Glycogen Synthase Kinase-3 Family Research Articles

Abstract P3056: Cardiac Fibroblast GSK-3α Contributes To Chronic Inflammation In The Ischemic Heart

Background: Myocardial Infarction (MI) is the leading cause of death worldwide. Glycogen Synthase Kinase-3 (GSK-3) has been identified as a promising therapeutic target to treat cardiovascular diseases. The GSK-3 family has 2 isoforms, α and β. Previously, our lab has identified cardiac fibroblast (FB) GSK-3β acts as a negative regulator of fibrosis in the ischemic heart. However, the role of FB-GSK-3α in MI-induced adverse cardiac remodeling is not defined. Methods and Results: To determine the role of FB-GSK-3α in MI-induced adverse cardiac remodeling, GSK-3α was deleted specifically in the activated fibroblast/myofibroblast using tamoxifen (TAM)-inducible Periostin promoter-driven Cre recombinase system. At 12 weeks of age, mice were fed with the TAM diet. After 1 week of TAM treatment, control (GSK-3α fl/fl Cre -/- ) and KO (GSK-3α fl/fl Cre +/- ) mice were subjected to permanent LAD ligation (MI) surgery. Serial echocardiographic analysis revealed that FB-GSK-3α KO mice were protected from MI-induced systolic dysfunction and dilative remodeling. To investigate the role of FB-GSK-3α in MI-induced chronic inflammation, hearts were harvested at 4 weeks post-MI, and expression of signature inflammatory genes (TNFα, IL-6, IL-1β) was analyzed by qPCR. Surprisingly, the KO heart displayed remarkably low levels of inflammatory gene expression. To delineate underlying mechanisms, the effect of FB-GSK-3α deletion on the pro-inflammatory Nuclear factor kappa B (NF-κB) pathway was examined in vitro . Western blot analysis confirmed a significant reduction in NF-κB activation in GSK-3α-KO FBs. Importantly, the co-culture experiment demonstrated that the expression of inflammatory genes was downregulated in immune cells when cultured with GSK-3α KO FBs. These results indicate that GSK-3α can modulate immune cell phenotype through FB-immune cell crosstalk. Conclusion: Our findings suggest a critical role of FB-GSK-3α in pathological cardiac remodeling and heart failure that could be therapeutically targeted for future clinical applications.

Inhibition of the Glycogen Synthase Kinase 3 Family by the Bikinin Alleviates the Long-Term Effects of Salinity in Barley.

Crops grown under stress conditions show restricted growth and, eventually, reduced yield. Among others, brassinosteroids (BRs) mitigate the effects of stress and improve plant growth. We used two barley cultivars with differing sensitivities to BRs, as determined by the lamina joint inclination test. Barley plants with the 2nd unfolded leaf were sprayed with a diluted series of bikinin, an inhibitor of the Glycogen Synthase Kinase 3 (GSK3) family, which controls the BR signaling pathway. Barley was grown under salt stress conditions up to the start of the 5th leaf growth stage. The phenotypical, molecular, and physiological changes were determined. Our results indicate that the salt tolerance of barley depends on its sensitivity to BRs. We confirmed that barley treatment with bikinin reduced the level of the phosphorylated form of HvBZR1, the activity of which is regulated by GSK3. The use of two barley varieties with different responses to salinity led to the identification of the role of BR signaling in photosynthesis activity. These results suggest that salinity reduces the expression of the genes controlling the BR signaling pathway. Moreover, the results also suggest that the functional analysis of the GSK3 family in stress responses can be a tool for plant breeding in order to improve crops’ resistance to salinity or to other stresses.

Glycogen synthase kinase GSK3\u03b1 promotes tumorigenesis by activating HIF1/VEGFA signaling pathway in NSCLC tumor

