Glycogen Synthase Kinase-3 Inhibitors Research Articles

Exploring pathological targets and advancing pharmacotherapy in autism spectrum disorder: Contributions of glial cells and heavy metals.

Autism spectrum disorder (ASD) is a globally recognized neurodevelopmental condition characterized by repetitive and restrictive behavior, persistent deficits in social interaction and communication, mental disturbances, etc., affecting approximately 1 in 100 children worldwide. A combination of genetic and environmental factors is involved in the etiopathogenesis of the disease, but specific biomarkers have not yet been identified. Due to the lack of clinical evidence, fluctuations in symptoms, and difficulties in in-vitro and in-vivo modeling, developing medications for ASD is quite difficult. Although several drugs are used to treat autism, only risperidone and aripiprazole have received FDA approval in the United States. Epidemiological studies have suggested that maternal exposure to valproic acid (VPA), acetaminophen, propionic acid, and metals, such as cadmium (Cd), lead (Pb), arsenic (As), and mercury (Hg), may contribute to the development of various neurodevelopmental disorders. Pathological targets directly implicated in the disease include excitatory-inhibitory (E/A) imbalance, hyperserotonemia, GSK-3 inhibition, and Akt pathway activation. However, while a combination of pharmacotherapy, behavioral, and nutritional/dietary interventions has been found to be the most effective conventional therapy to date, many patients have chosen to implement particular dietary supplements for reducing ASD symptoms. In this review, we briefly describe various pathological targets and their roles in the pathophysiology of ASD and treatment strategies, including some future research directions.

The role of the NOTCH1 signaling pathway in the maintenance of mesenchymal stem cell stemness and chondrocyte differentiation and its potential in the treatment of osteoarthritis

ObjectiveThis study aims to explore the potential role of mesenchymal stem cells (MSCs) in the treatment of osteoarthritis (OA), particularly the function of the NOTCH1 signaling pathway in maintaining the stemness of MSCs and in chondrocyte differentiation.MethodsUtilizing diverse analytical techniques on an osteoarthritis dataset, we unveil distinct gene expression patterns and regulatory relationships, shedding light on potential mechanisms underlying the disease. Techniques used include the culture of MSCs, induction of differentiation into chondrocytes, establishment of stable cell lines, Western Blot, and immunofluorescence. Through the construction of lentiviruses overexpressing and knocking out NOTCH1, the effects of NOTCH1 on the stemness of MSCs and chondrocyte differentiation were investigated. Additionally, the effects of NOTCH1 on chondrocyte homeostasis and apoptosis were evaluated by adding the EZH2 inhibitor GSK126 and the endoplasmic reticulum stress inducer tunicamycin.ResultsExperimental results demonstrated that NOTCH1 expression can influence the maintenance of MSC stemness and chondrocyte differentiation by regulating EZH2. Knockout of NOTCH1 decreased the expression of chondrocyte markers, while overexpression increased their expression. Under conditions of endoplasmic reticulum stress, NOTCH1 expression helped reduce the expression of stress-related proteins, maintain chondrocyte homeostasis, and inhibit apoptosis.ConclusionThe NOTCH1 signaling pathway plays a crucial role in maintaining the stemness of MSCs, differentiating into chondrocytes, and in the treatment of osteoarthritis. NOTCH1 influences the differentiation fate of MSCs and the homeostasis of chondrocytes by regulating EZH2 and other related genes, offering new targets and strategies for the treatment of diseases like osteoarthritis.

Inhibition of RIPK1 or RIPK3 kinase activity post ischemia-reperfusion reduces the development of chronic kidney injury.

