Sporadic pediatric vestibular schwannoma: a case report in a 4-year-old boy.

Sporadic vestibular schwannomas (VSs) are rare in children. When occurred in the pediatric population, they usually appear bilaterally and are related to neurofibromatosis type 2 (NF2). The current study reports a 4-year-old boy without family history of VS or NF2 who presented with a large (5.7-cm) VS involving the right cerebellopontine angle and internal auditory canal. Through seven-staged surgical interventions and two stereotactic γ­knife radiosurgery, the disease was stabilized. At 2-year follow-up, the child had right ear hearing loss, grade IV facial palsy, and normal motor function and gait. No definite evidence of gene mutation regarding NF2 can be identified after sequence analysis and deletion/duplication testing. This case highlights the significance of considering the possibility of sporadic VSs, even in very young children. It emphasizes the importance of not overlooking initial symptoms, as they may indicate the presence of a large tumor and could potentially result in delayed diagnosis.

Role of Glycogen Synthase Kinase-3 in Interferon-γ-Mediated Immune Hepatitis.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase, is a vital glycogen synthase regulator controlling glycogen synthesis, glucose metabolism, and insulin signaling. GSK-3 is widely expressed in different types of cells, and its abundant roles in cellular bioregulation have been speculated. Abnormal GSK-3 activation and inactivation may affect its original bioactivity. Moreover, active and inactive GSK-3 can regulate several cytosolic factors and modulate their diverse cellular functional roles. Studies in experimental liver disease models have illustrated the possible pathological role of GSK-3 in facilitating acute hepatic injury. Pharmacologically targeting GSK-3 is therefore suggested as a therapeutic strategy for liver protection. Furthermore, while the signaling transduction of GSK-3 facilitates proinflammatory interferon (IFN)-γ in vitro and in vivo, the blockade of GSK-3 can be protective, as shown by an IFN-γ-induced immune hepatitis model. In this study, we explored the possible regulation of GSK-3 and the potential relevance of GSK-3 blockade in IFN-γ-mediated immune hepatitis.

Mutant glucocorticoid receptor binding elements on the interleukin-6 promoter regulate dexamethasone effects.

BACKGROUND: Glucocorticoids (GCs) have been extensively used as essential modulators in clinical infectious and inflammatory diseases. The GC receptor (GR) is a transcription factor belonging to the nuclear receptor family that regulates anti-inflammatory processes and releases pro-inflammatory cytokines, such as interleukin (IL)-6. RESULTS: Five putative GR binding sites and other transcriptional factor binding sites were identified on theIL-6 promoter, and dexamethasone (DEX) was noted to reduce the lipopolysaccharide (LPS)-induced IL-6 production. Among mutant transcriptional factor binding sites, nuclear factor-kappa B (NF-κB), activator protein (AP)-1, and specificity protein (Sp)1-2 sites reduced basal and LPS-induced IL-6 promoter activities through various responses. The second GR binding site (GR2) was noted to play a crucial role in both basal and inducible promoter activities in LPS-induced inflammation. CONCLUSIONS: We concluded that selective GR2 modulator might exert agonistic and antagonistic effects and could activate crucial signaling pathways during the LPS-stimulated inflammatory process.

How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase.

Protein kinases are important cellular signaling molecules involved in cancer and a multitude of other diseases. It is well-known that inactive kinases display a remarkable conformational plasticity; however, the molecular mechanisms remain poorly understood. Conformational heterogeneity presents an opportunity but also a challenge in kinase drug discovery. The ability to predictively model various conformational states could accelerate selective inhibitor design. Here we performed a proton-coupled molecular dynamics study to explore the conformational landscape of a c-Src kinase. Starting from a completely inactive structure, the simulations captured all major types of conformational states without the use of a target structure, mutation, or bias. The simulations allowed us to test the experimental hypotheses regarding the mechanism of DFG flip, its coupling to the αC-helix movement, and the formation of regulatory spine. Perhaps the most significant finding is how key titratable residues, such as DFG-Asp, αC-Glu, and HRD-Asp, change protonation states dependent on the DFG, αC, and activation loop conformations. Our data offer direct evidence to support a long-standing hypothesis that protonation of Asp favors the DFG-out state and explain why DFG flip is also possible in simulations with deprotonated Asp. The simulations also revealed intermediate states, among which a unique DFG-out/α-C state formed as DFG-Asp is moved into a back pocket forming a salt bridge with catalytic Lys, which can be tested in selective inhibitor design. Our finding of how proton coupling enables the remarkable conformational plasticity may shift the paradigm of computational studies of kinases which assume fixed protonation states. Understanding proton-coupled conformational dynamics may hold a key to further innovation in kinase drug discovery.

