Glycogen Synthase Kinase-3 Inhibitors Research Articles

Preventing erosion of X-chromosome inactivation in human embryonic stem cells.

X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.

Metabolic Dysfunction Governs Regulatory T Cell Inflammatory Response during Inflammatory Bowel Disease

Abstract Background Significant proportion of inflammatory bowel disease (IBD) patients respond inconsistently to therapies, underscoring disease complexity. Interleukin 21 (IL21) is highly expressed during IBD. In addition to T helper (Th) cells, inflammatory regulatory T cells (Tregs) have been linked to refractory IBD. Thus, we explored the metabolic role of IL21 in inducing Treg dysfunction and IBD. Methods Human Tregs and Th cells were subjected to transcriptional profiling and metabolic phenotyping. Colitis was induced in Rag1−/− mice by naïve CD4 T cell adoptive transfer. Results IL21 stimulation of human Tregs induced glycolysis and OXPHOS. In agreement, IL21 enhanced the expression of genes associated with glycolysis, anabolism, and OXPHOS, leading to inflammatory cytokine production. Mechanistically, we found disruption to mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent mitochondrial pyruvate metabolism. IL21-induced GSK3β activation was accompanied by pyruvate and lactate buildup. Notably, GSK3 inhibition or supplementation with membrane-permeable methyl pyruvate abrogated metabolic wiring of and inflammatory responses by IL21-stimulated Tregs and effector Th cells. These results suggest that impaired mitochondrial pyruvate metabolism is a feature of inflammatory CD4 T cells. Lastly, GSK3 inhibition prevented pathogenic CD4 T cell-induced colitis in mice, as evidenced by reduced Mouse Colon Histology Index and serum inflammatory cytokines. Conclusions IL21 engages Tregs in a hypermetabolic state that augments inflammatory cytokine production. Therefore, desensitizing CD4 T cells to IL21 may also augment Treg function during IBD. Supported by NIDDK award K01DK124358, the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery.

Interleukin 21‐induced Mitochondrial Dysfunction Drives Regulatory T Cell Inflammatory Response during Intestinal Inflammation

BackgroundSignificant proportion of inflammatory bowel disease (IBD) patients continue to respond inconsistently to therapies, underscoring disease complexity and the need for efficacious treatment. Interleukin 21 (IL‐21), which is known to support T helper (Th) cell function, is highly expressed within inflamed intestinal tissues of IBD patients compared to healthy controls. In addition, inflammatory regulatory T cells (Tregs) have been linked to refractory human IBD. Given that healthy Tregs are critical for self‐tolerance and prevention of IBD, we investigated the metabolic role of IL‐21 in instigating Treg dysfunction and the therapeutic ramifications of targeting metabolism pathways during IBD pathogenesis.MethodsHuman Tregs as well as relevant control effector Th cells were generated from naïve CD4+ T cells isolated from healthy blood donors. Microarray analysis was utilized for targeted metabolic transcriptional profiling. Immune phenotyping was assessed by fluorescence‐activated cell sorting. Metabolic phenotyping of cells was assessed by Seahorse flux analysis and mass spectrometry‐based metabolomics. Ultrastructural analysis of mitochondria was performed by confocal and transmission electron microscopy. Intestinal inflammation was induced in Rag1‐/‐ (T and B cell deficient) mice by the adoptive transfer of pathogenic naïve CD4+ T cells.ResultsAcute IL‐21 stimulation of human Tregs induced glycolysis and fluctuations in mitochondrial respiration (i.e. oxidative phosphorylation – OXPHOS), as assessed by Seahorse flux analysis. In agreement, microarray analysis, validated by qPCR, revealed an IL‐21‐mediated increase in the expression of genes associated with glycolysis and pathways known to support anabolic and OXPHOS metabolism, thus resembling a hypermetabolic state. Furthermore, IL‐21 stimulation rendered Tregs susceptible to inflammatory response, as evidenced by the production of effector Th cell‐associated cytokines such as interferon γ, tumor necrosis factor, IL‐17A, and IL‐17F. Exploring the mechanisms underlying IL‐21‐induced effects, we found significant disruption of mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent pyruvate entry into the mitochondria. IL‐21‐induced GSK3β activation was accompanied by a marked increase in intracellular and extracellular metabolites such as pyruvate and lactate, as assessed by metabolomics. Importantly, GSK3 inhibition or supplementation with mitochondrial membrane‐permeable methyl pyruvate broadly abrogated metabolic wiring of and inflammatory responses by IL‐21‐stimulated Tregs and effector Th cells. Collectively, these results suggest that impaired mitochondrial pyruvate metabolism is a feature of inflammatory CD4+ T cells. Lastly, GSK3 inhibition prevented pathogenic CD4+ T cell‐induced colitis in mice as evidenced by reduced Disease Activity Index, Mouse Colon Histology Index, and serum inflammatory cytokines.ConclusionsIL‐21 potently engages human Tregs in a hypermetabolic state that augments inflammatory cytokine production via induction of mitochondrial dysfunction. Therefore desensitizing CD4+ T cells to detrimental cues, such as IL‐21, may also augment Treg function during human IBD.

