Glycogen Synthase Kinase-3 Activity Research Articles

Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells.

Skin barrier damage is present in the patients with hereditary disorders of the magnesium channel, but the molecular mechanism has not been fully understood. We found that the expressions of hyaluronan synthase (HAS), HAS2 and HAS3 are influenced by MgCl2 concentration in human keratinocyte-derived HaCaT cells. The exposure of cells to a high concentration (5.8 mM) of MgCl2 induced the elevation of HAS2/3 expression, which was inhibited by mRNA knockdown of nonimprinted in Prader-Willi/Angelman syndrome-like domain containing 4 (NIPAL4). Similarly, the content of hyaluronic acid (HA) was changed according to MgCl2 concentration and the expression of NIPAL4. The MgCl2 supplementation increased the reporter activities of HAS2/3, which were inhibited by NIPAL4 knockdown, indicating that the expressions of HAS2/3 are up-regulated at the transcriptional level. The reporter activities and mRNA levels of HAS2/3, and the production of HA were inhibited by CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor, and naphthol AS-E, a cyclic AMP-response element binding protein (CREB) inhibitor. Furthermore, the mutation in putative CREB-binding sites of promoter region in HAS2/3 genes inhibited the MgCl2 supplementation-induced elevation of promoter activity. Our results indicate that the expressions of HAS2/3 are up-regulated by MgCl2 supplementation in HaCaT cells mediated through the activation of GSK3 and CREB. Magnesium may play a pivotal role in maintaining the skin barrier function and magnesium supplementation may be useful to enhance moisturization and wound repair in the skin.

Glycogen synthesis and beyond, a comprehensive review of GSK3 as a key regulator of metabolic pathways and a therapeutic target for treating metabolic diseases.

Glycogen synthase kinase‐3 (GSK3) is a highly evolutionarily conserved serine/threonine protein kinase first identified as an enzyme that regulates glycogen synthase (GS) in response to insulin stimulation, which involves GSK3 regulation of glucose metabolism and energy homeostasis. Both isoforms of GSK3, GSK3α, and GSK3β, have been implicated in many biological and pathophysiological processes. The various functions of GSK3 are indicated by its widespread distribution in multiple cell types and tissues. The studies of GSK3 activity using animal models and the observed effects of GSK3‐specific inhibitors provide more insights into the roles of GSK3 in regulating energy metabolism and homeostasis. The cross‐talk between GSK3 and some important energy regulators and sensors and the regulation of GSK3 in mitochondrial activity and component function further highlight the molecular mechanisms in which GSK3 is involved to regulate the metabolic activity, beyond its classical regulatory effect on GS. In this review, we summarize the specific roles of GSK3 in energy metabolism regulation in tissues that are tightly associated with energy metabolism and the functions of GSK3 in the development of metabolic disorders. We also address the impacts of GSK3 on the regulation of mitochondrial function, activity and associated metabolic regulation. The application of GSK3 inhibitors in clinical tests will be highlighted too. Interactions between GSK3 and important energy regulators and GSK3‐mediated responses to different stresses that are related to metabolism are described to provide a brief overview of previously less‐appreciated biological functions of GSK3 in energy metabolism and associated diseases through its regulation of GS and other functions.

Comparison of the chemical constituents and anti-Alzheimer\u2019s disease effects of Uncaria rhynchophylla and Uncaria tomentosa

BackgroundUncaria tomentosa, which has similar chemical constituents with Uncaria rhynchophylla, has been reported to alleviate cognitive impairments in Alzheimer’s disease (AD) animal models. This study aimed to compare the chemical constituents and anti-AD effect of the ethanol extracts of U. tomentosa (UTE) and U. rhynchophylla (URE).MethodsThe high-performance liquid chromatography (HPLC) was used to compare the chemical constituents of UTE and URE. Streptozotocin (STZ) was intracerebroventricularly (ICV) injected into adult male Sprague–Dawley (SD) rats to establish AD model. UTE (400 mg/kg) or URE (400 mg/kg) was administrated intragastrically once daily to the rats for 6 consecutive weeks. Morris water maze (MWM) test was conducted to assess the neurological functions in the STZ-induced AD rats. The brain tissues of the rats were harvested for further biochemical assay.ResultsThe MWM test results showed both UTE and URE could significantly improve the learning and memory impairments induced by STZ in rats. Both UTE and URE could significantly inhibit the hyperphosphorylation of tau protein, reduce the elevated levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), enhance activities of antioxidant enzymes (SOD, CAT and GPx) and increase the protein expression of HO-1. In addition, UTE could decrease the malondialdehyde (MDA) level. Furthermore, both UTE and URE significantly enhanced Akt activation, down regulated the activation of glycogen synthase kinase 3β (GSK-3β), and induced the nuclear translocation of Nrf2 in the STZ-induced AD rats.ConclusionsUTE and URE contained similar chemical constituents. We found for the first time that both of them could ameliorate cognitive deficits in the STZ-induced AD rats. The underlying molecular mechanism involve suppression of tau hyperphosphorylation, anti-oxidant and anti-neuroinflammation via modulating Akt (Ser473)/GSK3β (Ser9)-mediated Nrf2 activation. These findings amply implicate that both of UTE and URE are worthy of being developed clinically into pharmaceutical treatment for AD.

