Aquaporin-1 Knockdown Research Articles

Aquaporin-1 Facilitates Macrophage M1 Polarization by Enhancing Glycolysis Through the Activation of HIF1α in Lipopolysaccharide-Induced Acute Kidney Injury.

This study aimed to investigate how aquaporin 1 (AQP1) modulates hypoxia-inducible factor-1α (HIF1α) to promote glycolysis and drive the M1 polarization of macrophages. Within 12 h post-treatment with LPS to induce acute kidney injury in rats, a significant upregulation of AQP1 and HIF1α protein levels was noted in serum and kidney tissues. This elevation corresponded with a decrease in blood glucose concentrations and an enhancement of glycolytic activity relative to the control group. Furthermore, there was a pronounced reduction in the circulating levels of the anti-inflammatory cytokine IL-10, accompanied by an upregulation in the levels of the pro-inflammatory cytokines IL-6 and TNF-α. The administration of an HIF1α inhibitor reversed these effects, which did not affect the production of AQP1 protein. In cellular assays, AQP1 knockdown mitigated the increase in HIF1α expression induced by LPS. Furthermore, the suppression of HIF1α with PX-478 led to decreased expression levels of Hexokinase 2 (HK2) and Lactate Dehydrogenase A (LDHA), indicating that AQP1 regulates glycolysis through HIF1α. M1 polarization of macrophages was reduced by AQP1 knockdown and was further diminished by the addition of an HIF1α inhibitor. Inhibition of the glycolytic process not only weakened M1 polarization but also promoted M2 polarization, thereby reducing the release of inflammatory cytokines. These findings provide a novel perspective for developing therapeutic strategies that target AQP1 and HIF1α, potentially improving the treatment of sepsis-associated AKI.

Alpinetin alleviates LPS-induced lung epithelial cell injury by inhibiting p38 and ERK1/2 signaling via aquaporin-1

Alpinetin has been reported to play a protective role in lung diseases, while its special mechanisms remain indistinct. In this study, acute lung injury (ALI) model was constructed by inducing MLE-12 cells with lipopolysaccharide (LPS). Cell activity together with apoptosis was judged employing cell counting kit-8 (CCK-8), flow cytometry along with western blot. Oxidative stress levels were measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining and corresponding kits. In addition, enzyme-linked immunosorbent assay (ELISA) was to examine the levels of inflammatory factors. The protein expressions of aquaporin-1 (AQP1), p38 and extracellular signal-regulated kinase (ERK) 1/2 pathway were estimated utilizing western blot. The data showed that alpinetin increased the viability, reduced the apoptosis, oxidative stress and inflammation and inactivated p38 and ERK1/2 signaling in LPS-induced MLE-12 cells. Moreover, alpinetin also increased AQP1 expression and AQP1 knockdown reversed the impacts of alpinetin on LPS-induced MLE-12 cells. Additionally, AQP1 agonist AqF026 also exerted anti-apoptotic and anti-inflammatory activities in LPS-treated MLE-12 cells. Evidently, alpinetin may exert its protective role in LPS-induced ALI by inactivation of p38 and ERK1/2 signaling through regulating AQP1.

