Using a pathway-specific PCR array and Gene Ontology analysis, we identified that Wnt/β-catenin pathway was associated with the suppressive effect of SJC on HCC cell proliferation and cell cycle progression. SJC repressed transcription activity of Wnt/β-catenin pathway and reduced expression of β-catenin in GSK-3β-independent but promoter-specific transcription inhibition mechanism. The suppressive effect of SJC on β-catenin expression and its transcription activity was associated with Wilms’ tumor 1 (WT1) protein. WT1 was overexpressed in HCC tissues, and was negatively correlated to the overall survival of HCC patients. WT1 promoted proliferation and invasion of HCC cells, as well as β-catenin-dependent transcription activation of Wnt products, while knockdown of WT1 had the opposite effect. Docking experiment revealed that protocatechualdehyde (PCA) might be the active component of the herb. PCA suppressed transcription activity of Wnt/β-catenin pathway in WT1-dependent manner. Our study sheds light on the potential of PCA from commonly used anti-cancer Chinese herbal Medicine SJC as a lead compound targeting WT1 in the discovery of anti-HCC drugs.Candidates from Chinese herbal Medicine might be preferable in drug discovery as the abundant experiences of traditional use usually hint the clinical efficacy. In this study, we screened the anti-tumour effect of several commonly used Chinese herbal Medicines on human hepatocellular carcinoma cells (HCC). We identified that Salvia chinensia Benth.  exhibited prominent  Hepatocellular carcinoma is one of the most malignant human cancers that results in increasing annual cancer death, ranking the second highest mortality rate all over the world . AlthougSome of the Chinese herbal Medicines have been widely reported for their inhibitory effect on HCC at pre-clinical and clinical levels , 13. It SJC) was the most prominent suppressor of HCC growth. We systemically evaluated the in vitro and in vivo anti-HCC activity of SJC, and identified by disease-specific array that WT1-associated Wnt/β-catenin was involved in the pharmacological action of SJC. As a novel oncogene in HCC, the clinical significance and biological function of WT1 was determined in this study. In addition, prediction and validation of biologically active ingredient of SJC was performed using computational molecular docking combined with bioassay validation.In this study, we first of all screened several rarely studied Chinese herbal Medicines that have been extensively used in clinical treatment of HCC by Chinese Medicine practitioners. We identified that Salvia chinensia Benth.  had minimal effect on body weight, indicating no observational toxicity of the herb Figure . in vivot Figure . This wat Figure . This prsiveness , we measTo make a global understanding on the pharmacological effect of SJC, we introduced HCC-specific pathway PCR array. A series of genes were regulated by SJC in HCC cells, nine of which were down-regulated while six were induced Figure . EnrichmTo further understand the action and action of mechanism underlying regulation of Wnt/β-catenin pathways by SJC, we exposed HCC cells with SJC in the presence or absence of Wnt3a, a stimulator of canonical Wnt pathway. Treatment of SJC could significantly suppress the expression of Wnt pathway product genes Figure . The inhhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14323). WT1 was significantly overexpressed in HCC tissue in compared with non-tumour liver . Compounds with favourable pharmacokinetic properties (OB ≥ 33% and Caco2 ≥ 0.4 cm/s) were selected. Five compounds  were shortlisted. As β-sitosterol has wide distribution in a lot of natural plants and may not be a specific component of SJC, only four phenolic compounds were performed molecular docking analysis with particular structure of several proteins identified in the regulatory network of Figure To further understand the molecular basis of the anti-HCC action, we tried to explore the possible active components of SJC with combination of computational and experimental approaches. First, we searched for chemical ingredients SJC on Traditional Chinese Medicine Systems Pharmacology Database  in HCC. We screened and identified that SJC was one of most prominent herbs that possessed inhibition on HCC. SJC exhibited cytotoxicity in HCC cells, and suppressed the NX), Salvia chinensia Benth. , Fruit of Fiverleaf Akebia , Portulaca Grandiflora , Solanum nigrum , Radix Trichosanthis , Paris polyphylla  and Smilax china L. , were collected from the clinical centre of School of Chinese Medicine, The University of Hong Kong. All the herbs were authenticated by Dr. Feng Yibin. Species of individual herb were kept and voucher numbers were given. To obtain the aqueous extracts of Chinese herbal Medicines, minced herb was boiled twice with 10 times of water for 2 h, follow by freeze-dry of the extracts. The dry powder was kept in −20°C and reconstituted in appropriate vehicle before use. Doses used in this study were presented in equal weight of raw herb per millilitre of vehicle.Dried herbs of eight Chinese herbal Medicines, including Achyranthes bidentata Bl. . MHCC97L cells expressing luciferase reporter gene were kindly gifted by Prof. Man Kwan in Department of Surgery, The University of Hong Kong. All cells were maintained by Dulbecco's Modified Eagle's Medium  supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin in humidified condition with 5% COsiRNA against human β-catenin was purchased from Santa Cruz. pAd/WT1-IRES-nAmCyan was a gift from Edward McCabe (Addgene plasmid # 29756). shRNA against WT1 and β-catenin promoter reporter were from Genecopoeia (USA). Transfection was conducted with FuGene Transfection reagent . In brief, corresponding nucleotide was mixed with FuGene reagent in serum free medium  followed by 15 min incubation at room temperature. The mixture was then added to the cells and incubated for 24 to 72 h. Treatment was applied during the incubation if necessary.Protocol of animal experiment has been approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong (Ref: 3776-15). Orthotopic HCC implantation was performed on 6-week nude mice with surgical procedure reported by our previous study . In brie2 First Strand Kit . For PCR array, the pre-coated Liver Cancer RT2 Profiler kit  was used; for real-time PCR, cDNA was amplified in SYBR Green I reagent  with specific primer sets  under manufacturer's instruction. First strand cDNA was prepared with RTmer sets  for partFor analysis of cell cycle, synchronized cells were treated with SJC and fixed in 70% Ethanol, followed by propidium iodide  staining in dark for 40 min. For analysis of cell proliferation, SJC-treated cells were incubated with BrdU reagents for 1 hr. Staining was performed with FITC BrdU Flow Kit  according to manufacturer's instruction. Cell cycle and proliferation were then analysed on Canto II flow cytometer .Cells transfected with GLuc-ON containing promoter region of human β-catenin  were treated with SJC for 48 h. Cells were then lysed and the firefly luciferase activity was detected. The lysate was detected by adding 150 μg/mL luciferin  as substrate and measuring the signal intensity on IVIS imager.http://lsp.nwsuaf.edu.cn/). Only compounds which meet the criteria of oral availability (OB) ≥ 33% and predicted permeability on Caco2 cells (Caco2) ≥ 0.4 cm/s were shortlisted. Structure of the shortlisted compounds as well as protein PDB IDs were input into the on-line docking software systemDocks (http://systemsdock.unit.oist.jp/iddp/home/index/). Binding portfolio of compound-protein pairs was shown when the predicted active binding score was larger than default threshold (5.22 in the case).List of ingredients in SJC was extracted from on Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, Results were analysed using One-way ANOVA and expressed as mean ± SD.


Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates β-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling. The adrenal cortex undergoes functional zonation to generate an outer zona glomerulosa (ZG) and inner zona fasciculata (ZF), but how this is regulated at a molecular level is unclear. Here, the authors show that ZG differentiation is stimulated by WNT signalling and that PKA blocks WNT signalling to allow ZF differentiation and also prevents WNT-induced cancer development. Cyp11b2 have shown that the preferred pathway for adrenal renewal involves initial differentiation to ZG and subsequent lineage conversion to ZF, along centripetal cell migrationIn vivo and in vitro, β-catenin activation is also associated with upregulation of AT1R and CYP11B2, two key determinants of ZG identity6The adrenal cortex plays essential roles in body homeostasis through secretion of mineralocorticoids, essential for sodium and potassium homeostasis and glucocorticoids, which are involved in stress response, glucose homeostasis and immune suppression. The production of these two distinct classes of steroids is the result of functional adrenal cortex zonation in the perinatal period3Expression of MC2R, the receptor for ACTH and MRAP, an accessory protein essential to trigger cAMP/PKA signalling pathway activation is higher in ZF than in ZGIn this paper, we postulated that reciprocally, cAMP/PKA signalling played a role in repressing WNT/β-catenin signalling to allow lineage conversion and functional adrenal cortex zonation. Indeed, by a combination of hormonal manipulations and genetic alterations, we show that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. We further demonstrate that PKA activation is able to restrain WNT-mediated malignant adrenal cortex tumorigenesis. These data suggest an all-encompassing role of PKA-dependent WNT signalling inhibition in adrenal homeostasis and disease.Akr1b7, an ACTH/cAMP/PKA responsive ZF differentiation markerLef1 and Axin2, two canonical WNT target genes, in ACTH-treated animals  to block endogenous ACTH secretion or dexamethasone for 3 days, followed by ACTH for 2 days to evaluate the effect of exogenous ACTH administration . As expected, the expression of s ANOVA, . Converse ANOVA, . In situt groups . Howevert groups . This wat groups . Altogett groups  and a cot groups . Altogets Prkar1a. Analysis Prkar1a. RT–qPCR Prkar1a . This wa , these in vitro findings were further confirmed by a significant increase in pT41/S45 β-catenin accumulation in the adrenals of Sf1:Cre;Prkar1aFl/Fl mice compared with their wild-type littermates  in knockout compared with wild-type adrenals  for 3 days. As expected, ACTH treatment resulted in robust repression of Axin2 and Lef1 mRNA accumulation in control adrenals  miceΔCat;Sf1:Cre mice). However, consistent with previously published dataΔCat;Sf1:Cre mice at one month of age, which precluded analysis of adrenal differentiation and proliferation  had significantly higher overall survival, when compared with patients with low StAR accumulation , APC or ZNRF3 (inactivating) and evaluated a potential negative association between StAR expression and WNT signature. Interestingly, WNT activation signature was inversely correlated with StAR . Altogether, these observations suggested that PKA activity could repress