Aquaporins—new players in cancer biology

The aquaporins (AQPs) are small, integral-membrane proteins that selectively transport water across cell plasma membranes. A subset of AQPs, the aquaglyceroporins, also transport glycerol. AQPs are strongly expressed in tumor cells of different origins, particularly aggressive tumors. Recent discoveries of AQP involvement in cell migration and proliferation suggest that AQPs play key roles in tumor biology. AQP1 is ubiquitously expressed in tumor vascular endothelium, and AQP1-null mice show defective tumor angiogenesis resulting from impaired endothelial cell migration. AQP-expressing cancer cells show enhanced migration in vitro and greater local tumor invasion, tumor cell extravasation, and metastases in vivo. AQP-dependent cell migration may involve AQP-facilitated water influx into lamellipodia at the front edge of migrating cells. The aquaglyceroporin AQP3, which is found in normal epidermis and becomes upregulated in basal cell carcinoma, facilitates cell proliferation in different cell types. Remarkably, AQP3-null mice are resistant to skin tumorigenesis by a mechanism that may involve reduced tumor cell glycerol metabolism and ATP generation. Together, the data suggest that AQP expression in tumor cells and tumor vessels facilitates tumor growth and spread, suggesting AQP inhibition as a novel antitumor therapy.     

Role of aquaporins in lung liquid physiology

Aquaporins (AQPs) are small, integral membrane proteins that facilitate water transport across cell membranes in response to osmotic gradients. Water transport across epithelia and endothelia in the peripheral lung and airways occurs during airway hydration, alveolar fluid transport and submucosal gland secretion. Several AQPs are expressed in the lung and airways: AQP1 in microvascular endothelia, AQP3 and AQP4 in airway epithelia, and AQP5 in type I alveolar epithelial cells, submucosal gland acini, and a subset of airway epithelial cells. Phenotype analysis of transgenic knockout mice lacking AQPs has defined their roles in the lung and airways. AQP1 and AQP5 provide the principal route for osmotically driven water transport between airspace and capillary compartments; however, alveolar fluid clearance in the neonatal and adult lung is not affected by their deletion, nor is lung fluid accumulation in experimental models of lung injury. In the airways, though AQP3 and AQP4 facilitate osmotic water transport, their deletion does not impair airway hydration, regulation of airway surface liquid, or fluid absorption. In contrast to these negative findings, AQP5 deletion in submucosal glands reduced fluid secretion by >50%. The substantially slower fluid transport in the lung compared to renal and secretory epithelia probably accounts for the lack of functional significance of AQPs in the lung and airways. Recent data outside of the lung implicating the involvement of AQPs in cell migration and proliferation suggests possible new roles for lung AQPs to be explored.     

水通道蛋白与肿瘤血管新生

肿瘤血管新生对于肿瘤的生长具有重要作用。水通道蛋白在肿瘤组织中常出现表达异常，并且可以促进肿瘤血管的新生及包括肿瘤血管内皮在内的多种细胞的迁移，与此同时，水通道蛋白与肿瘤血管的高通透性有关，这可能是肿瘤组织对水通道蛋白缺失具有较高敏感性的原因。鉴于水通道蛋白对于肿瘤特别是肿瘤血管新生的影响，其抑制剂可能成为新的肿瘤治疗的目标靶点。     

Aquaporin-1 in breast cancer

The canonical function of aquaporin (AQP) water channels is to facilitate passive transport of water across cellular membranes making them essential in the regulation of body water homeostasis. Moreover, AQPs, including AQP1, have been found to be overexpressed in multiple cancer types, including breast cancer, where AQP1 overexpression is associated with poor prognosis. AQPs have been shown to affect cellular processes associated with cancer progression and spread including cell migration, angiogenesis, and proliferation. Moreover, AQPs can regulate levels of adhesion proteins at cell–cell junctions, a regulatory role, which is still largely unexplored in cancer. Understanding the molecular mechanisms of how AQP1 contributes to breast cancer progression and metastatic processes is essential to establish AQP1 as a biomarker and to develop targeted anticancer treatments for breast cancer patients. This mini-review focuses on the role of AQP1 in breast cancer.     

Upregulation of AQP3 and AQP5 induced by dexamethasone and ambroxol in A549 cells

Aquaporins (AQPs) are membrane channel proteins that play roles in the regulation of water permeability in many tissues. AQP1 and AQP5 expressed in lung provide the principal route for osmotically driven water transport. In the airways, AQP3 and AQP4 facilitate water transport. Dexamethasone and ambroxol are often used to treat patients with pulmonary diseases accompanied by airway hypersecretion. The role of AQPs in these effective treatments has not been addressed. In this study, we analyzed the expression of AQPs in a human airway epithelial cell line (A549 cells) and showed that AQP3 and 5, but not AQP1 and 4, were expressed in A549 cells. Both dexamethasone and ambroxol stimulated the expression of AQP3 and 5 at the mRNA and protein levels. The data suggest potential roles of AQP3 and 5 in the regulation of airway hypersecretion, perhaps ultimately providing a target for treating such diseases.     

