Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport  in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10−12 cm3/s or 4.17 (±1.38)×1010 H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium,  High water transport rates across some biological membranes such as renal collecting ducts and red blood cell plasma membranes were not well understood until the discovery of the first water channel protein in red blood cells E. coli aquaporin, EcAqpZ, a reduction in growth rate was reported for knockout mutants indicating EcAqpZ mediated water uptake in the logarithmic growth phase M. marburgensis was shown to be functionally expressed and responsible for cytoplasmic shrinkage of the cell exposed to hypertonic shock Synecoccous sp. PCC7942, transports CO2 as well as water and has an apparent role in CO2 uptake during photosynthesis cerevisiae, AQY1 and AQY2 were shown to improve the freeze tolerance of yeast under rapid freezing conditions Higher organisms including vertebrate, invertebrate, and plants have multiple copies of aquaporin genes to regulate water transport activities in different tissues. The regulation and function of these gene products are well understood in many cases, e.g. controlled regulation of AQP2 in renal collecting duct Rhodobacter sphaeroides is a purple non-sulphur photosynthetic bacterium, commonly studied as a model organism in photosynthesis research. It is characterized by a wide capability for growth modes including aerobic and anaerobic respiration, fermentation, photoheterotrophy and chemolithotrophy. In the photosynthetic growth mode, membrane surface area of R. sphaeroides increases extensively due to biosynthesis of intracellular membranes accommodating the photosynthetic apparatus. These features have been used to employ R. sphaeroides in expression of membrane proteins for structural and functional studies Rhodobacter sphaeroides ATCC 17023 (RsAqpZ) utilizing a native expression system. The protein was fully functional, as determined by stopped-flow light scattering experiments and showed high water permeability as well as low activation energy, similar to the well-studied E. coli homolog (EcAqpZ). However, higher insertion efficiency was observed for this protein at higher protein to lipid weight ratios (PLR) starting from 0.005. The large decrease in permeability of proteoliposomes with high PLR in some aquaporin studies, for example Borgnia et al. Rhodobacter expression platform, we demonstrate a high level expression (∼20 mg protein/L culture) of functionally active and highly permeable RsAqpZ and show that it remains active in lipids and amphiphilic block copolymers.Aquaporins (in particular EcAqpZ) are being studied extensively for application to biomimetic desalination membranes and their scale-up is quite challenging n-octyl β-D-glucoside (OG) was purchased from Affymetrix, Inc., Santa Clara, CA. Phosphatidylcholine  and phosphatidylserine  were purchased from Avanti Polar Lipids, Inc., Alabaster, AL. Polyclonal His-tag antibody and anti-rabbit-HRP antibody were purchased from GenScript USA, Inc., Piscataway, NJ. Co-NTA resin was purchased from Thermo Fisher Scientific, Rockford, IL. All other chemicals were purchased from Sigma Aldrich or Thermo Fisher Scientific. Restriction enzymes, DNA polymerases, ligase and markers were purchased from New England Biolabs.Rhodobacter sphaeroides ATCC 17023 (ABA78939.1), Escherichia coli K12 , Methanobacter marburgensis , Synecoccocus elongatus , Pichia pastoris , Spinacia oleracea  and Homo sapiens . The alignment was performed using Clustal-Ω multiple sequence alignment software Aquaporin amino acid sequences were retrieved from Genbank for R. sphaeroides has been used as a membrane protein expression platform due to its abundant inducible intracellular membranes (ICM) accommodating the photosynthetic apparatus, which is comprised of core complexes (photosynthetic reaction center [RC] and first tier light harvesting antenna [LHI]) and peripheral complexes (second tier light harvesting antenna [LHII]) PUC705-BApRKPLHT7, was a derivative of broad-host-range plasmid pRK404 puc promoter. In Rhodobacter, puc promoter controls the expression of the puc operon, which encodes for LHII complex and gets activated by low light intensity as well as reduced oxygen tension R. sphaeroides ATCC 17023 RsAqpZ gene. RsAqpZ was expressed from a pRKPLHT7-based plasmid in the PUC705-BA strain. PUC705-BA bearing the empty pRKPLHT7 plasmid was used as a negative control in expression screening with western-blot. Unless otherwise noted, R. sphaeroides strains were cultivated in minimal (MR26) Wild type strain, Rhodobacter sphaeroides 2.4.1 revealed a single putative aquaporin gene (RSP_2782) showing 44% identity and 58% similarity to EcAqpZ. We designed forward (5′CCACGGACTAGTGGAGGCCATTCATGACCAAGAAGCTC3′) and reverse primers (5′TAAATAAGATCTGAGCGTGGCCGCGCCGGTTGCGGG3′) to amplify this gene sequence from genomic DNA of R. sphaeroides ATCC 17023 and cloned it into the pRKLHT7 expression vector between SpeI and BglII restriction sites. The cloning strategy is described in RsAqpZ, a ribosome binding site (GGAGG) exists that is highly conserved in the photosynthetic gene cluster of R. sphaeroides. This RBS was included to provide a bias towards protein overexpression that is observed for the native light-harvesting genes E.coli DH5α by electroporation. After selection of transformants in the presence of 15 µg/mL tetracycline (tet), the transfer of pRKLHT7-RsAqpZ plasmid was verified by colony PCR. The plasmid sequence was confirmed by DNA sequencing.A BLAST search performed on the genome sequence of Rhodobacter has not been achieved for the pRK404 - based plasmids by chemical or electroporative transformation, likely due to prolific restriction endonuclease activity E.coli, S17-λ-pirpRKLHT7-RsAqpZ plasmid into PUC570BA. Rhodobacter exconjugates were selected on a proline dropout media, MR26 S17-λ-pir, and grown photo-heterotrophically Plasmid DNA transfer into R. sphaeroides was grown under photoheterotrophic conditions using high power IR-LEDs  with a peak emission wavelength of 850 nm at a light intensity of approximately 4 W/m2. It was recently shown that low light intensity increases ICM biosynthesis and expression of LH2, which is controlled by puc promoter driving the expression of recombinant proteins in our expression system Rhodobacter picked from a YCC agar plate containing 1.5 µg/mL tet was inoculated into a 50 mL conical tube containing 15 mL YCC+tet medium. After 24 hrs of growth at 34°C with shaking at 200 rpm, the inoculum was transferred in a 250 mL Erlenmeyer flask containing an additional 50 mL of fresh YCC media with 1 µg/mL tetracycline to culture for another 24 hr at 34°C, 100 rpm. Anaerobic photoheterotrophic growth was fully implemented by transferring 50 mL of the semi-aerobic culture into the T-flask  and topped off with approximately 700 mL YCC media with 1 µg/mL tet. The culture in T-flask was placed horizontally below an IR-LED array, while the culture temperature was maintained at 25°C by partial submergence in a simple plastic water bath on top of a stir plate to provide agitation from the stir bar inside the T-flask. A miniature axial box fan blowing across the T-flask also helped to maintain temperature by dissipating the radiant heat of the IR panel.E.coli JM109 cells harboring an N-terminal 10-His-tagged version of E.coli K12 Aquaporin Z were grown following a previously published procedure R. sphaeroides and E. coli cells were harvested by centrifugation at 8000 rpm for 30 min and frozen at least one hour at −80°C prior to cell lysis. Cell lysis was performed in a buffer containing 100 mM K2HPO4, 1 mM MgSO4, 10 mM imidazole, 1 mM PMSF, 0.1 mg/ml DNase I, 0.02 mg/ml RNase, Halt protease inhibitor cocktail (Pierce), 1 mg/ml lysozyme at pH = 7.0 by sonication 3 times at 4°C in pulsed mode for 3 min at 50% duty cycle using a horn sonicator . Cells were then incubated at 37°C for 1 hr and sonicated again for 3 times. Cell debris and unbroken cells were pelleted at 18,000×g, 4°C for 30 min and the supernatant was centrifuged at 240,000×g for 2 hrs to pellet the membrane fraction. The supernatant was removed and membrane fraction was solubilized overnight in a buffer containing 5% n-octyl-β-D-glucopyranoside (OG), 100 mM K2HPO4 (pH = 7.0), 200 mM NaCl, 10% glycerol, 20 mM imidazole, and 2 mM β-mercaptoethanol (BME). Insoluble material was pelleted at 240,000×g, 4°C for one hour and supernatant was incubated with His-pur Co-NTA resin (Pierce) for 2 hrs with gentle agitation. The resin was loaded onto an affinity column, and the column was washed with up to 30 bed volumes of buffer containing 100 mM K2HPO4 (pH = 7.0), 1% OG, 200 mM NaCl, 10% glycerol, 50 mM imidazole, and 2 mM BME. The protein was eluted with 100 mM K2HPO4 (pH = 7.0), 1% OG, 200 mM NaCl, 10% glycerol, 500 mM imidazole, and 2 mM BME. Sample purity and loss was estimated by running eluted fractions on a SDS-PAGE gel.8) coronal blocks and hydrophobic poly-dimethylsiloxane (PDMS55) core block was synthesized as described previously 1H NMR. Specific block composition was found to be PMOXA8PDMS55PMOXA8. GPC analysis in chloroform, using polystyrene calibration, revealed a polydispersity index (PDI) value of 1.34 .The triblock copolymer (ABA55) consisting of hydrophilic poly-2-methyloxazoline  covered with a 12–14 kDa MWCO dialysis membrane  and dialyzed for four days at 4°C to remove detergent and form proteoliposomes. For pH experiments, buttons were moved into different buffers with pH values between 4.5 and 8.5 and dialyzed for two extra days to equilibrate pH within the vesicles. Buffering agents used at different pH values were 20 mM sodium acetate for pH 5.0; 20 mM MES for pH 6.0; and 20 mM HEPES for pH 7.0, 7.4 and 8.0. Proteoliposomes were extruded through a 200 nm track-etched nucleopore membrane  to obtain unilamellar vesicles with low polydispersity index (PDI <0.2).The functionality of the purified RsAqpZ and EcAqpZ was evaluated by measuring osmotic water permeability of proteoliposomes embedded with purified protein as described elsewhere 3 and added on the film followed by a 3 hr incubation for rehydration of the film. The flask was placed on a stir plate and the protein/polymer/detergent ternary mixture was stirred overnight. The mixture was transferred into a Slide-A-Lyser dialysis cassette  and dialyzed against the sample buffer without OG for 2 days until proteopolymersomes were formed. The sample was extruded 10 times through a 0.4 µm and 10 times through 200 nm track-etched filters  to obtain unilamellar, monodisperse vesicles. The size and PDI of proteoliposome and proteopolymersome samples were measured in a dynamic light scattering instrument .ABA55-RsAqpZ and ABA55-EcAqpZ proteopolymersomes were formed by film rehydration k. fP is water permeability; k is rate constant; S/V0 is initial surface to volume ratio of liposome; Vw is molar volume of water, 18 cm3; Δπ is osmotic pressure gradient.Aquaporin-mediated water transport across proteoliposomes was measured in a stopped-flow light scattering device  and compared to control liposomes prepared identically but devoid of RsAqpZ. Liposomes were rapidly mixed with its corresponding dialysis buffer consisting of additional 0.2M NaCl to provide an outwardly directed gradient to drive water transport outside of the liposome. This exposure to hypertonic solution results in shrinkage of the vesicles and increase in light scattering at 90° according to the Rayleigh-Gans theory pRKPLHT7 expression vector. This construct was codon optimized for Rhodobacter expression by GenScript . The purified fusion protein was used in a fluorescence correlation spectroscopy (FCS) based method to determine the average number of RsAqpZ-mBanana molecules incorporated per proteoliposome. To obtain proteoliposomes that are free of non-incorporated RsAqpZ, a final separation from non-incorporated protein was achieved by gel filtration chromatography using a Superdex 200 column with a buffer containing 20 mM HEPES, 100 mM NaCl and 0.02% NaN3. Column fractions rich in proteoliposomes were collected and pooled for stopped-flow light scattering and FCS experiments.We created a C-terminal fusion of RsAqpZ protein with mBanana fluorescent protein 2 by measuring light intensity at the back of the objective aperture. The dimensions of the confocal volume were determined using a fluorescent dye standard with known diffusion coefficient (Rhodamine B). Proteoliposome samples were diluted in HEPES buffer containing a final concentration of 2.5% OG. Data