Out of the breast cancer subtypes, triple-negative breast cancer (TNBC) has the poorest prognosis without effective targeted therapies. Metformin, a first-line drug for type 2 diabetes mellitus, was demonstrated to target breast cancer stem cells selectively. However, the efficiency and the mechanism of action of metformin in TNBC are unclear. In this study, we demonstrated that metformin decreased the percentage of TNBC stem cells partially through the downregulation of the expression of the stem cell transcription factor Krüppel-like factor 5 (KLF5) and its downstream target genes, such as Nanog and FGF-BP1, in TNBC cell lines. Metformin induced glycogen synthase kinase-3β (GSK3β)-mediated KLF5 protein phosphorylation and degradation through the inhibition of protein kinase A (PKA) activity in TNBC cells. Consistently, PKA activators increased the expression levels of KLF5. We observed a positive correlation between p-CREB, p-GSK3β, KLF5 and FGF-BP1 protein levels in human TNBC samples. These findings suggest that metformin suppresses TNBC stem cells partially through the PKA-GSK3β-KLF5 signaling pathway.

Synthetic lethality and collateral lethality are two well-validated conceptual strategies for identifying therapeutic targets in cancers with tumour-suppressor gene deletions. Here, we explore an approach to identify potential synthetic-lethal interactions by screening mutually exclusive deletion patterns in cancer genomes. We sought to identify 'synthetic-essential' genes: those that are occasionally deleted in some cancers but are almost always retained in the context of a specific tumour-suppressor deficiency. We also posited that such synthetic-essential genes would be therapeutic targets in cancers that harbour specific tumour-suppressor deficiencies. In addition to known synthetic-lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic-essential gene in PTEN-deficient cancers. In PTEN-deficient prostate and breast cancers, CHD1 depletion profoundly and specifically suppressed cell proliferation, cell survival and tumorigenic potential. Mechanistically, functional PTEN stimulates the GSK3β-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via the β-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in stabilization of CHD1, which in turn engages the trimethyl lysine-4 histone H3 modification to activate transcription of the pro-tumorigenic TNF-NF-κB gene network. This study identifies a novel PTEN pathway in cancer and provides a framework for the discovery of 'trackable' targets in cancers that harbour specific tumour-suppressor deficiencies.

P63 is a p53 family member involved in multiple facets of biology, including embryonic development, cell proliferation, differentiation, survival, apoptosis, senescence and aging. The p63 gene encodes multiple protein isoforms either with (TAp63) or without (ΔNp63) the N-terminal transactivation domain. Amounting evidence suggests that p63 can function as a tumor suppressor, yet the precise molecular mechanisms, and particularly the specific roles of TAp63 and ΔNp63 in cancer progression, are still largely unclear. Here, we demonstrated that ΔNp63α, the predominant isoform expressed in epithelial cells and squamous cell carcinomas, inhibits cell invasion. Affymetrix gene expression profiling, combined with gain- and loss-of-function analyses and chromatin immunoprecipitation, indicated that cluster of differentiation 82 (CD82), a documented metastasis suppressor, is a direct transcriptional target of ΔNp63α. Expression of ΔNp63α inhibited outgrowth in Matrigel and cancer cell invasion, which was largely reversed by specific ablation of CD82. Conversely, ΔNp63α knockdown led to increased cell invasion, which was reversed by ectopic expression of CD82. Moreover, inhibition of glycogen synthase kinase-3β (GSK3β) by either pharmacological inhibitors or by RNA interference resulted in the downregulation of ΔNp63α and CD82 expression, concomitant with increased cell invasion, independently of β-catenin. Furthermore, decreased expression of p63 and CD82 is correlated with cancer progression. Taken together, this study reveals that ΔNp63α upregulates CD82 to inhibit cell invasion, and suggests that GSK3β can regulate cell invasion by modulating the ΔNp63α-CD82 axis.

Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). Recent data strongly suggests the important role of miRNAs in cancer progression and therapeutic response. Here, we have established a radioresistant human GBM cell line U87R derived from parental U87 and found miR-135b expression was upregulated in U87R cells. miR-135b knockdown reversed radioresistance of U87R cells, and miR-135b overexpression enhanced radioresistance of U87 cells. Mechanically, bioinformatics analysis combined with experimental analysis demonstrated GSK3β (Glycogen synthase kinase 3 beta) was a novel direct target of miR-135b. Moreover, GSK3β protein expression was downregulated in U87R cells and restored expression of GSK3β increased radiosensitivity of U87R cells. In addition, clinical data indicated that the expression of miR-135b or GSK3β was significantly association with IR resistance of GBM samples. Our findings suggest miR-135b is involved in the radioresistance of human GBM cells and miR-135b-GSK3β axis may be a novel candidate for developing rational therapeutic strategies for human GBM treatment.

BACKGROUND

A growing amount of evidence has indicated that PSAT1 is an oncogene that plays an important role in cancer progression and metastasis. In this study, we explored the expression and function of PSAT1 in estrogen receptor (ER)-negative breast cancer.

METHODS

The expression level of PSAT1 in breast cancer tissues and cells was analyzed using real-time-PCR (RT-PCR), TCGA datasets or immunohistochemistry (IHC). The overall survival of patients with ER-negative breast cancer stratified by the PSAT1 expression levels was evaluated using Kaplan-Meier analysis. The function of PSAT1 was analyzed using a series of in vitro assays. Moreover, a nude mouse model was used to evaluate the function of PSAT1 in vivo. qRT-PCR and western blot assays were used to evaluate gene and protein expression, respectively, in the indicated cells. In addition, we demonstrated that PSAT1 was activated by ATF4 by chromatin immunoprecipitation (ChIP) assays.

RESULTS

mRNA expression of PSAT1 was up-regulated in ER-negative breast cancer. A tissue microarray that included 297 specimens of ER-negative breast cancer was subjected to an immunohistochemistry assay, which demonstrated that PSAT1 was overexpressed and predicted a poor clinical outcome of patients with this disease. Our data showed that PSAT1 promoted cell proliferation and tumorigenesis in vitro and in vivo. We further found that PSAT1 induced up-regulation of cyclin D1 via the GSK3β/β-catenin pathway, which eventually led to the acceleration of cell cycle progression. Furthermore, ATF4 was also overexpressed in ER-negative breast cancers, and a positive correlation between the ATF4 and PSAT1 mRNA levels was observed in ER-negative breast cancers. We further demonstrated that knockdown of ATF4 by siRNA reduced PSAT1 expression. Finally, chromatin immunoprecipitation (ChIP) assays showed that PSAT1 was a target of ATF4.

CONCLUSIONS

PSAT1, which is overexpressed in ER-negative breast cancers, is activated by ATF4 and promotes cell cycle progression via regulation of the GSK3β/β-catenin/cyclin D1 pathway.

Adverse environmental conditions such as hypobaric hypoxia (HH) cause memory impairment by affecting cellular machinery leading to neurodegeneration. Providing enriched environment (EE) is found to be beneficial for curing several neurodegenerative disorders. The protective role of EE in preventing HH induced neuronal death has been reported previously but the involved mechanism is still not clearly understood. The present study is an attempt to verify the impact of EE on spatial memory during HH and also to explore the possible role of neurotrophin in EE mediated neuroprotection. Signaling mechanism involved in neuroprotection was also explored. Male Sprague Dawley rats were simulated to HH condition in an Animal Decompression Chamber at an altitude of 25000 feet in standard and enriched cages for 7 days. Spatial memory was assessed through Morris Water Maze. Role of different neurotrophins was explored by gene silencing and inhibitors for their respective receptors. Further, using different blockers signaling pathway was also explored. Finding of the present study suggested that EE prevents HH mediated memory impairment and neurodegeneration. Also brain-derived neurotrophic factor (BDNF) plays a major role in EE mediated neuroprotection and it effectively prevented neurodegeneration by activating PI3K/AKT pathway resulting in GSK3β inactivation which further inhibits apoptosis. Moreover GSK3β phosphorylation and hence its inactivation upregulates CREB phosphorylation which may also accounts for activation of survival machinery in cells and provides neuroprotection. From these observations it can be postulated that EE has a therapeutic potential in amelioration of HH induced memory impairment and neurodegeneration. Hence it may be used as a non invasive and non pharmacological intervention against various neurological disorders.