Glycogen synthase kinase (GSK-3) synergizes with PD-1/PDL1 blockade to generate super-armed CD8 killers against tumors

Abstract Immune checkpoint blockade (ICB) of negative co-receptors on T-cells such as programmed cell death-1 (PD-1) is promising for the treatment of cancer. Despite success, the poor prognosis for most patients highlights the need for novel clinical interventions. We have shown that the kinase, glycogen synthase kinase-3 (GSK-3) negatively regulates T-cell activation due to altered PD-1 and LAG-3 expression (Taylor et al., 2016 Immunity; Rudd et al., 2019 Cell Reports). GSK-3 inhibition (GSK-3i) is as effective as anti-PD-1 in controlling the growth of melanoma (Taylor et al., 2017 Can Res; Krueger and Rudd, Immunity 2017; Stelle et al., 2021 iScience). GSK-3i increases Tbet (Tbx21) transcription, which inhibits PD-1/LAG-3 transcription, while increasing granzyme B (GZMB) and interferon gamma (IFNγ). Here, we show that Gsk3−/− mice and small molecule inhibitors (SMIs) synergize with anti-PD-1 to eliminate melanomas that are resistant to anti-PD-1 monotherapy. Transcriptomic profiling showed that GSK-3 × PD-1 cooperativity was characterized by a specific increase in a family of different granzymes (7/12 GZM genes out of a data base of 20,500 potential genes). Some GZMs have been characterized and others not, but as a family, this increased armory of GZMs in CD8+ T-cells is expected to greatly enhance tumor killing. Overall, our data shows the PD-1 × GSK-3 synergy in limiting tumor growth is due to a specific set of cytolysis mediators needed for tumor killing.

Baicalin Coadministration with Lithium Chloride Enhanced Neurogenesis via GSK3β Pathway in Corticosterone Induced PC-12 Cells.

Accumulating studies suggest that hippocampal neurogenesis plays a crucial role in the pathological mechanism of depression. As a classic antidepressant, lithium chloride can play an antidepressant role by inhibiting glycogen synthase kinase 3β (GSK3β) and promoting neurogenesis. Correspondingly, baicalin is a compound extracted from natural plants, which shows potential antidepressant effect, however, whether baicalin exerts antidepressant effects by promoting neurogenesis still needs further investigation. In the current study, we established an in vitro depression model through corticosterone induced PC-12 cells, and explored the potential mechanism of baicalin's antidepressant effect by comparing it with lithium chloride alone and the coadministration with lithium chloride. We used Cell Counting Kit-8 (CCK-8) assay, 5-ethynil-2'-deoxyuridine (EdU) staining and cell cycle analysis to evaluate the state of cell survival and cell proliferation. The protein expression levels of neurodevelopmental related factors Doublecortin (DCX), brain-derived neurotrophic factor (BDNF), and the GSK3β pathway-related proteins and mRNA were detected by Western blot and Real-time PCR. The results showed that baicalin could decrease the expression level of GSK3β, while upregulate the expression level of DCX, BDNF, Cyclin D1-cyclin dependent kinase 4/6 (CDK4/6), thus promoted cell proliferation and survival in corticosterone (CORT) induced PC-12 cells. Moreover, this effect was enhanced when baicalin and lithium chloride were coadministration. Taking the above results together, we conclude that baicalin can promote the proliferation and development of PC-12 cells by regulating GSK3β pathway, so as to reverse the depressive-like pathological changes induced by corticosterone.