collection was kept under 3 minutes to minimize background interference. The number of RsAqpZ-mBanana tetramers embedded per proteoliposome was calculated by taking the ratio of number of particles in confocal volume before and after solubilization of proteoliposomes, which is a method that has recently been reported elsewhere for a potassium channel FCS experiments were performed on a time correlated single photon counting (TCSPC) module . The detailed instrumentation setup is described elsewhere After measurement of the water permeability for the proteoliposomes and control liposomes by stopped-flow experiment, total water permeability contributed by RsAqpZ in proteoliposomes was calculated. Knowing the average number of tetramers per liposome, the subunit permeability of RsAqpZ was determined by combining the results from stopped-flow light scattering and FCS experiments.E.coli AqpZ, as well as the archeal aquaporin, AqpM. It is located in a cluster that is distinct from the aquaglyceroporins, E.coli GlpF and human AQP9 , which provides a significantly higher hydrophobicity index. Earlier studies with Thr183 Cys mutant of EcAqpZ did not show any difference in water transport RsAqpZ gene (Genbank ID: CP000143) was amplified from genomic DNA of Rhodobacter sphaeroides ATCC 17023 by colony PCR. This amplicon of 764 bp was then cloned into the multiple cloning site of pRKPLHT7 expression vector using SpeI and BglII sites. The plasmid, pRKPLHT7-RsAqpZ, was maintained in E. coli DH5α strain and transformed into E. coli S17-λ-pir for mobilization into Rhodobacter by conjugation. Rhodobacter exconjugants were verified by colony PCR  affinity chromatography at 5 mg recovered protein/L; higher expression levels of up to 20 mg/L could be purified at a higher yield using Ni-NTA resin but consistently higher purity was obtained with Co-NTA resins and therefore utilized for the remainder of this study. 5 and 0.5 µg of protein eluted from the Co column was run on a 12% polyacryamide gel for SDS-PAGE and western-blot experiment, respectively. The majority of RsAqpZ remained in tetrameric form as detected around 70 kDa in SDS-PAGE gel, where the monomer band was not noticeable  as determined by dynamic light scattering. Water permeability of proteoliposomes embedded with RsAqpZ and EcAqpZ as well as empty liposomes was measured in a stopped-flow light scattering apparatus. An outwardly directed 0.2M NaCl gradient was imposed on the proteoliposomes and change in light scattering due to vesicles shrinkage was monitored on a millisecond time scale . The resE. coli aquaporin was 586.2±141.7 µm/sec that occurred at a lower protein loading (PLR = 0.02). We observed visible precipitation for EcAqpZ samples during dialysis, which implied lower incorporation efficiency leading to lower water permeability compared to RsAqpZ samples at the same PLR  from 0.001 up to a PLR of 0.01 and 0.02 for EcAqpZ and RsAqpZ, respectively . RsAqpZ same PLR  and S3. same PLR .We evaluated a possible pH gating property of RsAqpZ by measuring water permeability at different pH values .. No staAlthough aquaporin single channel transport rates have been reported in the literature, these have assumed 100% incorporation of the purified protein into liposomes In an FCS experiment, time-dependent changes in fluorescent intensity within a small observation  volume (∼ 1 femtoliter) are monitored and the fluctuations are fit to an autocorrelation function described below .(2)t. When τ = 0, the term on the right side of the Here, G(τ) is the normalized autocorrelation function; δF(t) is the fluorescence intensity fluctuation at time t; δF(t+τ) is the fluorescence intensity fluctuation after a time lag τ, and F(t) is the average fluorescence intensity at time M is the number of fluorescent species in the confocal volume; r and z are radius and half height of the confocal volume, respectively, which is often assumed to have 3D Gaussian illumination profile Di is 2D lateral diffusion time of fluorescent species i across the confocal volume; if is the fraction of fluorescent species i. We used a single species fitting for free RsAqpZ-mBanana, whereas for proteoliposomes autocorrelation curves were fit to the two species model.Here, RsAqpZ. In this method, free RsAqpZ-mBanana tetramer in HEPES buffer with 1% OG was used as a standard. Molecular brightness (photons emitted per molecule at standard excitation rate) of RsAqpZ-mBanana, εRsAqpZ-mBanana, was measured by dividing total number of collected photons throughout the experiment by the duration of the measurement and dividing that number by the number of RsAqpZ-mBanana in the confocal volume, NRsAqpZ-mBanana, as obtained by fitting of the time-shifted autocorrelation curve to a 3D diffusion model , in principle yield an estimate of the number of RsAqpZ-mBanana tetramers per liposome, NRsAqpZ. However, this method did not result in consistent numbers for RsAqpZ tetramers in proteoliposomes at a fixed protein to lipid ratio, likely due to significantly altered fluorescence lifetime of mBanana in detergent micelles versus an aqueous environment surrounding the proteoliposomes  by fitting the autocorrelation curves constructed from TCSPC data to a 2 species, 3D diffusion equation , represent the length of time that the fluorescent species spends within the confocal volume. Since the confocal volume has a precise dimension, this is then related to the diffusion time of the species, with larger species having longer diffusion times. As expected, the diffusion time of the tagged aquaporin in proteoliposomes is much longer than the free protein, and the increased size of the tagged protein (∼204 kDa in tetrameric form) relative to the mBanana fluorophore (27 kDa) is also readily observed . The difequation  [51]. A prot) was likewise determined. Thus, the number of the RsAqpZ tetramers per liposome (NRsAqpZ) was calculated taking the ratio of Nprot over Nlip, which then allows calculation of the incorporation efficiency based on the initial purified protein level used to produce the proteoliposomes  and found the most sensitive experimental range to be between PLR 0.004 and PLR 0.04 for mBanana fusion of RsAqpZ. At lower PLR ratios resolution from FCS and stopped-flow experiments was lost due to low photon count and relatively high background permeability of PC-PS lipid vesicles. We evaluated the efficiency of protein incorporation into the phospholipid liposomes by comparing the number of RsAqpZ-mBanana tetramers per liposome obtained from FCS experiments and the theoretical number corrected for lipid displacement by assuming 100% RsAqpZ incorporation and the geometric characteristics of the liposomes, lipids and tetramers at a given PLR (described in The water channel activity of EcAqpZ and RsAqpZ-mBanana was measured in ABA55 triblock copolymer vesicles and compared to polymersomes without these proteins. Both proteins showed water transport activity as evidenced by significantly higher permeability of proteopolymersomes as compared to control polymersomes . Water p9–1010 molecules per channel per second). Aquaporins from plant and animal species have well-established physiological roles. However, the function of microbial aquaporins is not well understood, as many species do not have identifiable aquaporins. Characterization of additional aquaporins will enable deciphering the function of this important class of proteins in microorganisms.Aquaporins facilitate passive transport of water through membranes at extremely high rates (10Rhodobacter species have a multiplicity of growth modes and have been extensively studied as model organisms in photosynthesis research. They have gained importance as novel expression platforms for membrane proteins due to their intracellular membranes providing large available membrane areas for recombinantly expressed membrane proteins R. sphaeroides ATCC 17023 that was homologously overexpressed under anaerobic photoheterotrophic growth conditions. We used a knockout strain that is incapable of synthesizing the second tier light-harvesting complex (LH-II) in the photosynthetic apparatus, which may comprise up to 10% of the actual biomass in this photosynthetic growth mode. Thus, more membrane area was available for recombinantly expressed membrane proteins. RsAqpZ was purified at 5 mg/L by metal affinity chromatography, although actual expression level was estimated to be around 20 mg/mL via western-Blot analysis. The purified protein was functional, which allowed assessment of channel mediated water transport rates by stopped-flow light scattering. We could determine the amount of protein incorporated in each liposome by using a fluorescently tagged version of RsAqpZ. From these measurements, we found the permeability of RsAqpZ to be ∼40×109 H2O molecules per channel per second, which is the highest value reported thus far for aquaporins.−12 cm3/s based on the average of 4 data points from PLR 0.004 to 0.04 (−14 cm3/s) and MD simulation values (4.2×10−14 cm3/s) for EcAqpZ. However, previous E.coli aquaporin experimental permeability studies presumed 100% incorporation of the purified protein into the liposomes, which might lead to underestimation of the single channel permeability. The single channel water permeability of RsAqpZ was calculated to be 1.23±0.43×10 to 0.04 . This vaIncorporation of RsAqpZ into liposomes enhanced the water permeability by 18 times compared to empty liposomes and by 2.9 times compared to EcAqpZ proteoliposomes at a PLR of 0.02. Water permeability values measured in this study are higher than those reported by Borgnia et al for EcAqpZ. Energy of activation for water transport was determined as 2.93±0.6 kcal/mol for RsAqpZ proteoliposomes, is consistent with RsAqpZ mediated water transport rather than diffusive transport through lipid bilayers. This activation energy falls at the lower end of the reported range of activation energy values for other orthodox aquaporins. Water transport through RsAqpZ in proteoliposomes did not seem to be affected by pH below or above neutral pH .Aquaporins have been proposed for use in biomimetic water desalination membranes to reduce energy costs in desalination processes Understanding the physiological roles of bacterial aquaporins will require characterization of more aquaporins from different classes. In particular the physiological role of the RsAqpZ needs to be further explored through phenotype characterization of knockouts. To the best of our knowledge, this study represents the first study showing biophysical characterization of an orthodox aquaporin from a photosynthetic bacterium.Figure S1Multiple cloning sites of the expression vectors for RsAqpZ and RsAqpZ-mBanana.(TIF)Click here for additional data file.Figure S2Increasing PLRs during EcAqpZ reconstitution into liposomes leads to increased precipitation. a) Photograph of dialysis buttons prepared from a 3.8 mg/mL protein stock at increasing PLRs. b) Photograph of dialysis buttons prepared from a 0.66 mg/mL protein stock at increasing PLRs. c) Dialyzed samples at increasing PLRs after 18,000×g centrifugation show increasing amounts of precipitation. d) SDS page analysis of precipitates and supernatants from C show that precipitates are high molecular weight aggregates, which did not enter in the gel, whereas supernatant did not have a significant amount of aggregates.(TIF)Click here for additional data file.Figure S3Electron microscopy of samples with increasing PLRs during EcAqpZ reconstitution into liposomes also shows increased precipitation and thus lowered incorporation of proteins.(TIF)Click here for additional data file.Figure S4Water permeability of RsAqpZ proteoliposomes (PLR = 0.01) at different pH levels. RsAqpZ proteoliposomes were formed at pH = 7.4 by detergent dialysis and moved into the buffer solutions at pH between 5.0 and 8.0 to investigate the pH effect on water permeability. There was not a significant change in water permeability as evidenced by ANOVA.(TIF)Click here for additional data file.Figure S5Fluorescence Lifetime of mBanana in different surroundings. RsAqpZ-mBanana: in elution buffer, mBanana-TEV: RsAqpZ-mBanana treated with TEV and filtered through 30 kDa MWCO filter, PLR 0.004–0.04: RsAqpZ-mBanana in proteoliposomes.(TIF)Click here for additional data file.Figure S6The calculation of the number of RsAqpZ tetramers per proteoliposome.(TIF)Click here for additional data file.Table S1Analysis of variance for water permeability at different pH values.(TIF)Click here for additional data file.File S1Supporting Information.(DOCX)Click here for additional data file.