Ischemia-reperfusion injury (IRI) occurs when the blood supply to an organ is temporarily reduced and then restored. Kidney IRI is a form of acute kidney injury, which often progresses to kidney fibrosis. Necroptosis is a regulated necrosis pathway that has been implicated in kidney IRI. Necroptotic cell death involves recruitment of the RIPK1 and RIPK3 kinases and activation of the terminal effector, the MLKL pseudokinase. Phosphorylated MLKL causes cell death by plasma membrane rupture, driving "necroinflammation". Owing to their apical role in the pathway, RIPK1 and RIPK3 have been implicated in the development of kidney fibrosis. Here, we used a mouse model of unilateral kidney IRI to assess whether inhibition of RIPK1 or RIPK3 kinase activity reduces acute kidney injury and the progression to kidney fibrosis. Mice treated with the RIPK1 inhibitor Nec-1s, either before or after IR, showed reduced kidney injury at 24 hours compared to controls, whereas no protection was offered by the RIPK3 inhibitor GSK´872. In contrast, treatment with either inhibitor from days 3-9 post-IR reduced the degree of kidney fibrosis at day 28. These findings further support the role of necroptosis in IRI and provide important validation for the contribution of both RIPK1 and RIPK3 catalytic activities in the progression of kidney fibrosis. Targeting the necroptosis pathway could be a promising therapeutic strategy to mitigate kidney disease following IR.

Glycogen synthase kinase-3 inhibition and insulin enhance proliferation and inhibit maturation of human iPSC-derived cardiomyocytes via TCF and FOXO signaling.

Embryonic signaling pathways exert stage-specific effects during cardiac development, yet the precise signals for proliferation or maturation remain elusive. To uncover the cues for proliferation, we performed a combinatory cell-cycle screen for insulin and glycogen synthase kinase-3 (GSK3) inhibition in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our analysis for proliferation, and subsequential downstream sarcomere development, gene expression analysis, and molecular interventions identified a temporal interplay between insulin/Akt/FOXO and CHIR99021/Wnt/GSK3/TCF signaling. Combined pathway activation led to proliferation of immature hiPSC-CMs with low sarcomere and mitochondria content, while, in the absence of pathway activators, cardiomyocytes rapidly exited the cell cycle and fetched higher organization of sarcomeres and mitochondria. Our data demonstrate two important pathways, which enhance proliferation and inhibit maturation, and provide molecular mechanistic understanding of these cell fate decisions in immature hiPSC-CMs.

Epigenetic Suppression of miR-137 Induces RNF4 Expression, Facilitating Wnt Signaling in Colorectal Cancer.

Colorectal cancer (CRC) is a significant health issue worldwide. Recent studies highlight the critical role of miRNAs in CRC development, particularly miR-137, which acts as a key tumor suppressor. Despite its known role, further exploration of miR-137's downstream signaling is needed to understand its biology and therapeutic potential. We examined the methylation status of miR-137 using one TCGA data and three GEO data sets. A clinical validation cohort of 78 samples was analyzed using MSP for miR-137 promoter methylation. Various in vitro molecular/cellular and animal experiments were conducted to elucidate miR-137's role in CRC. Bioinformatic analysis indicated frequent methylation of miR-137 in CRC tissues, correlating with suppressed expression. EZH2-mediated H3K27 trimethylation silences miR-137 in CRC cells by increasing chromatin compaction, reversible by EZH2 siRNA or inhibitor GSK343. miR-137 inhibits CRC cell proliferation, migration, invasion, and xenograft tumor growth, confirming its tumor-suppressive role. Using the miRWalk repository showed that miR-137 regulates the Wnt signaling pathway by reducing typical protein expression in HCT116 and SW480 cells. miR-137 directly targets RNF4, leading to its downregulation at transcriptional and protein levels, with an observed inverse correlation in CRC tissues. miR-137 accelerates c-Myc and β-catenin degradation by inhibiting RNF4, impacting protein stability and Wnt pathway inhibition. miR-137 is epigenetically silenced through DNA methylation and EZH2-mediated H3K27 trimethylation. It regulates the Wnt signaling pathway by targeting RNF4, leading to c-Myc and β-catenin destabilization. Restoring miR-137 or inhibiting RNF4 suppresses CRC cell proliferation, migration, invasion, and tumor growth, highlighting its therapeutic potential in CRC.

GSK3β Deficiency Expands Obese Adipose Vasculature to Mitigate Metabolic Disorders.

