Famine and viral infection, as well as interferon therapy have been reported to increase the risk of developing bipolar disorder. In addition, almost 100 polymorphic genes have been associated with this disease. Several form most of the components of a phosphatidyl-inositol signalling/AKT1 survival pathway (PIK3C3, PIP5K2A, PLCG1, SYNJ1, IMPA2, AKT1, GSK3B, TCF4) which is activated by growth factors (BDNF, NRG1) and also by NMDA receptors (GRIN1, GRIN2A, GRIN2B). Various other protein products of genes associated with bipolar disorder either bind to or are affected by phosphatidyl-inositol phosphate products of this pathway (ADBRK2, HIP1R, KCNQ2, RGS4, WFS1), are associated with its constituent elements (BCR, DUSP6, FAT, GNAZ) or are downstream targets of this signalling cascade (DPYSL2, DRD3, GAD1, G6PD, GCH1, KCNQ2, NOS3, SLC6A3, SLC6A4, SST, TH, TIMELESS). A further pathway relates to endoplasmic reticulum-stress (HSPA5, XBP1), caused by problems in protein glycosylation (ALG9), growth factor receptor sorting (PIK3C3, HIP1R, SYBL1), or aberrant calcium homoeostasis (WFS1). Key processes relating to these pathways appear to be under circadian control (ARNTL, CLOCK, PER3, TIMELESS). DISC1 can also be linked to many of these pathways. The growth factor pathway promotes protein synthesis, while the endoplasmic reticulum stress pathway, and other stress pathways activated by viruses and cytokines (IL1B, TNF, Interferons), oxidative stress or starvation, all factors associated with bipolar disorder risk, shuts down protein synthesis via control of the EIF2 alpha and beta translation initiation complex. For unknown reasons, oligodendrocytes appear to be particularly prone to defects in the translation initiation complex (EIF2B) and the convergence of these environmental and genomic signalling pathways on this area might well explain their vulnerability in bipolar disorder.

An elevated DNA-repair capacity in cancer cells leads to radiation resistance and severely limits the efficacy of radiation therapy. Activation of Akt is tightly associated with resistance to radiotherapy, and Mre11 protein has important role during the repair of DNA double-strand breaks (DSBs). In this report, our results showed that inhibition of Akt activity impaired the repair of DSBs in CNE2 cells, whereas activated Akt promoted the repair of DSBs in HeLa cells. Knockdown of Mre11 also impaired the process of DSB repair in both these two cell lines. More importantly, we found that Akt could regulate Mre11 expression. Inhibition of Akt activity by small interfering RNA or LY294002 efficiently downregulated the Mre11 expression in CNE2 cells, and transfection with myr-Akt plasmid in HeLa cells upregulated the Mre11 expression. In addition, luciferase reporter analysis revealed that Mre11 reporter activity increased after transfection with myr-Akt1 plasmids, and this myr-Akt1-induced transcriptional activity was blocked in the presence of LY294002. Further study showed GSK3β/β-catenin/LEF-1 pathway was involved in this regulation. Knockdown of β-catenin or LEF-1 led to the downregulation of Mre11, whereas overexpression of β-catenin led to upregulation of Mre11. The chromatin immunoprecipitation assay assay showed β-catenin/LEF-1 heterodimer could directly bind to the promoter of Mre11 in vivo. And the luciferase activity of the pGL3-Mre11 and pGL3-Lef increased in HeLa cells following β-catenin plasmid co-transfected, but was abolished when the LEF-1-binding conserved sequences of Mre11 promoter were mutated. These results together support Akt can upregulate the expression of Mre11 through GSK3β/ β-catenin/LEF pathway to elevate DSB-repair capacity in cancer cells.

Alzheimer's disease (AD) affects millions of people world-wide and new effective and safe therapies are needed. Cotinine, the main metabolite of nicotine, has a long half-life and does not have cardiovascular or addictive side effects in humans. We studied the effect of cotinine on amyloid-β (Aβ) aggregation as well as addressed its impact on working and reference memories. Cotinine reduced Aβ deposition, improved working and reference memories, and inhibited Aβ oligomerization in the brains of transgenic (Tg) 6799 AD mice. In vitro studies confirmed the inhibitory effect of cotinine on Aβ1-42 aggregation. Cotinine stimulated Akt signaling, including the inhibition of glycogen synthase kinase 3β (GSK3β), which promotes neuronal survival and the synaptic plasticity processes underlying learning and memory in the hippocampus and cortex of wild type and Tg6799 AD mice. Simulation of the cotinine-Aβ1-42 complex using molecular dynamics showed that cotinine may interact with key histidine residues of Aβ1-42, altering its structure and inhibiting its aggregation. The good safety profile in humans and its beneficial effects suggest that cotinine may be an excellent therapeutic candidate for the treatment of AD.