While investigating the role of p38 MAPK in regulating melanogenesis, we found that pyridinyl imidazole inhibitors class compounds as well as the analog compound SB202474, which does not inhibit p38 MAPK, suppressed both α-MSH-induced melanogenesis and spontaneous melanin synthesis. In this study, we demonstrated that the inhibitory activity of the pyridinyl imidazoles correlates with inhibition of the canonical Wnt/β-catenin pathway activity. Imidazole-treated cells showed a reduction in the level of Tcf/Lef target genes involved in the β-catenin signaling network, including ubiquitous genes such as Axin2, Lef1, and Wisp1 as well as cell lineage-restricted genes such as microphthalmia-associated transcription factor and dopachrome tautomerase. Although over-expression of the Wnt signaling pathway effector β-catenin slightly restored the melanogenic program, the lack of complete reversion suggested that the imidazoles interfered with β-catenin-dependent transcriptional activity rather than with β-catenin expression. Accordingly, we did not observe any significant change in β-catenin protein expression. The independence of p38 MAPK activity from the repression of Wnt/β-catenin signaling pathway was confirmed by small interfering RNA knockdown of p38 MAPK expression, which by contrast, stimulated β-catenin-driven gene expression. Our data demonstrate that the small molecule pyridinyl imidazoles possess two distinct and opposite mechanisms that modulate β-catenin dependent transcription: a p38 inhibition-dependent effect that stimulates the Wnt pathway by increasing β-catenin protein expression and an off-target mechanism that inhibits the pathway by repressing β-catenin protein functionality. The p38-independent effect seems to be dominant and, at least in B16-F0 cells, results in a strong block of the Wnt/β-catenin signaling pathway. Mitf promoter Mitf gene transcription but also functionally interacts with the Mitf protein Melanocytes are specialized cells located at the basal layer of the epidermis that produce and transfer melanin pigments to surrounding keratinocytes, thereby contributing to the appearance of skin color. Within keratinocytes, melanins provide a primary defense system against UV radiation by preventing cellular injury and consequential DNA damage that can cause cancer and aging of the skin One of the key factors in β-catenin regulation is the control of its stability, which in turn influences its translocation into the nucleus and its binding to T-cell factor (Tcf)/lymphoid enhancer factor (Lef) family transcription factors Even if Mitf, that is considered the master regulator of pigmentation, is a target of the Wnt pathway Small cell-permeant inhibitors of protean kinases have become precious reagents with which to investigate the physiological roles of protein kinases. ATP-competitive antagonists of the p38 stress-activated protein kinases have been used to assess the physiological role of p38 MAPK in melanogenesis Small-molecule inhibitors of p38 protein kinases have contributed greatly to our understanding of MAP kinase biological signaling. However, some evidence showed that pyridinyl imidazole compounds unexpectedly may have unexpected targets in addition to p38 MAPK Mitf gene transcription led to consequent effects on melanogenic enzyme production and was responsible for the attenuation of melanogenesis in the presence of PI compounds. As pyrimidinyl imidazole compounds showed an inhibitory effect on Mitf mRNA levels, we also measured the generation of cAMP and the phosphorylation of CREB, two downstream events rapidly generated by the activation of the melanogenic pathway through α-MSH-dependent stimulation of melanocortin 1 receptor (MC1R). The intracellular elevation of cAMP levels was not decreased by co-treatment with pyridinyl imidazole compounds  or a control construct carrying Mitf cDNA in antisense orientation (pCAAG-mi-AS). As shown in cis-acting elements of a network of HMG-containing proteins including Sox10, Pax3, and Lef1 (Lef1/β-catenin complex). Moreover, Mitf functions as a non-DNA binding cofactor of Lef1 on its own promoter In contrast to other Mitf isoforms widely expressed in many cell types, Mitf-M has been established as a specific marker for melanocyte-lineage cells Previous studies have demonstrated that Wnt/β-catenin signaling controls melanocyte differentiation primarily through the direct regulation of Mitf A possible intersection of the MAPK pathway with the Wnt/β-catenin signaling pathway has been suggested in mouse F9 teratocarcinoma cells and HEK293 human embryonic cells 50 values against p38 MAP kinase activity (see 50 = 380 nM) and PD169316 (IC50 = 89 nM) at doses of 10 and 5 µM reduced melanin synthesis more strongly than SB202190 (IC50 = 50 nM) and SB220025 (IC50 = 60 nM).Previous studies demonstrated that despite being used widely, the pyridinyl imidazole class of p38 MAPK inhibitors as well as the inactive compound SB202474 are involved in many different p38-independent cellular activities, such as cyclic nucleotide accumulation vity see . For exain vitro, GSK3β activity In this study, all pyridinyl imidazole compounds tested suppressed melanin synthesis as well as the expression of Mitf, and consequently TRP1 and Dct, by blocking β-catenin-dependent transcription. The centrality of Mitf protein regulation in imidazole-dependent melanogenesis inhibition was also confirmed by transfection experiments that clearly demonstrated restoration of melanin synthesis by forced exogenous Mitf expression. Four transcription factors are known that regulate expression of the melanocyte-specific Mitf-M isoforms: Sox10, Pax3, CREB and Lef1. Data obtained from HeLa cells indicate that imidazoles act on ubiquitous factors, such as CREB, Lef1, and related Tcf factors more than on melanocyte-specific proteins such as Sox10 and Pax3. Moreover, the level of cAMP as well as the hyperphosphorylation of CREB suggested a compensatory mechanism trigged by Mitf expression failure, excluding a defect in the cAMP/PKA/CREB axis responsiveness. Based on these results, we restricted our investigation to Lef1. Lef1 transcription factor is responsive to Wnt signaling through its interaction with β-catenin in vitroN-methylpyrrole and N-methylimidazole amino acid contained in pyridinyl imidazole derivatives suppress the recruitment of Lef1 or other co-activators on Wnt target gene promoters. Further studies to elucidate the exact mechanism of action of pyridinyl imidazoles are currently on-going in our laboratory.It is becoming increasingly clear that a variety of routes exist that modulate the Wnt/β-catenin pathway and that β-catenin signaling regulation may be more complicated than the simplified view of β-catenin cellular homeostasis. Accordingly, we previously demonstrated that over-expression of β-catenin slightly activates Tcf/Lef-mediated transcription in melanocyte lineages In this paper, we described possible mechanisms by which pyridinyl imidazole derivates may suppress melanin synthesis by acting on Wnt signaling through unknown target molecules. Nevertheless, it is evident that the PI-dependent interference with Wnt signaling leads to low level of Mitf expression. However, is it also important to consider that the decreased Lef1 expression could trigger a negative loop reinforcing the inhibition of β-catenin-dependent genes transcription. Importantly, attenuation of Wnt signaling and decreased level of melanin synthesis were also confirmed in normal human epidermal melanocyte cell cultures (data not shown) supporting a possible topical application to treat disorders of hyperpigmentation. A prerequisite of further exploring the possibility of using PI compounds as skin-whitening agents is the knowledge of any additional effects. For example, we observed that while SB202474, SB202190, SB203580, PD169319 and MAPK inhibitor III all caused a strong p38-independent retention of melanin in the intracellular compartment, SB220025 did not alter melanin secretion (data not shown).From the therapeutic point of view, there is an increasing interest in small molecules capable of regulating the Wnt signaling cascade as this pathway is involved in cancer cell proliferation and migration. In addition, reduction of Mitf activity sensitizes melanoma, a highly chemotherapy-resistant neoplasm, to chemotherapeutic agents, and it has been suggested that targeting Mitf in combination with B-RAF or cyclin dependent kinases inhibitors may offer a rational therapeutic avenue into melanoma 6, were transfected with Amaxa Nucleofector System using Amaxa Nucleofector Cell Line Kit R (program P-031). Cells were transfected by nucleofection in a single cuvette and plated immediately to ensure identical transfection efficiency. For siRNA experiments, we transferred 200 pmols of either p38 specificsiRNA, Mitf specific siRNA or scrambled siRNA as a negative control. Twenty-four hours later, cells were treated with compounds or DMSO. For Mitf or β-catenin (wild-type or mutated) exogenous over-expression cells, 1.5×106, were transfected with 5 µg of the corresponding construct using Amaxa Nucleofector System. For luciferase experiments cells, 1.5×106, were transfected with 1 µg of p-Mitf-Luc containing the Mitf melanocyte-specific promoter sequence upstream of the luciferase reporter gene or pTopFlash luciferase reporter plasmids containing three copies of the optimal Tcf/Lef-binding site (AAGATCAAAGGGGGT) upstream of a TK minimal promoter. A pTK-Renilla–expressing vector  was included as an internal control to avoid non-specific effects on luciferase expression treatment and to normalize data. After 24 hours, cells were treated with compounds (or not) and 6 hours later cells were lyzed and resuspended in reporter lysis buffer. Firefly and Renilla luciferase activity were determined with Dual-Luciferase reporter assay  from duplicate plates. The results represent data of duplicates from three independent experiments.The B16-F0 murine melanoma and HeLa cells were purchased from ATCC (American Type Culture Collection) and were maintained in Dulbecco's modified Eagle's medium (DMEM)+7% (B16-F0) or 10% (HeLa) heat-inactivated fetal bovine serum (FBS) and antibiotics . For the induction studies, cells were plated for 24 hours, and then the medium was removed and the cells cultured in DMEM with 2% heat-inactivated FBS and antibiotics with or without pharmacological treatment in the absence of phenol red. SB202190, SB203580, α-MSH, DMSO and L-DOPA were purchased from Sigma , while MAPK inhibitor III, PD169316, SB202474, and SB220025 were purchased from Calbiochem . For transient transfection experiments cells, 1.5×104 cells/cm2 and left to grow overnight. Cells were treated with increasing concentrations of PI compounds in DMEM containing 2% FBS and antibiotics in quadruplicate, and were left to grow for 72 h before being incubated with 3--2,5-diphenyl tetrazolium bromide (MTT) for 60 min. After this time, the medium was removed and the resulting crystals were solubilized in DMSO. The absorbance was measured at 570 nm with a reference wavelength of 650 nm. Absorbance readings were subtracted from the value of blank wells, and the reduction in cell growth was calculated as a percentage of control absorbance in the absence of any drug (DMSO).Cells were plated in a 24-well plate at a density of 2×10Extracellular melanin release was measured as previously described Proteins extracts were prepared with RIPA buffer  containing Complete Mini protease inhibitor cocktail . Aliquots of cell lysates were separated by electrophoresis on SDS-polyacrylamide gels, transferred to nitrocellulose membranes and then treated with the appropriate antibodies: anti-β-catenin 1∶3000, anti-Mitf 1∶500, anti-p38 1∶500  and anti-tubulin 1∶5000 . Horseradish peroxidase-conjugated goat anti-mouse and bovine anti-goat immunoglobulin  were used at 1∶5,000 and 1∶3,000, respectively. Antibody complexes were detected by chemiluminescence . Western blot assays were representative of at least three experiments. Densitometric analysis was performed using a GS-800 Calibrated Image Densitometer .t value of each