GSK-3β Inhibitor LiCl Research Articles

ED-71 Ameliorates Bone Loss in Type 2 Diabetes Mellitus by Enhancing Osteogenesis Through Upregulation of the Circadian Rhythm Coregulator BMAL1.

Bone loss is a common complication of type 2 diabetes mellitus (T2DM). Circadian rhythms play a significant role in T2DM and bone remodeling. Eldecalcitol (ED-71), a novel active vitamin D analog, has shown promise in ameliorating T2DM. We aimed to investigate whether the circadian rhythm coregulator BMAL1 mediates the anti-osteoporotic effect of ED-71 in T2DM and its associated mechanisms. A T2DM mouse model was established using high-fat diet (HDF) and streptozotocin (STZ) injection, and blood glucose levels were monitored weekly. HE staining, Masson staining, and Micro-CT were performed to assess the changes in bone mass. IHC staining and IF staining were used to detect osteoblast status and BMAL1 expression and RT-qPCR was applied to detect the change of oxidative stress factors. In vitro, high glucose (HG) stimulation was used to simulate the cell environment in T2DM. RT-qPCR, Western blot, IF, ALP staining and AR staining were used to detect osteogenic differentiation and SIRT1/GSK3β signaling pathway. DCFH-DA staining was used to detect reactive oxygen species (ROS) levels. ED-71 increased bone mass and promoted osteogenesis in T2DM mice. Moreover, ED-71 inhibited oxidative stress and promoted BMAL1 expression in osteoblasts The addition of STL1267, an agonist of the BMAL1 transcriptional repressor protein REV-ERB, reversed the inhibitory effect of ED-71 on oxidative stress and the promotional effect on osteogenic differentiation. In addition, ED-71 facilitated SIRT1 expression and reduced GSK3β activity. The inhibition of SIRT1 with EX527 partially attenuated ED-71's effects, whereas the GSK3β inhibitor LiCl further enhanced ED-71's positive effects on BMAL1 expression. ED-71 ameliorates bone loss in T2DM by upregulating the circadian rhythm coregulator BMAL1 and promoting osteogenesis through inhibition of oxidative stress. The SIRT1/GSK3β signaling pathway is involved in the regulation of BMAL1.

Taste papilla cell differentiation requires the regulation of secretory protein production by ALK3-BMP signaling in the tongue mesenchyme.

Taste papillae are specialized organs, each of which comprises an epithelial wall hosting taste buds and a core of mesenchymal tissue. In the present study, we report that during early taste papilla development in mouse embryos, bone morphogenetic protein (BMP) signaling mediated by type 1 receptor ALK3 in the tongue mesenchyme is required for epithelial Wnt/β-catenin activity and taste papilla differentiation. Mesenchyme-specific knockout (cKO) of Alk3 using Wnt1-Cre and Sox10-Cre resulted in an absence of taste papillae at E12.0. Biochemical and cell differentiation analyses demonstrated that mesenchymal ALK3-BMP signaling governed the production of previously unappreciated secretory proteins, i.e. it suppressed those that inhibit and facilitated those that promote taste papilla differentiation. Bulk RNA-sequencing analysis revealed many more differentially expressed genes (DEGs) in the tongue epithelium than in the mesenchyme in Alk3 cKO versus control. Moreover, we detected downregulated epithelial Wnt/β-catenin signaling and found that taste papilla development in the Alk3 cKO was rescued by the GSK3β inhibitor LiCl, but not by Wnt3a. Our findings demonstrate for the first time the requirement of tongue mesenchyme in taste papilla cell differentiation.

Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3β/Nrf2 signalling pathway in vitro.

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia‐induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3‐kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling pathway in SH‐SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3β inhibitor LiCl were employed in this study. Pre‐treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)‐induced viability loss, reduced the proportion of apoptosis and regulated OGD‐mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD‐caused mitochondrial membrane potential and decomposition of caspase‐3 to cleaved caspase‐3, and heightened the B‐cell lymphoma 2 (Bcl‐2)/Bax ratio. PI3K/Akt/GSK3β/Nrf2 signalling pathway activation in SH‐SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p‐Akt, p‐GSK3β, nucleus Nrf2 and haeme oxygenase 1 (HO‐1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD‐induced apoptosis via the PI3K/Akt/GSK3β/Nrf2 signalling pathway.

