The efficient synthesis of 7-substituted pyrido[2',3':4,5]furo[3,2-d]pyrimidin-4-amines and their N-aryl analogues is described. 3,5-Dibromopyridine was converted into 3-amino-6-bromofuro[3,2-b]pyridine-2-carbonitrile intermediate which was formylated with DMFDMA. Functionalization at position 7 of the tricyclic scaffold was accomplished, before or after cyclisation step, by palladium-catalyzed Suzuki-Miyaura cross-coupling while the pyrimidin-4-amines and N-aryl counterparts were synthesized by microwave-assisted formamide degradation and Dimroth rearrangement, respectively. The final products were evaluated for their potent inhibition of a series of five Ser/Thr kinases (CDK5/p25, CK1δ/ε, CLK1, DYRK1A, GSK3α/β). Compound 35 showed the best inhibitory activity with an IC50 value of 49 nM and proved to be specific to CLK1 among the panel of tested kinases.

BACKGROUND

Cancer stem cells (CSCs) are responsible for multi-drug resistance in tumors. CD133 is a known biomarker of CSCs. The aim of this study is to screen for drug-resistant differentially expressed genes in CD133+ and CD133- lung cancer cells and to identify novel lung tumor drug-resistant genes.

METHODS

Magnetic activated cell sorting was used to isolate CD133+ and CD133- cells from human lung cancer cell line A549, and drug-resistant microarray was used to detect drug-resistant genes in the these cells. RT-qPCR was used to examine the expression of six lung tumor drug-resistant genes in pre- and post-chemotherapeutic A549 cells.

RESULTS

A total of 31 differentially expressed genes were screened by microarray analysis. Of these genes, 30 were upregulated and one was downregulated in CD133+ cells compared with CD133- cells. Results were verified by RT-qPCR. CYP2C19, CYP2D6, CYP2E1, GSK3α, PPARα, and PPARβ/δ were significantly upregulated after the A549 cells were treated with 1.97 μg/mL DDP or 0.61 μg/mL doxorubicin for 48 h.

CONCLUSIONS

The drug resistance of lung adenosarcoma may be correlated with 31 differentially expressed genes screened by drug-resistant microarray. CYP2C19, CYP2D6, CYP2E1, GSK3α, PPARα, and PPARβ/δ might be novel lung adenosarcoma drug-resistant genes.

Promotion of insulin-secreting β-cell regeneration in patients with diabetes is a promising approach for diabetes therapy, which can contribute to rescue the uncontrolled hyperglycemia. Low-power laser irradiation (LPLI) has been demonstrated to regulate multiple physiological processes both in vitro and in vivo through activation of various signaling pathways. In the present study, we showed that LPLI promoted β-cell replication and cell cycle progression through activation of Akt1/GSK3β isoform-specific signaling axis. Inhibition of PI3-K/Akt or GSK3 with specific inhibitors dramatically reduced or increased LPLI-induced β-cell replication, revealing Akt/GSK3 signaling axis was involved in β-cell replication and survival upon LPLI treatment. Furthermore, the results of shRNA-mediated knock down of Akt/GSK3 isoforms revealed that Akt1/GSK3β isoform-specific signaling axis regulated β-cell replication and survival in response to LPLI, but not Akt2/GSK3α. The mechanism by which LPLI promoted β-cell replication through Akt1/GSK3β signaling axis involved activation of β-catenin and down-regulation of p21. Taken together, these observations suggest that Akt1/GSK3β isoform signaling axis play a key role in β-cell replication and survival induced by LPLI. Moreover, our findings suggest that activation of Akt1/GSK3β isoform signaling axis by LPLI may provide guidance in practical applications for β-cell regenerative therapies.