particular sample. The median ΔCt value, calculated as the differences between the Ct value for the gene of interest and that for the endogenous control β-actin, was used to calculate ΔΔCt = ΔCt treated sample−ΔCt control sample, and this, in turn, was used to calculate the fold difference of the genes of interest between treated and control samples: Normalized expression ratio = 2−(ΔΔCt). Values represent the means±SD of normalized fold-difference (increase or decrease). Data were analyzed with iQ5 Optical System Software (BioRad Laboratories).the average was then used to calculate the C Sequences of oligonucleotide primers indicated with an F correspond to sense strands and with an R correspond to anti-sense. In preliminary tests the amplification efficiency of all primer pair was showed to be ≥99.5%. Reverse primers can be found in Total RNA was extracted using an RNeasy mini kit . cDNA was synthesized from 1 µg of total RNA using the Improm II™ Reverse Transcription System . First-strand cDNA (1 µl) was amplified in a reaction mixture (15 µl) containing BioRad Master SYBR Green and 25 pmol of forward and reverse primers. All samples were run in triplicate, and Experiments included the following negative controls (no antibody) to confirm staining specificity for single color analysis with anti-CREB-PE-conjugated (pSer133) antibody; goat-anti-mouse-Alexa488 and goat-anti-mouse-Alexa488 plus CREB-PE-conjugated (pSer133) for in double staining experiments. Data from 1×104 cells were acquired from each sample. Median Fluorescence Intensity (MFI) was evaluated on a linear scale.Cells were fixed and permeabilized with Cytofix/Citoperm™. Cells were stained with anti-Mitf  primary antibody followed by an incubation step with goat-anti-mouse-Alexa488 secondary antibody; anti-CREB-PE-conjugated (pSer133) antibody  and then analyzed by flow cytometry using a FACSCalibur. To confirm staining specificy each experiment included a negative control incubated exclusively with Alexa-Fluor-488-conjugated goat anti-mouse IgG in absence of no primary antibody. Nuclei were stained with DAPI . Images were captured using a CCD camera .For indirect immunofluorescence experiments, cells were grown on coverslips and after treatment were fixed with 3% paraformaldehyde in PBS for 15 min at room temperature and then permeabilized with 0.05% Trition X-100 in PBS for 5 min. Cells were rinsed in PBS and incubated for 2 h with mouse anti-β-catenin  primary antibody (1∶1000 in PBS). Cells were rinsed three times with PBS and incubated for 60 min with an Alexa-Fluor-488-conjugated goat anti-mouse IgG (1∶800 in PBS) (Molecular Probe). t-test was used to assess statistical significance with thresholds of * p≤0.05 and # p≤0.01.Student's Figure S1Effect of pyridinyl imidazoles on cell viability. Cells were left to grow for 72 in presence of increasing concentrations of pyridinyl imidazoles compounds before being incubated with 3--2,5-diphenyl tetrazolium bromide (MTT) for 2 hrs. The resulting crystals were solubilized in DMSO. The absorbance was measured at 570 nm with a reference wavelength of 650 nm. Values reported as O.D. decrease over untreated control represent the means±SD of two experiments performed in triplicate.(TIF)Click here for additional data file.Figure S2Time-dependent α-MSH-dependent stimulation of Mitf and melanogenic enzymes. Semi-quantitative real-time-PCR to measure the kinetics of Mitf, tyrosinase, TRP1 and DCT mRNA increase following α-MSH treatment was performed by using the real-time detection system. The graphs show fold differences in transcripts abundance in α-MSH-stimulated cells compared with untreated cells. The results shown were normalized by the β-actin mRNA levels. The data show the mean±SD of four experiments performed in triplicate.(TIF)Click here for additional data file.Table S1Pyridinyl Imidazole Compounds. All PI included in the study are listed with the corresponding chemical name and IC50.(DOCX)Click here for additional data file.Table S2Sequences real-time PCR oligonucleotide primers list.(DOCX)Click here for additional data file.


Beta-catenin is a key nuclear effector of Wnt signaling which could be antagonized by dickkopf-1(DKK1). Beta-catenin and DKK1 are involved in a variety of biological processes; however, their expression in the placenta with severe preeclampsia (PE) has not been elucidated. This study was aimed to detect the localization and compare the expression of beta-catenin and DKK1 in normal and preeclamptic placenta.Sixty pregnant women who underwent cesarean section were enrolled in this study, including 30 healthy pregnant women in the control group and 30 preeclamptic women in the severe PE group. Real-time polymerase chain reaction  and western blot were employed to detect the beta-catenin and DKK1 mRNA and protein expression levels, respectively, and their locations were evaluated by immunohistochemistry (IHC).Our results indicated that beta-catenin and DKK1 were expressed predominantly in the syncytiotrophoblast and the extravillous trophoblast (EVT). The beta-catenin mRNA and protein expressions were significantly decreased, whereas the DKK1 significantly increased in preeclamptic placental tissues compared to normal placental controls.In conclusion, decreased beta-catenin expression, as well as DKK1 over-expression might be associated with the process of the pathogenesis of PE. Further studies would elucidate their exact roles in the pathogenesis of PE. Preeclampsia (PE) is a common, pregnancy-specific disease that belongs to the family of hypertensive disorders in pregnancy and is characterized by new onset of hypertension and proteinuria after 20 weeks of gestation. PE is a major contributor to maternal and fetal morbidity and mortality. The preWnt signaling has been identified as an essential pathway which can direct cell proliferation, migration, and tissue homeostasis. The canonical Wnt signaling is activated when a Wnt ligand binds to the Frizzled receptors and their co-receptors. The signal is then transmitted into the cytoplasm by a series of cellular factors, and these events lead to the stabilization of β-catenin in the cytoplasm. Consequently, β-catenin accumulates and travels into the nucleus to form complexes with T cell-specific factors (TCFs)/lymphoid enhancer-binding factor-1(LEF-1), and activate target gene expression-6. β-catSixty pregnant women who had undergone cesarean section at the Third Affiliated Hospital of Zhengzhou University, from January 2010 to January 2012, were enrolled in this study. This sample included 30 normal pregnant women, who constituted the control group, and 30 preeclamptic women, who were classified as the severe PE group. This study was approved by the Ethics Committee of Zhengzhou University School of the Third Clinical Medicine, China. The informed consent was obtained from all subjects. The criteria for diagnosis of severe PE were strictly based on the American College of Obstetricians and Gynecologists Practice Bulletin (ACOG 2002). Normal The placental biopsies were collected from the maternal aspect of the placenta within 15 min of delivery. Five small separate biopsies (2 cm × 2 cm × 1 cm) were excised from the placental center, as well as from each quadrant  to avoid sampling bias. Placenta sampling included full-thickness placental blocks biopsies were not collected from the placental periphery or areas of calcification and necrosis. A portion of the samples were fixed in 10% formalin for immunohistochemistry, and the other portion of samples were flash frozen in liquid nitrogen and stored at −80°C until the tissue was processed. In this study, these samples were studied pooled.The total RNA was extracted from placental biopsies using the Trizol reagent  according to the manufacturer's protocol. RNA was fractionated on agarose/ethidium bromide gels to confirm the integrity. cDNA synthesis was performed using a Reverse Transcriptase M-MLV kit as per the manufacturer’s instructions . Quantitative real-time PCR assays of β-catenin and DKK1 were performed using the Ultra SYBR Mixture (with ROX)  in an ABI 7500 Fast Real-time PCR System . The PCR primers were designed and synthesized by Sangon Biotech ; utilized primers are shown in Table 2O 8.2 μl to compose the final volume. A sample without cDNA was subjected to an identical protocol as a negative control. The PCR amplification was accomplished with initial denaturation at 95°C for 10 min, followed by 35 cycles at 95°C for 20 s and 60°C for 1 min. During the melt cycle, the temperature was increased by increments of 1°C from 60°C to 95°C. The CT values for the targets (β-catenin and DKK1) and GAPDH genes were provided by real-time PCR instrumentation. The comparative method 2-ΔΔCT was used for the relative quantification of β-catenin and DKK1 transcription between the control and the severe PE groups.The real-time PCR was performed in a final volume of 20 μl, which contained 10 μl of 2×Ultra SYBR Mixture (With ROX), 0.4 μl of forward and reverse primers, respectively, 1 μl of template cDNA, and RNA free HThe fixed biopsies were deparaffinized, and the paraffin blocks were cut into 4 μm sections and mounted onto microscope slides. The sections were then hydrated by sequential immersion in xylene and graded alcohol solutions. Prior to staining, antigen retrieval was accomplished by boiling tissue slides in a citrate buffer solution. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 20 min. After blocking the tissue with goat serum, the sections were incubated for 1 h at room temperature with the primary antibodies specific to β-catenin , DKK1 and HLA-G . The phenotype characteristic of EVT was confirmed with the use of serial sections stained with HLA-G. Negative control sections were incubated for 1 h at room temperature with phosphate buffer solution (PBS). The polink-2 plus® polymer HRP detection system for rabbit and mouse primary antibody kits and DAB detection kit  were used following the manufacturer' s protocols. Stained slides were examined with an Olympus microscope . Images for analysis were captured by a digital camera using Image Pro 6.0 software.The sections were assessed by two observers separately. The immunohistochemical staining was graded on a semiquantitative scale. Briefly, staining intensities were documented according to the following categories: 0(absent staining/ no color), 1, 2 (distinct staining/dark brown color), 3 (strong staining/brownish-black color).The frozen placental tissue was directly homogenized with the use of RIPA buffer containing a protease inhibitor cocktail . The total protein concentration was determined with a BCA assay . Following quantification, the samples containing 100 μg of protein were separated by 10% SDS polyacrylamide gel electrophoresis, and then they were electrophoretically transferred to the nitrocellulose membranes. The membranes were blocked for 90 min at room temperature with blocking buffer  and incubated overnight at 4°C with mouse monoclonal to DKK1  and rabbit polyclonal to β-catenin , respectively. Then the membranes were incubated for 1 h at room temperature with their respective secondary antibodies. The peroxidase-conjugated goat anti-mouse IgG and the goat anti-rabbit IgG were purchased from Dingguo Biotech . The chemiluminescent detection was performed using a Pro-light HRP chemiluminescent detection kit . Image J analysis software was utilized to estimate the relative density of the proteins of interest. β-actin was detected by rabbit polyclonal anti-β-actin antibody , and the expression of β-actin was used for verifying the protein loading variations.