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The myristoylated zeta inhibitory peptide (ZIP), which was originally developed as a protein kinase C/Mζ (PKCζ/PKMζ) inhibitor, is known to produce the loss of different forms of memories. However, ZIP induces memory loss even in the absence of PKMζ, and its mechanism of action, therefore, remains elusive. Here, through a kinome-wide screen, we found that glycogen synthase kinase 3 beta (GSK-3β) was robustly activated by ZIP in vitro. ZIP induced depotentiation (a cellular substrate of memory erasure) of conditioning-induced potentiation at LA synapses, and the ZIP-induced depotentiation was prevented by a GSK-3β inhibitor, 6-bromoindirubin-3-acetoxime (BIO-acetoxime). Consistently, GSK-3β inhibition by BIO-acetoxime infusion or GSK-3β knockdown by GSK-3β shRNA in the LA attenuated ZIP-induced disruption of learned fear. Furthermore, conditioned fear was decreased by expression of a non-inhibitable form of GSK-3β in the LA. Our findings suggest that GSK-3β activation is a critical step for ZIP-induced disruption of memory.

Neurons synthesizing gonadotropin-inhibitory hormone (GnIH) have been implicated in the control of reproduction, food intake and stress. Serotonin (5-HT) receptors have been shown in GnIH neurons; however, their functional role in the regulation of GnIH neurons remains to be elucidated. In this study, we measured intracellular calcium ion levels following 5-HT treatment to hypothalamic primary cultures of enhanced fluorescent green protein-tagged GnIH (EGFP-GnIH) neurons from Wistar rat pups of mixed sex. Three days after initial seeding of the primary cultures, the test groups were pre-treated with lithium chloride to selectively inhibit glycogen synthase kinase 3 beta to promote intracellular calcium levels, whereas the control groups received culture medium with no lithium chloride treatment. 24 h later, the cultures were incubated with rhodamine-2AM (rhod-2AM) calcium indicator dye for one hour prior to imaging. 5-HT was added to the culture dishes 5 min after commencement of imaging. Analysis of intracellular calcium levels in EGFP-GnIH neurons showed that pre-treatment with lithium chloride before 5-HT treatment resulted in significant increase in intracellular calcium levels, two times higher than the baseline. This suggests that lithium chloride enhances the responsiveness of GnIH neurons to 5-HT.

Lithium is an effective, well-established treatment for bipolar disorder (BD). However, the mechanisms of its action, and reasons for variations in clinical response, are unclear. We used neural precursor cells (NPCs) and lymphoblastoid cell lines (LCLs), from BD patients characterized for clinical response to lithium (using the “Alda scale” and “NIMH Retrospective Life chart method”), to interrogate cellular phenotypes related to both disease and clinical lithium response. NPCs from two biologically related BD patients who differed in their clinical response to lithium were compared with healthy controls. RNA-Seq and analysis, mitochondrial membrane potential (MMP), cell viability, and cell proliferation parameters were assessed, with and without in vitro lithium. These parameters were also examined in LCLs from 25 BD patients (16 lithium responders and 9 non-responders), and 12 controls. MMP was lower in both NPCs and LCLs from BD; but it was reversed with in vitro lithium only in LCLs, and this was unrelated to clinical lithium response. The higher cell proliferation observed in BD was unaffected by in vitro lithium. Cell death was greater in BD. However, LCLs from clinical lithium responders could be rescued by addition of in vitro lithium. In vitro lithium also enhanced BCL2 and GSK3B expression in these cells. Our findings indicate cellular phenotypes related to the disease (MMP, cell proliferation) in both NPCs and LCLs; and those related to clinical lithium response (cell viability, BCL2/GSK3B expression) in LCLs.

