Phosphorylation Of GSK-3β Research Articles

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1392.

Abstract 7192: Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is a lethal malignancy that is partly attributable to the acquisition of chemoresistance to conventional chemotherapies. Hence, developing strategies to overcome such chemoresistance is essential. Teriflunomide, an active metabolite of leflunomide, has been widely utilized in managing autoimmune disorders, and emerging evidence suggests that it can augment the efficacy of chemotherapy in cancer. We investigated the therapeutic effect of teriflunomide in overcoming resistance to Gemcitabine-based therapy in PC. Methods: We performed docking and genomewide transcriptomic profiling analyses to identify therapeutic targets of Teriflunomide associated with Gemcitabine resistance (Gem-R) in PC. Subsequently, we analyzed the TCGA dataset to determine the prognostic significance of candidate genes in PC patients. A series of experiments were performed in PC cells, and the key findings were validated in patient-derived 3D organoids and a Gem-R Mia-PaCa2 xenograft model. Results: Docking and transcriptomic profiling studies identified GSK3β, MAPK8, and MAPK9 as potential targets of Teriflunomide, which were significantly upregulated in Gem-R cell lines (P < 0.05). GSK3β was independently associated with poor overall and disease-free survival in PC patients. Combined Teriflunomide and Gemcitabine treatment exhibited superior synergistic anti-tumor potential, as evidenced by reducing PC cell proliferation, clonogenicity, invasion, and migration. The combined treatment with Teriflunomide and Gemcitabine primarily inhibited GSK3β phosphorylation, resulting in the silencing of the downstream AKT/mTOR pathway. Lentiviral-based silencing of GSK3β inhibited the AKT/mTOR pathway and reduced the IC50s of Gemcitabine in PC cells. Furthermore, co-administrating both drugs to Gem-R Mia-PaCa-2 xenograft mice significantly reduced tumor growth rates and volumes. These findings were recapitulated in patient-derived 3D-organoids, where the combined treatment resulted in fewer and smaller organoids vs. each drug individually (P < 0.05). Conclusions: We provide novel evidence that Teriflunomide helps overcome resistance to Gemcitabine by inhibiting the GSK3β/AKT/mTOR pathway, which could improve treatment outcomes in PC. Citation Format: Caiming Xu, Yuan Li, Silei Sui, Meiling Chen, Laleh Melstorm, Haiyong Han, Steven T. Rosen, Ajay Goel. Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7192.

The redox-sensitive GSK3β is a key regulator of glomerular podocyte injury in type 2 diabetic kidney disease

Emerging evidence suggests that GSK3β, a redox-sensitive transducer downstream of insulin signaling, acts as a convergent point for myriad pathways implicated in kidney injury, repair, and regeneration. However, its role in diabetic kidney disease remains controversial. In cultured glomerular podocytes, exposure to a milieu of type 2 diabetes elicited prominent signs of podocyte injury and degeneration, marked by loss of homeostatic marker proteins like synaptopodin, actin cytoskeleton disruption, oxidative stress, apoptosis, and stress-induced premature senescence, as shown by increased staining for senescence-associated β-galactosidase activity, amplified formation of γH2AX foci, and elevated expression of mediators of senescence signaling, like p21 and p16INK4A. These degenerative changes coincided with GSK3β hyperactivity, as evidenced by GSK3β overexpression and reduced inhibitory phosphorylation of GSK3β, and were averted by tideglusib, a highly-selective small molecule inhibitor of GSK3β. In agreement, post-hoc analysis of a publicly-available glomerular transcriptomics dataset from patients with type 2 diabetic nephropathy revealed that the curated diabetic nephropathy-related gene set was enriched in high GSK3β expression group. Mechanistically, GSK3β-modulated nuclear factor Nrf2 signaling is involved in diabetic podocytopathy, because GSK3β knockdown reinforced Nrf2 antioxidant response and suppressed oxidative stress, resulting in an improvement in podocyte injury and senescence. Conversely, ectopic expression of the constitutively active mutant of GSK3β impaired Nrf2 antioxidant response and augmented oxidative stress, culminating in an exacerbated diabetic podocyte injury and senescence. Moreover, IRS-1 was found to be a cognate substrate of GSK3β for phosphorylation at IRS-1S332, which negatively regulates IRS-1 activity. GSK3β hyperactivity promoted IRS-1 phosphorylation, denoting a desensitized insulin signaling. Consistently, in vivo in db/db mice with diabetic nephropathy, GSK3β was hyperactive in glomerular podocytes, associated with IRS-1 hyperphosphorylation, impaired Nrf2 response and premature senescence. Our finding suggests that GSK3β is likely a novel therapeutic target for treating type 2 diabetic glomerular injury.