ANGI-03. NOVEL ROLE OF ENDOTHELIAL CELL AQUAPORIN-1 IN GLIOBLASTOMA VASCULAR PERMEABILITY AND TUMOR-ASSOCIATED EDEMA

Abstract Glioblastomas are the most common malignant brain tumors in adults, with a median survival of only 12-15 months. Glioblastomas are marked by an aberrant, leaky vasculature that promotes a hypoxic, immunosuppressive microenvironment. This aberrant vascularity not only affects treatment sensitivity but the integrity of the blood brain barrier (BBB). Disruption of the BBB can lead to increased flow of water into intercellular spaces, causing vasogenic edema. Increased intracranial pressure due to vasogenic edema in glioblastoma patients is a significant cause of morbidity and mortality. Severity of edema not only correlates with patient survival, but causes numerous harmful side effects, such as aphasia, cognitive decline, and acute herniation syndrome. Currently, the mainstay of treatment for brain tumor edema is glucocorticosteroids, such as dexamethasone. However, this treatment causes immune suppression and severe long-term adverse effects. To determine the potential role of vascular endothelial cells (ECs) in edema formation, we performed single cell RNAseq on a genetically engineered glioblastoma mouse model, which recapitulates the edema seen in human patients. We found that ECs from glioblastomas and normal brain tissue had distinct expression patterns, with selective expression of a water channel protein called Aquaporin-1 (AQP1) in glioblastoma ECs. In addition, siRNA-mediated AQP1 knockdown decreased GBM EC monolayer permeability. Inhibiting the water channel function of AQP1 alone through Bacopaside II treatment, however, did not alter EC monolayer permeability. Several tight junctions are downregulated in GBM ECs compared to normal brain ECs, and knocking down AQP1 via siRNA restored expression of the tight junction protein Claudin-5. These data suggest that AQP1 induces brain tumor edema by inhibiting tight junction protein expression via its non-water-channel function and that targeting AQP1 in glioblastoma ECs would be a novel therapeutic strategy to improve the BBB and decrease edema levels in vivo.

Shen Qi Wan attenuates renal interstitial fibrosis through upregulating AQP1.

Renal interstitial fibrosis (RIF) is the crucial pathway in chronic kidney disease (CKD) leading to the end-stage renal failure. However, the underlying mechanism of Shen Qi Wan (SQW) on RIF is not fully understood. In the current study, we investigated the role of Aquaporin 1 (AQP1) in SQW on tubular epithelial-to-mesenchymal transition (EMT). A RIF mouse model induced by adenine and a TGF-β1-stimulated HK-2 cell model were etablished to explore the involvement of AQP 1 in the protective effect of SQW on EMT in vitro and in vivo. Subsequently, the molecular mechanism of SQW on EMT was explored in HK-2 cells with AQP1 knockdown. The results indicated that SQW alleviated kidney injury and renal collagen deposition in the kidneys of mice induced by adenine, increased the protein expression of E-cadherin and AQP1 expression, and decreased the expression of vimentin and α-smooth muscle actin (α-SMA). Similarly, treatmement with SQW-containing serum significantly halted EMT process in TGF-β1 stimulated HK-2 cells. The expression of snail and slug was significantly upregulated in HK-2 cells after knockdown of AQP1. AQP1 knockdown also increased the mRNA expression of vimentin and α-SMA, and decreased the expression of E-cadherin. The protein expression of vimentin increased, while the expression of E-cadherin and CK-18 significantly decreased after AQP1 knockdown in HK-2 cells. These results revealed that AQP1 knockdown promoted EMT. Furthermore, AQP1 knockdown abolished the protective effect of SQW-containing serum on EMT in HK-2 cells. In sum, SQW attentuates EMT process in RIF through upregulation of the expression of AQP1.

Differential endothelial senescence elicited by AQP1 regulation of epigenetic/metabolic responses

Abstract Introduction Accumulation of senescent endothelial cells with age is an important driver of vascular aging and related cardiovascular disease. However, little is known about the mechanisms and signaling pathways that underpin endothelial cell senescence. Aquaporin-1 (AQP1) has been implicated in sensing hydrogen peroxide (H2O2), which differentially modulates endothelial signal transduction. Nonetheless, the role of AQP1 in the regulation of metabolism and function in young and old vascular cells remains elusive. Purpose We sought to investigate whether 1) AQP1 differentially regulates vascular senescence and angiogenesis through epigenetic/metabolic response, senescence-associated secretory phenotype (SASP), and 2) AQP1 knockdown/inhibition rejuvenates senescent endothelial cells and restores angiogenesis. Methods We assessed the role of AQP1 in endothelial cell senescence and impaired angiogenesis through mitochondrial metabolic disorder, epigenetic alteration, and the SASP in human aortic endothelial cells and aortas from wild-type C57BL/6 mice. For these, we conducted AQP1 knockdown (with siRNA) or blockade of AQP1 (with Bacopaside-II, 10 μM) to inhibit H2O2 transport. Results Here we show the upregulation of AQP1 in the senescent endothelial cells (SEC, p.15) and aorta from old mice (>24 months) (p<0.01 and p<0.0001, respectively), facilitating outside-in H2O2 transport. Interestingly, AQP1 knockdown/inhibition reverses both replicative (increased SA-β-galactosidase positive cells) and stress-induced senescence (increased cell-cycle arrest markers p16, p19, and p21; and SASP markers IL1α, IL1β, and IL6) in SEC, whereas it triggers the senescence processes in the proliferating endothelial cells (PEC, p.5) (for each, p<0.05, n=6). Unlike in PEC or young aorta (3-month-old), AQP1 knockdown/inhibition restores angiogenesis in both SEC and old aortas. The interventions increased mitochondrial OXPHOS (represented by enhanced oxygen consumption rate (OCR)) in SEC, which is accompanied by marked increase in CaMKKβ-regulated AMPKT172 phosphorylation (for each, p<0.05, n=6) and in histone deacetylase-4 (HDAC4) phosphorylation at Ser632 (p<0.05) and further histone 3 (H3) acetylation (p<0.01). The HDAC4 phosphorylation leads to significant increase in Mef2A-mediated eNOS phosphorylation at Ser1177 (p<0.05). By contrast, AQP1 knockdown/inhibition induced a senescent-like state by a decrease in mitochondrial OCR and the phosphorylation of CaMKKβ-AMPK and HDAC4-Mef2A regulation of eNOS activity. Conclusion Our studies identify AQP1 as essential regulator of EC proliferation that differentially regulates angiogenesis via epigenetic/metabolic response and SASP modulation. Moreover, genetic or pharmacological inhibition of AQP1 reverses endothelial senescence and restores angiogenesis, representing a potential therapeutic target for vascular rejuvenation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)SwissLife Jubiläumsstiftung