One of the unique characteristics of cellular signaling pathways is that a common signaling pathway can selectively regulate multiple cellular functions of a hormone; however, this selective downstream control through a common signaling pathway is poorly understood. Here we show that the insulin-dependent AKT pathway uses temporal patterns multiplexing for selective regulation of downstream molecules. Pulse and sustained insulin stimulations were simultaneously encoded into transient and sustained AKT phosphorylation, respectively. The downstream molecules, including ribosomal protein S6 kinase (S6K), glucose-6-phosphatase (G6Pase), and glycogen synthase kinase-3β (GSK3β) selectively decoded transient, sustained, and both transient and sustained AKT phosphorylation, respectively. Selective downstream decoding is mediated by the molecules' network structures and kinetics. Our results demonstrate that the AKT pathway can multiplex distinct patterns of blood insulin, such as pulse-like additional and sustained-like basal secretions, and the downstream molecules selectively decode secretion patterns of insulin.

Overexpression of GSK3β in transgenic mice induces learning deficits and some features associated with Alzheimer's disease (AD), including dentate gyrus (DG) atrophy. Here, we assessed whether these mice also recapitulate DG atrophy as well as impaired neurogenesis reported in AD. Ultrastructural analysis revealed that there were fewer and more disorganized neurogenic niches in these animals, coupled with an increase in the proportion of immature neurons. Indeed, the maturation of granule cells is delayed as witnessed by the alterations to the length and patterning of their dendritic trees and to the mossy fiber terminals. Together with an increase in neuronal death, these phenomena lead to a marked decrease in the number and disorganization of granule cells of the DG. Our results suggest that GSK3β overexpression perturbs proliferation and maturation, resulting in the loss of immature neurons. In turn, the activation of microglia is stimulated in conjunction with a decrease in the birth of new functional neurons, leading to the deterioration of this structure. These data support the idea that by inducing degeneration of the DG, GSK3β could be involved in the pathogenesis of AD.

OBJECTIVE

Recent reports have suggested that salidroside could protect cardiomyocytes from oxidative injury and stimulate glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase (AMPK). The aim of this study was to evaluate the therapeutic effects of salidroside on diabetic mice and to explore the underlying mechanisms.

METHODS

The therapeutic effects of salidroside on type 2 diabetes were investigated. Increasing doses of salidroside (25, 50 and 100 mg·kg(-1) ·day(-1)) were administered p.o. to db/db mice for 8 weeks. Biochemical analysis and histopathological examinations were conducted to evaluate the therapeutic effects of salidroside. Primary cultured mouse hepatocytes were used to further explore the underlying mechanisms in vitro.

RESULTS

Salidroside dramatically reduced blood glucose and serum insulin levels and alleviated insulin resistance. Hypolipidaemic effects and amelioration of liver steatosis were observed after salidroside administration. In vitro, salidroside dose-dependently induced an increase in the phosphorylations of AMPK and PI3K/Akt, as well as glycogen synthase kinase 3β (GSK3β) in hepatocytes. Furthermore, salidroside-stimulated AMPK activation was found to suppress the expression of PEPCK and glucose-6-phosphatase. Salidroside-induced AMPK activation also resulted in phosphorylation of acetyl CoA carboxylase, which can reduce lipid accumulation in peripheral tissues. In isolated mitochondria, salidroside inhibited respiratory chain complex I and disturbed oxidation/phosphorylation coupling and moderately depolarized the mitochondrial membrane potential, resulting in a transient increase in the AMP/ATP ratio.

CONCLUSIONS

Salidroside exerts an antidiabetic effect by improving the cellular metabolic flux through the activation of a mitochondria-related AMPK/PI3K/Akt/GSK3β pathway.

