Glycogen Synthase Kinase-3 Activity Research Articles

Intraneuronal Aβ accumulation induces hippocampal neuron hyperexcitability through A-type K+ current inhibition mediated by activation of caspases and GSK-3

Amyloid β-protein (Aβ) pathologies have been linked to dysfunction of excitability in neurons of the hippocampal circuit, but the molecular mechanisms underlying this process are still poorly understood. Here, we applied whole-cell patch-clamp electrophysiology to primary hippocampal neurons and show that intracellular Aβ42 delivery leads to increased spike discharge and action potential broadening through downregulation of A-type K+ currents. Pharmacologic studies showed that caspases and glycogen synthase kinase 3 (GSK-3) activation are required for these Aβ42-induced effects. Extracellular perfusion and subsequent internalization of Aβ42 increase spike discharge and promote GSK-3-dependent phosphorylation of the Kv4.2 α-subunit, a molecular determinant of A-type K+ currents, at Ser-616. In acute hippocampal slices derived from an adult triple-transgenic Alzheimer's mouse model, characterized by endogenous intracellular accumulation of Aβ42, CA1 pyramidal neurons exhibit hyperexcitability accompanied by increased phosphorylation of Kv4.2 at Ser-616. Collectively, these data suggest that intraneuronal Aβ42 accumulation leads to an intracellular cascade culminating into caspases activation and GSK-3-dependent phosphorylation of Kv4.2 channels. These findings provide new insights into the toxic mechanisms triggered by intracellular Aβ42 and offer potentially new therapeutic targets for Alzheimer's disease treatment.

Inhibition by lithium of glycogen synthase kinase-3 (GSK-3): Possible mechanism of therapeutic action of lithium

Lithium ion (Li+) is used as mood stabilizer in mood disorder for more than 60 years, but its mode of action remains largely obscure. Due to similarities with Mg2+, Li+ affects many biological processes dependent on Mg2+. During the past 10 years, a body of evidence has highlighted the inhibition of glycogen synthase kinase-3 (GSK-3) as a possible mechanism of therapeutic action of Li+. GSK-3 corresponds to two kinase-type enzymes (GSK-3α and GSK-3β), able to phosphorylate many proteins in neuronal and non-neuronal cells and, thereby, to exert a regulatory role in many cellular functions. GSK-3 itself is negatively regulated by phosphorylation produced by several enzymes, including Akt. It is currently believed that direct inhibition of GSK-3 by Li+ has no therapeutic relevance since only observed with Li+ concentrations toxic in humans. In contrast, Li+ concentrations consistent with therapeutic action in human activate Akt and, thereby, strongly inhibit the activity of GSK-3 [1]. Experimental animal studies have shown that the inhibitory effects of Li+ on responses to psychostimulants are related to an action on GSK-3 [2]. These behavioral responses can be compared to manic episodes and these results suggest that the antimanic effect of Li+ is mediated through GSK-3 inhibition. Several other studies suggest that antidepressant-like responses of Li+, assessed by behavioral tests in animal, depend on the GSK-3 inhibition [3]. Altogether, these preclinical data tend to attribute to the GSK-3 inhibition both antimanic and antidepressant effects, and perhaps a mood stabilizing effect. However, in the absence of clear understanding of mood disorder etiology, evaluating the exact contribution of GSK-3 inhibition to clinical effects of Li+ remains a complex issue.

Rescue of impaired late–phase long-term depression in a tau transgenic mouse model

Cognitive decline, the hallmark of Alzheimer's disease, and accompanying neuropsychiatric symptoms share dysfunctions of synaptic processes as a common cellular pathomechanism. Long-term potentiation has proven to be a sensitive tool for the “diagnosis” of such synaptic dysfunctions. Much less, however, is known about how long-term depression (LTD), an alternative mechanism for the storage of memory, is affected by Alzheimer's disease progression. Here, we demonstrate that impaired late LTD (>3 hours) in THY-Tau22 mice can be rescued by either inhibition of glycogen synthase kinase-3 (GSK3β) activity or by application of the protein-phosphatase 2A agonist selenate. In line with these findings, we observed increased phosphorylation of GSK3β at Y216 and reduced total phosphatase activity in biochemical assays of hippocampal tissue of THY-Tau22 mice. Interestingly, LTD induction and pharmacologic inhibition of GSK3β appeared to downregulate GSK3ß activity via a marked upregulation of phosphorylation at the inhibitory Ser9 residue. Our results point to alterations in phosphorylation and/or dephosphorylation homeostasis as key mechanisms underlying the deficits in LTD and hippocampus-dependent learning found in THY-Tau22 mice.