Maintaining a well-developed vascular system alongside adipose tissue (AT) expansion significantly reduces the risk of metabolic complications. Although GSK3β (glycogen synthase kinase-3 beta) is known for its role in various cellular processes, its specific functions in AT and regulation of body homeostasis have not been reported. GSK3β-floxed and GSK3α-floxed mice were crossed with adiponectin-Cre mice to generate GSK3β or GSK3α adipocyte-specific knockout mice (GSK3βADKO and GSK3αADKO). A comprehensive whole-body metabolism analysis was performed on obese GSK3βADKO mice induced by a high-fat diet. RNA sequencing was conducted on AT of both obese GSK3βADKO and GSK3αADKO mice. Various analyses, including vessel perfusion studies, lipolysis analysis, multiplex protein assays, in vitro protein phosphorylation assays, and whole-mount histology staining, were performed on AT of obese GSK3βADKO mice. Tube-formation experiments were performed using 3B-11 endothelial cells cultured in the conditional medium of matured adipocytes under hypoxic conditions. Chromatin precipitation and immunofluorescence studies were conducted using cultured adipocytes with GSK3 inhibition. Our findings provide the first evidence that adipocyte-specific knockout of GSK3β expands AT vascularization and mitigates obesity-related metabolic disorders. GSK3β deficiency, but not GSK3α, in adipocytes activates AMPK (AMP-activated protein kinase), leading to increased phosphorylation and nuclear accumulation of HIF-2α, resulting in enhanced transcriptional regulation. Consequently, adipocytes increased VEGF (vascular endothelial growth factor) expression, which engages VEGFR2 on endothelial cells, promoting angiogenesis, expanding the vasculature, and improving vessel perfusion within obese AT. GSK3β deficiency promotes AT remodeling, shifting unhealthy adipocyte function toward a healthier state by increasing insulin-sensitizing hormone adiponectin and preserving healthy adipocyte function. These effects lead to reduced fibrosis, reactive oxygen species, and ER stress in obese AT and improve metabolic disorders associated with obesity. Deletion of GSK3β in adipocytes activates the AMPK/HIF-2α/VEGF/VEGFR2 axis, promoting vasculature expansion within obese AT. This results in a significantly improved local microenvironment, reducing inflammation and effectively ameliorating metabolic disorders associated with obesity.

Characterization of the BH1406 non-small cell lung cancer (NSCLC) cell line carrying an activating SOS1 mutation.

Approximately 30% of the non-small cell lung cancer (NSCLC) patients which harbor no recognizable oncogenic driver mutation are not eligible for targeted therapy. Functional drug screening of tumor cells helps to identify susceptible drug targets not recognized by gene panels for targeted mutation analysis. The aim of this study is to characterize the BH1406 cell line carrying an activating SOS1 mutation and to check its sensitivity to cognate inhibitors. The NSCLC cell line BH1406 was established from a pleural effusion and found to be sensitive to the SOS1 inhibitor BAY-293 in initial viability screenings. Since in a limited next-generation sequencing (NGS) lung cancer mutation panel no driver could be detected, the patient underwent chemotherapy with poor outcome. This cell line was further characterized by exome sequencing, SOS1 Western blotting, comparison of two-dimensional (2D) and three-dimensional (3D) chemosensitivity assays and phosphoprotein arrays. In whole-exome sequencing (WES) the SOS1 mutation P481delinsLFFL, positioned near the known P478L activating mutation was detected. Besides BAY-293, BH1406 cells proved to be sensitive to the SOS1 inhibitors MRTX0902 and BI-3406. The sensitivity of BH1406 cells to BI-3406 was increased under 3D conditions compared to 2D cultures. Western blot phosphoprotein arrays revealed reduced phosphorylation of CREB, GSK3, CHK-2 and STAT3 in BH1406 by BAY-293 treatment in 2D culture. In 3D conditions, cells switched from GSK3α to elevated ERK1/2 signaling, again blocked by the SOS1 inhibitor BAY-293. Similar results were obtained for the SOS1 inhibitors MRTX0902 and BI3406. Additionally, the PI3K inhibitor dactolisib, the GSK-3 inhibitor BI-5521 as well as the bromodomain protein-directed PROTAC ARV-771 inhibited the growth of BH1406 cells significantly and showed synergistic interaction with BAY-293. Furthermore, Western blots demonstrated reduced expression of SOS1 and MYC proteins in response to BAY-293 treatment. The rare SOS1 P481delinsLFFL mutation in lung cancer may be targetable with corresponding inhibitors, alone or in combination with GSK3/PI3K/BET inhibitors. BH1406 cells represent a novel cellular model suitable for the molecular characterization of SOS1 druggability. Such rare oncogenic driver genes are not included in standard NGS panels and need to be detected by expanded assays like WES.

