AQP4-A25Q Point Mutation in Mice Depolymerizes Orthogonal Arrays of Particles and Decreases Polarized Expression of AQP4 Protein in Astrocytic Endfeet at the Blood-Brain Barrier.

Aquaporin-4 (AQP4) is characterized by the formation of orthogonal arrays of particles (OAPs) comprising its M1 and M23 isoforms in the plasma membrane. However, the biological importance of OAP formation is obscure. Here, we developed an OAP depolymerization male mouse model by transgenic knock-in of an AQP4-A25Q mutation. Analyses of the mutant brain tissue using blue native polyacrylamide gel electrophoresis, super-resolution imaging, and immunogold electron microscopy revealed remarkably reduced OAP structures and glial endfeet localization of the AQP4-A25Q mutant protein without effects on its overall mRNA and protein expression. AQP4A25Q/A25Q mice showed better survival and neurologic deficit scores when cerebral edema was induced by water intoxication or middle cerebral artery occlusion/reperfusion. The brain water content and swelling of pericapillary astrocytic endfeet processes in AQP4A25Q/A25Q mice were significantly reduced, functionally supporting decreased AQP4 protein expression at the blood-brain barrier. The infarct volume and neuronal damage were also reduced in AQP4A25Q/A25Q mice in the middle cerebral artery occlusion/reperfusion model. Astrocyte activation in the brain was alleviated in AQP4A25Q/A25Q mice, which may be associated with decreased cell swelling. We conclude that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.SIGNIFICANCE STATEMENT Aquaporin-4 (AQP4) is characterized by orthogonal arrays of particles (OAPs) comprising the M1 and M23 isoforms in the membrane. Here, an OAP depolymerization male mouse model induced by AQP4-A25Q mutation was first established, and the functions of OAP depolymerization in cerebral edema have been studied. The results revealed that AQP4 lost its OAP structure without affecting AQP4 mRNA and protein levels in AQP4-A25Q mice. AQP4-A25Q mutation mice has neuroprotective effects on cerebral edema induced by water intoxication and middle cerebral artery occlusion/reperfusion through relieving the activation of astrocytes and suppressed microglia-mediated neuroinflammation. We concluded that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.

Tubeimoside-1 induces oxidative stress-mediated apoptosis and G0/G1 phase arrest in human prostate carcinoma cells in vitro.

AIM: Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from the Chinese herbal medicine Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), has shown anticancer activities in various cancer cell lines. The aim of this study was to investigate the anticancer activity and molecular targets of TBMS1 in human prostate cancer cells in vitro. METHODS: DU145 and P3 human prostate cancer cells were treated with TBMS1. Cell viability and apoptosis were detected. ROS generation, mitochondrial membrane potential and cell cycle profile were examined. Western blotting was used to measure the expression of relevant proteins in the cells. RESULTS: TBMS1 (5-100 µmol/L) significantly suppressed the viability of DU145 and P3 cells with IC50 values of approximately 10 and 20 µmol/L, respectively. Furthermore, TBMS1 dose-dependently induced apoptosis and cell cycle arrest at G0/G1 phase in DU145 and P3 cells. In DU145 cells, TBMS1 induced mitochondrial apoptosis, evidenced by ROS generation, mitochondrial dysfunction, endoplasmic reticulum stress, modulated Bcl-2 family protein and cleaved caspase-3, and activated ASK-1 and its downstream targets p38 and JNK. The G0/G1 phase arrest was linked to increased expression of p53 and p21 and decreased expression of cyclin E and cdk2. Co-treatment with Z-VAD-FMK (pan-caspase inhibitor) could attenuate TBMS1-induced apoptosis but did not prevent G0/G1 arrest. Moreover, co-treatment with NAC (ROS scavenger), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor) or salubrinal (ER stress inhibitor) significantly attenuated TBMS1-induced apoptosis. CONCLUSION: TBMS1 induces oxidative stress-mediated apoptosis in DU145 human prostate cancer cells in vitro via the mitochondrial pathway.

Osmotic water permeability diversification in primary trophoblast cultures from aquaporin 1-deficient pregnant mice.

