Fractionated radiotherapy (RT) is widely used in cancer treatment, because it preserves normal tissues. However, repopulation of radioresistant tumors during fractionated RT limits the efficacy of RT. We recently demonstrated that a moderate level of long-term fractionated radiation confers acquired radioresistance to tumor cells, which is caused by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. The resulting cyclin D1 overexpression leads to forced progression of the cell cycle to S-phase, concomitant with induction of DNA double-strand breaks (DSBs). In this study, we investigated the molecular mechanisms underlying cyclin D1 overexpression-induced DSBs during DNA replication in acquired radioresistant cells. DNA fiber data demonstrated that replication forks progressed slowly in acquired radioresistant cells compared with corresponding parental cells in HepG2 and HeLa cell lines. Slowly progressing replication forks were also observed in HepG2 and HeLa cells that overexpressed a nondegradable cyclin D1 mutant. We also found that knockdown of Mus81 endonuclease, which is responsible for resolving aberrant replication forks, suppressed DSB formation in acquired radioresistant cells. Consequently, Mus81 created DSBs to remove aberrant replication forks in response to replication perturbation triggered by cyclin D1 overexpression. After treating cells with a specific inhibitor for DNA-PK or ATM, apoptosis rates increased in acquired radioresistant cells but not in parental cells by inhibiting the DNA damage response to cyclin D1-mediated DSBs. This suggested that these inhibitors might eradicate acquired radioresistant cells and improve fractionated RT outcomes.

Here we confirmed that metastasis-associated in colon cancer 1 (MACC1) and β-catenin expression were higher in colorectal cancer (CRC) cells and tissues than those in normal colonic epithelial cell line and adjacent non-tumour colorectal mucosa (ANM) tissues, respectively. MACC1 expression was significantly related to histological differentiation (p<0.001), UICC stage (p=0.029), T classification (p=0.017), and N classification (p=0.023). Cox regression analysis demonstrated that high MACC1/abnormal β-catenin expression was the strongest independent prognostic indicator for reduced overall survival in CRC patients. Significant positive correlation between MACC1 expression and abnormal β-catenin expression was found in CRC tissues. MACC1 knockdown dramatically inhibited cellular proliferation, migration, invasion, colony formation, and tumorigenesis, both in vitro and in vivo, but induced apoptosis in CRC cells. Further MACC1 over-expression increased Met, β-catenin, and its downstream genes including c-Myc, cyclin D1, and MMP9 expression, and its upstream gene phos-GSK3β (Ser9) expression. In addition, MACC1 increased vimentin and suppressed E-cadherin in HCT116 cells. Silencing of MACC1 reversed all these changes. Our results firstly suggest that MACC1 plays an important role in carcinogenesis and progression of CRC through β-catenin signaling pathway and mesenchymal-epithelial transition.

Gastric cancer (GC) remains a threat to public health with high incidence and mortality worldwide. Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) play critical regulatory roles in cancer biology, including GC. Previous profiling study showed that lncRNA linc00261 was aberrantly expressed in GC. However, the role of linc00261 in GC progression and the precise molecular mechanism remain unknown. In this study, we report that linc00261 was significantly down-regulated in GC tissues and the expression level of linc00261 negatively correlated with advanced tumour status and clinical stage as well as poor prognostic outcome. In vitro functional assays indicate that ectopic expression of linc00261 suppressed cell invasion by inhibiting the epithelial-mesenchymal transition (EMT). By RNA pull-down and mass spectrum experiments, we identified Slug as an RNA-binding protein that binds to linc00261. We confirmed that linc00261 down-regulated Slug by decreasing the stability of Slug proteins and that the tumour-suppressive function of linc00261 can be neutralized by Slug. linc00261 may promote the degradation of Slug via enhancing the interaction between GSK3β and Slug. Moreover, linc00216 overexpression repressed lung metastasis in vivo. Together, our findings suggest that linc00261 acts a tumour suppressor in GC by decreasing the stability of Slug proteins and suppressing EMT. By clarifying the mechanisms underlying GC progression, these findings may facilitate the development of novel therapeutic strategies for GC.