P405. Selective GSK3α and β Inhibition During Early Postnatal Development Alter Working Memory-Related Behavior in a Sex-Biased Manner in a Mouse Model of 22q11.2 Deletion Syndrome

Overactivation of the Glycogen Synthase Kinase (GSK3) pathway is implicated in a variety of neurological and psychiatric disorders. Non-selective GSK3 (α and β isoform) inhibitors have been found to rescue cognitive deficits in a variety of preclinical models, but their non-selective profiles hamper their preclinical research utility.

Progranulin regulates the development and function of NKT2 cells through EZH2 and PLZF.

T helper 2 (Th2) cytokine production by invariant natural killer T (iNKT) cells is involved in the development of asthma, but the regulation of Th2 cytokines in iNKT cells remains unknown. Although it is known that progranulin (PGRN) induces the production of Th2 cytokines in iNKT cells in vivo, the underlying mechanism is not clear. This study aims to investigate the role of PGRN in iNKT cells. The effects of PGRN on the differentiation of iNKT cells was detected by flow cytometry. Then stimulation of iNKT cells and airway resistance were carried out to evaluate the function of PGRN on iNKT cells. Furthermore, the mechanisms of PGRN in regulating iNKT cells was investigated by RT-PCR, WB, confocal and luciferase reporter assays. The absolute number of iNKT cells decreased in PGRN KO mice despite an increase in the percentage of iNKT cells. Furthermore, analyzing the subsets of iNKT cells, we found that NKT2 cells and their IL-4 production were reduced. Mechanistically, the decrease in NKT2 cells in the PGRN KO mice was caused by increased expression of enhancer of zeste homolog 2 (EZH2), that in turn caused increased degradation and altered nuclear localization of PLZF. Interestingly, PGRN signaling decreased expression of EZH2 and treatment of the PGRN KO mice with the EZH2 specific inhibitor GSK343 rescued the defect in NKT2 differentiation, IL-4 generation, and PLZF expression. Altogether, We have revealed a new pathway (PGRN-EZH2-PLZF), which regulates the Th2 responses of iNKT cells and provides a potentially new target for asthma treatment.

Chemically defined and small molecules-based generation of sinoatrial node-like cells

BackgroundExisting methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably.MethodsWe induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/β-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs.ResultsWNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro.ConclusionWe provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction.

Activation of FXR and inhibition of EZH2 synergistically inhibit colorectal cancer through cooperatively accelerating FXR nuclear location and upregulating CDX2 expression

Our previous study indicated that colon cancer cells varied in sensitivity to pharmacological farnesoid X receptor (FXR) activation. Herein, we explore the regulatory mechanism of FXR in colorectal cancer (CRC) development and aim to design effective strategies of combined treatment based on the regulatory axis. We found that the expression of FXR was negatively correlated with enhancer of zeste homolog 2 (EZH2) in colon cancer tissues. EZH2 transcriptionally suppressed FXR via H3K27me3. The combination of FXR agonist OCA plus EZH2 inhibitor GSK126 acted in a synergistic manner across four colon cancer cells, efficiently inhibiting clonogenic growth and invasion in vitro, retarding tumor growth in vivo, preventing the G0/G1 to S phase transition, and inducing caspase-dependent apoptosis. Benign control cells FHC were growth-arrested without apoptosis induction, but retained long-term proliferation and invasion capacity. Mechanistically, the drug combination dramatically accelerated FXR nuclear location and cooperatively upregulated caudal-related homeobox transcription factor 2 (CDX2) expression. The depletion of CDX2 antagonized the synergistic effects of the drug combination on tumor inhibition. In conclusion, our study demonstrated histone modification-mediated FXR silencing by EZH2 in colorectal tumorigenesis, which offers useful evidence for the clinical use of FXR agonists combined with EZH2 inhibitors in combating CRC.