BackgroundLung cancer is one of the most common cancers and the leading cause of cancer-related death. Glycogen synthase kinase-3 (GSK-3) α, a member of the glycogen synthase kinase-3 family, reportedly plays a role in tumorigenesis. However, its biological function in tumorigenesis requires deeper exploration. Hypoxia is a major feature of solid tumor, along with decreasing availability of oxygen, inducing treatment resistance, and tumor progress.MethodsLevels of GSK3α expression in clinical samples were detected using western blot and IHC assays, while its biological function and underlying mechanism of action in tumor progression were investigated using western blot, CCK8, cell cycle, colony formation, Transwell, ELISA and tube formation assays. Furthermore, we investigated the relationship between GSK3α expression and the HIF1α/VEGFA signaling pathway in vivo using a mouse xenograft model.ResultsGSK3α was significantly upregulated in NSCLC patients with cases that exhibited high GSK3α levels recording shorter survival times. Moreover, GSK3α overexpression promoted proliferation, migration, invasion and clone formation ability of NSCLC cells, while its silencing resulted in an opposite phenomenon. Moreover, GSK3α not only activated the HIF1α/VEGFA signaling pathway, but also regulated HIF1α stabilization independently via the PHDs-pVHL signaling pathway. Moreover, GSK3α-mediated tumor angiogenesis depended on HIF1α expression both in vitro and in vivo.ConclusionGSK3α functioned as an oncogene in NSCLC tumorigenesis by regulating the HIF1/VEGFA signaling pathway in an independent manner through the PHDs-pVHL signaling pathway. These findings were expected to provide novel sights to guide future development of therapies for effective treatment of NSCLC.BdenY92FCzrTCxoDyoPgeDVideo abstract

GSK-3 Inhibitors as New Leads to Treat Type-II Diabetes

In India as well as globally, diabetes is in a state of high risk and next to cardiovascular disease. As per the WHO, the risk of diabetes is expected to rise about 511 million by 2030. In quest of novel targets for type-2 diabetes, many targets were elucidated, such as Glycogen Synthase Kinase-3 (GSK-3), Dipeptidyl Peptidase (DPP-IV), PPAR-γ, α-Glucosidase, α-Amylase, GLP-1, and SGLT. Among the targets, GSK-3 was reported to be an effective target for the treatment of diabetes. In the metabolism of glycogen, GSK is a regulatory enzyme for the biosynthesis of glycogen (glycogenesis). It catalyzes the synthesis of a linear unbranched molecule with 1,4-α-glycosidic linkages. GSK-3 family has two isoenzymes, namely α and β, which differ in their Nand C- terminal sequences and are semi-conservative multifunctional serine/threonine kinase enzymes. In this chapter, we discuss an overview of general diabetic mechanisms and how GSK-3 modulation may influence these processes. Mutation in the GSK-3 complex causes diabetes. Synthetic and natural scaffolds modulate GSK-3 against diabetes and leading to its optimization for the development of GSK-3 inhibitors. This review mainly focuses on the development of GSK-3 inhibitors and highlights current and potential future therapeutic approaches that support the notion of targeting glucose metabolism with novel antidiabetic agents.

AtSK11 and AtSK12 Mediate the Mild Osmotic Stress-Induced Root Growth Response in Arabidopsis.

Although most osmotic stresses are harmful to plant growth and development, certain drought- or polyethylene glycol (PEG)-induced mild osmotic stresses promote plant root growth. The underlying regulatory mechanisms of this response remain elusive. Here, we report that the GLYCOGEN SYNTHASE KINASE 3 (GSK3) genes ARABIDOPSIS THALIANA SHAGGY-RELATED KINASE 11 (AtSK11) (AT5G26751) and AtSK12 (AT3G05840) are involved in the mild osmotic stress (−0.4 MPa) response in Arabidopsis thaliana. When grown on plant medium infused with different concentrations of PEG to mimic osmotic stress, both wild-type (WT) and atsk11atsk12 plants showed stimulated root growth under mild osmotic stress (−0.4 MPa) but repressed root growth under relatively strong osmotic stress (−0.5, −0.6, −0.7 MPa) as compared to the mock condition (−0.25 MPa). The root growth stimulation of atsk11atsk12 was more sensitive to −0.4 MPa treatment than was that of WT, indicating that AtSK11 and AtSK12 inhibit the mild stress-induced root growth response. RNA-seq analysis of WT and atsk11atsk12 plants under three water potentials (−0.25 MPa, −0.4 MPa, −0.6 MPa) revealed 10 differentially expressed candidate genes mainly involved in cell wall homeostasis, which were regulated by AtSK11 and AtSK12 to regulate root growth in response to the mild stress condition (−0.4 MPa). Promoter motif and transcription factor binding analyses suggested that the basic helix-loop-helix (bHLH) transcription factor bHLH69/LJRHL1-LIKE 2 (LRL2) may directly regulate the expression of most −0.4 MPa-responsive genes. These findings indicate that mild osmotic stress (−0.4 MPa) promotes plant growth and that the GSK3 family kinase genes AtSK11 and AtSK12 play a negative role in the induction of root growth in response to mild osmotic stress.