Therapeutic potential of salidroside in preserving rat cochlea organ of corti from gentamicin-induced injury through modulation of NRF2 signaling and GSK3β/NF-κB pathway.

Salidroside (SAL) is a phenol glycoside compound found in plants of the Rhodiola genus which has natural antioxidant and free radical scavenging properties. SAL are able to protect against manganese-induced ototoxicity. However, the molecular mechanism by which SAL reduces levels of reactive oxygen species (ROS) is unclear. Here, we established an in vitro gentamicin (GM) ototoxicity model to observe the protective effect of SAL on GM-induced hair cells (HC) damage. Cochlear explants of postnatal day 4 rats were obtained and randomly divided into six groups: two model groups (treatment with 0.2 mM or 0.4 mM GM for 24 h); two 400 μmol/L SAL-pretreated groups pretreatment with SAL for 3 h followed by GM treatment (0.2 mM or 0.4 mM) for 24 h; 400 μmol/L SAL group (treatment with SAL for 24 h); control group (normal cultured cochlear explants). The protective effects of SAL on GM-induced HC damage, and on mRNA and protein levels of antioxidant enzymes were observed. HC loss occurred after 24 h of GM treatment. Pretreatment with SAL significantly reduced GM-induced OHC loss. In cochlear tissues, mRNA and protein levels of NRF2 and HO-1 were enhanced in the GM alone group compared with the SAL pretreatment GM treatment group. SAL may protect against GM-induced ototoxicity by regulating the antioxidant defense system of cochlear tissues; SAL can activate NRF2/HO-1 signaling, inhibit NF-κB activation, activate AKT, and increase inhibitory phosphorylation of GSK3β to decrease GSK3 activity, all of which exert antioxidant effects.

RND1 inhibits epithelial-mesenchymal transition and temozolomide resistance of glioblastoma via AKT/GSK3-β pathway

ABSTRACT GBM is one of the most malignant tumor in central nervous system. The resistance to temozolomide (TMZ) is inevitable in GBM and the characterization of TMZ resistance seriously hinders clinical treatment. It is worthwhile exploring the underlying mechanism of aggressive invasion and TMZ resistance in GBM treatment. Bioinformatic analysis was used to analyze the association between RND1 and a series of EMT-related genes. Colony formation assay and cell viability assay were used to assess the growth of U87 and U251 cells. The cell invasion status was evaluated based on transwell and wound-healing assays. Western blot was used to detect the protein expression in GBM cells. Treatment targeted RND1 combined with TMZ therapy was conducted in nude mice to evaluate the potential application of RND1 as a clinical target for GBM. The overexpression of RND1 suppressed the progression and migration of U87 and U251 cells. RND1 knockdown facilitated the growth and invasion of GBM cells. RND1 regulated the EMT of GBM cells via inhibiting the phosphorylation of AKT and GSK3-β. The promoted effects of RND1 on TMZ sensitivity was identified both in vitro and in vivo. This research demonstrated that the overexpression of RND1 suppressed the migration and EMT status by downregulating AKT/GSK3-β pathway in GBM. RND1 enhanced the TMZ sensitivity of GBM cells both in vitro and in vivo. Our findings may contribute to the targeted therapy for GBM and the understanding of mechanisms of TMZ resistance in GBM.