AQP1 mediates water transport in the carotid body

In this study, we explored the presence of aquaporins (AQPs), a family of membrane water channel proteins, in carotid body (CB) type I chemoreceptor cells. The CB is a polymodal chemoreceptor whose major function is to detect changes in arterial O₂ tension to elicit hyperventilation during hypoxia. The CB has also been proposed to function as a systemic osmoreceptor, thus we hypothesized that the presence of AQPs in type I cell membrane may confer higher sensitivity to osmolarity changes and hence accelerate the activation of chemoreceptor cells. We detected the expression of AQP1, AQP7, and AQP8 in the CB and confirmed the location of AQP1 in type I cells. We have also shown that inhibition of AQP1 expression clearly reduced type I cell swelling after a hyposmotic shock, demonstrating that AQP1 has a major contribution in transmembrane water movement in these chemoreceptor cells. Interestingly, CB AQP1 expression levels change during postnatal development, increasing during the first postnatal weeks as the organ matures. In conclusion, in this study, we report the novel observation that AQPs are expressed in the CB. We also show that AQP1 mediates water transport across the cell membrane of type I cells, supporting the contribution of this protein to the osmoreception function of the CB.     

Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing

Healing of skin wounds is a multi-step process involving the migration and proliferation of basal keratinocytes in epidermis, which strongly express the water/glycerol-transporting protein aquaporin-3 (AQP3). In this study, we show impaired skin wound healing in AQP3-deficient mice, which results from distinct defects in epidermal cell migration and proliferation. In vivo wound healing was approximately 80% complete in wild-type mice at 5 days vs approximately 50% complete in AQP3 null mice, with remarkably fewer proliferating, BrdU-positive keratinocytes. After AQP3 knock-down in keratinocyte cell cultures, which reduced cell membrane water and glycerol permeabilities, cell migration was slowed by more than twofold, with reduced lamellipodia formation at the leading edge of migrating cells. Proliferation of AQP3 knock-down keratinocytes was significantly impaired during wound repair. Mitogen-induced cell proliferation was also impaired in AQP3 deficient keratinocytes, with greatly reduced p38 MAPK activity. In mice, oral glycerol supplementation largely corrected defective wound healing and epidermal cell proliferation. Our results provide evidence for involvement of AQP3-facilitated water transport in epidermal cell migration and for AQP3-facilitated glycerol transport in epidermal cell proliferation.     

水通道蛋白与肿瘤

水通道蛋白（AQPs）是一类小分子质量的、膜内嵌蛋白,它所介导的自由水快速被动的跨生物膜转运,是水进出细胞的主要途径。AQPs大量表达于不同组织来源的肿瘤细胞,特别是侵袭性肿瘤。AQPs高表达的肿瘤细胞在体外表现为较强的迁移能力,体内则表现为局部侵袭力增强,渗出增多以及转移灶增多。抑制AQPs的表达将有可能为肿瘤的治疗提供帮助。     

Aquaporin‐1 expression in the chick embryo chorioallantoic membrane

The chick embryo chorioallantoic membrane (CAM) is commonly used in vivo to study both angiogenesis and anti‐angiogenesis. Rapid membrane water transport is mediated by a family of molecular water channels, called aquaporins (AQPs), which have been identified in the epithelial and endothelial cells of higher vertebrates. AQP1, expressed in adsorptive and secretory epithelia, is also expressed in endothelial cells of capillaries and arteries. Its mRNA has been found in vascular smooth muscle cells (VSMCs) of arteries and capillaries, as well as in a subset of VSMCs of human atherosclerotic plaques. This study investigated the developmental expression of AQP1 in the chick CAM by Western blot and immunohistochemistry. Western blot results show that a major nonglycosylated band was observed with electrophoretic mobility of approximately 28 kDa in the three developmental stages examined. Immunohistochemistry data demonstrate that AQP1 was clearly expressed in the ectodermal and endodermal epithelia, the vascular endothelium, and the VSMCs. Because little information is available on the behavior of microvessel AQP1 during angiogenesis in normal and pathological conditions, our data relative to the pattern of expression of AQP1 in CAM blood vessels in normal conditions may be considered a useful tool to further investigate its modifications in several experimental conditions implying a stimulation or an inhibition of angiogenesis in the CAM assay. Anat Rec 268:85–89, 2002. © 2002 Wiley‐Liss, Inc.     