Mycophenolate antagonizes IFN-γ-induced catagen-like changes via β-catenin activation in human dermal papilla cells and hair follicles.

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce β-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3β, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-β2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased β-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3β and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-β2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/β-catenin signaling, and that MPA stabilizes β-catenin by inhibiting GSK3β leading to increased β-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.

The antimicrobial protein S100A7/psoriasin enhances the expression of keratinocyte differentiation markers and strengthens the skin's tight junction barrier.

S100A7/psoriasin is a member of the S100 protein family and is encoded in the epidermal differentiation complex, which contains genes for markers of epidermal differentiation. S100A7/psoriasin is overexpressed in hyperproliferative skin diseases, where it is believed not only to exhibit antimicrobial functions, but also to induce immunomodulatory activities, including chemotaxis and cytokine/chemokine production. To evaluate the effect of S100A7/psoriasin on keratinocyte differentiation and regulation of the tight junction (TJ) barrier. Expression of differentiation markers and TJ proteins in human keratinocytes was determined by real-time polymerase chain reaction and Western blot. The changes in TJ barrier function were assessed by transepithelial electrical resistance and paracellular permeability assays. Glycogen synthase kinase-3 (GSK-3) and mitogen-activated protein kinase (MAPK) activation was analysed by Western blot, whereas β-catenin and E-cadherin activation was evaluated by Western blot and immunofluorescence. S100A7/psoriasin enhanced the expression of several differentiation markers and selectively increased the expression of TJ proteins (e.g. claudins and occludin), which are known to strengthen the TJ barrier. Furthermore, S100A7/psoriasin increased β-catenin and E-cadherin accumulation at cell-cell contact, and enhanced transepithelial electrical resistance while reducing the paracellular permeability of keratinocyte layers. The data suggest that S100A7/psoriasin-mediated regulation of the TJ barrier was via both the GSK-3 and MAPK pathways, as evidenced by the inhibitory effects of inhibitors for GSK-3 and MAPKs. Our finding that S100A7/psoriasin regulates differentiation and strengthens TJ barrier function provides novel evidence that, in addition to antimicrobial and immunoregulatory activities, S100A7/psoriasin is involved in skin innate immunity.

Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis.

Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

Snail regulated by PKC/GSK-3β pathway is crucial for EGF-induced epithelial-mesenchymal transition (EMT) of cancer cells.

Cancer metastasis is considered a major challenge in cancer therapy. Recently, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling has been shown to induce epithelial-mesenchymal transition (EMT) and thereby to promote cancer metastasis. However, the underlying mechanism has not been fully elucidated. We demonstrate that EGF can induce EMT in human prostate and lung cancer cells and thus promote invasion and migration. EGF-induced EMT has been characterized by the cells acquiring mesenchymal spindle-like morphology and increasing their expression of N-cadherin and fibronectin, with a concomitant decrease of E-cadherin. Both protein and mRNA expression of transcription factor Snail rapidly increases after EGF treatment. The knockdown of Snail significantly attenuates EGF-induced EMT, suggesting that Snail is crucial for this process. To determine the way that Snail is accumulated, we demonstrate (1) that EGF promotes the stability of Snail via inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3β), (2) that protein kinase C (PKC) rather than the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is responsible for GSK-3β inhibition and (3) that GSK-3β inhibition promotes the transcription of Snail. Taken together, these results reveal that the PKC/GSK-3β signaling pathway controls both the stability and transcription of Snail, which is crucial for EMT induced by EGF in PC-3 and A549 cells. Our study suggests a novel signaling pathway for Snail regulation and provides a better understanding of growth-factor-induced tumor EMT and metastasis.