A useful and rapid access to libraries of N-arylbenzo[b]thieno[3,2-d]pyrimidin-4-amines and their pyrido and pyrazino analogues was designed and optimized for the first time via microwave-accelerated condensation and Dimroth rearrangement of the starting anilines with N'-(2-cyanoaryl)-N,N-dimethylformimidamides obtained by reaction of thiophene precursors with dimethylformamide dimethylacetal. The inhibitory potency of the final products against five protein kinases (CDK5/p25, CK1δ/ɛ, GSK3α/β, DYRK1A and CLK1) was estimated. N-arylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine series of compounds (4a-j) turned out to be particularly promising for the development of new pharmacological inhibitors of CK1 and CLK1 kinases.

Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca(2+)-concentration ([Ca(2+)]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3(KI) ). Factors affecting [Ca(2+)]i include Ca(2+)-release from intracellular stores (CRIS), store-operated Ca(2+)-entry (SOCE) through STIM1/STIM2-regulated Orai1, K(+)-dependent Na(+)/Ca(2+)-exchangers (NCKX), K(+)-independent Na(+)/Ca(2+)-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3(KI) mice and respective wild-type mice (gsk3(WT) ), [Ca(2+)]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1-10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3(WT) than in gsk3(KI) DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3(KI) than in gsk3(WT) DCs. Activity of NCKX and NCX was significantly higher in gsk3(KI) than in gsk3(WT) DCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3(WT) DCs with SB216763 (1 µM, 4-24 h) or GSK-XIII (10 µM, 4-24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca(2+) in DCs. Acute inhibition of GSK3 blunted the increase of [Ca(2+)]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca(2+)]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na(+)/Ca(2+)-exchanger activity and calbindin D28k expression.

The multifaceted glycogen synthase kinase (GSK3) has an essential role in sperm and male fertility. Since cyclic AMP (cAMP) plays an important role in sperm function, we investigated whether GSK3 and cAMP pathways may be interrelated. We used GSK3 and soluble adenylyl cyclase (sAC) knockout mice and pharmacological modulators to examine this relationship. Intracellular cAMP levels were found to be significantly lower in sperm lacking GSK3α or GSK3β. A similar outcome was observed when sperm cells were treated with SB216763, a GSK3 inhibitor. This reduction of cAMP levels was not due to an effect on sperm adenylyl cyclase but was caused by elevated phosphodiesterase (PDE) activity. The PDE4 inhibitor RS25344 or the general PDE inhibitor IBMX could restore cAMP levels in sperm lacking GSK3α or β-isoform. PDE activity assay also showed that hyperactivated PDE4 contributes in lowering of cAMP levels in GSK3α null sperm suggesting that in wild-type sperm PDE4 activity is kept in check by GSK3. Conversely, PKA being triggered by cAMP, affected GSK3 activity through increasing its phosphorylation. Increased GSK3 phosphorylation also occurred by inhibition of sperm specific protein phosphatase type 1, PP1γ2. The relationship between cAMP, GSK3, and PP1γ2 activities was also confirmed in sperm from sAC null mice. Pull-down assay using recombinant PP1γ2 indicated that PKA, GSK3, and PP1γ2 could exist as a complex. Pharmacological inhibition of GSK3 in mature spermatozoa resulted in significantly reduced fertilization of eggs in vitro. Our results show that cAMP, PKA, and GSK3 are interrelated in regulation of sperm function.

Clinical studies have revealed that testosterone supplementation had a positive effect on glucose homeostasis in type 2 diabetes mellitus (T2DM), but did not address how testosterone supplementation affected insulin responsiveness in the liver, a key glucose homeostatic organ. In this study, we aimed to study the effect of testosterone supplementation on hepatic insulin responsiveness and glucose homeostasis through liver in male high-fat diet-induced T2DM mice. Testosterone treatment to T2DM animals showed reduced hepatic glucose output. Testosterone inhibited the insulin signaling in liver, thus increased insulin resistance. However, testosterone treatment inactivated GSK3α independent of PI3K/AKT pathway and inhibited FOXO1 By interaction of androgen receptor to FOXO1 and downregulated PEPCK, causing repression of gluconeogenic pathway, which is otherwise upregulated in T2DM, resulted in better glucose homeostasis.