Insulin and insulin-like growth factor 1 (IGF-1) receptor signaling pathways differentially modulate cardiac growth under resting conditions and following exercise training. These effects are mediated by insulin receptor substrate 1 (IRS1) and IRS2, which also differentially regulate resting cardiac mass. To determine the role of IRS isoforms in mediating the hypertrophic and metabolic adaptations of the heart to exercise training, we subjected mice with cardiomyocyte-specific deletion of either IRS1 (CIRS1 knockout [CIRS1KO] mice) or IRS2 (CIRS2KO mice) to swim training. CIRS1KO hearts were reduced in size under basal conditions, whereas CIRS2KO hearts exhibited hypertrophy. Following exercise swim training in CIRS1KO and CIRS2KO hearts, the hypertrophic response was equivalently attenuated, phosphoinositol 3-kinase (PI3K) activation was blunted, and prohypertrophic signaling intermediates, such as Akt and glycogen synthase kinase 3β (GSK3β), were dephosphorylated potentially on the basis of reduced Janus kinase-mediated inhibition of protein phosphatase 2a (PP2A). Exercise training increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein content, mitochondrial capacity, fatty acid oxidation, and glycogen synthesis in wild-type (WT) controls but not in IRS1- and IRS2-deficient hearts. PGC-1α protein content remained unchanged in CIRS1KO but decreased in CIRS2KO hearts. These results indicate that although IRS isoforms play divergent roles in the developmental regulation of cardiac size, these isoforms exhibit nonredundant roles in mediating the hypertrophic and metabolic response of the heart to exercise.

OBJECTIVE

Growing evidences indicate that microRNAs (miRNAs) are involved in cardiac hypertrophy development. Multiple miRNAs have been identified as diagnostic and prognostic biomarkers of cardiac hypertrophy, as well as potential therapeutic tools. The present study aimed to investigate the functions and regulatory mechanisms of miR-21-3p in cardiac hypertrophy.

RESULTS

Decreased expression of miR-21-3p was observed in cardiac hypertrophy induced by transverse aortic constriction (TAC) and angiotensin (Ang) II infusion in mice. To further explore the role of miR-21-3p in cardiac hypertrophy, rAAV-miR-21-3p was administered intravenously in mice. Overexpression of miR-21-3p markedly suppressed TAC-induced cardiac hypertrophy and also blocked Ang II-induced cardiac hypertrophy as determined by cardiac function measurement and biomarker detection. Furthermore, western blot assays showed that histone deacetylase-8 (HDAC8) was silenced by miR-21-3p, and luciferase reporter assays showed that miR-21-3p binds to the 3' UTR of HDAC8. Moreover, re-expression of HDAC8 attenuated miR-21-3p-mediated suppression of cardiac hypertrophy by enhancing phospho-Akt and phospho-Gsk3β expression.

CONCLUSIONS

Our data reveal a role of miR-21-3p in regulating HDAC8 expression and Akt/Gsk3β pathway, and suggest that modulation of miR-21-3p levels may provide a therapeutic approach for cardiac hypertrophy.