Knockdown of ACOT4 alleviates gluconeogenesis and lipid accumulation in hepatocytes

Acyl-CoA thioesterase 4 (ACOT4) has been reported to be related to acetyl-CoA carboxylase activity regulation; However, its exact functions in liver lipid and glucose metabolism are still unclear. Here, we discovered explored the regulatory roles of ACOT4 in hepatic lipid and glucose metabolism in vitro. We found that the expression level of ACOT4 was significantly increased in the hepatic of db/db and ob/ob mice as well as obese mice fed a high fat diet. Adenovirus-mediated overexpression of ACOT4 promoted gluconeogenesis and high-glucose/high-insulin-induced lipid accumulation and impaired insulin sensitivity in primary mouse hepatocytes, whereas ACOT4 knockdown notably suppressed gluconeogenesis and decreased the triglycerides accumulation in hepatocytes. Furthermore, ACOT4 knockdown increased insulin-induced phosphorylation of AKT and GSK-3β in primary mouse hepatocytes. Mechanistically, we found that upregulation of ACOT4 expression inhibited AMP-activated protein kinase (AMPK) activity, and its knockdown had the opposite effect. However, activator A769662 and inhibitor compound C of AMPK suppressed the impact of the change in ACOT4 expression on AMPK activity. Our data indicated that ACOT4 is related to hepatic glucose and lipid metabolism, primarily via the regulation of AMPK activity. In conclusion, ACOT4 is a potential target for the therapy of non-alcoholic fatty liver (NAFLD) and type 2 diabetes.

In Vitro and In Vivo Models for the Development of Hair Growth Materials By Regulating the β-Catenin Signaling Pathways.

Although hair loss contributes to various social and economic, research methods for material development are currently limited. In this study, we established a research model for developing materials for hair growth through the regulation of β-catenin. We confirmed that 100 nM tegatrabetan (TG), a β-catenin inhibitor, decreased the proliferation of human hair follicle dermal papilla cells (HFDPCs) at 72 h. In addition, TG-induced apoptosis suppressed the phosphorylation of GSK-3β and Akt, translocation of β-catenin from the cytosol to the nucleus, and the expression of cyclin D1. Interestingly, TG significantly increased the G2/M arrest in HFDPCs. Subcutaneous injection of TG suppressed hair growth and the number of hair follicles in C57BL/6 mice. Moreover, TG inhibited the expression of cyclin D1, β-catenin, keratin 14, and Ki67. These results suggest that TG-induced inhibition of hair growth can be a promising model for developing new materials for enhancing β-catenin-mediated hair growth.

Elevated levels of alcohol dehydrogenase aggravate ethanol-evoked cardiac remodeling and contractile anomalies through FKBP5-yap-mediated regulation of ferroptosis and ER stress

Alcohol intake provokes severe organ injuries including alcoholic cardiomyopathy with hallmarks of cardiac remodeling and contractile defects. This study examined the toxicity of facilitated ethanol metabolism in alcoholism-evoked changes in myocardial morphology and contractile function, insulin signaling and various cell death domains using cardiac-selective overexpression of alcohol dehydrogenase (ADH). WT and ADH mice were offered an alcohol liquid diet for 12 weeks prior to assessment of cardiac geometry, function, ER stress, apoptosis and ferroptosis. Alcohol intake provoked pronounced glucose intolerance, cardiac remodeling and contractile anomalies with apoptosis, ER stress, and ferroptosis, the effects were accentuated by ADH with the exception of global glucose intolerance. Hearts from alcohol ingesting mice displayed dampened insulin-stimulated phosphorylation of insulin receptor (tyr1146) and IRS-1 (tyrosine) along with elevated IRS-1 serine phosphorylation, the effect was augmented by ADH. Alcohol challenge dampened phosphorylation of Akt and GSK-3β, and increased phosphorylation of c-Jun and JNK, the effects were accentuated by ADH. Alcohol challenge promoted ER stress, FK506 binding protein 5 (FKBP5), YAP, apoptosis and ferroptosis, the effects were exaggerated by ADH. Using a short-term ethanol challenge model (3 g/kg, i.p., twice in three days), we found that inhibition of FKBP5-YAP signaling or facilitated ethanol detoxification by Alda-1 alleviated ethanol cardiotoxicity. In vitro study revealed that the ethanol metabolite acetaldehyde evoked cardiac contractile anomalies, lipid peroxidation, and apoptosis, the effects of which were mitigated by Alda-1, inhibition of ER stress, FKBP5 and YAP. These data suggest that facilitated ethanol metabolism via ADH exacerbates alcohol-evoked myocardial remodeling, functional defects, and insulin insensitivity possibly through a FKBP5-YAP-associated regulation of ER stress and ferroptosis.