Hampshire pigs carrying the PRKAG3 mutation in the AMP-activated protein kinase (AMPK) γ3 subunit exhibit excessive skeletal muscle glycogen storage and an altered glycogen synthesis signalling response following exercise. AMPK plays an important role as a regulator of carbohydrate and fat metabolism in mammalian cells. Exercise-trained muscles are repeatedly exposed to glycogen degradation and resynthesis, to which the signalling pathways adapt. The aim of this study was to examine the effect of acute exercise on glycogen synthesis signalling pathways, and the levels of insulin and other substrates in blood in exercise-trained pigs with and without the PRKAG3 mutation. After 5 weeks of training, pigs performed two standardized treadmill exercise tests, and skeletal muscle biopsies were obtained immediately after exercise and 3 h postexercise in the first test, and 6 h postexercise in the second test. The PRKAG3 mutation carriers had higher glycogen storage, and resynthesis of glycogen was faster after 3 h but not after 6 h of recovery. Alterations in the concentrations of insulin, glucose, lactate and free fatty acids after exercise did not differ between the genotypes. The carriers showed a lower expression of AMPK and increased phosphorylation of Akt Ser(473) after exercise, compared with non-carriers. Acute exercise stimulated the phosphorylation of AS160 in both genotypes, and the phosphorylation of GSK3α Ser(21) and ACC Ser(79) in the non-carriers. In conclusion, exercise-trained pigs carrying the PRKAG3 mutation show an altered Akt and AMPK signalling response to acute exercise, indicating that glucose metabolism is associated with faster resynthesis of muscle glycogen in this group.

The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3) is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised.

GSK3α has been identified as a new target in the treatment of acute myeloid leukemia (AML). However, most GSK3 inhibitors lack specificity for GSK3α over GSK3β and other kinases. We have previously shown in lung cancer cells that GSK3α and to a lesser extent GSK3β are inhibited by the advanced clinical candidate tivantinib (ARQ197), which was designed as a MET inhibitor. Thus, we hypothesized that tivantinib would be an effective therapy for the treatment of AML. Here, we show that tivantinib has potent anticancer activity across several AML cell lines and primary patient cells. Tivantinib strongly induced apoptosis, differentiation and G2/M cell cycle arrest and caused less undesirable stabilization of β-catenin compared to the pan-GSK3 inhibitor LiCl. Subsequent drug combination studies identified the BCL-2 inhibitor ABT-199 to synergize with tivantinib while cytarabine combination with tivantinib was antagonistic. Interestingly, the addition of ABT-199 to tivantinib completely abrogated tivantinib induced β-catenin stabilization. Tivantinib alone, or in combination with ABT-199, downregulated anti-apoptotic MCL-1 and BCL-XL levels, which likely contribute to the observed synergy. Importantly, tivantinib as single agent or in combination with ABT-199 significantly inhibited the colony forming capacity of primary patient AML bone marrow mononuclear cells. In summary, tivantinib is a novel GSK3α/β inhibitor that potently kills AML cells and tivantinib single agent or combination therapy with ABT-199 may represent attractive new therapeutic opportunities for AML.

Increased levels of proinflammatory cytokines have been implicated in schizophrenia; however, their pathophysiological roles in abnormal brain dysfunctions remain unclear. We evaluated the effect of proinflammatory cytokines on a high-fat diet (HFD)-induced prepulse inhibition (PPI) deficits in the acoustic startle response. Eight-week-old male C57BL/6 J mice were fed a HFD for 3 weeks and then PPI was examined. HFD significantly induced PPI deficits and increased plasma IL-6, but not TNFα, levels. Interestingly, MR16-1 administration during the HFD period ameliorated PPI deficits. Further, in the striatum of HFD-fed mice, phosphorylation of GSK3β, but not GSK3α, was significantly increased; this increase was attenuated by MR16-1, although the protein levels of GSK3α and β were not altered. There were no significant differences in either phosphorylation or protein levels of GSK3α, β in the PFC during the HFD period. These results suggest that increased IL-6 levels during HFD may induce sensorimotor gating deficits, likely through the alteration of striatal GSK3β phosphorylation. MR16-1 might have a beneficial effect on such HFD-induced sensorimotor gating deficits.