Glycogen synthase kinase-3β inactivation promotes cervical cancerprogression, invasion, and drug resistance.

Human papillomavirus (HPV) infection-dependent cervical cancer is one of the most common gynecological cancers and often becomes aggressive, with rapid proliferation, invasion/migration, and drug resistance. Here, 135 fresh human cervical squamous cell carcinoma (CSCC) tissue specimens, comprising 21 adjacent normal (AN), 30 cervical intraepithelial neoplasia (CIN1-3 ), 45 CSCC, and 39 drugs (chemo-radiation)-resistant cervical tumor (DRCT) tissues were included. HPV-positive (HeLa, SiHa), HPV-negative (C33A), and cisplatin-resistant (CisR-HeLa/-SiHa/-C33A) cell lines were used for in vitro studies. HPV16/18 oncoproteins E6/E7, pERK1/2, and glycogen synthase kinase-3 (GSK3) and the matrix metalloproteinases (MMPs) MMP-9/-2 were assessed using immunohistochemistry, WB, and gelatin zymography. HPV16/18 infection was observed in 16.7% of the CIN1-3 , 77.8% of the CSCC, and 89.7% of DRCT samples. Total and inactive GSK3β expressions were associated with overall CSCC progression (p=0.039 and p=0.024, respectively) and chemoresistance (p=0.004 and p=0.014, respectively). Positive correlations were observed, between the expression of E6 and pGSK3β expression (p=0.013); E6 and CSCC progression (p<0.0001)/drug resistance (p=0.0001). CisR-HeLa/-SiHa was more dependent on pGSK3β, and activation of GSK3 by SMIs (iAkt), treatment with nimbolide, or knockdown of E6/E7 reduced cisplatin resistance and promoted apoptosis. Hence, the activation of GSK3β with nimbolide and iAkt can be exploited for therapeutic interventions of cervical cancer.

Testing Fmr1 KO Phenotypes in Response to GSK3 Inhibitors: SB216763 versus AFC03127.

Glycogen synthase kinase 3 (GSK3) is a proline-directed serine-threonine kinase that is associated with several neurological disorders, including Alzheimer’s disease and fragile X syndrome (FXS). We tested the efficacy of a novel GSK3 inhibitor AFC03127, which was developed by Angelini Pharma, in comparison to the metabotropic glutamate receptor 5 inhibitor 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the GSK3 inhibitor SB216763 in in vivo and in vitro assays in Fmr1KO mice, a mouse model useful for the study of FXS. The in vivo assay tested susceptibility to audiogenic-induced seizures (AGS) whereas the in vitro assays assessed biomarker expression and dendritic spine length and density in cultured primary neurons as a function of drug dose. MPEP and SB216763 attenuated AGS in Fmr1KO mice, whereas AFC03127 did not. MPEP and AFC03127 significantly reduced dendritic expression of amyloid-beta protein precursor (APP). All drugs rescued spine length and the ratio of mature dendritic spines. Spine density was not statistically different between vehicle and GSK3 inhibitor-treated cells. The drugs were tested over a wide concentration range in the in vitro assays to determine dose responses. A bell-shaped dose response decrease in APP expression was observed in response to AFC03127, which was more effective than SB216763. These findings confirm previous studies demonstrating differential effects of various GSK3 inhibitors on AGS propensity in Fmr1KO mice and confirm APP as a downstream biomarker that is responsive to GSK3 activity.

\u03b1-Synuclein Induces the GSK-3-Mediated Phosphorylation and Degradation of NURR1 and Loss of Dopaminergic Hallmarks

In Parkinson’s disease, the dysfunction of the dopaminergic nigrostriatal tract involves the loss of function of dopaminergic neurons of the substantia nigra pars compacta followed by death of these neurons. The functional recovery of these neurons requires a deep knowledge of the molecules that maintain the dopaminergic phenotype during adulthood and the mechanisms that subvert their activity. Previous studies have shown that transcription factor NURR1, involved in differentiation and maintenance of the dopaminergic phenotype, is downregulated by α-synuclein (α-SYN). In this study, we provide a mechanistic explanation to this finding by connecting α-SYN-induced activation of glycogen synthase kinase-3 (GSK-3) with NURR1 phosphorylation followed by proteasomal degradation. The use of sequential deletion mutants and single point mutants of NURR1 allowed the identification of a domain comprising amino acids 123-PSSPPTPSTPS-134 that is targeted by GSK-3 and leads to subsequent ubiquitination and proteasome degradation. This study provides a detailed analysis of the regulation of NURR1 stability by phosphorylation in synucleinopathies such as Parkinson’s disease.Graphical abstract

The novel potent GSK3 inhibitor AF3581 reverts fragile X syndrome phenotype.

Glycogen-synthase kinase 3 (GSK3) is a kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK3 has been linked to several disease conditions such as fragile X syndrome (FXS). Recent evidences demonstrating an increased activity of GSK3 in murine models of FXS, suggest that dysregulation/hyperactivation of the GSK3 path should contribute to FXS development. A likely possibility could be that in FXS there is a functional impairment of the upstream inhibitory input over GSK3 thus making overactive the kinase. Since GSK3 signaling is a central regulatory node for critical neurodevelopmental pathways, understanding the contribution of GSK3 dysregulation to FXS, may provide novel targets for therapeutic interventions for this disease. In this study we used AF3581, a potent GSK3 inhibitor that we recently discovered, in an in vivo FXS mouse model to elucidate the crucial role of GSK3 in specific behavioral patterns (locomotor activity, sensorimotor gating and social behavior) associated with this disease. All the behavioral alterations manifested by Fmr1 knockout mice were reverted after a chronic treatment with our GSK3 inhibitor, confirming the importance of this pathway as a therapeutic target.

GSK3β is involved in promoting Alzheimer’s disease pathologies following chronic systemic exposure to Porphyromonas gingivalis lipopolysaccharide in amyloid precursor proteinNL-F/NL-F knock-in mice

In line with the strong association between periodontitis and Alzheimer’s disease (AD) clinically, preclinical studies have shown that systemic exposure to Porphyromonas gingivalis (Pg) initiates AD pathologies. However, the involvement of periodontitis in promoting AD pathologies is unclear. In the present study, we provided evidence that chronic systemic exposure to lipopolysaccharide derived from Pg (PgLPS, 1 mg/kg, daily, intraperitoneally) prompted neuroinflammation and tau hyperphosphorylation in 10-month-old of amyloid precursor protein (APP) knock-in mice, a model of AD, carrying the Swedish and Beyreuther/Iberian mutation (APPNL-F/NL-F). The learning and memory function were assessed using the passive avoidance test. The production of APP, Amyloid (A)β1-42, cytokines, synaptic proteins and the activation of glycogen synthase kinase (GSK)-3β as well as phosphorylation of tau were analyzed by immunohistochemistry, Western blotting or an enzyme-linked immunosorbent assay (ELISA) in the cortex of APPNL-F/NL-F mice. We found that systemic exposure of PgLPS for three consecutive weeks induced learning and memory deficits with significantly reduced postsynaptic density protein (PSD95). Increased hyperphosphorylation of tau in multiple residues, including Ser202, Thr231 and Ser396, but not the accumulation of Aβ1-42 was detected in the neurons of APPNL-F/NL-F mice. Furthermore, PgLPS increased the GSK3β activity by reducing its phosphorylation of the serine residue at position 9 (Ser9) and promoted neuroinflammation by increasing the expression of interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) while decreasing that of interleukin-10 (IL-10) and transforming growth factor (TGFβ) in the cortex of APPNL-F/NL-F mice. Moreover, the PgLPS-increased GSK3β activity was detected in both microglia and neurons, while the PgLPS-increased TNF-α expression was mainly detected in the microglia in the cortex of APPNL-F/NL-F mice. In in vitro studies, PgLPS (1 µg/ml) stimulation increased the mRNA and protein level of TNF-α in MG6 microglia, which were significantly inhibited by the GSK3β-specific inhibitor TWS119. In contrast, the tau hyperphosphorylation and activation of GSK3β in N2a neurons were enhanced after treatment with conditioned medium from PgLPS-stimulated microglia, which was attenuated after pre-treatment with TNF-α inhibitor. Taken together, these findings indicate that GSK3β is involved in prompting microglia (TNF-α)-dependent tau hyperphosphorylation in neurons, resulting in learning and memory deficits in APPNL-F/NL-F mice without changes in the Aβ expression during chronic systemic exposure to PgLPS. We propose that dampening GSK3β activation may help delay the periodontitis-promoted pathological progression of AD.