Shen-Qi-Wan Protects the Renal Peritubular Capillary Injury from Adenine-mediated Damage by Upregulating Aquaporin 1

Shen-Qi-Wan (SQW), a commonly used prescription against chronic kidney disease (CKD) in Traditional Chinese Medicine (TCM), has a nephroprotective action in adenine-induced kidney injury. However, the mechanism of SQW in renal injury remains unclear. This study was undertaken to measure the effect of SQW on peritubular capillary injury both in vivo and in vitro assays. The effect of SQW on the peritubular capillary injury was evaluated according to measuring hypothalamic-pituitary-adrenal (HPA) function, inflammatory cytokines, VEGF (vascular endothelial growth factor), CD34 (Cluster of differentiation 34), and AQP1 (Aquaporin 1) levels in the kidney, cell migration, and lumen forming capacity. SQW supplementation could ameliorate dysfunction of the HPA and renal function loss induced by adenine. SQW also significantly inhibited the inflammatory cytokines including MCP-1 (monocyte chemotactic protein-1) and VCAM-1 (vascular cell adhesion molecule-1) level. Alternatively, SQW administration showed an ameliorating effect from the toxicity and alleviated the injury of capillaries around renal tubules instigated by adenine through increasing AQP1 mRNA and protein level. SQW medicated the serum enhanced the migration and lumen formation ability of HMEC-1 cells, and significantly increased AQP1 protein level. Moreover, AQP1 knockdown efficiently inhibited the migration and lumen formation ability in HMEC-1 cells, and weakened the effect of SQW medicated serum. These results suggested that SQW attenuated peritubular capillary injury in adenine-induced CKD model rats through boosting angiogenesis in endothelial cell, and AQP1 may be a potential target of SQW for treating the renal injury.

271 Critical role of Aquaporin-1 and telocytes in infantile hemangioma response to propranolol beta blockade

Propranolol is currently the first-line treatment for severe infantile hemangioma (IH), the most frequent tumor in childhood. Neither the molecular mechanism of action nor the cellular target of the drug has been identified with a strong proof of concept. We showed that propranolol antitumor activity was associated with alteration of Aquaporin 1 (AQP1), a water and ion channel involved in tumor cell migration and angiogenesis. In pathological context, elevated AQP1 is involved in the development and progression of tumors and associated with poor prognosis. We developed an IH in vitro model using lesional patient-derived endothelial cells (EC), pericytes (PER) and CD34+/ PDGFR-α+ stromal cells named telocytes (TC) that we first described in IH. Thus, we investigated the role of these perivascular cells in the antiangiogenic effect of propranolol in IH and tested AQP1 as a marker and target of the beta-blockade response in IH. Immunuhistofluorescence staining revealed a unique AQP1 expression in IH-TC compared to normal dermis. IH-TC maintain high in vitro AQP1 protein levels compared to foreskin control-TC. We performed a Matrigel based tube formation assay including IH-EC, IH-PER and IH-TC, which highlights migration and cell-cell interaction alterations. This 3D model has a unique response to low doses of propranolol. Indeed 3 μM of propranolol highly decreased IH angiogenesis in our in vitro model and this effect is abolished when control foreskin-TC are used instead of IH-TC. Interestingly, the knockdown of AQP1 in IH-TC shows the same anti-angiogenic effect as propranolol and the combination is not additive. Moreover, we show that this beta-blockade downregulates AQP1 in patient derived telocytes. All together, these data suggest that IH sensitivity to propranolol rely at least in part to a cross talk between lesional vascular cells and stromal telocytes and reveal a critical role of AQP1 in this antitumor response.