Assessing Lysine and Cysteine Reactivities for Designing Targeted Covalent Kinase Inhibitors.

Targeted covalent inhibitor design is gaining increasing interest and acceptance. A typical covalent kinase inhibitor design targets a reactive cysteine; however, this strategy is limited by the low abundance of cysteine and acquired drug resistance from point mutations. Inspired by the recent development of lysine-targeted chemical probes, we asked if nucleophilic (reactive) catalytic lysines are common on the basis of the published crystal structures of the human kinome. Using a newly developed p Ka prediction tool based on continuous constant pH molecular dynamics, the catalytic lysines of eight unique kinases from various human kinase groups were retrospectively and prospectively predicted to be nucleophilic, when kinase is in the rare DFG-out/αC-out type of conformation. Importantly, other reactive lysines as well as cysteines at various locations were also identified. On the basis of the findings, we proposed a new strategy in which selective type II reversible kinase inhibitors are modified to design highly selective, lysine-targeted covalent inhibitors. Traditional covalent drugs were discovered serendipitously; the presented tool, which can assess the reactivities of any potentially targetable residues, may accelerate the rational discovery of new covalent inhibitors. Another significant finding of the work is that lysines and cysteines in kinases may adopt neutral and charged states at physiological pH, respectively. This finding may shift the current paradigm of computational studies of kinases, which assume fixed solution protonation states.

Electrical Programming of Soft Matter: Using Temporally Varying Electrical Inputs To Spatially Control Self Assembly.

The growing importance of hydrogels in translational medicine has stimulated the development of top-down fabrication methods, yet often these methods lack the capabilities to generate the complex matrix architectures observed in biology. Here we show that temporally varying electrical signals can cue a self-assembling polysaccharide to controllably form a hydrogel with complex internal patterns. Evidence from theory and experiment indicate that internal structure emerges through a subtle interplay between the electrical current that triggers self-assembly and the electrical potential (or electric field) that recruits and appears to orient the polysaccharide chains at the growing gel front. These studies demonstrate that short sequences (minutes) of low-power (∼1 V) electrical inputs can provide the program to guide self-assembly that yields hydrogels with stable, complex, and spatially varying structure and properties.

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet.

OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.

Conformational dynamics of cathepsin D and binding to a small-molecule BACE1 inhibitor.

BACE1 is a major therapeutic target for prevention and treatment of Alzheimer's disease. Developing inhibitors that can selectively target BACE1 in favor of other proteases, especially cathepsin D (CatD), has presented significant challenges. Here, we investigate the conformational dynamics and protonation states of BACE1 and CatD using continuous constant pH molecular dynamics with pH replica-exchange sampling protocol. Despite similar structure, BACE1 and CatD exhibit markedly different active site dynamics. BACE1 displays pH-dependent flap dynamics that controls substrate accessibility, while the CatD flap is relatively rigid and remains open in the pH range 2.5-6. Interestingly, although each protease hydrolyzes peptide bonds, the protonation states of the catalytic dyads are different within the active pH range. The acidic and basic components of the BACE1 catalytic dyad are clear, while either aspartic acid of the CatD catalytic dyad could play the role of acid or base. Finally, we investigate binding of the inhibitor LY2811376 developed by Eli Lilly to BACE1 and CatD. Surprisingly, in the enzyme active pH range, LY2811376 forms a stronger salt bridge with the catalytic dyad in CatD than in BACE1, which might explain the retinal toxicity of the inhibitor related to off-target inhibition of CatD. This work highlights the complexity and challenge in structure-based drug design where receptor-ligand binding induces protonation state change in both the protein and the inhibitor. © 2017 Wiley Periodicals, Inc.

Comparison of radii sets, entropy, QM methods, and sampling on MM-PBSA, MM-GBSA, and QM/MM-GBSA ligand binding energies of F. tularensis enoyl-ACP reductase (FabI).

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM-PBSA, MM-GBSA, and QM/MM-GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM-GBSA using 6 ns MD simulation trajectories together with GB(neck2) , PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r(2) = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called "multiple independent sampling"), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GB(HCT) and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r(2) = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive "multiple independent sampling method" may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM-P(G)BSA were limited to inhibitors' relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program.

Glycogen synthase kinase-3 facilitates con a-induced IFN-γ-- mediated immune hepatic injury.