Fibroblast growth factor (Fgf) and Wnt signaling are necessary for the intertwined processes of tail elongation, mesodermal development and somitogenesis. Here, we use pharmacological modifiers and time-resolved quantitative analysis of both nascent transcription and protein phosphorylation in the tailbud, to distinguish early effects of signal perturbation from later consequences related to cell fate changes. We demonstrate that Fgf activity elevates Wnt signaling by inhibiting transcription of the Wnt antagonists dkk1 and notum1a. PI3 kinase signaling also increases Wnt signaling via phosphorylation of Gsk3β. Conversely, Wnt can increase signaling within the Mapk branch of the Fgf pathway as Gsk3β phosphorylation elevates phosphorylation levels of Erk. Despite the reciprocal positive regulation between Fgf and Wnt, the two pathways generally have opposing effects on the transcription of co-regulated genes. This opposing regulation of target genes may represent a rudimentary relationship that manifests as out-of-phase oscillation of Fgf and Wnt target genes in the mouse and chick tailbud. In summary, these data suggest that Fgf and Wnt signaling are tightly integrated to maintain proportional levels of activity in the zebrafish tailbud, and this balance is important for axis elongation, cell fate specification and somitogenesis.

Glycogen synthase kinase (GSK-3) is upregulated in many types of tumor, including prostate cancer. GSK-3 inhibitors reduce prostate tumor cell growth; however, it is not clear if both isoforms, GSK-3α and GSK-3β, are involved. Here, we compared their expression in prostate tumors and used gene silencing to study their functions in 22Rv1 prostate cancer cells. Compared to normal prostate, GSK-3α and GSK-3β were upregulated in 25/79 and 24/79 cases of prostate cancer, respectively, with GSK-3α elevated in low Gleason sum score tumors and GSK-3β expressed in high Gleason tumors, and both isoforms correlating with high expression of the androgen receptor (AR). Gene silencing of GSK-3α and, to a lesser extent, GSK-3β reduced AR transcriptional activity. In addition, silencing of GSK-3β, but not GSK-3α, reduced Akt phosphorylation. Acute and chronic silencing of either isoform reduced 22Rv1 growth in colony formation assays; however, this did not correlate with effects on AR activity. The GSK-3 inhibitor CHIR99021 reduced 22Rv1 colony formation by 50% in normal growth medium and by 15% in hormone-depleted medium, suggesting that GSK-3 is required both for hormone-dependent and hormone-independent proliferation. In addition, CHIR99021 enhanced growth inhibition by the AR antagonists bicalutamide and MDV3100. Finally, expression of GSK3A and GSK3B mRNAs correlated with a gene expression signature for androgen-regulated genes. Our observations highlight the importance of the GSK-3/AR signaling axis in prostate cancer and support the case for development of isoform-specific GSK-3 inhibitors and their use, in combination with AR antagonists, to treat patients with prostate cancer.

Regulator of calcineurin 1 (RCAN1) is a key regulator of the calcineurin-NFAT signaling network in organisms ranging from yeast to human, but its functional role is still under debate because different roles of RCAN1 have been suggested under various experimental conditions. To elucidate the mechanisms underlying the RCAN1 regulatory system, we used a systems approach by combining single-cell experimentation with in silico simulations. In particular, we found that the nuclear export of GSK3β, which switches on the facilitative role of RCAN1 in the calcineurin-NFAT signaling pathway, is promoted by PI3K signaling. Based on this, along with integrated information from previous experiments, we developed a mathematical model in which the functional role of RCAN1 changes in a dose-dependent manner: RCAN1 functions as an inhibitor when its levels are low, but as a facilitator when its levels are high. Furthermore, we identified a hidden incoherent regulation switch that mediates this role change, which entails negative regulation through RCAN1 binding to calcineurin and positive regulation through sequential phosphorylation of RCAN1.

