Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

Novel N-aryl-7-methoxybenzo[b]furo[3,2-d]pyrimidin-4-amines (1) and their N-arylbenzo[b]thieno[3,2-d]pyrimidin-4-amine analogues (2) were designed and prepared for the first time via microwave-accelerated multi-step synthesis. Various anilines were condensed with N'-(2-cyanaryl)-N,N-dimethylformimidamide intermediates obtained by reaction of 3-amino-6-methoxybenzofuran-2-carbonitrile (3) and 3-amino-6-methoxybenzothiophene-2-carbonitrile (4) precursors with dimethylformamide dimethylacetal. The inhibitory potency of the final products against five protein kinases (CDK5/p25, CK1δ/ε, GSK3α/β, DYRK1A and CLK1) was estimated. Compounds (2a-z) turned out to be particularly promising for the development of new pharmacological dual inhibitors of CLK1 and DYRK1A kinases.

We found that the coagulation and cytokine pathways were important mechanisms involve in the degeneration of intervertebral discs (IVD) using a microarray approach to analyze gene expression in different grades of specimens. Furthermore, using a cytokine/chemokine array, a significant increase in CXCL8 expression was observed in human nucleus pulposus (NP) cells after thrombin treatment. The enhancement of CXCL8 expression by thrombin was activated by the PAR1 receptor. Importantly, analysis of degenerated human NP tissue samples showed that EGFR expression positively correlated with the grade of tissue degeneration. In NP cells, thrombin caused an increase in phosphorylation of the EGFR at the Tyr1068, and treatment with the pharmacological EGFR inhibitor, AG1473 effectively blocked thrombin-enhanced CXCL8 production. Surprisingly, inhibition of STAT3 for 24 h decreased expression of EGFR. Treatment with thrombin also increased Akt and GSK3α/β activation; this activation was also blocked by EGFR inhibitor. Although c-Src, ERK, and FAK were activated by thrombin, only c-Src and ERK were involved in the STAT3/CXCL8 induction. Our findings indicate that stimulation of an inflammatory response in NP cells by thrombin is part of a specific pathophysiology that modulates the EGFR activation through activation of Src/ERK/STAT3 signaling.

BACKGROUND

Bipolar disorder (BD) is a severe and recurrent psychiatric disorder. The severity of prognosis in BD is mainly linked to the high rate of suicide in this population. Indeed, patients with BD commit suicide 20 to 30 times more frequently than the general population, and half of the BD population with an early age of onset have a history of suicide attempt. International therapeutic guidelines recommend lithium (Li) as the first-line treatment in BD for its prophylactic action on depressive or manic episodes. In addition, Li is the only mood stabilizer that has demonstrated efficacy in suicide prevention. This effect of Li is unfortunately often unknown to psychiatrists. Thus, this review aims to highlight evidence about the preventive action of Li on suicide in BD populations.

METHODS

We conducted a literature search between April 1968 and August 2014 in PubMed database using the following terms: "lithium" AND "suicide" OR "suicidality" OR "suicide attempt".

RESULTS

As confirmed by a recent meta-analysis, many studies show that Li has a significant effect on the reduction of suicide attempts and deaths by suicide in comparison to antidepressants or other mood-stabilisers in BD populations. Studies have demonstrated that long-term treatment with Li reduces suicide attempts by about 10% and deaths by suicide by about 20%. The combination of Li and an antidepressant could reduce suicidal behaviours by reducing suicidal ideation prior to depressive symptoms. It appears crucial for Li efficacy in suicide prevention to maintain the Li blood concentrations in the efficient therapeutic zone and to instate long-term Li treatment. The "impulsive-aggressive" endophenotype is associated with suicide in BD. The specific action of Li on the 5-HT serotoninergic system could explain the specific anti-suicidal effects of Li via the modulation of impulsiveness and aggressiveness. Furthermore, genetic variants of the glycogen synthase kinase 3α/β (GSK3α and β; proteins inhibited by Li) seem to be associated with more impulsiveness in BD populations.