Recent studies provided strong support for the view that ubiquitin-specific protease 22 (USP22) plays a central role in cell-cycle progression and also in pathological processes such as oncogenesis. We have recently shown that USP22 levels are elevated in colorectal carcinoma with associated increase in the expression of several cell-cycle-related genes. However, the precise mechanism for these functions of USP22 at molecular level has not been fully elucidated. Currently, we investigated the role of USP22 in human colorectal cancer (CRC). We observed that USP22 expression was statistically significantly correlated positively with that of BMI-1, c-Myc and both, pAkt (Ser473), and pAkt (Thr308), in primary tumor tissues from 43 CRC patients. Down-regulation of USP22 expression in HCT116 colorectal cancer cells by siRNA resulted in the accumulation of cells in the G1 phase of the cell cycle. RNAi-knockdown of USP22 in HCT16 cells also led to the repression of BMI-1 and was accompanied by the up-regulation of p16INK4a and p14ARF, with a consequent decrease in E2F1 and p53 levels. In addition, down-regulation of c-Myc-targeted cyclin D2 was also noticed in cells treated with USP22-siRNA. Furthermore, our results showed that USP22 deletion also caused down-regulation of Akt/GSK3β activity, which can also contribute to the reduction of cyclin D2. Collectively, our current results suggest that USP22 may act as an oncogene in CRC as it positively regulates cell cycle via both BMI-1-mediated INK4a/ARF pathway and Akt signaling pathway.

Wnts are secreted glycolipoproteins that play important roles in the regulation of embryonic development and tissue homeostasis. Binding of Wnt to receptors and co-receptors causes inactivation of the β-catenin destruction complex, which leads to the stabilization and nuclear translocation of β-catenin to initiate Wnt-responsive gene expression after associating with TCF in the nucleus. As its deregulation results in serious human diseases, especially cancers, the Wnt signaling pathway serves as a promising platform for screening anti-cancer drugs. Resveratrol was selected based on its ability to inhibit the β-catenin/TCF-mediated transcriptional activity. Resveratrol, a natural phytoalexin found in a variety of plants, possesses health-promoting properties including anti-aging, anti-inflammatory, anti-oxidant, anti-cancer, cardioprotective and neuroprotective activities. We found that resveratrol indeed exhibited dose-dependent suppression of Wnt signaling, reduced the expression of Wnt target genes such as cyclin D1 and conductin, and inhibited the growth of Wnt-stimulated cells and Wnt-driven colorectal cancer cells. Further studies indicated that resveratrol functions downstream of GSK3β. Treatment with resveratrol did not alter the amount of β-catenin and its distribution in the cytoplasm and nucleus, suggesting that resveratrol did not affect the accumulation and nuclear targeting of β-catenin. In contrast, co-immunoprecipitation and in vitro binding analyses substantiated that resveratrol was capable of disrupting the binding between β-catenin and TCF4, contributing to the decreased Wnt signaling. Our discoveries not only reveal a novel target of resveratrol in the Wnt signaling pathway but also show the potential of therapy with harmless resveratrol in colorectal cancer and other Wnt-related diseases.

Aberrant Ras/Raf/MEK/ERK signaling is one of the most prevalent oncogenic alterations and confers survival advantage to tumor cells. Inhibition of this pathway can effectively suppress tumor cell growth. For example, sorafenib, a multi-kinase inhibitor targeting c-Raf and other oncogenic kinases, has been used clinically for treating advanced liver and kidney tumors, and also has shown efficacy against other malignancies. However, how inhibition of oncogenic signaling by sorafenib and other drugs suppresses tumor cell growth remains unclear. In this study, we found that sorafenib kills cancer cells by activating PUMA (p53-upregulated modulator of apoptosis), a p53 target and a BH3-only Bcl-2 family protein. Sorafenib treatment induces PUMA in a variety of cancer cells irrespective of their p53 status. Surprisingly, the induction of PUMA by sorafenib is mediated by IκB-independent activation of nuclear factor (NF)-κB, which directly binds to the PUMA promoter to activate its transcription. NF-κB activation by sorafenib requires glycogen synthase kinase 3β activation, subsequent to ERK inhibition. Deficiency in PUMA abrogates sorafenib-induced apoptosis and caspase activation, and renders sorafenib resistance in colony formation and xenograft tumor assays. Furthermore, the chemosensitization effect of sorafenib is dependent on PUMA, and involves concurrent PUMA induction through different pathways. BH3 mimetics potentiate the anti-cancer effects of sorafenib, and restore sorafenib sensitivity in resistant cells. Together, these results demonstrate a key role of PUMA-dependent apoptosis in therapeutic inhibition of Ras/Raf/MEK/ERK signaling. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to the therapies that target oncogenic kinase signaling.