AIM: Aquaporins (AQP) are water channel proteins, and some play an important role in maternal-fetal fluid exchange. The present study aimed to measure the osmotic water permeability in primary cultures of trophoblast cells from AQP1-deficient (AQP1(-/-) ) pregnant mice and to define the quantitative role of AQP1 in water transport across the trophoblast plasma membrane. MATERIAL AND METHODS: Trophoblast cells were obtained from placental tissue cell culture of AQP1(-/-) pregnant mice and were characterized by cytokeratin 7 immunostaining. The expression of the AQP1 gene in trophoblast cells of wild-type (AQP1(+/+) ) mice was confirmed by immunofluorescence. The osmotic water permeability of trophoblast plasma membranes was measured by a calcein fluorescence quenching method in response to osmotic gradients. RESULTS: A primary cell culture system for trophoblasts was successfully established. Immunofluorescence showed the expression of AQP1 in the trophoblast cell membrane of AQP1(+/+) mice. The osmotic water permeability of AQP1(-/-) trophoblast cells was significantly lower than that in AQP1(+/+) trophoblast cells, in response to both hypotonic and hypertonic challenges. CONCLUSION: The results suggest an important role of AQP1-mediated plasma membrane water permeability in maternal-fetal fluid balance and also provide a potential direction for the identification of therapeutic targets for the treatment of abnormalities in amniotic fluid volume.

Measurable residual disease testing by next generation sequencing is more accurate compared with multiparameter flow cytometry in adults with B-cell acute lymphoblastic leukemia.

Results of measurable residual disease (MRD)-testing by next-generation sequencing (NGS) correlate with relapse risk in adults with B-cell acute lymphoblastic leukemia (ALL) receiving chemotherapy or an allotransplant from a human leukocyte antigen (HLA)-identical relative or HLA-matched unrelated donor. We studied cumulative incidence of relapse (CIR) and survival prediction accuracy using a NGS-based MRD-assay targeting immunoglobulin genes after 2 courses of consolidation chemotherapy cycles in 93 adults with B-cell ALL most receiving HLA-haplotype-matched related transplants. Prediction accuracy was compared with MRD-testing using multi-parameter flow cytometry (MPFC). NGS-based MRD-testing detected residual leukemia in 28 of 65 subjects with a negative MPFC-based MRD-test. In Cox regression multi-variable analyses subjects with a positive NGS-based MRD-test had a higher 3-year CIR (Hazard Ratio [HR] = 3.37; 95 % Confidence Interval [CI], 1.34-8.5; P = 0.01) and worse survival (HR = 4.87 [1.53-15.53]; P = 0.007). Some data suggest a lower CIR and better survival in NGS-MRD-test-positive transplant recipients but allocation to transplant was not random. Our data indicate MRD-testing by NGS is more accurate compared with testing by MPFC in adults with B-cell ALL in predicting CIR and survival. (Registered in the Beijing Municipal Health Bureau Registration N 2007-1007 and in the Chinese Clinical Trial Registry [ChiCTR-OCH-10000940 and ChiCTROPC-14005546]).

Molecular and clinical features of papillary thyroid cancer in adult patients with a non-classical phenotype.

Purpose: Genotyping is fundamental in papillary thyroid cancer (PTC) and helps to enhance diagnosis and prognosis and determine appropriate treatments. The phenotype-genotype association in PTC was previously studied, with BRAF V600E characterizing classic PTC and tall-cell PTC and RAS mutations characterizing follicular-variant PTC. In clinic, some non-classical histological subtypes of PTC were also identified, however, their genotype remains unclear. In this study, we collected samples of these non-classical PTC after the exclusion of classic phenotypes and examined their phenotypes, genotype and the relationship between phenotype and genotype. Methods: We screened out non-classical PTC by excluding classical PTC from 1,059 different thyroid samples, and a total of 24 cases was obtained and described from the morphological features, which is rare in differentiated PTC. DNA/RNA sequencing was performed using 18 available samples to describe the genetic features. Results: PTC with the non-classical phenotype were characterized cuboidal to low columnar tumor cells with subtle nuclear features of PTC and without discernible nuclear elongation, concurrently with dense microfollicles, delicate papillae or solid nodules with delicate fibrovascular cores. They were associated with lymphatic vessel invasion (P<0.001) but not with a worse prognosis (P=0.791). Gene fusions were identified in 14 of 18 (77.8%) cases, including eight fusions of NTRK and six fusions of RET. The high percentage of fusions in this papillary thyroid cancer subgroup suggested a correlation of gene fusions with the phenotype that does not belong to the BRAF V600E-mutant or RAS-mutant group. Conclusions: Our study retrospectively screened a large cohort of different thyroid tissue samples, and presented the histopathological and genetic features of a non-classical phenotype of PTC from 24 patients. It may contribute to diagnose in PTC, and patients of these non-classical phenotype may benefit from targeted therapy, compared to a natural patient cohort without selection.