CONCLUSIONS

The anti-suicidal effect of Li has been very well demonstrated. By its specific action on the serotoninergic system, treatment with Li significantly reduces "impulsive-aggressive" behaviour which is a vulnerability factor common to suicide and BD. Long-term appropriately modulated treatment with Li seems to have considerable impact on the reduction of suicidal behaviours, suicidal ideation and death by suicide in the BD population.

Proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the development of vascular restenosis. Our previous study showed that CCN4, namely Wnt1 inducible signaling pathway protein 1 (WISP1), significantly promotes proliferation and migration of rat VSMCs, but its mechanism remains unclear. This study aims to investigate whether and how WISP1 stimulates proliferation and migration of human VSMCs. Western blot analysis showed that FBS treatment increased WISP1 protein levels in human VSMCs in a dose-dependent manner. Overexpression of WISP1 using adenovirus encoding WISP1 (AD-WISP1) significantly increased proliferation rate of human VSMCs by 2.98-fold compared with empty virus (EV)-transfected cells, shown by EdU incorporation assay. Additionally, Scratch-induced wound healing assay revealed that adenovirus-mediated overexpression of WISP1 significantly increased cell migration compared with EV-transfected cells from 6h (4.56±1.14% vs. 11.23±2.25%, P<0.05) to 48h (25.25±5.51% vs. 97.54±13.12%, P<0.01) after injury. Transwell Migration Assay confirmed that WISP1 overexpression significantly promoted human VSMC migration by 2.25-fold compared with EV. Furthermore, WISP1 overexpression stimulated Akt signaling activation in human VSMCs. Blockage of Akt signaling by Akt inhibitor AZD5363 or PI3K inhibitor LY294002, led to an inhibitory effect of WISP1-induced proliferation and migration in human VSMCs. Moreover, we found that WISP1 overexpression stimulated GSK3α/β phosphorylation, and increased expression of cyclin D1 and MMP9 in human VSMCs, and this effect was abolished by AZD5363. Collectively, we demonstrated that Akt signaling pathway mediates WISP1-induced migration and proliferation of human VSMCs, suggesting that WISP1 may act as a novel potential therapeutic target for vascular restenosis.

Transient receptor potential melastatin type 2 (TRPM2) is a cation channel activated by free intracellular ADP-ribose and reactive oxygen species. TRPM2 signaling has been linked to the pathophysiology of CNS disorders such as neuropathic pain, bipolar disorder and Alzheimer's disease. In this manuscript, we describe the discovery of JNJ-28583113, a potent brain penetrant TRPM2 antagonist. Ca²⁺ flux assays in cells overexpressing TRPM2 and electrophysiological recordings were used to test the pharmacology of JNJ-28583113. JNJ-28583113 was assayed in vitro on GSK-3 phosphorylation levels, cell death, cytokine release in microglia and unbiased morphological phenotypic analysis. Finally, we dosed animals to evaluate its pharmacokinetic properties. Our results showed that JNJ-28583113 is a potent (126 ± 0.5 nM) TRPM2 antagonist. Blocking TRPM2 caused phosphorylation of GSK3α and β subunits. JNJ-28583113 also protected cells from oxidative stress induced cell death as well as morphological changes induced by non-cytotoxic concentrations of H₂O₂. In addition, inhibiting TRPM2 blunted cytokine release in response to pro-inflammatory stimuli in microglia. Lastly, we showed that JNJ-28583113 was brain penetrant but not suitable for systemic dosing as it was rapidly metabolized in vivo. While the in-vitro pharmacology of JNJ-28583113 is the best in class, its in-vivo properties would need optimization to assist in further probing key roles of TRPM2 in CNS pathophysiology.