WNT-mediated tumour development in mouse models and in humans.To further confirm the repressive action of PKA on WNT signalling, we decided to reduce catalytic PKA activity to assess a potential overactivation of WNT signalling. ild-type , suggesttermates . This watermates  and a sitermates . Accelery ANOVA, . The core ANOVA, . This wae ANOVA, . These dgnalling . To furtAdrenal cortex renewal after recruitment of subcapsular progenitors preferentially occurs through lineage conversion from ZG to ZF cell identityMc2r knockout micePrkaca allele becomes available.One apparent contradiction to our model is the lack of ZG expansion in Sf1:Cre-mediated Prkar1a inactivation. This inhibition can even partially rescue deleterious WNT-induced developmental phenotypes. (2) Combination of PKA activation (through Prkar1a inactivation) with WNT pathway activation mediated by the Akr1b7:Cre driver, partially inhibits WNT-induced tumorigenesis. (3) Inactivation of one allele of the catalytic subunit of PKA markedly accelerates WNT-induced tumorigenesis. Although we cannot completely rule out that species-specific mechanisms may account for these effects, it is more likely that the kinetics of pathway alterations play an essential role in phenotypic outcome. Indeed, in most PKA-dependent tumours that were analysed, PKA activation resulted from germline mutations of PRKAR1A or GNAS. Although β-catenin was found activated in these tumours on the basis of immunohistochemical analyses, CTNNB1 somatic mutations were mostly found in larger tumours or macronodules that developed in the context of micronodular lesions36In the adrenal, β-catenin activation is associated with both benign and malignant tumorigenesis. In contrast, abnormal PKA signalling has only been described in benign tumours. Observation of abnormal β-catenin accumulation within a number of benign tumours associated with PKA pathway activation suggested that some of the oncogenic effects of PKA could be mediated by WNT pathway activation36373839Prkaca heterozygosity is intriguing. Indeed, mice combining both alterations develop tumours with malignant characteristics as early as 12 months. This is in striking contrast with the 16 to 19 months period required for β-catenin activation alone, to induce a similar phenotype with the same Cre driver6StAR expression may not completely reflect the degree of PKA activity, our data suggest that decreased PKA signalling is associated with more aggressive tumours that exhibit higher levels of WNT target genes expression in ACC patients. This is consistent with published data showing that LOH at the ACTH receptor was associated with more aggressive adrenal cortex carcinoma (ACC) in a small cohort of patientsThe marked acceleration in WNT-induced tumorigenesis in response to Axin2 expression  and decreased activating β-catenin phosphorylation (pS552), which results in decreased accumulation of the active ABC form of the protein. Whether increased T41/S45 is a direct effect of PKA on β-catenin in adrenocortical cells is unclear, even though experimental evidence suggests that these residues could be targets of the catalytic activity of PKA, both in reconstructed in vitro kinase assays and in cell culturein vivo, is that PKA indirectly triggers activating Y216 phosphorylation of GSK3β , which would result in increased T41/S45 phosphorylation of β-catenin. Beyond regulation of critical phosphorylations, our unbiased microarray analysis of WNT pathway regulators expression in Sf1:Cre;Prkar1aFl/Fl adrenals suggests that PKA may exert its inhibitory effect at multiple levels of WNT signalling pathway by deregulating expression of a number of potential repressors and activators. In this paper, we provide genetic evidence that inhibition of WNT4 expression in response to PKA signalling activation is one important aspect of this phenomenon. Indeed, Sf1:Cre;Wnt4Fl/Fl mutants display a differentiation phenotype reminiscent of Sf1:Cre;Prkar1aFl/Fl mutants and Lef1 repression by ACTH is almost abrogated in Wnt4 mutants. One conundrum of our study is the observation that PKA activation is still capable of inhibiting WNT signalling in ΔCat mice Sf1:Cre mT/mG Ctnnb1Fl(ex3) Prkar1aFl/Fl Prkaca+/− (constitutive heterozygous deletion of Prkaca)50, Wnt4Fl/Fl Ctnnb1Fl(ex3)/+ or homozygous Ctnnb1Fl(ex3)/Fl(ex3) mice. We did not observe any discernible difference between both genotypes, which is consistent with the dominant effect of the mutationPrkar1aFl/Fl;Sf1:Cre and ΔCat;Prkar1aFl/Fl;Sf1:Cre mice were analysed from 4 to 6 weeks. ΔCat;Prkar1aFl/Fl;Akr1b7:Cre and Wnt4Fl/Fl;Sf1:Cre mice were analysed at 6 months. ΔCat;Prkaca+/−;Akr1b7:Cre mice were analysed at 12 months. For hormonal manipulations, 6-month-old C57Bl/6 mice were injected subcutaneously with vehicle for 5 days (sesame oil twice daily), dexamethasone actetate for 5 days, which depletes endogenous ACTH production (75 μg twice daily in sesame oil) or dexamethasone actetate for 3 days and Synacthene Retard for 2 days . Sf1:cre Wnt4Fl/Fl mice and littermate controls were treated for 3 days with Synacthene Retard (1.2 U). For acute ACTH stimulation, wild-type mice were treated for 24 h with Synacthene Retard (1.2 U). At the end of the experimental procedures, the mice were killed by decapitation and blood was collected in vacuum blood collection tubes . The adrenals were either frozen in liquid nitrogen or fixed in 4% PFA. Total mRNAs were extracted using RNAII nucleotide extraction kit (Macherey Nagel) according to the manufacturer's instructions. Total proteins were extracted with RIPA buffer in the presence of protease  and phosphatase inhibitors .All animal studies were approved by Auvergne Ethics committee and were conducted in agreement with international standards for animal welfare. Immunohistochemistry for β-catenin , DAB2 , CYP11B2 , AKR1B7 , tissues were incubated for 12 h in X-Gal staining solution 6, 5 mM K4Fe(CN)6, 1 mg ml−1 X-gal) at 37 °C. After staining, the adrenals were rinsed in PBS and fixed in 4% PFA for 1 h. The tissues were then cryoprotected in sucrose 20% in PBS overnight at 4 °C and embedded in OCT. Ten micrometre sections were cut on a cryostat and deposited on Superfrost slides for observation.X-Gal staining was performed on whole adrenals as described in ref. VegfA and Connexinα43 that were amplified with Taqman chemistry as described in ref. ΔCt method after normalization to 36b4 (mouse) or PPIB (human).One microgram of total mRNAs (from tissues or cell culture) was reverse transcribed for 1 h at 37 °C with 5 pmol of random hexamers primers, 200 units reverse transcriptase , 2 mM dNTPs and 20 units RNAsin . One microlitre of a one-tenth dilution of cDNA was used in each qPCR. Except for g and steroids contained in the supernatant were extracted with 10 volumes of dichloromethane. After evaporation, the samples were resuspended in 150 μl of buffer and aldosterone concentration was measured with Aldosterone ELISA Kit .Whole adrenal glands were disrupted in 1X PBS with tungsten beads (Qiagen) using a Tissue Lyser (Qiagen). Adrenal extract was then centrifuged at 12,000L-Glutamine , 50 U ml−1 penicillin, 100 μg ml−1 streptomycin and 1X insulin transferrin selenium . COS7 cell line was grown with DMEM supplemented supplemented with 5% FBS , 2 mM L-Glutamine , 50 U ml−1 penicillin and 100 μg ml−1 streptomycin. For PKA stimulation treatments, the cells were seeded at a density of 3 × 105 cells per well in six-well plates. The day after seeding, the cells were deprived of serum and growth factors for 12 h. They were then treated with vehicle (dimethyl sulfate (DMSO)) or with Forskolin (10−5 M) for the indicated amount of time. For inhibition of PKA signalling, H295R cells were deprived of serum and growth factors for 12 h. They were then treated with vehicle (DMSO), 10 μM H89, 10−5 M Forskolin or a combination of 10 μM H89 and 10−5 M Forskolin for 8 h, following 2 h pre-incubation with H89. For transfection experiments, the cells were seeded at a density of 3 × 105 cells per well and transfected with 1 μg pCMV-WNT4 or empty vector (pCMV5) with Effectene reagent (Qiagen) according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were deprived of serum and growth factors for 12 h and treated as above. Proteins were extracted with RIPA buffer in the presence of protease  and phosphatase inhibitors . mRNAs were extracted by TriReagent (Molecular Research Center Inc.) according to the manufacturer's instructions. All the experiments were performed at least three times in triplicate.Human adrenocortical cancer H295R cell line (obtained from ATCC as a mycoplasma free cell line) was grown with DMEM/Ham's F12 supplemented with 10% FBS , 2 mM Ten to forty micrograms of total proteins were loaded on 8% SDS–PAGE gel, transferred onto nitrocellulose and detected with the following antibodies: phospho-β-catenin S552 , phospho-β-catenin S675 , phospho-β-catenin T41/S45 , phospho-β-catenin S33/37/T41 , active β-catenin ABC , β-catenin , phospho-GSK3 , GSK3β , phospho-GSK3β Y216  and GAPDH . The signals were quantified with a DNR MF ChemiBis 3.2 camera system and Multi Gauge software suite (Fujifilm). Expression of the phospho-proteins was normalized to expression of the corresponding total protein. Full western-blot pictures are provided as Sf1:Cre;Prkar1aFl/Fl and four wild-type littermates were analysed using Affymetrix Mouse Gene 2.0 ST Arrays (Raw and processed data are deposited on NCBI GEO platform). Gene expression was normalized by RMA (Affy R package) and genotype comparisons were performed with t-test. All the P values were adjusted by the Benjamini–Hochberg correction method. Volcano plot was generated with R and displays −Log10 as a function of Log2(fold change in knockout versus WT). Genes with adjusted P value <0.05 and Log 2 fold changes <−1 (blue dots) or >1 (red dots) were considered significantly down and upregulated, respectively. The WNT pathway targets and regulators list was curated from WNT Homepage (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/) and Pubmed searches. They are appended to this manuscript as P values <0.05 are shown in these representations.Adrenal gene expression profiles for four 6-week-old http://gdac.broadinstitute.org). These include 79 adrenal cortex carcinomas. All the calculations were performed on Log2 values of RSEM normalized read counts. Mutational status was retrieved from cBioPortal . Heatmaps were generated with R. WNT gene signature was determined as the geometric mean of the Log2 values of RSEM normalized read counts for WNT target genes LEF1, AXIN2 and APCDD1.Gene expression and clinical data from adrenocortical carcinoma patients were extracted from mRNA sequencing data available from Broad Institute GDAC Firehose .Supplementary Figures 1-9http://web.stanford.edu/group/nusselab/cgi-bin/wnt/) and Pubmed searches.WNT signalling pathway targets and regulators. A list of 127 canonical WNT signalling targets and regulators was curated from the WNT homepage (Genes significantly down and up-regulated in Sf1:Cre; Prkar1a Fl/Fl adrenals.Sequences of the primers used for RTqPCR