AQP4 Aggravates Cognitive Impairment in Sepsis-Associated Encephalopathy through Inhibiting Nav 1.6-Mediated Astrocyte Autophagy.

The pathology of sepsis-associated encephalopathy (SAE) is related to astrocyte-inflammation associated with aquaporin-4 (AQP4). The aim here is to investigate the effects of AQP4 associated with SAE and reveal its underlying mechanism causing cognitive impairment. The in vivo experimental results reveal that AQP4 in peripheral blood of patients with SAE is up-regulated, also the cortical and hippocampal tissue of cecal ligation and perforation (CLP) mouse brain has significant rise in AQP4. Furthermore, the data suggest that AQP4 deletion could attenuate learning and memory impairment, attributing to activation of astrocytic autophagy, inactivation of astrocyte and downregulate the expression of proinflammatory cytokines induced by CLP or lipopolysaccharide (LPS). Furthermore, the activation effect of AQP4 knockout on CLP or LPS-induced PPAR-γ inhibiting in astrocyte is related to intracellular Ca2+ level and sodium channel activity. Learning and memory impairment in SAE mouse model are attenuated by AQP4 knockout through activating autophagy, inhibiting neuroinflammation leading to neuroprotection via down-regulation of Nav 1.6 channels in the astrocytes. This results in the reduction of Ca2+ accumulation in the cell cytosol furthermore activating the inhibition of PPAR-γ signal transduction pathway in astrocytes.

The D1 dopamine receptor agonist, SKF83959, attenuates hydrogen peroxide-induced injury in RGC-5 cells involving the extracellular signal-regulated kinase/p38 pathways.

PURPOSE: Oxidative stress is widely implicated in the death of retinal ganglion cells associated with various optic neuropathies. Agonists of the dopamine D(1) receptor have recently been found to be potentially neuroprotective against oxidative stress-induced injury. The goal of this study was to investigate whether SKF83959, a next-generation high-affinity D(1) receptor agonist, could protect retinal ganglion cell 5 (RGC-5) cells from H(2)O(2)-induced damage and the molecular mechanism involved. METHODS: We examined expression of the D(1) receptor in RGC-5 cells with reverse-transcription-PCR and immunoblotting and assessed neuroprotection using propidium iodide staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, we monitored the activation and involvement of members of mitogen-activated protein kinase family, extracellular signal-regulated kinase (ERK), p38 and c-Jun NH(2)-terminal kinase, with western blot and specific inhibitors. RESULTS: We found that the D(1) receptor was expressed in RGC-5 cells, but the sequence analysis suggested this cell line is from mouse and not rat origin. SKF83959 exhibited a remarkable neuroprotective effect on H(2)O(2)-damaged RGC-5 cells, which was blocked by the specific D(1) receptor antagonist, SCH23390. ERK and p38 were activated by SKF83959, and pretreatment with their inhibitors U0126 and SB203580, respectively, significantly blunted the SKF83959-induced cytoprotection. However, the specific c-Jun NH(2)-terminal kinase inhibitor, SP600125, had no effect on the SKF83959-induced protection. CONCLUSIONS: We conclude that SKF83959 attenuates hydrogen peroxide-induced injury in RGC-5 cells via a mechanism involving activation of the ERK and p38 pathways and the D(1) receptor is a potential molecular target for developing neuroprotective drugs.

Potent Inhibition of Human Cytochrome P450 3A4 by Biflavone Components from Ginkgo Biloba and Selaginella Tamariscina.