-ΔΔCT method[All statistical analyses were performed using SPSS 17.0 software. Quantitative data are presented as mean ± standard deviation (SD). Comparison of two groups was performed using either unpaired t test (for parametric data) or the Mann–Whitney U-test (for non-parametric data). The differences in enumeration data were detected with the χ2 test. The 2T method was usedWe employed real-time PCR to examine relative quantity of β-catenin and DKK1 mRNA in both groups (n=30 for each group). Our results indicated that β-catenin and DKK1 mRNA expression could be detected in both the severe PE and normal control groups, The β-catenin mRNA expression was decreased in the severe PE group(0.63±0.39) compared with the control group (1.25±0.56) (P<0.05) . The sections were examined by hematoxylin and eosin (H&E) staining before IHC analysis. The image of negative control section was shown in Figure The immunohistochemical staining for the β-catenin and DKK1 proteins was observed in the syncytiotrophoblast and extravillous trophoblasts (EVT). The phenotype characteristic of EVT was confirmed with the use of serial sections stained with HLA-G. Our results indicated that the staining intensity of β-catenin in the placental tissue of the severe PE group  and those with an AGA fetus . The β-catenin mRNA expression was decreased in the severe PE group who had an IUGR fetus (0.47±0.34) compared with those with an AGA fetus (0.76±0.40)  (P<0.05). The β-catenin protein expression was decreased in the severe PE group who had an IUGR fetus (0.34±0.12) compared with those with an AGA fetus (0.43±0.09)  (P<0.05). However, the DKK1 mRNA expression was increased in the severe PE group who had an IUGR fetus (2.35±0.86) compared with those with an AGA fetus (1.64±0.54)(P<0.05). The DKK1 protein expression was increased in the severe PE group who had an IUGR fetus (0.42±0.19) compared with those with an AGA fetus (0.27±0.13) (P<0.05).Besides, we analysed the β-catenin and DKK1 expression between patients with preterm and term preeclampsia. However, the results suggested that there was no difference in the β-catenin mRNA expression between patients with preterm (0.56±0.31) and term PE(0.67±0.45)  (P>0.05). There is was no difference in the β-catenin protein expression between patients with preterm (0.36±0.12) and term PE(0.41±0.11)  (P>0.05). The DKK1 mRNA expression was no difference between patients with preterm (2.14±0.61) and term PE (1.97±0.94) (P>0.05). The DKK1 protein expression was no difference between the preterm (0.35±0.20) and term PE (0.31±0.13) (P>0.05).The Wnt signaling is a complex pathway which is composed of the Wnt protein ligand, membrane receptors, extracellular inhibitory factors, intracellular signal switch and nuclear transcription factors. Wnt signaling has been identified as an essential pathway which is involved in development, tissue regeneration and cancer. Sondereβ-catenin is considered to be the intracellular signal switch in the canonical Wnt signaling pathway. It has been implicated in the genesis of many human cancers, including non-small cell lung cancer, colorectal carcinoma and many others cancer variants-20.Previous study reported that the β-catenin destabilization could be provoked and trophoblast motility reduced because of the gene silencing of protease activated receptor-1, PAR1. MoreoveDKK1, the founding member of the dickkopf (DKK) family, plays important roles in diverse developmental processes. It has been implicated in the process of tumorigenesis, such as in the case of colorectal cancer, medulloblastoma, and mesothelioma,27.Recent studies have suggested that DKK1 may negatively affect the implantation and adhesion of the trophoblast to the endometrium. The treatment of JAR spheroids with DKK1 was shown to impair the attachment to endometrial-like Ishikawa cells. DKK1 haPE is a multisystem disease, the subclassifications of PE are often defined as: mild, moderate, and severe, as well as early and late. The concept of early and late PE is more modern, and it is widely accepted that these two entities have different etiologies and should be regarded as different forms of the disease,32. In oClinical risk factors for intrauterine fetal growth contain severe PE, cardiopulmonary system disorders, umbilical cord and placenta dysfunction, etc. In this study, we also found that the β-catenin expression was decreased, and the DKK1expression was increased in the severe PE group who had an IUGR fetus compared with those with an AGA fetus (P<0.05). However, the results suggested that there is no difference in the expression of β-catenin and DKK1 between patients with preterm and term PE. Potential reasons for this finding may be that the premature birth contains spontaneous premature delivery and therapeutic premature delivery, the influence factors are complex.In summary, the pathogenesis of PE is highly complex, and the results of this study indicated that β-catenin and DKK1 are expressed in human third trimester placentas, decreased β-catenin expression, as well as DKK1 over-expression might be involved in the process of the pathogenesis of PE. Further studies would elucidate their exact roles in the pathogenesis of PE. Moreover, the expression of β-catenin and DKK1 during the first and second trimester, as well as the potential roles of the other associated proteins in Wnt signaling pathway in PE, awaits further study.DKK1: Dickkopf-1; EVT: Extravillous trophoblast; H&E: Hematoxylin and eosin; IHC: Immunohistochemistry; LEF-1: Lymphoid enhancer-binding factor-1; PE: Preeclampsia; TCF: T cell-specific factors.The authors declare that they have no competing interests.ZZ and HL participated in designing this study, writing and modifying this article, accomplishing the entire experiments. LLZ participated in collecting the samples, helping to accomplish the experiments. JLT and WP participated in helping to accomplish the experiments. All authors read and approved the final manuscript.


Accumulating evidence has suggested that Wnt/β-catenin exhibits a pivotal function in the progression of diabetic nephropathy (DN). In this review, we focused on discussing the dual role of Wnt/β-catenin in apoptosis and epithelial mesenchymal transition (EMT) formation of mesangial cells. Moreover, we also elucidated the effect of Wnt/β-catenin in podocyte dysfunction, tubular EMT formation, and renal fibrosis under DN conditions. In addition, the molecular mechanisms involved in this process are introduced. This information provides a novel molecular target of Wnt/β-catenin for the protection of kidney damage and in delay of the progression of DN.The Wnt family of proteins belongs to a group of secreted lipid-modified glycoproteins with highly conserved cysteine residues. Prior results indicate that Wnt/ Diabetic nephropathy (DN) is a chronic kidney disease and is a major complication of diabetes mellitus. DN is also the main cause of end-stage renal disease worldwide . It is kβ/Smad signaling pathway, a well-known profibrotic pathway in renal fibrosis, plays a key role in the progression of DN. In addition to the TGF-β/Smad pathway, many non-Smad pathways, such as PI3K/Akt, p38 MAPK, and JAK/STAT signaling, are also known to contribute to the development of DN . Th. Thβ-cat pathway , 14. Num pathway . Importadiseases –23, incldiseases , 23–25.β-catenin pathway is activated under diabetic conditions. Zhou et al. showed that the levels of β-catenin and WNT proteins are upregulated in the kidney tissues of both type 1 and type 2 diabetic animal models. However, insulin could attenuate the activation of WNT signaling via lowering blood glucose levels [β-catenin signaling pathway has been demonstrated to play a role in the pathogenesis and progression of DN, and multiple cells are thought to be involved in this process, including mesangial cells, podocytes, and tubular cells [Recently, emerging evidence has suggested that Wnt/e levels . In addie levels . Moreovear cells –10.β-catenin signaling pathway is involved in the regulation of morphological changes and pathogenesis in glomerular cells in DN, and its effect in HG-induced MCs apoptosis is particularly emphasized. Recently, Lin et al. found that HG inhibits the expression of Wnt4 and Wnt5a in cultured MCs, while increased GSK-3 expression and caspase-3 activities resulted in an increase in the level of MC apoptosis. In contrast, a reduction in HG-mediated caspase-3 cleavage and cell apoptosis was observed to inhibit GSK-3β activation or increase in nuclear β-catenin via transfection of Wnt4 or Wnt5a or stable β-catenin (S33Y) [β and restoration of Wnt4 and Wnt5a expression in vitro and in vivo [via activation of the Wnt signaling pathway [β-catenin destabilization and subsequently promotes caspase-3 and poly (ADP-ribose) polymerase cleavage, which subsequently caused apoptosis in cultured MCs [β-catenin signaling is beneficial in preventing HG-induced apoptosis of mesangial cells.Accumulating studies have shown that mesangial cells (MCs) play an important role in the structure and function of glomerular maintenance, which supports capillary loops and regulates glomerular filtration . Substann (S33Y) . Similar in vivo . Moreove pathway . In addiured MCs . These dβ-catenin signaling in MC cell survival induced by HG ambient. Lin et al. reported that HG can induce Ras and Rac1 activation, increase ROS production and destabilization of Wnt5a/β-catenin in MCs, and subsequently induce cellular apoptosis via the promotion of caspase-3 and poly (ADP-ribose) polymerase cleavage. However, this effect was abolished in cells transfected with either dominant-negative Ras (S17N) or dominant-negative Rac1 (T17N). Furthermore, stabilization of β-catenin via transfection of stable β-catenin (Delta45) and kinase-inactive GSK-3β has been shown to attenuate HG-mediated MCs apoptosis [β and inhibited Wnt5a/β-catenin signaling via the regulation of superoxide mediated by Ras and Rac1 activation was observed.Recent studies have demonstrated cross talk between oxidative stress and Wnt/poptosis  indicatiβ-catenin is involved in the epithelial-mesenchymal phenotypic transition (EMT) of MCs under DN conditions [β1 has been proposed as a prominent mediator of HG-induced EMT formation in various cells. In addition, β-catenin interacting protein 1 (CTNNBIP1) is a direct target of miR-215, which inhibits Wnt/β-catenin signaling. Mu et al. found that increased miR-215 expression suppressed CTNNBIP1 and activated Wnt/β-catenin, which subsequently promoted TGF-β-induced EMT formation of MCs in DN and is characterized by an increased expression of fibronectin and α-SMA. However, knockdown of miR-215 may reverse this phenotypic transition. Moreover, a previous study found that sustained Wnt signaling reduced c-Jun-dependent TGF-β1-mediated fibronectin accumulation in MCs [β1-mediated EMT formation of MCs via the CTNNBIP1/β-catenin pathway.It is known that the transition of MCs plays a key role in the progression of DN. In addition to apoptotic regulation, accumulating studies have also demonstrated that Wnt/nditions . TGF-β1 n in MCs , suggestXenopus Laevis embryos [β, which resulted in an accumulation of β-catenin and its nuclear localization, and activated the transcription factor TCF/LEF and increased MCs apoptosis via the regulation of the expression of Wnt targets. However, treatment with DKK-1, an endogenous LRP6 receptor antagonist or knockdown of LRP6 via siRNA, ameliorated CCN2-induced Wnt signaling activation in human MCs [Recent evidence suggests that there is a complex relationship between CTGF/CCN family protein 2 (CTGF/CCN2) and the Wnt signaling pathway. Previous studies have demonstrated that CCN2 can modulate Wnt signaling by binding to LRP5/6. In addition, Wnt ligands increased CCN protein expression in  embryos . Rooney uman MCs . Moreoveuman MCs . Taken tβ-catenin expression is essential to maintain the function of the glomerular filtration barrier [β-catenin signaling promoted podocyte dysfunction in DN [β-catenin expression in podocytes were also observed [β-catenin resulted in podocyte dysfunction, whereas blockade of Wnt signaling by DKK1 ameliorated podocyte dysfunction and albuminuria [β-catenin contributed to podocyte damage in DN pathogenesis. However, the underlying molecular mechanisms of this phenomenon remain poorly understood.Emerging studies have demonstrated that Wnt/CTNNBIP1 plays an important role in the regulation and integration of cell adhesion, motility, and cell death and differentiation of glomerular podocytes. Proper  barrier . Severalon in DN , 33, 34.on in DN . Furtherobserved . Moreoveuminuria , indicatIn vitro studies have shown that HG induced apoptosis and reduced the viability of differentiated mouse podocytes with an upregulation of TRPC6 and activation of the canonical Wnt signaling pathway, which was blocked when treated with DKK-1 [β-catenin pathway might potentially be activated during TRPC6-mediated podocyte injury in DN. Furthermore, angiotensin II (Ang II) plays an important role in promoting podocyte dysfunction and albuminuria. Encouragingly, there is some evidence demonstrating that Ang II induced Wnt1 expression and β-catenin nuclear translocation via calmodulin-dependent protein kinase II and cAMP response element-binding protein in cultured mouse podocytes. However, treatment with DKK-1 or β-catenin siRNA could partially block this effect [th DKK-1 . These fs effect . In addis effect .