Alzheimer's disease (AD) is the most common cause of dementia, affecting approximately 35 million people worldwide. The current treatment options for people with AD consist of drugs designed to slow the rate of decline in memory and cognition, but these treatments are not curative, and patients eventually suffer complete cognitive injury. With the substantial amounts of published data on targets for this disease, we proposed that machine learning software could be used to find novel small-molecule treatments that can supplement the AD drugs currently on the market. In order to do this, we used publicly available data in ChEMBL to build and validate Bayesian machine learning models for AD target proteins. The first AD target that we have addressed with this method is the serine-threonine kinase glycogen synthase kinase 3 beta (GSK3β), which is a proline-directed serine-threonine kinase that phosphorylates the microtubule-stabilizing protein tau. This phosphorylation prompts tau to dissociate from the microtubule and form insoluble oligomers called paired helical filaments, which are one of the components of the neurofibrillary tangles found in AD brains. Using our Bayesian machine learning model for GSK3β consisting of 2368 molecules, this model produced a five-fold cross validation ROC of 0.905. This model was also used for virtual screening of large libraries of FDA-approved drugs and clinical candidates. Subsequent testing of selected compounds revealed a selective small-molecule inhibitor, ruboxistaurin, with activity against GSK3β (avg IC₅₀ = 97.3 nM) and GSK3α (IC₅₀ = 695.9 nM). Several other structurally diverse inhibitors were also identified. We are now applying this machine learning approach to additional AD targets to identify approved drugs or clinical trial candidates that can be repurposed as AD therapeutics. This represents a viable approach to accelerate drug discovery and do so at a fraction of the cost of traditional high throughput screening.

Melanoma is an aggressive malignant skin tumor. Study found that miR-149* was abnormally expressed in melanoma. Adenosine deaminases acting on the RNA1 (ADAR1) is an RNA editing enzyme. It can change the structure and function of miRNA. In this study, we investigate the role of ADAR1 in regulation of miRNA-149* in melanoma. Western-blot analysis was used to analyze the expression of ADAR1p150, ADAR1p110 and GSK3α at protein level. The expression of ADAR1p150, miR-149* and GSK3α at mRNA level were detected using qRT-PCR. Co-immunoprecipitation test was then performed to determine the interaction between ADAR1 and Dicer. Target verification of miRNA-149*/GSK3α was carried out using luciferase reporter assay. CCK-8 was used to detect cell proliferation. Cell apoptosis was tested using Tunel assays. The expression level of ADAR1p150 was found to be increased in human melanoma tissues, but not ADAR1p110. There was a direct interaction between ADAR1p150 and Dicer in melanoma cells. MiRNA-149* was significantly up-regulated in melanoma tissues and melanoma cells. Luciferase reporter assay suggested that GSK3α was a directly target of miR-149*. The expression level of miR-149* showed a positive correlation with ADAR1p150. At the same time, ADAR1p150 expression was negatively correlated with the expression of GSK3α. ADAR1p150 promoted proliferation of melanoma cells and inhibited cell apoptosis. ADAR1p150 can promote the biosynthesis and function of miRNA-149* in melanoma cells which makes it be considered as both a bio-marker and a therapeutic target for treatment of melanoma.

WK234, a novel bisindolymaleimide derivative, was designed as a protein kinase Cβ (PKCβ) inhibitor. The objective of this study was to evaluate the anti-tumor activity of WK234 in the human chronic myelogenous leukemia (CML) K562 cell line and to investigate possible mechanisms of its action. The results show that WK234 inhibited K562 cell proliferation in a time- and dose-dependent manner. WK234 increased cytochrome C release and caspase-3 cleavage, which indicates that it induced apoptosis via mitochondria- and caspase-mediated pathways. Western blotting showed that PKCβ1, PKCβ2, and their phosphorylation levels were effectively decreased after 2-4 h of WK234 treatment. Meanwhile the phosphorylation status of PKCβ downstream proteins, glycogen synthase kinase 3α/β (GSK3α/β) and extracellular signal-regulated kinase (ERK), were inhibited. WK234 blocked phorbol myristate acetate (PMA)-induced Ser(660) phosphorylation of PKCβ2 located at the cell membrane, and increased Ser(660) PKCβ2 expression within the cytoplasm and the nucleus. These results indicate that WK234 inhibited cell proliferation and induced apoptosis through suppressing the PKCβ signal pathway. WK234 might be a promising candidate for the treatment of CML.