GSK3 inhibition improves skeletal muscle function and whole-body metabolism in male mouse models of Duchenne muscular dystrophy

Inhibiting glycogen synthase kinase 3 (GSK3) improves muscle function, metabolism, and bone health in many diseases and conditions; however, whether GSK3 should be targeted for Duchenne muscular dystrophy (DMD), a severe muscle wasting disorder with no cure, remains unknown. Here, we show the effects of GSK3 inhibition in male DBA/2J (D2) and C57BL/10 (C57) mdx mice. Treating D2 mdx mice with GSK3 inhibitors alone or in combination with aerobic exercise improves muscle strength, endurance, and morphology, attenuates the hypermetabolic phenotype, and enhances insulin sensitivity. GSK3 inhibition in C57 mdx mice also improves muscle fatigue resistance and increases cage ambulation. Moreover, muscle-specific GSK3 knockdown in mdx mice augments muscle force production and endurance. In both mdx strains, GSK3 inhibition increases bone mineral content and density. Overall, these improvements to muscle, metabolic, and bone health with GSK3 inhibition in mdx mice may have clinical implications for patients with DMD, where the current standard of care, glucocorticoids, delay the loss of ambulation but increase the risk for insulin resistance and osteoporosis. Along with our observation of lowered β-catenin content in DMD myoblasts, a known cellular target for GSK3, this study provides ample evidence in support of inhibiting GSK3 for this disease.

Abstract 4122717: An Epigenetic Drug, GSK126 Mitigates Endothelial to Mesenchymal Transition Attenuating Atherosclerosis in Diabetes

Background: Endothelial to mesenchymal transition (EndMT) involves transformation of an endothelial to mesenchymal like cellular state. Recent studies implicate EndMT in atherosclerosis development. Diabetes is associated with both EndMT as well as accelerated atherosclerosis. Experimental evidence supports the use of epigenetic drugs that act on relevant epigenetic mechanisms to attenuate atherosclerosis. Recently, inhibition of a histone methyltransferase Enhancer of zest homolog 2 (EZH2) with GSK-126 attenuated atherosclerosis development. However, the role of EZH2 in induction of EndMT in diabetes induced atherosclerosis is not known. Methods: An in-vitro model of EndMT was generated using high glucose (HG) and TNF-α in human aortic endothelial cells (HAECs). EZH2 methyltransferase activity was inhibited using EZH2 specific inhibitor GSK-126. RNA sequencing was performed in this setting. EZH2 genetic knockdown (shRNA) in HAECs was also performed to validate role of EZH2 in EndMT. In addition, EZH2 mediated H3K27me3 and EndMT was assessed in atheroprone diabetic mouse model. Results: In HAECs, morphological changes as well as gene and protein expression confirmed TNF-α+HG induced EndMT, which was blunted by GSK-126. Elevated EZH2 mediated H3K27me3 activity by the TNF-α + HG stimulation was blunted with GSK-126 treatment. Furthermore, RNA sequencing identified several enriched pathways including AGE-RAGE signaling pathway in diabetic complications, focal adhesion, and leukocyte trans-endothelial migration directly relevant to EndMT and commonly deregulated in diabetic complications, which were rescued by EZH2 inhibition. Transcriptomic analysis showed that 242 genes including MMP2, NOS3 linked to EndMT were dysregulated. Interestingly, expression of 76 dysregulated genes in EndMT were rescued by GSK-126. EZH2 knockdown studies in HAECs also validated the role of EZH2 in EndMT. Furthermore, immunofluorescence staining identified elevated levels of H3K27me3 in the aortic endothelial layer of diabetic Apoe -/- mice. Co-localization of EndMT markers was observed in the endothelial layer of aortic sinus of diabetic Apoe -/- mice which was blunted with GSK-126 treatment. Conclusion: The study identified EZH2 inhibition as a novel therapeutic target in diabetes induced EndMT in atherosclerosis. These findings highlighted ongoing efforts to develop targeted therapies for diabetic patients who are at risk to develop cardiovascular disease.