Here we report that microglia in the dentate gyrus (DG) of cx3cr1−/− mice displayed elevated microglial sirtuin 1 (SIRT1) expression levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 activation, despite unaltered morphology when compared to cx3cr1+/− or cx3cr1+/+ controls. This phenotype was restricted to the DG and accompanied by reduced adult neurogenesis in cx3cr1−/− mice. Remarkably, adult neurogenesis was not affected by the lack of the CX3CR1-ligand, fractalkine (CX3CL1). Mechanistically, pharmacological activation of SIRT1 improved adult neurogenesis in the DG together with an enhanced performance of cx3cr1−/− mice in a hippocampus-dependent learning and memory task. The reverse condition was induced when SIRT1 was inhibited in cx3cr1−/− mice, causing reduced adult neurogenesis and lowered hippocampal cognitive abilities. In conclusion, our data indicate that deletion of CX3CR1 from microglia under resting conditions modifies brain areas with elevated cellular turnover independent of CX3CL1.Homo and heterozygote  Microglial cells are fundamentally distinct from other brain cells in that they are derived from primitive myeloid progenitors that arise during embryogenesis [3CR1 ligand CX3CL1 . CX3CL1 is expressed at the cell membrane of selected neurons and binds to and activates CX3CR1 receptors on microglia [3CL1 exists in two distinct forms: a full-length membrane-bound form and a shed form that contains the N-terminal chemokine domain. The shed chemokine domain of CX3CL1 acts, when cleaved, as a signaling molecule and can bind microglial-expressed CX3CR1 receptors [3CL1 is abundantly expressed in the granular cell layer of the rat dentate gyrus (DG), where addition of recombinant CX3CL1 reverses age-related decline in adult neurogenesis [cx3cr1−/− and cx3cr1+/− mice display reduced hippocampal neurogenesis compared with wild-type controls [3CL1 is mandatory for proliferation and neurogenesis. In addition to the already mentioned reduced hippocampal neurogenesis, cx3cr1−/− mice were reported to exhibit excessive hippocampal IL-1β expression and either enhanced [3CR1 deficiency, which might impact synaptic plasticity and cognition. One of these pathways could include the NF-kB signaling pathway, which may trigger microglial activation and induce the release of inflammatory factors including IL-1β, as seen following irradiation [3CR1 because it can suppress inflammatory responses by inhibiting NF-kB signaling [CXicroglia . Surprisogenesis . They reogenesis . Microglogenesis , 8. Neuricroglia , 10. CX3eceptors , whereaseceptors . CX3CL1 ogenesis . Both, ccontrols , 14. Howenhanced  or attenenhanced  or impaienhanced , 14. It adiation , during adiation  or neuroadiation . Along tignaling , 20.3CR1 deletion on cell morphology, activation of the NF-kB signaling pathway, the expression of SIRT1, interference with neuroblasts, immature neurons and cognition. Our findings indicate that, under brain homeostasis, hippocampal microglia from cx3cr1−/− mice are very similar, if not identical, to microglia from adult wild-type animals in contrast to the situation in newborns or during development. However, hippocampal cx3cr1−/− microglia show activation of SIRT1 and the NF-kB pathway in areas of adult neurogenesis. We found that manipulation of SIRT1 activation in cx3cr1−/− mice directly impacts cognitive performance, while the same treatment had no detectable effect on cognition in wild-type littermates.Here we investigated the consequences of the microglial CXThis article does not contain any studies with human participants performed by any of the authors.cx3cr1gfp/gfp on a C57BL/6 J background were obtained from the Jackson Laboratory. cx3cl1−/− mice were described previously [For all animal experiments “Principles of laboratory animal care”  were followed. All experiments were approved by the Federal Ministry for Nature, Environment and Consumers’ Protection of the states of Baden-Württemberg (G12/71 and G11/50), and were carried out in accordance to the respective national, federal, and institutional regulations. Adult, 8–12 weeks old, male mice were used for the experiments. Mice were group housed up to five per cage with 12 h light/dark cycle with lights on at 6 a.m. Food and water were available ad libitum. eviously . In all th day, the platform was removed, and the mice were allowed to swim for 60 s to determine their search bias. On testing day 8, mice were trained to find a visible platform, which had a 10 cm high pole with a white flag and was changed every trial to a new position. Timing of the latency to find the visible platform was started and ended by the experimenter. A computer running the BIOBSERVE software (BIOBSERVE) analyzed all variables of the MWM test. All behavioral experiments were carried out in a double-blind fashion and mice were tested in random order.The MWM was used to measure spatial learning and memory . The appEX527 (cat. no. 2780) was purchased from Tocris Bioscience , dissolved in 4 % DMSO/10 % cyclodextrin in PBS and administered intraperitoneally  at a concentration of 250 ng/μL once daily for 21 days . ResveraAdult mice received two intraperitoneal (i.p.) BrdU-injections  over 3 days . AnimalsHistology was performed as described previously , 27. Micth section was selected. Every positive cell was counted on these sections, and, to obtain the relative total number of cells in the dentate gyrus, these counts were multiplied by 12 to account for the sampling frequency [Cells were counted using the optical fractionator, a method for unbiased stereological analysis. This method was performed using a computer-assisted image analysis system, consisting of a Leica DMRB/DIC fluorescence microscope equipped with a computer-controlled motorized stage, a video camera, and the Stereo Investigator software . The number of positive cells throughout the rostrocaudal extent of the dentate gyrus was counted with a coded one-in-16 series for frozen sections (40 μm). We used a modified version of the optical fractionator method as reported previously –37. The requency . In cont40-μm coronal cryo sections from adult brain tissue were stained with anti-Iba-1   for 48 hMicrodissection of microglia was performed using a Zeiss PALM MicroBeam as described previously, with modification . Fast imAdult microglia cells were harvested from dissected hippocampal tissue using density gradient separation and were prepared as described before . In shorFACS-sorted microglial cell populations were collected directly in cell lysis buffer and subsequently RNA was isolated with the Arcturus Pico Pure RNA Isolation Kit (Life Technologies) according to the manufacturer’s protocol. Reverse transcription and real-time PCR analysis were performed using high capacity RNA-to-cDNA-Kit and Gene Expression Master Mix reagents (Applied Biosystems) according to the manufacturer’s recommendations. RT-PCRs were analyzed with a LightCycler 480 (Roche) and carried out as described previously , 38. The2 × 250 μm3 spatial resolution with a cryogenic cooled resonator (CCR) at ultrahigh field (7 T) as described previously [Mice underwent diffusion tensor imaging (DTI) examination at 156eviously , 40. DifTo quantify sirtuin 1 (Sirt1) activity, nuclear extracts from sorted microglia (pooled from hippocampi of three mice per group) were prepared. Nuclear extracts were used to measure deacetylase activity of an acetylated histone using Epigenase Universal SIRT Activity/Inhibition Assay Kit  .t test was applied. If the data did not meet the criteria of normality, the Mann–Whitney U test was applied. To test for effects of treatment or genotype a two-factorial analysis of variance (ANOVA) with post hoc Bonferroni test or Tukey-Kramer HSD test was applied. Differences were considered significant when p < 0.05. Number of animals per group is given as “n”. To obtain unbiased data, experimental mice were all processed together by technicians and cell quantifications were performed blinded by two scientists independently and separately.Statistical analysis was performed using GraphPad Prism . In general, chosen sample sizes are similar to those reported in previous publications . All dat+ cells present in the subgranular zone (SGZ) of the DG at two months of age as detected by immunofluorescent stainings. In cx3cr1−/− mice, the number of DCX+ cells was reduced when compared to cx3cr1+/+ littermates  . The rattes Fig. , c. The ice Fig. , b. To deuN Fig. . Again, tes Fig. , resulti DG Fig. . The redMRI Fig. . Interesice Fig. -h, indicnts Fig. -n. We coice Fig. , b. In apes Fig. , d.Fig. 3CR1 is a Gi-protein coupled receptor, which inhibits cAMP signaling including the cAMP-dependent protein kinase A (PKA) [cx3cr1−/− and cx3cr1+/+ mice  from cx3cr1−/− mice in the DG, with a gradual decrease in SIRT1 signal strength, when microglia from cx3cr1+/− (GFP+) and cx3cr1+/+ mice (CD11b+) were analyzed  in cx3cr1−/− microglia within the DG  . All mam A (PKA) . The pho A (PKA) . Thus, wice Fig. . RT-PCR ice Fig. . When hictivator , the acpof SIRT1   via coupling to a Gi-protein coupled receptor [3CR1 from microglia might facilitate activation of PKA and subsequently NF-kB activation [cx3cr1−/− microglia, SIRT1 activity is amplified, most likely to counteract excessive NF-kB signaling. Activation of SIRT1 can induce deacetylation of the RelA/p65 component of the NF-kB complex. The deacetylation of Lys310 inhibits the transactivation capacity of RelA/p65 subunit and consequently suppresses the transcription of the NF-kB-dependent gene expression [cx3cr1−/− microglia SIRT1 activity, although elevated, appears to be insufficient to prevent NF-kB-dependent gene expression [cx3cr1−/− mice  or by members of the disintegrin and metalloproteinase (ADAM) family, ADAM-10 and ADAM-17 [3CL1 on neurons and CX3CR1 on microglia suggests that neurons may maintain microglia in a surveilling/ramified state through a repressive CX3CL1 signal [3CL1-deficient mice while the same process is impaired in mice lacking CX3CR1. If binding of CX3CL1 elicits a tonic inhibitory signal, which maintains microglia in a quiescent state, its absence should result in activated or pro-inflammatory microglia with negative impact on neurogenesis and neurodevelopment. While there is a consensus among studies for a neuroprotective role of CX3CL1 signaling in vitro, some in vivo studies even suggest a neurotoxic role of CX3CL1 as seen in animal models for Alzheimer’s- and Parkinson’s disease. Here, CX3CL1 can act as a repressor of microglial phagocytic activity and cause overall microglia activation [3CL1. This leads to the endothelial attachment of the subset mononuclear leukocytes that express the sole CX3CL1 receptor, CX3CR1 [cx3cl1−/− mice exhibit reduced expression of CX3CR1 and impaired NF-kB signaling in lung tissue when compared to wt controls [3CL1 is a molecule that may have various activities, with either no, beneficial or destructive potential, likely depending on the activation state of its main target cells. In support of our findings cx3cl1−/− mice do not have histologic abnormalities in any major organs (including the brain), hematopoietic lineages in blood and lymphoid tissue are essentially normal, and they do not exhibit any overt behavioral abnormalities [3CR1 ligand, which might act similar to CX3CL1. In humans eotaxin-3/CC chemokine ligand 26 was recently reported to be a functional ligand for CX3CR1 [CCL26 gene, however, may be a pseudogene since no cDNA or expressed sequence tag (EST) has been reported [3CR1 [cx3cr1−/− mice have improved hippocampal cognitive abilities compared to wild-type controls [We hypothesize that the NF-kB signaling pathway and SIRT1 enzyme in microglia interact to maintain cellular homeostasis in vivo. Since the fractalkine receptor CXreceptor , deletiotivation . Stimulipression . Howeverpression  as indicpression  or by inice Fig. . Only ad−/− mice . A simil−/− mice . Notablyeriments . During −/− mice . Howeverthe soma . While t ADAM-17 . The ana1 signal . From thtivation , 58. A f, CX3CR1 . Followicontrols . CX3CL1 malities . There ir CX3CR1 ; in micereported . Similared [3CR1 . These med [3CR1 . There acontrols , 54 withcontrols  generaticx3cr1−/− mice the number of DCX+ cells is reduced, independent of the CX3CR1 ligand CX3CL1. Enhanced microglial NF-kB-dependent gene expression in the DG results in elevated levels of chemokines such as IL-1β and consequently in the inhibition of neurogenesis and spatial cognitive function. Manipulation of SIRT1 activity interferes with NF-kB signaling, adult neurogenesis and ultimately hippocampal learning and memory.Our findings indicate that in the SGZ of the DG area in