β-catenin is a critical regulator of the maintenance of glomerular filtration barrier and its function.Podocyte-specific CTNNB1 knockout mice exhibited basement membrane damage, albuminuria, and an increased susceptibility to glomerular injury. Importantly, mice with either podocyte-specific deletion of CTNNB1 or podocyte-specific expression of DKK-1 showed an increased susceptibility to DN . These fβ-catenin signaling-induced podocyte dysfunction in DN are complex and may involve an increase in the expression of either Wnt1 or stabilized β-catenin, which induced the transcription factor Snail and suppressed expression of the nephrin gene. These events subsequently resulted in podocyte dysfunction [β-catenin and Snail transcriptional activities, which may be a potential mechanism in podocyte injury under diabetic conditions [Mechanisms of activated Wnt/function . Emerginfunction , 37. Durfunction , suggestnditions .β-catenin pathway-mediated podocyte injury via the activation of transient receptor potential channel 6 (TRPC6) is essential for the proper regulation of podocyte structure and function, whereas inhibition of the Wnt/β-catenin pathway by DKK-1 downregulated HG-induced TRPC6 expression and ameliorated podocyte injury in DN [β-catenin in podocytes, which inhibits β-catenin nuclear translocation and regulates target gene transcription [Furthermore, Li et al. indicated thatWnt/ry in DN . In addicription .in vitro and in vivo [β-catenin also contributes to the EMT formation of tubular cells in DN.In addition to mesangial cells and podocytes, emerging studies have suggested that the renal tubule also exhibits a critical effect in DN development , 42. Tub in vivo , 44. Recβ-catenin activation in tubular epithelial cells [β-catenin and induce β-catenin target gene expression, including Snail and Twist, which suppressed E-cadherin expression and increased fibronectin, α-SMA and vimentin expression in tubular cells [β-catenin reversed HG-induced EMT of tubular cells [β-catenin mediated HG-induced EMT formation in tubular cells.Zhou et al. showed that HG increased the expression of WNT proteins and induced Wnt/al cells . Liu et ar cells . Howeverar cells , suggestβ-catenin signaling and the TGF-β/Smad or non-Smad signaling pathway in TGF-β induced EMT formation in DN [β1 from cultured tubular cells and altered the expression of adhesion and adherens junction proteins, associated with decreased E-cadherin and connexin-43 production. In addition, HG also caused an impairment in cell adhesion and also decreased cell-to-cell communication [β-catenin is required as a cofactor of Smad in TGF-β1-induced EMT of renal epithelial cells [β-catenin signaling pathway participated in HG-induced renal tubular epithelial cell EMT formation, although this effect was inhibited by treatment with tanshinone IIA, which downregulated the activities of Wnt/β-catenin signaling [β-catenin siRNA partially blocked HG-induced EMT relative protein expression in cultured primary proximal tubular cells. They also demonstrated that HG decreased PPARγ activation, while the PPARγ agonist troglitazone reversed HG-stimulated expression of GSK-3β, β-catenin and Snail1, and EMT relative proteins [Interestingly, it has been reported there is cross talk between Wnt/on in DN . Hills enication . Tian etal cells . Furtherignaling . Lee et proteins .β-catenin activation in unilateral ureteral obstruction (UUO) mice was associated with matrix metalloproteinases-7, fibronectin, Twist, and c-Myc. However, treatment using recombinant sFRP4, an inhibitor of Wnt signaling, resulted in a decreased expression of these extracellular matrix (ECM) related genes and alleviated the progression of renal fibrosis [β-catenin was involved in the cyst formation of polycystic kidney disease [β-catenin might be involved in various kidney injuries and renal fibrosis. Recent studies have also suggested that Wnt/β-catenin plays a pivotal role in renal fibrosis in DN.Surendran et al. found that Wnt/fibrosis . In addi disease . These dβ-catenin pathway participated in TGF-β/Smad signaling-modulated ECM protein accumulation in the DN state [β-catenin, which was accompanied with an increased expression of TGF-β and fibronectin in MCs exposed to HG, while this effect was alleviated in cells transfected with Wnt4, Wnt5a, and stable β-catenin [β and fibronectin expression in both the renal tissue of STZ-induced diabetes rats and in cultured mesangial cells exposed to HG. However, knockdown of DKK1 attenuated mesangial matrix accumulation via the restoration of the activation of Wnt/β-catenin signaling under hyperglycemia conditions [Accumulation of ECM components in the mesangial area is a prominent hallmark of DN, which ultimately results in renal fibrosis , 51. SevDN state , 52. Ho -catenin . In addiβ-catenin were up-regulated in proximal tubular epithelial cells under DN conditions in vitro and in vivo, which was accompanied by an increased expression of CTGF and fibronectin. Injection of LRP5 and LRP6 antibodies suppressed activation of the WNT pathway and decreased ECM formation in DN animal models, suggesting that WNT/β-catenin signaling might be involved in tubular-interstitial fibrosis in DN [β-catenin signaling via competition of TCF-4 and activated vitamin D receptor, then attenuated renal interstitial fibrosis [β was sufficient to activate the Wnt/β-catenin via p38-dependent mechanism and decreased the expression of DKK-1. A selective TBRI inhibitor could reverse the effect of TGF-β on Wnt/β-catenin and potentially reduce renal fibrosis [In addition, Zhou et al. found that the expression of Wnt and cytoplasmic is in DN . Surendris in DN . In addifibrosis . In addifibrosis .β-catenin signaling pathway has been shown to be activated under DN conditions, which was involved in the apoptosis and EMT formation of mesangial cells, podocyte dysfunction, and tubular cells EMT, subsequently resulting in renal fibrosis and interstitial fibrosis. It has been suggested that sustained Wnt/β-catenin expression is essential for its protective role against cellular damage, while abnormal activation of Wnt/β-catenin results in adverse effects and promotes the progression of DN, suggesting that Wnt/β-catenin plays a dual role in cellular damage under DN conditions. The mechanism underlying the regulation of Wnt/β-catenin and its crosstalk with other signaling pathways involved in DN is very complex and requires further studies. On the basis of these data, targets of the Wnt/β-catenin signaling pathway may provide a new therapeutic method to prevent DN progression.The Wnt/


Microglia are resident mononuclear phagocytes of the brain that become activated in response to insults including neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and prion disease. In the central nervous system the chemokine Cx3cl1  is expressed by neurons and its exclusive receptor Cx3cr1 is expressed solely on microglia. Cx3cl1/Cx3cr1 signalling is thought to maintain microglia in their resting state and disrupting this equilibrium may allow microglia to become activated. In prion disease, microglial proliferation has been suggested to contribute to overall disease progression, however, in different mouse models of neurodegeneration, loss of Cx3cr1 has been shown to either worsen or improve the phenotype depending on the paradigm.Cx3cr1 null mice with three different strains of prions. Following challenge with Chandler/RML, ME7 and MRC2 prion strains, Cx3cr1 knockout mice showed highly significant reductions in incubation time. No differences were seen in the pattern and localisation of activated microglia in the brain or in the mRNA expression levels of chemokines/cytokines .To investigate the role of Cx3cl1/Cx3cr1 signalling in prion disease we infected Our data suggest a protective role for Cx3cl1/Cx3cr1 cross-talk in prion disease. Neuroinflammation within the brain is a common feature of neurodegeneration and one of the hallmarks of these changes is the activation of microglia. Microglia are CNS  mononuclear phagocytes that under normal conditions are surveillant but are activated in response to various brain insults ,2. ActivNeurons express the chemokine Cx3cl1  in a membrane bound or soluble form. Its receptor within the CNS, Cx3cr1, is expressed solely on microglia -6. Cx3clPS1M146V knock-in, transgenic APPSwe and tauP301L) model of Alzheimer’s disease (AD) it was shown that loss of Cx3cr1 resulted in the increase of microglial migration and prevented neuronal loss while levels of Aβ deposition remained unaltered [Cx3cr1 in a toxin-induced model of Parkinson’s disease (PD) and a genetic model of Amyotrophic Lateral Sclerosis (ALS) (Tg SOD1G93A) worsened the phenotype, in both cases producing more neuronal loss and, for the ALS model, accelerated deterioration of hind limb grip function and reduced survival were also seen [MAPT transgenic mouse also lacking Cx3cr1 showed altered microglial activation, enhanced tau phosphorylation and aggregation as well as poorer spatial working memory [The role of Cx3cr1 has been extensively studied in many models of neurodegeneration and brain injury through the use of Cx3cr1 knockout mice, although often with conflicting results. In a triple transgenic  to abnormal isoforms, designated PrPSc and constitute lethal infectious agents. Prion infection is accompanied by spongiform change and neuronal loss in the brain. In contrast to other infectious diseases little if any evidence of inflammation is seen in the periphery. However, even before significant neuronal loss occurs, there is widespread activation of microglia, glia and astrocytes in the brain [Prion diseases or transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases that include Creuzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep . In commhe brain -16. Proihe brain ,17.In wild type mice infected with a mouse-adapted scrapie prion strain (ME7) Cx3cl1 is reported to be upregulated in astrocytes but there is no change in surviving neurons and Cx3cr1 expression is also upregulated on microglia . AnalysiCx3cr1+/- mice  on a Balb/c background were obtained as embryos from the European Mouse Mutant Archive, resurrected and bred to homozygosity [zygosity . Wild ty2, female mice were inoculated intra-cerebrally with 30 μl of inocula into the right parietal lobe as previously described [Inocula for three different prion strains were made from the brains of terminally sick mice as 1% (weight/volume) homogenates in sterile D-PBS. Chandler/RML (I9900) and ME7 (I9459) are two different mouse-adapted strains of scrapie and MRC2 (I9468) is a mouse-passaged strain of BSE ,20. Follescribed . Mice weescribed . Incubatescribed . EthicalMouse brains were fixed in 10% buffered formal saline (BFS) and prion-infected tissue was treated in 98% formic acid for one hour to remove infectivity. Tissues were paraffin wax embedded, sagitally sectioned and stained as previously described . HaematoCxcl10, Il-12b, Il-1b, Arg-1 and Cx3cl1. Each reaction was duplexed independently with two endogenous controls . All reactions were carried out in triplicate. Statistical tests were carried out using GraphPad InStat .RNA was extracted using a TRIzol Plus RNA Purification kit (Life Technologies) from half brains taken from terminally sick prion infected mice and uninfected controls (n = 4 or 5 per group). cDNA