Gene Expression Profiling in Mouse Embryonic Stem Cells Reveals Glycogen Synthase Kinase-3-Dependent Targets of Phosphatidylinositol 3-Kinase and Wnt/β-Catenin Signaling Pathways.

Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways. Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling. Genetic deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells (ESCs) has previously been shown to lead to the constitutive activation of the Wnt/β-catenin signaling pathway. However, in addition to Wnt signaling, all Gsk-3-regulated pathways, such as insulin signaling, are also affected simultaneously in Gsk-3α−/−; Gsk-3β−/−ESCs. In an effort to better understand how specific signaling pathways contribute to the global pattern of gene expression in Gsk-3α−/−; Gsk-3β−/−ESCs, we compared the gene expression profiles in Gsk-3α−/−; Gsk-3β−/− ESCs to mouse ESCs in which either Wnt/β-catenin signaling or phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling are constitutively active. Our results show that Wnt signaling has a greater effect on up-regulated genes in the Gsk-3α−/−; Gsk-3β−/−ESCs, whereas PI3K-dependent insulin signaling is more responsible for the down-regulation of genes in the same cells. These data show the importance of Gsk-3 activity on gene expression in mouse ESCs, and that these effects are due to the combined effects of multiple signaling pathways.

AKT/GSK-3β regulates stability and transcription of snail which is crucial for bFGF-induced epithelial–mesenchymal transition of prostate cancer cells

BackgroundEpithelial–mesenchymal transition (EMT) plays a pivotal role in the development of metastatic cancers. Basic fibroblast growth factor (bFGF) is significantly elevated in metastatic prostate cancers, which has been mentioned mainly to induce EMT in normal cells. However, there is no description about bFGF induced EMT and its underlying mechanism in prostate cancer cells. MethodsWestern blotting, immunofluorescence and qRT-PCR assays were used to study protein or mRNA expression profiles of the EMT. Wound healing scratch, migration and invasion assays were used to test the motility of cells undergoing EMT. More methods were used to explore the underlying mechanisms. ResultsWe demonstrated that bFGF promoted EMT and motility of human prostate cancer PC-3 cells. Both protein and mRNA expression of Snail were rapidly increased after bFGF treatment. Ectopic expression of Snail triggered EMT and enhanced cell motility in PC-3 cells, and knockdown of Snail almost abolished bFGF induced EMT, suggesting the critical role of Snail. Mechanistic study demonstrated that bFGF promoted the stability, nuclear localization and transcription of Snail by inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3β) through phosphatidylinositide 3 kinases (PI3K)/protein kinase B (AKT) signaling pathway. ConclusionsIt is concluded that bFGF can promote EMT and motility of PC-3 cells, and AKT/GSK-3β signaling pathway controls the stability, localization and transcription of Snail which is crucial for this bFGF induced EMT. General significanceTo our knowledge, this is the first study to demonstrate that bFGF can induce EMT via AKT/GSK-3β/Snail signaling pathway in prostate cancer cells.

High glucose reduces hepatic glycogenesis by suppression of microRNA‑152.

MicroRNAs (miRNAs) are a class of non‑coding 18‑25-nucleotide endogenous RNA molecules. miRNAs act as specific gene silencers to regulate target gene expression at the posttranscriptional level, by base pairing to the 3'‑untranslated region of the target mRNA. miR‑152 is an miRNA that was originally identified in cancer cells, and was shown to be able to modulate the expression of specific oncogenes and tumor suppressor genes, leading to enhanced carcinoma growth. However, little is known regarding the role of miR‑152 in the regulation of hepatic insulin resistance and glucose metabolism. In the present study, it was identified that the activation of AKT and glycogen synthase kinase 3 (GSK‑3), and the expression levels of glycogen, were reduced in mouse NCTC 1469 hepatocytes and mouse primary hepatocytes, following exposure to 25 mM glucose for 48 h. Furthermore, it was demonstrated that high glucose levels suppressed the expression of miR‑152 in hepatocytes. In order to further assess the effects of miR‑152 on the glucose‑induced reduction of glycogen synthesis and activation of AKT and GSK, miR‑152 mimic and inhibitor were transfected into the NCTC 1469 cells, respectively. The transfection of the miR‑152 inhibitor resulted in reduced expression of glycogen, accompanied by impaired phosphorylation of AKT and GSK in the NCTC 1469 cells treated with or without glucose. Conversely, upregulation of miR‑152 by transfection of an miR‑152 mimic reversed the glucose‑induced decrease in glycogen synthesis and reduced AKT and GSK phosphorylation in hepatocytes. This indicated that miR‑152 could modulate the AKT/GSK pathway and glycogen synthesis. In conclusion, to the best of our knowledge, this study was the first to indicate that high glucose impaired the activation of the AKT/GSK pathway and the synthesis of glycogen in mouse hepatocytes, in part through the downregulation of miR‑152.