CYP3A4-mediated Phase I biotransformation is the rate-limiting step of elimination for many commonly used clinically agents. The modulatory effects of herbal medicines on CYP3A4 activity are one of the risk factors affecting the safe use of drug and herbal medicine. In the present study, the inhibitory effects of nearly hundred kinds of herbal medicines against CYP3A4 were evaluated based on a visual high-throughput screening method. Furthermore, biflavone components including bilobetin (7-demethylginkgetin, DGK), ginkgetin (GK), isoginkgetin (IGK), and amentoflavone (AMF) were identified as the main inhibitory components of Ginkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST), which displayed very strong inhibitory effects toward CYP3A4. The inhibitory effects of these biflavones on clinical drugs that mainly undergo CYP3A4-dependent metabolism were evaluated. The IC 50 of GK toward tamoxifen, gefitinib and ticagrelor were found to be of 0.478 ± 0.003, 0.869 ± 0.001, and 1.61 ± 0.039 µM, respectively. These results suggest the potential pharmacokinetic interactions between the identified biflavones and clinical drugs undergoing CYP3A4-mediated biotransformation. The obtained information is important for guiding the rational use of herbal medicine in combination with synthetic pharmaceuticals.

Profiling of gene fusion involving targetable genes in Chinese gastric cancer.

BACKGROUND: Approximately half of all new cases of gastric cancer (GC) and related deaths occur in China. More than 80% of patients with GC are diagnosed at an advanced stage, which results in poor prognosis. Although HER2-directed therapy and immune checkpoint inhibitors have been somewhat successful, new drugs are still needed for the treatment of GC. Notably, several gene fusion-targeted drugs have been approved by the United States Food and Drug Administration for solid tumors, including GC, such as larotrectinib for NTRK fusion-positive cancers and zenocutuzumab for NRG1 fusion-positive cancers. However, gene fusions involving targetable genes have not been well characterized in Chinese patients with GC. AIM: To identify the profile of fusions involving targetable genes in Chinese patients with GC using clinical specimens and determine the distribution of patients with gene fusion variants among the molecular subtypes of GC. METHODS: We retrospectively analyzed gene fusion events in tumor tissue samples from 954 Chinese patients with GC. Clinicopathological characteristics were obtained from their medical records. Genetic alterations, such as single nucleotide variants, indels, amplifications, and gene fusions, were identified using a targeted sequencing panel containing 825 genes. Fusions were validated by fluorescence in situ hybridization (FISH) using break-apart probes. The microsatellite instability (MSI) status was evaluated using MSIsensor from the targeted sequencing panel data. Tumor mutational burden (TMB) was calculated using the total number of nonsynonymous mutations divided by the total genomic targeted region. Chi-square analysis was used to determine the enrichment of gene fusions associated with the molecular subtypes of GC. RESULTS: We found that 1.68% (16/954) of patients harbored 20 fusion events involving targetable genes. RARA fusions (n = 5) were the most common, followed by FGFR2, BRAF, MET, FGFR3, RET, ALK, EGFR, NTRK2, and NRG1 fusions. Two of the RARA fusions, EML4-ALK (E6:E20) and EGFR-SEPTIN14 (E7:E10), have been identified in other tumors but not in GC. Surprisingly, 18 gene fusion events were previously not reported in any cancer types. Twelve of the eighteen novel gene fusions included complete exons encoding functional domains of targetable genes, such as the tyrosine kinase domain of receptor tyrosine kinases and the DNA- and ligand-binding domains of RARA. Consistent with the results of detection using the targeted sequencing fusion panel, the results of FISH (fluorescence in situ hybridization) confirmed the rearrangement of FGFR2 and BRAF in tumors from patients 04 and 09, respectively. Genetic analysis indicated that the fusion genes were significantly enriched in patients with ERBB2 amplification (P = 0.02); however, there were no significant differences between fusion-positive and fusion-negative patients in age, sex, MSI status, and TMB. CONCLUSION: We characterized the landscape of fusions involving targetable genes in a Chinese GC cohort and found that 1.68% of patients with GC harbor potential targetable gene fusions, which were enriched in patients with ERBB2 amplification. Gene fusion detection may provide a potential treatment strategy for patients with GC with disease progression following standard therapy.

Visible light may directly induce nuclear DNA damage triggering the death pathway in RGC-5 cells.