Akt and mammalian target of rapamycin (mTOR) are both activated after traumatic brain injury (TBI), however complex interplay between the two hampers deciphering their functional implications in vivo. We examined the effects of single and combination inhibitors of Akt/mTOR in a mouse controlled cortical impact (CCI) model. Following CCI, phospho-Akt-473 (p-Akt) and -S6 ribosomal protein (p-S6RP), a downstream substrate of mTOR, were increased in cortical and hippocampal brain homogenates (P<0.05 versus sham). At 24 hours, p-S6RP was detected in neurons and was robustly induced in microglia and astrocytes in injured hippocampus. In vivo activity of Akt and mTOR inhibitors administered separately was confirmed by reduced expression of p-GSK3β (P<0.01) or p-S6RP (P<0.05), respectively, after CCI. Importantly, administration of Akt and mTOR inhibitors together (but not of either alone) improved postinjury motor (P=0.02) and cognitive deficits (hidden platform trials, P=0.001; probe trials, P<0.05), decreased propidium iodide-positive cells in CA1 and CA3 (P<0.005), and unexpectedly increased p-GSK3β in hippocampus. Although the roles of Akt and mTOR in the pathogenesis of TBI remain to be fully elucidated, dual inhibition of Akt and mTOR may have therapeutic potential for TBI.

The ubiquitous serine/threonine kinase glycogen synthase kinase 3 beta (Gsk3β) differentially regulates macrophage Toll-like receptor (TLR)-triggered pro- and anti-inflammatory cytokine programs. This study was designed to determine the in vivo role and therapeutic potential of Gsk3β modulation in tissue inflammation and injury in a murine model of liver partial warm ischemia/reperfusion injury (IRI). As a constitutively activated liver kinase, Gsk3β became quickly inactivated (phosphorylated) following IR. The active Gsk3β, however, was essential for the development of IRI pathology, as administration of its specific inhibitor, SB216763, ameliorated the hepatocellular damage, evidenced by reduced serum alanine aminotransferase (sALT) levels and well-preserved liver architecture compared with controls. The liver protective effect of Gsk3β inhibition was dependent on an immune regulatory mechanism, rather than direct cytoprotection via mitochondria permeability transition pores (MPTP). Indeed: (1) coadministration of SB216763 and atractyloside (MPTP opener) failed to abrogate a local cytoprotective Gsk3β inhibition effect; (2) SB216763 selectively inhibited IR-triggered liver pro-inflammatory, but spared interleukin (IL)-10, gene induction programs; and (3) IL-10 neutralization restored liver inflammation and IRI in SB216763-treated mice. Gsk3β inactivation by IR was a self-regulatory mechanism in liver homeostasis, critically dependent on phosphoinositide 3 (PI3)-kinase activation, as administration of a PI3 kinase inhibitor, wortmannin, reduced Gsk3 phosphorylation and augmented liver damage. In vitro, IL-10 was critical for the suppression of pro-inflammatory gene programs by Gsk3 inhibition in bone marrow-derived macrophages in response to TLR4 stimulation.

CONCLUSIONS

Our novel findings document the key immune regulatory function of Gsk3β signaling in the pathophysiology of liver IRI, and provide a rationale to target Gsk3β as a refined therapeutic strategy to ameliorate liver IRI.

In rodents, acute brain insulin action reduces blood glucose levels by suppressing the expression of enzymes in the hepatic gluconeogenic pathway, thereby reducing gluconeogenesis and endogenous glucose production (EGP). Whether a similar mechanism is functional in large animals, including humans, is unknown. Here, we demonstrated that in canines, physiologic brain hyperinsulinemia brought about by infusion of insulin into the head arteries (during a pancreatic clamp to maintain basal hepatic insulin and glucagon levels) activated hypothalamic Akt, altered STAT3 signaling in the liver, and suppressed hepatic gluconeogenic gene expression without altering EGP or gluconeogenesis. Rather, brain hyperinsulinemia slowly caused a modest reduction in net hepatic glucose output (NHGO) that was attributable to increased net hepatic glucose uptake and glycogen synthesis. This was associated with decreased levels of glycogen synthase kinase 3β (GSK3β) protein and mRNA and with decreased glycogen synthase phosphorylation, changes that were blocked by hypothalamic PI3K inhibition. Therefore, we conclude that the canine brain senses physiologic elevations in plasma insulin, and that this in turn regulates genetic events in the liver. In the context of basal insulin and glucagon levels at the liver, this input augments hepatic glucose uptake and glycogen synthesis, reducing NHGO without altering EGP.

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease characterized by skeletal muscle wasting, weakness, and myotonia. DM1 is caused by the accumulation of CUG repeats, which alter the biological activities of RNA-binding proteins, including CUG-binding protein 1 (CUGBP1). CUGBP1 is an important skeletal muscle translational regulator that is activated by cyclin D3-dependent kinase 4 (CDK4). Here we show that mutant CUG repeats suppress Cdk4 signaling by increasing the stability and activity of glycogen synthase kinase 3β (GSK3β). Using a mouse model of DM1 (HSA(LR)), we found that CUG repeats in the 3' untranslated region (UTR) of human skeletal actin increase active GSK3β in skeletal muscle of mice, prior to the development of skeletal muscle weakness. Inhibition of GSK3β in both DM1 cell culture and mouse models corrected cyclin D3 levels and reduced muscle weakness and myotonia in DM1 mice. Our data predict that compounds normalizing GSK3β activity might be beneficial for improvement of muscle function in patients with DM1.

Clinical use of nonsteroidal anti-inflammatory drugs (NSAIDs) like diclofenac (DCLF) is limited by multiple adverse effects, including renal toxicity leading to acute kidney injury. In mice with DCLF-induced nephrotoxicity, TDZD-8, a selective glycogen synthase kinase (GSK)3β inhibitor, improved acute kidney dysfunction and ameliorated tubular necrosis and apoptosis associated with induced cortical expression of cyclooxygenase-2 (COX-2) and prostaglandin E2. This renoprotective effect was blunted but still largely preserved in COX-2-null mice, suggesting that other GSK3β targets beyond COX-2 functioned in renal protection. Indeed, TDZD-8 diminished the mitochondrial permeability transition in DCLF-injured kidneys. In vitro, GSK3β inhibition reinstated viability and suppressed necrosis and apoptosis in DCLF-stimulated tubular epithelial cells. DCLF elicited oxidative stress, enhanced the activity of the redox-sensitive GSK3β, and promoted a mitochondrial permeability transition by interacting with cyclophilin D, a key component of the mitochondrial permeability transition pore. TDZD-8 blocked GSK3β activity and prevented GSK3β-mediated cyclophilin D phosphorylation and the ensuing mitochondrial permeability transition, concomitant with normalization of intracellular ATP. Conversely, ectopic expression of a constitutively active GSK3β abolished the effects of TDZD-8. Hence, inhibition of GSK3β ameliorates NSAID-induced acute kidney injury by induction of renal cortical COX-2 and direct inhibition of the mitochondrial permeability transition.

BACKGROUND

Distant metastasis is the major cause of cancer-related death, and epithelial-to-mesenchymal transition (EMT) has a critical role in this process. Accumulating evidence indicates that EMT can be regulated by microRNAs (miRNAs). miR-29c has been implicated as a tumor suppressor in several human cancers. However, the role of miR-29c in the progression of colorectal cancer (CRC) metastasis remains largely unknown.

METHODS

The expression of miR-29c was examined by qRT-PCR in a cohort of primary CRC (PC) and distant liver metastasis (LM) tissues. A series of in vivo and in vitro assays were carried out in order to elucidate the functions of miR-29c and the molecular mechanisms underlying the pathogenesis of metastatic CRC.

RESULTS

miR-29c was markedly downregulated in PCs with distant metastasis and determined to be an independent predictor of shortened patient survival. But LM tissues showed higher levels of miR-29c than that in PC tissues. In CRC cells, miR-29c dramatically suppressed cell migration and invasion abilities in vitro and cancer metastasis in vivo. In addition, miR-29c inhibited EMT and negatively regulated Wnt/β-catenin signaling pathway. Guanine nucleotide binding protein alpha13 (GNA13) and protein tyrosine phosphatase type IVA (PTP4A) were identified as direct targets of miR-29c, which acted through ERK/GSK3β/β-catenin and AKT/GSK3β/β-catenin pathways, respectively, to regulate EMT. Furthermore, significant associations between miR-29c, its target genes (GNA13 and PTP4A) and EMT markers were validated in both PC and LM tissues.