Immune hepatic injury induced by Con A results primarily from IFN-γ-mediated inflammation, followed by hepatic cell death. Glycogen synthase kinase (GSK)-3, which acts proapoptotically and is proinflammatory, is also important for facilitating IFN-γ signaling. We hypothesized a pathogenic role for GSK-3 in Con A hepatic injury. Con A stimulation caused GSK-3 activation in the livers of C57BL/6 mice. Inhibiting GSK-3 reduced Con A hepatic injury, including hepatic necrosis and apoptosis, inflammation, infiltration of T cells and granulocytes, and deregulated expression of adhesion molecule CD54. Con A induced hepatic injury in an IFN-γ receptor 1-dependent manner. Con A/IFN-γ induced activation and expression of STAT1 in a GSK-3-dependent manner. GSK-3 facilitated IFN-γ-induced inducible NO synthase, but had limited effects on CD95 upregulation and CD95-mediated hepatocyte apoptosis in vitro. Notably, inhibiting GSK-3 decreased Con A-induced IFN-γ production in both wild-type and IFN-γ receptor 1-deficient C57BL/6 mice. In Con A-activated NKT cells, GSK-3 was also activated and was required for nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, but it was not required for CD95 ligand expression or activation-induced cell death. These results demonstrate the dual and indispensable role of GSK-3 in Con A hepatic injury by facilitating IFN-γ-induced hepatopathy.

Inhibiting glycogen synthase kinase-3 decreases 12-O-tetradecanoylphorbol-13-acetate-induced interferon-γ-mediated skin inflammation.

Glycogen synthase kinase-3 (GSK-3) facilitates interferon (IFN)-γ signaling. Because IFN-γ is involved in inflammatory skin diseases, such as psoriasis, the aim of this study was to investigate the pathogenic role of GSK-3 in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IFN-γ-mediated ear skin inflammation. TPA (3 µg per ear) induced acute skin inflammation in the ears of C57BL/6 mice, including edema, infiltration of granulocytes but not T cells, and IFN-γ receptor 1-mediated deregulation of intercellular adhesion molecule 1 (CD54). TPA/IFN-γ induced GSK-3 activation, which in turn activated signal transducer and activator of transcription 1. Inhibiting GSK-3 pharmacologically, by administering 6-bromoindirubin-3'-oxime (1.5 µg per ear), and genetically, with lentiviral-based short-hairpin RNA, reduced TPA-induced acute skin inflammation but not T-cell infiltration. It is noteworthy that inhibiting GSK-3 decreased TPA-induced IFN-γ production and the nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, in CD3-positive T cells. In chronic TPA-induced skin inflammation, inhibiting GSK-3 attenuated epidermis hyperproliferation and dermis angiogenesis. These results demonstrate the dual role of GSK-3 in TPA-induced skin inflammation that is not only to facilitate IFN-γ signaling but also to regulate IFN-γ production. Inhibiting GSK-3 may be a potential treatment strategy for preventing such effects.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis.

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance.

Anesthetic propofol causes glycogen synthase kinase-3ß-regulated lysosomal/mitochondrial apoptosis in macrophages.

BACKGROUND: Overdose propofol treatment with a prolong time causes injury to multiple cell types; however, its molecular mechanisms remain unclear. Activation of glycogen synthase kinase (GSK)-3ß is proapoptotic under death stimuli. The authors therefore hypothesize that propofol overdose induces macrophage apoptosis through GSK-3ß. METHODS: Phagocytic analysis by uptake of Staphylococcus aureus showed the effects of propofol overdose on murine macrophages RAW264.7 and BV2 and primary human neutrophils in vitro. The authors further investigated cell apoptosis in vitro and in vivo, lysosomal membrane permeabilization, and the loss of mitochondrial transmembrane potential (MTP) by propidium iodide, annexin V, acridine orange, and rhodamine 123 staining, respectively. Protein analysis identified activation of apoptotic signals, and pharmacologic inhibition and genetic knockdown using lentiviral-based short hairpin RNA were further used to clarify their roles. RESULTS: A high dose of propofol caused phagocytic inhibition and apoptosis in vitro for 24 h (25 µg/ml, in triplicate) and in vivo for 6 h (10 mg/kg/h, n = 5 for each group). Propofol induced lysosomal membrane permeabilization and MTP loss while stabilizing MTP and inhibiting caspase protected cells from mitochondrial apoptosis. Lysosomal cathepsin B was required for propofol-induced lysosomal membrane permeabilization, MTP loss, and apoptosis. Propofol decreased antiapoptotic Bcl-2 family proteins and then caused proapoptotic Bcl-2-associated X protein (Bax) activation. Propofol-activated GSK-3ß and inhibiting GSK-3ß prevented Mcl-1 destabilization, MTP loss, and lysosomal/mitochondrial apoptosis. Forced expression of Mcl-1 prevented the apoptotic effects of propofol. Decreased Akt was important for GSK-3ß activation caused by propofol. CONCLUSIONS: These results suggest an essential role of GSK-3ß in propofol-induced lysosomal/mitochondrial apoptosis.

A Proof-of-Concept Inhibitor of Endothelial Lipase Suppresses Triple-Negative Breast Cancer Cells by Hijacking the Mitochondrial Function.

Triple-negative breast cancer (TNBC) cells reprogram their metabolism to provide metabolic flexibility for tumor cell growth and survival in the tumor microenvironment. While our previous findings indicated that endothelial lipase (EL/LIPG) is a hallmark of TNBC, the precise mechanism through which LIPG instigates TNBC metabolism remains undefined. Here, we report that the expression of LIPG is associated with long non-coding RNA DANCR and positively correlates with gene signatures of mitochondrial metabolism-oxidative phosphorylation (OXPHOS). DANCR binds to LIPG, enabling tumor cells to maintain LIPG protein stability and OXPHOS. As one mechanism of LIPG in the regulation of tumor cell oxidative metabolism, LIPG mediates histone deacetylase 6 (HDAC6) and histone acetylation, which contribute to changes in IL-6 and fatty acid synthesis gene expression. Finally, aided by a relaxed docking approach, we discovered a new LIPG inhibitor, cynaroside, that effectively suppressed the enzyme activity and DANCR in TNBC cells. Treatment with cynaroside inhibited the OXPHOS phenotype of TNBC cells, which severely impaired tumor formation. Taken together, our study provides mechanistic insights into the LIPG modulation of mitochondrial metabolism in TNBC and a proof-of-concept that targeting LIPG is a promising new therapeutic strategy for the treatment of TNBC.

Autophagy facilitates IFN-gamma-induced Jak2-STAT1 activation and cellular inflammation.

Autophagy is regulated for IFN-gamma-mediated antimicrobial efficacy; however, its molecular effects for IFN-gamma signaling are largely unknown. Here, we show that autophagy facilitates IFN-gamma-activated Jak2-STAT1. IFN-gamma induces autophagy in wild-type but not in autophagy protein 5 (Atg5(-/-))-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-gamma induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5(-/-) or Atg7(-/-) MEFs are, independent of changes in IFN-gamma receptor expression, resistant to IFN-gamma-activated Jak2-STAT1, which suggests that autophagy is important for IFN-gamma signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-gamma-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-gamma-induced activation of STAT1 in Atg5(-/-) MEFs. Our study provides evidence that there is a link between autophagy and both IFN-gamma signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.

Increased galectin-3 facilitates leukemia cell survival from apoptotic stimuli.

Galectin-3 is regulated for cancer cell survival and apoptosis depending upon the cell type and stimulus. We investigated a glycogen synthase kinase (GSK)-3ß/galectin-3-regulated mechanism used by leukemia cells to escape from apoptotic stimuli. Galectin-3 expression was time- and transcription-dependently deregulated in K562 chronic myeloid leukemia cells stimulated for apoptosis by cisplatin (a platinum-based chemotherapy drug), sphingolipid ceramide analog C(2)-ceramide, and LY294002 (a phosphatidylinositol 3-kinase inhibitor). Notably, galectin-3 was upregulated in survival cells. Forced galectin-3 expression caused resistance to apoptosis, whereas knockdown galectin-3 expression increased susceptibility to apoptosis. Sub-cellular distribution of inducible galectin-3 was mitochondria-specific. Apoptotic stimuli decreased pro-survival Bcl-2 family protein expression (especially Mcl-1), whereas galectin-3 overexpression reversed but it was enhanced by a galectin-3 expression knockdown. Under apoptotic stimulation, GSK-3ß was activated after Akt was inactivated and GSK-3ß was inhibited-either pharmacologically or using short hairpin RNA to abolish galectin-3, increase apoptosis, and inhibit colony formation-which suggests a pro-survival role for GSK-3ß. We found that GSK-3ß upregulated galectin-3 and stabilized anti-apoptotic Bcl-2 family proteins, which is important for the escape of leukemia cells from apoptotic stimuli.

Glycogen synthase kinase-3beta facilitates IFN-gamma-induced STAT1 activation by regulating Src homology-2 domain-containing phosphatase 2.

Glycogen synthase kinase-3beta (GSK-3beta)-modulated IFN-gamma-induced inflammation has been reported; however, the mechanism that activates GSK-3beta and the effects of activation remain unclear. Inhibiting GSK-3beta decreased IFN-gamma-induced inflammation. IFN-gamma treatment rapidly activated GSK-3beta via neutral sphingomyelinase- and okadaic acid-sensitive phosphatase-regulated dephosphorylation at Ser(9), and proline-rich tyrosine kinase 2 (Pyk2)-regulated phosphorylation at Tyr(216). Pyk2 was activated through phosphatidylcholine-specific phospholipase C (PC-PLC)-, protein kinase C (PKC)-, and Src-regulated pathways. The activation of PC-PLC, Pyk2, and GSK-3beta was potentially regulated by IFN-gamma receptor 2-associated Jak2, but it was independent of IFN-gamma receptor 1. Furthermore, Jak2/PC-PLC/PKC/cytosolic phospholipase A(2) positively regulated neutral sphingomyelinase. Inhibiting GSK-3beta activated Src homology-2 domain-containing phosphatase 2 (SHP2), thereby preventing STAT1 activation in the late stage of IFN-gamma stimulation. All these results showed that activated GSK-3beta synergistically affected IFN-gamma-induced STAT1 activation by inhibiting SHP2.

Pharmacologically Inhibiting Glycogen Synthase Kinase-3ß Ameliorates Renal Inflammation and Nephrotoxicity in an Animal Model of Cisplatin-Induced Acute Kidney Injury.

The adverse effect of cisplatin administration causes acute kidney injury (AKI) following renal inflammation and nephrotoxicity, characterized by proximal tubular cell apoptosis and necrosis. Pro-apoptotic and pro-inflammatory roles of glycogen synthase kinase (GSK)-3ß have been reported. This study investigated the therapeutic blockade of GSK-3ß in cisplatin-induced AKI. A renal cisplatin nephrotoxicity model showed activation of GSK-3ß in vivo, particularly in proximal tubular epithelial cells. Pharmacologically inhibiting GSK-3ß abolished cisplatin nephrotoxicity, including proximal tubular injury, cell cytotoxicity, and biochemical dysfunction. Additionally, GSK-3ß inhibitor treatment ameliorated renal inflammation by reducing immune cell infiltration, cell adhesion molecule expression, and pro-inflammatory cytokine/chemokine production. Cisplatin treatment caused GSK-3ß activation in vitro in the human renal proximal tubular epithelial cell line HK-2, whereas either pharmacological administration of GSK-3ß inhibitors or genetic transduction of GSK-3ß short-hairpin RNA impeded cisplatin-induced cytotoxicity. These results indicate that cisplatin activates GSK-3ß followed by GSK-3ß-mediated renal inflammation and nephrotoxicity, contributing to AKI.

Glycogen synthase kinase-3ß indirectly facilitates interferon-γ-induced nuclear factor-κB activation and nitric oxide biosynthesis.

Either glycogen synthase kinase (GSK)-3ß or nuclear factor (NF)-κB regulates interferon (IFN)-γ-induced nitric oxide (NO) biosynthesis; however, the inter-regulation between GSK-3ß and NF-κB is unknown. We have previously shown that IFN-γ-activated GSK-3ß negatively regulates Src homology-2 domain-containing phosphatase (SHP) 2 to facilitate Janus kinase (Jak) 2-signal transducer and activator of transcription (STAT) 1 activation. Because Jaks-IFN-inducible dsRNA-activated serine-threonine protein kinase (PKR) axis signaling is essential for IFN-γ-activation of NF-κB, in this study we investigate the potential mechanism for GSK-3ß-facilitated NF-κB activation in IFN-γ-stimulated RAW264.7 murine macrophages. Pharmacological inhibitors of GSK-3ß or NF-κB signaling, such as the inhibitor of κB (IκB) kinase ß (IKKß) and IκBα, inhibited IFN-γ-induced inducible NO synthase (iNOS) and thus NO biosynthesis. Inhibiting GSK-3ß decreased IFN-γ-induced NF-κB phosphorylation (Ser536) and activation. The upstream regulators for GSK-3ß activation, including okadaic acid-sensitive protein phosphatase and proline-rich tyrosine kinase 2, were also important for IFN-γ-induced IκBα phosphorylation (Ser32) and degradation. Under IFN-γ stimulation, Jak2-PKR axis signaling induced IκBα inactivation as well as iNOS/NO biosynthesis. It is notable that inhibiting GSK-3ß caused SHP2-mediated dephosphorylation of PKR (Thr446), IKKß (Ser180), and NF-κB (Ser536). Taken together, we provide the first evidence to demonstrate that GSK-3ß indirectly facilitates IFN-γ-induced NF-κB activation by inhibiting SHP2, in turn activating the PKR-IKKß-IκBα axis signaling pathway.