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

Glycogen Synthase Kinase 3 (GSK3) is one of the Serine/Threonine protein kinases that has gained a lot of attention for its role in a variety of pathways. It has two isoforms, GSK3α and GSK3β. However, GSK3β is highly expressed in different areas of the brain and has been implicated in Alzheimer's disease as it is involved in tau phosphorylation. Due to its high specificity concerning substrate recognition, GSK3 has been considered as an important target. In the last decade, several GSK3 inhibitors have been reported and two molecules are in clinical trials. This review collates the information published in the last decade about the role of GSK3 in Alzheimer's disease and progress in the development of its inhibitors. Using this collated information, medicinal chemists can strategize and design novel GSK3 inhibitors that could be useful in the treatment of Alzheimer's disease.

BACKGROUND

The C-type lectin-like receptor 2 (CLEC-2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen-activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC-2 signaling is not known.

OBJECTIVE

We sought to investigate the role of the Akt and MAPK pathways in platelet activation by CLEC-2.

RESULTS

The CLEC-2 agonist rhodocytin stimulated phosphorylation of Akt and p38 and extracellular signal-related kinase (ERK) MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A2 in CLEC-2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31-8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/β (GSK3α/β). Phosphorylation of GSK3α/β was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/β inhibitors.

CONCLUSIONS

These results demonstrate that CLEC-2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/β, and enhanced platelet aggregation and secretion.

Sepsis is one of the leading causes of mortality in intensive care units besides causing profound alterations in the brain. One of the structures notably affected during sepsis is the hypothalamus, resulting in important physiopathological consequences. Recently, we provided evidence that the presence of neuroinflammation, oxidative stress, and apoptosis in the hypothalamus of septic rats, is accompanied by impairment of arginine vasopressin (AVP) secretion. We had also demonstrated that sepsis survivor animals present attenuated AVP secretion after osmotic challenge, suggesting a persistent inflammation in the hypothalamus. However, the long-term course of inflammation in the hypothalamus remains unclear. Thus, we induced sepsis by cecal ligation and puncture (CLP) in Wistar rats and, five days after sepsis induction, the hypothalamus of each animal was collected for analysis. Nonmanipulated animals (naive) were used as controls. We found that CLP-induced morphological alterations in microglial cells are accompanied by an increase in Iba-1 immunoreactivity. Moreover, we observed enhanced expression of NF-κB and CREB transcription factors, which are well known to modulate the immune response. Additionally, we found that phosphorylation of GSK3α/β (a kinase upstream to the CREB signaling pathway) was increased, as well as COX-2, iNOS, and IL-6 that are canonic inflammatory proteins. Thus, our results indicated the presence of sustained activation of resident glial cells that may result in neuroinflammation and cholinergic neurotransmission disruptions in the hypothalamus.

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.

Glycogen synthase kinase 3 (GSK3) has been linked to the mechanisms of stress, mood regulation, and the effects of antidepressants. The functions of the GSK3β isoform have been extensively investigated, but little is known about the α-isoform, although they may functionally related. In a recently established modified swim test with a third delayed swim exposure, brain GSK3β mRNA expression positively correlated with floating behaviour on the third test. A two-week-long pretreatment regime with imipramine (7.5mg/kg/day) or thiamine (200mg/kg/day), which is known to have antidepressant properties, reduced the GSK3β over-expression and decreased floating behaviour on Day 5. GSK3α mRNA levels were measured in the hippocampus and prefrontal cortex on Days 1, 2 and 5. GSK3α expression was decreased in the prefrontal cortex on Day 2 and increased on Day 5. In this model, GSK3α mRNA changes were prevented by imipramine or thiamine treatment. There was a significant correlation between the expression of the two isoforms in the prefrontal cortex on Day 2 in untreated group. These results provide the first evidence for the potential involvement of GSK3α in depressive-like behaviours and as a target of anti-depressant therapy. Furthermore, the correlations suggest some cross-talk may exist between the two GSK3 isoforms.