MiR-557 inhibits hepatocellular carcinoma progression through Wnt/β-catenin signaling pathway by targeting RAB10.

Accumulating evidence suggests that aberrant miRNAs participate in carcinogenesis and progression of hepatocellular carcinoma (HCC). Abnormal miR-557 expression is reported to interfere with the progression of several human cancers. However, the potential roles of miR-557 in HCC remain largely unknown. In the current study, we found that miR-557 was down-regulated in HCC tissues and cell lines, and was closely related to recurrence and metastasis of HCC. Notably, overexpression of miR-557 inhibited proliferation, migration, invasion, epithelial-to-mesenchymal transition (EMT) progression, blocked cells in G0/G1 phase of MHCC-97H cells in vitro, and suppressed tumor growth in vivo. However, loss of miR-557 facilitated these parameters in Huh7 cells both in vitro and in vivo. Moreover, RAB10 was identified as a direct downstream target of miR-557 through its 3'-UTR. Furthermore, RAB10 re-expression or knockdown partially abolished the effects of miR-557 on proliferation, migration, invasion, and EMT progression of HCC cells. Mechanistically, overexpression of miR-557 suppressed Wnt/β-catenin signaling by inhibiting GSK-3β phosphorylation, increasing β-catenin phosphorylation, and decreasing β-catenin transport to the nucleus, while knockdown of miR-557 activated Wnt/β-catenin signaling. Moreover, the TOP/FOP-Flash reporter assays showed that miR-557 overexpression or knockdown significantly suppressed or activated Wnt signaling activity, respectively. Additionally, low expression of miR-557 and high expression of RAB10 in HCC tissues was closely associated with tumor size, degree of differentiation, TNM stage and poor prognosis in HCC patients. Taken together, these results demonstrate that miR-557 blocks the progression of HCC via the Wnt/β-catenin pathway by targeting RAB10.

Montelukast, an available and safe anti-asthmatic drug, prevents maladaptive remodelling and maintains cardiac functionality following myocardial infarction

Preclinical and clinical data indicate that the 5-lipoxygenase pathway becomes activated in cardiovascular diseases suggesting an important role of CysLTs in atherosclerosis and in its ischemic complications. This study aims to investigate the effects of montelukast, a CysLTR-1 antagonist, in a mouse model of myocardial infarction (MI). C57BL/6N female mice were subjected to coronary artery ligation and received montelukast (10 mg/kg/day, intraperitoneal) or vehicle. Montelukast exerted beneficial effects in the infarcted area, decreasing mRNA expression of inflammatory genes, such Il1β and Ccl2 (p < 0.05), at 48 h after MI, and reducing infarct size and preventing ischemic wall thinning (p < 0.05) at 4 weeks. Furthermore, montelukast counteracted maladaptive remodelling of whole heart. Indeed, montelukast reduced LV mass (p < 0.05) and remote wall thickening (p < 0.05), and improved cardiac pumping function, as evidenced by increased global ejection fraction (p < 0.01), and regional contractility in infarcted (p < 0.05) and in remote non-infarcted (p < 0.05) myocardium. Finally, montelukast prevented cardiomyocytes hypertrophy (p < 0.05) in remote myocardium, reducing the phosphorylation of GSK3β, a regulator of hypertrophic pathway (p < 0.05). Our data strongly demonstrate the ability of montelukast to contrast the MI-induced maladaptive conditions, thus sustaining cardiac contractility. The results provide evidences for montelukast “repurposing” in cardiovascular diseases and in particular in myocardial infarction.

D-pinitol alleviates diabetic cardiomyopathy by inhibiting the optineurin-mediated endoplasmic reticulum stress and glycophagy signaling pathway.