PURPOSE: Visible light has been previously demonstrated to induce retinal ganglion cell (RGC)-5 cell death through the mitochondrial pathway. The present study was designed to determine whether visible light might also directly trigger the death pathway by damaging nuclear DNA. METHODS: RGC-5 cells were exposed to various intensities and durations of visible light exposure. Cell viability and death were monitored with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide staining. Nuclear DNA damage caused by light was determined with the plasmid assay, genome DNA assay, and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling. The subsequent activation of nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was measured with western blot, and PARP-1's role in the death pathway was assessed by using specific inhibitors. Poly (ADP-ribose) glycohydrolase and apoptosis-inducing factor (AIF) inhibitors were used to show their influence on light-induced cell death. Calcium influx was examined with the fura-2 assay and calcium channel blocker. RESULTS: We found that visible light induced RGC-5 cell death in a time- and intensity-dependent manner. After the light intensity was increased to 2,600 lx, activation of the death pathway in RGC-5 cells was clearly observed by detecting double-strand DNA breaks and nuclear DNA damage in vitro. Nuclear enzyme PARP-1 was promptly activated after exposure to 2,600 lx of light for 2 days, and specific inhibitors of PARP-1 had significant neuroprotective effects. The poly(ADP-ribose) glycohydrolase inhibitor tannic acid and AIF inhibitor N-phenylmaleimide partially protected RGC-5 cells from light injury. A massive calcium influx was detected after 2 days of light exposure, and a calcium channel blocker partially protected cells against light injury. CONCLUSIONS: These results suggest that visible light exposure may directly cause nuclear DNA damage, which consequently activates PARP-1. In addition, RGC-5 cells damaged by 2,600 lx of light exposure can be used as an appropriate cell death model for screening neuroprotective drugs, since this treatment induced remarkable cell death within 2 days. Moreover, these results show that 2,600 lx of light exposure provides a more apparent activation of the death pathway than 1,000 lx of light exposure, which was used in a previous study.

Expression and function of aquaporins in peripheral nervous system.

The expression and role of the aquaporin (AQP) family water channels in the peripheral nervous system was less investigated. Since 2004, however, significant progress has been made in the immunolocalization, regulation and function of AQPs in the peripheral nervous system. These studies showed selective localization of three AQPs (AQP1, AQP2, and AQP4) in dorsal root ganglion neurons, enteric neurons and glial cells, periodontal Ruffini endings, trigeminal ganglion neurons and vomeronasal sensory neurons. Functional characterization in transgenic knockout mouse model revealed important role of AQP1 in pain perception. This review will summarize the progress in this field and discuss possible involvement of AQPs in peripheral neuropathies and their potential as novel drug targets.

Maternal-fetal fluid balance and aquaporins: from molecule to physiology.

Maternal-fetal fluid balance is critical during pregnancy, and amniotic fluid is essential for fetal growth and development. The placenta plays a key role in a successful pregnancy as the interface between the mother and her fetus. Aquaporins (AQPs) form specific water channels that allow the rapid transcellular movement of water in response to osmotic/hydrostatic pressure gradients. AQPs expression in the placenta and fetal membranes may play important roles in the maternal-fetal fluid balance.

Pregnant phenotype in aquaporin 8-deficient mice.

AIM: Aquaporin 8 (AQP8) is expressed within the female reproductive system but its physiological function reminds to be elucidated. This study investigates the role of AQP8 during pregnancy using AQP8-knockout (AQP8-KO) mice. METHODS: Homozygous AQP8-KO mice were mated, and the conception rate was recorded. AQP8-KO pregnant mice or their offspring were divided into 5 subgroups according to fetal gestational day (7, 13, 16, 18 GD) and newborn. Wild type C57 pregnant mice served as the control group. The number of pregnant mice, total embryos and atrophic embryos, as well as fetal weight, placental weight and placental area were recorded for each subgroup. The amount of amniotic fluid in each sac at 13, 16, and 18 GD was calculated. Statistical significance was determined by analysis of variance of factorial design and chi-square tests. RESULTS: Conception rates did not differ significantly between AQP8-KO and wild type mice. AQP8-KO pregnant mice had a significantly higher number of embryos compared to wild type controls. Fetal/neonatal weight was also significantly greater in the AQP8-KO group compared to age-matched wild type controls. The amount of amniotic fluid was greater in AQP8-KO pregnant mice than wild type controls, although the FM/AFA (fetal weight/amniotic fluid amount) did not differ. While AQP8-KO placental weight was significantly larger than wild type controls, there was no evidence of placental pathology in either group. CONCLUSION: The results suggest that AQP8 deficiency plays an important role in pregnancy outcome.

CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives.

