Glycogen Synthase Kinase-3: A Focal Point for Advancing Pathogenic Inflammation in Depression.
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ABSTRACT:
SUBMITTER: McCallum RT
PROVIDER: S-EPMC8470361 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the activation of Rac were suppressed in GSK-3 or h-prune knocked-down cells. GSK-3 inhibitors suppressed the disassembly of paxillin and the activation of FAK and Rac. Furthermore, h-prune was highly expressed in colorectal and pancreatic cancers, and the positivity of the h-prune expression was correlated with tumor invasion. These results suggest that GSK-3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration and that h-prune is useful as a marker for tumor aggressiveness.
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Project description:IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation.
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Project description:CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3β protein levels in Th17 cells, whereas GSK3β was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.
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Project description:The glycogen synthase kinase 3B (GSK3B) is an important target protein of several antidepressants, such as lithium, a mood stabilizer. Recent studies associated structural variations of the GSK3B gene to bipolar disorder (BP), although replications were not conclusive. Here we present data on copy number variations (CNVs) of the GSK3B gene probing the 9th exon region in 846 individuals (414 controls, 172 patients with major depressive disorder (MDD) and 260 with BP). A significant accumulation (odds ratio: 5.5, P = 0.00051) of the amplified exon 9 region was found in patients (22 out of 432) compared to controls (4 of 414). Analyzing patient subgroups, GSK3B structural variants were found to be risk factors of BP particularly (P = 0.00001) with an odds ratio of 8.1 while no such effect was shown in the MDD group. The highest odds (19.7 ratio) for bipolar disorder was observed in females with the amplified exon 9 region. A more detailed analysis of the identified GSK3B CNV by a set of probes covering the GSK3B gene and the adjacent NR1I2 and C3orf15 genes showed that the amplified sequences contained 3' (downstream) segments of the GSK3B and NR1I2 genes but none of them involved the C3orf15 gene. Therefore, the copy number variation of the GSK3B gene could be described as a complex set of structural variants involving partial duplications and deletions, simultaneously. In summary, here we confirmed significant association of the GSK3B CNV and bipolar disorder pointing out that the copy number and extension of the CNV varies among individuals.
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Project description:Functional differentiation of the two isoforms of the protein-serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is an unsettled area of research. The isoforms are highly similar in structure and are largely redundant, though there is also evidence for specific roles. Identification of isoform-specific protein interactors may elucidate the differences in function and provide insight into isoform-selective regulation. We therefore sought to identify novel GSK-3 interaction partners and to examine differences in the interactomes of the two isoforms using both affinity purification and proximity-dependent biotinylation (BioID) mass spectrometry methods. While the interactomes of the two isomers are highly similar in HEK293 cells, BioID in HeLa cells yielded a variety of preys that are preferentially associated with one of the two isoforms. DCP1B, which favored GSK-3α, and MISP, which favored GSK-3β, were evaluated for reciprocal interactions. The differences in interactions between isoforms may help in understanding the distinct functions and regulation of the two isoforms as well as offer avenues for the development of isoform-specific strategies.
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Project description:The regulation of glycogen metabolism is critical for the maintenance of glucose and energy homeostasis in mammals. Glycogen synthase, the enzyme responsible for glycogen production, is regulated by multisite phosphorylation in yeast and mammals. We have previously identified PAS kinase as a physiological regulator of glycogen synthase in Saccharomyces cerevisiae. We provide evidence here that PAS kinase is an important regulator of mammalian glycogen synthase. Glycogen synthase is efficiently phosphorylated by PAS kinase in vitro at Ser-640, a known regulatory phosphosite. Efficient phosphorylation requires a region of PAS kinase outside the catalytic domain. This region appears to mediate a direct interaction between glycogen synthase and PAS kinase, thereby targeting kinase activity to this substrate specifically. This interaction is regulated by the PAS kinase PAS domain, raising the possibility that this interaction (and phosphorylation event) is modulated by the cellular metabolic state. This mode of regulation provides a mechanism for metabolic status to impinge directly on the cellular decision of whether to store or use available energy.
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Project description:Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162-5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.
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Project description:Metabolic Dysfunction Associated Steatohepatitis (MASH) is characterized by excess circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to the liver sinusoidal endothelial cells (LSEC) and transendothelial migration (TEM) are crucial in the inflammatory process. LSEC under lipotoxic stress develops a pro-inflammatory phenotype known as endotheliopathy. However, the mediators of endotheliopathy remain obscure. Primary mouse LSEC isolated from C57BL/6J mice on chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH, due to a choline-deficient high-fat diet (CDHFD) or FFC diet, were treated with two structurally distinct GSK3 inhibitors [LY2090314, elraglusib (9-ING-41)]. Integrated pathway analysis of mouse LSEC proteome and transcriptome indicates that leukocyte TEM and focal adhesion are major pathways altered in MASH. Kinome profiling of the LSEC phospho-proteome identified GSK3β as the major kinase hub in MASH. GSK3β activating phosphorylation was increased in primary human LSEC treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependant mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC and CDHFD-fed mice, respectively. Immunophenotyping of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib using cytometry by the time of flight showed reduced proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells infiltration.GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and may serve as a potential therapeutic strategy in human MASH.
2024-06-22 | PXD048073 | Pride
Project description:Objective:To investigate the role of glycogen metabolism in regulating rheumatoid fibroblast-like synoviocyte (FLS)-mediated synovial inflammation and its underlying mechanism. Methods:FLSs were separated from synovial tissues (STs) obtained from rheumatoid arthritis (RA) patients. Glycogen content was determined by periodic acid Schiff staining. Protein expression was analyzed by Western blot or immunohistochemistry. Gene expression of cytokines and matrix metalloproteinases (MMPs) was evaluated by quantitative real-time PCR. FLS proliferation was detected by EdU incorporation. Migration and invasion were measured by Boyden chamber assay. Results:Glycogen levels and glycogen synthase 1 (GYS1) expression were significantly increased in the ST and FLSs of RA patients. TNF-? or hypoxia induced GYS1 expression and glycogen synthesis in RA FLSs. GYS1 knockdown by shRNA decreased the expression of IL-1?, IL-6, CCL-2, MMP-1, and MMP-9 and proliferation and migration by increasing AMP-activated protein kinase (AMPK) activity in RA FLS. AMPK inhibitor or knockdown AMPK could reverse the inhibitory effect of GYS1 knockdown on the inflammatory response in RA FLSs; however, an AMPK agonist blocked RA FLS activity. We further determined that hypoxia-inducible factor-1? mediates TNF-?- or hypoxia-induced GYS1 expression and glycogen levels. Local joint depletion of GYS1 or intraperitoneal administration with an AMPK agonist ameliorated the severity of arthritis in rats with collagen-induced arthritis. Conclusion:Our data demonstrate that GYS1-mediated glycogen accumulation contributes to FLS-mediated synovial inflammation in RA by blocking AMPK activation. In our knowledge, this is a first study linking glycogen metabolism to chronic inflammation. Inhibition of GYS1 might be a novel therapeutic strategy for chronic inflammatory arthritis, including RA.
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