Diabetic cardiomyopathy (DCM) is an important complication resulting in heart failure and death of diabetic patients. However, there is no effective drug for treatments. This study investigated the effect of D-pinitol (DP) on cardiac injury using diabetic mice and glycosylation injury of cardiomyocytes and its molecular mechanisms. We established the streptozotocin-induced SAMR1 and SAMP8 mice and DP (150 mg/kg/day) intragastrically and advanced glycation end-products (AGEs)-induced H9C2 cells. H9C2 cells were transfected with optineurin (OPTN) siRNA and overexpression plasmids. The metabolic disorder indices, cardiac dysfunction, histopathology, immunofluorescence, western blot, and immunoprecipitation were investigated. Our results showed that DP reduced the blood glucose and AGEs, and increased the expression of heart OPTN in diabetic mice and H9C2 cells, thereby inhibiting the endoplasmic reticulum stress (GRP78, CHOP) and glycophagy (STBD1, GABARAPL1), and alleviating the myocardial apoptosis and fibrosis of DCM. The expression of filamin A as an interaction protein of OPTN downregulated by AGEs decreased OPTN abundance. Moreover, OPTN siRNA increased the expression of GRP78, CHOP, STBD1, and GABARAPL1 and inhibited the expression of GAA via GSK3β phosphorylation and FoxO1. DP may be helpful to treat the onset of DCM. Targeting OPTN with DP could be translated into clinical application in the fighting against DCM.

Ddx5 Targeted Epigenetic Modification of Pericytes in Pulmonary Hypertension After Intrauterine Growth Restriction.

Newborns with intrauterine growth restriction (IUGR) have a higher likelihood of developing pulmonary arterial hypertension (PAH) in adulthood. Although there is increasing evidence suggesting that pericytes play a role in regulating myofibroblast transdifferentiation and angiogenesis in malignant and cardiovascular diseases, their involvement in the pathogenesis of IUGR-related pulmonary hypertension and the underlying mechanisms remain incompletely understood. To address this issue, a study was conducted using a Sprague-Dawley rat model of IUGR-related pulmonary hypertension. Our investigation revealed increased proliferation and migration of pulmonary microvascular pericytes in IUGR-related pulmonary hypertension, accompanied by weakened endothelial-pericyte interactions. Through whole-transcriptome sequencing, Ddx5 (DEAD-box protein 5) was identified as one of the hub genes in pericytes. DDX5, a member of the RNA helicase family, plays a role in the regulation of ATP-dependent RNA helicase activities and cellular function. MicroRNAs have been implicated in the pathogenesis of PAH, and microRNA-205 (miR-205) regulates cell proliferation, migration, and angiogenesis. The results of dual-luciferase reporter assays confirmed the specific binding of miR-205 to Ddx5. Mechanistically, miR-205 negatively regulates Ddx5, leading to the degradation of β-catenin by inhibiting the phosphorylation of Gsk3β at serine 9. In vitro experiments showed the addition of miR-205 effectively ameliorated pericyte dysfunction. Furthermore, invivo experiments demonstrated that miR-205 agomir could ameliorate pulmonary hypertension. Our findings indicated that the downregulation of miR-205 expression mediates pericyte dysfunction through the activation of Ddx5. Therefore, targeting the miR-205/Ddx5/p-Gsk3β/β-catenin axis could be a promising therapeutic approach for IUGR-related pulmonary hypertension.

Effects of 625 nm light-emitting diode irradiation on preventing ER stress-induced apoptosis via GSK-3β phosphorylation in MC3T3-E1.

Prolonged endoplasmic reticulum (ER) stress contributes to cell apoptosis and interferes with bone homeostasis. Although photobiomodulation (PBM) might be used for ER stress-induced diseases, the role of PBM in relieving cell apoptosis remains unknown. During ER stress, glycogen synthase kinase-3β (GSK-3β) is critical; however, its functions in PBM remain uncertain. Thus, this study aimed to investigate the role of GSK-3β in 625 nm light-emitting diode irradiation (LEDI) relieving tunicamycin (TM)-induced apoptosis. Based on the results, pre-625 nm LEDI (Pre-IR) phosphorylated GSK-3β via ROS production. Compared with the TM group, Pre-IR + TM group reduced the phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF-2α) and B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax)/Bcl-2 ratio through regulating GSK-3β. Furthermore, a similar tendency was observed between Pre-IR + TM and Pre-LiCl+TM groups in preventing TM-induced early and late apoptosis. In summary, this study suggests that the Pre-IR treatment in TM-induced ER stress is beneficial for preventing cell apoptosis via GSK-3β phosphorylation.