Synergistic Killing Effect and Molecular Mechanism of Dual Targeting of Lymphoma By GSK-3 Inhibitor (9-ING-41) and BCL2 Inhibitor (ABT-199)

Synergistic Killing Effect and Molecular Mechanism of Dual Targeting of Lymphoma By GSK-3 Inhibitor (9-ING-41) and BCL2 Inhibitor (ABT-199)

Elraglusib Induces Cytotoxicity via Direct Microtubule Destabilization Independently of GSK3 Inhibition.

Elraglusib was designed as a GSK3 inhibitor and is currently in clinical trials for several cancers. We show conclusively that the target of elraglusib that leads to cytotoxicity is MTs and not GSK3. This has significant implications for ongoing clinical trials of the compound and will help in understanding off-target side effects, inform future clinical trial design, and facilitate the development of biomarkers to predict response.

Single-cell transcriptomics reveal diverging pathobiology and opportunities for precision targeting in scleroderma-associated versus idiopathic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) involves progressive cellular and molecular change within the pulmonary vasculature, leading to increased vascular resistance. Current therapies targeting nitric oxide (NO), endothelin, and prostacyclin pathways yield variable treatment responses. Patients with systemic sclerosis-associated PAH (SSc-PAH) often experience worse outcomes than those with idiopathic PAH (IPAH). Lung tissue samples from four SSc-PAH, four IPAH, and four failed donor specimens were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) lung tissue bank. Single-cell RNA sequencing (scRNAseq) was performed using the 10X Genomics Chromium Flex platform. Data normalization, clustering, and differential expression analysis were conducted using Seurat. Additional analyses included gene set enrichment analysis (GSEA), transcription factor activity analysis, and ligand-receptor signaling. Pharmacotranscriptomic screening was performed using the Connectivity Map. SSc-PAH samples showed a higher proportion of fibroblasts and dendritic cells/macrophages compared to IPAH and donor samples. GSEA revealed enriched pathways related to epithelial-to-mesenchymal transition (EMT), apoptosis, and vascular remodeling in SSc-PAH samples. There was pronounced differential gene expression across diverse pulmonary vascular cell types and in various epithelial cell types in both IPAH and SSc-PAH, with epithelial to endothelial cell signaling observed. Macrophage to endothelial cell signaling was particularly pronounced in SSc-PAH. Pharmacotranscriptomic screening identified TIE2, GSK-3, and PKC inhibitors, among other compounds, as potential drug candidates for reversing SSc-PAH gene expression signatures. Overlapping and distinct gene expression patterns exist in SSc-PAH versus IPAH, with significant molecular differences suggesting unique pathogenic mechanisms in SSc-PAH. These findings highlight the potential for precision-targeted therapies to improve SSc-PAH patient outcomes. Future studies should validate these targets clinically and explore their therapeutic efficacy.

Glycogen synthase kinase 3 (GSK3) inhibition: a potential therapeutic strategy for Alzheimer's disease.

Alzheimer's disease (AD), the most common type of dementia among older adults, is a chronic neurodegenerative pathology that causes a progressive loss of cognitive functioning with a decline of rational skills. It is well known that AD is multifactorial, so there are many different pharmacological targets that can be pursued. According to estimates from the World Health Organization (WHO), 18 million individuals worldwide suffer from AD. Major initiatives to identify risk factors, enhance care giving, and conduct basic research to delay the beginning of AD were started by the USA, France, Germany, France, and various other nations. Widely recognized as a key player in the development and subsequent progression of AD pathogenesis, glycogen synthase kinase-3 (GSK-3) controls a number of crucial targets associated with neuronal degeneration. GSK-3 inhibition has been linked to reduced tau hyperphosphorylation, β-amyloid formation, and neuroprotective benefits in Alzheimer's disease. Lithium, the very first inhibitor ofGSK-3βthat was used therapeutically,has been successfully used for many years with remarkable results. A great variety of structurally varied strong GSK-3β blockers have been identified in recent years. The purpose of this thorough review is to cover the biological and structural elements of glycogen synthase kinase, as well as the medicinal chemistry aspects of GSK inhibitors that have been produced in recent years.