CONCLUSIONS

Our findings highlight the important role of miR-29c in regulating CRC EMT via GSK-3β/β-catenin signaling by targeting GNA13 and PTP4A and provide new insights into the metastatic basis of CRC.

Myocardial ischemia is the major cause of morbidity and mortality due to cardiovascular diseases. This disease is a severe stress condition that causes extensive biochemical changes which trigger cardiac cell death. Stress conditions such as deprivation of glucose and oxygen activate the endoplasmic reticulum in the cytoplasm of cells, including cardiomyocytes, to generate and propagate apoptotic signals in response to these conditions. microRNAs (miRNAs) are a class of small non-coding RNAs that mediate posttranscriptional gene silencing. The miRNAs play important roles in regulating cardiac physiological and pathological events such as hypertrophy, apoptosis, and heart failure. However, the roles of miRNAs in reactive oxygen species (ROS)-mediated injury on cardiomyocytes are uncertain. In this study, we identified at the apoptotic concentration of H(2)O(2), miR-26a expression was increased. To determine the potential roles of miR-26a in H(2)O(2)-mediated cardiac apoptosis, miR-26a expression was regulated by a miR-26a or an anti-miR-26a. Overexpression of miR-26a increased apoptosis as determined by upregulation of Annexin V/PI positive cell population, caspase-3 activity and expression of pro-apoptotic signal molecules, whereas inhibition of miR-26a reduced apoptosis. We identified GSK3B as a direct downstream target of miR-26a. Furthermore, miR-26a attenuated viability and increased caspase-3 activity in normal cardiomyocytes. This study demonstrates that miR-26a promotes ROS-induced apoptosis in cardiomyocytes. Thus, miR-26a affects ROS-mediated gene regulation and cellular injury response.

BACKGROUND

Interstitial fibrosis and fibrotic scar formation contribute to cardiac remodeling and loss of cardiac function in myocardial infarction (MI) and heart failure. Recent studies showed that histone deacetylase (HDAC) inhibitors retard fibrosis formation in acute MI settings. However, it is unknown whether HDAC inhibition can reverse cardiac fibrosis in ischemic heart failure. In addition, specific HDAC isoforms involved in cardiac fibrosis and myofibroblast activation are not well defined. Thus, the purpose of this study is to determine the effects of selective class I HDAC inhibition on cardiac fibroblasts activation and cardiac fibrosis in a congestive heart failure (CHF) model secondary to MI.

METHODS

MI was created by left anterior descending (LAD) coronary artery occlusion. Class I HDACs were selectively inhibited via Mocetinostat in CD90+ fibroblasts isolated from atrial and ventricular heart tissue in vitro. In vivo, Class I HDACs were inhibited in 3 weeks post MI rats by injecting Mocetinostat for the duration of 3 weeks. Cardiac function and heart tissue were analyzed at 6 weeks post MI.

RESULTS

In sham hearts, HDAC1 and HDAC2 displayed differential expression patterns where HDAC1 mainly expressed in cardiac fibroblast and HDAC2 in cardiomyocytes. On the other hand, we showed that HDAC1 and 2 were upregulated in CHF hearts, and were found to co-localize with CD90+ cardiac fibroblasts. In vivo treatment of CHF animals with Mocetinostat improved left ventricle end diastolic pressure and dp/dt max and decreased the total collagen amount. In vitro treatment of CD90+ cells with Mocetinostat reversed myofibroblast phenotype as indicated by a decrease in α-Smooth muscle actin (α-SMA), Collagen III, and Matrix metalloproteinase-2 (MMP2). Furthermore, Mocetinostat increased E-cadherin, induced β-catenin localization to the membrane, and reduced Akt/GSK3β signaling in atrial cardiac fibroblasts. In addition, Mocetinostat treatment of atrial CD90+ cells upregulated cleaved-Caspase3 and activated the p53/p21 axis.

CONCLUSIONS

Taken together, our results demonstrate upregulation of HDAC1 and 2 in CHF. In addition, HDAC inhibition reverses interstitial fibrosis in CHF. Possible anti-fibrotic actions of HDAC inhibition include reversal of myofibroblast activation and induction of cell cycle arrest/apoptosis.