Glycogen synthase kinase 3 inhibition stimulates human cartilage destruction and exacerbates murine osteoarthritis.

To assess the role of glycogen synthase kinase 3 (GSK-3) as a regulator of cartilage destruction in human tissue and a murine model of osteoarthritis (OA). Surgical destabilization of the medial meniscus (DMM) was performed to induce experimental murine OA, and joint damage was assessed histologically. Bovine nasal and human OA cartilage samples were incubated with interleukin-1 (IL-1) plus oncostatin M (OSM) and GSK-3 inhibitor. Collagen and proteoglycan release was assessed by hydroxyproline measurement and dye binding assay, collagenase activity was assessed by bioassay, and gene expression was analyzed by real-time polymerase chain reaction. Human articular chondrocytes were isolated by enzymatic digestion and cultured prior to gene silencing and immunoblotting of cell lysates and nuclear fractions. Mice treated with GSK-3 inhibitor exhibited significantly greater cartilage damage compared with sham-operated control mice. GSK-3 inhibition in bovine cartilage dramatically accelerated IL-1 plus OSM-stimulated degradation, concomitant with a profound increase in collagenase activity. GSK-3 inhibitor induced collagen release from human OA cartilage in the presence of IL-1 plus OSM and increased proteoglycan loss. Gene expression profiling of resorbing OA cartilage revealed a marked procatabolic switch in gene expression upon GSK-3 inhibition. This was mirrored in human articular chondrocytes following GSK3 silencing, particularly with the GSK-3β isoform. GSK-3 inhibition or silencing led to enhanced IL-1 plus OSM-stimulated abundance and activity of Jun, and silencing of c-jun ameliorated GSK-3 inhibitor-mediated procatabolic gene expression. GSK-3 is an important regulator of matrix metalloproteinase (MMP)-mediated joint destruction, the inhibition of which by proinflammatory stimuli de-represses catabolic gene expression. Therapeutic strategies that maintain cartilage GSK-3 activity may therefore help curtail aberrant MMP activity during pathologic joint destruction.

Reduced striatal dopamine DA D2 receptor function in dominant-negative GSK-3 transgenic mice

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with constitutive activity involved in cellular architecture, gene expression, cell proliferation, fate decision and apoptosis, among others. GSK-3 expression is particularly high in brain where it may be involved in neurological and psychiatric disorders such as Alzheimer׳s disease, bipolar disorder and major depression. A link with schizophrenia is suggested by the antipsychotic drug-induced GSK-3 regulation and by the involvement of the Akt/GSK-3 pathway in dopaminergic neurotransmission. Taking advantage of the previous development of dominant negative GSK-3 transgenic mice (Tg) showing a selective reduction of GSK-3 activity in forebrain neurons but not in dopaminergic neurons, we explored the relationship between GSK-3 and dopaminergic neurotransmission in vivo. In microdialysis experiments, local quinpirole (DA D2-R agonist) in dorsal striatum reduced dopamine (DA) release significantly less in Tg mice than in wild-type (WT) mice. However, local SKF-81297 (selective DA D1-R agonist) in dorsal striatum reduced DA release equally in both control and Tg mice indicating a comparable function of DA D1-R in the direct striato-nigral pathway. Likewise, systemic quinpirole administration – acting preferentially on presynaptic DA D2- autoreceptors to modulate DA release-reduced striatal DA release similarly in both control and Tg mice. Quinpirole reduced locomotor activity and induced c-fos expression in globus pallidus (both striatal DA D2-R-mediated effects) significantly more in WT than in Tg mice. Taking together, the present results show that dominant negative GSK-3 transgenic mice show reduced DA D2-R-mediated function in striatum and further support a link between dopaminergic neurotransmission and GSK-3 activity.

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.

Abstract 67: GSK-3 Dephosphorylation by PP2A Modulates Age-dependent Cardiac Growth and Hypertrophic Response

Glycogen synthase kinase-3 (GSK-3) is a known negative regulator of cardiac hypertrophic response and much remains unknown about its dephosphorylation process that activates GSK-3. Since GSK-3 function is regulated by PI3K, age based assessment of cardiac hypertrophic response was measured in PI3Kγ knockout (PI3Kγ KO) mice. Interestingly, we observed marked reduction in heart size in PI3Kγ KO mice compared to littermate controls. Consistent with the reduced size, we observed elevated PP2A activity and dephosphorylation mediated activation of GSK-3 in PI3Kγ-KO hearts. Mechanistically, we found that higher PP2A activity in PI3Kγ-KO was due to PP2A methylation mediated by elevated PP2A methyl transferase (PPMT-1) activity. To test in vivo whether PI3K activity regulates GSK-3 dephosphorylation mediated cardiac growth, we generated mice with cardiac specific overexpression of inactive PI3Kγ (PI3Kγinact) and constitutively active PI3Kγ (myrPI3Kγ) in the PI3Kγ-KO background. Age based hypertrophic response and cardiac function was assessed by heart weight/body weight ratios and echocardiography. The heart size was significantly increased in PI3Kγinact (LVEDD- 3.41+/- 0.12) and myrPI3Kγ (LVEDD- 3.73 +/- 0.14) overexpressing mice, when compared to WT (LVEDD- 3.00 +/- 0.15) mice at 18 months age, indicating that kinase activity of PI3Kγ is inconsequential for age-dependent regulation of cardiac growth. Similar cardiac growth due to overexpression of active and inactive form of PI3Kγ suggests that the kinase independent function potentially overrides the kinase activity of PI3Kγ. Correspondingly, PP2A activity was normalized and was associated with decreased GSK-3 dephosphorylation with overexpression of PI3Kγinact. This led to normalization of heart size in these mice compared to PI3Kγ-KO littermates correlating with the decreased GSK-3 dephosphorylation and consequent inhibition of GSK-3 activity. Regulation of PP2A mediated dephosphorylation of GSK-3 by PI3Kγ combined with similar level of increased cardiac growth in PI3Kγ-KO mice with cardiac overexpression of PI3Kγinact or myrPI3Kγ, suggests a non-canonical role of PI3Kγ in hypertrophy of the heart due to age related increase in mechanical demand.

Regulation of methylglyoxal-elicited leukocyte recruitment by endothelial SGK1/GSK3 signaling

Excessive levels of the glycolysis metabolite methylglyoxal (MG) elicit enhanced expression of adhesion molecules which foster leukocyte–endothelial cell interactions. The signaling mechanisms involved remain elusive. To address this, we investigated the signal transduction of leukocyte- and endothelial-expressed phosphoinositide 3-kinase (PI3K) effector kinases glycogen synthase kinase 3 (GSK3) and serum- and glucocorticoid-inducible kinase 1 (SGK1) in the regulation of MG-elicited leukocyte recruitment. Using intravital microscopy of mouse cremasteric microvasculature, we demonstrate that GSK3 inhibitors lithium and SB216763 mitigate MG-elicited leukocyte recruitment and microvascular hyperpermeability. In SVEC4-10EE2 endothelial cells, but not in neutrophils, MG transiently activates GSK3 by reducing inhibitory phospho-GSK3α/β (Ser21/9) which parallels decrease of phospho-Akt at early time points (<30min). At later time points (≥1h), MG induces GSK3 deactivation which is dissipated by siRNA silencing of SGK. MG treatment potentiates endothelial SGK1 mRNA, total SGK1, phospho-SGK1 and phospho-NDRG1. The SGK1 inhibitor GSK650394 attenuates MG-elicited leukocyte recruitment. Pharmacological inhibition or silencing endothelial GSK3 or SGK attenuates MG-triggered nuclear factor (NF)-κB activity. Furthermore, silencing SGK blunts MG-triggered redox-sensitive phosphorylation of endothelial transcription factor CREB. Inhibition of SGK1 or GSK3 mitigates the expression of endothelial adhesion molecules P- and E-selectins and ICAM-1. Moreover, SGK1-dependent CREB activation participates in MG-elicited ICAM-1 upregulation. We conclude that temporal activation of endothelial SGK1 and GSK3 is decisive in MG-elicited upregulation of transcription factors, adhesion molecule expression, and leukocyte-vascular endothelium interactions. This novel signaling pathway may link excessive MG levels in vivo to inflammation, thus, unraveling potential therapeutic targets.