AIM: To clarify whether CFTR is a molecular target of intestinal fluid secretion caused by the anthraquinone compounds from laxative herbal plants. METHODS: A cell-based fluorescent assay to measure I(-) influx through CFTR chloride channel. A short-circuit current assay to measure transcellular Cl(-) current across single layer FRT cells and freshly isolated colon mucosa. A closed loop experiment to measure colon fluid secretion in vivo. RESULTS: Anthraquinone compounds rhein, aloe-emodin and 1,8-dihydroxyanthraquinone (DHAN) stimulated I(-) influx through CFTR chloride channel in a dose-dependent manner in the presence of physiological concentration of cAMP. In the short-circuit current assay, the three compound enhanced Cl(-) currents in epithelia formed by CFTR-expressing FRT cells with EC(50) values of 73 ± 1.4, 56 ± 1.7, and 50 ± 0.5 µmol/L, respectively, and Rhein also enhanced Cl(-) current in freshly isolated rat colonic mucosa with a similar potency. These effects were completely reversed by the CFTR selective blocker CFTR(inh)-172. In in vivo closed loop experiments, rhein 2 mmol/L stimulated colonic fluid accumulation that was largely blocked by CFTR(inh)-172. The anthraquinone compounds did not elevate cAMP level in cultured FRT cells and rat colonic mucosa, suggesting a direct effect on CFTR activity. CONCLUSION: Natural anthraquinone compounds in vegetable laxative drugs are CFTR potentiators that stimulated colonic chloride and fluid secretion. Thus CFTR chloride channel is a molecular target of vegetable laxative drugs.

NPA motifs play a key role in plasma membrane targeting of aquaporin-4.

The two highly conserved NPA motifs (asparagine-proline-alanine, NPA) are the most important structural domains that play a crucial role in water-selective permeation in aquaporin water channels. However, the functions of NPA motifs in aquaporin (AQP) biogenesis remain largely unknown. Few AQP members with variations in NPA motifs such as AQP11 and AQP12 do not express in the plasma membrane, suggesting an important role of NPA motifs in AQP plasma membrane targeting. In this study, we examined the role of the two NPA motifs in AQP4 plasma membrane targeting by mutagenesis. We constructed a series of AQP4 mutants with NPA deletions or single amino acid substitutions in AQP4-M1 and AQP4-M23 isoforms and analyzed their expression patterns in transiently transfected FRT and COS-7 cells. Western blot analysis showed similar protein bands of all the AQP4 mutants and the wild-type AQP4. AQP4 immunofluorescence indicated that deletion of one or both NPA motifs resulted in defective plasma membrane targeting, with apparent retention in endoplasmic reticulum (ER). The A99T mutant mimicking AQP12 results in ER retention, whereas the A99C mutant mimicking AQP11 expresses normally in plasma membrane. Furthermore, the AQP4-M1 but not the M23 isoform with P98A substitution in the first NPA motif can target to the plasma membrane, indicating an interaction of N-terminal sequence of AQP4-M1 with the first NPA motif. These results suggest that NPA motifs play a key role in plasma membrane expression of AQP4 but are not involved in AQP4 protein synthesis and degradation. The NPA motifs may interact with other structural domains in the regulation of membrane trafficking during aquaporin biogenesis.

Reduced lung water transport rate associated with downregulation of aquaporin-1 and aquaporin-5 in aged mice.