All therapies were provided intravenously at 3h after AIS procedure. BIZ determined by histopathology (by day-60) and brain MRI (by day-28) were highest in group 2, lowest in group 1, higher in groups 3 and 4 than in group 5, but they showed no difference between groups 3 and 4 . By day-28, sensorimotor functional results exhibited an opposite pattern to BIZ among the five groups (p < 0.005). Protein expressions of inflammatory , oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/ Poly-ADP-ribose polymerase), and fibrotic (Smad3/transforming growth factor-β) biomarkers, and cellular expressions of brain-damaged (γ-H2AX+/ XRCC1-CD90+/p53BP1-CD90+), inflammatory  and brain-edema (aquaporin-4+) markers showed a similar pattern of BIZ among the groups . In conclusion, xenogenic ADMSC/ADMSC-derived exosome therapy was safe and offered the additional benefit of reducing BIZ and improving neurological function in rat AIS.We tested the hypothesis that combined xenogenic (from mini-pig) adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy could reduce brain-infarct zone (BIZ) and enhance neurological recovery in rat after acute ischemic stroke (AIS) induced by 50-min left middle cerebral artery occlusion. Adult-male Sprague-Dawley rats ( Acute ischemic stroke (AIS) is the leading cause of long-term disability and the second cause of death worldwide –4. CurrePlentiful data have demonstrated that stem cell therapies were effective and promising for many diseases refractory to traditional management strategies –21. Of tAs expected, H&E microscopy revealed no abnormal histopathological findings in these five organs. IF microscopy also did not illustrate any ADMS/ADMSC-derived exosomes present in these organs. Our findings highlighted that exogenic AMDMS/ADMSC-derived exosomes administered to rodent animals should maintain immune privilege without immune reaction and without harm to the healthy animals.By day 3 after AIS, the brain MRI findings exhibited that the ratio of left brain volume (LBV) to total brain volume (TBV) was significantly higher in AIS only, than in SC, AIS-ADMSC, AIS-Ex and AIS-ADMSC-Ex, and significantly higher in groups AIS-ADMSC, AIS-Ex and AIS-ADMSC-Ex than in SC, but no difference was found among AIS-ADMSC, AIS-Ex and AIS-ADMSC-Ex. Additionally, by day 28 after AIS, the ratio of LBV/TBV was significantly lower in AIS only than in AIS-ADMSC, AIS-Ex and AIS-ADMSC-Ex, and significantly lower in AIS-ADMSC and AIS-Ex than in SC and AIS-ADMSC-Ex, but it showed no difference between AIS-ADMSC and AIS-Ex or between SC and AIS-ADMSC-Ex  and the ratio of LIV/TBV were lowest in SC and highest in AIS only, and significantly lower in AIS-ADMSC-Ex than in AIS-ADMSC and AIS-Ex, but they showed no difference between AIS-ADMSC and AIS-Ex Figure . AdditioBy day 60, the left brain infarct area  showed an identical pattern of LIV by 28 after AIS among the five groups Figure . ImportaCorner test Figure  demonstrThe protein expressions of MMP-9, IL-1β, TNFα, RANTES, PAI-1, NF-κB and iNOS, seven indicators of inflammation, were highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC. This identical pattern was found among the five groups for protein expression of NOX-1, NOX-2 and oxidized protein, three indicators of oxidative stress.The protein expressions of cleaved caspase 3 and cleaved PARP, two indicators of apoptosis, were highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC. The protein expressions of Smad3 and TGF-β, two indices of fibrosis, exhibited an identical pattern of apoptosis among the five groups. The protein expressions of Smad1/5 and BMP-2, two indicators of anti-fibrosis, displayed an opposite pattern of apoptosis among the five groups.The protein expressions of γ-H2AX, a DNA-damage marker, and cytosolic cytochrome C, a mitochondrial-damage marker, was highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC. On the other hand, protein expression of mitochondrial cytochrome C, an indicator of mitochondrial integrity, showed an opposite pattern to γ-H2AX among the five groups. Additionally, the protein expressions of CD31 and eNOS, two indicators of endothelial function, exhibited an identical pattern to mitochondrial cytochrome C among the five groups. Furthermore, the protein expressions of VEGF and CXCR4, two indices of angiogenesis, exhibited significantly progressive increases from groups SC to AIS-ADMSC-Ex, suggesting an intrinsic response to ischemic stimulation.The IF microscopic findings demonstrated that the cellular expressions Figure  of CD11 The cellular expressions of CD31 and vWF Figure , two indThe number of small vessels in BIZ, an indicator of angiogenesis, was highest in SC, lowest in AIS only, significantly higher in AIS-ADMSC-Ex than in AIS-ADMSC and AIS-Ex, and significantly higher in AIS-ADMSC than in AIS-Ex. The γ-H2AX+ cell expression, another DNA-damage biomarker, was highest in AIS only, lowest in SC, significantly higher in AIS-ADMSC and AIS-Ex than in AIS-ADMSC-Ex, and significantly higher in AIS-Ex than in AIS-ADMSC.Our recent study showed that allogenic ADMSC/ADMSC-derived exosome therapy significantly protected kidney architecture, glomerular ultrastructure and renal function from acute kidney ischemia-reperfusion injury in rodent . In addiIt has long been known that innate immune reactions are quickly activated following tissue/organ ischemic injury and necrosis, which, in turn, elicit the complement cascade, inflammation and the generation of oxidative stress and reactive oxygen species (ROS) , 32–35. A strong correlation between inflammation/oxidative stress and cellular apoptosis/death, fibrosis of organ and tissues, as well as DNA/mitochondria damage in the setting of ischemic injury has been keenly investigated by previous studies , 37–39. Another important finding in the present study was that the angiogenesis biomarkers  were significantly higher in AIS animals with xenogenic ADMSC/ADMSC-derived exosome treatment than in those without. Intriguingly, our recent studies , 39 haveThis study has limitations. First, this study only considered the management of AIS and therefore did not provide any information regarding the impact of xenogenic ADMSC/ADMSC-derived exosome therapy on chronic IS. Second, this study did not test the optimal dosage of ADMSCs or ADMSC-derived exosomes on improving the neurological outcome. Accordingly, we remain uncertain whether ADMSC therapy or ADMSC-derived exosome therapy was superior at improving the outcomes in rat after AIS, when both dosage regimens have been optimized.In conclusion, the present study provided evidence that xenogenic cell-based therapy was safe and efficacious for improving outcomes in rat after AIS.All animal experimental protocols and procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital  and performed in accordance with the Guide for the Care and Use of Laboratory Animals [The Eighth Edition of the Guide for the Care and Use of Laboratory Animals (NRC 2011)].Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved animal facility in our hospital with controlled temperature and light cycles (24°C and 12/12 light cycle).®) with food and water for 24 hours.The protocol and procedure for the experimental rodent AIS model have previously been described , 36. In The sensorimotor functional test (Corner test) was conducted for each rat at baseline and on days 1, 3, 14 and 28 after AIS induction as we recently described , 36. Bri6 cells) by intravenous administration at 3 h after AIS procedure], AIS + exosomes [(AIS-Ex) by intravenous administration of 100 μg at 3 h after AIS procedure] and AIS-Ex-ADMSC. The utilized dosages of Ex and ADMSC were based on our recent investigations [Sixty rats were equally categorized into sham control (SC) (by incision of the neck skin and dissection of the LCCA only), AIS only, AIS + ADMSC [ would be harmful to the normal organs/tissues, an additional eight healthy animals (4 per group) were utilized to receive these two different therapies intravenously. By day 21, all animals were euthanized and specimens were collected. Microscopic investigations  study were based on our recent report . MRI wasn = 6 for each group) was promptly removed, immersed in cold saline, snap-frozen in liquid nitrogen and then stored at −80°C for individual study. For immunofluorescent (IF) and immunohistochemical (IHC) staining studies, the brains of 6 other animals in each group were reperfused with normal saline via the carotid artery, removed, fixed with 4% paraformaldehyde in 1x PBS (pH7.4), and soaked in 20% sucrose in 1x PBS (freshly prepared) until the brain took on a completely sunken appearance. The sucrose was then discarded and the brain soaked in 30% sucrose in 1x PBS (freshly prepared) for 48 h. The infarcted and non-infarcted parts were then collected. Finally, the OCT block  was prepared for IHC and IF staining.Animals were euthanized by day 60 after the AIS procedure. For examination of protein expression, the brain of each rat .The procedure and protocol of IF and IHC staining were based on our previous reports –38. In dThree brain sections were analyzed for each rat. For quantification, three randomly selected high-power fields  were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.The procedure and protocol for identifying small vessels in the BIZ were based on our previous reports , 36–38. The procedure and protocol for Western blot analysis were based on our recent reports , 32, 36.The procedure and protocol for evaluating the protein expression of oxidative stress have been described in detail in our previous reports , 32, 36.Quantitative data are expressed as means ± SD. Statistical analysis was adequately performed by ANOVA, followed by Bonferroni multiple-comparison post hoc test. SAS statistical software for Windows version 8.2  was utilized. A P value of less than 0.05 was considered statistically significant.