Downregulation of MEIS1 mediated by ELFN1-AS1/EZH2/DNMT3a axis promotes tumorigenesis and oxaliplatin resistance in colorectal cancer

Oxaliplatin is widely used in the frontline treatment of colorectal cancer (CRC), but an estimated 50% of patients will eventually stop responding to treatment due to acquired resistance. This study revealed that diminished MEIS1 expression was detected in CRC and harmed the survival of CRC patients. MEIS1 impaired CRC cell viabilities and tumor growth in mice and enhanced CRC cell sensitivity to oxaliplatin by preventing DNA damage repair. Mechanistically, oxaliplatin resistance following MEIS1 suppression was critically dependent on enhanced FEN1 expression. Subsequently, we confirmed that EZH2-DNMT3a was assisted by lncRNA ELFN1-AS1 in locating the promoter of MEIS1 to suppress MEIS1 transcription epigenetically. Based on the above, therapeutics targeting the role of MEIS1 in oxaliplatin resistance were developed and our results suggested that the combination of oxaliplatin with either ELFN1-AS1 ASO or EZH2 inhibitor GSK126 could largely suppress tumor growth and reverse oxaliplatin resistance. This study highlights the potential of therapeutics targeting ELFN1-AS1 and EZH2 in cell survival and oxaliplatin resistance, based on their controlling of MEIS1 expression, which deserve further verification as a prospective therapeutic strategy.

Beneficial effects of aloperine on inflammation and oxidative stress by suppressing necroptosis in lipopolysaccharide-induced acute lung injury mouse model

RationaleAlveolar epithelial cell death, inflammation, and oxidative stress are typical features of acute lung injury (ALI). Aloperine (Alo), an alkaloid isolated from Sophora alopecuroides, has been reported to display various biological effects, such as anti-inflammatory, immunoregulatory, and anti-oxidant properties. In this study, we investigated the effects and mechanisms of Alo in treating a lipopolysaccharide (LPS)-induced ALI in a murine model. MethodsThe effects of Alo in LPS-induced ALI were investigated in C57BL/6 mice. The RIPK1 inhibitor (Nec-1) and the RIPK3 inhibitor (GSK’872) were used to evaluate the relationship of necroptosis, NF-κB activation, and PDC subunits in LPS-treated mouse alveolar epithelial cells (MLE-12). Then the effects of Alo on necroptosis, inflammation, and oxidative stress of LPS-stimulated MLE-12 cells were evaluated. ResultsAlo significantly attenuated histopathological lung injuries and reduced lung wet/dry ratio in LPS-induced ALI mice. Alo also remarkedly reduced total protein and neutrophils recruitment in bronchoalveolar lavage fluid of ALI mice. Meanwhile, Alo ameliorated the LPS-induced necroptosis in the lungs of ALI mice. The RIPK3 inhibitor GSK’872, but not the RIPK1 inhibitor Nec-1, reversed LPS-induced p65 phosphorylation and translocation to the nucleus in MLE-12 cells. GSK’872 also reversed the LPS-induced increase in ROS and binding of RIPK3 and PDC subunits in MLE-12 cells. Moreover, Alo down-regulated the levels of p-RIPK1, p-RIPK3, p-MLKL, p-p65, the translocation of p65 to the nucleus, and reduced the expression of IL-6 and IL-8 in LPS-stimulated MLE-12 cells. Alo also inhibited the binding of RIPK3 and PDC-E1α, PDC-E1β, PDC-E2, and PDC-E3 and the ROS production in LPS-treated MLE-12 cells. ConclusionThe present study validated the beneficial effects of Alo on LPS-induced ALI , suggesting Alo may be a new drug candidate against ALI.