Phosphatidylinositol 3 kinase (PI3K) is a major player in platelet activation and regulates thrombus formation and stabilization. The β isoform of PI3K is implicated in integrin αIIbβ3 outside-in signaling, is required for the phosphorylation of Akt, and controls efficient platelet spreading upon adhesion to fibrinogen. In this study we found that during integrin αIIbβ3 outside-in signaling PI3Kβ-dependent phosphorylation of Akt on Serine473 is mediated by the mammalian target of rapamycin complex 2 (mTORC2). The activity of mTORC2 is stimulated upon platelet adhesion to fibrinogen, as documented by increased autophosphorylation. However, mTORC2 activation downstream of integrin αIIbβ3 is PI3Kβ-independent. Inhibition of mTORC2, but not mTORC1, also prevents Akt phosphorylation of Threonine308 and affects Akt activity, resulting in the inhibition of GSK3α/β phosphorylation. Nevertheless, mTORC2 or Akt inhibition does not alter PI3Kβ-dependent platelet spreading on fibrinogen. The activation of the small GTPase Rap1b downstream of integrin αIIbβ3 is regulated by PI3Kβ but is not affected upon inhibition of either mTORC2 or Akt. Altogether, these results demonstrate for the first time the activation of mTORC2 and its involvement in Akt phosphorylation and stimulation during integrin αIIbβ3 outside-in signaling. Moreover, the results demonstrate that the mTORC2/Akt pathway is dispensable for PI3Kβ-regulated platelet spreading on fibrinogen.

Anaplastic thyroid cancer (ATC) is a rare neoplasia with a poor prognosis. Proliferation and apoptosis assays were performed on ATC cell lines (8305C, 8505C) exposed to vinorelbine, lenvatinib, as well as to concomitant combinations. ABCB1, ABCG2 and CSF-1 mRNA expression was evaluated by real time PCR. The relative levels of pospho Akt were investigated as part of a human phospho-kinase array analysis, and CSF-1 and VEGFR-2 protein levels were measured by ELISA. The intracellular concentration of lenvatinib in ATC cells was measured by combined reversed-phase liquid chromatography-tandem mass spectrometry. An ATC subcutaneous xenograft tumor model in nude mice was treated with vinorelbine, lenvatinib, or vinorelbine plus lenvatinib. After treatment with vinorelbine, lenvatinib, a significant antiproliferative effect in ATC cell lines was observed. The concomitant treatment of vinorelbine and lenvatinib revealed synergism for all the fractions of affected cells. A decrease in ABCB1 expression was reported in both ATC cell lines treated with the lenvatinib plus vinorelbine combination, as was an increase in the intracellular concentration of lenvatinib. The combination caused a decrease in Akt, GSK3α/β, PRAS40 and Src phosphorylation, and in both CSF-1 mRNA and protein levels. In the subcutaneous tumor model, the combination reduced the tumor volume during the treatment period. Our results establish the synergistic ATC antitumor activity of a vinorelbine and lenvatinib combination.

