporin 3 (AQP3) gene was isolated, and its structural organization was characterized. The gene appeared to exist
as a single copy in the human genome and to comprise six exons distributing over 7 kilobases. The sizes of the exons are 171,
127, 138, 119, 218, and 1035 base pairs, and those of introns are approximately 3530, 300, 350, 330, and 90 base pairs, respectively.
The initiation site of transcription was identified to locate 64 base pairs upstream of the first ATG codon by primer extension
analysis and ribonuclease protection assay. The 5′-flanking region has a TATA box, two Sp1 sequences, and some consensus sequences
including AP2 sites. With luciferase assay, the 5′-flanking region was demonstrated to have a promoter activity, which is
up-regulated 4-fold by phorbol ester. These findings about the genomic clone of human AQP3 will contribute to elucidate the
molecular mechanism of transcriptional regulation of AQP3.
A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.
In searching for a basolateral membrane water transporter in rat kidney with homology to channel forming integral protein (CHIP28), water channel-collecting duct (WCH-CD), and mercurial-insensitive water channel (MIWC), we cloned a new member of the major intrinsic protein family (GLIP, GLycerol Intrinsic Protein). GLIP cDNA had an 855-base pair open reading frame encoding a 30.5-kDa protein with 19-23% amino acid identity to the water channels and 36% identity to the bacterial glycerol facilitator GlpF. Northern blot analysis showed a 5.5-kilobase mRNA encoding GLIP in kidney, brain, and lung; RT-PCR/Southern blot analysis indicated expression of GLIP in kidney, brain, lung, eye, colon, stomach, and skeletal muscle, but not in heart, liver, and spleen. In situ hybridization in rat kidney showed GLIP mRNA expression in medullary collecting duct. Immunofluorescence with a peptide-derived polyclonal antibody showed GLIP protein expression in basolateral membrane of kidney collecting duct principal cells and brain meningeal cells. Functional measurements in Xenopus oocytes expressing GLIP cRNA showed a > 20-fold increase in [3H]glycerol uptake compared with water-injected oocytes; glycerol uptake was inhibited 88% by diisothiocyanodisulfonic stilbene (0.2 mM) and 36% by phloretin (0.25 mM). GLIP did not function as a transporter for water, urea, inositol, glucose, lactate, and monovalent ions. Glycerol uptake in oocytes expressing CHIP28 and MIWC was not different from that in water-injected controls. GLIP represents the first mammalian water channel homolog that selectively transports a solute other than water. The physiological substrate(s) and role(s) of GLIP remain to be elucidated.
Tissue-specific expression of the human adenosine deaminase (ADA) gene Is mediated by transcriptlonal activation over a thousand-fold
range. Cis-regulatory regions responsible for high and basal levels of activation Include an enhancer and the proximal promoter region.
While analyses of the T-cell specific enhancer have been carried out, detailed studies of the the promoter region or promoter-enhancer
Interactions have not. Examination of the promoter region by homology searches revealed six putative Sp1 binding sites. DNase
I footprinting showed that Sp1 Is able to bind these sites. Deletion analysis indicated that the proximal Sp1 site Is required
for activation of a reporter gene to detectable levels and that the more distal Sp1 sites further activate the level of expression.
Inclusion of an ADA enhancer-containing fragment in these deletion constructions demonstrated that Sp1 sites are also essential
for enhancer function. Apparently Sp1 controls not only low level expression but is also an integral part of the mechanism
by which the enhancer achieves high level ADA expression. Muta-genesis of a potential TBP binding site at base pairs −21 to
−26 decreased activity only two-fold indicating that It is not essential for transcriptional activation or enhancement.