The environmental bacterium Burkholderia cenocepacia causes opportunistic lung infections in immunocompromised individuals, particularly in patients with cystic fibrosis. Infections in these patients are associated with exacerbated inflammation leading to rapid decay of lung function, and in some cases resulting in cepacia syndrome, which is characterized by a fatal acute necrotizing pneumonia and sepsis. B. cenocepacia can survive intracellularly in macrophages by altering the maturation of the phagosome, but very little is known on macrophage responses to the intracellular infection. In this study, we have examined the role of the PI3K/Akt signaling pathway in B. cenocepacia-infected monocytes and macrophages. We show that PI3K/Akt activity was required for NF-κB activity and the secretion of proinflammatory cytokines during infection with B. cenocepacia. In contrast to previous observations in epithelial cells infected with other Gram-negative bacteria, Akt did not enhance IκB kinase or NF-κB p65 phosphorylation, but rather inhibited GSK3β, a negative regulator of NF-κB transcriptional activity. This novel mechanism of modulation of NF-κB activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection.

Okadaic acid (OKA) is a potent inhibitor of protein phosphatases 1/2A (PP2A). Inhibition of PP2A leads to hyperphosphorylation of Tau protein. Hyperphosphorylated Tau protein is present in intraneuronal neurofibrillary tangles a characteristic feature of neuropathology of Alzheimer's disease. Intracerebroventricular (ICV) administration of OKA causes neurotoxicity, which is associated with increased intracellular Ca(2+) level, oxidative stress, and mitochondrial dysfunction in the brain areas. The present study explored Tau phosphorylation in OKA-treated rats in relation to memory function, PP2A activity, intracellular Ca(2+), glycogen synthase kinase-3β (GSK-3β) and N-methyl-d-aspartate (NMDA) receptor after 13days of OKA (200ng, ICV) administration in rats, memory was found impaired in the water maze test. OKA-induced memory-impaired rats showed increased mRNA and protein expression of Tau, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Calpain and GSK3β in the hippocampus and cerebral cortex. On the other hand, mRNA expression and activity of PP2A was reduced in these brain areas. OKA treatment also, resulted in decrease in mRNA expression of C and N terminals of Tau. Treatment with NMDA antagonist, MK801 (0.05mg/kg, i.p.) for 13days significantly prevented OKA-induced changes in the expression of PP2A, Tau, GSK3β, CaMKII and Calpain. Further, daily administration of anticholinergic drug, donepezil (5mg/kg, p.o.), and the NMDA receptor antagonist, memantine (10mg/kg, p.o.) initiated after OKA administration for 13days significantly attenuated OKA-induced variation in Tau, Tau-C terminal, Tau-N terminal CaMKII, Calpain, PP2A and GSK3β. These results infer that NMDA antagonist MK801 and memantine are effective against OKA-induced neurotoxicity. Therefore, the present study clearly indicates the involvement of NMDA receptor in OKA (ICV)-induced Tau hyperphosphorylation.

Renal proximal tubular damage and repair are hallmarks of acute kidney injury. As glycogen synthase kinase-3β (GSK3β) is an important cellular regulator of survival and proliferation, we determined its role during injury and recovery of proximal tubules in a mercuric chloride-induced nephrotoxic model of acute kidney injury. Renal proximal tubule-specific GSK3β knockout mice exposed to mercuric chloride had improved survival and renal function compared to wild-type mice. Apoptosis, measured by TUNEL staining, Bax activation, and caspase 3 cleavage, was reduced in the knockout mice. The restoration of renal structure, function, and cell proliferation was also accelerated in the GSK3β knockout mice. This enhanced repair, evidenced by increased Ki-67 and BRDU staining, along with increased cyclin D1 and c-myc levels, was recapitulated by treatment of wild-type mice with the small-molecule GSK3 inhibitor TDZD-8 following injury. This confirmed that hastened repair in the knockout mice was not merely due to lower initial injury levels. Thus, inhibition of GSK3β prior to nephrotoxic insult protects from renal injury. Such treatment after acute kidney injury may accelerate repair and regeneration.