The impact of mosquito-borne flavivirus infections worldwide is significant, and many critical aspects of these viruses' biology, including virus-host interactions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent reemergence and spread of flaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlight the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression posttranscriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and antiflaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on the replication of three flaviviruses, DENV, WNV, and Japanese encephalitis virus (JEV), using a high-content immunofluorescence screen. Several families of miRNAs were identified as inhibiting multiple flaviviruses, including the miRNA miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/β-catenin signaling, demonstrated strong antiflaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and confirmed that these miRNAs potentiate interferon regulatory factor 3 (IRF3) phosphorylation and translocation to the nucleus, the induction of IFN-responsive genes, and the release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of glycogen synthase kinase 3β (GSK3β)-TANK-binding kinase 1 (TBK1) binding, inducing TBK1 to phosphorylate IRF3 and initiate downstream IFN signaling. In this way, we have identified a novel cellular signaling network with a critical role in regulating the replication of multiple virus families. These findings highlight the opportunities for using miRNAs as tools to discover and characterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research.IMPORTANCE MicroRNAs are a class of small regulatory RNAs that modulate cellular processes through the posttranscriptional repression of multiple transcripts. We hypothesized that individual miRNAs may be capable of inhibiting viral replication through their effects on host proteins or pathways. To test this, we performed a high-content screen for miRNAs that inhibit the replication of three medically relevant members of the flavivirus family: West Nile virus, Japanese encephalitis virus, and dengue virus 2. The results of this screen identify multiple miRNAs that inhibit one or more of these viruses. Extensive follow-up on members of the miR-34 family of miRNAs, which are active against all three viruses as well as the closely related Zika virus, demonstrated that miR-34 functions through increasing the infected cell's ability to respond to infection through the interferon-based innate immune pathway. Our results not only add to the knowledge of how viruses interact with cellular pathways but also provide a basis for more extensive data mining by providing a comprehensive list of miRNAs capable of inhibiting flavivirus replication. Finally, the miRNAs themselves or cellular pathways identified as modulating virus infection may prove to be novel candidates for the development of therapeutic interventions.

Insulin rapidly suppresses hepatic glucose production and slowly decreases expression of genes encoding gluconeogenic proteins. In this study, we show that an immediate effect of insulin is to redirect newly synthesized glucose-6-phosphate to glycogen without changing the rate of gluconeogenesis. This process requires hepatic Akt2, as revealed by blunted insulin-mediated suppression of glycogenolysis in the perfused mouse liver, elevated hepatic glucose production during a euglycemic-hyperinsulinemic clamp, or diminished glycogen accumulation during clamp or refeeding in mice without hepatic Akt2. Surprisingly, the absence of Akt2 disrupted glycogen metabolism independent of GSK3α and GSK3β phosphorylation, which is thought to be an essential step in the pathway by which insulin regulates glycogen synthesis through Akt. These data show that (1) the immediate action of insulin to suppress hepatic glucose production functions via an Akt2-dependent redirection of glucose-6-phosphate to glycogen, and (2) insulin increases glucose phosphorylation and conversion to glycogen independent of GSK3.

BACKGROUND

Hepatocellular carcinoma (HCC) is a common and aggressive cancer, and the treatment options are limited for patients with advanced HCC. Bufalin, the major digoxin-like component of the traditional Chinese medicine Chansu, exhibits significant anti-tumor activities in many tumor cell lines. In the present study, we investigated the effect of bufalin on the inhibition of an AKT-related signaling pathway, and examined the relationship between regulatory proteins and anti-tumor effects in hepatoma cells.

METHODS

Proliferation, wound healing, transwell-migration/invasion and adhesion assays were performed in HCCLM3 and HepG2 cell lines. The protein levels of pAKT, AKT, pGSK3β, GSK3β, pβ-catenin, β-catenin, E-cadherin, MMP-9, and MMP-2 were measured by western blot analysis. E-Cadherin and β-catenin expression levels were also evaluated by immunofluorescence.

RESULTS

Bufalin inhibited hepatoma cell proliferation, migration, invasion and adhesion. In addition, treatment with bufalin significantly decreased the levels of pAKT, pGSK3β, MMP-9, and MMP-2, while increasing the levels of GSK3β and E-cadherin and suppressing the nuclear translocation of β-catenin.

CONCLUSIONS

Bufalin is a potential anti-HCC therapeutic candidate through its inhibition of the AKT/GSK3β/β-catenin/E-cadherin signaling pathway. Further studies with bufalin are warranted in patients with HCC, especially those with the disease at advanced stages.