A GSK3-SRF Axis Mediates Angiotensin II Induced Endothelin Transcription in Vascular Endothelial Cells.

Endothelin, encoded by ET1, is a vasoactive substance primarily synthesized in vascular endothelial cells (VECs). Elevation of endothelin levels, due to transcriptional hyperactivation, has been observed in a host of cardiovascular diseases. We have previously shown that serum response factor (SRF) is a regulator of ET1 transcription in VECs. Here we report that angiotensin II (Ang II) induced ET1 transcription paralleled activation of glycogen synthase kinase 3 (GSK3) in cultured VECs. GSK3 knockdown or pharmaceutical inhibition attenuated Ang II induced endothelin expression. Of interest, the effect of GSK3 on endothelin transcription relied on the conserved SRF motif within the ET1 promoter. Further analysis revealed that GSK3 interacted with and phosphorylated SRF at serine 224. Phosphorylation of SRF by GSK3 did not influence its recruitment to the ET1 promoter. Instead, GSK3-mediated SRF phosphorylation potentiated its interaction with MRTF-A, a key co-factor for SRF, which helped recruit the chromatin remodeling protein BRG1 to the ET1 promoter resulting in augmented histone H3 acetylation/H3K4 trimethylation. Consistently, over-expression of a constitutively active GSK enhanced Ang II-induced ET1 transcription and knockdown of either MRTF-A or BRG1 abrogated the enhancement of ET1 transcription. In conclusion, our data highlight a previously unrecognized mechanism that contributes to the transcriptional regulation of endothelin. Targeting this GSK3-SRF axis may yield novel approaches in the intervention of cardiovascular diseases.

Abstract LB207: Glutamine transporter SLC38A3 promotes breast cancer migration via GSK3beta/beta-catenin/EMT pathway

Abstract Breast cancer is one of the most commonly diagnosed cancers and the leading cause of cancer-related death among women. Triple negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. TNBC shows a high capacity for early metastasis and leads to worse clinical outcomes than other breast cancer subtypes due to the lack of specific therapeutic targets. We are looking to develop cancer-specific therapeutic targets for TNBC. Many types of cancer cells including TNBC cells rely on glutamine as carbon and nitrogen source to fuel unchecked growth. We screened for key genes in regulating glutamine metabolism in a panel of breast cancer cell lines. This screen identified the mRNA and protein levels of solute carrier family 38 member 3 (SLC38A3), a glutamine transporter, to be upregulated in human breast cancer cell lines, especially in TNBC cell lines. The TCGA breast cancer patient database also showed that SLC38A3 mRNA is overexpressed in invasive ductal breast carcinoma tissues, and it is even higher in TNBC relative to other breast cancer subtypes. To test the biological role of SLC38A3 protein in TNBC cells, we performed loss-of-function experiments in multiple TNBC cell lines. Silencing of SLC38A3 decreased cellular glutamate, glutamine and alanine levels. Silencing of SLC38A3 also activated apoptosis, and suppressed cell viability, migration and invasion in several TNBC cell lines. Interestingly, silencing of SLC38A3 increased the activity of glycogen synthase kinase 3-β (GSK3β) which promoted the degradation of β-catenin, leading to the decrease of the mRNA levels of epithelial-to-mesenchymal-transition (EMT)-associated transcription factors in TNBC cell lines. In summary, we showed that SLC38A3 is overexpressed in TNBC and promotes breast cancer migration and invasion via GSK3β/β-catenin/EMT pathway, which could be a novel therapeutic target for breast cancer. Citation Format: Zheqiong Tan, Caitlin M. Tressler, Kanchan Sonkar, Kristine Glunde. Glutamine transporter SLC38A3 promotes breast cancer migration via GSK3beta/beta-catenin/EMT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB207.

