GSK3β Activity Research Articles

Novel Role of Glycogen Synthase Kinase-3β in Determining Cancer Cell Response to Genotoxic Stress through Regulation of 53BP1 Function

Glycogen synthase kinase-3β (GSK3β) is a multifunctional serine/threonine kinase involved in various cellular processes and signaling pathways. Accumulating evidence suggests GSK3β plays a role in cancer treatment with effects on tumorigenesis and treatment response. We have previously shown that inhibition of GSK3β protects neurons from ionizing radiation-induced apoptosis through upregulation of non-homologous end joining (NHEJ) mediated repair of double strand breaks (DSBs). Here, we investigate the molecular mechanism underlying GSK3β regulation of NHEJ. Using biochemical, molecular, and genetic approaches, we investigated the physical and functional interaction between GSK3β and 53BP1. Cultured human tumor cells were utilized as model system to further characterize how GSK3β controls 53BP1 function in DNA DSB recognition and repair. Finally, we employed a cell biology approach to genetically and pharmacologically manipulate GSK3β activity and test how the GSK3β-53BP1 axis impacts tumor cytotoxic response to PARP inhibitor (PARPi) and radiation therapy. We illustrate that GSK3β directly interacts with 53BP1 and phosphorylates 53BP1 at threonine 334 amino acid (T334) within a region heavily phosphorylated by several stress kinases. Phosphorylation at T334 inhibits 53BP1's function in recruitment to DNA DSB sites as well as canonical NHEJ. Furthermore, our results identify GSK3β regulation of 53BP1 function in NHEJ is achieved through suppression of downstream mediators, RIF1 and PTIP, and their function. In contrast, GSK3β enhances single strand DNA resection and promotes homologous recombination (HR) repair. Most importantly, genetic and pharmacologic inhibition of GSK3β-53BP1 signaling axis dramatically enhances the cytotoxic response of BRCA1-deficit cancer cells to PARPi. The assessment of the effect of GSK3β-53BP1 axis on tumor cell response to radiation treatment is underway. This study establishes the connection between GSK3β and DSB repair through its phosphorylation and regulation of 53BP1 pathways. Moreover, it demonstrates that GSK3β kinase activity uniquely results in inhibition of 53BP1 as opposed to other kinases that enhance 53BP1 function. Importantly, this novel signaling axis provides a strategy for targeting cancer cell resistance to PARPi.

Mitochondrial aldehyde dehydrogenase rescues against diabetic cardiomyopathy through GSK3β-mediated preservation of mitochondrial integrity and Parkin-mediated mitophagy.

Mitochondrial aldehyde dehydrogenase (ALDH2) offers proven cardiovascular benefit, although its impact on diabetes remains elusive. This study examined the effects of ALDH2 overexpression and knockout on diabetic cardiomyopathy and the mechanism involved with a focus on mitochondrial integrity. Mice challenged with streptozotocin (STZ, 200mg/kg, via intraperitoneal injection) exhibited pathological alterations, including reduced respiratory exchange ratio, dampened fractional shortening and ejection fraction, increased left ventricular end-systolic and diastolic diameters, cardiac remodeling, cardiomyocyte contractile anomalies, intracellular Ca2+ defects, myocardial ultrastructural injury, oxidative stress, apoptosis, and mitochondrial damage, which were overtly attenuated or accentuated by ALDH2 overexpression or knockout, respectively. Diabetic patients also exhibited reduced plasma ALDH2 activity, cardiac remodeling, and diastolic dysfunction. In addition, STZ challenge altered expression levels of mitochondrial proteins (PGC-1α and UCP2) and Ca2+ regulatory proteins (SERCA, Na+-Ca2+ exchanger, and phospholamban), dampened autophagy and mitophagy (LC3B ratio, TOM20, Parkin, FUNDC1, and BNIP3), disrupted phosphorylation of Akt, GSK3β, and Foxo3a, and elevated PTEN phosphorylation, most of which were reversed or worsened by ALDH2 overexpression or knockout, respectively. Furthermore, the novel ALDH2 activator torezolid, as well as the classical ALDH2 activator Alda-1, protected against STZ- or high glucose-induced in vivo or in vitro cardiac anomalies, which was nullified by inhibition of Akt, GSK3β, Parkin, or mitochondrial coupling. Our data discerned a vital role for ALDH2 in diabetic cardiomyopathy possibly through regulation of Akt and GSK3β activation, Parkin mitophagy, and mitochondrial function.

