Xuechao Feng

Aquaporins as potential drug targets

AbstractThe aquaporins (AQP) are a family of integral membrane proteins that selectively transport water and, in some cases, small neutral solutes such as glycerol and urea. Thirteen mammalian AQP have been molecularly identified and localized to various epithelial, endothelial and other tissues. Phenotype studies of transgenic mouse models of AQP knockout, mutation, and in some cases humans with AQP mutations have demonstrated essential roles for AQP in mammalian physiology and pathophysiology, including urinary concentrating function, exocrine glandular fluid secretion, brain edema formation, regulation of intracranial and intraocular pressure, skin hydration, fat metabolism, tumor angiogenesis and cell migration. These studies suggest that AQP may be potential drug targets for not only new diuretic reagents for various forms of pathological water retention, but also targets for novel therapy of brain edema, inflammatory disease, glaucoma, obesity, and cancer. However, potent AQP modulators for in vivo application remain to be discovered.

Localization of aquaporin-1 water channel in glial cells of the human peripheral nervous system.

The aquaporins (AQPs) are a family of water channel proteins with at least 13 mammalian members (AQPs 0–12) expressed in diverse fluid transporting tissues. AQP1, AQP4, and AQP9 have been identified in the central nervous system and demonstrated or proposed to play important roles in brain water homeostasis. Aquaporin expression in the peripheral nervous system is poorly studied. Here we report that the AQP1 water channel is specifically localized to glial cells of the peripheral nervous system by immunohistochemistry, RT‐PCR, and immunoblotting. Paraffin‐embedded biopsies of human pancreas, esophagus, and sciatic nerves were accessed by immunoperoxidase staining using affinity‐purified AQP1, AQP4, and AQP9 antibodies. Strong AQP1 expression was identified in pancreatic nerve plexuses and in the submucosal and myenteric nerve plexuses in the esophagus. AQP1 was localized to the same cell population expressing glial fibrillary acidic protein (GFAP), but not to the neurons in the plexuses, indicating glial cell‐specific expression. RT‐PCR and immunoblot analysis of microdissected pancreatic ganglia confirmed the expression of AQP1 transcript and protein. Pancreatic and sciatic nerve bundles, which contain nonmyelinating and myelinating Schwann cells, respectively, were also selectively labeled by AQP1 antibody. AQP4 and AQP9, which are broadly expressed in astroglial cells in brain and spinal cord, were not localized in glial cells in the peripheral nerve plexuses. These results suggest that AQPs are differentially expressed in the peripheral versus central nervous system and that channel‐mediated water transport mechanisms may be involved in peripheral neuronal activity by regulating water homeostasis in nerve plexuses and bundles.

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.

Sporadic obstructive hydrocephalus in Aqp4 null mice.

Aquaporin‐4 (Aqp4) is a water transport protein expressed in glia and ependymocytes in brain. We report here the unexpected occurrence of severe obstructive hydrocephalus in a random subset of Aqp4 knockout mice. Of 612 Aqp4 knockout mice produced by heterozygote–heterozygote or knockout–knockout breedings, 9.6% of offspring manifested progressive encephalomegaly. Encephalomegaly was never seen in wild‐type or Aqp4 heterozygous mice. Examination of the subset encephalomegalic mice revealed marked triventricular hydrocephalus (lateral ventricle size ∼500 mm3), elevated intracranial pressure (19 ± 3 vs. 6.1 ± 0.6 mm Hg), and death by age 6 weeks, with a median survival of 28 days. Intraventricular dye injection studies revealed total obstruction of the cerebral aqueduct. Evans blue extravasation studies indicated an intact blood–brain barrier in the hydrocephalic mice. Brain histology revealed reduced ventricular size and ependymocyte disorganization in some nonhydrocephalic Aqp4 null mice. Our studies establish Aqp4 deletion as a predisposing factor for the development of congenital obstructive hydrocephalus in mice. We suggest that AQP4 polymorphisms might also contribute to the development of aqueduct stenosis in humans.