Tobacco smoking has been correlated with a lower incidence of Alzheimer's disease (AD). This negative correlation has been attributed to nicotine's properties. However, the undesired side-effects of nicotine and the absence of clear evidence of positive effects of this drug on the cognitive abilities of AD patients have decreased the enthusiasm for its therapeutic use. In this review, we discuss evidence showing that cotinine, the main metabolite of nicotine, has many of the beneficial effects but none of the negative side-effects of its precursor. Cotinine has been shown to be neuroprotective, to improve memory in primates as well as to prevent memory loss, and to lower amyloid-beta (Aβ)) burden in AD mice. In AD, cotinine's positive effect on memory is associated with the inhibition of Aβ aggregation, the stimulation of pro-survival factors such as Akt, and the inhibition of pro-apoptotic factors such as glycogen synthase kinase 3 beta (GSK3β). Because stimulation of the α7 nicotinic acetylcholine receptors (α7nAChRs) positively modulates these factors and memory, the involvement of these receptors in cotinine's effects are discussed. Because of its beneficial effects on brain function, good safety profile, and nonaddictive properties, cotinine may represent a new therapeutic agent against AD.

Translationally controlled tumor protein (TCTP), is a highly conserved protein involved in fundamental processes, such as cell proliferation and growth, tumorigenesis, apoptosis, pluripotency, and cell cycle regulation. TCTP also inhibits Na,K-ATPase whose subunits have been suggested as a marker of epithelial-to-mesenchymal transition (EMT), a crucial step during tumor invasiveness, metastasis and fibrosis. We hypothesized that, TCTP might also serve as an EMT inducer. This study attempts to verify this hypothesis. We found that overexpression of TCTP in a porcine renal proximal tubule cell line, LLC-PK1, induced EMT-like phenotypes with the expected morphological changes and appearance of EMT related markers. Conversely, depletion of TCTP reversed the induction of these EMT phenotypes. TCTP overexpression also enhanced cell migration via activation of mTORC2/Akt/GSK3β/β-catenin, and invasiveness by activating MMP-9. Moreover, TCTP depletion in melanoma cells significantly reduced pulmonary metastasis by inhibiting the development of mesenchymal-like phenotypes. Overall, these findings support our hypothesis that TCTP is a positive regulator of EMT and suggest that modulation of TCTP expression is a potential approach to inhibit the invasiveness and migration of cancer cells and the attendant pathologic processes including metastasis.

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and a systemic pro-inflammatory response. Here we show that tumour necrosis factor receptor-associated factor 3 (TRAF3) is upregulated in mouse and human livers with hepatic steatosis. After 24 weeks on a high-fat diet (HFD), obesity, insulin resistance, hepatic steatosis and inflammatory responses are significantly ameliorated in liver-specific TRAF3-knockout mice, but exacerbated in transgenic mice overexpressing TRAF3 in hepatocytes. The detrimental effects of TRAF3 on hepatic steatosis and related pathologies are confirmed in ob/ob mice. We further show that in response to HFD, hepatocyte TRAF3 binds to TGF-β-activated kinase 1 (TAK1) to induce TAK1 ubiquitination and subsequent autophosphorylation, thereby enhancing the activation of downstream IKKβ-NF-κB and MKK-JNK-IRS1(307) signalling cascades, while disrupting AKT-GSK3β/FOXO1 signalling. The TRAF3-TAK1 interaction and TAK1 ubiquitination are indispensable for TRAF3-regulated hepatic steatosis. In conclusion, hepatocyte TRAF3 promotes HFD-induced or genetic hepatic steatosis in a TAK1-dependent manner.

Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase involved in human cancers including glioblastoma. We have previously demonstrated that GSK3β inhibition enhances temozolomide effect in glioma cells. In this report, we investigated the molecular mechanisms of sensitization of glioblastoma cells to temozolomide by GSK3β inhibition, focusing on O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing. Glioblastoma tissues from patients treated with the GSK3β-inhibiting drugs were subjected to immunohistochemistry and methylation-specific PCR assay. Human glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3β inhibitor, AR-A014418 or GSK3β-specific small interfering RNA. The combined effect of temozolomide and AR-A014418 on cell proliferation was determined by AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by methylation-specific PCR and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative reverse transcriptase-PCR. c-Myc and DNA (cytosine-5)-methyltransferase 3A binding to the MGMT promoter was estimated by chromatin immunoprecipitation assay. GSK3β inhibition decreased phosphorylation of glycogen synthase and reduced MGMT expression and increased MGMT promoter methylation in clinical tumors. In glioblastoma cell lines, GSK3β inhibition decreased cell viability, enhanced temozolomide effect and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of DNA (cytosine-5)-methyltransferase 3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3β inhibition enhances temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation.