Of note, despite the abundant expression of CFTR in mammalian kidney, the role of CFTR in kidney disease development is unclear. Here, we report that CFTR expression is downregulated in the UUO -induced kidney fibrosis mouse model and human fibrotic kidneys. Dysfunction or downregulation of CFTR in renal epithelial cells leads to alteration of genes involved in Epithelial-Mesenchymal Transition (EMT) and kidney fibrosis. In addition, dysregulation of CFTR activates canonical Wnt/β-catenin signaling pathways, whereas the β-catenin inhibitor reverses the effects of CFTR downregulation on EMT marker. More interestingly, CFTR interacts with Dishevelled 2 (Dvl2), a key component of Wnt signaling, thereby suppressing the activation of β-catenin. Compared to wild type, deltaF508 mice with UUO treatment exhibit significantly higher β-catenin activity with aggregated kidney fibrogenesis, which is reduced by forced overexpression of CFTR. Taken together, our study reveals a novel mechanism by which CFTR regulates Wnt/β-catenin signaling pertinent to progression of kidney fibrosis and indicates a potential treatment target.Cystic fibrosis transmembrane conductance regulator (CFTR), known as a cAMP-activated Cl While the cellular origin of myofibroblasts remains to be a subject of intensive debate, numerous animal and clinical studies have pointed to epithelial-to-mesenchymal-transition (EMT) from the tubular epithelial cells as one of the major contributors leading to the generation of myofibroblasts and overproduction of interstitial ECM9. A multitude of biochemical signaling pathways has been proposed to be associated with the development of kidney fibrosis12. While Wnt/β-catenin signaling is functionally important in normal kidney development14, aberrant activation of Wnt/β-catenin signaling and subsequent downstream targets, such as matrix metalloproteinases (MMPs), fibronectin and snail, has been observed in the progression of tubulointerstitial kidney fibrosis in unilateral ureteral obstruction (UUO) model17 and different types of kidney diseases21. Of interest, β-catenin is predominantly up-regulated in renal tubular epithelium of the fibrotic kidneys, suggesting that tubular cells are the major targets of canonical Wnt signaling. In addition, activation of β-catenin in tubular epithelial cells induces EMT, as well as the expression of several fibrosis-related genes in vitro24. These results suggest that activation of tubular β-catenin probably plays a critical role in the pathogenesis and progression of kidney fibrosis16. However, it remains unclear how the Wnt/β-catenin signaling pathway is over-activated in fibrotic kidney, and how this signal initiated from the tubular epithelial cells is converted into pathological changes in the tubulo-interstitial space leading to excessive deposition of ECM and fibrogenesis.Kidney fibrosis is a common histological manifestation of functional decline in most cases of end-stage kidney diseases. Fibrosis is a reactive process that develops in response to excessive epithelial injury and inflammation, leading to myofibroblast activation and an accumulation of extracellular matrix (ECM)− channel, is widely expressed at the apical membrane of epithelial cells in a wide variety of tissues26. Dysfunction of CFTR causes cystic fibrosis (CF), the most common lethal autosomal recessive disease in Caucasians27. Of note, despite the abundant expression of CFTR in mammalian kidney which is localized at the apical surface of proximal tubules and distal tubules28, no overwhelming primary renal disease has been associated with CF. Nevertheless, as CF patients age and have an increased exposure to potentially nephrotoxic agents, such as antibiotics and high glucose, development of chronic renal disease has been commonly recognized30. Given that CF-related nephropathies are frequently compounded by increasing age, pulmonary infection, malnutrition, liver dysfunction and insulin insufficiency31, chronic kidney damage has been considered as secondary manifestation due to the abnormalities in salt transport, infection, and development of cystic fibrosis related diabetes (CFRD)32. One study involving 12 000 patients followed up for a median of 4 years reported an overall annual prevalence of stage 3 chronic kidney disease of 2.3%33. In addition, permanent microalbuminuria occurs in more than 6% of patients with cystic fibrosis30. Despite all these link, the exact role of CFTR in the pathogenesis of chronic kidney diseases has never been investigated.Cystic fibrosis transmembrane conductance regulator (CFTR), known as a cAMP-activated Cl34. In addition, both our group and other groups have reported that CFTR interacts with β-catenin in intestinal epithelial cells, dysregulation of which leads to aggregated inflammation and development of cancer36. On the other hand, TGF-β and hypoxia inducing factor α (HIFα), key inducers of kidney fibrosis, have been demonstrated to suppress the expression and/or localization of CFTR in the context of epithelial cells39. Based on these observations, we hypothesized that dysregulation of CFTR might lead to aberrant activation of Wnt/β-catenin pathway and development of kidney fibrosis. We undertook the present study to investigate this possibility using kidney epithelial cell lines, UUO mouse model and CFTR mutant mice in conjunction with genetic manipulation in vitro and in vivo.Interestingly, a recent comprehensive study on human lung epithelial cells has found that the CFTR interactome includes several hundred proteins, with β-catenin listing as a potential interacting protein2 at 37 °C, and seeded into 6 well plates at 5 × 104/well and incubated in growth medium supplemented with 2% FBS during treatment. CFTRinh-172  was used at the final concentration of 10 µM, whereas GlyH101  was used at 2.5 μM, 5 μM and 10 μM for 2 or 3 days as indicated. DMSO (0.1%) at the same volume was used as vehicle control. The effect of hypoxia was achieved by incubating cells in 1% O2–5% CO2 - balance N2 chamber for 1 and 2 days before further assay. Cells incubated under normoxia condition were used as control.Renal distal tubular Madin–Darby canine kidney (MDCK)  cells were cultured in α-MEM medium , supplemented with 10% Fetal Bovine Serum (FBS). Human proximal tubule (HK-2) cells were cultured in DMEM/F12 medium , supplemented with 10% FBS. Cells were maintained in 5% COTM 6.2-GW/EmGFP encoding a pre-miRNA sequence was constructed using BLOCK-iTTM Pol II miR RNAi Expression Vector kit (Life technologies). MDCK cells were seeded on 35 mm culture dishes and 3 μg of vectors were transiently transfected with 6 μl Lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction. The GFP signals were used to evaluate the transfection efficiency. 48 hours after transfection, cell lysates were collected for protein assay to confirm the transfection efficiency.To knockdown CFTR expression in MDCK cells, miRNAs duplex specific to canis familiaris CFTR was synthesized by Life Technologies . The siRNA sequence is as follows: 5′- ACT TGT TAC TCC TTT CCA A -3′. MiRNA against Lac-z was used as the control sequence. pcDNA40. For knockdown experiments, 3 μg DNA was transfected into HK-2 cells with 6 μl Lipofectamine 2000 (Invitrogen) and selected by 8 μg/ml Blasticidin S for 2–3 weeks. The stable transfectants were maintained in medium containing 4 μg/ml Blasticidin S.The ribozymes were generated and used for CFTR knockdown in HK-2 cells as described in our previous studyTotal RNA was isolated and reversely transcribed using standard protocols. For quantitative PCR, assays were performed in triplicate on an Applied Biosystems 7500 Fast Real-Time PCR System. The primers were listed in Supplementary Tables Protein was extracted using lysis buffer , 2 mM EDTA and protease inhibitor). Cell lysates was incubated with primary antibody: anti-CFTR  or anti-Dvl2 . After incubation, the complexes were precipitated using the protein A/G plus-agarose immunoprecipitation reagent, washed and eluted for Western blot assay.3000 cells/well in 200 μL growth medium were seeded into 96-well plate. Following the appropriate incubation period, medium was removed and 100 μL full medium with 20 μL MTS -5-(3carboxymethoxyohenyl)-2-(4-sulfop henyl)-2H-tetrazolium, Promega) reagent was added. The experimental plate was further incubated for 2 hours to allow the bio-reduction of metabolically active cells. The intensity of soluble product was measured by spectrophotometer at 490 nm. Three replicated wells were measured for each experimental group.TM-P; Amersham Biosciences, Piscataway, NJ). The transferred membrane was blocked with TBS containing 0.2% Tween-20 (TBST) and 5% non-fat dry milk, and incubated with primary antibodies as follow: anti-CFTR ; anti-HIF-1α ; EMT markers including anti-E-cadherin , anti-Occludin , anti-ZO-1 , anti-Vimentin , anti-N-cadherin  and anti-α-SMA , anti-Fibronectin , anti-Collagen1 ; Wnt/β-catenin signaling including Wnt signaling antibody sampler kit , Wnt/β-catenin activated targets antibody sampler kit , anti-β-catenin  at 4 °C overnight. Antibody against GAPDH , β-tubulin  or Histone H1 were used for protein loading controls. The membrane was subsequently washed with TBST and incubated for 1 h with peroxidase-conjugated secondary antibodies. The membrane was washed three times with TBST and then detected by enhanced chemiluminescence .Cell or tissue protein were extracted using lysis buffer  supplemented with protease inhibitors including phenylmethylsulfonyl fluoride and pimix. The samples were incubated with RIPA at room temperature for 30 minutes for CFTR detection, or boiled at 100 °C for 3 minutes for other protein detection. Total lysates of cells (20 µg per lane) or tissues (40 µg per lane) were then subjected to SDS–polyacrylamide gel electrophoresis and were transferred onto nitrocellulose membranes (HybondMDCK cells were seeded into the 0.4 mm pore size transwell (upper chamber) and allowed to reach full confluence, after which fresh medium was replaced for further experiments. TER of epithelial monolayer was measured using EVOM electrovoltohmeter  every two days following the standard procedure. Three replicated wells were used for measurement at each time point.5 cells/well in 6-well plate one day before creating a wound across the cell monolayer with a plastic tip. Closure of the wound via the migration of cells was tracked and recorded using a Time Lapse Imaging System (CarlZwiss). Five 100× views from each well were documented and the results were expressed as reduced areas of wound. The experimental procedure was repeated three independent times.Cells were seeded at 5 × 10The plasmids pcDNA3.1 and pcDNA3.1-CFTR were transiently co-transfected to HK-2 with TOPflash reporter gene (0.05 ug/ml)  by Lipofectamine 2000 (Invitrogen). Renilla-Luc (0.005 ug/ml) was included in all transfection assays as an internal control. Firefly and renilla luciferase activity was assessed with respective assay kit (Promega). The relative luciferase activity was defined as the ratio of readout for firefly luciferase to that for renilla luciferase with that of control group set as 1.0. Triplicate wells were used for measurement at each group. The experimental procedure was repeated three independent times.deltaF508 mouse model  generated on C57BL/6 background41 was used in the study. KAPA Mouse Genotyping Kits (KAPA Biosystem) are used for genotyping followed standard protocol. Briefly, DNA was extracted from 3–5 mm mouse tail by the KAPA Express Extract (a thermostable protease and buffer system). The supernatant was then diluted 10-fold prior to use as template in PCR reaction  were reduced in size compared to that of normal wild type mice (wt) and heterozygous (deltaF508 hetero). Mutant mice showed an increased mortality within the first month after birth (~60%), due to bowel obstructions, bowel strictures and peritonitis. Regardless of the survival rate, the animal lungs, pancreas, gall bladder, male reproductive tract, lacrimal gland and submandibular glands from deltaF508 mice appear normal.All mice were provided by the Laboratory Animal Service Center of the Chinese University of Hong Kong. All animal experiments were conducted in accordance with the University Laboratory Animals Service Center’s guidelines on animal experimentation with approval from the Animal Ethnics Committee of the University. CFTR n Figure . HomozygdeltaF508 heterozygous (hetero), deltaF508 