Mechanical regulation of glycogen synthase kinase 3β (GSK3β) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3β at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3β at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3β, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3β inactivation was prevented, whereas insulin inhibition of GSK3β was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3β. Thus, mechanical regulation of GSK3β downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.     

DRG2 knockdown induces Golgi fragmentation via GSK3β phosphorylation and microtubule stabilization

The perinuclear stacks of the Golgi apparatus maintained by dynamic microtubules are essential for cell migration. Activation of Akt (protein kinase B, PKB) negatively regulates glycogen synthase kinase 3β (GSK3β)-mediated tau phosphorylation, which enhances tau binding to microtubules and microtubule stability. In this study, experiments were performed on developmentally regulated GTP-binding protein 2 (DRG2)-stably knockdown HeLa cells to determine whether knockdown of DRG2 in HeLa cells treated with epidermal growth factor (EGF) affects microtubule dynamics, perinuclear Golgi stacking, and cell migration. Here, we show that DRG2 plays a key role in regulating microtubule stability, perinuclear Golgi stack formation, and cell migration. DRG2 knockdown prolonged the EGF receptor (EGFR) localization in endosome, enhanced Akt activity and inhibitory phosphorylation of GSK3β. Tau, a target of GSK3β, was hypo-phosphorylated in DRG2-knockdown cells and showed greater association with microtubules, resulting in microtubule stabilization. DRG2-knockdown cells showed defects in microtubule growth and microtubule organizing centers (MTOC), Golgi fragmentation, and loss of directional cell migration. These results reveal a previously unappreciated role for DRG2 in the regulation of perinuclear Golgi stacking and cell migration via its effects on GSK3β phosphorylation, and microtubule stability.     

MicroRNA‐21 regulates intestinal epithelial tight junction permeability

Increased tight junction (TJ) barrier permeability, induced by tumour necrosis factor (TNF)‐α, may lead to the defects in TJ barrier and subsequent development of inflammation. Recent evidence suggests that miR‐21 is implicated in inflammatory diseases. However, the physiological role of miR‐21 in intestinal permeability remains elusive. This study aimed to explore the role of miR‐21 in intestinal epithelial tight junction permeability. The filter‐grown Caco‐2 monolayers model system was established to mimic intestinal barrier defect. The tight junction proteins were detected by immunofluorescence and western blot analysis. The expression of miR‐21 was assessed by real‐time polymerase chain reaction (PCR). We found that the expression of miR‐21 was increased significantly in TNF‐α induced intestinal TJ barrier defect model. miR‐21 overexpression significantly enhanced while miR‐21 knockdown significantly decreased intestinal permeability. In addition, miR‐21 overexpression significantly increased while miR‐21 knockdown significantly decreased the levels of interleukin‐6, interleukin‐8 and prostaglandin E2 in cell culture medium. Furthermore, miR‐21 positively regulated Akt phosphorylation and negatively regulated Phosphatase and tensin homolog (PTEN) expression in Caco‐2 cells. Our results suggest that miR‐21 may regulate intestinal epithelial tight junction permeability through PTEN/PI3K/Akt signalling pathway. This promotes the feasibility of targeting miR‐21 in the clinical to preserve the intestinal barrier. Copyright © 2015 John Wiley & Sons, Ltd.     

Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF^+/− animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.     

Complex formation and reciprocal regulation between GSK3β and C3G

GSK3β, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3β. In vivo and in vitro interaction assays demonstrated that GSK3β and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3β, and overlaps with its nuclear export sequence. GSK3β phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3β. Huntingtin aggregate formation, dependent on GSK3β inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3β phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3β inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3β phosphorylation, does not alter S9-GSK3β phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3β and C3G. We have identified a novel function of C3G as a negative regulator of GSK3β, a property important for its ability to induce myogenic differentiation.     

Glycogen synthase kinase-3β is a prosurvival signal for the maintenance of human mast cell homeostasis

Homeostasis of mature tissue-resident mast cells is dependent on the relative activation of pro- and antiapoptotic regulators. In this study, we investigated the role of glycogen synthase kinase 3β (GSK3β) in the survival of neoplastic and nonneoplastic human mast cells. GSK3β was observed to be phosphorylated at the Y(216) activating residue under resting conditions in both the neoplastic HMC1.2 cell line and in peripheral blood-derived primary human mast cells (HuMCs), suggesting constitutive activation of GSK3β in these cells. Lentiviral-transduced short hairpin RNA knockdown of GSK3β in both the HMC1.2 cells and HuMCs resulted in a significant reduction in cell survival as determined with the MTT assay. The decrease in stem cell factor (SCF)-mediated survival in the GSK3β knockdown HuMCs was reflected by enhancement of SCF withdrawal-induced apoptosis, as determined by Annexin V staining and caspase cleavage, and this was associated with a pronounced reduction in SCF-mediated phosphorylation of Src homology 2 domain-containing phosphatase 2 and ERK1/2 and reduced expression of the antiapoptotic proteins Bcl-xl and Bcl-2. These data show that GSK3β is an essential antiapoptotic factor in both neopastic and nontransformed primary human mast cells through the regulation of SCF-mediated Src homology 2 domain-containing phosphatase 2 and ERK activation. Our data suggest that targeting of GSK3β with small m.w. inhibitors such as CHIR 99021 may thus provide a mechanism for limiting mast cell survival and subsequently decreasing the intensity of the allergic inflammatory response.     

The protective effect of trefoil factor 3 on the intestinal tight junction barrier is mediated by toll-like receptor 2 via a PI3K/Akt dependent mechanism

Trefoil factor peptides are highly conserved secreted molecules characterized by heat and enzymatic digestion resistance. Intestinal trefoil factor 3 (TFF3) protects and repairs the gastrointestinal mucosa and restores normal intestinal permeability, which is dependent on the integrity of the tight junction (TJ) barrier and the TJ associated proteins claudin-1, zona occludens-1 (ZO-1) and occludin. Despite the important role of intestinal barrier dysfunction in the pathogenesis of inflammatory bowel diseases, the underlying mechanisms and associated molecules remain unclear. In the present study, we show that TFF3 and toll-like receptor 2 (TLR2) are functionally linked and modulate intestinal epithelial permeability via a mechanism that involves the PI3K/Akt pathway. We used the Caco-2 cell model to show that TLR2 and TFF3 inhibit the IL-1β induced increase in permeability and release of proinflammatory cytokines, and that this effect is mediated by activation of PI3K/Akt signaling. TLR2 silencing downregulated the expression of TFF3 and overexpression of TLR2 and TFF3 increased the levels of phospho-Akt. TFF3 overexpression significantly upregulated the expression of ZO-1, occludin and claudin-1 and this effect was abrogated by inhibition of the PI3K/Akt pathway. Taken together, our results indicate that TLR2 signaling selectively enhances intestinal TJ barrier integrity through a mechanism involving TFF3 and the activation of the PI3K/Akt pathway.     

Functional significance of glycogen synthase kinase-3 regulation by serotonin

Serotonin modulates brain physiology and behavior and has major roles in brain diseases involving abnormal mood and cognition. Enhancing brain serotonin has been found to regulate glycogen synthase Kinase-3 (GSK3), but the signaling mechanism and functional significance of this regulation remain to be determined. In this study, we tested the signaling mechanism mediating 5-HT1A receptor-regulated GSK3 in the hippocampus. Using mutant GSK3 knock-in mice, we also tested the role of GSK3 in the behavioral effects of 5-HT1A receptors and the serotonin reuptake inhibitor fluoxetine. The results showed that activation of 5-HT1A receptors by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) increased phosphorylation of the N-terminal serine of both GSK3α and GSK3β in several areas of the hippocampus. The effect of 8-OH-DPAT was accompanied by an increase in the active phosphorylation of Akt, and was blocked by LY294002, an inhibitor of phosphoinositide 3-kinases (PI3K). Phosphorylation of GSK3β, but not GSK3α, was necessary for 5-HT1A receptors to suppress the hippocampus-associated contextual fear learning. Furthermore, acute fluoxetine treatment up-regulated both phospho-Ser21-GSK3α and phospho-Ser9-GSK3β in the hippocampus. Blocking phosphorylation of GSK3α and GSK3β diminished the anti-immobility effect of fluoxetine treatment in the forced swim test, wherein the effect of GSK3β was more prominent. These results together suggest that PI3K/Akt is a signaling mechanism mediating the GSK3-regulating effect of 5-HT1A receptors in the hippocampus, and regulation of GSK3 is an important intermediate signaling process in the behavioral functions of 5-HT1A receptors and fluoxetine.     

Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells

The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of hepatic diseases involving oxidative stress such as alcoholic liver disease (ALD). One consequence of ALD is increased insulin resistance in hepatocytes. To understand the effects of 4-HNE on insulin signaling in liver cells, we employed a model using hepatocellular carcinoma cell line HepG2. Previously, we have demonstrated an increase in the level of Akt phosphorylation is mediated by 4-HNE inhibition of PTEN, a direct regulator of Akt. In this work, we evaluated the effects of 4-HNE on insulin-dependent stimulation of the Akt2 pathway. We demonstrate that 4-HNE selectively leads to phosphorylation of Akt2. Although Akt2 is phosphorylated following 4-HNE treatment, the level of downstream phosphorylation of Akt substrates such as GSK3β and MDM2 is significantly decreased. Pretreatment with 4-HNE prevented insulin-dependent Akt signaling and decreased intracellular Akt activity by 87%. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of Akt2, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt2 (rAkt2) with 20 or 40 μM 4-HNE inhibited rAkt2 activity by 30 or 85%, respectively. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF) identified Michael addition adducts of 4-HNE with His196, His267, and Cys311 of rAkt2. Computation-based molecular modeling analysis of 4-HNE adducted to His196 and Cys311 of Akt2 suggests inhibition of GSK3β peptide binding by 4-HNE in the Akt2 substrate binding pocket. The inhibition of Akt by 4-HNE provides a novel mechanism for increased insulin resistance in ALD. These data provide a potential mechanism of dysregulation of Akt2 during events associated with sustained hepatocellular oxidative stress.     

Hydroxysafflor Yellow A Protects Against Cerebral Ischemia–Reperfusion Injury by Anti-apoptotic Effect Through PI3K/Akt/GSK3β Pathway in Rat

Hydroxysafflor yellow A (HSYA) is the major active chemical component of the flower of the safflower plant, Carthamus tinctorius L. Previously, its neuroprotection against cerebral ischemia–reperfusion (I/R) injury was reported by anti-oxidant action and suppression of thrombin generation. Here, we investigate the role of HSYA in cerebral I/R-mediated apoptosis and possible signaling pathways. Male Wistar rats were subjected to transient middle cerebral artery occlusion for 2 h, followed by 24 h reperfusion. HSYA was administered via tail-vein injection just 15 min after occlusion. The number of apoptotic cells was measured by TUNEL assay, apoptosis-related proteins Bcl-2, Bax and the phosphorylation levels of Akt and GSK3β in ischemic penumbra were assayed by western blot. The results showed that administration of HSYA at the doses of 4 and 8 mg/kg significantly inhibited the apoptosis by decreasing the number of apoptotic cells and increasing the Bcl-2/Bax ratio in rats subjected to I/R injury. Simultaneously, HSYA treatment markedly increased the phosphorylations of Akt and GSK3β. Blockade of PI3K activity by wortmannin dramatically abolished its anti-apoptotic effect and lowered both Akt and GSK3β phosphorylation levels. Taken together, these results suggest that HSYA protects against cerebral I/R injury partly by reducing apoptosis via PI3K/Akt/GSK3β signaling pathway.     