Glycogen Synthase Kinase 3 activation status in soleus and EDL muscles from space‐flown mice aboard the BION‐M1 biosatellite

Background A significant limitation to long-term spaceflight and space habitation is the deterioration of human musculoskeletal health. It is well known that prolonged exposure to microgravity leads to pronounced muscle and bone loss, with consequences mimicking several major effects observed in human aging (i.e. osteoporosis and sarcopenia). It is therefore critical to elucidate the underlying molecular mechanisms of these processes to develop meaningful intervention strategies. Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase initially defined in its role of regulating glycogen homeostasis, though it's now known to interact with more than 100 target proteins. GSK3 has notably proven to negatively regulate muscle mass by inhibiting hypertrophy-related mechanisms, myoblast fusion, and muscle regeneration, while also promoting ubiquitin-mediated protein degradation and muscle inflammation. In this study, we explored GSK3 expression and its serine phosphorylation status, known to control its activation/inactivation, in soleus and extensor digitorum longus (EDL) muscles obtained from the BION-M1 mission. Methods Male C57BL6/J mice (3 months old) were flown in space for 1 month aboard the BION-M1 biosatellite (F) and were compared to a ground control (GC, space housing and diet conditions) and a flight control (FC, standard housing conditions) group. Soleus and EDL muscles were extracted and homogenized in protein extraction buffer. Western blotting was used to examine total and phosphorylated (i.e. inactivated) forms of GSK3 (ser21 GSK3α; ser9 GSK3β). Results In the soleus muscle, total GSK3α and GSK3β were reduced in the F group. Though we did not observe any changes in ser21 (GSK3α) or ser9 (GSK3β) phosphorylation, the ratio of phosphorylated:total GSK3β was significantly elevated in the F group compared with GC and FC. We did not observe any differences in soleus phosphorylated:total GSK3α. Further, we did not observe any differences in total, phosphorylated or phosphorylated:total GSK3α/β content in the EDL F group compared to GC and FC. Conclusions Altogether, our data suggest that in response to 1 month of spaceflight, GSK3 is less active in the soleus muscle through changes in expression and phosphorylation. GSK3 may therefore not be involved in the later stages of soleus atrophy in flown mice. This may be explained by a slow-to-fast fibre shift in soleus muscle, as GSK3 presence and activity is typically reduced in fast-twitch muscle fibres compared to slow-twitch. Future studies with soleus muscles from two other NASA-flown missions (RR1 and RR9) will be conducted to confirm these effects.

GSK-3: a multifaceted player in acute leukemias.

Glycogen synthase kinase 3 (GSK-3) consists of two isoforms (α and β) that were originally linked to glucose metabolism regulation. However, GSK-3 is also involved in several signaling pathways controlling many different key functions in healthy cells. GSK-3 is a unique kinase in that its isoforms are constitutively active, while they are inactivated mainly through phosphorylation at Ser residues by a variety of upstream kinases. In the early 1990s, GSK-3 emerged as a key player in cancer cell pathophysiology. Since active GSK-3 promotes destruction of multiple oncogenic proteins (e.g., β-catenin, c-Myc, Mcl-1) it was considered to be a tumor suppressor. Accordingly, GSK-3 is frequently inactivated in human cancer via aberrant regulation of upstream signaling pathways. More recently, however, it has emerged that GSK-3 isoforms display also oncogenic properties, as they up-regulate pathways critical for neoplastic cell proliferation, survival, and drug-resistance. The regulatory roles of GSK-3 isoforms in cell cycle, apoptosis, DNA repair, tumor metabolism, invasion, and metastasis reflect the therapeutic relevance of these kinases and provide the rationale for combining GSK-3 inhibitors with other targeted drugs. Here, we discuss the multiple and often conflicting roles of GSK-3 isoforms in acute leukemias. We also review the current status of GSK-3 inhibitor development for innovative leukemia therapy.