Fagopyrum tataricum (L.) Gaertn interacts with Gsk-3β/Nrf-2 signalling to protect neurotoxicity in a zebrafish model

Ethnopharmacological relevanceFagopyrum tataricum (L.) Gaertn is used as a folk medicine in many Asian countries due to its anti-inflammatory, antioxidant, and several other health-promoting properties. It is also prescribed to improve neurocognitive functions and alleviate inflammatory conditions. Aim of the studyOxidative stress and neuroinflammation plays a crucial role in neurodegenerative conditions. Hence, based on the ethnomedical claims and available literature, the present study investigated neuroprotective efficacy of a seed extract (ft-ext) of Fagopyrum tataricum against acrylamide (ACR)-induced neurotoxicity. Materials and methodsThe phytochemical characterization of ft-ext was performed by a high-performance liquid chromatography method. Molecular interactions of the identified compounds of ft-ext were studied using an in-silico docking tool. An in-vitro protein denaturation assay was done to check anti-inflammatory activity. The 5 days’ post-fertilized zebrafish larvae were exposed to 1 mM and 2.5 mM ACR with or without ft-ext for 72 h to study its neuroprotective efficacy. Real-time polymerase chain reaction and western blotting studies were performed to analyse the oxidative stress-related gene and protein expressions respectively. ResultsThe extract showed the presence of chlorogenic acid, rutin, caffeic acid, vitexin, syringic acid, quercetin, p-coumaric acid, kaempferol, and ferulic acid. In-vitro protein denaturation assay of ft-ext showed a potent anti-inflammatory effect. The ft-ext improved ACR-mediated locomotor deficit and reduced overall mortality in the larvae. The brain lipid peroxidation and protein carbonylation results revealed an elevated level of oxidative stress in the ACR-treated group, which was reduced in ft-ext-treated larvae. The extract treatment increased the expression of nrf2, gpx, and hmox1a, while simultaneously downregulated trxr2 levels in the brain of larvae exposed to ACR. The treatment also showed inactivation of Gsk-3β, thus maintaining a normal pool of Nrf2 and β-catenin. Molecular docking of identified compounds of ft-ext showed possible hydrogen and hydrophobic interactions with Gsk-3β. ConclusionThe ft-ext prevents ACR-mediated neurotoxicity by suppressing Gsk-3β mediated oxidative stress.

Diabetic Endothelial Cell Glycogen Synthase Kinase 3β Activation Induces VCAM1 Ectodomain Shedding.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1's role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1's release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3β, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3β activation in control ECs increased ADAM10/17 and VCAM1. A GSK3β inhibitor reduced active GSK3β and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3β activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3β inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.

Acteoside ameliorates learning and memory impairment in APP/PS1 transgenic mice by increasing Aβ degradation and inhibiting tau hyperphosphorylation.

Alzheimer's disease (AD) is a neurodegenerative disease. Senile plaques and intracellular neurofibrillary tangles are pathological hallmarks of AD. Recent studies have described the improved cognitive and neuroprotective functions of acteoside (AS). This study aimed to investigate whether the improved cognition of AS was mediated by Aβ degradation and tau phosphorylation in APP/PS1 mice. The open field, Y maze, and novel object recognition tests were used to assess cognitive behavioral changes. We evaluated the levels of Aβ40 and Aβ42 in serum, cortex, and hippocampus, and Aβ-related scavenging enzymes, phosphorylated GSK3β and hyperphosphorylated tau in the cortex and hippocampus of APP/PS1 mice by western blotting. Our results revealed that AS treatment ameliorated anxious behaviors, spatial learning, and memory impairment in APP/PS1 mice and significantly reduced Aβ deposition in their serum, cortex, and hippocampus. AS significantly increased Aβ degradation, inhibited the hyperphosphorylation of tau, and significantly decreased the activity of GSK3β, which is involved in tau phosphorylation. Altogether, these findings indicated that the beneficial effects of AS on AD-associated anxious behaviors and cognitive impairments could be attributed to promoting Aβ degradation and inhibiting tau hyperphosphorylation, which might be partly mediated by GSK3β.