Defective macrophage function in aquaporin-3 deficiency

Macrophages play an essential role in innate immunity. We found that mouse resident peritoneal macrophages (mRPMs) express the aquaglyceroporin aquaporin‐3 (AQP3) in a plasma membrane pattern. AQP3‐deficient (AQP3–/–) mice showed significantly greater mortality than wild‐type (AQP3+/+) mice in a model of bacterial peritonitis. To establish the cellular mechanism of the peritonitis phenotype, measurements were made of mRPM phagocytosis, migration, and water/glycerol permeability. We found significantly impaired engulfment of Escherichia coli and chicken erythrocytes in AQP3–/– vs. AQP3+/+ mRPMs, as well as impaired migration of AQP3–/– mRPMs in response to a chemotactic stimulus. In AQP3+/+ mRPMs, AQP3 was polarized to pseudopodia at the leading edge during migration and around the phagocytic cup during engulfment. Water and glycerol permeabilities in mRPMs from AQP3–/– mice were reduced compared to mRPMs from AQP3+/+ mice. Cellular glycerol and ATP content were remarkably lower in AQP3–/– vs. AQP3+/+ mRPMs, and glycerol supplementation partially rescued the reduced ATP content and impaired function of AQP3–/– mRPMs. These data implicate AQP3 as a novel determinant in macrophage immune function by a cellular mechanism involving facilitated water and glycerol transport, and consequent phagocytic and migration activity. This is the first study demonstrating involvement of an aquaporin in innate immunity. Our results suggest AQP3 as a novel therapeutic target in modulating the immune response in various infectious and inflammatory conditions.—Zhu, N., Feng, X., He, C., Gao, H., Yang, L., Ma, Q., Guo, L., Qiao, Y., Yang, Y., Ma, T. Defective macrophage function in aquaporin‐3 deficiency. FASEB J. 25, 4233–4239 (2011). www.fasebj.org

Increased Female Fertility in Aquaporin 8-Deficient Mice

Aquaporin‐8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8−/− mothers compared with wild‐type mothers in cross‐mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8−/− ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8−/− ovaries, suggesting increased maturation and ovulation of follicles. By RT‐PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8−/− mice showed 45% of decreased membrane water permeability than wild‐type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild‐type and AQP8−/− mice. The results indicated significantly lower apoptosis rate in AQP8−/− granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8+/+ granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.

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 condrocyte 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.

Mild processing defect of porcine ΔF508-CFTR suggests that ΔF508 pigs may not develop cystic fibrosis disease ☆

Recent efforts have made significant progress in generating transgenic pigs with the DeltaF508-CFTR mutation to model the lung and pancreatic disease of human cystic fibrosis. However, species differences in the processing and function of human, pig and mouse DeltaF508-CFTR reported recently raise concerns about the phenotypic consequence of the gene-targeted pig model. The purpose of the present study was to characterize the DeltaF508 mutant of porcine CFTR to evaluate the severity of its processing defect. Biochemical and immunofluorescence analysis in transfected COS7 and FRT cells indicated that pig DeltaF508-CFTR efficiently targets to the plasma membrane and is present mainly as the mature glycosylated protein. Functional characterization in stably transfected FRT cells by fluorometric and electrophysiological assays supported efficient plasma membrane targeting of pig DeltaF508-CFTR. The mild cellular processing defect of pig DeltaF508-CFTR suggests that its gene-targeted pig model may not develop the lung and pancreatic phenotypes seen in CF patients.

Increased differentiation capacity of bone marrow-derived mesenchymal stem cells in aquaporin-5 deficiency.

Mesenchymal stem cells (MSCs) are adult stem cells with a self-renewal and multipotent capability and express extensively in multitudinous tissues. We found that water channel aquaporin-5 (AQP5) is expressed in bone marrow-derived MSCs (BMMSCs) in the plasma membrane pattern. BMMSCs from AQP5(-/-) mice showed significantly lower plasma membrane water permeability than those from AQP5(+/+) mice. In characterizing the cultured BMMSCs from AQP5(-/-) and AQP5(+/+) mice, we found no obvious differences in morphology and proliferation between the 2 genotypes. However, the multiple differentiation capacity was significantly higher in AQP5(-/-) than AQP5(+/+) BMMSCs as revealed by representative staining by Oil Red O (adipogenesis); Alizarin Red S and alkaline phosphatase (ALP; osteogenesis); and type II collagen and Safranin O (chondrogenesis) after directional induction. Relative mRNA expression levels of 3 lineage differentiation markers, including PPARγ2, C/EBPα, adipsin, collagen 1a, osteopontin, ALP, collagen 11a, collagen 2a, and aggrecan, were significantly higher in AQP5(-/-) -differentiating BMMSCs, supporting an increased differentiation capacity of AQP5(-/-) BMMSCs. Furthermore, a bone-healing process was accelerated in AQP5(-/-) mice in a drill-hole injury model. Mechanistic studies indicated a significantly lower apoptosis rate in AQP5(-/-) than AQP5(+/+) BMMSCs. Apoptosis inhibitor Z-VAD-FMK increased the differentiation capacity to a greater extent in AQP5(+/+) than AQP5(-/-) BMMSCs. We conclude that AQP5-mediated high plasma membrane water permeability enhances the apoptosis rate of differentiating BMMSCs, thus decreasing their differentiation capacity. These data implicate AQP5 as a novel determinant of differentiation of BMMSCs and therefore a new molecular target for regulating differentiation of BMMSCs during tissue repair and regeneration.

