Suppressive Regulatory T Cell Activity Is Potentiated by Glycogen Synthase Kinase 3β Inhibition

The mechanism by which regulatory T (Treg) cells suppress the immune response is not well defined. A recent study has shown that β-catenin prolongs Treg cell survival. Because β-catenin is regulated by glycogen synthase kinase 3β (GSK-3β)-directed phosphorylation, we focused on GSK-3β and the role it plays in Treg cell function. Inhibition of GSK-3β led to increased suppression activity by Treg cells. Inhibitor-treated Treg cells exhibited prolonged FoxP3 expression and increased levels of β-catenin and of the antiapoptotic protein Bcl-xL. Systemic administration of GSK-3β inhibitor resulted in prolonged islet survival in an allotransplant mouse model. Our data suggest that GSK-3β could be a useful target in developing strategies designed to increase the stability and function of Treg cells for inducing allotransplant tolerance or treating autoimmune conditions.     

人肝内胆管癌细胞PRL-3 的表达与侵袭转移研究

目的:探讨PRL-3在人肝内胆管癌侵袭转移中的作用.方法:利用小RNA技术干扰肝内胆管癌细胞株PRL-3表达,并采用细胞划痕实验和Transwell体外侵袭实验评价PRL-3对肝内胆管癌细胞侵袭转移能力的影响.结果:RT-PCR和Western blot结果均显示转染PRL-3特异性siRNA-2组PRL-3表达明显降低(P＜0.05).PRL-3 siRNA-2组在划痕培养24h后划痕区域宽度占初始划痕区域宽度的百分比为(62.12±6.28)％,阴性对照组为(23.88±2.55)％,空白对照HCCC-9810组为(21.20±6.07)％.PRL-3siRNA-2组细胞的划痕两端距离相比明显较宽,分别与阴性对照组细胞和空白对照HCCC-9810组细胞相比均有统计学意义(P＜0.05),后两者无显著性差异(P＞0.05).Transwell体外侵袭实验结果显示,PRL-3 SiRNA-2组细胞侵袭能力明显减弱,穿膜细胞数为(19.40±2.30)个/HP,明显少于阴性对照组(64.00±2.73)个/HP和正常HCCC-9810组(67.20±3.l1)个/HP,差异有显著性(P＜0.05);正常HCCC-9810组和阴性对照组无明显差异(P＞0.05).结论:PRL-3特异性siRNA能够抑制肝内胆管癌细胞HC-CC-9810中内源性PRL-3的表达,并可以明显抑制肝内胆管癌细胞的迁移侵袭能力.     

Axil, a Member of the Axin Family, Interacts with Both Glycogen Synthase Kinase 3β and β-Catenin and Inhibits Axis Formation of Xenopus Embryos

Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3β (GSK-3β). This protein had 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway.We designated this protein Axil for Axin like. Like Axin, Axil ventralized Xenopus embryos and inhibited Xwnt8-induced Xenopus axis duplication. Axil was phosphorylated by GSK-3β. Axil bound not only to GSK-3β but also to β-catenin, and the GSK-3β-binding site of Axil was distinct from the β-catenin-binding site. Furthermore, Axil enhanced GSK-3β-dependent phosphorylation of β-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3β-dependent phosphorylation of β-catenin, thereby inhibiting axis formation.     

Activation of Wnt/β-Catenin Signaling Increases Apoptosis in Melanoma Cells Treated with Trail

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation.     

Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway

Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.     

The Wnt pool of glycogen synthase kinase 3beta is critical for trophic-deprivation-induced neuronal death

Glycogen synthase kinase 3 (GSK-3) is implicated in neuronal death through a causal role, and precise mechanisms have not been unambiguously defined. We show that short hairpin RNA (shRNA) knockdown of GSK-3β, but not GSK-3α, protects cerebellar granule neurons from trophic-deprivation-induced death. Using compartment-targeted inhibitors of the Wnt-regulated GSK-3 pool, NLS-FRAT1, NES-FRAT1, and axin-GSK-3-interacting domain (axin-GID), we locate proapoptotic GSK-3 action to the cytosol and regulation of Bim protein turnover despite constitutive cycling of GSK-3 between the cytosol and nucleus, revealed by leptomycin B. We examine the importance of Ser21/9 (GSK-3α/β) phosphorylation on proapoptotic GSK-3 function. Neurons isolated from GSK-3α/β^S21A/S9A knock-in mice survive normally and are fully sensitive to trophic-deprivation-induced death. Nonetheless, inhibition of GSK-3 catalytic activity with lithium or SB216763 protects GSK-3α/β^S21A/S9A neurons from death. This indicates that dephosphorylation of GSK-3β/Ser9 and GSK-3α/Ser21 is insufficient for GSK-3 proapoptotic function and that another level of regulation is required. Gel filtration reveals a stress-induced loss of neuronal GSK-3β from a high-molecular-mass complex with a concomitant decrease in axin-bound GSK-3β. These data imply that Wnt-regulated GSK-3β plays a nonredundant role in trophic-deprivation-induced death of neurons.     

MicroRNA-3646 Contributes to Docetaxel Resistance in Human Breast Cancer Cells by GSK-3β/β-Catenin Signaling Pathway

Acquisition of resistance to docetaxel (Doc) is one of the most important problems in treatment of breast cancer patients, but the underlying mechanisms are still not fully understood. In present study, Doc-resistant MDA-MB-231 and MCF-7 breast cancer cell lines (MDA-MB-231/Doc and MCF-7/Doc) were successfully established in vitro by gradually increasing Doc concentration on the basis of parental MDA-MB-231 and MCF-7 cell lines (MDA-MB-231/S and MCF-7/S). The potential miRNAs relevant to the Doc resistance were screened by miRNA microarray. We selected 5 upregulated miRNAs (has-miR-3646, has-miR-3658, has-miR-4438, has-miR-1246, and has-miR-574-3p) from the results of microarray for RT-qPCR validation. The results showed that expression level of miR-3646 in MDA-MB-231/Doc cells was significantly higher than in MDA-MB-231/S cells. Compared to MCF-7/S cells, miR-3646 expression was up-regulated in MCF-7/Doc cells. Further studies revealed that transfection of miR-3646 mimics into MDA-MB-231/S or MCF-7/S cells remarkably increased their drug resistance, in contrast, transfection of miR-3646 inhibitors into MDA-MB-231/Doc or MCF-7/Doc cells resulted in significant reduction of the drug resistance. By the pathway enrichment analyses for miR-3646, we found that GSK-3β/β-catenin signaling pathway was a significant pathway, in which GSK-3β was an essential member. RT-qPCR and Western blot results demonstrated that miR-3646 could regulate GSK-3β mRNA and protein expressions. Furthermore, a marked increase of both nuclear and cytoplasmic β-catenin expressions (with phosphorylated-β-catenin decrease) was observed in MDA-MB-231/Doc cells compared with MDA-MB-231/S cells, and their expression were positively related to miR-3646 and negatively correlated with GSK-3β. Taken together, our results suggest that miR-3646-mediated Doc resistance of breast cancer cells maybe, at least in part, through suppressing expression of GSK-3β and resultantly activating GSK-3β/β-catenin signaling pathway.     

Extracellular α-Synuclein Leads to Microtubule Destabilization via GSK-3β-Dependent Tau Phosphorylation in PC12 Cells

α-Synuclein (ASN) plays an important role in pathogenesis of Parkinson''s disease (PD) and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.     

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.     

Polarized Regulation of Glycogen Synthase Kinase-3β Is Important for Glioma Cell Invasion

Glioma malignancy greatly depends on its aggressive invasion. The establishment of cell polarity is an important initial step for cell migration, which is essential for cell-directional translocation. However, our understanding of the molecular mechanisms underlying cell polarity formation in glioma cell invasion remains limited. Glycogen synthase kinase-3 (GSK-3) has a critical role in the formation of cell polarity. We therefore investigated whether localized GSK-3β, a subtype of GSK-3, is important for glioma cell invasion. We reported here that the localized phosphorylation of GSK-3β at the Ser9 (pSer9-GSK-3β) was critical for glioma cell invasion. Scratching glioma cell monolayer up-regulated pSer9-GSK-3β specifically at the wound edge. Inhibition of GSK-3 impaired the cell polarity and reduced the directional persistence of cell migration. Consistently, down-regulation of GSK-3α and 3β by specific small interfering RNAs inhibited glioma cell invasion. Over-expressing wild-type or constitutively active forms of GSK-3β also inhibited the cell invasion. These results indicated the polarized localization of GSK-3 regulation in cell migration might be also important for glioma cell migration. Further, EGF regulated both GSK-3α and 3β, but only pSer9-GSK-3β was enriched at the leading edge of scratched glioma cells. Up- or down-regulation of GSK-3β inhibited EGF-stimulated cell invasion. Moreover, EGF specifically regulated GSK-3β, but not GSK-3α, through atypical PKC pathways. Our results indicated that GSK-3 was important for glioma cell invasion and localized inhibition of GSK-3β was critical for cell polarity formation.