Physalin B Reduces Tau Phosphorylation and Cell Apoptosis in HEK293 Cells by Activating FoxO1.

Physalin B (PB) is one of the main active compounds of Solanaceae plants, with a wide range of biological activities. PB reportedly has the potential to treat Alzheimer's disease (AD). In this study, we investigated the effect of PB on Tau phosphorylation and cell apoptosis using Tau-expressing HEK293 cells (HEK293/Tau) as a cellular model. The optimum concentration of PB to treat HEK293/Tau cells was determined using the CCK-8 assay. Additionally, the expression of FoxO1, Tau-5, p-Tau (T231, S262, and S404), ERK, p-ERK, GSK-3β, and p-GSK-3β was detected using western blotting to determine the effect of PB on Tau phosphorylation. The apoptosis rate was detected using flow cytometry, and the expression of Bax and Bcl-2 was detected using western blotting and verified using real-time quantitative polymerase chain reaction (RT-qPCR). Moreover, cells were transfected with FoxO1 siRNA to downregulate FoxO1 expression, and the expression of the above-mentioned proteins was detected to verify the effect of PB on Tau phosphorylation and cell apoptosis. After 24 h of PB treatment, the phosphorylation levels of Tau at S404, S262, and T231 sites decreased significantly, and the activities of GSK-3β and ERK were inhibited. PB also reduced cell apoptosis by reducing the expression of Bax and increasing the expression of Bcl-2. In addition, PB decreased Tau phosphorylation and cell apoptosis by upregulating FoxO1. The natural compound PB exhibited a protective effect in the AD cell model by increasing FoxO1 expression and reducing Tau phosphorylation and cell apoptosis.

The Axin scaffold protects the kinase GSK3β from cross-pathway inhibition.

Multiple signaling pathways regulate the kinase GSK3β by inhibitory phosphorylation at Ser9, which then occupies the GSK3β priming pocket and blocks substrate binding. Since this mechanism should affect GSK3β activity towards all primed substrates, it is unclear why Ser9 phosphorylation does not affect other GSK3β-dependent pathways, such as Wnt signaling. We used biochemical reconstitution and cell culture assays to evaluate how Wnt-associated GSK3β is insulated from cross-activation by other signals. We found that the Wnt-specific scaffold protein Axin allosterically protects GSK3β from phosphorylation at Ser9 by upstream kinases, which prevents accumulation of pS9-GSK3β in the Axin-GSK3β complex. Scaffold proteins that protect bound proteins from alternative pathway reactions could provide a general mechanism to insulate signaling pathways from improper crosstalk.

Epithelial cell adhesion molecule (EpCAM) regulates HGFR signaling to promote colon cancer progression and metastasis