Tanshinones Increase Fibrinolysis through Inhibition of Plasminogen Activator Inhibitor-1

Danshen, the dried roots of Salvia miltiorrhiza, is a well-known traditional Chinese medicine used for the treatment of stroke since 1970. A similar plant called Salvia columbariae was also used by Californian Indians to treat people suffering from stroke [1]. Pharmacological studies indicated that their active ingredients, tanshinones, and salvianolic acids exhibited anticoagulant, vasodilatory, anti-inflammatory, antioxidant, neuroprotective, and other activities underlying the therapeutic effect [2]. Thrombosis secondary to atherosclerosis is a major cause of stroke and coronary artery disease. Inhibition of clot formation and potential clot dissolution has been demonstrated in many clinical trials of S. miltiorrhiza [1]. Early in vitro studies found that S. miltiorrhiza extract can increase the proteolysis of fibrinogen to fibrinogen degradation products [3], suggesting a unique mechanism of antithrombosis comparing to other anticoagulant drugs that prevent clot formation by interfering with platelet aggregation, thrombin activity or vitamin K function. However, the molecular basis of the fibrinolytic action of S. miltiorrhiza remains unknown. Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of both tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), is a key physiological regulator of the fibrinolytic system and is implicated in thrombotic pathophysiology in ischemic stroke and coronary artery disease [4,5]. Pharmacological inhibition of PAI-1 showed antithrombotic benefits devoid of bleeding effect in rodents and monkey [6,7]. In the present study, we tested the hypothesis that S. miltiorrhiza contains active compounds that inhibit PAI-1 activity. Using a chromogenic substrate-based PAI-1 activity assay kit (Milllipore, MA, USA), we analyzed the PAI-1 inhibitory effect of seven natural compounds from S. miltiorrhiza: Tanshinone I, Tanshinone IIA, Sodium tanshinone IIA sulfonate, Cryptotanshinone, Sodium Danshensu, Protocatechuic aldehyde and β-sitosterol (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). The purity of the compounds was determined to be >98% by high-performance liquid chromatography analysis. For the uPA-mediated assay, PAI-1 (Molecular Innovations, MI, USA) was incubated with compounds at 23◦C for 15 min, then PAI-1 activity was assayed according to the protocol of the kit. For the tPA-mediated PAI-1 activity assay, uPA and its chromogenic substrate in the kit was replaced by tPA (Molecular Innovations, MI, USA) and its substrate S-2288 (Chromogenix, NC, USA). As shown in Figure 1(A), sodium tanshinone IIA sulfonate and cryptotanshinone inhibited PAI-1 activity in a dosedependent manner. The IC50 values for sodium tanshinone IIA sulfonate and cryptotanshinone are 41 and 98 μM, respectively, in uPA/PAI-1 assay and 59 and 152 μM, respectively, in tPA/ PAI-1 assay. Tanshinone IIA exhibited much weaker effect than its salt form derivative sodium tanshinone IIA sulfonate, likely due to its lower solubility. To further confirm the PAI-1 inhibitory activity by the tanshinone compounds, we performed biochemical analysis to determine if sodium tanshinone IIA sulfonate and cryptotanshinone can block the formation of PAI-1/uPA complex. In a 25 μL reaction volume in tris-buffered saline buffer, recombinant human PAI-1 (Molecular Innovations, MI, USA) at 1 μM final concentration was first incubated with various concentrations of sodium tanshinone IIA sulfonate and cryptotanshinone for 15 min at room temperature. Then uPA (Molecular Innovations, MI, USA) was added to a final concentration of 0.8 μM and incubated for 10 min at 37◦C. The samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10% gel and stained with Coomassie blue. As shown in Figure 1(B), both sodium tanshinone IIA sulfonate and cryptotanshinone dose dependently prevented the formation of PAI-1/uPA complex. The results of enzymatic and