Lithium salts as a treatment for COVID-19: Pre-clinical outcomes

IntroductionIdentifying effective drugs for Coronavirus disease 2019 (COVID-19) is urgently needed. An efficient approach is to evaluate whether existing approved drugs have anti-SARS-CoV-2 effects. The antiviral properties of lithium salts have been studied for many years. Their anti-inflammatory and immune-potentiating effects result from the inhibition of glycogen synthase kinase-3. AimsTo obtain pre-clinical evidence on the safety and therapeutic effects of lithium salts in the treatment of COVID-19. ResultsSix different concentrations of lithium, ranging 2–12 mmol/L, were evaluated. Lithium inhibited the replication of SARS-CoV-2 virus in a dose-dependent manner with an IC50 value of 4 mmol/L. Lithium-treated wells showed a significantly higher percentage of monolayer conservation than viral control, particularly at concentrations higher than 6 mmol/L, verified through microscopic observation, the neutral red assay, and the determination of N protein in the supernatants of treated wells. Hamsters treated with lithium showed less intense disease with fewer signs. No lithium-related mortality or overt signs of toxicity were observed during the experiment. A trend of decreasing viral load in nasopharyngeal swabs and lungs was observed in treated hamsters compared to controls. ConclusionsThese results provide pre-clinical evidence of the antiviral and immunotherapeutic effects of lithium against SARS-CoV-2, which supports an advance to clinical trials on COVID-19′s patients.

Re-Expression of ERα and AR in Receptor Negative Endocrine Cancers via GSK3 Inhibition.

DNA methylation, catalyzed by DNA methyltransferase (DNMT), is a well-characterized epigenetic modification in cancer cells. In particular, promoter hypermethylation of AR and ESR1 results in loss of expression on Androgen Receptor (AR) and Estrogen Receptor (ER), respectively, and is associated with a hormone refractory state. We now report that Glycogen Synthase Kinase 3 (GSK3) phosphorylates DNMT1 at S714, which is localized to a 62 amino acid region referred to as auto-inhibitory linker, which functions to occlude the DNA from the active site of DNMT1 to prevent the methylation of unmethylated DNA. Molecular Dynamics simulation indicates that phosphorylation at S714 resulted in conformational rearrangement of the autoinhibitory domain that inactivated its ability to block the methylation of unmethylated DNA and resulted in enhanced DNA binding. Treatment with a novel and more selective inhibitor of GSK3 resulted in decreased methylation of the promoter region of genes encoding the Androgen Receptor (AR) and Estrogen Receptor alpha (ERa) and re-expression of the AR and ERa in AR negative prostate cancer and ER negative breast cancer cells, respectively. As a result, concurrent treatment with the GSK3 inhibitor resulted in responsiveness of AR negative prostate cancer and ER negative breast cancer cells to inhibitors of the AR or ER, respectively, in in vitro and in vivo experimental models.

Quantification of Hepatocellular Mitoses in a Toxicological Study in Rats Using a Convolutional Neural Network.

Convolutional neural networks (CNNs) have been recognized as valuable tools for rapid quantitative analysis of morphological changes in toxicologic histopathology. We have assessed the performance of CNN-based (Halo-AI) mitotic figure detection in hepatocytes in comparison with detection by pathologists. In addition, we compared with Ki-67 and 5-bromodesoxyuridin (BrdU) immunohistochemistry labeling indices (LIs) obtained by image analysis. Tissues were from an exploratory toxicity study with a glycogen synthase kinase-3 (GSK-3) inhibitor. Our investigations revealed that (1) the CNN achieved similarly accurate but faster results than pathologists, (2) results of mitotic figure detection were comparable to Ki-67 and BrdU LIs, and (3) data from different methods were only moderately correlated. The latter is likely related to differences in the cell cycle component captured by each method. This highlights the importance of considering the differences of the available methods upon selection. Also, the pharmacology of our test item acting as a GSK-3 inhibitor potentially reduced the correlation. We conclude that hepatocyte cell proliferation assessment by CNNs can have several advantages when compared with the current gold standard: it relieves the pathologist of tedious routine tasks and contributes to standardization of results; the CNN algorithm can be shared and iteratively improved; it can be performed on routine histological slides; it does not require an additional animal experiment and in this way can contribute to animal welfare according to the 3R principles.

Yes-associated protein activation potentiates glycogen synthase kinase-3 inhibitor-induced proliferation of neonatal cardiomyocytes and iPS cell-derived cardiomyocytes.