1. The purpose of the present study was to examine lung water transport properties and the expression and regulation of the alveolar endothelial water channel aquaporin (AQP)-1 and the epithelial water channel AQP-5 in aged mouse lung using gene expression analysis and water permeability measurements. 2. In aged (20-24-month-old) mice, AQP-1 and AQP-5 mRNA expression decreased by 55.5 and 50.3%, respectively, compared with that in young (8-10-week-old) mice (P < 0.01). In addition, AQP-1 and AQP-5 protein expression decreased in aged mice by 36.9 and 44.6%, respectively, compared with that in young mice (P < 0.01). 3. The osmotically driven water transport rate between the airspace and capillary compartments was reduced by 31.7% in aged mice compared with young mice (2.8 +/- 0.3 vs 4.1 +/- 0.3 mg/s, respectively; P < 0.01). The hydrostatically driven lung water accumulation rate in response to a 10 cmH(2)O increase in pulmonary artery pressure was also reduced in aged mice by 21.9% compared with young mice (0.32 +/- 0.06 vs 0.41 +/- 0.04 mg/s, respectively; P < 0.01). 4. There was a 62.7% decrease in serum glucocorticoids in aged mice compared with young mice (67.6 +/- 26.8 vs 181.3 +/- 44.4 nmol/L, respectively; P < 0.01). In vivo administration of dexamethasone (4 mg/kg) for 5 consecutive days to aged mice increased lung AQP-1 mRNA and protein expression by 2.1 +/- 0.1 fold (P < 0.01) and 1.8 +/- 0.2 fold (P < 0.01), respectively. Accordingly, osmotically and hydrostatically driven water transport rates increased by 35.6% (P < 0.01) and 31.2% (P < 0.01), respectively. 5. The present study provides the first evidence of altered lung water transport associated with downregulation of AQPs in aged lung. Blood glucocorticoid hormone levels are important to maintain normal AQP-1 expression in the lung microvascular endothelium. Corticosteroid-induced AQP-1 upregulation may contribute to the role of corticosteroids in accelerating oedema clearance in aged lung.

Water channel activity of plasma membrane affects chondrocyte migration and adhesion.

1. Recent studies indicate that the aquaporin-1 (AQP1) water channel is expressed in human and equine articular chondrocytes. The role of AQP1 in chondrocyte function has not been characterized. In the present study, we investigated the expression of the AQP1 water channel in cultured articular chondrocytes from wild-type (AQP1(+/+)) and AQP1-knockout (AQP1(-/-)) mice and characterized its function in chondrocyte proliferation, migration and adhesion. 2. Expression of AQP1 mRNA and protein was identified in freshly isolated neonatal AQP(+/+) chondrocytes. Immunofluorescence localized the AQP1 protein to the plasma membrane of AQP(+/+) chondrocytes in primary cultures. Relative plasma membrane water permeability of AQP1(+/+) chondrocytes was approximately 1.6-fold higher than that of AQP1(-/-) chondrocytes. 3. The chondrocyte proliferation rate was not affected by AQP1 deletion. However, the serum-induced transwell migration rate of AQP1(-/-) chondrocytes was markedly reduced compared with AQP1(+/+) chondrocytes (16.2 +/- 0.2 vs 27.1 +/- 0.3%, respectively; P < 0.01). Cell adhesion to type II collagen-coated plates was also significantly reduced in AQP1(-/-) chondrocytes compared with AQP1(+/+) chondrocytes (38.1 +/- 0.3 vs 51 +/- 1%, respectively; P < 0.01). 4. The results provided direct evidence that AQP1-mediated plasma membrane water permeability plays an important role in chondrocyte migration and adhesion.

Identification of natural coumarin compounds that rescue defective DeltaF508-CFTR chloride channel gating.

1. Deletion of phenylalanine at position 508 (DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the DeltaF508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects. 2. We identified a class of natural coumarin compounds that can correct the defective DeltaF508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing DeltaF508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L). 3. Dose-dependent potentiation of defective DeltaF508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTR(inh)-172. Two potent compounds, namely imperatorin and osthole, have activation K(d) values of approximately 10 micromol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of DeltaF508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of DeltaF508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout. 4. In conclusion, the natural coumarin DeltaF508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the DeltaF508 mutation.

Hypermethylation of the CHRDL1 promoter induces proliferation and metastasis by activating Akt and Erk in gastric cancer.

CHRDL1 (Chordin-like 1) is a secreted protein that acts as an antagonist of bone morphogenetic protein (BMP). BMP plays a role as an activator of BMP receptor II (BMPR II), which mediates extracellular to intracellular signal transmission and is involved in carcinogenesis and metastasis. Herein, we report that CHRDL1 expression was significantly down-regulated in gastric cancer tissues and associated with poor survival. Clinic-pathological parameters demonstrated a close relationship between low CHRDL1 expression and metastasis. In vitro, CHRDL1 knockdown promoted tumor cell proliferation and migration through BMPR II by activating Akt, Erk and ß-catenin. Furthermore, we observed the hypermethylation of the CHRDL1 promoter in gastric cancer, which induced low expression of CHRDL1 and decreased its secretion to the supernatant. Finally, in vivo experiments confirmed that CHRDL1 acted as a tumor suppressor gene in suppressing tumor growth and metastasis.