LAIR1-mediated resistance of hepatocellular carcinoma cells to T cells through a GSK-3β/β-catenin/MYC/PD-L1 pathway

BackgroundAn increasing number of studies have reported the involvement of oncogenes in the regulation of the immune system. LAIR1 is an immunosuppressive molecule and its role in immune-related diseases has been mainly reported. To date, it is unclear whether LAIR1 in tumor cells is involved in immune regulation. Therefore, the aim of this study was to investigate the role of LAIR1 in the immune microenvironment of hepatocellular carcinoma (HCC) to seek the novel therapeutic discoveries. MethodsTumor Immune Dysfunction and Exclusion database was used to predict the response of LAIR1 expression to immune checkpoint blockade. CD8+ T cells were co-cultured with HCC cells, and the killing efficiency of leukocytes on HCC cells was detected by flow cytometry. Flow cytometry was also used to detect the expression of inhibitory receptors. In addition, Western blot, immunofluorescence, and nucleus/cytoplasm fractionation experiments were performed to explore the molecular mechanisms by which LAIR1 created a suppressive tumor microenvironment. ResultsLAIR1 expression in HCC was associated with worse immune prognosis and T-cell dysfunction. HCC cells overexpressing LAIR1 co-cultured with CD8+ T cells induced exhaustion of latter. Mechanism studies indicated that LAIR1 in HCC cells up-regulated the phosphorylation of β-catenin by inducing the phosphorylation of GSK-3β, leading to the impairment of the expression and the nuclear localization signal of β-catenin. Low β-catenin expression and nuclear localization signal inhibited MYC-mediated PD-L1 expression. Therefore, PD-L1 up-regulated by LAIR1 caused the exhaustion of infiltrating CD8+ T cells in HCC, which aggravated the malignant progression of HCC. ConclusionLAIR1 increased PD-L1 expression through the GSK-3β/β-catenin/MYC/PD-L1 pathway and promoted immune evasion of HCC cells. Targeted inhibition of LAIR1 helped to enhance the immune killing effect of CD8+ T cells in HCC.

The AMPK and AKT/GSK3β pathways are involved in recombinant proteins fibroblast growth factor 1 (rFGF1 and rFGF1a) improving glycolipid metabolism in rainbow trout (Oncorhynchus mykiss) fed a high carbohydrate diet

Fibroblast growth factor 1 (FGF1) regulates vertebrate cell growth, proliferation and differentiation, and energy metabolism. In this study, we cloned rainbow trout (Oncorhynchus mykiss) fgf1 and fgf1a, prepared their recombinant proteins (rFGF1 and rFGF1a), and described the molecular mechanisms by which they improve glycolipid metabolism in carnivorous fish. A 31-d feeding trial was conducted to investigate whether they could enhance glycolipid metabolism in rainbow trout on high-carbohydrate diets (HCD). A total of 720 rainbow trout (8.9 ± 0.5 g) were equally divided into 4 groups: the chow diet (CD) group injected with PBS, the HCD group injected with PBS, the HCD group injected with rFGF1 (400 ng/g body weight), and the HCD group injected with rFGF1a (400 ng/g body weight). The results showed that short-term HCD had a significant positive effect on the specific growth rate (SGR) of rainbow trout (P < 0.05). However, it led to an increase in crude fat, serum triglyceride (TG) and glucose content, as well as serum glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) contents (P < 0.05), suggesting a negative health effect of HCD. Nevertheless, rFGF1 and rFGF1a showed beneficial therapeutic effects. They significantly reduced the crude fat content of the liver, serum TG, GOT, and GPT contents caused by HCD (P < 0.05). The upregulation in atgl, hsl, and acc2 mRNAs implied the promotion of TG catabolism. Moreover, rFGF1 and rFGF1a contributed to promoting lipolysis by activating the AMPK pathway and reducing lipid accumulation in the liver caused by HCD. In addition, the rFGF1 and rFGF1a-treated groups significantly reduced serum glucose levels and elevated hepatic glycogen content under HCD, and increased glucose uptake by hepatocytes. We observed a decrease in mRNA levels for pepck, g6pase, and pygl, along with an increase in mRNA levels for gys, glut2, and gk in the liver. Furthermore, these proteins regulated hepatic gluconeogenesis and glycogen synthesis by increasing the phosphorylation level of AKT, ultimately leading to an increase in GSK3β phosphorylation. In conclusion, this study demonstrates that rFGF1 and rFGF1a can enhance lipolysis and glucose utilization in rainbow trout by activating the AMPK pathway and AKT/GSK3β axis.