The hallmark of Alzheimer's disease (AD) pathology is an accumulation of amyloid β (Aβ) and phosphorylated tau, which are encoded by the amyloid precursor protein (APP) and microtubule-associated protein tau (MAPT) genes, respectively. Less than 5% of all AD cases are familial in nature, i.e. caused by mutations in APP, PSEN1 or PSEN2. Almost all mutations found in them are related to an overproduction of Aβ1-42, which is prone to aggregation. While these genes are mutation free, their function, or those of related genes, could be compromised in sporadic AD as well. In this study, pyrosequencing analysis of post-mortem brains revealed aberrant CpG methylation in APP, MAPT and GSK3B genes of the AD brain. These changes were further evaluated by a newly developed in vitro-specific DNA methylation system, which in turn highlighted an enhanced expression of APP and MAPT. Cell nucleus sorting of post-mortem brains revealed that the methylation changes of APP and MAPT occurred in both neuronal and non-neuronal cells, whereas GSK3B was abnormally methylated in non-neuronal cells. Further analysis revealed an association between abnormal APP CpG methylation and apolipoprotein E ε4 allele (APOE ε4)-negative cases. The presence of a small number of highly methylated neurons among normal neurons contribute to the methylation difference in APP and MAPT CpGs, thus abnormally methylated cells could compromise the neural circuit and/or serve as 'seed cells' for abnormal protein propagation. Our results provide a link between familial AD genes and sporadic neuropathology, thus emphasizing an epigenetic pathomechanism for sporadic AD.

BACKGROUND

Although ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification.

RESULTS

We have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling.

CONCLUSIONS

Our findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

The pathological hallmarks of Alzheimer's disease (AD) include amyloid beta (Aβ) accumulation, neurofibrillary tangle formation, synaptic dysfunction and neuronal loss. In this study, we investigated the neuroprotection of novel osmotin, a plant protein extracted from Nicotiana tabacum that has been considered to be a homolog of mammalian adiponectin. Here, we observed that treatment with osmotin (15 μg/g, intraperitoneally, 4 hr) at 3 and 40 days post-intracerebroventricular injection of Aβ1-42 significantly ameliorated Aβ1-42-induced memory impairment in mice. These results revealed that osmotin reverses Aβ1-42 injection-induced synaptic deficits, Aβ accumulation and BACE-1 expression. Treatment with osmotin also alleviated the Aβ1-42-induced hyperphosphorylation of the tau protein at serine 413 through the regulation of the aberrant phosphorylation of p-PI3K, p-Akt (serine 473) and p-GSK3β (serine 9). Moreover, our western blots and immunohistochemical results indicated that osmotin prevented Aβ1-42-induced apoptosis and neurodegeneration in the Aβ1-42-treated mice. Furthermore, osmotin attenuated Aβ1-42-induced neurotoxicity in vitro.To our knowledge, this study is the first to investigate the neuroprotective effect of a novel osmotin against Aβ1-42-induced neurotoxicity. Our results demonstrated that this ubiquitous plant protein could potentially serve as a novel, promising, and accessible neuroprotective agent against progressive neurodegenerative diseases such as AD.

The library of integrated network-based cellular signatures (LINCS) L1000 data set currently comprises of over a million gene expression profiles of chemically perturbed human cell lines. Through unique several intrinsic and extrinsic benchmarking schemes, we demonstrate that processing the L1000 data with the characteristic direction (CD) method significantly improves signal to noise compared with the MODZ method currently used to compute L1000 signatures. The CD processed L1000 signatures are served through a state-of-the-art web-based search engine application called L1000CDS2. The L1000CDS2 search engine provides prioritization of thousands of small-molecule signatures, and their pairwise combinations, predicted to either mimic or reverse an input gene expression signature using two methods. The L1000CDS2 search engine also predicts drug targets for all the small molecules profiled by the L1000 assay that we processed. Targets are predicted by computing the cosine similarity between the L1000 small-molecule signatures and a large collection of signatures extracted from the gene expression omnibus (GEO) for single-gene perturbations in mammalian cells. We applied L1000CDS2 to prioritize small molecules that are predicted to reverse expression in 670 disease signatures also extracted from GEO, and prioritized small molecules that can mimic expression of 22 endogenous ligand signatures profiled by the L1000 assay. As a case study, to further demonstrate the utility of L1000CDS2, we collected expression signatures from human cells infected with Ebola virus at 30, 60 and 120 min. Querying these signatures with L1000CDS2 we identified kenpaullone, a GSK3B/CDK2 inhibitor that we show, in subsequent experiments, has a dose-dependent efficacy in inhibiting Ebola infection in vitro without causing cellular toxicity in human cell lines. In summary, the L1000CDS2 tool can be applied in many biological and biomedical settings, while improving the extraction of knowledge from the LINCS L1000 resource.