was generated using a QuantiTect Reverse Transcription Kit (Qiagen). Contaminating genomic DNA was removed during the extraction with a DNase I digestion and this was confirmed by including a no reverse-transcription control for each sample. Real-time RT-PCR reactions were carried out using Taqman Gene Expression Master Mix enzyme (Life Technologies) on a 7500 Fast Real-time PCR System (Life Technologies). Gene specific Taqman Gene Expression assays (Life Technologies) were used for Cx3cr1 knockout mice were generated by replacing the Cx3cr1 gene with the green fluorescent protein (GFP) reporter gene [Cx3cr1 mRNA transcript and the generation of a chimeric transcript of an untranslated Cx3cr1 exon spliced to the GFP exon. The mice have a normal lifespan with no visible abnormal phenotype. The presence of GFP has not been reported to interfere with microglial function and microglia are still able to become activated in the absence of Cx3cr1 [Cx3cr1-/- mice and Balb/c wild type controls intracerebrally with three different mouse-adapted prion strains. Chandler/RML and ME7 are two distinct scrapie-derived prion strains and MRC2 was generated from the passage of cattle BSE in mice [Sc distribution, gliosis and spongiosis [Cx3cr1-/- mice relative to controls for all three prion strains  and for ME7 the mean incubation time was reduced by 7% from 178 ± 1 in Cx3cr1+/+ to 168 ± 1 in Cx3cr1-/- mice . For MRC2 the mean incubation time was reduced by 7% from 206 ± 2 in Cx3cr1+/+ to 200 ± 1 in Cx3cr1-/- mice .ter gene . The genf Cx3cr1 . To inve in mice ,20. Eachongiosis . Onset oCx3cr1-/- groups. Even in the absence of Cx3cl1/Cx3cr1 signalling, microglia show extensive proliferation and based on morphology appear to be activated and similarly distributed to that seen in the controls  [Cxcl10 expression was significantly increased in all three prion strains relative to uninfected controls with 5-fold increases for Chandler/RML and ME7 and a 15-fold increase for MRC2 . No significant differences were seen between knockout and wild type groups . However, a 2-fold increase was seen in Cx3cr1-/- relative to Cx3cr1+/+ mice (P = 0.01) but only with the ME7 prion strain , however, no significant differences were seen between Cx3cr1-/- and wild type controls  mRNA expression and saw no significant differences between Cx3cr1 knockout and wild type mice or between infected and uninfected groups  . Cxcl10 We have shown that Cx3cr1 deficiency results in an acceleration in the onset of the clinical signs of prion disease, as illustrated by shorter incubation times, with three different prion strains. These data suggest that in wild type animals intact Cx3cl1/Cx3cr1 signalling is partially protective and may act to somewhat restrain the microglial response. The difference in incubation time observed in knockout mice is quantitatively modest but highly statistically significant with a reduction of 4% for Chandler/RML and 7% for both ME7 and MRC2 prion strains and no overall differences observed in microglial activation either by neuropathology or cytokine/chemokine expression in terminally sick mice. These data suggest that Cx3cl1/Cx3cr1 signalling plays only a minor role in modulating microglial activation in prion disease.Cx3cr1-/- mice the earlier exposure to neurotoxic factors may contribute to the earlier onset of clinical disease. We observed a 2-fold increase in Il-12b expression in Cx3cr1-/- relative to Cx3cr1+/+ mice (P = 0.01) but only with the ME7 prion strain which suggests a prion strain specific effect. Given that this was not observed with other proinflammatory cytokines or with the other prion strains it is difficult to assess the relevance of this finding.Although microglial activation was unaltered in our study it is worth noting that for both neuropathological examination and cytokine/chemokine production, we looked only at brains from terminally sick animals. It is possible that microglia are activated earlier in the absence of Cx3cr1 but by end stage disease microglia are activated and distributed as seen in wild type mice. If microglia are activated earlier in SOD1G93A), tauopathy and PD [The role of Cx3cl1/Cx3cr1 signalling in neurodegeneration is variable, highly dependent on the model used and the phenotype measured and probably reflects the nature of the neuroinflammatory response triggered by each disease process. Our data suggest that for prion disease the neuronal-microglia cross talk provided by this interaction is partially protective but is not sufficient to override other pathways of microglial activation or neuronal loss. The protective role of Cx3cl1/Cx3cr1 is also seen in models of AD (APPPS1 and R1.40), ALS  suggesting that intact Cx3cl1/Cx3cr1 signalling is partially protective in prion disease. This occurs in the context of unchanged microglial activation, as assessed by cellular distribution and cytokine profiles. The role of Cx3cl1/Cx3cr1 signalling in different models of neurodegeneration is highly variable suggesting that its influence is dependent on the precise cytokine environment created by each disease process.AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; BFS: Buffered formal saline; BSE: Bovine spongiform encephalopathy; CJD: Creuzfeldt-Jakob disease; CNS: Central nervous system; D-PBS: Dulbecco’s phosphate buffered saline; GFAP: Glial fibrillary acid protein; GFP: Green fluorescent protein; H&E: Haematoxylin and eosin; PD: Parkinson’s disease; PrPc: Normal cellular prion protein; PrPSc: Abnormal disease associated isoform of prion protein; RT-PCR: Reverse transcriptase-polymerase chain reaction; TSE: Transmissible spongiform encephalopathy.JC is a director and shareholder of D-Gen Limited, an academic spin-out company in the field of prion diagnosis, decontamination and therapeutics. D-Gen markets the monoclonal antibody ICSM35 used in this study.SL planned the experiments; SA, GJ, and SL carried out the experimental work. SB and SL analysed the data; SL and JC wrote the manuscript. All authors read and approved the final manuscript.Methods-western blotting and additional figure legends.Click here for fileSc from infected mouse brains.Western blots of PrPClick here for fileCx3cl1 mRNA expression.Click here for file


In cultured human microvascular endothelial cells, LiCl as well as SB 216763, another small molecule that activates Wnt/β-catenin signaling, induced proliferation, survival and migration, which are all common parameters for angiogenic properties in vitro. Moreover, treatment with both agents caused an increase in the levels of β-catenin and their translocation to nuclei while quercetin, an inhibitor of Wnt/β-catenin signaling, completely blocked the effects of LiCl on proliferation. In mice with OIR, intraperitonal or intravitreal treatment with LiCl markedly increased the retinal levels of β-catenin, but did not improve capillary repair. In contrast, repair was significantly improved following intravitreal treatment with Norrin. The effects of LiCl on HDMEC in vitro have minor relevance for OIR in vivo, and the influence of the Norrin/Frizzled-4 pathway on capillary repair in OIR is not reproducible upon enhancing Wnt/β-catenin signaling by LiCl treatment strongly indicating the presence of additional and essential mechanisms.Wnt/β-catenin signaling induced by the Norrin/Frizzled-4 pathway has been shown to improve capillary repair following oxygen induced retinopathy (OIR) in the mouse, a model for retinopathy of prematurity. Here we investigated if treatment with the monovalent cation lithium that has been shown to augment Wnt/β-catenin signaling  Ischemic retinal diseases, such as diabetic retinopathy or retinopathy of prematurity, are among the leading cause of blindness in developed countries During development, the formation of retinal capillaries depends on signaling pathways that differ between the different capillary beds of the retina. The initial outgrowth of capillaries on the retinal surfaces is largely driven by VEGF which is mainly released from retinal astrocytes in vitro and in vivo, an effect which is thought to involve the inhibition of the enzyme glycogen synthase kinase-3β (GSK-3β) which decreases the cytoplasmic β-catenin levels through phosphorylation-dependent proteasomal degradation Since the protective effects of Norrin in OIR required the activity of canonical Wnt/β-catenin signaling, we wondered if this pathway has the potential to be used for capillary regrowth and/or maintenance in the treatment of retinal ischemic diseases. A molecule that activates canonical Wnt/β-catenin signaling in the mouse brain upon oral administration ad libitum.All procedures in this study conformed to the Policies on the Use of Animals and Humans in Neuroscience Research of the Society for Neurosciences, and were approved by the local authority . All surgery was performed under isofluorane anesthesia, and all efforts were made to minimize suffering. Mice were housed under standard laboratory conditions  with food and water At postnatal day (P) 7, FVB/n mice including their mother were exposed to 75% oxygen for 5 days 2. For all experiments unsupplemented cell culture medium was used. Confluent HDMEC were incubated for 3 h with LiCl  or SB 216763  in unsupplemented cell culture medium and subjected to western blot analyses or immunocytochemistry (see below).Human dermal microvascular endothelial cells (HDMEC) were purchased from Promocell  and cultured in supplemented Microvascular Endothelial Cell Growth Medium (Promocell) containing penicillin (100 U/ml) and streptomycin (100 g/ml) at 37°C and 5% CO2) per length of the scratch (µm) by using the Axiovision software 4.8.2 (Carl Zeiss).Cell proliferation assays were performed by BrdU labeling of dividing cells according to the manufacturer’s instructions (Roche). In brief, 4000 HDMEC were seeded in 96-well tissue culture plates, allowed to attach overnight and incubated with an unsupplemented cell culture medium that contained BrdU (10 µM) and LiCl  with or without quercetin , or BrdU (10 µM) and SB 216763 . In addition, control cells were treated with 0.2 or 1 mM NaCl, 50 ng/ml VEGF  or DMSO. After 24 h, cells were fixed and BrdU incorporation was measured by ELISA at 450 nm . Cell viability was investigated by measuring the NADH- and NADPH-dependent turnover of WST-1 dye according to the manufacturer’s instructions (Roche). In brief, 4000 HDMEC were seeded in 96-well tissue culture plates, allowed to attach overnight and incubated in unsupplemented cell culture medium that contained LiCl  or SB 216763 . Again, additional control cells were treated with 0.2 or 1 mM NaCl, 50 ng/ml VEGF or DMSO. After 72 h, the medium was removed and unsupplemented cell culture medium containing WST-1 was added for 30 min. Extinction was detected at 450 nm on an ELISA reader (Tecan). Cell migration was quantified by standard scratch assays as described previously VEGF ELISA was purchased from Biorbyt  and was performed in accordance with the manufacturer’s instructions. In brief, 10.000 HDMEC were cultured on a 96-well cell culture dish, allowed to attach, starved overnight in unsupplemented cell culture medium and incubated in unsupplemented cell culture medium with 0.2 mM NaCl or 0.2 mM LiCl. After 24 h, 100 µl of conditioned cell culture medium were subjected to VEGF ELISA that was measured by an ELISA plate reader at 450 nm (Tecan).For immunocytochemistry, HDMEC were fixed with methanol for 5 min 3 h after treatment with 1 mM LiCl or 10 µM SB 216763 in unsupplemented cell culture medium. For immunohistochemistry, LiCl-treated FVB/n mice were perfused with 4% PFA. Enucleated eyes were fixed in 4% PFA for 2 h, embedded in Tissue Tek (Sakura Finetek) according to standard protocols and subjected to cryosectioning. Prior to incubation with rabbit anti-β-catenin antibodies  overnight at 4°C, samples were blocked for 45 min with 0.1 M phosphate buffer containing 3% bovine serum albumin (BSA) and 0,1% Triton X-100. After three washes (10 min each) with 0.1 M phosphate buffer, samples were treated with biotin-labeled goat anti-rabbit antibodies  for 1 h. Following three additional washes, cells