Stanniocalcin 2 Promotes Neuronal Differentiation in Neural Stem/Progenitor Cells of the Mouse Subventricular Zone Through Activation of AKT Pathway.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone (SVZ) throughout life, responding to various pathophysiological stimuli and playing a crucial role in central nervous system repair. Although numerous studies have elucidated the role of stanniocalcin 2 (STC2) in regulating cell differentiation processes, its specific function in NSPCs differentiation remains poorly understood. Clarifying the role of STC2 in NSPCs is essential for devising novel strategies to enhance the intrinsic potential for brain regeneration postinjury. Our study revealed the expression of STC2 in NSPCs derived from the SVZ of the C57BL/6N mouse. In cultured SVZ-derived NSPCs, STC2 treatment significantly increased the number of Tuj1 and DCX-positive cells. Furthermore, STC2 injection into the lateral ventricle promoted the neuronal differentiation of NSPCs and migration to the olfactory bulb. Conversely, the STC2 knockdown produced the opposite effect. Further investigation showed that STC2 treatment enhanced AKT phosphorylation in cultured NSPCs, whereas STC2 inhibition hindered AKT activation. Notably, the neuronal differentiation induced by STC2 was blocked by the AKT inhibitor GSK690693, whereas the AKT activator SC79 reversed the impact of STC2 knockdown on neuronal differentiation. Our findings indicate that enhancing STC2 expression in SVZ-derived NSPCs facilitates neuronal differentiation, with AKT regulation potentially serving as a key intracellular target of STC2 signaling.

Animal models of haploinsufficiency revealed the isoform-specific role of GSK-3 in HFD-induced obesity and glucose intolerance.

Glycogen synthase kinase 3 (GSK-3), a serine-threonine kinase with two isoforms (α and β) is implicated in the pathogenesis of type 2 diabetes mellitus (T2D). Recently, we reported the isoform-specific role of GSK-3 in T2D using homozygous GSK-3α/β knockout mice. Although the homozygous inhibition models are idealistic in a preclinical setting, they do not mimic the inhibition seen with pharmacological agents. Hence, in this study, we sought to investigate the dose-response effect of GSK-3α/β inhibition in the pathogenesis of obesity-induced T2D. Specifically, to gain insight into the dose-response effect of GSK-3 isoforms in T2D, we generated tamoxifen-inducible global GSK-3α/β heterozygous mice. GSK-3α/β heterozygous and control mice were fed a high-fat diet (HFD) for 16 wk. At baseline, the body weight and glucose tolerance of GSK-3α heterozygous and controls were comparable. In contrast, at baseline, a modest but significantly higher body weight (higher lean mass) was seen in GSK-3β heterozygous compared with controls. Post-HFD, GSK-3α heterozygous and controls displayed a comparable phenotype. However, GSK-3β heterozygous were significantly protected against obesity-induced glucose intolerance. Interestingly, the improved glucose tolerance in GSK-3β heterozygous animals was dampened with chronic HFD-feeding, likely due to significantly higher fat mass and lower lean mass in the GSK-3β animals. These findings suggest that GSK-3β is the dominant isoform in glucose metabolism. However, to avail the metabolic benefits of GSK-3β inhibition, it is critical to maintain a healthy weight.NEW & NOTEWORTHY The precise isoform-specific role of GSK-3 in obesity-induced glucose intolerance is unclear. To overcome the limitations of pharmacological GSK-3 inhibitors (not isoform-specific) and tissue-specific genetic models, in the present study, we created novel inducible heterozygous mouse models of GSK-3 inhibition that allowed us to delete the gene globally in an isoform-specific and temporal manner to determine the isoform-specific role of GSK-3 in obesity-induced glucose intolerance.