Aquaporin-1 expression in the chick embryo chorioallantoic membrane

The chick embryo chorioallantoic membrane (CAM) is commonly used in vivo to study both angiogenesis and anti-angiogenesis. Rapid membrane water transport is mediated by a family of molecular water channels, called aquaporins (AQPs), which have been identified in the epithelial and endothelial cells of higher vertebrates. AQP1, expressed in adsorptive and secretory epithelia, is also expressed in endothelial cells of capillaries and arteries. Its mRNA has been found in vascular smooth muscle cells (VSMCs) of arteries and capillaries, as well as in a subset of VSMCs of human atherosclerotic plaques. This study investigated the developmental expression of AQP1 in the chick CAM by Western blot and immunohistochemistry. Western blot results show that a major nonglycosylated band was observed with electrophoretic mobility of approximately 28 kDa in the three developmental stages examined. Immunohistochemistry data demonstrate that AQP1 was clearly expressed in the ectodermal and endodermal epithelia, the vascular endothelium, and the VSMCs. Because little information is available on the behavior of microvessel AQP1 during angiogenesis in normal and pathological conditions, our data relative to the pattern of expression of AQP1 in CAM blood vessels in normal conditions may be considered a useful tool to further investigate its modifications in several experimental conditions implying a stimulation or an inhibition of angiogenesis in the CAM assay.     

Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization

Neutrophils are of prime importance in the host innate defense against invading microorganisms by using two primary mechanisms-locomotion toward and phagocytosis of the prey. Recent research points to pivotal roles for water channels known as AQPs in cell motility. Here, we focused on the role of AQP9 in chemoattractant-induced polarization and migration of primary mouse neutrophils and neutrophil-like HL60 cells. We found that AQP9 is phosphorylated downstream of fMLFR or PMA stimulation in primary human neutrophils. The dynamics of AQP9 were assessed using GFP-tagged AQP9 constructs and other fluorescent markers through various live-cell imaging techniques. Expression of WT or the phosphomimic S11D AQP9 changed cell volume regulation as a response to hyperosmotic changes and enhanced neutrophil polarization and chemotaxis. WT AQP9 and S11D AQP9 displayed a very dynamic distribution at the cell membrane, whereas the phosphorylation-deficient S11A AQP9 failed to localize to the plasma membrane. Furthermore, we found that Rac1 regulated the translocation of AQP9 to the plasma membrane. Our results show that AQP9 plays an active role in neutrophil volume regulation and migration. The display of AQP9 at the plasma membrane depends on AQP9 phosphorylation, which appeared to be regulated through a Rac1-dependent pathway.     

Intracellular aquaporins: clues for intracellular water transport?

Aquaporins (AQPs) are usually present at the plasma membrane to regulate influx and outflow of water and small molecules. They are important for the regulation of water homeostasis for the cells and organisms. AQPs are also present inside the cell, at the membranes of intracellular organelles. The roles of such AQPs have not yet been established. They will be clues to clarify the mechanisms of water and small solutes movements inside the cell. Recently, a new AQP subfamily has been identified with highly deviated asparagine-proline-alanine boxes, signature sequences for AQP. With limited homology less than 20%, this subfamily will be a superfamily of AQPs. Accordingly, it was tentatively named "superaquaporin subfamily," which is so far only present in multicellular organisms including plants, insects, nematodes, and vertebrates. Some superaquaporins are functionally water channels and localized intracellularly. AQP11, one of the two superaquaporins in mammals, has been shown to be important for the development of the proximal tubule as its disruption produced neonatally fatal polycystic kidneys in mice. Hence, recent identification of intracellular AQPs will open new areas of research on cell biology and expand the scope of AQPs.     

Aquaporin 1 expression in invasive breast carcinomas

Aquaporin1 (AQP1) is a water channel protein which facilitates water flux across cell membranes. AQP1 is found in epithelial and endothelial cells in various tissues. There is increasing evidence that AQP1 is expressed in malignant tumours and that it may play a role in tumour angiogenesis, cell migration and metastasis. We studied the immunohistochemical expression of AQP1 in a cohort of 203 invasive breast carcinomas with long-term follow up. AQP1 expression was detected in 11 cases (5.4%), and showed a significant correlation with high tumour grade, medullary-like histology, "triple-negativity", as well a cytokeratin 14 and actin expression. In univariate analysis, AQP1 was associated with a significantly poorer prognosis. Multivariate analysis showed that AQP1 expression has an independent predictive value for outcome if stratified by age, tumour size, lymph node status, histological grade and ER status. AQP1 expression in invasive breast carcinomas is associated with a basal-like phenotype and poor prognosis.     

Aquaporin-8 mediates human esophageal cancer Eca-109 cell migration via the EGFR-Erk1/2 pathway

Abnormal expression of aquaporins (AQPs) has been reported in several human cancers. Epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinases1/2 (ERK1/2) are associated with tumorigenesis and cancer progression and may upregulate AQPs expression. In this study, we investigated acquaporin-8 expression and signaling via epidermal growth factor receptor-extracellular signal-regulated kinases1/2 in human esophageal cancer Eca-109 cells by western blot, immunofluorescence and wound healing (scratch) assays. Our results showed that epidermal growth factor (EGF) induced both Eca-109 migration and AQP8 expression. Wound healing results showed that cell migration was increased by 1.23-1.10-fold at 24 h and 48 h after EGF treatment. AQP8 expression was significantly increased (1.19-fold) at 48 h after EGF treatment in Eca-109. The EGFR kinase inhibitor, PD153035, blocked EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 3.65-fold (EGF-treated) to 0.55-fold (PD153035-treated) in Eca-109. Furthermore, the MEK [MAPK (mitogen-activated protein kinase)/Erk1/2]/Erk1/2 inhibitor U0126 also inhibited EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 3.92-fold (EGF-treated) to 1.38-fold (U0126-treated) in Eca-109. In conclusions, EGF induces AQP8 expression and cell migration in Eca-109 cells via the EGFR/Erk1/2 signal transduction pathway.     

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K⁺–Cl⁻ cotransporter 1 (KCC1), believed to provide the pathways for Cl⁻ and K⁺ secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.     

Tanshinone IIA ameliorates seawater exposure-induced lung injury by inhibiting aquaporins (AQP) 1 and AQP5 expression in lung

Aquaporins (AQPs), a family of transmembrane water channels, mediate physiological response to changes of fluid volume and osmolarity. It is still unknown what role of AQPs plays in seawater drowning-induced acute lung injury (ALI) and whether pharmacologic modulation of AQPs could alleviate the severity of ALI caused by seawater aspiration. In our study, the results from RT-PCR and Western blotting showed that intratracheal installation of seawater up-regulated the mRNA and protein levels of AQP1 and AQP5 in lung tissues. Furthermore, we found that treatment of tanshinone IIA (TIIA, one of the main active components from Chinese herb Danshen) significantly reduced the elevation of AQP1 and AQP5 expression induced by seawater in rats, A549 cells and primary alveolar type II cells. Treatment of TIIA also improved lung histopathologic changes and blood-gas indices, and reduced lung edema and vascular leakage. These findings demonstrated that AQP1 and AQP5 might play an important role in the development of lung edema and lung injury, and that treatment with TIIA could significantly alleviate seawater exposure-induced ALI, which was probably through the inhibition of AQP1 and AQP5 over-expression in lungs.     

Expression and localization of epithelial aquaporins in the adult human lung

Aquaporins (AQPs) facilitate water transport across epithelia and play an important role in normal physiology and disease in the human airways. We used in situ hybridization and immunofluorescence to determine the expression and cellular localization of AQPs 5, 4, and 3 in human airway sections. In nose and bronchial epithelia, AQP5 is expressed at the apical membrane of columnar cells of the superficial epithelium and submucosal gland acinar cells. AQP4 was detected in basolateral membranes in ciliated ducts and by in situ in gland acinar cells. AQP3 is present on basal cells of both superficial epithelium and gland acinus. In these regions AQPs 5, 4, and 3 are appropriately situated to permit transepithelial water permeability. In the small airways (proximal and terminal bronchioles) AQP3 distribution shifts from basal cell to surface expression (i.e., localized to the apical membrane of proximal and terminal bronchioles) and is the only AQP identified in this region of the human lung. The alveolar epithelium has all three AQPs represented, with AQP5 and AQP4 localized to type I pneumocytes and AQP3 to type II cells. This study describes an intricate network of AQP expression that mediates water transport across the human airway epithelium.     

Downregulation of aquaporin 5 induced by vector-based short hairpin RNA and its effect on MUC5AC gene expression in human airway submucosal gland cells

The aquaporins (AQPs) are a family of homologous water channels expressed in many epithelial and endothelial cells, however no reliable and non-toxic inhibitors of AQPs have been reported yet. Our researchers have analyzed the changes of AQP5 expression induced by vector-based short hairpin RNA (shRNA) in the human airway submucosal gland cell line (SPC-A1) and observed its regulation on the expression of MUC5AC gene. Localizations of AQP5 and MUC5AC in SPC-A1cells were detected by Immunofluorescence. AQP5 mRNA was significantly reduced by 75.1% one day after transfection with specific shRNA, named shAQP5. However, the significant suppression of AQP5 protein did not appear until day 5 after transfection. MUC5AC mRNA was remarkably increased by 119.9% On day 3 after shAQP5 transfection, while comparable MUC5AC protein changes were not found in SPC-A1 cells with flow cytometry analysis. These results indicate that vector-based shRNA could be used as a potential tool to inhibit the expression of AQP5. This is the first investigation providing evidence demonstrating the regulation of the mucin gene by AQP5 gene silencing.