Neuroprotective brain-derived neurotrophic factor signaling in the TAU-P301L tauopathy zebrafish model

Brain-derived neurotrophic factor (BDNF) dysregulations contribute to the neurotoxicity in neurodegenerative pathologies and could be efficiently targeted by therapies. In Alzheimer’s disease (AD), although the relationship between BDNF and amyloid load has been extensively studied, how Tau pathology affects BDNF signaling remains unclear. Using the TAU-P301L transgenic zebrafish line, we investigated how early Tau-induced neurotoxicity modifies BDNF signaling. Alterations in BDNF expression levels were observed as early as 48 h post fertilization in TAU-P301L zebrafish embryos while TrkB receptor expression was not affected. Decreasing BDNF expression, using a knockdown strategy in wild-type embryos to mimic Tau-associated decrease, did not modify TrkB expression but promoted neurotoxicity as demonstrated by axonal outgrowth shortening and neuronal cell death. Moreover, the TrkB antagonist ANA-12 reduced the length of axonal projections. Rescue experiments with exogenous BDNF partially corrected neuronal alterations in TAU-P301L by counteracting primary axonal growth impairment but without effect on apoptosis. Importantly, the axonal rescue was proved functionally effective in a behavioral test, at a similar level as obtained with the GSK3β inhibitor LiCl, known to decrease TAU phosphorylation. Finally, treatment with a TrkB agonist, 7,8-dihydroxyflavone, led to comparable results and allowed full rescue of locomotor response. We provided here strong evidence that Tau neurotoxicity provoked alterations in BDNF system and that BDNF pathway might represent an efficient therapeutic target.

Electroacupuncture pretreatment prevents ischemic stroke and inhibits Wnt signaling-mediated autophagy through the regulation of GSK-3β phosphorylation

Electroacupuncture (EA), a traditional Chinese replacement therapy, is widely accepted to treat ischemic stroke. Increasing evidence show that autophagy is involved in the process of cerebral ischemia injury and the Wnt/GSK3β pathway, playing an important role in protecting central nervous system. In this study, rats were treated with EA prior to focal ischemia by middle cerebral artery occlusion (MCAO). Deficit score, infarct volumes and levels of autophagy markers, such as LC3I, LC3II and p62, were assessed with either PI3K inhibitor wortmannin or a GSK-3β inhibitor LiCl. Oxygen-glucose deprivation/re-oxygenation (OGD/R) was made in the primitive neuron in vitro, and was respectively treated with autophagy inhibitors 3-MA, LiCl, GSK3β siRNA, or mTOR inhibitor rapamycin. The results indicated that EA pretreatment increased the levels of autophagy marker LC3-II and reduced the levels of p62. Meanwhile, deficit outcome was improved, and infarct volumes were reduced by EA pretreatment. Furthermore, the beneficial effects of EA pretreatment were reversed by wortmannin. LiCl and GSK3β siRNA can mimic the neuroprotective effects of EA pretreatment by downregulating autophagy, and increasing protein levels of p-mTOR, p-GSK3β and β-catenin in OGD/R neurons. However, the protective effects of GSK3β siRNA were blocked by rapamycin. These results suggest that EA pretreatment induces tolerance to cerebral ischemia by inhibiting autophagy via the Wnt pathway through the inhibition of GSK3β.

Metformin promotes survivin degradation through AMPK/PKA/GSK-3β-axis in non-small cell lung cancer.