The GSK3-NRF2 Axis in Suicide

Mutations in the genes coding for tryptophan-hydrolase-2 and the scaffold protein FKBP5 are associated with an increased risk of suicide. The mutation in both cases enhances the enzymatic activity of glycogen synthase kinase-3 (GSK3). Conversely, anti-suicidal medications, such as lithium, clozapine, and ketamine, indirectly inhibit the activity of GSK3. When GSK3 is active, it promotes the metabolic removal of the transcription factor NRF2 (nuclear factor erythroid 2-related factor-2), which suppresses the transcription of multiple genes that encode anti-oxidative and anti-inflammatory proteins. Notably, several suicide-biomarkers bear witness to an ongoing inflammatory process. Moreover, alterations in serum lipid levels measured in suicidal individuals are mirrored by data obtained in mice with genetic deletion of the NRF2 gene. Inflammation is presumably causally related to both dysphoria and anger, two factors relevant for suicide ideation and attempt. Preventing the catabolism of NRF2 could be a strategy to obtain novel suicide-prophylactic medications. Possible candidates are minocycline and nicotinic-α7 agonists. The antibiotic minocycline indirectly activates NRF2-transcriptional activity, whereas the activation of nicotinic-α7 receptors indirectly inhibits GSK3.

Dysregulation of GSK3β-Target Proteins in Skin Fibroblasts of Myotonic Dystrophy Type 1 (DM1) Patients.

Myotonic dystrophy type 1 (DM1) is the most common monogenetic muscular disorder of adulthood. This multisystemic disease is caused by CTG repeat expansion in the 3'-untranslated region of the DM1 protein kinase gene called DMPK. DMPK encodes a myosin kinase expressed in skeletal muscle cells and other cellular populations such as smooth muscle cells, neurons and fibroblasts. The resultant expanded (CUG)n RNA transcripts sequester RNA binding factors leading to ubiquitous and persistent splicing deregulation. The accumulation of mutant CUG repeats is linked to increased activity of glycogen synthase kinase 3 beta (GSK3β), a highly conserved and ubiquitous serine/threonine kinase with functions in pathways regulating inflammation, metabolism, oncogenesis, neurogenesis and myogenesis. As GSK3β-inhibition ameliorates defects in myogenesis, muscle strength and myotonia in a DM1 mouse model, this kinase represents a key player of DM1 pathobiochemistry and constitutes a promising therapeutic target. To better characterise DM1 patients, and monitor treatment responses, we aimed to define a set of robust disease and severity markers linked to GSK3βby unbiased proteomic profiling utilizing fibroblasts derived from DM1 patients with low (80- 150) and high (2600- 3600) CTG-repeats. Apart from GSK3β increase, we identified dysregulation of nine proteins (CAPN1, CTNNB1, CTPS1, DNMT1, HDAC2, HNRNPH3, MAP2K2, NR3C1, VDAC2) modulated by GSK3β. In silico-based expression studies confirmed expression in neuronal and skeletal muscle cells and revealed a relatively elevated abundance in fibroblasts. The potential impact of each marker in the myopathology of DM1 is discussed based on respective function to inform potential uses as severity markers or for monitoring GSK3β inhibitor treatment responses.

Lycium ruthenicum Murray anthocyanins effectively inhibit α-glucosidase activity and alleviate insulin resistance

Reducing blood glucose level is an important strategy to control type 2 diabetes mellitus (T2DM). This study aimed to explore the potential of Lycium ruthenicum Murray (LM) anthocyanins to reduce blood glucose levels. LM anthocyanins effectively inhibited the activity of α-glucosidase of Caco-2 cells (IC50 value was 25.3 μg/mL), and promoted glucose consumption and uptake of Hep-G2 cells with insulin resistance (IR). Furthermore, LM anthocyanins effectively improved glycometabolism by alleviating IR through 1) activating the phosphatidylinositol 3-kinase/serine-threonine specific protein kinase B pathway, and inhibiting the activity of downstream glycogen synthase kinase-3 β and forkhead box protein O1; and 2) improving mitochondrial function by reducing cell and mitochondrial reactive oxygen species and restoring ATP production and the mitochondrial membrane potential. The results suggested that LM anthcoyanins are helpful in reducing blood glucose levels and therefore could be used to control T2DM.

Macrophage Function and the Role of GSK3.

Macrophages are present in nearly all vertebrate tissues, where they respond to a complex variety of regulatory signals to coordinate immune functions involved in tissue development, metabolism, homeostasis, and repair. Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed protein kinase that plays important roles in multiple pathways involved in cell metabolism. Dysregulation of GSK3 has been implicated in several prevalent metabolic disorders, and recent findings have highlighted the importance of GSK3 activity in the regulation of macrophages, especially with respect to the initiation of specific pathologies. This makes GSK3 a potential therapeutic target for the development of novel drugs to modulate immunometabolic responses. Here, we summarize recent findings that have contributed to our understanding of how GSK3 regulates macrophage function, and we discuss the role of GSK3 in the development of metabolic disorders and diseases.