BackgroundEpithelial cell adhesion molecule (EpCAM) is known to highly expression and promotes cancer progression in many cancer types, including colorectal cancer. While metastasis is one of the main causes of cancer treatment failure, the involvement of EpCAM signaling in metastatic processes is unclear. We propose the potential crosstalk of EpCAM signaling with the HGFR signaling in order to govern metastatic activity in colorectal cancer.MethodsImmunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) was conducted to explore the extracellular domain of EpCAM (EpEX) and HGFR interaction. Western blotting was taken to determine the expression of proteins in colorectal cancer (CRC) cell lines. The functions of EpEX in CRC were investigated by proliferation, migration, and invasion analysis. The combined therapy was validated via a tail vein injection method for the metastasis and orthotopic colon cancer models.ResultsThis study demonstrates that the EpEX binds to HGFR and induces downstream signaling in colon cancer cells. Moreover, EpEX and HGF cooperatively mediate HGFR signaling. Furthermore, EpEX enhances the epithelial-to-mesenchymal transition and metastatic potential of colon cancer cells by activating ERK and FAK-AKT signaling pathways, and it further stabilizes active β-catenin and Snail proteins by decreasing GSK3β activity. Finally, we show that the combined treatment of an anti-EpCAM neutralizing antibody (EpAb2-6) and an HGFR inhibitor (crizotinib) significantly inhibits tumor progression and prolongs survival in metastatic and orthotopic animal models of colon cancer.ConclusionOur findings illuminate the molecular mechanisms underlying EpCAM signaling promotion of colon cancer metastasis, further suggesting that the combination of EpAb2-6 and crizotinib may be an effective strategy for treating cancer patients with high EpCAM expression.

Huannao Yicong decoction ameliorates cognitive deficits in APP/PS1/tau triple transgenic mice by interfering with neurotoxic interaction of Aβ-tau

Ethnopharmacological relevanceHuannao Yicong decoction (HYD) has been used in the study of AD for many years, which consists of Polygonum multiflorum Thunb., Panax ginseng C.A.Mey., Acorus gramineus Aiton, Coptis chinensis Franch., and Conioselinum acuminatum (Franch.) Lavrova. Previous studies have found that HYD could reduce β-Amyloid (Aβ) deposition and tau hyperphosphorylation which are the two critical pathological factors of AD. However, the mechanism of the neurotoxic interaction between Aβ and tau in AD remains unclear. Thus, the underlying mechanisms for HYD improving cognitive function of AD by interfering with the neurotoxic interaction between Aβ and tau remain to be explored. Aim of the studyThe main objective of this study is to clarify the specific mechanisms of HYD on interfering with the neurotoxic interaction between Aβ and tau of AD both in vivo and in vitro. Materials and methodsAPP/PS1/tau triple transgenic mice were randomly divided into 4 groups, namely model group, memantine group, HYD low-dose group (HYD-L), and HYD high-dose group (HYD-H) with 28 mice in each group, while 28 C57BL/6J mice as the control group. Gavage was applied to all the mice daily for 24 weeks. SH-SY5Y model cells overexpressing Aβ and tau proteins as the intervention object in vitro experiments. Morris water maze was used to observe the learning and memory ability of APP/PS1/tau mice. Aβ deposition was detected by immunohistochemistry, and the levels of Aβ1-40 and Aβ1-42 were detected by enzyme-linked immunosorbent assay (ELISA). Neurofibrillary tangles (NFTs) were observed by silver staining and the levels of phosphorylated tau proteins were detected by Western blot. The GSK-3β and CDK-5 mRNA expression were detected by real-time polymerase chain reaction (RT-PCR). Besides, the levels of PSD95, GluR1, NR2A, and NR2B were detected by Western blot. Meanwhile, cell experiments were performed to further verify the effect of HYD on tau phosphorylation related kinases (GSK-3β, CDK-5, and PP2A), which further to clarify the mechanism of HYD intervention on the neurotoxic interaction between Aβ and tau. ResultsHYD improved the learning and memory ability of APP/PS1/tau mice. HYD decreased the levels of Aβ1-40 and Aβ1-42 and inhibited tau hyperphosphorylation, which reduced Aβ deposition and NFTs forming. In addition, HYD inhibited the activity of kinases GSK-3β and CDK-5, and enhancing the activity of kinase PP2A. Moreover, HYD inhibited the overexpression of NR2A and NR2B, and increased the expression of GluR1 and postsynaptic density protein-95 (PSD95). ConclusionsHYD can improve the cognitive deficits by interfering with the neurotoxic interaction between Aβ and tau. In addition, HYD can inhibit the overactivation of NMDARs and increase the levels of GluR1 and PSD95, which may play a role in alleviating neuronal excitotoxicity and improving synaptic function.