S100A4 negatively regulates β-catenin by inducing the Egr-1-PTEN-Akt-GSK3β degradation pathway

S100A4, also known as the mts1 gene, has been reported as an invasive and metastatic marker for many types of cancers. S100A4 interacts with various target genes that affect tumor cell metastasis; however, little is known about cellular signaling pathways elicited by S100A4. In the current study, we demonstrate an inhibitory effect of S100A4 on β-catenin signaling in breast cancer cells. By overexpressing S100A4 in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, we observed the down-regulation of β-catenin expression and β-catenin-dependent TCF/LEF transcriptional activities. The activity of GSK3β, which phosphorylates β-catenin and induces proteasomal degradation of β-catenin, was increased in S100A4-overexpressing cell lines. Blocking Glycogen Synthase Kinase (GSK3β) activity by lithium chloride or Dvl gene overexpression restored β-catenin expression. We also found that increased GSK3β activity was due to decrease in Akt activity resulting from Egr-1-induced phosphatase and tensin homolog (PTEN) expression. S100A4 induced Egr-1 nuclear localization by increasing the association between Egr-1 and importin-7 and this effect was reduced in S100A4 mutants that harbored a defect in nuclear localization signals. Collectively, we verify herein that S100A4 may act as a tumor suppressor in breast cancers by down-regulating the central signaling axis for tumor cell survival.

Beneficial synergistic effects of microdose lithium with pyrroloquinoline quinone in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is a complicated, neurodegenerative disorder involving multifactorial pathogeneses and still lacks effective clinical treatment. Recent studies show that lithium exerts disease-modifying effects against AD. However, the intolerant side effects at conventional effective dosage limit the clinical use of lithium in treating AD. To explore a novel AD treatment strategy with microdose lithium, we designed and synthesized a new chemical, tri-lithium pyrroloquinoline quinone (Li3PQQ), to study the synergistic effects of low-dose lithium and pyrroloquinoline quinone, a native compound with powerful antioxidation and mitochondrial amelioration. The results showed that Li3PQQ at a relative low dose (6 and 12 mg/kg) exhibited more powerful effects in restoring the impairment of learning and memory, facilitating hippocampal long-term potentiation, and reducing cerebral amyloid deposition and phosphorylated tau level in APP/PS1 transgenic mice than that of lithium chloride at both low and high dose (5 and 100 mg/kg). We further found that Li3PQQ inhibited the activity of glycogen synthase kinase-3 and increased the activity of β-amyloid-binding alcohol dehydrogenase, which might underlie the beneficial effects of Li3PQQ on APP/PS1 transgenic mice. Our study demonstrated the efficacy of a novel AD therapeutic strategy targeting at multiple disease-causing mechanisms through the synergistic effects of microdose lithium and pyrroloquinoline quinone.

Clinicopathological and biological significance of aberrant activation of glycogen synthase kinase-3 in ovarian cancer.