The present study was undertaken to explore the functional involvement of Hh signaling and its regulatory mechanism in endometrial hyperplasia. Differential expression of Hh signaling molecules i.e., Ihh, Shh, Gli1 or Gsk3β was observed in endometrial hyperplasial (EH) cells as compared to normal endometrial cells. Estradiol induced the expression of Hh signaling molecules and attenuated the expression of Gsk3β whereas anti-estrogen (K1) or progestin (MPA) suppressed these effects in EH cells. Cyclopamine treatment or Gli1 siRNA knockdown suppressed the growth of EH cells and reduced the expression of proliferative markers. Estradiol also induced the nuclear translocation of Gli1 which was suppressed by both MPA and K1 in EH cells. While exploring non-canonical mechanism, LY-294002 (Gsk3β activator) caused a decrease in Gli1 expression indicating the involvement of Gsk3β in Gli1 regulation. Further, Gsk3β silencing promoted the expression and nuclear translocation of Gli1 demonstrating that Gsk3β serves as a negative kinase regulator of Gli1 in EH cells. Similar attenuation of Hh signaling molecules was observed in rats with uterine hyperplasia undergoing anti-estrogen treatment. The study suggested that Hh/Gli1 cascade (canonical pathway) as well as Gsk3β-Gli1 crosstalk (non-canonical pathway) play crucial role in estrogen-dependent cell proliferation in endometrial hyperplasia.

The molecular mechanisms of thymosin beta-4 (TB4) involved in regulating hepatic stellate cell (HSC) functions remain unclear. Therefore, we hypothesize that TB4 influences HSC activation through hedgehog (Hh) pathway. HSC functions declined in a TB4 siRNA-treated LX-2. TB4 suppression down-regulated both integrin linked kinase (ILK), an activator of smoothened, and phosphorylated glycogen synthase kinase 3 beta (pGSK-3B), an inactive form of GSK-3B degrading glioblastoma 2 (GLI2), followed by the decreased expression of both smoothened and GLI2. A TB4 CRISPR also blocked the activation of primary HSCs, with decreased expression of smoothened, GLI2 and ILK compared with cells transfected with nontargeting control CRISPR. Double immunostaining and an immunoprecipitation assay revealed that TB4 interacted with either smoothened at the cytoplasm or GLI2 at the nucleus in LX-2. Smoothened suppression in primary HSCs using a Hh antagonist or adenovirus transduction decreased TB4 expression with the reduced activation of HSCs. Tb4-overexpressing transgenic mice treated with CCl4 were susceptible to the development hepatic fibrosis with higher levels of ILK, pGSK3b, and Hh activity, as compared with wild-type mice. These findings demonstrate that TB4 regulates HSC activation by influencing the activity of Smoothened and GLI2, suggesting TB4 as a novel therapeutic target in liver disease.

Tuberous sclerosis (TS) is a multi-organ autosomal dominant disorder that is best characterized by neurodevelopmental deficits and the presence of benign tumors. TS pathology is caused by mutations in tuberous sclerosis complex (TSC) genes and is associated with insulin resistance, decreased glycogen synthase kinase 3β (GSK3β) activity, activation of the mammalian target of rapamycin complex 1 (mTORC1), and subsequent increase in protein synthesis. Here, we show that extracellular signal–regulated kinases (ERK1/2) respond to insulin stimulation and integrate insulin signaling to phosphorylate and thus inactivate GSK3β, resulting in increased protein synthesis that is independent of Akt/mTORC1 activity. Inhibition of ERK1/2 in Tsc2−/− cells—a model of TS—rescues GSK3β activity and protein synthesis levels, thus highlighting ERK1/2 as a potential therapeutic target for the treatment of TS.

Bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate diverse processes including cell proliferation, differentiation, and migration. However, their roles in cardiac differentiation and cardiomyocyte proliferation have not been explored. Using a 96-well differentiation platform for generating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) we found that S1P and LPA can independently enhance cardiomyocyte generation when administered at an early stage of differentiation. We showed that the combined S1P and LPA treatment of undifferentiated hiPSCs resulted in increased nuclear accumulation of β-catenin, the canonical Wnt signaling pathway mediator, and synergized with CHIR99021, a glycogen synthase kinase 3 beta inhibitor, to enhance mesodermal induction and subsequent cardiac differentiation. At later stages of cardiac differentiation, the addition of S1P and LPA resulted in cell cycle initiation in hiPSC-CMs, an effect mediated through increased ERK signaling. Although the addition of S1P and LPA alone was insufficient to induce cell division, it was able to enhance β-catenin-mediated hiPSC-CM proliferation. In summary, we demonstrated a developmental stage-specific effect of bioactive lipids to enhance hiPSC-CM differentiation and proliferation via modulating the effect of canonical Wnt/β-catenin and ERK signaling. These findings may improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.