Sevoflurane anaesthesia induces cognitive impairment in young mice through sequential tau phosphorylation

BackgroundThe volatile anaesthetic sevoflurane induces time (single or multiple exposures)-dependent effects on tau phosphorylation and cognitive function in young mice. The underlying mechanism for this remains largely undetermined. MethodsMice received 3% sevoflurane for 0.5 h or 2 h daily for 3 days on postnatal day (P) 6, 9, and 12. Another group of mice received 3% sevoflurane for 0.5 h or 1.5 h (3 × 0.5) on P6. We investigated effects of sevoflurane anaesthesia on tau phosphorylation on P6 or P12 mice, on cognitive function from P31 to P37, and on protein interactions, using in vivo studies, in vitro phosphorylation assays, and nanobeam single-molecule level interactions in vitro. ResultsAn initial sevoflurane exposure induced CaMKIIα phosphorylation (132 [11]% vs 100 [6]%, P<0.01), leading to tau phosphorylation at serine 262 (164 [7]% vs 100 [26]%, P<0.01) and tau detachment from microtubules. Subsequent exposures to the sevoflurane induced GSK3β activation, which phosphorylated detached or free tau (tau phosphorylated at serine 262) at serine 202 and threonine 205, resulting in cognitive impairment in young mice. In vitro phosphorylation assays also demonstrated sequential tau phosphorylation. Nanobeam analysis of molecular interactions showed different interactions between tau or free tau and CaMKIIα or GSK3β, and between tau and tubulin at a single-molecule level. ConclusionsMultiple exposures to sevoflurane can induce sequential tau phosphorylation, leading to cognitive impairment in young mice, highlighting the need to investigate the underlying mechanisms of anaesthesia-induced tau phosphorylation in developing brain.

Sustained release of a highly specific GSK3β inhibitor SB216763 in the PCL scaffold creates an osteogenic niche for osteogenesis, anti-adipogenesis, and potential angiogenesis.

The safe and effective use of Wnt signaling is a hot topic in developing osteogenic drugs. SB216763 (S33) is a widely used highly specific GSK3β inhibitor. Here, we show that S33 initiates canonical Wnt signaling by inhibiting GSK3β activity in the bone marrow stromal cell line ST2 and increases osteoblast marker alkaline phosphatase activity, osteoblast marker gene expression including Alpl, Col1α1, and Runx2, promoting osteogenic differentiation and mineralization of ST2 cells. In addition, S33 suppressed the expression of adipogenic transcription factors Pparg and Cebpa in ST2 cells to suppress adipogenesis. ICRT-14, a specific transcriptional inhibitor of Wnt signaling, reversed the effects of S33 on the differentiation of ST2 cells. S33 also increased the expression of osteoclast cytokines RANKL and Opg but decreased the RANKL/Opg ratio and had the potential to inhibit osteoclast differentiation. In addition, we printed the PSCI3D (polycaprolactone, S33, cell-integrated 3D) scaffolds using a newly established integrated 3D printing system for hard materials and cells. S33 sustained release in the hydrogel of the scaffold with 25.4% release on day 1% and 81.7% release over 7days. Cells in the scaffolds had good cell viability. The ratio of live/dead cells remained above 94% for 7days, while the cells in the scaffolds proliferated linearly, and the proliferative activity of the PSCI3D scaffold group increased 1.4-fold and 1.7-fold on days 4 and 7, respectively. Similarly, in PSCI3D scaffolds, osteogenic differentiation of st2 cells was increased. The alkaline phosphatase activity increased 1.4- and 4.0-fold on days 7 and 14, respectively, and mineralization increased 1.7-fold at 21days. In addition, PSCI3D conditioned medium promoted migration and tubulogenesis of HUVECs, and S33 upregulated the expression of Vegfa, a key factor in angiogenesis. In conclusion, our study suggests that S33 functions in osteogenesis, anti-adipogenesis, and potential inhibition of osteoclast differentiation. And the sustained release of S33 in PSCI3D scaffolds creates a safe osteogenic niche, which promotes cell proliferation, osteogenesis, and angiogenesis and has application prospects.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3β, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3β activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3β activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3β. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.