Metformin, a first-line antidiabetic drug, has been reported with anticancer activities in many types of cancer. However, its molecular mechanisms remain largely unknown. As a member of inhibitor of apoptosis proteins, survivin plays an important role in the regulation of cell death. In the present study, we investigated the role of survivin in metformin-induced anticancer activity in non-small cell lung cancer in vitro. Metformin mainly induced apoptotic cell death in A549 and H460 cell lines. It remarkably suppressed the expression of survivin, decreased the stability of this protein, then promoted its proteasomal degradation. Moreover, metformin greatly suppressed protein kinase A (PKA) activity and induced its downstream glycogen synthase kinase 3β (GSK-3β) activation. PKA activators, both 8-Br-cAMP and forskolin, significantly increased the expression of survivin. Consistently both GSK-3β inhibitor LiCl and siRNA restored the expression of survivin in lung cancer cells. Furthermore, metformin induced adenosine 5'-monophosphate-activated protein kinase (AMPK) activation. Suppression of the activity of AMPK with Compound C reversed the degradation of survivin induced by metformin, and meanwhile, restored the activity of PKA and GSK-3β. These results suggest that metformin kills lung cancer cells through AMPK/PKA/GSK-3β-axis-mediated survivin degradation, providing novel insights into the anticancer effects of metformin.

Rosmarinic acid attenuates β-amyloid-induced oxidative stress via Akt/GSK-3β/Fyn-mediated Nrf2 activation in PC12 cells.

Oxidative stress is an important pathogenic factor in Alzheimer's disease (AD). Recently, nuclear factor E2-related factor 2 (Nrf2) has emerged as a master regulator for the endogenous antioxidant response, and thus represents an attractive therapeutic target against AD. The aim of this study is to test the hypothesis that rosmarinic acid (RosA) attenuates amyloid-β (Aβ)-evoked oxidative stress through activating Nrf2-inducible cellular antioxidant defense system. Here, we reported that RosA attenuated Aβ-induced cellular reactive oxygen species (ROS) generation and lipid hydroperoxides (LPO). Interestingly, knockdown of Nrf2 by plasmid-based short hairpin RNA (shRNA) abrogated, at least in part, RosA-mediated neuroprotection in Aβ-challenged PC12 cells. Mechanistically, RosA enhanced the nuclear translocation of Nrf2 and binding to antioxidant response element (ARE) core element but did not induced Nrf2 transcription. Simultaneously, RosA induced a set of Nrf2 downstream target genes encoding phase-II antioxidant enzymes. Furthermore, RosA enhanced protein kinase B (Akt) phosphorylation, glycogen synthase kinase-3β (GSK-3β) phosphorylation at Ser9, and Fyn phosphorylation. Noteworthy, pharmacological inhibition or gene knockdown studies demonstrated that Akt locate upstream of GSK-3β and regulate Nrf2 through Fyn in the context of PC12 cells pre-incubated with RosA following exposed to Aβ. Conversely, the antioxidant effects of RosA could be blocked by Akt inhibitors LY294002, GSK-3β inhibitor LiCl, Nrf2 shRNA, or Fyn shRNA in Aβ-challenged PC12 cells. Consequently, the antioxidant effects of RosA are mediated predominantly by Akt/GSK-3β/Fyn pathway through increased activity of Nrf2. These results suggest, although do not prove, that RosA can be a promising candidate for neuroprotective treatment of AD.

Inhibition of GSK-3β Activation Protects SD Rat Retina Against N-Methyl-N-Nitrosourea-Induced Degeneration by Modulating the Wnt/β-Catenin Signaling Pathway.

Retinal degenerative diseases are characterized by photoreceptor cell loss. Photoreceptor cell loss leading to retinal degeneration can be induced by N-methyl-N-nitrosourea (MNU), which was widely used to mimic the pathology. However, the mechanism by which MNU induces photoreceptor cell loss is still largely unknown. The purpose of the present study was to investigate whether phosphorylation of glycogen synthase kinase-3β (p-GSK-3β) is a potent mediator of MNU-induced retinal degeneration and how p-GSK-3β affects the process. MNU-induced photoreceptor cell loss was evaluated in Sprague-Dawley (SD) rat retinas. GSK-3β and Akt expression levels did not change during MNU-induced retinal degeneration but the phosphorylation of GSK-3β and Akt was decreased by MNU treatment. Lithium chloride (LiCl), which increases p-GSK-3β level and active-β-catenin level, reversed retinal degeneration induced by MNU treatment. These results suggest that GSK-3β activation is closely related to photoreceptor cell loss and that the application of the GSK-3β inhibitor LiCl could activate Wnt/β-catenin signaling pathway and reduce photoreceptor cell loss induced by MNU. Our findings indicate that inhibition of GSK-3β activation may be a potential therapeutic target for retinal degeneration induced by photoreceptor cell loss.