Keywords: Anaplastic thyroid cancer; Lenvatinib; Lenvatinib - IUPAC name: 4-[3-chloro-4-(cyclopropylcarbamoylamino)phenoxy]-7-methoxyquinoline-6-carboxamide - PubChem CID: 9823820; Sorafenib; Synergism; Vinorelbine; Vinorelbine - IUPAC namemethyl (1R, 9R, 10S, 11R, 12R, 19R)-11-acetyloxy-12-ethyl-4-[(12S, 14R,)-16-ethyl-12-methoxycarbonyl-1, 10-diazatetracyclo[12.3.1.0, (3), (11).0, (4), (9),]octadeca-3(11), 4, 6, 8, 15-pentaen-12-yl]-10-hydroxy-5-methoxy-8-methyl-8, 16-diazapentacyclo[10.6.1.0, (1), (9).0, (2), (7).0, (16), (19),]nonadeca-2, 4, 6, 13-tetraene-10-carboxylate- PubChem CID 5311497.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

Organogenesis, including renal development, requires an appropriate retinoic acid concentration, which is established by differential expression of aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and cytochrome P450 family 26 subfamily A/B/C member 1 (CYP26A1/B1/C1). In the fetal kidney, ALDH1A2 expresses in the developing stroma and renal vesicle and its derivatives but does not present in the ureteric bud. It remains unclear what may contribute to this expression pattern. Here we show that the glycogen synthase kinase 3 alpha/beta (GSK3A/B) inhibitor CHIR99021 significantly represses ALDH1A2 expression in WiT49, which is a Wilms' tumor cell line that exhibits "triphasic" differential potential and is used as a fetal kidney cell model. CHIR99021 fails to suppress ALDH1A2 as β-catenin is inhibited, suggesting that the downregulation of ALDH1A2 by CHIR99021 is through Wnt/β-catenin signaling. Ectopic expression of mouse Wnt1, Wnt3a, Wnt4, and Wnt9b represses ALDH1A2 expression in WiT49 cells. Using immunohistochemistry, we show an inverse correlation of Aldh1a2 expression with β-catenin in rat E18.5 kidney. ChIP demonstrated that β-catenin is recruited to the ALDH1A2 promoter, the conserved intron1G, and another site within intron 1 of ALDH1A2. Using a luciferase assay, we further show that the ALDH1A2 promoter and the intron1G element are involved in the repression of ALDH1A2 expression by CHIR99021. Our work demonstrates that ALDH1A2 expression can be directly repressed by the Wnt/β-catenin signaling in fetal kidney cells, suggesting that Wnt/β-catenin may play a role in maintaining the expression pattern of ALDH1A2 in the fetal kidney, thus controlling the availability and localization of retinoic acid and regulating aspects of kidney development.

Human sperm motility is essential for fertilization and among pathologies underlying male infertility is asthenozoospermia. Nevertheless, mechanisms regulating sperm motility are not completely unraveled. This work investigates phosphoproteins underlying human sperm motility by using differential phosphoproteomic in two human sperm subpopulations: high (HM) and low (LM) motility, obtained by centrifugation in a density gradient. Phosphoproteomics (HPLC-MS/MS triple TOF), comparing human LM and HM phosphoproteomes, identified 210 phosphopeptides with different abundance that correspond with 119 sperm proteins. Analysis showed that 40% of phosphoproteins in LM spermatozoa are involved in metabolism, (catabolism, protein transport, lipid biosynthesis), 25% in spermatogenesis and sperm function, 8% in immune system and 6% in DNA repair. In HM spermatozoa, 48% of phosphoproteins are related to spermatogenesis and sperm function (motility), whereas 8% are associated to metabolism. GSK3α resulted one of the most abundant phosphoproteins in HM spermatozoa. Western blot confirmed that GSK3α phosphorylation is higher in HM spermatozoa. Summarizing, this study i) identified phosphoproteins in two human spermatozoa populations, ii) supports that human spermatozoa rely in protein phosphorylation, such as GSK3 α, to regulate sperm motility, iv) raises the challenge of using some identified human sperm phosphorylated proteins (GSK3α) as targets to develop into clinically relevant biomarkers. SIGNIFICANCE: Human sperm phosphoproteome analyzed by nano HPLC-MS/MS triple TOF identifies the differential abundance of sperm phosphoproteins in two human sperm populations exhibiting high motility (HM) and/or low motility (LM) that were isolated from normozoospermic healthy donors. Majority of human phosphoproteins found in LM spermatozoa are involved in sperm metabolism (40%), whereas those in HM spermatozoa are associated to spermatogenesis and sperm function, as motility (48%), and only 8% are associated to metabolism. One of the most abundant phosphoproteins found in HM spermatozoa is GSK3α, kinase directly involved in the regulation of sperm motility that was also validated by western blot. The biological relevance of this study is based in the fact that supports that mature human sperm cells rely in protein phosphorylation to efficiently regulate sperm motility and allows identifying those regulatory human sperm phosphoproteins. This work will clearly impacts the human reproductive field as it raises the challenge of consider identified human sperm phosphoproteins, such as GSK3α, as potential biological targets to develop into relevant biomarkers for the human clinic or assisted reproductive technology.