Aquaporin 1 knockdown inhibits triple-negative breast cancer cell proliferation and invasion in vitro and in vivo.

Aquaporin 1 (AQP1) contributes to the progression of several cancer types, but its potential involvement in triple-negative breast cancer (TNBC) is unclear. The aim of the present study was to examine the role of AQP1 in cell proliferation and invasion in TNBC. Reverse transcription-quantitative PCR analysis and western blotting were used to detect AQP1 expression in different cell lines. A short hairpin (sh)RNA targeting AQP1 was established and transfected into MDA-MB-231 breast cancer cells. To investigate the effects of AQP1 knockdown, breast cancer cell proliferation, migration and invasion were evaluated by Cell Counting Kit-8 and Transwell assays. Furthermore, the volume and weight of tumor xenografts in mice were measured to evaluate breast cancer growth ability. The results revealed that the levels of AQP1 were higher in the MDA-MB-231 cell line compared with those in other breast cancer cell lines (MCF-7 and SK-BR-3) and a normal mammary epithelial cell line (MCF-10A). The shRNA targeting AQP1 effectively downregulated AQP1 expression at the mRNA and protein levels, and markedly suppressed TNBC cell proliferation, migration and invasion in vitro, and tumor growth in vivo. These results suggested that AQP1 may serve as a potential therapeutic target in TNBC.

Abstract 14736: Aquaporin 1 Modulates H 2 o 2 -mediated Endothelial Inflammaging

Background: Aquaporin 1 (AQP1), a key regulator of endothelial cell function, transports hydrogen peroxide (H 2 O 2 ) into the cells. H 2 O 2 -mediated inflammation has been implicated in endothelial dysfunction, yet specific roles of AQP1-mediated signaling pathways underlying age-dependent endothelial dysfunction remains incompletely understood. Methods: In these studies, we dissected AQP1-regulated inflammation modulation of eNOS signaling pathways in human aortic endothelial cells (HAEC) from passages 5 to 15 by exploiting siRNA approaches, live cell fluorescence imaging with genetically-encoded H 2 O 2 biosensor HyPer, biochemical and in vitro endothelial function assays. Results: We discovered that AQP1 expression remarkably increases in senescent HAEC (P.15) in association with significant increased SA-βgal activity compared to young cells (P.5). H 2 O 2 levels were increased in senescent cell cytosol revealed by fluorescence HyPer imaging. We found that senescence-associated increase in AQP1-mediated H 2 O 2 led to enhanced TNF-α, unlike HO-1, transcription (p<0.01). Moreover, immunofluorescence assay documented that AQP1 gene knock-down significantly ameliorated senescence-associated elevation of adhesion molecule ICAM-1. Immunoblot analyses demonstrated that increased AQP1 protein levels in senescent HAEC leads to significant increase in caveolin-1 phosphorylation (2-fold) and decreases in phosphorylation of AMPK (Thr172; 4-fold) and eNOS (Ser1177; 2-fold) compared to young ECs (for each, p<0.05, n>6). We discovered that AQP1 knock-down improved the reduced angiogenesis and wound healing capacity in association with eNOS down-regulation in senescent endothelial cells. Conclusion: These results establish that AQP1 plays a crucial role in the regulation of H 2 O 2 -mediated inflammation-associated endothelial senescence, and AQP1 deletion improves endothelial dysfunction by ameliorating ROS-modulated inflammaging.

The Effect of Shen Qi Wan Medicated Serum on NRK-52E Cells Proliferation and Migration by Targeting Aquaporin 1 (AQP1).