Recent advances in genomics, proteomics, cell biology and biochemistry of tumors have revealed new pathways that are aberrantly activated in numerous cancer types. However, the enormous amount of data available in this field may mislead scientists in focused research. As cancer cell growth and progression is often dependent upon the phosphoinositide 3-kinase (PI3K)/AKT pathway, there has been extensive research into the proteins implicated in the PI3K pathway. Using data available in the Human Protein Atlas database, the current study investigated the expression of 25 key proteins that are known to be involved with PI3K pathway activation in a distinct group of 20 cancer types. These proteins are AKTIP, ARP1, BAD, GSK3A, GSK3B, MERTK-1, PIK3CA, PRR5, PSTPIP2, PTEN, FOX1, RHEB, RPS6KB1, TSC1, TP53, BCL2, CCND1, WFIKKN2, CREBBP, caspase-9, PTK2, EGFR, FAS, CDKN1A and XIAP. The analysis revealed pronounced expression of specific proteins in distinct cancer tissues, which may have the potential to serve as targets for treatments and provide insights into the molecular basis of cancer.

OBJECTIVE

Though CCR4-NOT2 (CNOT2), one of CCR4-NOT complex subunits, was known to be involved in metastasis and apoptosis through transcription and mRNA degradation, its other biological function is poorly understood so far. The aim of this study is to elucidate the molecular role of CNOT2 in the differentiation process of 3T3-L1 preadipocytes.

RESULTS

CNOT2 was overexpressed during the differentiation process of 3T3-L1 preadipocytes. Consistently, mRNA levels of CNOT2, adiponectin, adiponectin 2, PPARx03B3; and CEBPα were enhanced in 3T3-L1 adipocytes. Conversely, CNOT2 depletion by siRNA transfection also reversed the activation of PPARx03B3; and CEBPα and inhibition of GSK3α/β and β-catenin at the protein level in 3T3-L1 preadipocytes. Immunofluorescence assay revealed that CNOT2 was colocalized with PPARx03B3;, but not with CEBPα in 3T3-L1 adipocyte. Consistently, IP western blots revealed that CNOT2 interacted with PPARx03B3; in 3T3-L1 adipocyte.

CONCLUSIONS

Our findings demonstrate that CNOT2 promotes the differentiation of 3T3-L1 preadipocytes via upregulation of PPARx03B3;, and CEBPα and inhibition of GSK3α/β and β-catenin signaling as a potent molecular target for obesity.

IKBKE (IKKε) has emerged as a key modulator of multiple substrates, controlling oncogenic pathways in various malignancies. mTOR signaling, required for cellular growth, proliferation, and vascular angiogenesis in cancer, is potentially one of the pathways regulated by IKKε. Upon activation by various stimuli, PI3K/AKT or similar effectors can relieve the inhibitory effect of the TSC1/TSC2 complex through their phosphorylation to favor mTOR/S6K activation in the downstream. Therefore, any activity that interferes with PI3K/AKT or their downstream targets, such as TSC1/2 or GSK3α/β, may activate the mTOR/S6K pathway for oncogenic transformation in normal cells. Previous studies have shown that PI3K/AKT can be directly phosphoregulated by IKKε. Here, we propose a new regulatory function for IKKε in the mTOR/S6K pathway through its direct interaction with TSC1, leading to TSC1 phosphorylation, which is vital to suppress its inhibitory role in mTOR activation. Experimentally, upon IKKε deficiency in colorectal cancer cells, we observed that S6K activity was diminished while TSC1 levels were found to be stabilized. We hypothesized that these observations may result from direct interaction between IKKε and TSC1. Indeed, the interaction of these two proteins involves the phosphoregulation of TSC1 in various cell lines. Therefore, we propose a mechanism where IKKε, through regulating TSC1 stability in cancer cells, may create an alternative regulatory loop for the activation of mTOR signaling. These results can potentially be important for the development of novel therapeutic strategies targeting mTOR signaling.

The effects of dipeptide cyclo-[His-Pro] (CHP), known to participate in the appetite behavior and food intake control, have been investigated using PC12 cells in culture as model system. We found that only in the presence of experimental conditions that cause cellular stress the cyclic dipeptide affect cellular proliferation and protects from apoptosis. It greatly enhances the phosphorylation of hsp27, alpha-B-crystallin, Cdc2, and p-38 MAPK, whereas it decreases the phosphorylation of MEK1, Cav 2, GSK3a, PKB/Akt, PKCdelta, PKCgamma, and Erk2. PKA and PKG are involved in ERK1/2 deactivation via a receptor that appears to be dually coupled to Gs and Gq protein subfamilies.

Glycogen synthase kinase-3 (GSK3) is a constitutively active serine threonine kinase with 1) two isoforms (GSK3A and GSK3B) that have unique and overlapping functions, 2) multiple molecular intracellular mechanisms that involve phosphorylation of diverse substrates, and 3) implications in pathogenesis of many diseases. Insulin causes phosphorylation and inactivation of GSK3 and mammalian oocytes have a functional insulin-signaling pathway whereby prolonged elevated insulin during follicle/oocyte development causes GSK3 hyperphosphorylation, reduced GSK3 activity, and altered oocyte chromatin remodeling. Periconceptional diabetes and chronic hyperinsulinemia are associated with congenital malformations and onset of adult diseases of cardiovascular origin. Objectives were to produce transgenic mice with individual or concomitant loss of GSK3A and/or GSK3B and investigate the in vivo role of oocyte GSK3 on fertility, fetal development, and offspring health. Wild-type males bred to females with individual or concomitant loss of oocyte GSK3 isoforms did not have reduced fertility. However, concomitant loss of GSK3A and GSK3B in the oocyte significantly increased neonatal death rate due to congestive heart failure secondary to ventricular hyperplasia. Individual loss of oocyte GSK3A or GSK3B did not induce this lethal phenotype. In conclusion, absence of oocyte GSK3 in the periconceptional period does not alter fertility yet causes offspring cardiac hyperplasia, cardiovascular defects, and significant neonatal death. These results support a developmental mechanism by which periconceptional hyperinsulinemia associated with maternal metabolic syndrome, obesity, and/or diabetes can act on the oocyte and affect offspring cardiovascular development, function, and congenital heart malformation.

Repeated administration of cocaine induces heightened behavioral hyperactivity termed sensitization. Although NK-3 receptors have been shown to modulate acute cocaine-induced behaviors, their role in behavioral sensitization is unknown. The present study investigated whether NK-3 receptor blockade altered behavioral sensitization to cocaine. Additionally, glycogen synthase kinase-3 (GSK3) has been shown to be involved in dopamine receptor signaling and in development of sensitization; therefore regulation of GSK3 activity in the nucleus accumbens was also investigated. Administration of the NK-3 receptor antagonist SB 222200 (5 mg/kg, s.c.) prior to repeated cocaine (20 mg/kg, i.p.) prevented the development of sensitized responses after a cocaine challenge. Pre-treatment with SB 222200 before a cocaine challenge also blocked expression of sensitization. Decrease in GSK3 activity demonstrated by increased phosphorylation of GSK3α and GSK3β was detected 20 mins after an acute cocaine injection. In contrast, a cocaine challenge failed to alter phosphorylation of GSK3α and GSK3β in sensitized mice. SB 222200 prior to repeated cocaine resulted in increased phosphorylation of GSK3α and GSK3β akin to changes following acute cocaine. Collectively, these findings demonstrate the involvement of NK-3 receptors in development and expression of behavioral sensitization and in regulation of GSK3 activity in the nucleus accumbens after repeated cocaine.