Moderate calorie restriction (CR) (∼60% of ad libitum, AL, intake) has been associated with numerous favorable physiological outcomes in many species, and the insulin/IGF-1 and mTOR signaling pathways have each been proposed as potential mediators for many of CR's bioeffects. However, few studies have assessed the widely held idea that CR induces the down-regulation of the insulin/IGF-1 and/or mTOR pathways in multiple tissues. Accordingly, we analyzed the phosphorylation status of 11 key signaling proteins from the insulin/IGF-1 (IR(Tyr1162/1163), IGF-1R(Tyr1135/1136), IRS-1(Ser312), PTEN(Ser380), Akt(Ser473), GSK3α(Ser21), GSK3β(Ser9)) and mTOR (TSC2(Ser939), mTOR(Ser2448), P70S6K(Thr412), RPS6(Ser235/236)) pathways in 11 diverse tissues [liver, kidney, lung, aorta, two brain regions (cortex and cerebellum), and two slow-twitch and three fast-twitch skeletal muscles] from 9-month-old male AL and CR Fischer 344 x Brown Norway rats. The rats were studied under two conditions: with endogenous insulin levels (i.e., AL>CR) and with insulin infused during a hyperinsulinemic-euglycemic clamp so that plasma insulin concentrations were matched between the two diet groups. The most striking and consistent effect of CR was greater pAkt in 3 of the 5 skeletal muscles of CR vs. AL rats. There were no significant CR effects on the mTOR signaling pathway and no evidence that CR caused a general attenuation of mTOR signaling across the tissues studied. Rather than supporting the premise of a global downregulation of insulin/IGF-1 and/or mTOR signaling in many tissues, the current results revealed clear tissue-specific CR effects for the insulin signaling pathway without CR effects on the mTOR signaling pathway.

BACKGROUND

The epithelial-mesenchymal transition (EMT) induced by growth factors or cytokines, particularly transforming growth factor-β (TGF-β1), plays an important role in kidney tubulointerstitial injury. However, signaling pathways mediating TGF-β1-induced EMT are not precisely known. In this study, we examined the role of Akt2 on EMT.

METHODS

HK-2 cells were exposed to 10 ng/ml TGF-β1 to establish a model of EMT. The expression of proteins were detected by western blot assay and Immunofluorescence. The levels of genes were tested by RT-PCR.

RESULTS

We found that treatment of HK-2 cells, a human proximal tubular cell line, with 10 ng/ml TGF-β1 resulted in activation of phosphatidylinositol 3-kinase (PI3K)/Akt2 signaling as evidenced by increased p-PI3K, Akt2 and p-Akt (Ser 473) expression. Importantly, TGF-β1 treatment decreased zona occludins 1 (ZO-1) and E-cadherin (epithelial markers) expression, increased fibronectin and vimentin (mesenchymal makers) expression, which were prevented by Ly294002 (the inhibitor of PI3K) or small interfering RNA (siAkt2), suggesting that Akt2 mediated TGF-β1-induced EMT. Meanwhile, RNA and protein levels of Snail1, the key inducer of EMT, were significantly elevated in TGF-β1-treated HK-2 cells. TGF-β1 also induced inactivation of glycogen synthase kinase-3β (GSK3β), an endogenous inhibitor of Snail. Knockdown of Akt2 using siRNAs or the PI3K inhibitor Ly294002 inhibited TGF-β1-induced phosphorylation of GSK3β and expression of Snail1.

CONCLUSIONS

These findings revealed that knockdown of Akt2 antagonized TGF-β1-induced EMT by inhibiting GSK3β/Snail signaling pathway.

Chemokine (C-C motif) ligand 18 (CCL18), which is derived from tumour-associated macrophages (TAMs), plays a critical role in promoting breast cancer metastasis via its receptor, PYK2 N-terminal domain interacting receptor 1 (Nir1). However, the molecular mechanism by which Nir1 promotes breast cancer metastasis by binding to CCL18 remains elusive. In this study, Nir1 expression was associated with lymph node and distant metastasis in patients with invasive ductal carcinoma. For the first time, we report that Nir1 binding to CCL18 promotes the phosphorylation of Akt, LIN-11, Isl1 and MEC-3 protein domain kinase (LIMK), and cofilin, which is a critical step in cofilin recycling and actin polymerisation. Interestingly, Nir1 binding to CCL18 can enhance cell mesenchymal properties and induce epithelial-mesenchymal transition (EMT). Mechanistically, Nir1 binding to CCL18 stabilises Snail via the Akt/GSK3β signalling pathway. In support of these observations, Nir1 binding to CCL18 promoted lung metastasis and LY294002 could inhibit it in vivo. In summary, our in vitro and in vivo results indicate that Nir1 binding to CCL18 plays an important role in breast cancer invasion/metastasis. This study identified both Nir1 and CCL18 as potential anti-invasion targets for therapeutic intervention in breast cancer.

Three theories of regeneration dominate neuroscience today, all purporting to explain why the adult central nervous system (CNS) cannot regenerate. One theory proposes that Nogo, a molecule expressed by myelin, prevents axonal growth. The second theory emphasizes the role of glial scars. The third theory proposes that chondroitin sulfate proteoglycans (CSPGs) prevent axon growth. Blockade of Nogo, CSPG, and their receptors indeed can stop axon growth in vitro and improve functional recovery in animal spinal cord injury (SCI) models. These therapies also increase sprouting of surviving axons and plasticity. However, many investigators have reported regenerating spinal tracts without eliminating Nogo, glial scar, or CSPG. For example, many motor and sensory axons grow spontaneously in contused spinal cords, crossing gliotic tissue and white matter surrounding the injury site. Sensory axons grow long distances in injured dorsal columns after peripheral nerve lesions. Cell transplants and treatments that increase cAMP and neurotrophins stimulate motor and sensory axons to cross glial scars and to grow long distances in white matter. Genetic studies deleting all members of the Nogo family and even the Nogo receptor do not always improve regeneration in mice. A recent study reported that suppressing the phosphatase and tensin homolog (PTEN) gene promotes prolific corticospinal tract regeneration. These findings cannot be explained by the current theories proposing that Nogo and glial scars prevent regeneration. Spinal axons clearly can and will grow through glial scars and Nogo-expressing tissue under some circumstances. The observation that deleting PTEN allows corticospinal tract regeneration indicates that the PTEN/AKT/mTOR pathway regulates axonal growth. Finally, many other factors stimulate spinal axonal growth, including conditioning lesions, cAMP, glycogen synthetase kinase inhibition, and neurotrophins. To explain these disparate regenerative phenomena, I propose that the spinal cord has evolved regenerative mechanisms that are normally suppressed by multiple extrinsic and intrinsic factors but can be activated by injury, mediated by the PTEN/AKT/mTOR, cAMP, and GSK3b pathways, to stimulate neural growth and proliferation.

Rhizomelic chondrodysplasia punctata (RCDP) is a developmental disorder characterized by hypotonia, cataracts, abnormal ossification, impaired motor development, and intellectual disability. The underlying etiology of RCDP is a deficiency in the biosynthesis of ether phospholipids, of which plasmalogens are the most abundant form in nervous tissue and myelin; however, the role of plasmalogens in the peripheral nervous system is poorly defined. Here, we used mouse models of RCDP and analyzed the consequence of plasmalogen deficiency in peripheral nerves. We determined that plasmalogens are crucial for Schwann cell development and differentiation and that plasmalogen defects impaired radial sorting, myelination, and myelin structure. Plasmalogen insufficiency resulted in defective protein kinase B (AKT) phosphorylation and subsequent signaling, causing overt activation of glycogen synthase kinase 3β (GSK3β) in nerves of mutant mice. Treatment with GSK3β inhibitors, lithium, or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) restored Schwann cell defects, effectively bypassing plasmalogen deficiency. Our results demonstrate the requirement of plasmalogens for the correct and timely differentiation of Schwann cells and for the process of myelination. In addition, these studies identify a mechanism by which the lack of a membrane phospholipid causes neuropathology, implicating plasmalogens as regulators of membrane and cell signaling.

By virtue of its unique interactions with kidney cells, lithium became an important research tool in renal physiology and pathophysiology. Investigators have uncovered the intricate relationships of lithium with the vasopressin and aldosterone systems, and the membrane channels or transporters regulated by them. While doing so, their work has also led to 1) questioning the role of adenylyl cyclase activity and prostaglandins in lithium-induced suppression of aquaporin-2 gene transcription; 2) unraveling the role of purinergic signaling in lithium-induced polyuria; and 3) highlighting the importance of the epithelial sodium channel (ENaC) in lithium-induced nephrogenic diabetes insipidus (NDI). Lithium-induced remodeling of the collecting duct has the potential to shed new light on collecting duct remodeling in disease conditions, such as diabetes insipidus. The finding that lithium inhibits glycogen synthase kinase-3β (GSK3β) has opened an avenue for studies on the role of GSK3β in urinary concentration, and GSK isoforms in renal development. Finally, proteomic and metabolomic profiling of the kidney and urine in rats treated with lithium is providing insights into how the kidney adapts its metabolism in conditions such as acquired NDI and the multifactorial nature of lithium-induced NDI. This review provides state-of-the-art knowledge of lithium as a versatile tool for understanding the molecular physiology of the kidney, and a comprehensive view of how this tool is challenging some of our long-standing concepts in renal physiology, often with paradigm shifts, and presenting paradoxical situations in renal pathophysiology. In addition, this review points to future directions in research where lithium can lead the renal community.

Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16-22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a(+)-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription.

The high rate of non-responders to initial treatment with antidepressants requires subsequent treatment strategies such as augmentation of antidepressants. Clinical guidelines recommend lithium augmentation as a first-line treatment strategy for non-responding depressed patients. The objectives of this review were to discuss the current place of lithium augmentation in the management of treatment-resistant depression and to review novel findings concerning lithium's mechanisms of action. We conducted a comprehensive and critical review of randomized, placebo-controlled trials, controlled and naturalistic comparator studies, and continuation-phase and discontinuation studies of lithium augmentation in major depression. The outcomes of interest were efficacy, factors allowing outcome prediction and results from preclinical studies investigating molecular mechanisms of lithium action. Substantial efficacy of lithium augmentation in the acute treatment of major depression has been demonstrated in more than 30 open-label studies and 10 placebo-controlled trials. In a meta-analysis addressing the efficacy of lithium in 10 randomized, controlled trials, it had a significant positive effect versus placebo, with an odds ratio of 3.11 corresponding to a number-needed-to-treat (NNT) of 5 and a mean response rate of 41.2% (versus 14.4% in the placebo group). The main limitations of these studies were the relatively small numbers of study participants and the fact that most studies included augmentation of tricyclic antidepressants, which are not in widespread use anymore. Evidence from continuation-phase studies is sparse but suggests that lithium augmentation should be maintained in the lithium-antidepressant combination for at least 1 year to prevent early relapses. Concerning outcome prediction, single studies have reported associations of better outcome rates with more severe depressive symptomatology, significant weight loss, psychomotor retardation, a history of more than three major depressive episodes and a family history of major depression. Additionally, one study suggested a predictive role of the -50T/C single nucleotide polymorphism of the glycogen synthase kinase 3 beta (GSK3B) gene in the probability of response to lithium augmentation. With regard to novel mechanisms of action, GABAergic, neurotrophic and genetic effects might explain the effects of lithium augmentation. In conclusion, augmentation of antidepressants with lithium remains a first-line, evidence-based management option for patients with major depression who have not responded adequately to antidepressants. While the mechanisms of action are currently widely studied, further clinical research on the role of lithium potentiation of the current generation of antidepressants is warranted to reinforce its role as a gold-standard treatment for patients who respond inadequately to antidepressants.

Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.