homozygous (homo); male wild type and smad3 knockout mice  at 8-week-old. Six mice were used in each group. UUO model was established by left ureter ligation as previously described42. Mice were euthanized at Day 3 or 7, after the ligation. Kidney tissue samples were collected for histology, immunohistochemistry and real-time PCR.Unilateral ureteric obstruction (UUO) model was established in female wild type and CFTR The male C57 mice at 8-week-old were provided for the Ultrasound-mediated gene transfer within the kidney during fibrosis. The plasmid peGFP-C3 and peGFP-CFTR for injection were prepared using the EndoFree plasmid kit  according to the manufacturer’s instructions. Before injection, 200 μg of peGFP-C3 or peGFP-CFTR plasmid were mixed with sulphur hexafluoride microbubbles  at a ratio of 1:1 (v/v). The mixed solution (200 μl) was injected into tail vein. Ultrasound was performed immediately after injection with a continuous-wave output of 1 MHZ for 5 min on the back of left kidney location. Unilateral ureteric obstruction (UUO) model was established following the ultrasound treatment. A group of 9 or 10 mice were euthanized at Day 7, after the ligation. Kidney tissue samples were collected for histology, immunohistochemistry, western blot and real-time PCR.Changes in renal morphology were examined in paraformaldehyde fixed, paraffin-embedded tissue sections (5 μm) stained with haematoxylin and eosin or Modified Masson’s Trichrome Stain Kit (ScyTek Laboratories). Briefly, deparaffinized sections were incubated in Bouin’s Fluid overnight. After wash by tap water, slides were stained sequentially by working Weigert’s Iron Hematoxylin for 5 mins, Biebrich Scarlet/Acid Fuchsin solution for 30 s, and Aniline Blue Solution for 45 mins.43 were performed before incubation with primary antibody against CFTR or HIF-1α. Then secondary Alexa fluor 568 goat anti-mouse IgG, fluor 488 goat anti-rabbit IgG, fluor 488 donkey anti-goat IgG or fluor 568 donkey anti-rabbit IgG at a dilution of 1:500 was loaded on cells or tissues for 1 hour. Samples were all co-stained with Hoechst for nuclei detection. Eventually, the slides were mounted with ProLong Gold Antifade Reagent (Invitrogen) and visualized with fluorescence microscope (Nikon Intersilight C-HGF1) using 4×, 10×, 20× and 40× objectives.MDCK or HK-2 cells were seeded into 24-well plate with cover slips for immunofluorescent assay. After treatment, cells were fixed with 4% paraformaldehyde and blocked with 1% bovine serum albumin, and incubated overnight with primary antibodies at a dilution of 1:100 in 1% BSA against ZO-1, Vimentin, N-cadherin, CFTR  or Dvl2 at 4 °C. For paraffin embedded tissues, steps for rehydration and microwave-based antigen retrieval43. The primary antibodies used in this study include Collagen I and F4/80. All slides were further stained with haematoxylin for the nuclei. 15 to 20 views were taken randomly by the Nikon 20× and 40× objectives of every slide for the quantitative analysis. Area of Collagen I staining was quantitated by the image analysis software (Meta Imaging series 7.5) on 200× fields. Briefly, the area of the positive staining pattern in tubulointerstitium was identified. Then, the percentage of positive area in the examined view was measured. The number of macrophages with the positive staining of F4/80 was counted in 400× fields, and the numbers were shown as cells/mm2. All analysis data was expressed as the Mean ± S.E.M. from three animals at indicated time point for each group.Immunohistochemistry was also performed in paraffin sections following the similar antigen retrieval procedureAll morphometric and staining data were collected blindly. Statistical significance for comparison between two measurements was determined using the unpaired Two-tailed Student t-test. One-way or Two-way ANOVA was used for evaluation of the significance among three or more measurements. All statistical analysis was completed by Prism 5 . Values of p < 0.05 were regarded as statistically significant, and 0.01 < p < 0.05 was labeled as ‘*’, 0.001 < p < 0.01 was labeled as ‘**’ and p < 0.001 was labeled as ‘***’. All statistical data were shown as Mean ± S.E.M.To test this hypothesis, we first examined whether CFTR expression was altered during the development of renal interstitial fibrosis by using unilateral ureteral obstruction (UUO) mouse model. Our western blot result showed that the expression of CFTR was dramatically decreased in fibrotic kidneys compared to normal kidneys Fig. . Consist44, we investigated the influence of this pathophysiological important factor on the expression of CFTR in Madin-Darby canine kidney cell line MDCK and human tubular epithelial cell line HK-2, which are widely used as in vitro models to study EMT and renal dysfunction45. Our result showed that when subjected to hypoxia (1% O2), MDCK cells took on an elongated, fibroblastic morphology, which is reminiscent of a mesenchymal transition phenotype  mouse model. Intriguingly, we found that the UUO treatment-induced effect on HIF-1α and CFTR was similar in both Smad3 KO mice and WT mice  or GlyH101, and found marked morphological changes indicative of EMT in MDCK cells. CFTR inhibitor-treated cells had a greater predilection to break away as single cells and lose cell-cell contacts  mice, developed more intensive tubule-interstitial damage including tubular atrophy, inflammatory cell invasion and interstitial extracellular matrix accumulation as demonstrated by Masson trichrome staining, collagen I deposition and macrophage infiltration in the fibrotic area of tubule-interstitium  protein in the urine53. Originally, it was reported that most of the 19 Wnt proteins and 10 Fzd receptors were increased in renal tubular cells in the UUO model of kidney fibrosis15. Subsequent mechanistic studies further indicated that Wnt and β-catenin were not only regulated in chronic kidney diseases, but also contributed to fibrosis development15. For instance, genetic overexpression of active β-catenin in tubular cells induced fibrosis feature, including epithelial dedifferentiation and EMT in mice53. Most importantly, it has been shown recently that suppression of Wnt/β-catenin signaling, by either targeting upstream signaling factors or downstream targets ameliorates inflammation and tubulointerstitial fibrosis in mouse models55. In the present study, we have clearly shown that β-catenin pathway is upregulated in both CFTR- knockdown renal epithelial cells and UUO-treated kidney of deltaF508 mice . While we cannot entirely rule out other possibilities, these results suggest that the interaction between CFTR and Dvl via PDZ domain is likely to contribute to the effect of CFTR on β-catenin activity in kidney epithelial cells. As a result, disruption of the interaction due to loss of CFTR results in aberrant activation of the Wnt/β-catenin-dependent EMT, leading to renal fibrosis. Of note, CFTR has also been found to negatively regulate the expression and activation of β-catenin in colon crypt cells and lung epithelial cells58. Taken together, it appears that CFTR may exert differential effect on β-catenin signaling in different cellular contexts, probably via interacting with different components of the Wnt/β-catenin pathway.Over-activation of β-catenin pathway has been shown to be associated with the development of kidney fibrosis and chronic kidney diseasesice Figs  and 6a. ice Figs . These r44. As CFTR is expressed in a large variety of tissues, the presently observed hypoxia-induced CFTR downregulation provides a missing link between micro-environmental signaling i.e. hypoxia, and intracellular signaling pathways, i.e. Wnt/β-catenin, leading to EMT process during physiological and/or pathological conditions, such as fibrosis and cancer. On the other hand, it is very likely that dysfunction of CFTR itself augments the pathological process of kidney fibrosis in response to certain hostile environment, such as hypoxia and high glucose in CF patients. It should be noted that previous studies have established a link between TGF-β pathway and β-catenin pathway in the context of renal diseases59. For instance, Takenaka et al., showed that klotho protein suppressed epithelial-mesenchymal transition in Adriamycin-induced nephropathy by inhibiting TGF-β and Wnt signaling59. However, in our study, we clearly showed that TGF-β, one of the key mediators of tissue fibrosis did not affect CFTR expression. Moreover, UUO-induced downregulation of CFTR was also observed in Smad3 KO mice, excluding the involvement of TGF-β signaling since Smad3 is a key player of the TGF-β signaling60. These results indicate that the TGF-β pathway is dissociated with the CFTR-mediated regulatory pathway in the development of hypoxia-induced renal fibrosis. Indeed, while TGF-β1 inhibitors have been used in the clinic to treat chronic kidney diseases; the therapeutic effect is not significant63. This observation indicates other potential players might be involved. The presently observed downregulation of CFTR expression in human fibrotic kidneys and the suppression of kidney fibrosis progression in UUO mice upon forced CFTR overexpression indicate the possibility of using CFTR as a new or combined treatment target along with TGF-β in the fibrotic kidney diseases, which warrants future investigations.Decreases in tissue oxygen levels are commonly associated with injury and repairSupplementary materials


Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is a kinase principally known as a positive regulator of the ubiquitous transcription factor family Nuclear Factor-kappa B (NF-κB). In addition, IKKβ also phosphorylates a number of other proteins that regulate many cellular processes, from cell cycle to metabolism and differentiation. As a consequence, IKKβ affects cell physiology in a variety of ways and may promote or hamper tumoral transformation depending on hitherto unknown circumstances. In this article, we give an overview of the NF-κB-dependent and -independent functions of IKKβ. We also summarize the current knowledge about the relationship of IKKβ with cellular transformation and cancer, obtained mainly through the study of animal models with cell type-specific modifications in IKKβ expression or activity. Finally, we describe the most relevant data about IKKβ implication in cancer obtained from the analysis of the human tumoral samples gathered in The Cancer Genome Atlas (TCGA) and the Catalogue of Somatic Mutations in Cancer (COSMIC). IKBKB, which encodes a Ser/Thr kinase named Inhibitor of nuclear factor kappa-B kinase subunit beta, which is usually abbreviated in the literature as IKK2, IKKb or IKKβ. IKKβ is a protein able to phosphorylate a number of substrates not completely known yet  [ [2]). Soiew, see ). In theIKKβ was first identified as part, along with the kinase IKKα and the regulatory subunit NF-κB essential modulator (NEMO)/IKKγ, of the IKK complex, a master regulator of the ubiquitous family of dimeric transcription factors NF-κB . As theIκB (inhibitor of κB) is a family of inhibitory proteins of the NF-κB pathway that sequester NK-κB dimers in the cytoplasm. IκB members contain an ankyrin repeats domain, functionally implicated in the interaction with NF-κB proteins. The IKK complex is responsible for phosphorylation at specific Ser residues of members of the IκB family . In 68 phosphorylation leads to modification of the activity of the IKK complex, which possibly represents a form of fine-tuning of NF-κB activity [The control of NF-κB activity by IKKβ also expands to other proteins different from IκB proteins . This isactivity . B-cell activity .In summary, IKKβ is fundamental in regulating NF-κB activity by phosphorylation of proteins with a leading role in the regulation of NF-κB activity, such as p65, IκB members and NEMO and also other NF-κB family members . By this important function, changes in the expression or activity of IKKβ can turn out in changes in the expression of many other proteins with different cellular outcomes. But, as discussed in the following section, this is not the only way that IKKβ exploits for modifying cellular proliferation and physiology.As previously mentioned, IKKβ also phosphorylates a number of molecules that leads to changes in fundamental signaling and developmental pathways such as mechanistic target of rapamycin (mTOR), insulin and Wnt signaling. IKKβ also affects cellular metabolism, autophagy, cellular responses to