Targeted inhibition of glycogen synthase kinase-3 using 9-ING-41 (elraglusib) enhances CD8 T-cell-reactivity against neuroblastoma cells

The prognosis of patients with high-risk neuroblastoma remains poor, partly due to inadequate immune recognition of the tumor. Neuroblastomas display extremely low surface MHC-I, preventing recognition by cytotoxic T lymphocytes (CTLs) and contributing to an immunosuppressive tumor microenvironment. Glycogen synthase kinase-3 beta (GSK-3β) is involved in pathways that may affect the MHC-I antigen processing and presentation pathway. We proposed that therapeutic inhibition of GSK-3β might improve the surface display of MHC-I molecules on neuroblastoma cells, and therefore tested if targeting of GSK-3β using the inhibitor 9-ING-41 (Elraglusib) improves MHC-I-mediated CTL recognition. We analyzed mRNA expression data of neuroblastoma tumor datasets and found that non-MYCN-amplified neuroblastomas express higher GSK-3β levels than MYCN-amplified tumors. In non-MYCN-amplified cells SH-SY5Y, SK-N-AS and SK-N-SH 9-ING-41 treatment enhanced MHC-I surface display and the expression levels of a subset of genes involved in MHC-I antigen processing and presentation. Further, 9-ING-41 treatment triggered increased STAT1 pathway activation, upstream of antigen presentation pathways in two of the three non-MYCN-amplified cell lines. Finally, in co-culture experiments with CD8 + T cells, 9-ING-41 improved immune recognition of the neuroblastoma cells, as evidenced by augmented T-cell activation marker levels and T-cell proliferation, which was further enhanced by PD-1 immune checkpoint inhibition. Our preclinical study provides experimental support to further explore the GSK-3β inhibitor 9-ING-41 as an immunomodulatory agent to increase tumor immune recognition in neuroblastoma.

Quantitative label-free digital holographic imaging of cardiomyocyte optical volume, nucleation, and cell division

Cardiac regeneration in newborn rodents depends on the ability of pre-existing cardiomyocytes to proliferate and divide. This capacity is lost within the first week of postnatal development when these cells rapidly switch from hyperplasia to hypertrophy, withdraw from the cell cycle, become binucleated, and increase in size. How these dynamic changes in cell size and nucleation impact cardiomyocyte proliferative potential is not well understood. In this study, we innovate the application of a commercially available digital holographic imaging microscope, the Holomonitor M4, to evaluate the proliferative responses of mononucleated and binucleated cardiomyocytes after CHIR99021 treatment, a model proliferative stimulus. This system enables long-term label-free quantitative tracking of primary cardiomyocyte dynamics in real-time with single-cell resolution. Our results confirm that chemical inhibition of glycogen synthase kinase 3 with CHIR99021 promotes complete cell division of both mononucleated and binucleated cardiomyocytes with high frequency. Quantitative tracking of cardiomyocyte volume dynamics during these proliferative events revealed that both mononucleated and binucleated cardiomyocytes reach a similar size-increase threshold prior to attempted cell division. Binucleated cardiomyocytes attempt to divide with lower frequency than mononucleated cardiomyocytes, which may be associated with inadequate increases in cell size. By defining the interrelationship between cardiomyocyte size, nucleation, and cell cycle control, we may better understand the cellular mechanisms that drive the loss of mammalian cardiac regenerative capacity after birth.

Wnt signaling activation confers a syncytiotrophoblast progenitor state on trophoblast stem cells of cynomolgus monkey†.

Trophoblast stem cells, derived from the trophectoderm of the blastocyst, are used as an in vitro model to reveal the mechanisms underlying placentation in mammals. In humans, suitable culture conditions for trophoblast stem cell derivation have recently been established. The established human trophoblast stem cells differentiate efficiently toward two trophoblast subtypes: syncytiotrophoblasts and extravillous trophoblasts. However, the efficiency of differentiation is lower in macaque trophoblast stem cells than in human trophoblast stem cells. Here, we demonstrate that the activation of Wnt signaling downregulated the expression of inhibitory G protein and induced trophoblastic lineage switching to the syncytiotrophoblast progenitor state. The treatment of macaque trophoblast stem cells with a GSK-3 inhibitor, CHIR99021, upregulated syncytiotrophoblast progenitor markers and enhanced proliferation. Under the Wnt signaling-activated conditions, macaque trophoblast stem cells effectively differentiated to syncytiotrophoblasts upon dibutyryl cyclic AMP (dbcAMP) and forskolin treatment. RNA-seq analyses revealed the downregulation of inhibitory G protein, which may make macaque trophoblast stem cells responsive to forskolin. Interestingly, this lineage switching appeared to be reversible as the macaque trophoblast stem cells lost responsiveness to forskolin upon the removal of CHIR99021. The ability to regulate the direction of macaque trophoblast stem cell differentiation would be advantageous in elucidating the mechanisms underlying placentation in non-human primates.