Hsp90α/β associates with the GSK3β/axin1/phospho-β-catenin complex in the human MCF-7 epithelial breast cancer model

Hsp90α/β, the signal transduction chaperone, maintains intracellular communication in normal, stem, and cancer cells. The well characterised association of Hsp90α/β with its client kinases form the framework of multiple signalling networks. GSK3β, a known Hsp90α/β client, mediates β-catenin phosphorylation as part of a cytoplasmic destruction complex which targets phospho-β-catenin to the 26S proteasome. The canonical Wnt/β-catenin pathway promotes stem cell self-renewal as well as oncogenesis. The degree of Hsp90α/β involvement in Wnt/β-catenin signalling needs clarification. Here, we describe the association of Hsp90α/β with GSK3β, β-catenin, phospho-β-catenin and the molecular scaffold, axin1, in the human MCF-7 epithelial breast cancer cell model using selective inhibition of Hsp90α/β, confocal laser scanning microscopy and immunoprecipitation. Our findings suggest that Hsp90α/β modulates the phosphorylation of β-catenin by interaction in common complex with GSK3β/axin1/β-catenin.     

ERK/GSK3β/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition

Radiotherapy is one of the major treatment regimes for thoracic malignancies, but can lead to severe lung complications including pneumonitis and fibrosis. Recent studies suggest that epithelial-to-mesenchymal transition (EMT) plays an important role in tissue injury leading to organ fibrosis. To investigate whether radiation can induce EMT in lung epithelial cells and also to understand the potential mechanism(s) associated with this change, rat alveolar type II lung epithelial RLE-6TN cells were irradiated with 8 Gy of (137)Cs γ-rays. Western blot and immunofluorescence analyses revealed a time-dependent decrease in E-cadherin with a concomitant increase in α-smooth muscle actin (α-SMA) and vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. Protein levels and nuclear translocation of Snail, the key inducer of EMT, were significantly elevated in the irradiated cells. Radiation also induced a time-dependent inactivation of glycogen synthase kinase-3β (GSK3β), an endogenous inhibitor of Snail. A marked increase in phosphorylation of ERK1/2, but not JNK or p38, was observed in irradiated RLE-6TN cells. Silencing ERK1/2 using siRNAs and the MEK/ERK inhibitor U0126 attenuated the radiation-induced phosphorylation of GSK3β and altered the protein levels of Snail, α-SMA, and E-cadherin in RLE-6TN cells. Preincubating RLE-6TN cells with N-acetylcysteine, an antioxidant, abolished the radiation-induced phosphorylation of ERK and altered protein levels of Snail, E-cadherin, and α-SMA. These findings reveal, for the first time, that radiation-induced EMT in alveolar type II epithelial cells is mediated by the ERK/GSK3β/Snail pathway.     

Curcumin prevents leptin-induced tight junction dysfunction in intestinal Caco-2 BBe cells

Maintaining tight junction (TJ) integrity in the intestine is critical for nutrient absorption, host defense, and host immunity. While leptin secreted from adipose tissue is associated with obesity and obesity-related intestinal inflammation, the role of luminal leptin in intestinal TJ function is elusive. Here, we examined the role of leptin in intestinal TJ function in Caco-2 BBe cells and further explored the function of curcumin (CCM) in leptin-induced TJ dysfunction. Apical leptin, but not basolateral leptin, treatment at a concentration of 100 ng/ml deteriorated TJ function in Caco-2 BBe cells. Leptin-impaired TJ alteration was resulted from induction of leptin receptor-dependent JAK2/STAT3 signaling pathway and its-related PI3K/Akt/ERK1/2 signaling pathways. Apical leptin also lowered the expression levels of genes encoding TJ-associated proteins such as zonula occludens-3, claudin-5, and occludin, and elevated expression of pro-inflammatory genes such as IL-6 and TNF-α. Leptin-impaired TJ junction in Caco-2 BBe cells was blunted by a 30-min CCM pretreatment through inhibition of leptin receptor-dependent signaling pathway, and its-associated induction of expression of genes encoding TJ-associated proteins and pro-inflammatory cytokines. Our results elucidate a novel function of luminal leptin in intestinal TJ dysfunction, and further identify CCM as an effective dietary compound that prevents leptin-impaired TJ function in intestinal cells.     

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

BackgroundWhile Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.MethodsGain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.ResultsSyk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.ConclusionsSyk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.General significanceSyk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.