GSK-3β orchestrates the inhibitory innervation of adult-born dentate granule cells in vivo

Adult hippocampal neurogenesis enhances brain plasticity and contributes to the cognitive reserve during aging. Adult hippocampal neurogenesis is impaired in neurological disorders, yet the molecular mechanisms regulating the maturation and synaptic integration of new neurons have not been fully elucidated. GABA is a master regulator of adult and developmental neurogenesis. Here we engineered a novel retrovirus encoding the fusion protein Gephyrin:GFP to longitudinally study the formation and maturation of inhibitory synapses during adult hippocampal neurogenesis in vivo. Our data reveal the early assembly of inhibitory postsynaptic densities at 1 week of cell age. Glycogen synthase kinase 3 Beta (GSK-3β) emerges as a key regulator of inhibitory synapse formation and maturation during adult hippocampal neurogenesis. GSK-3β-overexpressing newborn neurons show an increased number and altered size of Gephyrin+ postsynaptic clusters, enhanced miniature inhibitory postsynaptic currents, shorter and distanced axon initial segments, reduced synaptic output at the CA3 and CA2 hippocampal regions, and impaired pattern separation. Moreover, GSK-3β overexpression triggers a depletion of Parvalbumin+ interneuron perineuronal nets. These alterations might be relevant in the context of neurological diseases in which the activity of GSK-3β is dysregulated.

PD-L1 dimerisation induced by biphenyl derivatives mediates anti-breast cancer activity via the non-immune PD-L1–AKT–mTOR/Bcl2 pathway

Recent studies on biphenyl-containing compounds, a type of PD-1/PD-L1 blocker which binds to PD-L1 and induces dimerisation, have focussed on its immune function. Herein, 10 novel biphenyl derivatives were designed and synthesised. The results of the CCK-8 showed that compounds have different anti-tumour activities for tumour cells in the absence of T cells. Particularly, 12j–4 can significantly induce the apoptosis of MDA-MB-231 cells (IC50 = 2.68 ± 0.27 μM). In further studies, 12j–4 has been shown to prevent the phosphorylation of AKT by binding to cytoplasmic PD-L1, which induces apoptosis in MDA-MB-231 cells through non-immune pathways. The inhibition of AKT phosphorylation restores the activity of GSK-3β, ultimately resulting in the degradation of PD-L1. Besides, in vivo study indicated that 12j–4 repressed tumour growth in nude mice. As these biphenyls exert their anti-tumour effects mainly through non-immune pathways, they are worthy of further study as PD-L1 inhibitors.

Inhibition of GSK3β activity alleviates acute liver failure via suppressing multiple programmed cell death

BackgroundAcute liver failure (ALF) is one of the most common life-threatening diseases in adults without previous liver disease. Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase that is widely distributed in the cells. Inhibition of its activity can inhibit cell death and promote autophagy through various pathways, thus providing a protective effect. In this study, we aimed to investigate the effect on ALF after inhibition of GSK3β and its potential mechanisms.Methods D- galactosamine(D-Gal) in combination with lipopolysaccharide(LPS) was used to induce ALF in vitro and in vivo. And then GSK3β inhibitor TDZD-8 was used to explore the protective effect against ALF. After TDZD-8 treatment TUNEL staining and flow techniques were used to detect the proportion of apoptosis in liver tissues and cells respectively, while western blotting and immunofluorescence assays were performed to detect the expression levels of apoptosis, pyroptosis and necroptosis-related proteins in tissues and cells. In addition, western blotting was performed to explore the specific mechanism of hepatoprotective effect after GSK3β inhibition to detect the expression levels of TAK1, TRAF6 and HDAC3 after TRAF6 and HDAC3 inhibition alone. The co-localization of TRAF6 and HDAC3 in vitro was detected by immunofluorescence, while the interaction between TRAF6 and HDAC3 was detected by immunoprecipitation assay.ResultsBoth in vivo and in vitro experiments, GSK3β inhibitor TDZD-8 can significantly alleviate the progression of ALF. Inhibition of GSK3β activity could significantly reduce the level of hepatocyte apoptosis, pyroptosis, necroptosis and improve liver dysfunction and tissue damage. Furthermore, we found that hepatocyte TAK1 and TRAF6 levels decreased and HDAC3 levels increased in ALF, whereas inhibition of GSK3β upregulated TAK1 and TRAF6 levels and decreased HDAC3 expression.ConclusionGSK3β inhibitor TDZD-8 can prevent the progression of ALF, and its action may involve the TRAF6/HDAC3/TAK1 pathway.

Chronic stress accelerates glioblastoma progression via DRD2/ERK/β-catenin axis and Dopamine/ERK/TH positive feedback loop

BackgroundAfter diagnosis, glioblastoma (GBM) patients undertake tremendous psychological problems such as anxiety and depression, which may contribute to GBM progression. However, systematic study about the relationship between depression and GBM progression is still lacking.MethodsChronic unpredictable mild stress and chronic restrain stress were used to mimic human depression in mice. Human GBM cells and intracranial GBM model were used to assess the effects of chronic stress on GBM growth. Targeted neurotransmitter sequencing, RNA-seq, immunoblotting and immunohistochemistry were used to detect the related molecular mechanism.ResultsChronic stress promoted GBM progression and up-regulated the level of dopamine (DA) and its receptor type 2 (DRD2) in tumor tissues. Down-regulation or inhibition of DRD2 abolished the promoting effect of chronic stress on GBM progression. Mechanistically, the elevated DA and DRD2 activated ERK1/2 and consequently inhibited GSK3β activity, leading to β-catenin activation. Meanwhile, the activated ERK1/2 up-regulated tyrosine hydroxylase (TH) level in GBM cells and then promoted DA secretion, forming an autocrine positive feedback loop. Remarkably, patients with high-depression exhibited high DRD2 and β-catenin levels, which showed poor prognosis. Additionally, DRD2 specific inhibitor pimozide combined with temozolomide synergistically inhibited GBM growth.ConclusionsOur study revealed that chronic stress accelerates GBM progression via DRD2/ERK/β-catenin axis and Dopamine/ERK/TH positive feedback loop. DRD2 together with β-catenin may serve as a potential predictive biomarker for worse prognosis as well as therapeutic target of GBM patients with depression.

Lithium co-administration with rutin improves post-stroke neurological outcomes via suppressing Gsk-3β activity in a rat model

Cerebral ischemic stroke is one of the leading causes of adult disability worldwide. Reperfusion is the only therapeutic option with a lot of side effects. In the current study, we investigated the efficacy of rutin and lithium co-treatment in improving post-stroke neurological outcomes in a transient global cerebral ischemia-reperfusion injury rat model. Middle-aged male rats were subjected to transient global cerebral ischemia-reperfusion. NORT and Y-maze were used to assess the cognitive processes. Lipid peroxidation, protein carbonylation, and nitric oxide assays were performed to study oxidative stress. The excitotoxicity index was calculated by HPLC. Real time-PCR and western blotting were performed to study gene and protein expressions. The co-administration of rutin and lithium improved the overall survival, recognition memory, spatial working memory, and neurological score following cerebral ischemia-reperfusion in rats. Further, a marked decrease in malonaldehyde, protein carbonyls, and nitric oxide levels was observed following combined treatment. The mRNA expression of antioxidant (Hmox1 and Nqo1) and pro-inflammatory (Il2, Il6, and Il1β) markers were significantly attenuated in the rutin and lithium co-administrated group. The treatment inhibited the Gsk-3β and maintained a normal pool of the downstream β-catenin and Nrf2 proteins. The results revealed that co-administration of rutin and lithium had a neuroprotective potential, suggesting it to be a viable treatment to overcome post-stroke deaths and neurological complications.

Glycogen synthase kinase-3β mediates toll-like receptors 4/nuclear factor kappa-B-activated cerebral ischemia-reperfusion injury through regulation of fat mass and obesity-associated protein.

Glycogen synthase kinase-3β (GSK3β), fat mass and obesity-associated protein (FTO), and toll-like receptors 4 (TLR4) take on critical significance in different biological processes, whereas their interactions remain unclear. The objective was the investigation of the interaction effect in cerebral ischemia-reperfusion (I/R) injury. The function of the cerebral cortex in the mouse middle cerebral artery occlusion (MCAO) model (each group n = 6) and P12 cells oxygen-glucose deprivation/reoxygenation (OGD/R) model was analyzed using short hairpin GSK3β lentivirus and overexpression of FTO lentivirus (in vitro), TLR4 inhibitor (TAK242), and LiCl to regulate GSK3β, FTO, TLR4 expression, and GSK3β activity, respectively. After GSK3β knockdown in the OGD/R model of PC12 cells, the levels of TLR4 and p-p65 were lower than in the control, and the level of FTO was higher than in the control. Knockdown GSK3β reversed the OGD/R-induced nuclear factor kappa-B transfer to the intranuclear nuclei. As indicated by the result, TLR4 expression was down-regulated by overexpressed FTO, and TLR4 expression was up-regulated notably after inhibition of FTO with the use of R-2HG. After the inhibition of the activity of GSK3β in vivo, the reduction of FTO in mice suffering from MCAO was reversed. Our research shows that GSK3β/FTO/TLR4 pathway contributes to cerebral I/R injury.

Role of insulin-like growth factor 1 (IGF1) in the regulation of mitochondrial bioenergetics in zebrafish oocytes: lessons from in vivo and in vitro investigations.

Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished reproductive potential and impaired fertility. The role of the intra-ovarian IGF system in ovarian follicular dynamics has been implicated earlier. Although several studies have demonstrated the role of the IGF axis in facilitating mitochondrial function over a multitude of cell lines, its role in oocyte energy metabolism remains largely unexplored. Here using zebrafish, the relative importance of IGF1 in modulating oocyte mitochondrial bioenergetics has been investigated. A dramatic increase in ovarian lhcgr and igf1 expression accompanied heightened ATP levels and mitochondrial polarization in full-grown (FG) oocytes resuming meiotic maturation and ovulation in vivo. Concomitant with elevated igf1 expression and IGF1R phosphorylation, hCG (LH analog) stimulation of FG follicles in vitro prompted a sharp increase in NRF-1 and ATP levels, suggesting a positive influence of gonadotropin action on igf1 expression vis-à-vis oocyte bioenergetics. While recombinant IGF1 administration enhanced mitochondrial function, IGF1R immunodepletion or priming with PI3K inhibitor wortmannin could abrogate NRF-1 immunoreactivity, expression of respiratory chain subunits, ΔΨM, and ATP content. Mechanistically, activation of PI3K/Akt signaling in IGF1-treated follicles corroborated well with the rapid phosphorylation of GSK3β at Ser9 (inactive) followed by PGC-1β accumulation. While selective inhibition of GSK3β promoted PGC-1β, Akt inhibition could abrogate IGF1-induced p-GSK3β (Ser9) and PGC-1β immunoreactive protein indicating Akt-mediated GSK3β inactivation and PGC-1β stabilization. The IGF1-depleted follicles showed elevated superoxide anions, subdued steroidogenic potential, and attenuated G2-M1 transition. In summary, this study highlights the importance of IGF1 signaling in oocyte bioenergetics prior to resumption of meiosis.

REDD1-dependent GSK3β dephosphorylation promotes NF-κB activation and macrophage infiltration in the retina of diabetic mice

Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor, NF-κB. The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16 weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3β and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3β variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3β knockdown inhibited NF-κB activation and proinflammatory cytokine expression by preventing inhibitor of κB kinase complex autophosphorylation and inhibitor of κB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in proinflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3β to promote canonical NF-κB signaling and the development of retinal inflammation.