Cyclin D1, an oncogenic G1 cyclin which can be induced by environmental carcinogens and whose over-expression may cause dysplasia and carcinoma, has been shown to be a target for cancer chemoprevention and therapy. In this study, we investigated the effects and underlying mechanisms of action of a polyprenylated xanthone, gambogenic acid (GEA) on gefitinib-sensitive and -resistant lung cancer cells. We found that GEA inhibited proliferation, caused G1 arrest and repressed colony-forming activity of lung cancer cells. GEA induced degradation of cyclin D1 via the proteasome pathway, and triggered dephosphorylation of GSK3β which was required for cyclin D1 turnover, because GSK3β inactivation by its inhibitor or specific siRNA markedly attenuated GEA-caused cyclin D1 catabolism. GEA induced autophagy of lung cancer cells, possibly due to activation of GSK3β and inactivation of AKT/mTOR signal pathway. These results indicate that GEA is a cyclin D1 inhibitor and a GSK3β activator which may have chemopreventive and therapeutic potential for lung cancer.

Wnt/β-catenin and EGFR pathways are important in cancer development and often aberrantly activated in human cancer. However, it is very important to understand the mechanism responsible for this activation and the relation between them. Here, we report the mechanism of EGFR expression by transcriptionally active β-catenin in GSK3β-inactivated prostate cancer cells that eventually leads to its enhanced proliferation and survival. Expressions of β-catenin and EGFR are elevated in various cancers specifically in prostate cancer cells, DU145. When GSK3β is inactivated in these cells, β-catenin gets stabilized, phosphorylated at Ser-552 by protein kinase A, accumulates in the nucleus, and regulates the expression of its target genes that include EGFR. Chromatin immunoprecipitation (ChIP) and promoter analysis revealed that the EGFR promoter gets occupied by transcriptionally active β-catenin when elevated in GSK3β-inactivated cells. This phenomenon not only leads to increased expression of EGFR but also initiates the activation of its downstream molecules such as ERK1/2 and Stat3, ultimately resulting in up-regulation of multiple genes involved in cell proliferation and survival.

Sphingadienes (SDs) derived from soy and other natural sphingolipids are cytotoxic to colon cancer cells via an Akt-dependent mechanism and reduce adenoma formation in Apc(Min/+) mice. Wnt signaling is fundamental to colon carcinogenesis and is the basis for spontaneous tumorigenesis in Apc(Min/+) mice and patients with familial adenomatous polyposis. In the present study, we investigated the impact of SDs on Wnt signaling. Oral SD administration reduced levels of active β-catenin and Wnt targets c-Myc and cyclin D1 in Apc(Min/+) mouse intestinal tissues. Colon cancer cells treated with SDs exhibited reduced Wnt transcriptional activity, as well as reduced nuclear β-catenin localization and subsequent reduction in active-β-catenin levels. Further, we observed a decrease in phosphorylated (inactive) GSK3β in SD-treated mice and colon cancer cells. Expression of constitutively active myristoylated-Akt or inactivation of GSK3β using LiCl attenuated SD-mediated inhibition of Wnt transcriptional activity and active-β-catenin levels. SDs exhibited additive effects with inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway to induce cytotoxicity. Further, a combination regime of SDs and low-dose rapamycin decreased visible polyps in Apc(Min/+) mice and reduced the levels of Wnt target gene expression and mTOR target activation. SD-mediated inhibition of Akt and Wnt pathways and cytotoxicity in colon cancer cells was dependent upon the activity of protein phosphatase 2A, as shown by reversal of these effects by pretreatment with okadaic acid or calyculin A. Our cumulative findings indicate that SDs inhibit Wnt signaling through a protein phosphatase 2A/Akt/GSK3β-dependent mechanism that may contribute to their chemopreventive effects in intestinal tumorigenesis.

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.