LW-213 induces G2/M cell cycle arrest through AKT/GSK3β/β-catenin signaling pathway in human breast cancer cells.

LW-213 is a derivative of Wogonin and the anticancer activities of Wogonin have been reported. To study whether LW-213 inhibits cancer cells and explore a possible mechanism, we investigate the compound in several cancer cell lines. We found LW-213 arrests G2/M cycle in breast cancer cells by suppression of Akt/Gsk3β/β-catenin signaling pathway. In compound treated cells, cell cycle-related proteins cyclin A, cyclin B1, p-CDK1, p-Cdc25C, and p-Chk2 (Thr68) were upregulated, and β-catenin nuclear translocation was inhibited. Electrophoretic mobility shift assay revealed LW-213 inhibits binding of β-catenin/LEF complex to DNA. GSK3β inhibitor LiCl and siRNA against GSK3β partially reversed G2/M arrest in breast cancer MCF-7 cells. These results suggest LW-213 triggered G2/M cell cycle arrest through suppression of β-catenin signaling. In BALB/c mice, growth of xenotransplanted MCF-7 tumor was also inhibited after treatment of LW-213. Regulation of cyclin A, cyclin B1, and β-catenin by LW-213 in vivo was the same as in vitro study. In conclusion, we found LW-213 exerts its anticancer effect on cell proliferation and cell cycle through repression of Akt/Gsk3β/β-catenin signaling pathway. LW-213 could be a potential candidate for anticancer drug development.

Ginsenoside Rb1 protects hippocampal neurons from high glucose-induced neurotoxicity by inhibiting GSK3β-mediated CHOP induction

Ginsenoside Rb1 is generally recognized as one of the principal bioactive ingredients in ginseng and shows neuroprotective effects in various neurons. Endoplasmic reticulum (ER) stress is considered to play an important role in numerous neurodegenerative disorders. Recently, glucogen synthase kinase 3β (GSK3β) was reported to regulate ER stress-induced C/EBP homologous protein (CHOP) in neuronal cells. Therefore, in this study, we investigated the effects of ginsenoside Rb1 on GSK3β-mediated ER stress in high glucose-treated hippocampal neurons. Results from the MTT assay showed that treatment with 1 µM Rb1 for 72 h protected neurons from high glucose-induced cell injury. Using western blot analysis, we found that treatment with Rb1 effectively inhibited the phosphorylation of the high glucose-induced protein kinase RNA-like ER kinase (PERK) and of GSK3β, and reduced the level of the CHOP protein. The levels of these proteins were also decreased by treatment with the GSK3β inhibitor Licl. Rb1 also significantly decreased the mRNA expression of the gene CHOP, as shown by quantitative RT-PCR analysis. Taken together, the present results suggested that Rb1 may protect neurons from high glucose-induced cell damage by inhibiting GSK3β‑mediated CHOP induction, providing a potentially new strategy for preventing and treating cognitive impairment caused by diabetes.

Inhibition of glycogen synthase kinase-3β by lithium chloride suppresses 6-hydroxydopamine-induced inflammatory response in primary cultured astrocytes

An increasing amount of evidence has emerged to suggest that neuroinflammatory process is involved in the pathogenesis of Parkinson’s disease (PD). Activated microglia and astrocytes are found in the substantia nigra (SN) of Parkinson’s disease brains as well as in animal models of Parkinson’s disease. Although reactive astrocytes are involved in the progression of PD, the role of reactive astrocytes in neuroinflammation of PD has received limited attention to date. Recently, Glycogen synthase kinase-3β (GSK-3β) was identified as a crucial regulator of the inflammatory response. The purpose of this study was to explore the mechanism by which 6-hydroxydopamine (6-OHDA) induces inflammatory response in astrocytes and observe the anti-inflammatory effect of lithium chloride (LiCl) on 6-OHDA-treated astrocytes.In the present study, we found that glial fibrillary acidic protein (GFAP) was markedly upregulated in the presence of 6-OHDA. Moreover, our results revealed that proinflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2(COX-2), prostaglandins E2 (PGE2), and tumor necrosis factor-α (TNF-α) were obviously increased in astrocytes exposed to 6-OHDA. Western blot analysis revealed that 6-OHDA significantly increased dephosphorylation/activation of GSK-3β as well as the nuclear translocation of nuclear factor-κB (NF-κB) p65. Besides, GSK-3β inhibitor LiCl and SB415286 inhibited the GSK-3β/NF-κB signaling pathway, leading to the reduction of proinflammatory molecules in 6-OHDA-activated astrocytes. These results confirmed that GSK-3β inhibitor LiCl and SB415286 provide protection against neuroinflammation in 6-OHDA-treated astrocytes. Therefore, GSK-3β may be a potential therapeutic target for the treatment of PD.

Enhanced apoptotic effects by downregulating Mcl-1: Evidence for the improvement of photodynamic therapy with Celecoxib

Tumor cells exposed to sub-lethal photodynamic therapy (PDT) cause cellular rescue responses that lead to resistance to the therapy, including expression of angiogenic factors and survival molecules. However, the mechanisms contributing to the resistance are yet to be fully understood. Here, we show for the first time that Mcl-1, an anti-apoptotic protein, plays an important role in protecting cells from PDT-induced apoptosis. In contrast to the reduction in the anti-apoptotic proteins Bcl-2 and Bcl-xl, sub-lethal PDT induces an increase in Mcl-1 expression. Silencing Mcl-1 sensitizes tumor cells to PDT-induced apoptosis, and ectopic expression of Mcl-1 significantly delays Bax translocation to mitochondria and inhibits caspase-3 activity following PDT. Mcl-1 expression is associated closely with activated AKT signaling following PDT. AKT can regulate Mcl-1 expression through GSK-3β and NF-κB at the protein and transcriptional levels, respectively. Inhibition of AKT by Wortmannin or siRNA significantly reduces the levels of Mcl-1 mRNA and protein and enhances PDT-induced apoptosis. Treatment with Celecoxib, a non-steroidal anti-inflammatory drug (NSAID), is shown to downregulate Mcl-1 expression, and enhances PDT-induced apoptosis both in vitro and in vivo. This down-regulation is closely related to the inhibition effect of Celecoxib on the AKT/GSK-3β pathway, and was blocked upon addition of GSK-3β inhibitor LiCl or the proteasome inhibitor MG132. These results suggest that Mcl-1 is a potential target for improving the antitumor efficiency of PDT. A loss in Mcl-1 by inhibiting AKT promotes PDT-induced apoptosis through the mitochondrial pathway. This also provides a novel rationale for utilizing Celecoxib to improve the efficacy of PDT.

Resveratrol Inhibits Inflammation Induced by Heat-Killed Listeria monocytogenes

Resveratrol is a polyphenolic compound in red wine that has antioxidant and cardioprotective effects in animal models. Listeria monocytogenes is a pathogen that mainly affects immunocompromised individuals and is initially detected at the cell surface or in phagosomes by toll-like receptor 2. Many antioxidants also exert anti-inflammatory activities; therefore, we evaluated the anti-inflammatory properties of resveratrol by studying the various inflammatory responses induced by heat-killed L. monocytogenes (HKLM). Resveratrol strongly blocked HKLM-induced NADPH oxidase-1 mRNA and reactive oxygen species production by macrophages. Resveratrol also suppressed monocyte chemotactic protein-1 expression, cyclooxygenase-2 expression, prostaglandin production, inducible nitric oxide (NO) synthase expression, and NO production induced by HKLM. We investigated the signaling pathway involved in the resveratrol effect. HKLM stimulated glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The involvement of GSK3β and ERK1/2 was tested using inhibitors. While the GSK3β inhibitor LiCl potentiated the effect of HKLM, the MEK inhibitor U0126 blocked these responses. Additionally, pretreatment with resveratrol blocked phosphorylation of both kinases induced by HKLM. These results suggest that HKLM is strong inducer of inflammatory mediators, and that the inhibitory effect of resveratrol may be mediated by the GSK3β and ERK1/2 pathways.

Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition

To identify the mechanisms underlying the elevation of intracellular Ca(2+) level ([Ca(2+)](i)) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons. Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca(2+)](i) was measured using fura-2 AM. Ca(2+) current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay. Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca(2+)](i) in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca(2+)](i) in ARC neurons depended on extracellular Ca(2+), and was blocked by P/Q-type Ca(2+)channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca(2+) channel blocker nifedipine (10 μmol/L) or N-type Ca(2+)channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca(2+) current in ARC neurons. The low-glucose induced elevation of [Ca(2+)](i) in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L). Lowering glucose level enhances the activity of P/Q type Ca(2+)channels and elevates [Ca(2+)](i) level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β.

Migfilin Regulates Esophageal Cancer Cell Motility through Promoting GSK-3β–Mediated Degradation of β-Catenin

Migfilin, a protein component of focal adhesions, has been implicated in regulation of cell-extracellular matrix adhesion and motility but the underlying mechanisms are not fully elucidated. In this study, we have determined the functions of migfilin in esophageal cancer cells and the mechanisms involved. We show that the expression level of migfilin is negatively associated with clinical metastasis, and enforced expression of migfilin suppressed cell motility through decreased free β-catenin level. Overexpression of migfilin resulted in destabilization of β-catenin in concomitance with reduction of its transcriptional activity. Knockdown of migfilin by siRNA, transfection of a mutant β-catenin at Ser37 which is a critical phosphorylation site of GSK-3β, GSK-3β inhibitor LiCl, or proteasome inhibitor MG132 reversed the migfilin-mediated β-catenin degradation and transcription inhibition. Moreover, migfilin promoted β-catenin degradation by reinforcing the association between β-catenin and GSK-3β. In addition, exogenously expressed β-catenin partially restored migfilin-induced suppression of cell invasion. Collectively, these results suggest that the expression level of migfilin in ESCCs is inversely correlated with clinical metastasis status, and migfilin inhibits ESCC cell invasion at least in part through promoting degradation of β-catenin.

PTEN Is a Key Regulator of Collagen-Induced Platelet Activation and Is Involved in αIIbβ3 -Mediated Outside-in Signaling,

Abstract 3252PTEN (Phosphatase and tensin homologue deleted in chromosome 10) is a well-known tumor suppressor. PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 and thereby negatively regulates PIP3-downstream signaling. According to present studies, PTEN is also a protein tyrosine phosphatase. In 2010 (Blood. 2010;116(14):2579–81), we reported that hematopoietic specific PTEN deficiency causes an increased platelet count, a shortened tail vein bleeding time, and enhanced platelet activation in response to stimulation by collagen and other agonists along with augmented collagen-induced phosphorylation of platelet Akt at Ser473. The PI3K specific inhibitor LY294002 almost completely blocked collagen-induced WT platelet aggregation, but had less effect on PTEN-deficient platelets. This result implies that PTEN may regulate collagen-induced platelet activation via both PI3K/Akt signaling and an yet to be identified pathway. In this study, we investigated the mechanism underlying PTEN regulation of collagen-induced platelet activation.It is well-known that the GPVI receptor signaling cascade is similar to that of T- and B-cell immune receptors. This signaling cascade relies on the formation of a signalosome composed of the transmembrane adapter LAT (linker for activation of T cells), the two cytosolic adapters SLP-76 and Gads, and the activation of PLCγ2. Accordingly, we investigated the phosphorylation of LAT, SLP-76 and PLCγ2 in PTEN-deficient and WT platelets in response to low level collagen (2μg/ml), with and without LY294002 pre-incubation. The platelet lysate was immunoprecipitated with 4G10, a specific anti-phosphotyrosine monoclonal antibody, to detect tyrosine phosphorylation. Interestingly, we found that in the presence of LY294002, the phosphorylation levels of LAT, SLP-76 and PLCγ2 were very high in PTEN-deficient platelets while the phosphorylation of these molecules was hardly detectable in WT platelets, all in response to low level collagen. These results suggested that PTEN may also regulate platelet activation through LAT, SLP-76 and PLCγ2. This hypothesis was supported by co-immunopricipitation experiments demonstrating that PTEN interacts with LAT in platelets upon collagen stimulation. Moreover, we also found that PTEN Ser380 phosphorylation was correlated with collagen-induced WT platelet activation without LY294002, and PTEN Ser380 phosphorylation could be inhibited by CK2 inhibitor DMAT and PDK1 inhibitor II, but not by Akt inhibitor SH6 or the GSK3β inhibitor LiCl. In contrast, the PDK1 inhibitor II blocked collagen-induced activation of CK2. Radioactivity assays indicated that collagen-induced enzyme activity activation of CK2 was blocked by the PDK1 inhibitor II. Ser380 phosphorylation is thought to reflect the loss of PTEN phosphatase function. So, because inhibition of PI3K, and PDK1 do not result in phosphorylation of PTEN S380 and the resulting loss of PTEN phosphatase function, it may be true that PDK1 inactivates PTEN. Therefore, we conclude that the mechanism of PTEN involvement in collagen-induced platelet activation is complicated, and PTEN plays a key role in GPVI mediated platelet activation probably through down-regulating both the PI3K/Akt and PI3K-PDK1-LAT pathways.We also investigated PTEN’s function in αIIbβ3 mediated platelet activation. Platelet spreading and clot retraction experiments were performed. We found that PTEN deficiency significantly promoted clot retraction, but had no effect on platelet spreading on immobilized fibrinogen. Moreover, U46619 and thrombin induced PTEN ser380 phosphorylation was inhibited in β3 deficient platelet. These results indicate that PTEN is also involved in αIIbβ3 mediated outside-in signaling. Although the molecular mechanism of PTEN modulation of αIIbβ3 mediated outside-in signaling is unclear, our results may partially explain why PTEN deficiency also enhances platelet aggregation and secretion in response to agonists other than collagen. Disclosures:No relevant conflicts of interest to declare.

Dihydroartemisinin accelerates c-MYC oncoprotein degradation and induces apoptosis in c-MYC-overexpressing tumor cells

Artemisinin and its derivatives (ARTs) are effective antimalarial drugs and also possess profound anticancer activity. However, the mechanism accounted for its distinctive activity in tumor cells remains unelucidated. We computed Pair wise Pearson correlation coefficients to identify genes that show significant correlation with ARTs activity in NCI-55 cell lines using data obtained from studies with HG-U133A Affymetrix chip. We found c-myc is one of the genes that showed the highest positive correlation coefficients among the probe sets analyzed ( r = 0.585, P < 0.001). Dihydroartemisinin (DHA), the main active metabolite of ARTs, induced significant apoptosis in HL-60 and HCT116 cells that express high levels of c-MYC. Stable knockdown of c-myc abrogated DHA-induced apoptosis in HCT116 cells. Conversely, forced expression of c-myc in NIH3T3 cells sensitized these cells to DHA-induced apoptosis. Interestingly, DHA irreversibly down-regulated the protein level of c-MYC in DHA-sensitive HCT116 cells, which is consistent to persistent G1 phase arrest induced by DHA. Further studies demonstrated that DHA accelerated the degradation of c-MYC protein and this process was blocked by pretreatment with the proteasome inhibitor MG-132 or GSK 3β inhibitor LiCl in HCT116 cells. Taken together, ARTs might be useful in the treatment of c-MYC-overexpressing tumors. We also suggest that c-MYC may potentially be a biomarker candidate for prediction of the antitumor efficacies of ARTs.