BackgroundShen Qi Wan (SQW) as a well-known formula for the amelioration of kidney yang deficiency syndrome (KYDS), and it has been widely employed in traditional Chinese medicine (TCM). This study aimed to investigate the effect and underlying mechanism of SQW medicated serum on proliferation and migration in NRK-52E cells.Material/MethodsWe employed the real-time cell analysis (RTCA) system to investigate the effect of SQW medicated serum on proliferation and migration in NRK-52E cells. In addition, the migration was further investigated by using a wound-healing assay. The mRNA and protein expression level of aquaporin 1 (AQP1) of NRK-52E cells with SQW medicated serum-treated were quantified by real-time quantitative polymerase chain reaction (q-PCR) and western blot assay, respectively. Furthermore, NRK-52E cells were transfected with lentivirus AQP1-RNAi to assess migratory cell abilities in vitro.ResultsThe migratory abilities of NRK-52E cells were significantly increased after SQW medicated serum treatment (P<0.05), and no significant difference in cell proliferation. In addition, SQW medicated serum was significantly upregulated the mRNA and protein expression level of AQP1 in NRK-52E cells (P<0.05). Additionally, the in vitro metastasis test proved that knockdown of AQP1 suppressed migratory abilities according to RTCA and wound healing test while was reversed by SQW medicated serum (P<0.05).ConclusionsOur study demonstrates that SQW medicated serum effectively promotes the migration of NRK-52E cells by increasing AQP1 expression, and AQP1 may be as a therapeutic target of SQW for renal injury treatment under KYDS.

Neuronal Aquaporin 1 Inhibits Amyloidogenesis by Suppressing the Interaction Between Beta-Secretase and Amyloid Precursor Protein.

The accumulation of amyloid-β (Aβ) is a characteristic event in the pathogenesis of Alzheimer's disease (AD). Aquaporin 1 (AQP1) is a membrane water channel protein belonging to the AQP family. AQP1 levels are elevated in the cerebral cortex during the early stages of AD, but the role of AQP1 in AD pathogenesis is unclear. We first determined the expression and distribution of AQP1 in brain tissue samples of AD patients and two AD mouse models (3xTg-AD and 5xFAD). AQP1 accumulation was observed in vulnerable neurons in the cerebral cortex of AD patients, and in neurons affected by the Aβ or tau pathology in the 3xTg-AD and 5xFAD mice. AQP1 levels increased in neurons as aging progressed in the AD mouse models. Stress stimuli increased AQP1 in primary cortical neurons. In response to cellular stress, AQP1 appeared to translocate to endocytic compartments of β- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced β-secretase (BACE1)-mediated cleavage of APP and reduced Aβ production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aβ production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aβ decreases as the expression of AQP1 increases. These results suggest that the upregulation of AQP1 is an adaptive response of neurons to stress that reduces Aβ production by inhibiting the binding between BACE1 and APP.

Combined pharmacological administration of AQP1 ion channel blocker AqB011 and water channel blocker Bacopaside II amplifies inhibition of colon cancer cell migration

Aquaporin-1 (AQP1) has been proposed as a dual water and cation channel that when upregulated in cancers enhances cell migration rates; however, the mechanism remains unknown. Previous work identified AqB011 as an inhibitor of the gated human AQP1 cation conductance, and bacopaside II as a blocker of AQP1 water pores. In two colorectal adenocarcinoma cell lines, high levels of AQP1 transcript were confirmed in HT29, and low levels in SW480 cells, by quantitative PCR (polymerase chain reaction). Comparable differences in membrane AQP1 protein levels were demonstrated by immunofluorescence imaging. Migration rates were quantified using circular wound closure assays and live-cell tracking. AqB011 and bacopaside II, applied in combination, produced greater inhibitory effects on cell migration than did either agent alone. The high efficacy of AqB011 alone and in combination with bacopaside II in slowing HT29 cell motility correlated with abundant membrane localization of AQP1 protein. In SW480, neither agent alone was effective in blocking cell motility; however, combined application did cause inhibition of motility, consistent with low levels of membrane AQP1 expression. Bacopaside alone or combined with AqB011 also significantly impaired lamellipodial formation in both cell lines. Knockdown of AQP1 with siRNA (confirmed by quantitative PCR) reduced the effectiveness of the combined inhibitors, confirming AQP1 as a target of action. Invasiveness measured using transwell filters layered with extracellular matrix in both cell lines was inhibited by AqB011, with a greater potency in HT29 than SW480. A side effect of bacopaside II at high doses was a potentiation of invasiveness, that was reversed by AqB011. Results here are the first to demonstrate that combined block of the AQP1 ion channel and water pores is more potent in impairing motility across diverse classes of colon cancer cells than single agents alone.

Development of a Photoswitchable Lithium-Sensitive Probe to Analyze Nonselective Cation Channel Activity in Migrating Cancer Cells.

This is the first work to use a newly designed Li+-selective photoswitchable probe Sabrina Heng Lithium (SHL) in living colon cancer cells to noninvasively monitor cation channel activity in real time by the appearance of lithium hot spots detected by confocal microscopy. Punctate Li+ hot spots are clustered in the lamellipodial leading edges of HT29 human colon cancer cells and are colocalized with aquaporin-1 (AQP1) channels. AQP1 is a dual water and cyclic-nucleotide-gated cation channel located in lamellipodia and is essential for rapid cell migration in a subset of aggressive cancers. Both the Li+ hot spots and cell migration are blocked in HT29 cells by the AQP1 ion channel antagonist AqB011. In contrast, Li+ hot spots are not evident in a poorly migrating colon cancer cell line, SW620, which lacks comparable membrane expression of AQP1. Knockdown of AQP1 by RNA interference in HT29 cells significantly impairs Li+ hot spot activity. The SHL probe loaded in living cells shows signature chemical properties of ionic selectivity and reversibility. Dynamic properties of the Li+ hot spots, turning on and off, are confirmed by time-lapse imaging. SHL is a powerful tool for evaluating cation channel function in living cells in real time, with particular promise for studies of motile cells or interlinked networks not easily analyzed by electrophysiological methods. The ability to reset SHL by photoswitching allows monitoring of dynamic signals over time. Future applications of the Li+ probe could include high-throughput optical screening for discovering new classes of channels, or finding new pharmacological modulators for nonselective cation channels.

Knockdown of AQP1 inhibits growth and invasion of human ovarian cancer cells.

Aquaporin 1 (AQP1), which is a water channel protein, has been demonstrated to have an important role in cell proliferation and migration of various cancers. However, its specific role in ovarian cancer remains to be elucidated. The present study demonstrated that AQP1 expression was elevated in the majority of patients with ovarian cancer compared with normal ovarian tissues. In addition, a short interfering (si)RNA targeting AQP1 was established, and transfected into the SKOV3 ovarian cancer cell line, to investigate the effects on cell viability, apoptosis, migration and invasion in the ovarian cancer cells using an MTT assay, flow cytometry, wound healing and Transwell invasion chamber assays, respectively. The results of the present study demonstrated that siRNA targeting AQP1 effectively downregulated AQP1 expression at the mRNA and protein levels, markedly suppressed cell viability, migration and invasion and promoted apoptosis of ovarian cancers cells. These results suggested that AQP1 may serve as a novel target for ovarian cancer treatment in the future.

Elevated hydrostatic pressure enhances the motility and enlarges the size of the lung cancer cells through aquaporin upregulation mediated by caveolin-1 and ERK1/2 signaling.

The mechanical characteristics presented in cancer microenvironment are known to have pivotal roles in cancer metastasis, which accounts for the leading cause of death from malignant tumors. However, while a uniformly distributed high interstitial fluid pressure (IFP) is a common feature in solid tumors, the effects of high IFP on the motility and invasiveness of cancer cells remain obscure. Using cell-culture devices that simulated increased IFP conditions by applying hydrostatic pressure (HP) ranging from 0 to 20 mm Hg to the cells, we found that the elevated HPs increased the migration speeds, invasiveness, cell volume, filopodial number and aquaporin-1 (AQP1), Snail and vinculin expression levels, as well as phosphorylation of caveolin-1 and extracellular signal-regulated kinase1/2 (ERK1/2), in the lung cancer cells CL1-5 and A549. The increases of migration speed and cell volume correlated temporally with the increase of AQP1 expression. The elevated HP-induced migration acceleration was hindered by AQP1 knockdown using small interfering RNA (siRNA) transfection. Inhibition of ERK1/2 phosphorylation using the mitogen-activated protein kinase kinase inhibitor PD98059 abrogated the elevated HP-induced AQP1 upregulation and migration acceleration in the cancer cells. Caveolin-1 knockdown by siRNA transfection attenuated the HP-induced, ERK1/2-depedent AQP1 upregulation and migration acceleration. Further biochemical studies revealed that the caveolin-1 activation-driven ERK1/2 signaling is mediated by Akt1/2 phosphorylation. By contrast, the elevated HPs had negligible effects on the migration speed and volume of normal bronchial epithelial cells. These results disclose a novel mechanism relating high IFP to the invasiveness of cancer cells and highlight potential targets to impede cancer spreading.