DNA damage and immune responses, as discussed in the following paragraphs. Not surprisingly, changes in IKKβ activity can affect the majority of the biological capabilities considered as “hallmarks of cancer” . In thisEspecially important from the point of view of cancer is the activity of IKKβ over tumor suppressor proteins. p53 is one of such substrates, being phosphorylated by IKKβ at Ser 366, in the C-terminal regulatory domain. This phosphorylation marks p53 for ubiquitination and posterior degradation  and reprCDKN2A) gene. In studies performed in WI-38 human lung fibroblasts and other cell lines, it was demonstrated that IKKβ binds to and phosphorylates p16 at Ser 8. This modification inactivates p16, preventing the inhibitory function of p16 over Cyclin Dependent Kinase 4 (CDK4), thus allowing progression to the phase of the cell cycle in which DNA is replicated (S phase) [It is noteworthy that IKKβ can also act over p16, one of the tumor suppressor proteins encoded by the Cyclin Dependent Kinase Inhibitor 2A (S phase) .TSC1 is a tumor suppressor gene whose mutation leads to the tuberous sclerosis complex syndrome, a genetic disorder that causes benign tumors in skin, brain and other organs. TSC1 is part of a protein complex that negatively regulates the activity of the mTOR pathway. It has been demonstrated in breast cancer cell lines that IKKβ is a TSC1 kinase whose activity leads to TSC1 suppression, mTOR activation and enhanced angiogenesis, favoring tumor development. Interestingly, these IKKβ-mediated alterations are also found in clinical samples of breast cancer [t cancer .Taken together, these data indicate that IKKβ activity could favor tumor formation by means of its negative regulation of proteins with a fundamental role in cell cycle regulation and tumor suppression, as p53 family members, p16 and TSC1.The immune system is a key element to avoid the apparition and progression of tumoral lesions. IKKβ affects the immune system by multiple ways and correct IKKβ functioning is fundamental for the establishment of a fully functional immune system, a process probably implicating both NF-κB-dependent and -independent functions of IKKβ. Thus T cell-specific IKKβ activity is needed for the formation of regulatory and memory T cells  and it hThere are several links between IKKβ and metabolism. IRS1, a member of the insulin receptor substrate family, is an adaptor protein that transmits signaling from insulin and IGF1 receptors to the PI3K/AKT (Protein Kinase B) and mitogen-activated protein kinase (MAPK) pathways, among others. IRS1 mutations are associated to insulin resistance and type II diabetes. Phosphorylation of IRS1 by IKKβ can contribute to impairment of insulin signaling pathways and to the insulin resistance mediated by inflammatory processes .IKKβ also influences cellular metabolism by regulating the inhibition of PI3K/AKT pathway in response to starvation. This effect is mediated by phosphorylation of the p85 regulatory subunit of PI3K in response to nutrient deprivation. Interestingly, these results have been observed both in cultured cells and in a variety of tissues in response to metabolic restriction . AnotherCurrently, we know a number of additional IKKβ substrates that take part in fundamental cellular processes and whose deregulation is related to some forms of cancer . Also important from the point of view of cancer is the role that IKKβ plays in the regulation of Aurora A kinase, an important regulator of cell cycle progression and maintenance of spindle bipolarity. Reduction of IKKβ activity leads to an increase in the amount of Aurora A and concomitantly to spindle defects, aneuploidy and cellular transformation . RecentlFinally, phosphorylation of 14-3-3β by IKKβ results in modulation of posttranscriptional regulation of gene expression by affecting messenger RNA (mRNA) stability  and coulFOXO3a is a transcription factor that controls cell cycle progression and induces apoptosis. In some tumors, as a consequence of AKT activation, FOXO3a is excluded from the nucleus, thus being unable to regulate the transcription of its target genes and contributing to tumor development. Hu et al. have found that several breast tumors have nuclear exclusion of FOXO3a not in response to AKT activation but associated to expression of IKKβ: so, IKKβ inactivates and directs FOXO3a to degradation, thus inducing tumorigenesis in breast cells .Interestingly, the study of the TNFα-induced phosphoproteome in the MCF-7 breast cancer cell line has revealed MTDH as other IKKβ substrate . MTDH isIkbkb knock-out mouse embryos die at mid-gestation due to massive apoptosis in the hepatocytes [IKBKB gene is not embryonic lethal—at least in some patients—but leads to a lack of Treg and γδ T cells and to severe immunodeficiency [Experiments performed almost 20 years ago revealed that IKKβ functions cannot be assumed by IKKα or other proteins, as atocytes ,44,45, aficiency .In the last years, animal models with tissue-specific modified IKKβ expression have shed light on the divergent functions of IKKβ in tumoral transformation of different organs and cell types , as we describe in this section  studies, a somewhat more complex situation has been described: in an experimental model of hepatic carcinogenesis induced by diethylnitrosamine (DEN), a lack of IKKβ in hepatocytes results in an increase in reactive oxygen species (ROS) production and in the incidence of HCCs—both in number and size—indicating an antitumoral role for IKKβ in hepatocytes. By contrast, deletion of IKKβ in both hepatocytes and Kupffer cells (liver resident macrophages which produce mitogens in response to liver damage) led to the development of fewer HCCs than mice expressing normal amounts of IKKβ, indicating a IKKβ protumoral role in Kupffer cells . The effIkbkb deletion in intestinal epithelial cells by a Villin-Cre transgene resulted in lower colon cancer incidence in a carcinogen-induced model, by enhanced apoptosis during tumor promotion [Ikbkb deletion in myeloid cells by a Cre transgene directed by regulatory elements from the Lysozyme 2 gene had also an antitumoral effect but mediated by a decrease in expression of proinflammatory mediators [Regarding intestinal cancer, different animal models have been studied. romotion . Ikbkb dediators . The expediators . These eediators . The mecediators .Ikbkb deletion in mesenchymal cells by means of a ColVI-Cre transgene led to protection against inflammation-induced intestinal carcinogenesis [Ikbkb inactivation was accomplished by an inducible Col1a2Cre-ER transgene, intestinal tumor growth is promoted upon carcinogenic treatments [Ikbkb inactivation in both models. In addition, IKKβ could have distinct functions in different fibroblast populations.Recently, two reports about the role of IKKβ in mesenchymal cells over intestinal tumorigenesis have been published ,61. Thesogenesis , indicatIkbkb inactivation in skin keratinocytes suffer from a TNFα-mediated inflammatory skin disease that is rescued in the absence of TNFR1 [Ikbkb deletion in skin resulted in epidermal hyperplasia and activation of STAT3 and ERK1/2 pathways but not in tumor formation [Ikbkb genes aimed to study IKKβ functions in T cells and astrocytes led unexpectedly to cre-mediated Ikbkb deletion in skin keratinocytes. These mice showed a hyperplasic skin phenotype associated to inflammation that in some cases led to SCC development [Mice with of TNFR1 ; these rormation . Other aelopment ,65.The expression of a constitutively active form of IKKβ in epithelial cells of murine esophagus leads to esophagitis and increased angiogenesis in the esophageal stroma. These phenotypes were accompanied by increased esophageal infiltration of immune cells and increased levels of GM-CSF and TNF . UnfortuWe have studied mice overexpressing human IKKβ in stratified epithelia under the transcriptional control of Keratin K5-derived regulatory sequences; this model is also illustrative of the varying effects of IKKβ over different cell types. K5-IKKβ transgenic mice are prone to the appearance of oral epithelia lesions in palate and forestomach, associated to a higher presence of infiltrating cells. These spontaneous lesions were frequently benign (from hyperplastic dysplasia to carcinoma in situ) but SCCs were also observed in some animals . When K5Ink4a/Arf locus (p16 and p19), as the number of tumors generated in K5-IKKβ mice in the absence of p16 and p19 were similar to the number generated in control mice; interestingly, in Ink4a/Arf null background, K5-IKKβ mice developed a significantly higher amount of undifferentiated and spindle SCCs , indicating that the skin tumor suppressive function of IKKβ turns into an oncogenic role in absence of p16 and/or p19. In order to clarify which Ink4a/Arf-coded tumor suppressor protein mediates these IKKβ skin suppressive cancer functions, it will be needed to generate K5-IKKβ mice lacking p16 or p19 individually and to study their sensitivity to skin cancer.However, when we probed this transgenic line for the role of IKKβ in non-melanoma skin cancer, a completely opposing outcome was obtained, as mice overexpressing IKKβ in keratinocytes were refractory to development of skin tumors under carcinogenesis protocols, indicating a tumor suppressor role for IKKβ in skin . K5-IKKβIKBKB.In the last years, a huge amount of genomic and expression data from numerous human tumoral samples have been generated. These data show association in some cases between tumor development and genetic or expression alterations in For example, IKKβ expression seems to be a risk factor in ovarian cancer and higher expression of IKKβ in human ovarian cancer samples is associated to lower patient survival. In agreement with these data, pharmacological inhibition of IKKβ or downregulation by RNA interference in ovarian cancer cell lines led to a decrease in some characteristics related to tumoral aggressiveness (as anchorage-independent growth or invasion through basement membrane) .IKBKB was recognized as a cancer gene by COSMIC based on the finding of IKBKB activating mutations in Lys175 in around 8% of splenic marginal zone lymphomas of B cells; this mutation renders a constitutively active IKKβ protein [IKBKB have been found in skin basal cell carcinomas, although the interpretation of these data is difficult due to the high mutation rate found in this particular tumor type [IKBKB is rarely mutated and COSMIC finds only five tumor types with mutations and always at low proportions. Lys175 Glu is the most frequently found mutation, although apparently there are no hotspots. In The Cancer Genome Atlas  and head and neck SCCs (35%), which seems to indicate a tumor suppressive role of IKKβ in these cancer types. Interestingly, heterozygous loss or low expression of IKBKB is associated to mutations in TP53 and to impaired overall survival in breast tumors  and liver tumors (around 4%). No particular association with any tumor variable or mutation has been found. It is intriguing that some tumors show loss of one single t tumors A and in t tumors B.IKBKB and copy-number amplifications are common, being IKBKB amplified at high level in around 25% of neuroendocrine prostate tumors. The same holds true in 10% of breast tumors and in around 5% of several other types of tumors in the uterus, ovary, esophagus, lung and bladder. In these tumor types, the possible existence of correlations between IKKβ expression and any biological or clinical variable is not known but in concordance with the previously mentioned association between low expression of IKBKB and mutations in TP53 in breast cancer, overexpression of IKBKB in breast cancer correlates negatively with TP53 mutation but positively with ER-positive status  breast cancer cohorts).The analysis of the data included in cBioportal indicates that overexpression of Overall, these data add to the growing body of IKKβ protumorigenic and antitumorigenic evidence obtained using genetically modified animal models, indicating that the effect of IKKβ over tumoral transformation is cell-type specific, favoring carcinogenesis in many cell types but not in others. The knowledge of the biological determinants that define the pro- or anti-tumorigenic role of IKKβ is a challenge that remains open. Consequently, further work is needed, both in animal models and with human-derived tumoral samples, to unravel the cell-type specificity and complex activities of IKKβ in cancer.


Although cancer cells are known to be “addicted” to glucose, the effect of glucose in proliferation of these cells remains elusive. Here, we report that upon glucose induction, protein arginine methyltransferase 5 (PRMT5) exerts a profound effect on the G1-S cell cycle progression via directly interacting with cyclin dependent kinase 4 (CDK4) in hepatocellular carcinoma (HCC). Upregulation of both PRMT5 and CDK4 predicts more malignant characteristics in human HCC tissues. Mechanistically, glucose promotes the interaction between PRMT5 and CDK4, which leads to activation of CDK4-RB-E2F-mediated transcription via releasing CDKN2A from CDK4. Moreover, the PRMT5 competitive inhibition of the interaction between CDK4 and CDKN2A is important for glucose-induced growth of HCC cells. Furthermore, the CDK4 mutant R24A weakly binds to PRMT5, inhibiting HCC cell cycle progression and tumor growth. Thus, our findings uncover a critical function for PRMT5 and CDK4 and provide an improved therapeutic strategy against HCC. Liver cancer, especially hepatocellular carcinoma (HCC), causes high morbidity and mortality relative to other cancers . Major pINK4a as the major isoform) complex is more critical for growth suppression in hepatoma cells [cyclin E, cyclin A, CDK1, cell division cycle 6 (CDC6), origin recognition complex subunit 1 (ORC1), minichromosome maintenance complex component 3 (MCM3), myeloblastosis oncogene (MYB) and E2F1 [Since aberrant progression of the cyclin dependent kinase (CDK)-driven cell cycle is one of the hallmarks of cancers , many stma cells  or otherma cells . With CCma cells , allowinma cells . The trama cells . RB intema cells . The CDKand E2F1 , 17. By Protein arginine methyltransferase 5 (PRMT5) is an enzyme which can transfer methyl groups to the arginine residues of histones and some nonhistone proteins, and its methyltransferase activity is necessary for tumor cell proliferation . PRMT5 hHere, we confirmed that glucose is indispensable for PRMT5 to facilitate HCC cell growth. Under the high glucose condition, PRMT5-depleted cells were more sensitive to a CDK4 inhibitor. Importantly, we identified a direct glucose-induced interaction between PRMT5 and CDK4. Through that interaction, PRMT5 inhibited the interaction between CDK4 and CDKN2A and then activated the CDK4-RB-E2F pathway in HCC cells under glucose induction. Furthermore, we revealed that the CDK4 mutant R24A weakly bound with PRMT5 and inhibited HCC cell cycle progression. As a result, the protein levels of PRMT5 and CDK4 were found to positively correlate in HCC and stimulate HCC cell proliferation.P = 0.029). However, patient sex, age, degree of tumor differentiation and other parameters had no observable relationship with PRMT5 expression . As shown in Figure on Table . Analogoon Table  in HCC ton Table . The tumSince PRMT5 is essential for tumor cell cycle and proliferation, we investigated the role of PRMT5 in HCC cell cycle regulation by flow cytometry. As shown in Figure In the high glucose condition, HepG2 cells with both PRMT5 and CDK4 knocked down by siRNA showed slower growth than those with only PRMT5 or CDK4 interference Figure . MoreoveCCNE1, CDC6, MCM3, CDK1, MYBL2, ORC1) were more significantly down-regulated in siPRMT5 treated cells cultured in high glucose medium  and different quantities  of HA-tagged PRMT5 into high/low glucose cultured HEK293T cells, and the cell lysis solution was immunoprecipitated with an anti-Flag M2 antibody. Interestingly, p16INK4a weakly bound to CDK4 in the presence of PRMT5 in the high glucose condition  and symmetric dimethylation (SDMA) of CDK4 through the interaction were not detected Figure . Using tn Figure . Howevera Figure . Moreovea Figure . These dEscherichia coli BL21 and then purified : amino acids 1-70, 71-139, 140-215 and 216-303 Figure . GST alopurified . GST-pulpurified , which id Figure . Similard Figure . These rINK4a than the wild-type (WT) protein . The consistent expression levels of PRMT5 and CDK4 (WT and R24A) in this two cell lines were validated  with CDK4 , CDK8 orCommonly, PRMT5 contributes to tumorigenesis and cell cycle imbalance as an arginine methyltransferase. PRMT5 methylates arginine residues of histones or non-histones leading to transcriptional silencing, including the cell cycle regulators. For example, acting as a tumor suppressor, E2F1 methylated by PRMT5 can increase tumor cell growth and inhibit apoptosis . HoweverThere are varieties of factors that affect cell proliferation, in which an important one is regulation of cell cycle progression. Cell cycle activators promote cell cycle progression, the ratio of apoptosis cells decrease and then cells grow faster. This is a cascade amplification process. In our study, PRMT5 as an upstream regulator promoted cell cycle through CDK4 in performance at G1-S transition. We observe a limited change of the ratio of G1 and S phase when PRMT5 was short-term knockdown, but over time the effect of cell proliferation could present a large variation. On the other hand, PRMT5 can also promote cell growth in other ways, such as accelerating cell metabolites biosynthesis , and celAs a major nutrient, glucose is critically required for cellular biosynthesis and energy supply. Compared with normal cells, tumor cells metabolize more glucose to lactate via glycolysis, a phenomenon called the Warburg effect . Due to Since results of the GST-pull down and co-immunoprecipitation assay suggested an interaction between PRMT5 and CDK4, we predicted that PRMT5 impacts HCC cell proliferation via the effect of binding with CDK4. CDK4 is well known as an oncogene and a significant clinical marker in HCC progression . MoreoveIn addition, CDK4 mutations have been reported in several types of cancer. The phosphorylation of CDK4 at threonine172 was found to be essential for CDK4-CCND activating binding, while the CDK4 T172A mutant displayed very weak activity , 42. CDKIn summary, we have demonstrated that PRMT5 is a conditional regulator of CDK4 in HCC. In the high glucose condition, PRMT5 was shown to emphatically interact with CDK4, releasing it from CDKN2A and leading the CDK4-RB-E2F axis to an active state for tumor growth Figure . Our stu2. Stable PRMT5 knockdown and control cells were cultured in the same media with the addition of 1 μg/ml puromycin (Life Technologies). PCR-amplified human PRMT5, CDK4 and CDK4 mutations were cloned into pcDNA4TO-Flag/HA vectors. Human CCND1 and p16INK4a were cloned into the pcDNA3.0-Myc-6×His vector. Prokaryotic expression plasmids pEGX-4T1 and pET-28a were also used. All plasmids were verified by DNA sequencing. Lipofectamine 2000 (Invitrogen) for transfection and fascaplysin (Santa Cruz Biotechnology) were purchased.All cell lines were purchased through ATCC and cultured in DMEM  supplemented with 10% FBS (GIBCO), 100 mg/ml penicillin and 100 mg/ml streptomycin sulfate (GIBCO) at 37°C and 5% CO2O2 to block endogenous peroxidase activity and incubated with primary antibodies (anti-CDK4 at 1:2000 and anti-PRMT5 at 1:2000). The sections were then incubated with an HRP-conjugated secondary antibody, followed by treatment with diaminobenzidine chromogen to visualize the signals. Staining of CDK4 and PRMT5 was scored by two independent researchers based on the distribution and intensity of signals. The distribution (percentage of stained tissue area) was scored as 0 (0–5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), 4 (75–100%). The intensity of staining was scored as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The final scores of CDK4 or PRMT5 expression in HCC and adjacent tissues were on a scale of 0-12. Samples were defined as having high expression (score > 6) or low expression (score ≤ 6). Pearson's test was applied to determine the correlation between CDK4 and PRMT5 using SPSS 20 software.All experimental procedures using human tissues were approved by the Human Ethics Committee of Institute of Health Sciences. The samples were obtained by surgical resection from patients who gave consent at Renji Hospital. The tissue sections were treated with 3% Hg for 30 min. After SDS-PAGE, extracted proteins were transferred onto PVDF membranes  and probed with primary antibodies at 4°C overnight. The membranes were then incubated with secondary antibodies for 1 h, and the bands were captured through an ECL HRP substrate (Millipore). Anti-PRMT5 , anti-CDK4 , anti-β-actin , anti-β-tubulin , anti-Flag M2 , anti-HA , anti-6× His , anti-pRB S780 , anti-Mono-Methyl Arginine  and anti-SDMA , anti-CCNE1 , anti-CDC6  antibodies were purchased.Cells were lysed in RIPA buffer containing protease inhibitors for 30 min on ice, followed by centrifugation at 15000 × HepG2 and HuH-7 cells were infected by lentivirus with Control shRNA  and shPRMT5 . Then the cells were screened by puromysin.PRMT5 , CDK4 , and Negative Control .Cells were transfected with oligo small silencing RNAs using Lipofectamine 2000 in serum-free Opti-MEM  according to the manufacturer's instruction. The sequences of siRNA oligos used in this study are listed as following: CCNE1-Forward Primer (AGATGTAGGCCGGGTGATCT), CCNE1-Reverse Primer (CCGCCCTGGATCATGAAGTC), CCNA2-F (TGATGTTGGGCAACTCTGCG), CCNA2-R (GTGCAACCCGTCTCGTCTTC), CDK1-F (AGCCGG GATCTACCATACCC), CDK1-R (AGGAACCCCTTC CTCTTCACT), CDC6-F (TCATGCCTCAAACCCG ATCC), CDC6-R (TGTCATCGCCCAGACGTTTC), ORC1-F (GGCAGCAGCTTCGGTTTCTA), ORC1-R (TCTTTGGCACCTTCGTGAGG), MCM3-F (AGGTAG TTCTTTGGCAGCGG), MCM3-R (TCAGAAGCCGG TTAGCCCT), MYBL2-F (CTGGTGAGGCAGTTTG GACA), MYBL2-R (ACCAGCTCGATGACTTTTTGGT), E2F1-F (AAGAGCAAACAAGGCCCGAT), E2F1-R (AC AACAGCGGTTCTTGCTCC).Total RNA was isolated using a Trizol kit , and cDNA was synthesized using a cDNA synthesis kit . Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix  on the StepOnePlus Real-Time PCR System . The primers used in this study are listed as following: 4 cells per well. Twenty-four hours after transfection or fascaplysin treatment, cells were harvested every 24 h, and cells numbers were counted using a hemocytometer.HepG2 or HuH-7 cells were plated in 24-well plates with a density of 1 × 1012-well plates were covered with 0.4% agarose gel (Gibco), and then cells in 0.375% agarose gel were overlaid on the top of the 0.4% gel. 200 cells were suspended per well and cultured. After 3 weeks, the cells were stained with 0.1% crystal violet. The picture of each well was photographed and the colony number was calculated.Trypsin digested cells were washed with 1 × PBS, fixed in cold 70% ethanol for 24 h and then incubated with RNase A (Invitrogen) for 30 min at 37°C and 50 μg/ml propidium iodide (Sigma-Aldrich) for 15 min at 4°C shielded from light. Cells were analyzed with a Gallios (Beckman) fluorescence activated cell sorter.Extracts of cells overexpressing Flag- or HA-tagged proteins were incubated with antibodies -binding Protein A/G (Pierce) or anti-Flag M2 affinity gel (Sigma) for 3 h at 4°C. After being washed three times with IP buffer, the samples were analyzed by Western blot.E. coli BL21 and purified by glutathione-Sepharose 4B beads . Cell lysis solution or His-tagged proteins purified by Ni-affinity resins  were mixed with purified GST, GST-CDK2, GST-CDK4 or GST-CDK4-fragments beads for 3 h at 4°C. After being washed five times with IP buffer, the samples were analyzed by Western blot.GST-fused CDK2, CDK4 or CDK4-fragments proteins were expressed in All experimental procedures using animals were conducted in accordance with the guidelines provided by the Animal Ethics Committee of the Institute of Health Sciences. HepG2 cells stably transfected with CDK4 WT and PRMT5 (or CDK4 R24A and PRMT5) were subcutaneously inoculated in the left (or in the right side) of 4-week-old male BALB/c SCID mice . Tumor sizes were monitored and measured for growth over a period of 3 weeks, and the results are presented as the mean ± SEM. At the end of the experiments, mice were sacrificed, and tumor tissues were collected and weighed.P < 0.05 was considered significant.All data were statistically analyzed using Graphpad Prism 6 or SPSS 20 software. Two-tailed t-test was used to analyze the difference between two groups. Pearson's test was applied to determine the correlation between CDK4, PRMT5 and clinicopathological parameters. Data are presented as the mean ± SEM or SD, and