Aquaporin-CHIP is the first known molecular water channel. Originally identified in red cells and renal tubules, transcripts and proteins related to AQP-CHIP are also expressed in diverse epithelia with distinct developmental patterns. Northern analyses of RNA from several tissues revealed transcripts of 3.1 kilobases and other sizes. The nucleotide sequences of human kidney AQP-CHIP cDNAs are identical to the human bone marrow AQP-CHIP cDNA. The 17-kilobase human AQP-CHIP structural gene was isolated, and restriction maps were constructed and partially sequenced. The TATA consensus sequence is located 87 bp 5' to the translation initiation site, and sequences surrounding the polyadenylation consensus were determined. Four exons were identified corresponding to amino acids 1-128, 129-183, 184-210, and 211-269, separated by introns of 9.6, 0.43, and 0.80 kilobases. Genomic Southern analyses indicated the existence of a single AQP-CHIP gene which was located at human chromosome 7p14 by in situ hybridization. Sequence comparisons of AQP-CHIP and cDNAs of similar proteins from diverse species suggested a common evolutionary origin. At least three of these proteins are now known to function as membrane water pores and are referred to as the "Aquaporins." These genomic AQP-CHIP DNA sequences should permit molecular characterization of the complex patterns of AQP-CHIP expression.
The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation.
The aquaporin family of membrane water transport proteins are expressed in diverse tissues, and in brain the predominant water channel protein is AQP4. Here we report the isolation and characterization of the human AQP4 cDNAs and genomic DNA. Two cDNAs were isolated corresponding to the two initiating methionines (M1 in a 323-aa polypeptide and M23 in a 301-aa polypeptide) previously identified in rat [Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B. & Agre, P. (1994) Proc. Natl. Acad. Sci. USA 91, 13052-13056]. Similar to other aquaporins, the AQP4 gene is composed of four exons encoding 127, 55, 27, and 92 amino acids separated by introns of 0.8, 0.3, and 5.2 kb. Unlike other aquaporins, an alternative coding initiation sequence (designated exon 0) was located 2.7 kb upstream of exon 1. When spliced together, M1 and the subsequent 10 amino acids are encoded by exon 0; the next 11 amino acids and M23 are encoded by exon 1. Transcription initiation sites have been mapped in the proximal promoters of exons 0 and 1. RNase protection revealed distinct transcripts corresponding to M1 and M23 mRNAs, and AQP4 immunoblots of cerebellum demonstrated reactive polypeptides of 31 and 34 kDa. Using a P1 and a lambda EMBL subclone, the chromosomal site of the human AQP4 gene was mapped to chromosome 18 at the junction of q11.2 and q12.1 by fluorescence in situ hybridization. These studies may now permit molecular characterization of AQP4 during human development and in clinical disorders.
The recent cloning of two urea transporters will allow to better understand their role in the urinary concentrating mechanism. This physiological approach needs to be sustained by a knowledge of their functional characteristics. We compared the pharmacological properties of the human red blood cell and kidney urea transporters (HUT11 and HUT2) in the Xenopus oocyte expression system. Both proteins allow the rapid transfer of urea but not of water. Both are inhibited by phloretin, although with different half-maximal inhibitory concentrations (IC50; 75 microM, for HUT11 and 230 microM for HUT2). Whereas para-chloromercuribenzene sulfonate inhibits HUT11 with an IC50 of 150 microM, it does not inhibit HUT2, whatever the concentration used. We demonstrate that thiourea diffuses through HUT11 with a Michaelis constant (Km) of 40 mM, but not through HUT2. In contrast, it inhibits urea transport through both proteins. This identification of a substrate binding site independent from the transport activity is the first step in the understanding of the molecular events underlying urea transport.
A new member of the aquaporin (AQP) family has been identified from rat testis. This gene, referred as aquaporin 7 (AQP7), encodes a 269-amino acid protein that contained the conserved NPA motifs of MIP family proteins. AQP7 has the amino acid sequence homology with other aquaporins ( approximately 30%), and it is highest with AQP3 (48%), suggesting that both AQP3 and AQP7 belong to a subfamily in the MIP family. Injection of AQP7-cRNA into Xenopus oocytes expressed a 26-kDa protein detected by immunoblotting. The expression of AQP7 in oocytes stimulated the osmotic water permeability by 10-fold which was not inhibited by 0.3 mM mercury chloride. The Arrhenius activation energy for the stimulated water permeability was low (2.1 kcal/mol). AQP7 also facilitated glycerol and urea transport by 5- and 9-fold, respectively. The activation energy for glycerol was also low (5.3 kcal/mol after the correction of the endogenous glycerol permeability of oocytes). Northern blot analysis revealed a 1.5-kilobase pair transcript expressed abundantly in testis. In situ hybridization of testis revealed the expression of AQP7 at late spermatids in seminiferous tubules. The immunohistochemistry of testis localized the AQP7 expression at late spermatids and at maturing sperms. AQP7 may play an important role in sperm function.