Endothelial Aquaporin-1 (AQP1) Expression Is Regulated by Transcription Factor Mef2c.

Aquaporin 1 (AQP1) is expressed in most microvasculature endothelial cells and forms water channels that play major roles in a variety of physiologic processes. This study aimed to delineate the transcriptional regulation of AQP1 by Mef2c in endothelial cells. Mef2c cooperated with Sp1 to activate human AQP1 transcription by binding to its proximal promoter in human umbilical cord vein endothelial cells (HUVEC). Over-expression of Mef2c, Sp1, or Mef2c/Sp1 increased HUVEC migration and tube-forming ability, which can be abolished AQP1 knockdown. These data indicate that AQP1 is a direct target of Mef2c in regulating angiogenesis and vasculogenesis of endothelial cells.

RNAi-mediated silencing of AQP1 expression inhibited the proliferation, invasion and tumorigenesis of osteosarcoma cells

Aquaporin 1 (AQP1), a member of water channel proteins, functions as a water-selective transporting protein in cell membranes. In recent years, AQP1 has been found to be overexpressed in various tumors. However, the molecular mechanism underlying the biological function of AQP1 in osteosarcoma is still unclear. This study was aimed at elucidating the roles of AQP1 in regulating the biological behavior of osteosarcoma cells. In this study, we found that AQP1 mRNA was elevated in osteosarcoma tissue. High level of AQP1 was associated with poor prognosis in osteosarcoma. Then, we found that knockdown of AQP1 in osteosarcoma cells, U2OS or MG63 cells inhibited cell proliferation and significantly increased cells population in G1 phase. Additionally, suppressing AQP1 expression in osteosarcoma cells dramatically induced cell apoptosis. We also found that down-regulation of AQP1 significantly inhibited cell adhesion and invasion. More importantly, AQP1 knockdown inhibited tumor growth in vivo and prolonged the survival time of nude mice. Gene set enrichment analysis (GSEA) showed that transforming growth factor-β (TGF-β) signaling pathway and focal adhesion genes was correlatively with AQP1 expression. In addition, real time PCR and western blot analysis revealed that expression of TGF-β1/TGF-β2, RhoA and laminin β 2 (LAMB2) was remarkably impaired by AQP1 silencing. In conclusion, AQP1 may be a useful diagnosis and prognosis marker for osteosarcoma. AQP1 knockdown can effectively inhibit cell proliferation, adhesion, invasion and tumorigenesis by targeting TGF-β signaling pathway and focal adhesion genes, which may serve a promising therapeutic strategy for osteosarcoma.

Aquaporin 1 promotes the proliferation and migration of lung cancer cell invitro.

To examine the potential role of aquaporin1 (AQP1) in lung cancer progression, the effects of AQP1 expression and underlying mechanisms on cell proliferation and migration were investigated on LLC and LTEP-A2 cell lines invitro. LLC and LTEP-A2 lung cancer cells with a discrepant AQP1 expression level were used to determine the role of AQP1 in cancer cell proliferation and migration potential. An immuno-fluorescence assay was used to detect AQP1 expression levels in the LLC and LTEP-A2 cell lines. The method targeting the knockdown of AQP1 on lung cancer cell lines by siRNA was established and validated by RT-PCR and western blot analysis. The proliferation and migration abilities of AQP1 knockdown cell lines were detected by MTT, invasion and wound-healing assays. Moreover, the alteration of MMP-2, MMP-9, TGF-β and epidermal growth factor receptor (EGFR) expression, associated with the migration and metastasis potential of lung cancer cell lines, was identified by western blot analysis in transfected cells. In the tumor cell migration and invasion test, AQP1 knockdown significantly decreased the migration and invasion of AQP1-siRNA cells. Additionally, the expression levels of MMPs were markedly decreased after AQP1-siRNA treatment in the two cell lines. Moreover, the decrease of MMP-2/-9 expression on lung cancer cell lines was associated with AQP1-siRNA doses. However, AQP1 knockdown did not have a significant effect on TGF-β and EGFR. The results suggest that AQP1 may facilitate lung cancer cell proliferation and migration in an MMP-2 and-9-dependent manner.