were incubated for 1 h with streptavidin-Alexa 488 Fluor conjugates . Finally, specimens were washed again three times and mounted in fluorescent mounting medium containing 1∶10 DAPI (Vector Laboratories). Immunochemical staining was analyzed in the ApoTome mode of an Axiovision fluorescent microscope (Carl Zeiss).For western blot analyses of β-catenin levels, total protein was extracted using RIPA buffer. Homogenized mouse retinae or HDMEC were dissolved in RIPA buffer, and cellular debris was removed by centrifugation. Protein concentration was measured by the BCA (Interchim) method and up to 25 µg of total proteins were subjected to a 10% SDS-PAGE. Proteins were subsequently transferred on a PVDF membrane (Roche) by semidry blotting. After blocking with 5% low fat milk in TBS-T, the PVDF membranes were incubated overnight at 4°C with rabbit anti-β-catenin antibodies , diluted 1∶1000, in TBS-T with 0.5% low fat milk. HRP-conjugated chicken anti-rabbit antibodies (Santa Cruz Biotechnology) were used as secondary antibodies at a 1∶2000 dilution in TBS-T with 0.5% low fat milk. Antibody labeling was visualized using the Immobilon HRP substrate (Millipore). For documentation a LAS 3000 Imager work station (Fujifilm) was used. HRP-conjugated anti-GAPDH antibodies (Rockland) were applied as loading control.All values are expressed as mean ± SEM. Comparisons between the mean variables of 2 groups were made by a two-tailed Student’s t-test. P-values less than 0.05 were considered to be statistically significant.To analyze if LiCl or SB 216763 which similarly block the activity of GSK-3β on β-catenin degradation in vitro, proliferation, survival and migration of HDMEC were analyzed. Proliferation of HDMEC was investigated by BrdU ELISA. In cells that were incubated with 0.2 mM or 1 mM NaCl for negative control no increase in BrdU uptake was observed , an approximate 2-fold increase in the levels of β-catenin was detected in retinal proteins compared with control eyes that received PBS only . We now Finally, to rule out the possibility that the lack of vessel regrowth after induction of an OIR and activation of retinal Wnt/β-catenin signaling by LiCl is due to potential side effects of LiCl, SB 216763 was injected into the vitreous cavity after oxygen treatment. First we analyzed by immunohistochemistry the potential of SB 216763 to activate Wnt/β-catenin signaling in the retina. Six hours after SB 216763 injection and termination of oxygen treatment a moderate increase of β-catenin staining was observed in the inner retina compared with DMSO-treated controls . Quite nin vivo following OIR in the mouse eye. Our conclusions rest upon (1) the capability of LiCl to activate β-catenin signaling in cultured HDMEC and in retinal cells in vivo; (2) the finding that LiCl induces proliferation, migration and survival in HDMEC and (3) the observation that a LiCl-mediated retinal activation of β-catenin signaling has no influence on vascular repair following OIR.We conclude that treatment with LiCl mediates angiogenic properties on cultured HDMEC via an activation of β-catenin signaling, but not in vitro are characteristically seen with agents or molecules that have potent angiogenic properties in vivo such as VEGF in vitroThe capability to induce proliferation, migration and survival of microvascular endothelial cells in vivo and in vitro findings of our study. First, we might have missed to administer LiCl or SB 216763 at the most potent concentration following i.p. or intravitreal injection. We regard this possibility as unlikely as we observed an accumulation of β-catenin in retinal proteins at amounts similar to those observed in a previous study following the intravitreal injection of Norrin There are several explanations for the discrepancy between the in vivo angiogenic activity of LiCl or SB 216763 induced via GSK-3β inhibition and β-catenin accumulation.Another possibility relates to the lack of specificity of LiCl or SB 216763. LiCl may act on neuronal signal transmission and neurotransmitter release through disturbing the homeostasis of sodium, magnesium and calcium in vivo. In previous work, we could show that inhibition of canonical Wnt/β-catenin signaling by treatment with dickkopf-1 attenuated the effects of Norrin on capillary repair after OIR indicating that this pathway is essential for capillary repair Finally, both inhibitors of GSK-3β enhanced β-catenin levels, but failed to induce vessel regrowth into vaso-obliterated areas whereas treatment with Norrin improved vascular repair. It is tempting to speculate that an accumulation of β-catenin and the increase in activity of canonical Wnt/β-catenin signaling is essential, but not sufficient to induce retinal capillary repair


During neuronal development, the neuroepithelial stem cells (NSCs) initially undergo proliferative divisions, later switching to neurogenic ones whereby one NSC and a post-mitotic neuron are generated. We recently showed that a member of the PRDM family of transcriptional regulators, PRDM4/SC1, recruits a type II protein arginine methyltransferase, PRMT5, to maintain the “stem-like” cellular state of the embryonic mouse cortical NSCs. However, little is known about the regulation of activity of this complex under proliferation- or differentiation-inducing growth conditions.In the present work I investigate the regulation of SC1/PRMT5-mediated methylation activity in PC12 cells treated with EGF or NGF. I present evidence that NGF down-regulates SC1/PRMT5 methyltransferase (MTase) activity and that the reduction in SC1/PRMT5 MTase activity occurs mainly in the nucleus. I suggest that high levels of SC1/PRMT5 activity are associated with the proliferative state of the cells. For growth factor treatment the serum content was lowered to 1% HS. EGF (PeproTech) was used at 20 ng/ml, NGF (PeproTech) - at 50 ng/ml. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 48 h post-transfection cells were treated with the indicated growth factors, lysed and processed for the histone methyltransferase activity (HMTase) as described 2.2PC12 cells were harvested in immunoprecipitation (IP) buffer  Nonidet P-40, 300 mM NaCl) supplemented with the protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktails 1 and 2 (Sigma), processed as previously described and collected on protein A/G beads (Santa Cruz) 2.3+ pH7.5, 10 mM KCl, 1.5 mM MgCl2, 0.5 DTT) supplemented with protease (Sigma) and phosphatase inhibitors (Sigma) and pelleted by centrifugation at 1000 rpm for 5 min at 4 °C. HBA was supplemented with NP40 to a final concentration of 0.5% and added to the pellet to lyse the cells for 10 minutes are 4 °C. Nuclei were pelleted at 3000 rpm for 2 min at 4 °C and the supernatants collected as the cytosolic fraction, aliquoted and stored at −80 °C. Pellets containing nuclei were washed with HBA without NP40, followed by resuspension in buffer C  supplemented with protease and phosphatase inhibitors. Nuclear proteins were extracted for 30 min on ice with regular vortexing. The suspension was centrifuged at 4 °C for 10 min and the supernatant containing nuclear proteins collected, aliquoted and stored at −80 °C or used for HMTase assays immediately.PC12 cells treated as specified were harvested and processed for cytoplasmic and nuclear fractionation. Cells were rinsed in cold PBS on ice, scraped off the plates, resuspended in Hypotonic Buffer A (HBA) (10 mM HEPES-K3) for 10 min on ice, centrifuged at 2000 rpm for 10 min at 4 °C, the supernatants discarded, pellets washed again in half the volume of TEB and centrifuged as before. Pellets were resuspended in 0.2 N HCl and extracted overnight at 4 °C. Extracts were centrifuged at 2000 rpm for 10 min at 4 °C and the supernatants used for SDS-PAGE.Histones were extracted from PC12 cells as follows. Cells were washed twice in cold PBS, lysed in Triton extraction buffer , 2 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% (v/v) NaN2.4Western blotting was performed in TBST buffer  Tween 20). For IP's and Western blotting the following antibodies were used: anti-myc , anti-H3 , anti-H4 , anti-H4R3me2s , anti-H4R3me2a , anti-TBP (Abcam 1:2000), anti-α-tubulin . The following antibodies were used for immunocytochemistry: anti-TuJ1 , anti-PRMT5 , anti-SC1/PRDM4 (a gift from Pilar Perez and Moses Chao (1:40) and our own (1:100) 2.55 cells/13 mm on glass cover slips. NSCs were cultured in DMEM supplemented with 40 ng/ml Neurotrophin-3 (NT-3) (Pepro Tech), 0.25% FCS, B27 supplement, sodium pyruvate and glutamine . Animal experiments were approved by the University College London local ethical committee and conformed to the UK Animals (Scientific Procedures) Act 1986. Project license number PPL 70/6697.Primary cortical neural stem cells were isolated from time mated mouse E10.5 embryos according to a published protocol 33.1rd panel from the top). Moreover, an in vitro HMTase assay using the immunoprecipitated proteins revealed an increase in the amount of H4R3me2s methylation consistent with the presence of PRMT5 in mycSC1 immunoprecipitated complex (rd panel) and an in vitro radioactive HMTase assay with exogenously added purified calf thymus histone mixture was performed We recently demonstrated that SC1 recruits the protein arginine methyltransferase, PRMT5, to mediate symmetric dimethylation of histone H4 on arginine 3 (H4R3me2s) 3.2Both SC1 and PRMT5 are found in the cytosol and in the nucleus NGF is known to increase asymmetrically dimethylated proteins 3.3The observations, that NGF induced a selective reduction of the nuclear SC1/PRMT5-mediated MTase activity in PC12 cells, prompted me to investigate the subcellular distribution of SC1 and PRMT5 in differentiating neurons from the developing mouse cortex. Cortical NSCs isolated from E10.5 mouse embryos were cultured in NT3-containing media for 3 days to induce neurogenesis. Early-born neurons were detected by immunostaining cells with antibodies against Tuj1. Subcellular localization of SC1 and PRMT5 in early-born neurons was determined by immunostaining cells with appropriate antibodies. Both proteins were detected in the nucleus and cytosol of neurons , suggest4Presented results demonstrate that SC1/PRMT5-mediated MTase activity is reduced by NGF, and sustained by EGF in PC12 cells. These observations are in line with the previously published report that NGF dampens PRMT5 activity in PC12 cells Our previous work identified PRMT5-mediated deposition of H4R3me2s modifications as a “signature” of proliferating embryonic cortical NSCs suggesting a role for PRMT5 in the control of NSC proliferation and “stemness” Another interesting observation is the selective, NGF-induced reduction of SC1/PRMT5-mediated MTase activity in the nucleus of PC12 cells. The decrease in the nuclear SC1/PRMT5 MTase activity is likely to result in a diminution of proteins symmetrically dimethylated by the complex within this compartment. Interestingly, histones may not be the only targets of NGF-dependent reduction of SC1/PRMT5-mediated protein methylation as no notable change in the overall H4R3me2s was observed in PC12 cells overexpressing SC1 and treated with NGF. Perhaps other nuclear proteins are also substrates of SC1/PRMT5-mediated symmetric arginine dimethylation down-regulated by NGF. This possibility is consistent with the previously published report demonstrating NGF-dependent increase in the amount of asymmetrically dimethylated proteins in the nucleus and our previous observations that asymmetric dimethylation of H4R3 is detected in the post-mitotic neurons of the developing cortex, but not in the proliferating NSCs where H4R3me2s prevails The selective down-regulation of the nuclear SC1/PRMT5-mediated MTase activity by NGF induces a localized, specific response of the cells to different growth factors, suggesting that some of the decisions between proliferation and differentiation are regulated by the magnitude of symmetric protein arginine dimethylation within the nuclear compartment. Importantly, PRMT5 has been shown to control ES cell pluripotency by methylating the cytosolic pool of histone H2A in the embryonic stem (ES) cells