Aberrant activation of the Wnt/β-catenin signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the function and mechanism of Wnt/β-catenin signaling on the pathogenesis of teratocarcinoma, we used the mouse teratocarcinoma P19 cell line as a model in vitro. Gsk3β specific inhibitor (SB216763) was used to activate Wnt/β-catenin signaling. All trans-retinoic acid (RA) was used to induce P19 cell differentiation. At different culture times, gene expression was examined by immunofluorescence staining, quantitative real-time PCR, and Western-blotting; BrdU incorporation assays were performed to measure P19 cell proliferation. Small interference RNA technology was used to downregulate c-myc expression. The results showed that SB216763 induced the nuclear translocation of β-catenin, upregulated the expression of c-myc and pluripotency related genes, oct4, sox2 and nanog, and blocked cell differentiation induced by all trans-RA. The proliferation of P19 cells was significantly enhanced by SB216763, as well as c-myc overexpression. C-myc downregulation inhibited P19 cell proliferation caused by activation of Wnt/β-catenin signaling and induced P19 cell differentiation. In conclusion, activation of the Wnt/β-catenin pathway could promote the proliferation and inhibit the differentiation of mouse teratocarcinoma cells by upregulation of c-myc expression.

Disturbed Wnt signaling has been implicated in numerous diseases, including type 2 diabetes and the metabolic syndrome. In the present study, we have investigated cross-talk between insulin and Wnt signaling pathways using preadipocytes with and without knockdown of the Wnt co-receptors LRP5 and LRP6 and with and without knock-out of insulin and IGF-1 receptors. We find that Wnt stimulation leads to phosphorylation of insulin signaling key mediators, including Akt, GSK3β, and ERK1/2, although with a lower fold stimulation and slower time course than observed for insulin. These Wnt effects are insulin/IGF-1 receptor-dependent and are lost in insulin/IGF-1 receptor double knock-out cells. Conversely, in LRP5 knockdown preadipocytes, insulin-induced phosphorylation of IRS1, Akt, GSK3β, and ERK1/2 is highly reduced. This effect is specific to insulin, as compared with IGF-1, stimulation and appears to be due to an inducible interaction between LRP5 and the insulin receptor as demonstrated by co-immunoprecipitation. These data demonstrate that Wnt and insulin signaling pathways exhibit cross-talk at multiple levels. Wnt induces phosphorylation of Akt, ERK1/2, and GSK3β, and this is dependent on insulin/IGF-1 receptors. Insulin signaling also involves the Wnt co-receptor LRP5, which has a positive effect on insulin signaling. Thus, altered Wnt and LRP5 activity can serve as modifiers of insulin action and insulin resistance in the pathophysiology of diabetes and metabolic syndrome.

Muscle-invasive bladder cancer is associated with a high frequency of metastasis, and bone is the most common metastatic site outside the pelvis. To clarify its organ-specific characteristics, we generated a successive bone metastatic T24-B bladder cancer subline following tail vein injection of metastatic T24-L cells. Compared with parental T24-L cells, epithelial-like T24-B cells displayed increased adhesion but decreased migration or invasion abilities as well as up-regulation of cytokeratins and down-regulation of vimentin, N-cadherin and MMP2. Mechanically, phosphatidylinositol 3-kinase (PI3K)/Akt targets glycogen synthase kinase-3β (GSK3β)/β-catenin to control ZEB1 gene transcription, and then subsequently regulates the expression of cytokeratins, vimentin and MMP2. Importantly, ZEB1 is essential for bladder cancer invasion in vitro and distant metastasis in vivo, and ZEB1 overexpression was highly correlated with the expression of those downstream markers in clinical tumor samples. Overall, this study reveals a novel mechanism facilitating metastatic bladder cancer cell re-colonization into bone, and confirms the significance of mesenchymal-to-epithelial transition (MET) in formation of bone metastasis.