AQP1 expression alterations affect morphology and water transport in Schwann cells and hypoxia-induced up-regulation of AQP1 occurs in a HIF-1α-dependent manner

Aquaporin-1 (AQP1) is the principle water channel in the peripheral nervous system (PNS) and is specifically localized to Schwann cells in the PNS. However, the pathophysiological role of AQP1 in peripheral nerves is poorly understood. Here, we utilized RNA interference by lentiviral transduction to specifically down-regulate AQP1 expression and a lentiviral overexpression protocol to up-regulate AQP1 expression, in primary Schwann cell cultures. AQP1 gene silencing resulted in a cell shrinkage phenotype, while AQP1 gene overexpression caused a cell swelling phenotype, as validated by cell volume determinations. Secondly, we utilized an in vitro hypoxia model in Schwann cells to mimic in vivo facial nerve injury. We demonstrated that AQP1 expression was induced within 8h following hypoxia injury in vitro, and that AQP1 knockdown (KD) caused the cells to resist edema following hypoxia. Finally, we investigated the hypoxic regulation of the AQP1 gene, as well as the involvement of Hypoxia-inducible factor-1α (HIF-1α) in AQP1 modulation and we found that KD of HIF-1α decreased hypoxia-dependent induction of endogenous AQP1 expression at both the mRNA and protein levels. Taken together, these results indicate that (1) AQP1 is an important factor responsible for the fast water transport of cultured Schwann cells and is involved in cell plasticity; (2) AQP1 alterations may be a primary factor in hypoxia-induced peripheral nerve edema; (3) HIF-1α participates in the hypoxic induction of the AQP1 gene; (4) AQP1 inhibition might provide a new therapeutic alternative for the treatment of some forms of peripheral nerve edema.

Aquaporin-1 Plays a Crucial Role in Estrogen-Induced Tubulogenesis of Vascular Endothelial Cells

Aquaporin-1 (AQP1) has been proposed as a mediator of estrogen-induced angiogenesis in human breast cancer and endometrial cancer. Elucidation of the molecular mechanisms governing AQP1-mediated, estrogen-induced angiogenesis may contribute to an improved understanding of tumor development. Our objective was to identify the estrogen-response element (ERE) in the promoter of the Aqp1 gene and investigate the effects and mechanisms of AQP1 on estrogen-induced tubulogenesis of vascular endothelial cells. The study was conducted in a university hospital in eastern China. Immunohistological, real-time PCR and Western blot analyses were used to determine the expression AQP1 mRNA and protein in vascular endothelial cells. Chromatin immunoprecipitation analyses and luciferase reporter assays identified ERE-like motif in the promoter of the Aqp1 gene. Expression of AQP1 in blood vessels of human breast and endometrial carcinoma tissues were significantly higher than controls. Estradiol (E2) dose-dependently increased the expression levels of AQP1 mRNA and protein in human umbilical vein endothelial cells (HUVECs). A functional ERE-like motif was identified in the promoter of the Aqp1 gene. AQP1 colocalized with ezrin, a component of the ezrin/radixin/moesin protein complex, and, ezrin colocalized with filamentous actin in HUVECs. Knockdown of AQP1 or ezrin with specific small interfering RNA significantly attenuated the formation of transcytoplasmic filamentous actin stress fibers induced by E2 and inhibited E2-enhanced cell proliferation, migration, invasion, and tubule formation of HUVECs. Estrogen induces AQP1 expression by activating ERE in the promoter of the Aqp1 gene, resulting in tubulogenesis of vascular endothelial cells. These results provide new insights into the molecular mechanisms underpinning the angiogenic effects of estrogen.