Lithium alleviates paralysis in experimental autoimmune neuritis in Lewis rats by modulating glycogen synthase kinase-3β activity.

Guillain-Barré syndrome (GBS)-like neuropathy mimics the leading cause of sporadic acute nontraumatic limb paralysis in individuals from developed countries. Experimental autoimmune neuritis (EAN) is an animal model of GBS and of syndromes such as acute canine polyradiculoneuritis, seen in dogs and cats. The involvement of glycogen synthase kinase (GSK)-3β, a pro-inflammatory molecule, in rat EAN is not fully understood. This study evaluated the potential role of GSK-3β in EAN through its inhibition by lithium. Lewis rats were injected with SP26 antigen to induce EAN. Lithium was administered from 1 day before immunization to day 14 post-immunization (PI). Then the rats were euthanized and their neural tissues were prepared for histological and Western blotting analyses. Lithium, an inhibitor of GSK-3, significantly ameliorated EAN paralysis in rats, when administered from day 1 to day 14 PI. This corresponded with reduced inflammation in the sciatic nerves of EAN rats, where phosphorylation of GSK-3β was also upregulated, indicating suppression of GSK-3. These findings suggest that lithium, an inhibitor of GSK-3β, plays a significant role in ameliorating rat EAN paralysis, by suppressing GSK-3β and its related signals in EAN-affected sciatic nerves.

Middle-age abolishes cardioprotection conferred by thioredoxin-1 in mice

Thioredoxin-1 (Trx1) has cardioprotective effects on ischemia/reperfusion (I/R) injury, although its role in ischemic postconditioning (PostC) in middle-aged mice is not understood. This study aimed to evaluate if combining two cardioprotective strategies, such as Trx1 overexpression and PostC, could exert a synergistic effect in reducing infarct size in middle-aged mice. Young or middle-aged wild-type mice (Wt), transgenic mice overexpressing Trx1, and dominant negative (DN-Trx1) mutant of Trx1 mice were used. Mice hearts were subjected to I/R or PostC protocol. Infarct size, hydrogen peroxide (H2O2) production, protein nitration, Trx1 activity, mitochondrial function, and Trx1, pAkt and pGSK3β expression were measured. PostC could not reduce infarct size even in the presence of Trx1 overexpression in middle-aged mice. This finding was accompanied by a lack of Akt and GSK3β phosphorylation, and Trx1 expression (in Wt group). Trx1 activity was diminished and H2O2 production and protein nitration were increased in middle-age. The respiratory control rate dropped after I/R in Wt-Young and PostC restored this value, but not in middle-aged groups. Our results showed that Trx1 plays a key role in the PostC protection mechanism in young but not middle-aged mice, even in the presence of Trx1 overexpression.

Integrating Network Pharmacology and Experimental Validation to Investigate the Mechanism of Qushi Huatan Decoction Against Coronary Heart Disease.

This study was designed to evaluate the effect and mechanism of the Qushi Huatan (QSHT) decoction against coronary heart disease (CHD) through network pharmacology and experimental verification. In the present study, the active ingredients of the QSHT decoction were identified by ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS), then the potential ingredients and coronary heart disease targets were predicted using the SwissTarget Prediction database and the database of Genecards and OMIM database, respectively. A herb-compound-target network was constructed using Cytoscape. GO and KEGG enrichment analysis were performed using the ClusterProfiler data package of R software. Molecular docking was used to predict the core targets of QSHT against CHD. In addition, we used a myocardial infarction (MI) and high-fat diet ApoE-/- mice model to investigate the cardioprotective effects of QSHT. Western blotting and immunochemistry were used to verify the core targets and the signaling pathway. A total of 68 active ingredients were found in the QSHT decoction. Network pharmacology indicated 28 targets and 147 signal pathways, including AKT1, HIF-1α, GSK-3β, TLR4 and NF-κB, those key targets were also verified by molecular docking. The results of GO and KEGG enrichment analysis showed that the targets of QSHT against CHD were largely associated with inflammatory and oxidative stress, and AKT/HIF-1α and TLR4/NF-κB pathways might be key functional pathways. In vivo, QSHT significantly improved cardiac function and attenuated fibrosis and inflammation. Furthermore, QSHT could significantly inhibit the expression of HIF-1α, TLR4, phosphorylation of AKT1, GSK-3β and NF-κB after MI in ApoE-/- mice. Based on network pharmacology, molecular docking and experimental verification, this study demonstrated that QSHT could improve cardiac function and attenuate cardiac fibrosis by regulating TLR4/NF-κB and AKT/HIF-1α signaling pathway in post- MI and high-fat diet ApoE-/- mice.

Mechanisms of Actinidia chinensis Planch in treating colon cancer based on the integration of network pharmacology, molecular docking, and experimental verification

BackgroundAs an anticancer Chinese herbal medicine, the effective components and mechanism of Actinidia chinensis Planch (ACP, Tengligen) in the treatment of colon cancer are still unclear. In the present study, the integration of network pharmacology, molecular docking, and cell experiments was employed to study the effective mechanism of ACP against colon cancer.MethodsThe Venn diagram and STRING database were used to construct the protein–protein interaction network (PPI) of ACP-colon cancer, and further topological analysis was used to obtain the key target genes of ACP in colon cancer. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to visualize the related functions and pathways. Molecular docking between key targets and compounds was determined using software such as AutoDockTools. Finally, the effect of ACP on CT26 cells was observed in vitro.ResultsThe study identified 40 ACP-colon key targets, including CASP3, CDK2, GSK3B, and PIK3R1. GO and KEGG enrichment analyses found that these genes were involved in 211 biological processes and 92 pathways, among which pathways in cancer, PI3K-Akt, p53, and cell cycle might be the main pathways of ACP against colon cancer. Molecular docking verified that the key components of ACP could stably bind to the corresponding targets. The experimental results showed that ACP could inhibit proliferation, induce apoptosis, and downregulate the phosphorylation of PIK3R1, Akt, and GSK3B in CT26 cells.ConclusionACP is an anti-colon cancer herb with multiple components, and involvement of multiple target genes and signaling pathways. ACP can significantly inhibit proliferation and induce apoptosis of colon cancer cells, which may be closely related to the regulation of PI3K/AKT/GSK3B signal transduction.

ACTN1 promotes HNSCC tumorigenesis and cisplatin resistance by enhancing MYH9-dependent degradation of GSK-3β and integrin β1-mediated phosphorylation of FAK

BackgroundHead and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors globally. Understanding the molecular basis of tumor progression and drug resistance can offer innovative strategies to enhance clinical outcomes for HNSCC patients.MethodsThe cytoskeletal remodeling genes associated with cisplatin resistance were screened using a PCR array. The role of alpha-actinin 1 (ACTN1) in modulating cisplatin resistance and tumorigenesis in HNSCC was evaluated both in vitro and in vivo. Co-immunoprecipitation (Co-IP), IP-mass spectrometry (MS), western blotting, dual-luciferase assay, and bioinformatics analysis were performed to elucidate the underlying mechanisms involved.ResultsOur study identifies ACTN1 as a crucial contributor to cisplatin resistance and tumorigenesis in HNSCC, as evidenced across cellular, animal, and patient-derived xenograft models. From a clinical perspective, overexpression of ACTN1 significantly correlates with a suboptimal response to neoadjuvant chemotherapy and reduced overall survival in HNSCC patients. Mechanistically, ACTN1 predominantly activates β-catenin-mediated signaling by promoting the interaction between myosin heavy chain 9 (MYH9) and GSK-3β, leading to the ubiquitin-dependent degradation of GSK-3β. ACTN1 also interacts with integrin β1, subsequently activating the FAK/PI3K/AKT pathway, providing an additional avenue for the activation of β-catenin signaling. Our study also unveils that the β-catenin/c-Myc axis transcriptionally regulates ACTN1, thereby creating a positive feedback loop promoting HNSCC tumorigenesis and drug resistance.ConclusionsThese insights underscore the novel mechanisms that highlight ACTN1's pivotal role in driving HNSCC progression and resistance to chemotherapy, suggesting ACTN1 as a promising therapeutic target in HNSCC management.