Because mammalian cardiomyocytes largely cease to proliferate immediately after birth, the regenerative activity of the heart is limited. To date, much effort has been made to clarify the regulatory mechanism of cardiomyocyte proliferation because the amplification of cardiomyocytes could be a promising strategy for heart regenerative therapy. Recently, it was reported that the inhibition of glycogen synthase kinase (GSK)‐3 promotes the proliferation of neonatal rat cardiomyocytes (NRCMs) and human iPS cell‐derived cardiomyocytes (hiPSC‐CMs). Additionally, Yes‐associated protein (YAP) induces cardiomyocyte proliferation. The purpose of this study was to address the importance of YAP activity in cardiomyocyte proliferation induced by GSK‐3 inhibitors (GSK‐3Is) to develop a novel strategy for cardiomyocyte amplification. Immunofluorescent microscopic analysis using an anti‐Ki‐67 antibody demonstrated that the treatment of NRCMs with GSK‐3Is, such as BIO and CHIR99021, increased the ratio of proliferative cardiomyocytes. YAP was localized in the nuclei of more than 95% of cardiomyocytes, either in the presence or absence of GSK‐3Is, indicating that YAP was endogenously activated. GSK‐3Is increased the expression of β‐catenin and promoted its translocation into the nucleus without influencing YAP activity. The knockdown of YAP using siRNA or pharmacological inhibition of YAP using verteporfin or CIL56 dramatically reduced GSK‐3I‐induced cardiomyocyte proliferation without suppressing β‐catenin activation. Interestingly, the inhibition of GSK‐3 also induced the proliferation of hiPSC‐CMs under sparse culture conditions, where YAP was constitutively activated. In contrast, under dense culture conditions, in which YAP activity was suppressed, the proliferative effects of GSK‐3Is on hiPSC‐CMs were not detected. Importantly, the activation of YAP by the knockdown of α‐catenin restored the proproliferative activity of GSK‐3Is. Collectively, YAP activation potentiates the GSK‐3I‐induced proliferation of cardiomyocytes. The blockade of GSK‐3 in combination with YAP activation resulted in remarkable amplification of cardiomyocytes.

Combination of artesunate and WNT974 induces KRAS protein degradation by upregulating E3 ligase ANACP2 and \u03b2-TrCP in the ubiquitin\u2013proteasome pathway

BackgroundKRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin.MethodsThe synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or β-TrCP. Western blot assay was utilized for the protein levels of NRAS, HRAS, KRAS, ANAPC2, β-TrCP, GSK-3β, p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, β-TrCP, GSK-3β or ANAPC2 in tumor tissues.ResultsOur study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3β and E3 ligase β-TrCP that is known to degrade GSK-3β-phosphorylated KRAS protein. Knockdown of β-TrCP- and inhibition of GSK-3β abolish the combination treatment-induce KRAS ubiquitination and reduction in expression. Last but not least, combination treatment suppresses PI3K/Akt/m-TOR signaling pathway.ConclusionsOur data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin–proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment.Bh4nRY9p6vgUp6geXci3gHVideo

GSK3 inhibition circumvents and overcomes acquired lorlatinib resistance in ALK-rearranged non-small-cell lung cancer

Anaplastic lymphoma kinase (ALK) fusion is found in ~3%–5% of patients with non-small-cell lung cancers (NSCLCs). Although the third-generation ALK tyrosine kinase inhibitor (TKI) lorlatinib shows high clinical efficacy in ALK-positive NSCLC, most of the patients eventually relapse with acquired resistance. Recently, drug-tolerant persister (DTP) cells have been considered an important seed of acquired resistance cells. In this study, we established lorlatinib intermediate resistant cells from a patient-derived cell model. Glycogen synthase kinase 3 (GSK3) inhibitions significantly suppressed lorlatinib intermediate resistant cell growth. GSK3 inhibition also sensitized acquired resistance cells derived from alectinib-treated patients with or without secondary mutations to lorlatinib. Therefore, GSK3 plays a crucial role in developing acquired resistance against lorlatinib in ALK-positive NSCLC mediated by lorlatinib intermediate resistant cells and could be a potential molecular target to prevent acquired lorlatinib resistance and overcome ALK-TKI resistance.

Evidence for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19

A key component of severe COVID-19 is a “cytokine storm” i.e., the excessive expression of unneeded cytokines. Previous studies suggest that SARS-CoV-2 proteins can induce macrophages to secrete pro-inflammatory cytokines; a process that may involve Toll-like receptors (TLRs). Glycogen synthase kinase-3 (GSK-3) has been implicated in TLR signal transduction and a selective GSK-3 inhibitor, termed COB-187, dramatically attenuates cytokine expression induced by the TLR ligand lipopolysaccharide (LPS). In the present study, we provide evidence that the SARS-CoV-2 spike protein (S) and the S2 subunit (S2) induce production of CXCL10 (a chemokine elevated in severe COVID-19) by a human macrophage cell line. Further, we report that two clinically relevant GSK-3 inhibitors and COB-187 attenuate S and S2 protein-induced CXCL10 production. Combined, our observations provide impetus for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

Unraveling the Effects of GSK-3β Isoform Modulation against Limbic Seizures and in the 6 Hz Electrical Kindling Model for Epileptogenesis.

Two closely related glycogen synthase kinase-3 (GSK-3) isoforms have been identified in mammals: GSK-3α and GSK-3β. GSK-3β is the most prominent in the central nervous system and was previously shown to control neuronal excitability. We previously demonstrated that indirubin and its structural analogue and the nonselective GSK-3 inhibitor BIO-acetoxime exerted anticonvulsant effects in acute seizure models in zebrafish, mice, and rats. We here examined for the first time the anticonvulsant effect of TCS2002, a specific and potent inhibitor of GSK-3β, in two models for limbic seizures: the pilocarpine rat model for focal seizures and the acute 6 Hz corneal mouse model for refractory seizures. Next, we additionally used the 6 Hz kindling model to establish differences in seizure susceptibility and seizure progression in mice that either overexpress human GSK-3β (GSK-3β OE) or lack GSK-3β (GSK-3β-/-) in neurons. We demonstrate that TCS2002 exerts anticonvulsant actions against pilocarpine- and 6 Hz-evoked seizures. Compared to wild-type littermates, GSK-3β OE mice are less susceptible to seizures but are more rapidly kindled. Interestingly, compared to GSK-3β+/+ mice, neuronal GSK-3β-/- mice show increased susceptibility to 6 Hz-induced seizures. These contrasting observations suggest compensatory neurodevelopmental mechanisms that alter seizure susceptibility in GSK-3β OE and GSK-3β-/- mice. Although the pronounced anticonvulsant effects of selective and acute GSK-3β inhibition in the 6 Hz model identify GSK-3β as a potential drug target for pharmacoresistant seizures, our data on the sustained disruption of GSK-3β activity in the transgenic mice suggest a role for GSK-3 in kindling and warrants further research into the long-term effects of selective pharmacological GSK-3β inhibition.

Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway.

BACKGROUNDIntestinal ischemia/reperfusion (I/R) injury is a fatal syndrome that occurs under many clinical scenarios. The apoptosis of intestinal cells caused by ischemia can cause cell damage and provoke systemic dysfunction during reperfusion. However, the mechanism of I/R-induced apoptosis remains unclear. Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel. Few researchers have paid attention to its role in intestinal I/R injury, or the relationship between CFTR and intestinal apoptosis induced by hypoxia/reoxygenation (H/R).AIMTo investigate the effects of CFTR on I/R-induced intestinal apoptosis and its underlying molecular mechanisms.METHODSAn intestinal I/R injury model was established in mice with superior mesenteric artery occlusion, and Caco2 cells were subjected to H/R for the simulation of I/R in vivo.RESULTSThe results suggested that CFTR overexpression significantly increased the Caco2 cell viability and decreased cell apoptosis induced by the H/R. Interestingly, we found that the translocation of p65, an NF-κB member, from the cytoplasm to the nucleus after H/R treatment can be reversed by the overexpression of CFTR, the NF-κB P65 would return from the nucleus to the cytoplasm as determined by immunostaining. We also discovered that CFTR inhibited cell apoptosis in the H/R-treated cells, and this effect was significantly curbed by the NF-κB activator BA, AKT inhibitor GSK690693 and the PI3K inhibitor LY294002. Moreover, we demonstrated that CFTR overexpression could reverse the decreased PI3K/AKT expression induced by the I/R treatment in vivo or H/R treatment in vitro.CONCLUSIONThe results of the present study indicate that the overexpression of CFTR protects Caco2 cells from H/R-induced apoptosis; furthermore, it also inhibits H/R-induced apoptosis through the PI3K/AKT/NF-κB signaling pathway in H/R-treated Caco2 cells and intestinal tissues.