Our previous study demonstrated the translocation of Aβ1-42 to the nucleus in response to antibiotic treatment, and interpreted it as a possible transcriptional response of Aβ1-42 to antibiotics. The present study aims to investigate how amyloid acts on the key elements of neurodegeneration and the molecules involved in the induction of Aβ1-42 production. For this purpose, we investigated the acute effect of Aβ1-42 on the transcriptional levels of genes that have roles in the mechanisms that produce Aβ itself: alpha secretase (ADAM10), beta secretase (BACE1), the gamma secretase complex (PS-1, PS-2, Nicastrin), the substrate APP, APOE (the significant risk factor for sporadic form of the AD), TREM2 (recently indicated as a contributor to AD risk), NMDAR subunits and PKCzeta (contributors of memory and learning), and key elements of tau pathology such as tau, GSK3α, GSK3β, and Cdk5. Additionally, we examined cholecalciferol metabolism-related enzyme 1α-hydroxylase (1αOHase) in primary cortical neurons with qRT-PCR. Our results indicate that Aβ1-42 has an effect on most of the target genes. This effect involves regulation of the amyloidogenic pathway in a complex manner, specifically, a general downregulation in NMDARs, ApoE, Trem2, and 1αOHase genes, and general up-regulation of tau pathway-related genes. We speculate that the presence of Aβ impacts the neurons not only with toxic events but also at the transcriptional level. The nuclear localization of Aβ1-42 and its regulatory effects on the target genes that we investigated in present study indicates Aβ1-42 as a transcriptional regulator of genes related to neurodegeneration.

FGF13, a member of the FGF subfamily, has been found to be highly expressed in cancer cells such as prostate cancer, melanoma, glioma and multiple myeloma. However, the mechanism of FGF13 function during cancer cell proliferation remains to be unexplored, especially Non-small cell lung cancer (NSCLC). In this study, the cell proliferation effect of FGF13 on A549 cells was checked by CCK-8, clone formation, Ki67 immunofluorescence staining and Flow Cytometry assay. Localization of FGF13 within A549 cells was performed with confocal laser scanning microscope. The protein variations and interaction were measured by western blotting and co-immunoprecipitation analysis. It showed that FGF13 was mainly distributed in the cytoplasm and exhibited a high expression level in A549 cells. High expression of FGF13 activated AKT-GSK3 signaling pathway, and inhibited the activity of p21 and p27. Thus, FGF13 enhanced the process of transition from G1 to S phase and promoted A549 cells proliferation. Furthermore, the interaction between FGF13 and SHCBP1 was confirmed. Meanwhile, FGF13 and SHCBP1 had a cooperative effect to accelerate the cell cycle progression, especially the ability to promote cell proliferation is significantly enhanced via protein interaction. Hence, we conclude that FGF13 played a positive regulation role during A549 cells proliferation. FGF13 interacted with SHCBP1 to facilitate cell cycle progression, providing new insights into deep understanding of non-small cell lung cancer mechanisms of proliferation and regulation function of FGF13.

Keywords: FGF13; Non-small cell lung cancer; SHCBP1; cell proliferation.

Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules in these metabolic pathways. Yet, little is known on the role in the HCV life cycle of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism. Therefore, the impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype 1b and 2a replicons or producing cell culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in hepatitis E virus (HEV) replication was investigated to assess virus specificity of the observed findings. We found that both inhibition of GSK3 function by synthetic inhibitors as well as silencing of GSK3β gene expression resulted in a decrease of HCV replication and infectious particle production, whereas silencing of the GSK3α isoform had no relevant effect on the HCV life cycle. Conversely, overexpression of GSK3β resulted in enhanced HCV replication. In contrast, GSK3β had no effect on replication of subgenomic HEV replicon. The pro-viral effect of GSK3β on HCV replication was mediated by supporting expression of microRNA-122 (miR-122), a micro-RNA which is mandatory for wild-type HCV replication, as GSK3 inhibitors suppressed miR-122 levels and as inhibitors of GSK3 had no antiviral effect on a miR-122-independent HCV mutant. In conclusion, we have identified GSK3β is a novel host factor supporting HCV replication by maintaining high levels of hepatic miR-122 expression.

It is well known that patients with Alzheimer's disease (AD) have imbalances in blood thiamine concentrations and lower activity of thiamine-dependent enzymes. Benfotiamine, a more bioavailable thiamine analog, has been proposed as an alternative to counteract these changes related to thiamine metabolism. Thus, our study aimed to analyze the effects of benfotiamine supplementation on brain thiamine absorption, as well as on parameters related to neuronal energy metabolism and disease progression in an experimental model of sporadic AD induced by intracerebroventricular injection of streptozotocin (STZ) in rats. The supplementation with 150 mg/kg of benfotiamine for 30 days increased the concentrations of thiamine diphosphate in the hippocampus and entorhinal cortex. This led to an improvement in mitochondria enzymes and insulin signaling pathway, with inactivation of GSK3α/β and ERK1/2, which are two tau-kinases related to the progression of AD, which could decrease tau hyperphosphorylation and apoptosis signaling. Besides, we observed an increased amount of Glun2b subunit of NMDA receptors, decreased inflammation, and improvement of cognitive deficit. Together, these results suggest that benfotiamine could be a potential therapeutic approach in the treatment of sporadic AD.

Keywords: Apoptosis; Dementia; Insulin sensitivity; Mitochondrial dysfunction; Streptozotocin.

Neural circuit development involves the coordinated growth and guidance of axons. During this process, axons encounter many different cues, but how these cues are integrated and translated into growth is poorly understood. In this study, we report that receptor signaling does not follow a linear path but changes dependent on developmental stage and coreceptors involved. Using developing chicken embryos of both sexes, our data show that calcium-sensing receptor (CaSR), a G-protein-coupled receptor important for regulating calcium homeostasis, regulates neurite growth in two distinct ways. First, when signaling in isolation, CaSR promotes growth through the PI3-kinase-Akt pathway. At later developmental stages, CaSR enhances tropomyosin receptor kinase B (TrkB)/BDNF-mediated neurite growth. This enhancement is facilitated through a switch in the signaling cascade downstream of CaSR (i.e., from the PI3-kinase-Akt pathway to activation of GSK3α Tyr279). TrkB and CaSR colocalize within late endosomes, cotraffic and coactivate GSK3, which serves as a shared signaling node for both receptors. Our study provides evidence that two unrelated receptors can integrate their individual signaling cascades toward a nonadditive effect and thus control neurite growth during development.SIGNIFICANCE STATEMENT This work highlights the effect of receptor coactivation and signal integration in a developmental setting. During embryonic development, neurites grow toward their targets guided by cues in the extracellular environment. These cues are sensed by receptors at the surface that trigger intracellular signaling events modulating the cytoskeleton. Emerging evidence suggests that the effects of guidance cues are diversified, therefore expanding the number of responses. Here, we show that two unrelated receptors can change the downstream signaling cascade and regulate neuronal growth through a shared signaling node. In addition to unraveling a novel signaling pathway in neurite growth, this research stresses the importance of receptor coactivation and signal integration during development of the nervous system.