Angiotensin II receptor 1 controls profibrotic Wnt/β-catenin signalling in experimental autoimmune myocarditis.

AimsAngiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis.Methods and resultsEAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund’s adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts.ConclusionAng II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

Anti-inflammatory Potential of GSK-3 Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a protein kinase containing threonine or serine amino acid residues. GSK-3 was first discovered in 1980 as a regulatory protein kinase, Glycogen synthase (GS) enzyme, which is responsible for the conversion of glycogen from glucose with the help of uridine diphosphate glucose (UDP-Glu) residue. GSK-3 has two isoforms present in human beings, namely GSK-3 α (serine residue at 21 position) and GSK-3 β (serine residue at 9 position). GSK-3 has two terminals, namely C- terminal and N- terminal. C-terminal of GSK-3 resembles α- helix conformation, which acts as an activator loop and is responsible for positioning residues in ATP binding and catalysis of substrates. On the other hand, the N- terminal of GSK-3 resembles β- strand conformation, which acts as an inhibitory loop; having a tyrosine molecule at 216 positions, it is essential for the complete GSK-3 activity. N- terminal of GSK-3 is responsible for ATP binding activity and exhibits various biological activities like cell signaling, gene induction following activation of T cell receptor, apoptosis, protein translation, glycogen metabolism, and inflammatory process. Activation of GSK-3 leads to pro-inflammatory actions, i.e. an increase in the binding activity of NF-kB (pro-inflammatory genes), increase in the transactivation activity of NF-kB, increase in the phosphorylation of p105, and a decrease in the transactivation activity of C/EBPβ (anti- inflammatory genes), resulting in a large number of prevalent diseases such as diabetes, cancer, neurodegenerative diseases, psychiatric diseases, mood disorders, etc. Glycogen synthase kinase inhibitors (GSK-3 inhibitors) are various chemotypes and have different mechanisms of actions. They are obtained from different sources such as natural products, synthetic ATP as well as non-ATP competitive inhibitors along with substrate-competitive inhibitors. The inhibitors of GSK3 have proven to possess very potent anti-inflammatory action. GSK-3 inhibitors are useful for treating different prevalent disorders, such as neurodegenerative diseases, including Alzheimer's disease, hyperglycemia, cancer disease, and mood disorders like depression, etc. In this review, we have highlighted the evidence regarding the description and types of GSK, inflammation process, and the factors affecting inflammation, the relationship between inflammation and GSK, GSK3 inhibitors, and finally, the impact of various natural as well as synthetic GSK3 inhibitors having anti-inflammatory activity.

Translation of IRF-1 Restricts Hepatic Interleukin-7 Production to Types I and II Interferons: Implications for Hepatic Immunity.

Interleukin-7 (IL-7) is an important cytokine with pivotal pro-survival functions in the adaptive immune system. However, the role of IL-7 in innate immunity is not fully understood. In the present study, the impact of hepatic IL-7 on innate immune cells was assessed by functional experiments as well as in patients with different stages of liver cirrhosis or acute-on-chronic liver failure (ACLF). Human hepatocytes and liver sinusoidal endothelial cells secreted IL-7 in response to stimulation with interferons (IFNs) of type I and II, yet not type III. De novo translation of interferon-response factor-1 (IRF-1) restricted IL-7 production to stimulation with type I and II IFNs. LPS-primed human macrophages were identified as innate immune target cells responding to IL-7 signaling by inactivation of Glycogen synthase kinase-3 (GSK3). IL-7-mediated GSK3 inactivation augmented LPS-induced secretion of pro-inflammatory cytokines and blunted LPS tolerance of macrophages. The IFN-IRF-1-IL-7 axis was present in liver cirrhosis patients. However, liver cirrhosis patients with or without ACLF had significantly lower concentrations of IL-7 in serum compared to healthy controls, which might contribute to LPS-tolerance in these patients. In conclusion, we propose the presence of an inflammatory cascade where IFNs of type I/II induce hepatocellular IL-7 in an IRF-1-restriced way. Beyond its role in adaptive immune responses, IL-7 appears to augment the response of macrophages to LPS and to ameliorate LPS tolerance, which may improve innate immune responses against invading pathogens.