The GSK-3 family as therapeutic target for myocardial diseases.

Glycogen synthase kinase-3 (GSK-3) is one of the few signaling molecules that regulate a truly astonishing number of critical intracellular signaling pathways. It has been implicated in several diseases including heart failure, bipolar disorder, diabetes mellitus, Alzheimer disease, aging, inflammation, and cancer. Furthermore, a recent clinical trial has validated the feasibility of targeting GSK-3 with small molecule inhibitors for human diseases. In the current review, we will focus on its expanding role in the heart, concentrating primarily on recent studies that have used cardiomyocyte- and fibroblast-specific conditional gene deletion in mouse models. We will highlight the role of the GSK-3 isoforms in various pathological conditions including myocardial aging, ischemic injury, myocardial fibrosis, and cardiomyocyte proliferation. We will discuss our recent findings that deletion of GSK-3α specifically in cardiomyocytes attenuates ventricular remodeling and cardiac dysfunction after myocardial infarction by limiting scar expansion and promoting cardiomyocyte proliferation. The recent emergence of GSK-3β as a regulator of myocardial fibrosis will also be discussed. We will review our recent findings that specific deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Finally, we will examine the underlying mechanisms that drive the aberrant myocardial fibrosis in the models in which GSK-3β is specifically deleted in cardiac fibroblasts. We will summarize these recent results and offer explanations, whenever possible, and hypotheses when not. For these studies we will rely heavily on our models and those of others to reconcile some of the apparent inconsistencies in the literature.

Abstract P335: Is GSK-3α a Regulator of Aging?

The glycogen synthase kinase-3 (GSK-3) family of protein kinases consists of two highly related isoforms, α and β. The GSK-3 family has been reported to regulate an astonishing variety of physiological and pathophysiological processes, whereas virtually nothing is known as for whether they regulate aging. Aging, defined as the progressive loss of function accompanied by decreasing fertility and increasing mortality with advancing age, is a complex biological process, controlled by multiple genetic, epigenetic and environmental factors. We now present the first studies defining the role of GSK-3α in the aging process using germline GSK-3α knockout (KO) mice at age of 3, 6, 12, and 24 mos. Age-matched wildtype (WT) mice served as control. Kaplan-Meier analysis shows that percent survivals (WT vs. KO) was 73.33 vs. 42.11 ‡ at 24 mos of age. In the heart we find cardiac hypertrophy, marked cell loss with replacement fibrosis, contractile dysfunction with profound abnormalities of mitochondrial structure, and impaired autophagy (assessed by Beclin1/ATG6), all becoming evident after 6 mos of age. Echocardiographic LV mass (in mg) is as follows (4 time points): 124.0±3.15 vs. 132.4±3.93, 136.2±4.75 vs. 171.5±7.57 ‡ , 169.2±8.53 vs. 203.5±8.18 ‡ , 195.4±9.82 vs. 257.4±7.24 ‡ ; ejection factor: 60.6±1.08 vs. 62.5±1.52, 64.3±1.10 vs. 60.7±1.61*, 58.5±1.74 vs. 52.2±1.41 ‡ , 55.5±0.96 vs. 49.4±1.02 ‡ ; invasive hemodynamic dP/dt+: 6863.8±673.8 vs. 6999.4±473.2, 7115.4±392.9 vs. 6012.6±407.3*, 7509.6±419.6 vs. 4291.6±473.5*, 6889.0±460.9 vs. 3955.5±306.9 ‡ . We also observed increased BrdU positive nuclei in isolated cardiomyocytes from the KO at 6 mos of age, and increased stem cells at every time-point, compared with age-matched WT. In the small intestine, we find marked senescence-associated β-galactosidase (SA-β-GAL) activity associated with thinning of the crypts, consistent with reduced cell cycling in this tissue. The results imply accelerated development of age-related pathologies in the KO heart and small bowel. These phenotypes are associated with a reduced lifespan in the mouse. Thus GSK-3α is a novel regulator of aging which retards age-related pathologies and prolongs lifespan of the organism. (*, P<0.05; ‡, P<0.01 KO vs. WT)

Deletion of GSK-3β in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation

Based on extensive preclinical data, glycogen synthase kinase-3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b(-/-) embryoid bodies. While GSK-3alpha-deficient mice were born without a cardiac phenotype, no live-born Gsk3b(-/-) pups were recovered. The Gsk3b(-/-) embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation - GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3alpha and GSK-3beta in the heart using loss-of-function approaches, implicate GSK-3beta as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.

Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants.

BackgroundThe glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinases (GSKs) are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences.ResultsWe found that the structure of GSK genes is generally conserved in Arabidopsis and rice, although we documented examples of exon expansion and intron loss. Our phylogenetic analyses of 139 sequences revealed four major clades of GSK genes that correspond to the four subgroups initially recognized in Arabidopsis. ESTs from basal angiosperms were represented in all four major clades; GSK homologs from the basal angiosperm Persea americana (avocado) appeared in all four clades. Gymnosperm sequences occurred in clades I, III, and IV, and a sequence of the red alga Porphyra was sister to all green plant sequences.ConclusionOur results indicate that (1) the plant-specific GSK gene lineage was established early in the history of green plants, (2) plant GSKs began to diversify prior to the origin of extant seed plants, (3) three of the four major clades of GSKs present in Arabidopsis and rice were established early in the evolutionary history of extant seed plants, and (4) diversification into four major clades (as initially reported in Arabidopsis) occurred either just prior to the origin of the angiosperms or very early in angiosperm history.

Lithium inhibits glycogen synthase kinase-3 activity and mimics Wingless signalling in intact cells

Background Exposing eukaryotic cells to lithium ions (Li+) during development has marked effects on cell fate and organization. The phenotypic consequences of Li+ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption, respectively, of a highly conserved protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In Drosophila, the GSK-3 homologue is encoded by zw3sgg, a segment-polarity gene involved in embryogenesis that acts downstream of wg. In higher eukaryotes, GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases.Results We investigated the effect of Li+ on the activity of the GSK-3 family. At physiological doses, Li+ inhibits the activity of human GSK-3β and Drosophila Zw3Sgg, but has no effect on other protein kinases. The effect of Li+ on GSK-3 is reversible in vitro. Treatment of cells with Li+ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau. Li+ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/β-catenin, demonstrating that Li+ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3.Conclusions Li+ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a possible molecular mechanism of Li+ action on development and differentiation.

A Saccharomyces cerevisiae protein-serine kinase related to mammalian glycogen synthase kinase-3 and the Drosophila melanogaster gene shaggy product

The glycogen synthase kinase-3 (GSK-3) family of protein-serine kinases is implicated in the development and hormonal regulation of higher eukaryotes. GSK-3-related genes have been cloned and characterized in mammals (α and β forms), Drosophila melanogaster ( shaggy/zeste-white3) and Saccharomyces cerevisiae ( MCK1). Using the polymerase chain reaction and primers designed to hybridize to conserved catalytic domain sequences of this family, a genomic fragment was amplified from budding yeast DNA. Genomic clones encompassing the entire reading frame were subsequently isolated and sequenced. The protein encoded by this gene, termed ScGSK-3, displays high identity with members of the GSK-3 family, sharing several structural features including a regulatory Tyr residue. A phylogenetic analysis of the catalytic domains of these protein kinases suggests that ScGSK-3 represents the bona fide homologue of GSK-3 and the shaggy product, while the related MCK1 protein kinase is encoded by a paralogous gene which originated by a gene duplication event in the yeast lineage.

Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.

Glycogen synthase kinase-3 (GSK-3) is a protein serine kinase implicated in the cellular response to insulin. The enzyme is the mammalian homologue of the zeste-white3 (shaggy) homeotic gene of Drosophila melanogaster and has been implicated in the regulation of the c-Jun/AP-1 transcription factor. In mammals this protein serine kinase is encoded by two related genes termed GSK-3 alpha and beta. Here, we demonstrate that these two proteins and the fruit fly protein are phosphorylated on tyrosine in vivo. Moreover, GSK-3 beta activity and function are shown to be dependent on tyrosine phosphorylation. The modified tyrosine residue is conserved in all members of the GSK-3 family and is equivalent to that required for activity by mitogen-activated protein (MAP) kinases. However, unlike MAP kinases, GSK-3 is highly phosphorylated on tyrosine and thus active in resting cells.