ER stress triggers myocardial contractile dysfunction although the underlying mechanism is still elusive. Given that NADPH oxidase was recently implicated in ER stress-induced tissue injury, this study was designed to examine the role of NADPH oxidase in ER stress-induced cardiac mechanical defects and the impact of Akt activation on ER stress-induced cardiac anomalies. Wild-type and transgenic mice with cardiac-specific overexpression of an active mutant of Akt (MyAkt) were subjected to the ER stress inducer thapsigargin (1 and 3mg/kg, ip, for 48h). Thapsigargin compromised echocardiographic parameters, including elevating LVESD and reducing fractional shortening; suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, and cell survival; and enhanced carbonyl formation, apoptosis, superoxide production, NADPH oxidase expression, and mitochondrial damage. Interestingly, these anomalies were attenuated or mitigated by chronic Akt activation. Treatment with thapsigargin also dephosphorylated Akt and its downstream signal GSK3β (leading to activation of GSK3β), the effect of which was abrogated in MyAkt hearts. Knockdown of the cytosolic subunit of NADPH oxidase, p47(phox), using siRNA abrogated thapsigargin-induced apoptosis and cell death in H9C2 myoblasts. In vitro exposure to thapsigargin induced murine cardiomyocyte dysfunction reminiscent of the in vivo setting, the effects of which were ablated by the NADPH oxidase inhibitor apocynin and the mitochondrial Ca(2+) uptake inhibitor Ru360. In addition, apocynin abrogated thapsigargin-induced loss of mitochondrial membrane potential and permeability transition pore opening, similar to chronic Akt activation. In summary, these data suggest that ER stress interrupts cardiac contractile and intracellular Ca(2+) homeostasis, cell survival, and mitochondrial integrity through an Akt dephosphorylation- and NADPH oxidase-dependent mechanism.

β-Catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt signaling, and the centrosome cycle. Whereas the regulation of β-catenin in cell-cell adhesion and Wnt signaling are well understood, how β-catenin is regulated at the centrosome is not. NIMA-related protein kinase 2 (Nek2), which regulates centrosome disjunction/splitting, binds to and phosphorylates β-catenin. Using in vitro and cell-based assays, we show that Nek2 phosphorylates the same regulatory sites in the N-terminus of β-catenin as glycogen synthase kinase 3β (GSK3β), which are recognized by a specific phospho-S33/S37/T41 antibody, as well as additional sites. Nek2 binding to β-catenin appears to inhibit binding of the E3 ligase β-TrCP and prevents β-catenin ubiquitination and degradation. Thus β-catenin phosphorylated by Nek2 is stabilized and accumulates at centrosomes in mitosis. We further show that polo-like kinase 1 (Plk1) regulates Nek2 phosphorylation and stabilization of β-catenin. Taken together, these results identify a novel mechanism for regulating β-catenin stability that is independent of GSK3β and provide new insight into a pathway involving Plk1, Nek2, and β-catenin that regulates the centrosome cycle.

Insulin resistance is the major pathogenesis underlying type 2 diabetes mellitus (T2DM) and these patients have doubled risk of Alzheimer's disease (AD). Increasing evidence suggests that insulin resistance plays an important role in AD pathogenesis, possibly due to abnormal GSK3β activation, causing intra- and extracellular amyloid-beta (Aβ) accumulation. Adiponectin (APN) is an adipokine with insulin-sensitizing and anti-inflammatory effects. Reduced circulatory APN level is associated with insulin resistance and T2DM. The role of APN in AD has not been elucidated. In this study, we aim to examine if adiponectin deficiency would lead to cerebral insulin resistance, cognitive decline and Alzheimer's-like pathology in mice.

To study the role of adiponectin in cognitive functions, we employed adiponectin-knockout (APN-KO) mice and demonstrated chronic APN deficiency in their CNS. Behavioral tests were performed to study the cognitions of male APN-KO mice. Brains and tissue lysates were collected to study the pathophysiological and molecular changes in the brain of APN-KO mice. SH-SY5Y neuroblastoma cell line was used to study the molecular mechanism upon APN and insulin treatment.

Aged APN-deficient mice displayed spatial memory and learning impairments, fear-conditioned memory deficit as well as anxiety. These mice also developed AD pathologies including increased cerebral Aβ42 level, Aβ deposition, hyperphosphorylated Tau proteins, microgliosis and astrogliosis with increased cerebral IL-1β and TNFα levels that associated with increased neuronal apoptosis and reduced synaptic proteins levels, suggesting APN deficiency may lead to neuronal and synaptic loss in the brain. AD pathologies-associated APN-KO mice displayed attenuated AMPK phosphorylation and impaired insulin signaling including decreased Akt induction and increased GSK3β activation in the hippocampus and frontal cortex. Aged APN-KO mice developed hippocampal insulin resistance with reduced pAkt induction upon intracerebral insulin injection. Consistently, APN treatment in SH-SY5Y cells with insulin resistance and overexpressing Aβ induce higher pAkt levels through AdipoR1 upon insulin treatment whereas the induction was blocked by compound C, indicating APN can enhance neuronal insulin sensitivity through AMPK activation.

Our results indicated that chronic APN deficiency inactivated AMPK causing insulin desensitization and elicited AD-like pathogenesis in aged mice which also developed significant cognitive impairments and psychiatric symptoms.

The Wnt/β-catenin signalling pathway is known to play a vital role in the maintenance of cancer stem cells (CSCs), which are reported to be the origin of malignant cancers, and result in poor prognosis of multiple kinds of cancer. Therefore, it is of great importance to illuminate the mechanism by which the Wnt/β-catenin pathway regulates the cancer stem cell-like traits in cancers. Here, we report that miR-942 is significantly upregulated in esophageal squamous cell carcinoma (ESCC), and miR-942 levels are associated with poor prognosis in ESCC patients. Overexpression of miR-942 promotes, whereas inhibition of miR-942 decreases, the tumor sphere formation, the CD90+ subpopulation cells and the expression of pluripotency associated markers. Moreover, in vivo assay shows that miR-942 overexpressing cells form larger tumors and display higher tumourigenesis. Furthermore, we demonstrate that miR-942 upregulates the Wnt/β-catenin signaling activity via directly targeting sFRP4, GSK3β and TLE1, which are multiple level negative regulators of the Wnt/β-catenin signaling cascade. In addition, our results indicate that c-myc directly binds to the miR-942 promoter and promotes its expression. Taken together, our findings establish an oncogenic role of miR-942 in ESCC and indicate that miR-942 might be an effective therapeutic target for ESCC.

Application of the volatile anesthetic isoflurane during the early phase of reperfusion reduces ischemic heart and brain injury (anesthetic post-conditioning). We hypothesize that inhibition of glycogen synthase kinase 3β (GSK3β), a protein whose activation can lead to cell death, participates in anesthetic post-conditioning-induced neuroprotection. SH-SY5Y cells, a human neuroblastoma cell line, were induced by retinoic acid to differentiate into terminal neuron-like cells. The cells were then subjected to a 1-h oxygen-glucose deprivation (OGD), a condition to simulate ischemia in vitro, and a 20-h simulated reperfusion. Isoflurane, sevoflurane or desflurane, three commonly used volatile anesthetics, were applied for 1 h during the early phase of simulated reperfusion. Cell injury was quantified by lactate dehydrogenase (LDH) release. Phospho-GSK3β at Ser9 and total GSK3β were quantified at 1 or 3 h after the OGD. OGD increased LDH release, suggesting that OGD induced cell injury. Post-treatment with isoflurane, sevoflurane or desflurane reduced this cell injury. This protection was apparent when 2% isoflurane was applied within 1 h after the onset of reperfusion. Isoflurane post-treatment also significantly increased the phosphorylation of GSK3β at Ser9 at 1 h after the OGD. GSK3β inhibitors reduced OGD and simulated reperfusion-induced LDH release. The combination of GSK3β inhibitors and isoflurane post-conditioning did not cause a greater protection than isoflurane post-conditioning alone. These results suggest that volatile anesthetic post-conditioning reduces OGD and simulated reperfusion-induced cell injury. Since phospho-GSK3β at Ser9 decreases GSK3β activity, our results suggest that volatile anesthetic post-conditioning in human neuron-like cells may be mediated by GSK3β inhibition.

Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

The cell fate determination factor Dachshund (DACH1) functions as a novel suppressor in the progression of various neoplasms. Previous study has suggested that hypermethylation of promoter region was responsible for the reduction of DACH1 expression in hepatocellular carcinoma (HCC), and associated with the progression of HCC, but the clinical significance and the exact molecular mechanisms of DACH1 in the progression of HCC remain unclear. In this study, we employed public microarray data analysis and tissue microarrays (TMAs) technologies and showed that DACH1 expression was reduced in HCC even at early stage and associated with the tumor progression. Notably, Kaplan-Meier analysis further indicated DACH1 could be an independent prognostic factor for the overall survival of HCC. Further, mechanistic studies revealed that overexpression of DACH1 inhibited the growth and migration of HCC cell line, which were dependent in part on the inactivation of Wnt pathway via phosphorylation of GSK3β to suppress β-catenin. In agreement, the abundance of DACH1 was inversely correlated with several Wnt target genes. Collectively, our study indicated β-catenin is a novel target of DACH1 in HCC.

The floor plate is one of the major organizers of the developing nervous system through its secretion of sonic hedgehog (Shh). Although the floor plate is located within the neural tube, the derivation of the floor plate during development is still debatable and some studies suggest that floor plate cells are specified by Shh in a temporarily restricted window different to neuroepithelial cells. Using human embryonic stem cells (hESC) as a model of neurogenesis, we sought to determine how floor plate cells may be temporarily specified by SHH signaling during human embryogenesis. We found that inhibition of both GSK3β and activin/nodal pathways in hESC induces a cellular state of SOX2+/PAX6- expression, we describe as "pre-neuroepithelial." Exposure of SHH during this pre-neuroepithelial period causes the expression of GLI transcription factors to function as activators and consequently upregulate expression of the floor plate marker, FOXA2, while also supressing PAX6 expression to inhibit neuroepithelial fate. FOXA2+ cells were able to efficiently generate mesencephalic dopaminergic neurons, a floor plate derivative. Overall, this study demonstrates a highly efficient system for generating floor plate cells from hESC and, most importantly, reveals that specification of floor plate cells is temporally dependent, whereby it occurs prior to the onset of PAX6 expression, within a pre-neuroepithelial stage.

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Macrophages play pivotal roles in development, homeostasis, tissue repair and immunity. Macrophage proliferation is promoted by macrophage colony-stimulating factor (M-CSF)-induced Akt signaling; yet, how this process is terminated remains unclear. Here, we identify casein kinase 2-interacting protein-1 (CKIP-1) as a novel inhibitor of macrophage proliferation. In resting macrophages, CKIP-1 was phosphorylated at Serine 342 by constitutively active GSK3β, the downstream target of Akt. This phosphorylation triggers the polyubiquitination and proteasomal degradation of CKIP-1. Upon M-CSF stimulation, Akt is activated by CSF-1R-PI3K and then inactivates GSK3β, leading to the stabilization of CKIP-1 and β-catenin proteins. β-catenin promotes the expression of proliferation genes including cyclin D and c-Myc. CKIP-1 interacts with TRAF6, a ubiquitin ligase required for K63-linked ubiquitination and plasma membrane recruitment of Akt, and terminates TRAF6-mediated Akt activation. By this means, CKIP-1 inhibits macrophage proliferation specifically at the late stage after M-CSF stimulation. Furthermore, CKIP-1 deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and CKIP-1(-/-) mice spontaneously develop a macrophage-dominated splenomegaly and myeloproliferation. Together, these data demonstrate that CKIP-1 plays a critical role in the regulation of macrophage homeostasis by inhibiting TRAF6-mediated Akt activation.