Aberrant activation of the canonical Wingless type (Wnt) signaling pathway plays a key role in the development of hypertrophic scars and keloids, and this aberrant activation of Wnt pathway can be a potential target for the development of novel anti-fibrotic agents. In this study, we evaluated the anti-fibrotic potential of a soluble Wnt decoy receptor (sLRP6E1E2)-expressing non-replicating adenovirus (Ad; dE1-k35/sLRP6E1E2) on human dermal fibroblasts (HDFs), keloid fibroblasts (KFs), and keloid tissue explants. Higher Wnt3a and β-catenin expression was observed in the keloid region compared to the adjacent normal tissues. The activity of β-catenin and mRNA expression of type-I and -III collagen were significantly decreased following treatment with dE1-k35/sLRP6E1E2 in HDFs and KFs. The expression of LRP6, β-catenin, phosphorylated glycogen synthase kinase 3 beta, Smad 2/3 complex, and TGF-β1 were decreased in Wnt3a- or TGF-β1-activated HDFs, following administration of dE1-k35/sLRP6E1E2. Moreover, dE1-k35/sLRP6E1E2 markedly inhibited nuclear translocation of both β-catenin and Smad 2/3 complex. The expression levels of type-I and -III collagen, fibronectin, and elastin were also significantly reduced in keloid tissue explants after treatment with dE1-k35/sLRP6E1E2. These results indicate that Wnt decoy receptor-expressing Ad can degrade extracellular matrix in HDFs, KFs, and primary keloid tissue explants, and thus it may be beneficial for treatment of keloids.

Reprogramming of adult somatic cells into induced pluripotent stem cells holds great promise in clinical therapy. Increasing evidences have shown that p53 and its target genes play important roles in somatic cell reprogramming. In this study, we report that PHLDA3, a p53 target gene, functions as a blockage of iPSCs generation by activating the Akt-GSK3β pathway. Furthermore, PHLDA3 is found to be transcriptionally regulated by Oct4. These findings reveal that PHLDA3 acts as a new member of the regulatory network of somatic cell reprogramming.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable nonselective cation channel and can be activated during ischemia/reperfusion (I/R). This study tested whether blockade of TRPV4 can alleviate myocardial I/R injury in mice. TRPV4 expression began to increase at 1 h, reached statistically at 4 h, and peaked at 24–72 h. Treatment with the selective TRPV4 antagonist HC-067047 or TRPV4 knockout markedly ameliorated myocardial I/R injury as demonstrated by reduced infarct size, decreased troponin T levels and improved cardiac function at 24 h after reperfusion. Importantly, the therapeutic window for HC-067047 lasts for at least 12 h following reperfusion. Furthermore, treatment with HC-067047 reduced apoptosis, as evidenced by the decrease in TUNEL-positive myocytes, Bax/Bcl-2 ratio, and caspase-3 activation. Meanwhile, treatment with HC-067047 attenuated the decrease in the activation of reperfusion injury salvage kinase (RISK) pathway (phosphorylation of Akt, ERK1/2, and GSK-3β), while the activation of survival activating factor enhancement (SAFE) pathway (phosphorylation of STAT3) remained unchanged. In addition, the anti-apoptotic effects of HC-067047 were abolished by the RISK pathway inhibitors. We conclude that blockade of TRPV4 reduces apoptosis via the activation of RISK pathway, and therefore might be a promising strategy to prevent myocardial I/R injury.

Several studies indicate that erythropoietin (EPO) has remarkable neuroprotective effects in various central nervous system disorders, while little is known about the effects of EPO in diabetes-associated cognitive dysfunction. Therefore, the present study aimed to investigate whether EPO ameliorates diabetes-associated cognitive dysfunction in vivo and in vitro. We investigated the protective effects of EPO on high-glucose (HG)-induced PC12 cell death and oxidative stress. The effects of EPO (300 U/kg administered three times a week for 4 weeks) on diabetes-associated cognitive decline were investigated in diabetic rats. EPO significantly increased cell viability, increased the activity of superoxide dismutase, decreased the production of malondialdehyde and reactive oxygen species, and decreased the apoptosis rate. Additionally, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the protective effects of EPO in HG-treated PC12 cells. In diabetic rats, EPO prevented deficits in spatial learning and memory in the Morris water maze test. The results of real-time PCR and Western blotting showed that EPO upregulated EPO receptor, PI3K, and phosphorylated Akt2 relative to unphosphorylated Akt2 (p-Akt2/Akt2) and downregulated glycogen synthase kinase-3β (GSK-3β). These studies demonstrate that EPO is an effective neuroprotective agent in the context of diabetes-associated cognitive dysfunction and show that this effect involves the PI3K/Akt/GSK-3β pathway.

Glycogen synthase kinase 3β (GSK3β) is a multifunctional protein kinase involved in many cellular activities including development, differentiation and diseases. GSK3β is thought to be constitutively activated by autophosphorylation at Tyr216 and inactivated by phosphorylation at Ser9. The GSK3β activity has previously been evaluated by inhibitory Ser9 phosphorylation, but it does not necessarily indicate the kinase activity itself. Here, we applied the Phos-tag SDS-PAGE technique to the analysis of GSK3β phosphoisotypes in cells and brains. There were three phosphoisotypes of GSK3β; double phosphorylation at Ser9 and Tyr216, single phosphorylation at Tyr216 and the nonphosphorylated isotype. Active GSK3β with phosphorylation at Tyr216 represented half or more of the total GSK3β in cultured cells. Although levels of phospho-Ser9 were increased by insulin treatment, Ser9 phosphorylation occurred only in a minor fraction of GSK3β. In mouse brains, GSK3β was principally in the active form with little Ser9 phosphorylation, and the phosphoisotypes of GSK3β changed depending on the regions of the brain, age, sex and disease conditions. These results indicate that the Phos-tag SDS-PAGE method provides a simple and appropriate measurement of active GSK3β in vivo, and the activity is regulated by the mechanism other than phosphorylation on Ser9.

Levodopa (L-dopa) is the dominating therapy drug for exogenous dopaminergic substitution and can alleviate most of the manifestations of Parkinson’s disease (PD), but long-term therapy is associated with the emergence of L-dopa-induced dyskinesia (LID). Evidence points towards an involvement of Glycogen Synthase Kinase-3β (GSK-3β) in development of LID. In the present study, we found that animals rendered dyskinetic by L-dopa treatment, administration of TDZD8 (2mg/kg) obviously prevented the severity of AIM score, as well as improvement in motor function (P

The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3β phosphorylation and β-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1G93A ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration.

The NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a critical role in protecting against oxidative stress in brain ischemia and reperfusion injury. Glycogen synthase kinase 3β (GSK-3β) may play a critical role in regulating Nrf2 in a Kelch-like ECH-associated protein 1 (Keap1)-independent manner. However, the relationship between GSK-3β and Nrf2 in brain ischemia and reperfusion injury is not clear. In this study, we explored the mechanisms through which GSK-3β regulates Nrf2 and Nrf-2/ARE pathways in vitro and in vivo. We used oxygen and glucose deprivation/reoxygenation (OGD/R) in primary cultured cortical neurons and a middle cerebral artery occlusion-reperfusion (MCAO/R) rat model to mimic ischemic insult. In this study, GSK-3β siRNA and inhibitors (SB216763 and LiCl) were used to inhibit GSK-3β in vitro and in vivo. After inhibiting GSK-3β, expression of total and nuclear Nrf2, Nrf2-ARE binding activity and expression of Nrf2/ARE pathway-driven genes HO-1 and NQO-1 increased. Overexpression of GSK-3β yielded opposite results. These results suggest that GSK-3β downregulates Nrf2 and the Nrf2/ARE pathway in brain ischemia and reperfusion injury. GSK-3β may be an endogenous antioxidant relevant protein and may represent a new therapeutic target in treatment of ischemia and reperfusion injury.

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase is frequently inactivated by the oncogenic signalling kinases PI3K/Akt and MAPK/ERK in diverse malignancies. The present study was designed to investigate GSK-3β signalling circuits in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model and the therapeutic potential of the neem limonoid nimbolide. Inactivation of GSK-3β by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and β-catenin was associated with increased cell proliferation and apoptosis evasion during stepwise evolution of HBP carcinomas. Administration of nimbolide inhibited PI3K/Akt signalling with consequent activation of GSK-3β thereby inducing trafficking of β-catenin away from the nucleus and enhancing the expression of miR-126 and let-7. Molecular docking studies confirmed interaction of nimbolide with PI3K, Akt, ERK and GSK-3β. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1Thr286 and pro-apoptotic proteins. The present study has unravelled aberrant phosphorylation as a key determinant for oncogenic signalling and acquisition of cancer hallmarks in the HBP model. The study has also provided mechanistic insights into the chemotherapeutic potential of nimbolide that may be a useful addition to the armamentarium of natural compounds targeting PI3K for oral cancer treatment.

Aggregation of highly phosphorylated tau is a hallmark of Alzheimer’s disease and other tauopathies. Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase glycogen-synthase-kinase-3beta (GSK-3β). Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated and nor did they contain beta-pleated sheet structure. This may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others. Whether these are inert or actively protective remains to be established. Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.

Glycogen synthase kinase-3 beta (GSK-3β) has been investigated as a therapeutic target for numerous human diseases including cancer because of their diverse cellular functions. Although GSK-3β inhibitors have been investigated as anticancer reagents, precise biological mechanisms remain to be determined. In this study, we investigated the anticancer effects of GSK-3β inhibitors on cancer cell lines and observed centrosome dysregulation which resulted in abnormal mitosis. Mitotic checkpoints sensed the mitotic abnormalities and induced apoptosis. For cells that were inherently resistant to apoptosis, cell death distinct from apoptosis was induced. After GSK-3β inhibitor treatment, these cells exhibited characteristic features of mitotic catastrophe, including distended and multivesiculated nuclei and inappropriate reductions in cyclin B1 expression. This suggested that mitotic catastrophe was an alternative mechanism in cells resistant to apoptosis. Although the role of GSK-3β in centrosomes has not yet been clarified, phosphorylated GSK-3β was localised in centrosomes. From these data, GSK-3β seems to regulate centrosome function. Thus, we propose that centrosome dysregulation is an important mechanism for the anticancer effects of GSK-3β inhibitors and that mitotic catastrophe serves as a safe-guard system to remove cells with any mitotic abnormalities induced by GSK-3β inhibition.

We have previously reported that expression of NMI (N-myc and STAT interactor) is compromised in invasive breast cancers. We also demonstrated that loss of NMI expression promotes epithelial-mesenchymal-transition and results in enhanced invasive ability of breast cancer cells. Additionally we had demonstrated that restoration of NMI expression reduced breast cancer xenograft growth and downregulated Wnt and TGFβ/SMAD signaling. Here we present our observations that NMI expression drives autophagy. Our studies were promoted by our observation that NMI expressing breast cancer cells showed autophagic vacuoles and LC3 processing. Additionally, we found that NMI expression increased the cisplatin sensitivity of the breast cancer cells. Our mechanistic investigations show that NMI prompts activation of GSK3-β. This multifunctional kinase is an upstream effector of the TSC1/TSC2 complex that regulates mTOR signaling. Inhibition of GSK3-β activity in NMI expressing cells activated mTOR signaling and decreased the cells’ autophagic response. Additionally we demonstrate that a key component of autophagy, DNA-damage regulated autophagy modulator 1 (DRAM1), is regulated by NMI. Our TCGA database analysis reveals concurrent expression of NMI and DRAM1 in breast cancer specimens. We present evidence that NMI sensitizes breast cancer cells to cisplatin treatment through DRAM1 dependent autophagy.

Abnormal tau hyperphosphorylation is an early pathological marker of Alzheimer’s disease (AD), however, the upstream factors that regulate tau phosphorylation are not illustrated and there is no efficient strategy to arrest tau hyperphosphorylation. Here, we find that activation of endogenous EphB2 receptor by ligand stimulation (ephrinB1/Fc) or by ectopic expression of EphB2 plus the ligand stimulation induces a remarkable tau dephosphorylation at multiple AD-associated sites in SK-N-SH cells and human embryonic kidney cells that stably express human tau (HEK293-tau). In cultured hippocampal neurons and the hippocampus of human tau transgenic mice, dephosphorylation of tau proteins was also detected by stimulation of EphB2 receptor. EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase and activates phosphatidylinositol-3-kinase (PI3K)/Akt both in vitro and in vivo, whereas simultaneous inhibition of PI3K or upregulation of GSK-3β abolishes the EphB2 stimulation-induced tau dephosphorylation. Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation. We conclude that activation of EphB2 receptor kinase arrests tau hyperphosphorylation through PI3K-/Akt-mediated GSK-3β inhibition. Our data provide a novel membranous target to antagonize AD-like tau pathology.

Environment enrichment (EE) has a variety of effects on brain structure and function. Brain-derived neurotrophic factor (BDNF) is essential for EE-induced hippocampal neurogenesis and memory enhancement. However, the intracellular pathway downstream of BDNF to modulate EE effects is poorly understood. Here we show that integrin-linked kinase (ILK) levels are elevated upon EE stimuli in a BDNF-dependent manner. Using ILK-shRNA (siILK) lentivirus, we demonstrate that knockdown of ILK impairs EE-promoted hippocampal neurogenesis and memory by increasing glycogen synthase kinase-3β (GSK3β) activity. Finally, overexpressing ILK in the hippocampus could rescue the neurogenesis and memory deficits in BDNF+/− mice. These results indicate that ILK is indispensable for BDNF-mediated hippocampal neurogenesis and memory enhancement upon EE stimuli via regulating GSK3β activity. This is a new insight of the precise mechanism in EE-enhanced memory processes and ILK is a potentially important therapeutic target that merits further study.

Tert-butylhydroquinone (TBHQ) is an antioxidant compound which shows multiple cytoprotective actions. We evaluated the effects of TBHQ on pathological cardiac remodeling and dysfunction induced by chronic overload. Pressure overload was created by transverse aortic constriction (TAC) in male C57BL/6 mice. TBHQ was incorporated in the diet and administered for 4 weeks. TBHQ treatment prevented left ventricular dilatation and cardiac dysfunction induced by TAC and decreased the prevalence of myocardial apoptosis. The beneficial effects of TBHQ were associated with an increase in Akt activation, but not related to activations of Nrf2 or AMP-activated protein kinase. TBHQ-induced Akt activation was accompanied by increased phosphorylation of Bad, glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin (mTOR). Mechanistically, we showed that in cultured H9c2 cells and primary cardiac myocytes, TBHQ stimulated Akt phosphorylation and suppressed oxidant-induced apoptosis; this effect was abolished by wortmannin or an Akt inhibitor. Blockade of the Akt pathway in vivo accelerated cardiac dysfunction and abrogated the protective effects of TBHQ. TBHQ also reduced the reactive aldehyde production and protein carbonylation in stressed myocardium. We suggest that TBHQ treatment may represent a novel strategy for timely activation of the cytoprotective Akt pathway in stressed myocardium.

Abnormal hyperphosphorylation of tau is pivotally involved in the pathogenesis of Alzheimer's disease (AD) and related tauopathies. Glycogen synthase kinase 3β (GSK-3β) is a primary tau kinase that is most implicated in tau pathology in AD. However, the exact molecular nature of GSK-3β involved in AD is unclear. In the present study, we found that GSK-3β was truncated at C-terminus and correlated with over-activation of calpain I in AD brain. Truncation of GSK-3β was positively correlated with tau hyperphosphorylation, tangles score and Braak stage in human brain. Calpain I proteolyzed GSK-3β in vitro at C-terminus, leading to an increase of its kinase activity, but keeping its characteristic to preferentially phosphorylate the protein kinase A-primed tau. Excitotoxicity induced by kainic acid (KA) caused GSK-3β truncation at C-terminus and hyperphosphorylation of tau in mouse brain. Inhibition of calpain prevented the KA-induced changes. These findings suggest that truncation of GSK-3β by Ca2+/calpain I markedly increases its activity and involvement of this mechanism probably is responsible for up-regulation of GSK-3β and consequent abnormal hyperphosphorylation of tau and neurofibrillary degeneration in AD.

Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition.