Novel Reporter Alleles of GSK-3α and GSK-3β

Glycogen Synthase Kinase 3 (GSK-3) is a key player in development, physiology and disease. Because of this, GSK-3 inhibitors are increasingly being explored for a variety of applications. In addition most analyses focus on GSK-3β and overlook the closely related protein GSK-3α. Here, we describe novel GSK-3α and GSK-3β mouse alleles that allow us to visualise expression of their respective mRNAs by tracking β-galactosidase activity. We used these new lacZ alleles to compare expression in the palate and cranial sutures and found that there was indeed differential expression. Furthermore, both are loss of function alleles and can be used to generate homozygous mutant mice; in addition, excision of the lacZ cassette from GSK-3α creates a Cre-dependent tissue-specific knockout. As expected, GSK3α mutants were viable, while GSK3β mutants died after birth with a complete cleft palate. We also assessed the GSK-3α mutants for cranial and sternal phenotypes and found that they were essentially normal. Finally, we observed gestational lethality in compound GSK-3β^−/−; GSK3α^+/− mutants, suggesting that GSK-3 dosage is critical during embryonic development.     

GSK-3与精神障碍的关系

糖原合成酶激酶3(GSK-3)是一种丝/苏氨酸蛋白激酶,通过Wnt/β-catenin等多条信号通路来调节机体的代谢、生长发育和凋亡等过程,是机体生存必不可少的物质。大量研究表明,GSK-3调节异常可以激活特定细胞、通路和环路,从而诱发精神障碍,如双向情感障碍、抑郁症、孤独症和精神分裂症等。抑制GSK-3活性是精神疾病治疗方法的一个重要组分。本文对近年来国内外有关GSK-3在精神疾病中的作用机制予以综述,以期为临床治疗提供依据。     

E-pharmacophore-based virtual screening to identify GSK-3β inhibitors

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase which has attracted significant attention during recent years in drug design studies. The deregulation of GSK-3β increased the loss of hippocampal neurons by triggering apoptosis-mediating production of neurofibrillary tangles and alleviates memory deficits in Alzheimer’s disease (AD). Given its role in the formation of neurofibrillary tangles leading to AD, it has been a major therapeutic target for intervention in AD, hence was targeted in the present study. Twenty crystal structures were refined to generate pharmacophore models based on energy involvement in binding co-crystal ligands. Four common e-pharmacophore models were optimized from the 20 pharmacophore models. Shape-based screening of four e-pharmacophore models against nine established small molecule databases using Phase v3.9 had resulted in 1800 compounds having similar pharmacophore features. Rigid receptor docking (RRD) was performed for 1800 compounds and 20 co-crystal ligands with GSK-3β to generate dock complexes. Interactions of the best scoring lead obtained through RRD were further studied with quantum polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area. Comparing the obtained leads to 20 co-crystal ligands resulted in 18 leads among them, lead1 had the lowest docking score, lower binding free energy and better binding orientation toward GSK-3β. The 50?ns MD simulations run confirmed the stable nature of GSK-3β-lead1 docking complex. The results from RRD, QPLD, IFD and MD simulations confirmed that lead1 might be used as a potent antagonist for GSK-3β.     

GSK-3β as a driving force in ovarian cancer

Ovarian cancer is one of the most lethal malignancies in women. Identification of new therapeutic targets would provide opportunities for developing potentially more effective treatment regimes. In the July issue of Cell Research, Cao et al. reports that glycogen synthase kinase-3β （GSK-3β） plays an important role in positively regulating the proliferation of human ovarian cancer cells, and thus it may represent such a target [ 1 ]. GSK-3β is a serine/threonine kinase that is known to be involved in regulation of β-catenin signaling, where it participates in the formation of a multi-component destruction complex that promotes the phosphorylation and subsequent degradation of β-catenin. Given that overactive β-catenin signaling is involved in many forms of human cancer, this classic mode of GSK-3β action should qualify it as a ＂tumor suppressor＂. Intriguingly, however, two recent studies have implicated that GSK-3β may actually play a pro-tumor role in pancreatic and colorectal cancers [2, 3]. Since ovarian tumors often exhibit increased expression of GSK-3β, these recent findings prompted Cao et al. to examine the potential role of GSK-3β in ovarian cancer cells.     

GSK-3 in liver diseases: Friend or foe?

Liver diseases, including hepatitis due to hepatitis B or C virus infection, non-alcoholic fatty liver disease, and hepatocellular carcinoma pose major challenges for overall health due to limited curative treatment options. Thus, there is an urgent need to develop new therapeutic strategies for the treatment of these diseases. A better understanding of the signaling pathways involved in the pathogenesis of liver diseases can help to improve the efficacy of emerging therapies, mainly based on pharmacological approaches, which influence one or more specific molecules involved in key signal transduction pathways. These emerging therapies are very promising for the prevention and treatment of liver diseases. One promising druggable molecular target is the multifunctional serine/threonine kinase, glycogen synthase kinase 3 (GSK-3). In this review, we discuss conditions in which GSK-3 is implicated in liver diseases. In addition, we explore newly emerging drugs that target GSK-3β, as well as their potential use in and impact on the management of liver diseases.     

A mitotic role for GSK-3β kinase in Drosophila

Glycogen synthase kinase-3β (GSK-3β) is involved in a wide variety of cellular processes, and implicated in a growing list of human diseases. Recent drug inhibition studies have suggested a role for GSK-3β in mitosis in animals. Here, we take an alternative approach to understanding GSK-3β function in mitosis by genetic mutational analysis in Drosophila. GSK-3β function is well conserved between Drosophila (Zw3) and humans, frequently operating similarly in pathways, as diverse as the Wnt signaling and circadian rhythm pathways, and sharing a key role in the development of the neuromuscular junction. Unlike drug inhibitor studies, we find that loss of function mutations of zw3 result in markedly curved, or bent, metaphase spindles that exhibit metaphase delay. These defects do not routinely result in mitotic catastrophe, and argue that Zw3 plays a role in the maintenance of the mitotic spindle, rather than an essential role in spindle morphogenesis. Consistent with a mitotic function, we observe a complex and dynamic localization of Zw3 during cell division. These studies provide genetic data that validate and extend drug inhibition studies on a novel mitotic role for glycogen synthase kinase in the maintenance of the mitotic spindle.     

Atorvastatin inhibits GSK-3β phosphorylation by cardiac hypertrophic stimuli

In this study we examined the effect of the statin atorvastatin on the Akt/GSK-3β pathway. Our findings indicate that atorvastatin treatment for 15 days inhibited pressure overload-induced cardiac hypertrophy and prevented nuclear translocation of GATA4 and c-Jun and AP-1 DNA-binding activity. In addition, atorvastatin treatment prevented the increase in the phosphorylation of Akt and GSK-3β caused by cardiac hypertrophy, and this effect correlated with an increase in protein levels of phosphatase and tensin homolog on chromosome 10 (PTEN), which negatively regulates the phosphoinositide-3 kinase/Akt pathway. To test whether the inhibitory effect of atorvastatin on Akt and GSK-3β phosphorylation was direct we performed in vitro studies using embryonic rat heart-derived H9c2 cells, human AC16 cardiomyoblasts and neonatal rat cardiomyocytes. Preincubation of cells with atorvastatin prevented Akt/GSK-3β phosphorylation by different hypertrophic stimuli without affecting PTEN protein levels. However, atorvastatin prevented endogenous reactive oxygen species (ROS) generation and PTEN oxidation, a process that correlates with its inactivation, suggesting that atorvastatin prevents ROS-induced PTEN inactivation in acute treatments. These findings point to a new potential anti-hypertrophic effect of statins, which can prevent activation of the Akt/GSK-3β hypertrophic pathway by modulating PTEN activation by different mechanisms in chronic and acute treatments.     

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology

Background

Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.

Results

We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3β (GSK-3β) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3β is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.

Conclusion

These results suggest that phosphorylation of tau by GSK-3β promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.     

GSK-3β在阿尔茨海默症病理特征形成中的作用

糖原合成酶激酶3β(glycogen synthase kinase-3β,GSK-3β)是糖原合成酶激酶3的一种亚型。GSK-3β不仅参与淀粉样蛋白质前体(amyloid precursor protein,APP)代谢,还在tau蛋白过度磷酸化过程中发挥作用,GSK-3β表达及活性的异常会导致神经元细胞的凋亡。APP异常代谢和tau蛋白异常磷酸化是阿尔茨海默病(Alzheimer’s disease,AD)发展的重要因素,因此GSK-3β可能与AD的病理变化密切相关,明确其在AD中的作用及其机制对AD的治疗有重要的意义。     

GSK-3β Is Required for Memory Reconsolidation in Adult Brain

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimer''s disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.     

Dendritic cell maturation occurs through the inhibition of GSK-3β

Dendritic cell (DC) maturation results in changes in antigen processing and presentation, governing the fate of adaptive immunity. Understanding the intracellular signaling pathways governing DC maturation is therefore critical. In this study, we observed that the kinase, GSK-3β, is present in its active form in resting immature DCs isolated from the spleen and bone marrow of mice. Induction of DC maturation using GM-CSF, IL-4 and TNF-α resulted in GSK-3β inhibition, as reflected by increased phosphorylation of Serine 9 on the kinase, and concomitant stabilization of its substrate, β-catenin. Treatment of immature DCs with a GSK-3β inhibitor increased cell surface expression of CD80, CD86 and CD40 on DCs, enhancing their ability to present antigen and activating IL-2 secretion by T cells. GSK-3β inhibition also parallels dendritic cell maturation in vivo. Our results show that GSK-3β signaling controls DC maturation and suggest that this kinase could be manipulated to modulate adaptive immunity.     

Discovery of new GSK-3β inhibitors through structure-based virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an attractive therapeutic target for human diseases, such as diabetes, cancer, neurodegenerative diseases, and inflammation. Thus, structure-based virtual screening was performed to identify novel scaffolds of GSK-3β inhibitors, and we observed that conserved water molecules of GSK-3β were suitable for virtual screening. We found 14 hits and D1 (IC₅₀ of 0.71?μM) were identified. Furthermore, the neuroprotection activity of D1–D3 was validated on a cellular level. 2D similarity searches were used to find derivatives of high inhibitory compounds and an enriched structure–activity relationship suggested that these skeletons were worthy of study as potent GSK-3β inhibitors.     

PKCβ Positively Regulates RANKL-Induced Osteoclastogenesis by Inactivating GSK-3β

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-κB ligand (RANKL) signaling has remained elusive. We now demonstrate that PKCβ acts as a positive regulator which inactivates glycogen synthase kinase-3β (GSK-3β) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, PKCβ expression is increased by RANKL. Pharmacological inhibition of PKCβ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-3β was decreased by PKCβ inhibition. Likewise, down-regulation of PKCβ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-3β phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the PKCβ pathway, leading to GSK-3β inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for PKCβ’s therapeutic targeting to treat inflammation-related bone diseases.     

The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand) signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinase 3β) inhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.     

GSK-3β对缺血性脑卒中病理过程调控作用的研究进展

糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)作为机体最重要的激酶之一,广泛地参与了缺血性脑卒中的病理过程。因此,正确认识脑卒中后GSK-3β的功能并加以利用,由此寻求减轻组织损伤和改善神经功能的方法是提高脑卒中治疗的重要途径。该文就GSK-3β对缺血性脑卒中后的氧化应激、炎症、自噬、凋亡等病理过程的调控机制进行综述,为缺血性脑卒中提供新的研究方向和潜在的临床治疗靶点。     

Stimulatory effect of α-synuclein on the tau-phosphorylation by GSK-3β

Hyperphosphorylation of tau protein (tau) causes neurodegenerative diseases such as Alzheimer's disease (AD). Recent studies of the physiological correlation between tau and α-synuclein (α-SN) have demonstrated that: (a) phosphorylated tau is also present in Lewy bodies, which are cytoplasmic inclusions formed by abnormal aggregation of α-SN; and (b) the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) increases the phosphorylation of tau as well as the protein level of α-SN in cultured neuronal cells, and also in mice. However, the molecular mechanism responsible for the α-SN-mediated hyperphosphorylation of tau remains to be elucidated. In this in vitro study, we found that: (a) α-SN directly stimulates the phosphorylation of tau by glycogen synthase kinase-3β (GSK-3β), (b) α-SN forms a heterotrimeric complex with tau and GSK-3β, and (c) the nonamyloid beta component (NAC) domain and an acidic region of α-SN are responsible for the stimulation of GSK-3β-mediated tau phosphorylation. Thus, it is concluded that α-SN functions as a connecting mediator for tau and GSK-3β, resulting in GSK-3β-mediated tau phosphorylation. Because the expression of α-SN is promoted by oxidative stress, the accumulation of α-SN induced by such stress may directly induce the hyperphosphorylation of tau by GSK-3β. Furthermore, we found that heat shock protein 70 (Hsp70) suppresses the α-SN-induced phosphorylation of tau by GSK-3β through its direct binding to α-SN, suggesting that Hsp70 acts as a physiological suppressor of α-SN-mediated tau hyperphosphorylation. These results suggest that the cellular level of Hsp70 may be a novel therapeutic target to counteract α-SN-mediated tau phosphorylation in the initial stage of neurodegenerative disease.