The sodium/myo-inositol cotransporter is a plasma membrane protein responsible for concentrative cellular accumulation of myo-inositol in a variety of tissues. When cells in kidney and brain are exposed to a hyperosmolar salt condition (hypertonicity) due to the operation of urinary concentration mechanism and pathological conditions, respectively, they survive the stress of hypertonicity by raising the cellular concentration of myo-inositol. Transcription of the sodium/myo-inositol cotransporter gene is markedly stimulated in response to hypertonicity, leading to an increase in the activity of the cotransporter, which in turn drives the osmoprotective accumulation of myo-inositol. To understand the molecular mechanisms by which hypertonicity stimulates transcription, we analyzed the 5'-flanking region of the cotransporter gene for cis-acting regulatory sequences. We identified five tonicity-responsive enhancers that are scattered over 50 kilobase pairs. All the enhancers are variations of the same type of enhancer interacting with the transcription factor named tonicity-responsive enhancer binding protein. In vivo methylation experiments demonstrated that exposure of cells to hypertonicity increases the binding of tonicity-responsive enhancer binding protein to the enhancer sites, indicating that all of these enhancers are involved in the transcriptional stimulation. We conclude that the sodium/myo-inositol cotransporter gene is regulated by a large region (approximately 50 kilobase pairs) upstream of the gene.
Potassium channels play important roles in shaping the electrical properties of excitable cells. Toward understanding the
transcriptional regulation of a member of the inwardly rectifying potassium channel family, we have characterized the genomic
structure and 5′-proximal promoter of the murineKcnj2 gene (also referred to as IRK1 and Kir2.1). TheKcnj2 transcription unit is composed of two exons separated by a 5.5-kilobase pair intron. Deletion analysis of 5′-flanking sequences
identified a promiscuously active 172-base pair minimal promoter, whereas expression from a construct containing additional
upstream sequences was cell type-restricted. The minimal promoter contained an E box, a Y box, and three GC box consensus
elements but lacked both TATA and CCAAT box elements. The activity of the minimal promoter was found to be controlled by a
combination of the activities of the transcription factors Sp1, Sp3, and NF-Y. The interplay between Sp1, Sp3, and NF-Y within
the architecture of theKcnj2 promoter, the ubiquitous nature of thesetrans-acting factors, and the action of tissue-selective repressor element(s) may combine to enable a wide variety of cell types
to differentially regulate Kcnj2 expression through transcriptional control.
In all living cells, coordination of solute and water movement across cell membranes is of critical importance for osmotic
balance. The current concept is that these processes are of distinct biophysical nature. Here we report the expression cloning
of a liver cDNA encoding a unique promiscuous solute channel (AQP9) that confers high permeability for both solutes and water.
AQP9 mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in
a phloretin- and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na+, K+, Cl−, and deprotonated monocarboxylates are excluded. The properties of AQP9 define a new evolutionary branch of the major intrinsic
protein family of aquaporin proteins and describe a previously unknown mechanism by which a large variety of solutes and water
can pass through a single pore, enabling rapid cellular uptake or exit of metabolites with minimal osmotic perturbation.
Adaptation of cells to hypertonicity often involves changes in gene expression. Since the concentration of salt in the interstitial fluid surrounding renal inner medullary cells varies with operation of the renal concentrating mechanism and generally is very high, the adaptive mechanisms of these cells are of special interest. Renal medullary cells compensate for hypertonicity by accumulating variable amounts of compatible organic osmolytes, including sorbitol, myo-inositol, glycine betaine, and taurine. In this review we consider how these solutes help relieve the stress of hypertonicity and the nature of transporters and enzymes responsible for their variable accumulation. We emphasize recent developments concerning the molecular basis for osmotic regulation of these genes, including identification and characterization of osmotic response elements. Although osmotic stresses are much smaller in other parts of the body than in the renal medulla, similar mechanisms operate throughout, yielding important physiological and pathophysiological consequences.
A new member (AQP9) of the aquaporin family was identified from human leukocytes by homology cloning using PCR. A full length clone was obtained by screening human liver cDNA library. AQP9 encodes a 295-amino-acid protein with the amino acid sequence identity with AQP3 (48%), AQP7 (45%), and other aquaporins (approximately 30%), suggesting that AQP3, AQP7, and AQP9 belong to a subfamily of the aquaporin family. Injection of AQP9-cRNA into Xenopus oocytes stimulated the osmotic water permeability 7-folds with a low activation energy (4.2 kcal/mol) which was inhibited by 0.3 mM mercury chloride by 48%. AQP9 also facilitated urea transport 4-folds. However, in contrast to AQP3 and AQP7, AQP9 did not stimulate the glycerol permeability, suggesting a unique permeability character. Northern blot analysis revealed the high expression of 3.5-kb messages in peripheral leukocytes > liver > lung = spleen, but not in thymus. The possible role of AQP9 in the immunological function of leukocytes is intriguing and the identification of AQP9 with unique permeability profile may expand our understanding of water and small solute transport in the body.
Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although
selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane
protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions.
Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited
by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane
water channels.
Studies of transcription by RNA polymerase II have revealed two promoter elements, the TATA motif and the initiator (Inr), capable of directing specific transcription initiation. Although binding to the TATA motif by one of the components of the transcription machinery has been shown to be the initial recognition step in transcription complex formation, many promoters that lack a traditional TATA motif have recently been described. In such TATA-less promoters, the Inr element is critical in positioning RNA polymerase II. Various Inr elements have been described and classified according to sequence homology. These Inr elements are recognized specifically by Inr-binding proteins. Interaction between these Inr-binding proteins and components of the basal transcription machinery provides a means through which a transcription competent complex can be formed.
Major intrinsic protein (MIP, also called MP26) is the predominant fiber cell membrane protein of the ocular lens. MIP has been suggested to play a role in cell-cell communication in the lens. Its expression is tissue-specific and developmentally regulated. We have isolated and characterized the human gene encoding MIP and report here its genomic structure and entire nucleotide sequence. The gene is 3.6 kb, contains four exons separated by introns ranging in size from 0.4 to 1.6 kb, and is present in single copy per haploid human genome. Primer extension of human lens RNA indicates that transcription of the gene initiates from a single site 26 nt downstream from the TATA box. Three complete Alu repetitive elements are found in tandem in the 5'-flanking region of the gene, and a single complete Alu sequence is present in the third intron. The interspecies comparisons of the MIP gene coding sequence and homologies to other members of a putative transmembrane channel protein superfamily are also discussed.
The terminal part of the inner medullary collecting duct (terminal IMCD) is unique among collecting duct segments in part because its permeability to urea is regulated by vasopressin. The urea permeability can rise to extremely high levels (greater than 100 x 10(-5) cm/s) in response to vasopressin. Recent studies in isolated perfused IMCD segments have established that the rapid movement of urea across the tubule epithelium occurs via a specialized urea transporter, presumably an intrinsic membrane protein, present in both the apical and basolateral membranes. This urea transporter has properties similar to those of the urea transporters in mammalian erythrocytes and in toad urinary bladder, namely, inhibition by phloretin, inhibition by urea analogues, saturation kinetics in equilibrium-exchange experiments, and regulation by vasopressin. The urea transport pathway is distinct from and independent of the vasopressin-regulated water channel. The increase in transepithelial urea transport in response to vasopressin is mediated by adenosine 3',5'-cyclic monophosphate and is associated with an increase in the urea permeability of the apical membrane. However, little is known about the physical events associated with the activation or insertion of urea transporters in the apical membrane. Because of the importance of this transporter to the urinary concentrating mechanism, efforts toward understanding its molecular structure and the molecular basis of its regulation appear to be justified.
Primary structure of thousands of genes is being determined in many laboratories worldwide. While it is relatively easy to analyse the coding region(s) of genes, it is usually hard to understand what is located in non-coding regions. A non-coding region may contain very valuable information about the mode of functioning of a given gene, e. g. promoters, enhancers, silencers etc. The regulatory function of these sequences is determined by their interaction with certain sequence-specific proteins, i. e. the presence of a certain DNA sequence in a non-coding region of a gene may suggest that the gene is regulated by a specific protein factor. This minireview summarizes recent data on most known eukaryotic sequence-specific DNA-binding protein factors, including their origin, DNA consensus, and their role in expression of corresponding genes.
In situ hybridization and other data showed that all hepatocytes express glutathione-S-transferase (GST) Ya mRNA but that
specifically pericentral cells can be induced 15- to 20-fold with 3-methylcholanthrene (3-MC). In order to identify DNA sequences
involved in inducible expression (pericentral hepatocytes) and constitutive expression (all hepatocytes), the upstream regions
of the GST Ya gene were further analyzed by transient transfection and DNA-binding studies to identify the nature of proteins
involved in regulating this gene. The sequences from -980 to -650 were necessary and sufficient for cell-specific and inducible
expression. Within this enhancer region, four nuclear protein-binding sites were identified. One site required for inducible
expression was bound by a protein(s) induced by 3-MC. Two other sites were bound by proteins similar or identical to the constitutive
hepatocyte nuclear factors HNF1 and HNF4. The fourth site was shown to be bound by a non-liver-specific nuclear protein that
is also important in the function of the albumin gene enhancer.
To characterize the NF-kappa B binding factor in molecular terms and to facilitate the cloning of its gene, we have purified this protein from bovine spleen tissue. We have found it is a 42,000-dalton protein that exists in solution as a dimer. We were able to use the purified protein to show that the same polypeptide is able to recognize sites important for activation of genes in either B- or T-lymphocytes. Moreover, we were able to define a consensus sequence which allows ascertainment of a wider variety of sequences that are capable of interacting with this protein. The implication of the same protein in gene regulation in two different lineages of lymphoid cells reveals an unexpected unity in the mechanism of gene expression during B- and T-lymphocyte activation. This also suggests that other regulatory events must participate with NF-kappa B activation in determining B- or T-cell-specific expression.
The enhancer-binding protein AP-1 has been purified to greater than 95% homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and identified as a 47 kd polypeptide. Purified AP-1 activates transcription in vitro of the wild-type human metallothionein IIA (hMT IIA) gene but not mutant hMT IIA promoters lacking AP-1 recognition sites. DNAase I protection analysis indicates that genetically defined enhancer elements in hMT IIA, SV40, and the human collagenase gene contain high-affinity AP-1-binding sites, each with a conserved recognition motif, TGACTCA. These three genes are transcriptionally induced by treatment of cells with the tumor promoter TPA. Here we demonstrate that multiple synthetic copies of the consensus AP-1-binding site can act as TPA-inducible enhancers in various plasmid constructs after transfection into HeLa cells. These findings suggest that AP-1 is a transcription factor that functions by interacting with a specific enhancer element, and that its activities may be modulated by treatment of cells with TPA, known to stimulate protein kinase C.
We have characterized three distinct proteins present in HeLa cell extracts that specifically recognize different subsets of transcriptional elements containing the pentanucleotide sequence CCAAT. One of these CCAAT-binding proteins, CP1, binds with high affinity to CCAAT elements present in the human alpha-globin promoter and the adenovirus major late promoter (MLP). A second protein, CP2, binds with high affinity to a CCAAT element present in the rat gamma-fibrinogen promoter. Finally, the third CCAAT-binding protein is nuclear factor I (NF-I), a cellular DNA-binding protein that binds to the adenovirus origin of replication and is required for the initiation of adenoviral replication. CP1, CP2, and NF-I are distinct activities in that each binds to its own recognition site with an affinity that is at least three orders of magnitude higher than that with which it binds to the recognition sites of the other two proteins. Surprisingly, CP1, CP2, and NF-I each appear to recognize their binding site with highest affinity as a multisubunit complex composed of heterologous subunits. In the case of CP1, two different types of subunits form a stable complex in the absence of a DNA-binding site. Moreover, both subunits are present in the CP1-DNA complex. We thus propose the existence of a family of related multisubunit CCAAT-binding proteins that are composed of heterologous subunits.
Exon insertions and exon duplications, two major mechanisms of exon shuffling, are shown to involve modules that have introns of the same phase class at both their 5'- and 3'-ends. At the sites of intronic recombinations exon insertions and duplications create new introns which belong to the same phase class as the recipient introns. As a consequence of repeated exon insertions and exon duplications introns of a single phase class predominate in the resulting genes, i.e. gene assembly by exon shuffling is reflected both by this nonrandom intron phase usage and by the correlation between the domain organization of the proteins and exon-intron organization of their genes. Genes that appeared before the eukaryote-prokaryote split do not show these diagnostic signs of exon shuffling. Since ancestral introns (e.g. self-splicing introns) did not favour intronic recombination, exon shuffling may not have been significant in the early part of protein evolution.
Certain biological membranes, such as the erythrocyte plasma membrane, have a high osmotic water permeability, and such membranes have long been suspected of harboring water channels. The molecular identity of these channels has now been established with the purification of water-channel proteins and the cloning of the genes encoding them. Homologous water-channel proteins, called 'aquaporins', are present in plants and animals. These channels are water selective and do not allow ions or metabolites to pass through them. Their discovery is providing new insights into how plant and animal cells facilitate and regulate the passage of water through their membranes.
A cDNA encoding rat AQP3, a water channel and a member of the MIP family, that is expressed predominantly in kidney medulla and colon was cloned recently. To determine the structure, tissue distribution, and chromosomal localization of the human AQP3 gene, we screened a human kidney cDNA library with rat AQP3 probe and isolated a cDNA coding for human AQP3 protein. The deduced amino acid sequence of human AQP3 was 91% identical to rat AQP3. Human AQP3 mRNA was expressed in colon, kidney, liver, pancreas, lung, peripheral leukocytes, spleen, and prostate. The human AQP3 gene was mapped to 7q36.2-q36.3 by chromosome fluorescence in situ hybridization.
For the functional interpretation of genomic sequences, effective algorithms have to be developed that will recognize regions of specific function and thus will suggest experiments for their verification. As a first step, relevant data have to be collected in an appropriate database from which suitable training sets can be extracted. In this paper, I discuss the requirements for a database that collects information about regulatory DNA sequences and describe the structure and contents of such a database (TRANSFAC). This compiled information will serve as a basis for comprehensive analysis of sites that regulate transcription, e.g., by statistical methods. It will thus facilitate the recognition of regulatory genomic sequence information and the assignment of the corresponding regulators. Moreover, it will provide all relevant data about the regulating proteins which will allow to trace back transcriptional control cascades to their origin.
The Transcription Factors Database is a specialized database focusing on transcription factors and their properties. This
report describes the present status of this database and developments during the past year. Within this time, the size of
this database has Increased by a 2799 total records, and has become accessible through a number of new mechanisms.
Aquaporins (AQPs) are a newly recognized family of transmembrane proteins that function as molecular water channels. At least four aquaporins are expressed in the kidney where they mediate rapid water transport across water-permeable epithelia and play critical roles in urinary concentrating and diluting processes. AQP1 is constitutively expressed at extremely high levels in the proximal tubule and descending limb of Henle's loop. AQP2, -3 and -4 are expressed predominantly in the collecting duct system. AQP2 is the predominant water channel in the apical plasma membrane and AQP3 and -4 are found in the basolateral plasma membrane. Short-term regulation of collecting duct water permeability by vasopressin is largely a consequence of regulated trafficking of AQP2-containing vesicles to and from the apical plasma membrane.
This review summarizes recent progress in water-transporting mechanisms across cell membranes. Modern biophysical concepts of water transport and new measurement strategies are evaluated. A family of water-transporting proteins (water channels, aquaporins) has been identified, consisting of small hydrophobic proteins expressed widely in epithelial and nonepithelial tissues. The functional properties, genetics, and cellular distributions of these proteins are summarized. The majority of molecular-level information about water-transporting mechanisms comes from studies on CHIP28, a 28-kDa glycoprotein that forms tetramers in membranes; each monomer contains six putative helical domains surrounding a central aqueous pathway and functions independently as a water-selective channel. Only mutations in the vasopressin-sensitive water channel have been shown to cause human disease (non-X-linked congenital nephrogenic diabetes insipidus); the physiological significance of other water channels remains unproven. One mercurial-insensitive water channel has been identified, which has the unique feature of multiple overlapping transcriptional units. Systems for expression of water channel proteins are described, including Xenopus oocytes, mammalian and insect cells, and bacteria. Further work should be directed at elucidation of the role of water channels in normal physiology and disease, molecular analysis of regulatory mechanisms, and water channel structure determination at atomic resolution.
The physical locations on human metaphase chromosomes of over 950 yeast artificial chromosome (YAC) clones from the CEPH library have been determined by fluorescence in situ hybridization and described as fractional chromosome length relative to the terminus of the short arm. Collectively, these clones contain approximately 1 billion basepairs of human DNA, about one-third of the human genome. In addition, the locations of 337 of these clones were established in terms of cytogenetic bands for chromosomes 1-18, 20, and X. Since most clones are positive for one or more of the Généthon polymorphic STS markers with defined genetic linkage distances corresponding to their physical locations, these data facilitate the integration of the cytogenetic, genetic, and physical maps of the human genome. Use of these mapping data in conjunction with public database information on CEPH YACs greatly facilitates the identification of YACs or polymorphic markers at specific locations in the genome.
Bardet-Biedl syndrome (BBS) is a clinically and genetically heterogeneous autosomal recessive disorder characterized by retinitis pigmentosa, polydactyly, obesity, hypogenitalism, mental retardation, and renal anomalies. To detect linkage to BBS loci, 29 BBS families, of mixed but predominantly European ethnic origin, were typed with 37 microsatellite markers on chromosomes 2, 3, 11, 15, 16, and 17. The results show that an estimated 36-56% of the families are linked to the 11q13 chromosomal site (BBS1) previously described by M. Leppert et al. (1994, Nature Genet. 7, 108-112), with the gene order cen-D11S480-5 cM-BBS1-3 cM-D11S913/D11S987-qter. A further 32-35% of the families are linked to the BBS4 locus, reported by R. Carmi et al. (1995, Hum. Mol. Genet. 4, 9-13) in chromosomal region 15q22.3-q23, with the gene order cen-D15S125-5 cM-BBS4-2 cM-D15S131/D15S204-qter. Three consanguineous BBS families are homozygous for three adjacent chromosome 15 markers, consistent with identity by descent for this region. In one of these families haplotype analysis supports a localization for BBS4 between D15S131 and D15S114, a distance of about 2 cM. Weak evidence of linkage to the 16q21 (BBS2) region reported by A. E. Kwitek-Black et al. (1993, Nature Genet. 5, 392-396) was observed in 24-27% of families with the gene order cen-D16S408-2 cM-BBS2-5 cM-D16S400. A fourth group of families, estimated at 8%, are unlinked to all three of the above loci, showing that at least one other BBS locus remains to be found. No evidence of linkage was found to markers on chromosome 3, corresponding to the BBS3 locus, reported by V. C. Sheffield et al. (1994, Hum. Mol. Genet. 3, 1331-1335), or on chromosome 2 or 17, arguing against the involvement of a BBS locus in a patient with a t(2;17) translocation.
The cDNA for the seventh mammalian aquaporin (AQP7) was isolated from rat testis, and its expression demonstrated at the tail of late spermatids (Ishibashi et al., J. Biol. Chem. 272 (1997) 20,782-20,786). Here we report the isolation of the mouse and the human AQP7 cDNA and the human AQP7 gene. The human AQP7 gene is identical with human adipose AQP (AQPap or AQP7L). The deduced amino acid sequences of human and mouse AQP7 were 68% and 79% identical to those of rat AQP7, respectively. The mouse AQP7 is 67% identical to the human AQP7. Such a lower conservation of AQP7 among species is unusual in the aquaporin family. The human AQP7 gene is composed of six exons distributing over 6.5 kb. The exon-intron boundaries are identical to those of the human AQP3 gene. The intron sizes are also similar. Moreover, chromosomal localization of AQP7 was assigned to 9p13 by fluorescent in situ hybridization, where AQP3 is also localized, suggesting that 9p13 may be another site of an aquaporin cluster.
Facilitated urea transporters (UTs) are responsible for urea accumulation in the renal inner medulla of the mammalian kidney and therefore play a central role in the urinary concentrating process. Recently, the cDNAs encoding three members of the UT family, UT1, UT2, and UT3 have been cloned. These transporters are expressed in different structures of the mammalian kidney. In rat, UT1 resides in the apical membrane of terminal inner medullary collecting ducts, where it mediates vasopressin-regulated urea reabsorption. UT2 and UT3 are located in descending thin limbs of Henle's loop and descending vasa recta, respectively, and participate in urinary recycling processes, which minimize urea escape from the inner medulla. UT1 and UT2 are regulated independently and respond differently to changes in dietary protein content and hydration state. Identification and characterization of these urea transporters advances our understanding of the molecular basis and regulation of the urinary concentrating mechanism.
The cDNA and the gene of human aquaporin8 (AQP8) were cloned from human testis cDNA and a genomic library, respectively. The AQP8 cDNA encodes 261 amino acids. The identity of the amino acid sequence to other aquaporins is highest with a plant water channel, gamma-TIP (40.4%), while AQP2 and AQP3 are 28.9 and 29.5% identical to human AQP8, respectively. The human AQP8 is only 74.9% identical to rat AQP8 and 76.0% identical to mouse AQP8. In Northern blot analysis, approximately 1.35-kb human AQP8 mRNA was expressed in pancreas and colon, but not in other tissues. Absence of human AQP8 in testis is noteworthy as rat AQP8 was abundantly expressed in testis. The expression of human AQP8 cRNA in Xenopus oocytes increased osmotic water permeability by 10-fold. AQP8 was not permeable to urea nor to glycerol. The AQP8 gene has five introns, and the locations of exon-intron boundaries were different from those of the other mammalian aquaporins, suggesting its separate phylogenetic origin.
A new aquaporin was isolated from rat liver based on homology to known aquaporins. A 1408 bp cDNA was sequenced (designated rAQP9L) with a 885 bp open reading frame encoding a 295 amino acid hydrophobic protein. rAQP9L has the greatest amino-acid sequence identity with human AQP9 (75%) and a less homology with AQP3 (49%) and AQP7 (47%). Northern blot analysis indicated a 1.4-kb transcript expressed strongly in liver > testis > brain = lung. Expression of rAQP9L cRNA in Xenopus oocytes increased osmotic water permeability by 6-folds which was inhibited by 0.3 mM mercury chloride by 42%. rAQP9L also facilitated glycerol and urea transport by 2- and 5-folds, respectively. The large discrepancy of tissue distribution between hAQP9 and rAQP9L suggest that rAQP9L is a new aquaporin, which is involved in transport of urea as well as water in liver.
Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells
- K Ishibashi
- S Sasaki
- K Fushimi
- S Uchida
- M Kuwahara
- H Saito
- T Furukawa
- K Nakajima
- Y Yamaguchi
- T Gojobori
- F Marumo
Ishibashi, K., S. Sasaki, K. Fushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y. Yamaguchi, T.
Gojobori, and F. Marumo. Molecular cloning and expression of
a member of the aquaporin family with permeability to glycerol
and urea in addition to water expressed at the basolateral
membrane of kidney collecting duct cells. Proc. Natl. Acad. Sci.
USA 91: 6269-6273, 1994.
- M A Knepper
- J B Wade
- J Terris
- C A Ecelbarger
- D Marples
- B Mandon
- C L Chou
- B K Kishore
- S Nielsen
Knepper, M. A., J. B. Wade, J. Terris, C. A. Ecelbarger, D.
Marples, B. Mandon, C. L. Chou, B. K. Kishore, and S.
Nielsen. Renal aquaporins. Kidney Int. 49: 1712-1717, 1996.