BackgroundGlycogen synthase kinase-3 (GSK-3) plays an important role in human cancer. The aim of this study is to evaluate the clinicopathological significance of expression of GSK-3α/β and pGSK-3α/βTyr279/216 in patients with epithelial ovarian cancer and to investigate whether GSK-3 inhibition can influence cell viability and tumor growth of ovarian cancer.MethodsImmunohistochemistry was used to examine expression of GSK-3α/β and pGSK-3α/βTyr279/216 in 71 human epithelial ovarian cancer tissues and correlations between protein expression, and clinicopathological factors were analyzed. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following exposure of ovarian carcinoma cells to pharmacological inhibitors of GSK-3 or GSK-3 small interfering RNA. In vivo validation of tumor growth inhibition was performed with xenograft mice.ResultsThe expression levels of GSK-3α/β and pGSK-3α/βTyr279/216 in ovarian cancers were significantly higher than those in benign tumors. High expression of GSK-3α/β was more likely to be found in patients with advanced International Federation of Gynecology and Obstetrics (FIGO) stages and high serum cancer antigen 125. Higher expression of pGSK-3α/βTyr279/216 was associated with advanced FIGO stages, residual tumor mass, high serum cancer antigen 125, and poor chemoresponse. Worse overall survival was revealed by Kaplan–Meier survival curves in patients with high expression of GSK-3α/β or pGSK-3α/βTyr279/216. Multivariate analysis indicated that FIGO stage, GSK-3α/β expression, and pGSK-3α/βTyr279/216 expression were independent prognostic factors for overall survival. GSK-3 inhibition by lithium chloride, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), or GSK-3 small interfering RNA can decrease viability of SKOV3 and SKOV3-TR30 ovarian cancer cells. Additionally, lithium chloride-treated SKOV3 xenograft mice had a significant reduction in tumor growth compared with control-treated animals.ConclusionOur findings suggest that overexpression and aberrant activation of GSK-3 may contribute to progression and poor prognosis in ovarian cancer. Inhibition of GSK-3 may be a potential therapy for ovarian cancer.

GSK-3 as potential target for therapeutic intervention in cancer.

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified and studied in the regulation of glycogen synthesis. GSK-3 functions in a wide range of cellular processes. Aberrant activity of GSK-3 has been implicated in many human pathologies including: bipolar depression, Alzheimer's disease, Parkinson's disease, cancer, non-insulin-dependent diabetes mellitus (NIDDM) and others. In some cases, suppression of GSK-3 activity by phosphorylation by Akt and other kinases has been associated with cancer progression. In these cases, GSK-3 has tumor suppressor functions. In other cases, GSK-3 has been associated with tumor progression by stabilizing components of the beta-catenin complex. In these situations, GSK-3 has oncogenic properties. While many inhibitors to GSK-3 have been developed, their use remains controversial because of the ambiguous role of GSK-3 in cancer development. In this review, we will focus on the diverse roles that GSK-3 plays in various human cancers, in particular in solid tumors. Recently, GSK-3 has also been implicated in the generation of cancer stem cells in various cell types. We will also discuss how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTORC1, Ras/Raf/MEK/ERK, Wnt/beta-catenin, Hedgehog, Notch and others.

Abstract 659: PKR-Like Endoplasmic Reticulum Kinase and Glycogen Synthase Kinase-3α/ß Signaling Regulates Endoplasmic Reticulum Stress-Induced Foam Cell Formation

Background: Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. The molecular mechanisms by which conditions of ER stress promote pro-atherogenic processes are not understood. We have found that ER stress-inducing agents can activate glycogen synthase kinase (GSK)-3α/β, a protein involved in many metabolic pathways. The objective of this study is to investigate the role of GSK3α/β in pro-atherogenic ER stress signaling. Methods and Results: Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin or palmitate, in the presence or absence of the GSK3α/β inhibitor CT99021. GSK3α/β inhibition did not affect the adaptive unfolded protein response (UPR), but did block ER stress-induced lipid accumulation as well as the up regulation of genes associated with lipid biosynthesis and uptake. Using small molecule inhibitors of specific UPR pathways, we found that PERK, but not IRE1 or ATF6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition attenuated ER stress-induced expression of distal components of the PERK pathway, including CHOP and ATF4. Atherosclerotic plaques from ApoE-/- mice, fed a diet supplemented with the GSK3α/β inhibitor valproate, had reduced levels of CHOP within the macrophage foam cells. In primary mouse macrophages PERK inhibition blocked ER stress induced lipid accumulation whereas the overexpression of constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. Conclusions: Pharmacological inhibition of GSK3α/β attenuates ER stress-induced lipid accumulation and macrophage foam cell formation. These findings indicate that GSK3α/β is an important factor in the ER stress-PERK signaling pathway and may play a central role in the pro-atherogenic dysregulation of lipid metabolism.