Generation of human hepatobiliary organoids with a functional bile duct from chemically induced liver progenitor cells

BackgroundLiver disease imposes a significant medical burden that persists due to a shortage of liver donors and an incomplete understanding of liver disease progression. Hepatobiliary organoids (HBOs) could provide an in vitro mini-organ model to increase the understanding of the liver and may benefit the development of regenerative medicine.MethodsIn this study, we aimed to establish HBOs with bile duct (BD) structures and mature hepatocytes (MHs) using human chemically induced liver progenitor cells (hCLiPs). hCLiPs were induced in mature cryo-hepatocytes using a small-molecule cocktail of TGF-β inhibitor (A-83-01, A), GSK3 inhibitor (CHIR99021, C), and 10% FBS (FAC). HBOs were then formed by seeding hCLiPs into ultralow attachment plates and culturing them with a combination of small molecules of Rock-inhibitor (Y-27632) and AC (YAC).ResultsThese HBOs exhibited bile canaliculi of MHs connected to BD structures, mimicking bile secretion and transportation functions of the liver. The organoids showed gene expression patterns consistent with both MHs and BD structures, and functional assays confirmed their ability to transport the bile analogs of rhodamine-123 and CLF. Functional patient-specific HBOs were also successfully created from hCLiPs sourced from cirrhotic liver tissues.ConclusionsThis study demonstrated the potential of human HBOs as an efficient model for studying hepatobiliary diseases, drug discovery, and personalized medicine.

Shikonin from lithospermum erythrorhizon induces pyroptosis in trophoblast cells by activating the CTSB-NLRP3 inflammasome

Background With the decline of global fertility, drug therapeutic of ectopic pregnancy is of great significance. Lithospermum erythrorhizon is using for embryo killing as herbal medicine. Shikonin is the critical nucleus of Lithospermum erythrorhizon; however, the mechanism is still unclear. The study aimed to explore the mechanism of shikonin against ectopic pregnancy. Material and Methods In this study, we examined the viability and LDH release of HTR-8/SVneo cells by assays, observed pore formation in cell membranes by microscopy imaging and PI staining, and IL-1β release by WB and ELISA assay kit. Then, we used network pharmacology to analyse the potential interaction between shikonin, ectopic pregnancy and pyroptosis and used molecular docking techniques to verify interactions between shikonin and core common targets. Finally, western blotting and immunofluorescence assay were used to explore the mechanism of shikonin-inducing pyroptosis of HTR-8/SVneo cells. Results Shikonin could cause a significant inhibition of HTR-8/SVneo cell viability in a concentration- and time-dependent manner. In HTR-8/SVneo cells, shikonin-induced cell swelling, bubble formation, an increase in the release of lactate dehydrogenase (LDH) and up-regulation of several pyroptosis-associated factors. And network pharmacology showed that The main targets of shikonin-ectopic pregnancy-pyroptosis were IL-1β and caspase-1, and molecular docking results showed that shikonin can closely bind to IL-1β, caspase-1 and GSDMD. Additionally, the necroptosis inhibitor GSK’872 could not suppress the expression of mature-IL-1β and prevent the pyroptosis phenotype from developing. However, the nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inhibitor MCC-950 could downregulate the expression of pyroptosis-associated factors and prevent the pyroptosis phenotype from developing. Shikonin led to an elevation in the expression of cathepsin B (CTSB), and the CTSB inhibitor CA-074 abolished pyroptosis induced by shikonin; however, the NLRP3 inhibitor MCC-950 could not inhibit the expression of CTSB. Conclusions Our results suggest that shikonin activates CTSB to induce NLRP3-dependent pyroptosis in HTR-8/SVneo cells. This study has important clinical implications for the treatment of ectopic pregnancy.