Glycogen synthase kinase 3α/β (GSK3α/β) is a serine/threonine kinase that participates in numerous processes in many cell types. Importantly, the role of GSK3α/β in homeostatic maintenance of the pulmonary endothelial cell barrier to protein is not known. We tested the hypothesis that GSK3α/β regulates endothelial barrier function by measuring the permeability to albumin of a rat pulmonary microvessel endothelial cell monolayer (PMECM) treated with and without the selective GSK3α/β inhibitor SB 216763 (1.0, 5.0 and 10 uM) for 1 h. The treatment with the inhibitor SB 216763 caused a dose dependent decrease in phospho-β-catenin-Ser(33/37) levels indicating effective suppression of GSK3α/β. SB216763 caused an increase in both permeability to albumin and DCFDA (6-Carboxy-2',7'-Dichlorodihydrofluorescein Diacetate, Di(Acetoxymethyl Ester)) oxidation that were prevented by co-treatment with the anti-oxidant tiron or the nitric oxide synthase inhibitor L-NAME (Nω-nitro-l-arginine-methyl ester). In separate studies PMECMs were treated with the Akt inhibitor triciribine (12.5 uM) for 1 h to unmask Akt dependent constitutive suppression of GSK3α/β. Triciribine decreased phospho-GSK3α/β-Ser(21)/9 (i.e., the product of Akt) which was associated with an increase in phospho-β-catenin-Ser(33/37) (i.e., the product of GSK3α/β) indicating constitutive activity of Akt for GSK3α/β-Ser(21/9). The data indicates GSK3α/β inhibition causes increased endothelial monolayer protein permeability which is mediated by reactive oxygen/nitrogen species.

Homozygous nonsense mutations in WNT2B were identified in three individuals from two unrelated families with severe, neonatal-onset osmotic diarrhea after whole-exome sequencing was performed on trios from the two families. Intestinal biopsy samples from affected individuals were used for histology and immunofluorescence and to generate enteroids ex vivo. Histopathologic evaluation demonstrated chronic inflammatory changes in the stomach, duodenum, and colon. Immunofluorescence demonstrated diminished staining for OLFM4, a marker for intestinal stem cells (ISCs). The enteroids generated from WNT2B-deficient intestinal epithelium could not be expanded and did not survive passage. Addition of CHIR-99021 (a GSK3A and GSK3B inhibitor and activator of canonical WNT/β-CATENIN signaling) could not rescue WNT2B-deficient enteroids. Addition of supplemental recombinant murine WNT2B was able to perpetuate small enteroids for multiple passages but failed to expand their number. Enteroids showed a 10-fold increase in the expression of LEF1 mRNA and a 100-fold reduction in TLR4 expression, compared with controls by quantitative RT-PCR, indicating alterations in canonical WNT and microbial pattern-recognition signaling. In summary, individuals with homozygous nonsense mutations in WNT2B demonstrate severe intestinal dysregulation associated with decreased ISC number and function, likely explaining their diarrheal phenotype. WNT2B deficiency should be considered for individuals with neonatal-onset diarrhea.

Glycogen synthase kinases 3 (GSK3) α and β are expressed in the nervous system, and disruption of GSK3 signaling has been implicated in a wide range of neurodevelopmental and psychiatric disorders. Although several studies have established a role of GSK3 signaling in the nervous system, much less is known about isoform-specific functions. Here, we have examined the role of GSK3α and GSK3β in the developing neocortex by performing in utero electroporation with specific small interfering RNAs targeting each isoform. We found that depletion of either GSK3α or GSK3β commonly promoted the proliferation of neural progenitor cells in the ventricular zone, but at later stages, knocking down of each isoform resulted in distinct outcomes. In particular, the transformation of radial progenitors to intermediate progenitor cells was promoted in GSK3α-depleted cells, but markedly prevented in GSK3β-depleted cells. Moreover, knocking down of GSK3β but not GSK3α prevented the generation of upper-layer Cux1+ neurons. Consistent with the distinct outcomes, protein levels of c-Myc and β-catenin, well-known substrates of GSK3, were differentially affected by depletion of GSK3α and GSK3β. Together, these results suggest that GSK3α and GSK3β might play distinct roles in the genesis and differentiation of neuronal lineage cells during neocortex development by differential regulation of downstream signaling pathways.

Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix.