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Glycogen synthesis is a critical metabolic function of the endometrium to prepare for successful implantation and sustain embryo development. Yet, regulation of endometrial carbohydrate metabolism is poorly characterized. Whereas glycogen synthesis is attributed to progesterone, we previously found that the metabolic B isoform of the insulin receptor is maximally expressed in secretory-phase endometrium, indicating a potential role of insulin in glucose metabolism.

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We sought to determine whether insulin or progesterone regulates glycogen synthesis in human endometrium.

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Endometrial epithelial cells were isolated from 28 healthy women and treated with insulin, medroxyprogesterone (MPA), or vehicle. Intracellular glycogen and the activation of key enzymes were quantified.

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In epithelia, insulin induced a 4.4-fold increase in glycogen, whereas MPA did not alter glycogen content. Insulin inactivated glycogen synthase (GS) kinase 3α/β (GSK3α/β), relieving inhibition of GS. In a regulatory mechanism, distinct from liver and muscle, insulin also increased GS by 3.7-fold through increased GS 2 (GYS2) gene expression.

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We demonstrate that insulin, not progesterone, directly regulates glycogen synthesis through canonical acute inactivation of GSK3α/β and noncanonical stimulation of GYS2 transcription. Persistently elevated GS enables endometrium to synthesize glycogen constitutively, independent of short-term nutrient flux, during implantation and early pregnancy. This suggests that insulin plays a key, physiological role in endometrial glucose metabolism and underlines the need to delineate the effect of maternal obesity and hyperinsulinemia on fertility and fetal development.

Lung cancer is the most prevalent in cancer-related deaths, while breast carcinoma is the second most dominant cancer in women, accounting for the most number of deaths worldwide. Cancers are heterogeneous diseases that consist of several subtypes based on the presence or absence of hormone receptors and human epidermal growth factor receptor 2. Several drugs have been developed targeting cancer biomarkers; nonetheless, their efficiency are not adequate due to the high reemergence rate of cancers and fundamental or acquired resistance toward such drugs, which leads to partial therapeutic possibilities. Recent studies on cardiac glycosides (CGs) positioned them as potent cytotoxic agents that target multiple pathways to initiate apoptosis and autophagic cell death in many cancers. In the present study, our aim is to identify the anticancer activity of a naturally available CG (strophanthidin) in human breast (MCF-7), lung (A549), and liver cancer (HepG2) cells. Our results demonstrate a dose-dependent cytotoxic effect of strophanthidin in MCF-7, A549, and HepG2 cells, which was further supported by DNA damage on drug treatment. Strophanthidin arrested the cell cycle at the G2/M phase; this effect was further validated by checking the inhibited expressions of checkpoint and cyclin-dependent kinases in strophanthidin-induced cells. Moreover, strophanthidin inhibited the expression of several key proteins such as MEK1, PI3K, AKT, mTOR, Gsk3α, and β-catenin from MAPK, PI3K/AKT/mTOR, and Wnt/β-catenin signaling. The current study adequately exhibits the role of strophanthidin in modulating the expression of various key proteins involved in cell cycle arrest, apoptosis, and autophagic cell death. Our in silico studies revealed that strophanthidin can interact with several key proteins from various pathways. Taken together, this study demonstrates the viability of strophanthidin as a promising anticancer agent, which may serve as a new anticancer drug.

The early apoptotic events induced by environmental pollutants with carcinogenic properties are poorly understood. Here, we focus on the early cytotoxic effects of benzo[a]pyrene (B[a]P). In F258 rat hepatic epithelial cells, B[a]P induces intrinsic apoptosis via a mitochondrial dysfunction characterized by the release of hexokinase II (HKII) from the mitochondria. Cancer cells often have an anomalous cell energy metabolism; since HKII dysfunction regulates B[a]P-induced apoptosis in F258 cells, but may also alter cell energy metabolism, HKII release from the mitochondria may represent an important B[a]P-related carcinogenic issue. Thus in the present study, we aimed at deciphering the mechanisms underlying HKII dysfunction upon B[a]P exposure. We show that while glycogen synthase kinase 3 beta (GSK3β) regulated the expression of HKII at the transcriptional level, glycogen synthase kinase 3 alpha (GSK3α) was involved in B[a]P-induced apoptosis via a decrease in c-Myc expression. The reduced level of c-Myc caused the relocation of HKII from the mitochondria to the cytosol, thereby being involved in the formation of reactive oxygen species and apoptosis. In conclusion, we show that the couple GSK3α/c-Myc plays a key role in B[a]P-induced early apoptotic cell signaling via HKII dysfunction.

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca²⁺ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/β were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca²⁺ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/β after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca²⁺ and Tyr phosphorylation of GSK3α/β. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

Keywords: acrosomal exocytosis; in vitro capacitation; seminal plasma; spermatozoa.

The cellular and organismal phenotypic response to a small-molecule kinase inhibitor is defined collectively by the inhibitor's targets and their functions. The selectivity of small-molecule kinase inhibitors is commonly determined in vitro, using purified kinases and substrates. Recently, competitive chemical proteomics has emerged as a complementary, unbiased, cell-based methodology to define the target landscape of kinase inhibitors. Here, we evaluated and optimized a competitive multiplexed inhibitor bead mass spectrometry (MIB/MS) platform using cell lysates, live cells, and treated mice. Several clinically active kinase inhibitors were profiled, including trametinib, BMS-777607, dasatinib, abemaciclib, and palbociclib. MIB/MS competition analyses of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors abemaciclib and palbociclib revealed overlapping and unique kinase targets. Competitive MIB/MS analysis of abemaciclib revealed 83 target kinases, and dose-response MIB/MS profiling revealed glycogen synthase kinase 3 alpha and beta (GSK3α and β) and Ca2+/calmodulin-dependent protein kinase II delta and gamma (CAMKIIδ and γ) as the most potently inhibited. Cell-based and in vitro kinase assays show that in contrast to palbociclib, abemaciclib directly inhibits GSK3α/β and CAMKIIγ/δ kinase activity at low nanomolar concentrations. GSK3β phosphorylates β-catenin to suppress WNT signaling, while abemaciclib (but not palbociclib or ribociclib) potently activates β-catenin-dependent WNT signaling. These data illustrate the power of competitive chemical proteomics to define kinase target specificities for kinase inhibitors, thus informing clinical efficacy, dose-limiting toxicities, and drug-repurposing efforts.Implications: This study uses a rapid and quantitative proteomics approach to define inhibitor-target data for commonly administered therapeutics and provides a cell-based alternative to in vitro kinome profiling. Mol Cancer Res; 16(2); 333-44. ©2017 AACR.

Glycogen synthase kinase 3 (GSK3) is a monomeric serine-threonine kinase discovered in 1980 in a rat skeletal muscle. It has been involved in various cellular processes including embryogenesis, immune response, inflammation, apoptosis, autophagy, wound healing, neurodegeneration and carcinogenesis. GSK3 exists in two different isoforms, GSK3α and GSK3β, both containing seven antiparallel beta-plates, a short linking part and an alpha helix, but coded by different genes and variously expressed in human tissues. In the current review, we comprehensively appraise the current literature on the role of GSK3 in various cancers with emphasis on ovarian carcinoma. Our findings indicate that the role of GSK3 in ovarian cancer development cannot be decisively determined as the currently available data support both prooncogenic and tumor-suppressive effects. Likewise, the clinical impact of GSK3 expression on ovarian cancer patients and its potential therapeutic implications are also limited. Further studies are needed to fully elucidate the pathophysiological and clinical implications of GSK3 activity in ovarian cancer.

Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C (IL-17RC), a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration (AMD), was associated with altered activation of phosphatidylinositide 3-kinase (PI3K), Akt, and glycogen synthase kinase 3 (GSK3). We wondered whether or not altered PI3K, Akt, and GSK3 activities could be detected in peripheral blood mononuclear cells (PBMC) obtained from AMD patients. In the patients' PBMC, absent or reduced serine-phosphorylation of GSK3α or GSK3β was observed, which was accompanied with increased phosphorylation of GSK3 substrates (e.g. CCAAT enhancer binding protein α, insulin receptor substrate 1, and TAU), indicative of enhanced GSK3 activation. In addition, decreased protein mass of PI3K85α and tyrosine-phosphorylation of PI3K50α was present in PBMC of the AMD patients, suggesting impaired PI3K activation. Moreover, abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients. These data demonstrate that despite the presence of high levels of IL-17RC, Wnt-3a and vascular endothelial growth factor, the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients. Thus, measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.

Increasing attention has been focused on the role of microRNAs in post-transcription regulation during spermatogenesis. Recently, the miR-34 family has been shown to be involved in the spermatogenesis, but the clear function of the miR-34 family in spermatogenesis is still obscure. Here we analyzed the function of miR-34a, a member of the miR-34 family, during spermatogenesis using miR-34a knockout zebrafish generated by the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system. miR-34a knockout zebrafish showed no obvious defects on testis morphology and sperm quantity. However, we found a significant increase in progressive sperm motility that is one of the pivotal factors influencing in vitro fertilization rates, in the knockout zebrafish. Moreover, breeding experiments showed that, when miR-34a-knockout male zebrafish mated with the wide-type females, they had a higher fertilization rate than did the wide-type males. Glycogen synthase kinase-3a (gsk3a), a potential sperm motility regulatory gene was predicted to be targeted by miR-34a, which was further supported by luciferase reporter assays, since a significant decrease of luciferase activity was detected upon ectopic overexpression of miR-34a. Our findings suggest that miR-34a downregulates gsk3a by targeting its 3' untranslated region, and miR-34a/gsk3a interaction modulates sperm motility in zebrafish. This study will help in understanding in the role of the miR-34 family during spermatogenesis and will set paths for further studies.

Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.

New pyridazino[4,5-b]indol-4-ones and pyridazin-3(2H)-one analogs were synthesized and their inhibitory activities against DYRK1A, CDK5/p25, GSK3α/β and p110-α isoform of PI3K evaluated using harmine as reference. Both furan-2-yl 10 and pyridin-4-yl 19 from the two different series, exhibited submicromolar IC50 against DYRK1A with no activities against the three other kinases. In addition, compound 10 exhibited antiproliferative activities in the Huh-7, Caco2 and MDA-MB-231 cell lines.

Despite advances in the systemic treatment of patients with metastatic melanoma using immune checkpoint and tyrosine kinase inhibitors (TKI), the majority of stage IV melanoma patients eventually succumb to the disease. We have previously identified the transcription factor Sox10 as a crucial player in melanoma, yet the underlying molecular mechanisms mediating Sox10-dependent tumorigenesis remain largely uncharacterized. Here, we show that MEK and RAF inhibitors do not suppress levels of SOX10 protein in patient-derived cells in vitro, as well as in melanoma patients in vivo. In a search for pharmacological inhibitors of SOX10, we performed a mass spectrometry-based screen in human melanoma cells. Subsequent analysis revealed that SOX10 directly interacts with β-catenin, which is a key mediator of canonical Wnt/β-catenin signaling. We demonstrate that inhibitors of glycogen synthase kinase 3 alpha/beta (GSK3α/β) efficiently abrogate SOX10 protein in human melanoma cells in vitro and in melanoma mouse models in vivo. The mechanism of action of GSK3-mediated SOX10 suppression is transcription-independent and relies on the presence of a proteasome degradable form of β-catenin. Taken together, we provide evidence that activation of canonical Wnt signaling has a profound effect on melanoma growth and is able to counteract Sox10-dependent melanoma maintenance both in vitro and in vivo.

High frequency deep brain stimulation (DBS) of the lateral habenula (LHb) reduces symptoms of depression in severely treatment-resistant individuals. Despite the observed therapeutic effects, the molecular underpinnings of DBS are poorly understood. This study investigated the efficacy of high frequency LHb DBS (130Hz; 200μA; 90μs) in an animal model of tricyclic antidepressant resistance. Further, we reported DBS mediated changes in Ca(2+)/calmodulin-dependent protein kinase (CaMKIIα/β), glycogen synthase kinase 3 (GSK3α/β) and AMP-activated protein kinase (AMPK) both locally and in the infralimbic cortex (IL). Protein expressions were then correlated to immobility time during the forced swim test (FST). Antidepressant actions were quantified via FST. Treatment groups comprised of animals treated with adrenocorticotropic hormone alone (ACTH; 100μg/day, 14days, n=7), ACTH with active DBS (n=7), sham DBS (n=8), surgery only (n=8) or control (n=8). Active DBS significantly reduced immobility in ACTH-treated animals (p<0.05). For this group, western blot results demonstrated phosphorylation status of LHb CaMKIIα/β and GSK3α/β significantly correlated to immobility time in the FST. Concurrently, we observed phosphorylation status of CaMKIIα/β, GSK3α/β, and AMPK in the IL to be negatively correlated with antidepressant actions of DBS. These findings suggest that activity dependent phosphorylation of CaMKIIα/β, and GSK3α/β in the LHb together with the downregulation of CaMKIIα/β, GSK3α/β, and AMPK in the IL, contribute to the antidepressant actions of DBS.

Soluble epoxide hydrolase (sEH), an enzyme with COOH-terminal hydrolase and NH2-terminal lipid phosphatase activities, is expressed in regions of the brain such as the cortex, white matter, hippocampus, substantia nigra, and striatum. sEH is involved in the regulation of cerebrovascular and neuronal function upon pathological insults. However, the physiological significance of sEH and its underlying mechanism in modulating brain function are not fully understood. In this study, we investigated the role of sEH in anxiety and potential underlying mechanisms in mice. Western blot for protein phosphorylation and expression was performed. Immunohistochemical analyses and Nissl and Golgi staining were performed for histological examination. Mouse behaviors were evaluated by open field activity, elevated plus maze, classical fear conditioning, social preference test, and Morris water maze. Our results demonstrated that the expression of sEH was upregulated during postnatal development in wild-type (WT) mice. Genetic deletion of sEH (sEH-/-) in mice resulted in anxiety-like behavior and disrupted social preference. Increased olfactory bulb (OB) size and altered integrity of neurites were observed in sEH-/- mice. In addition, ablation of sEH in mice decreased protein expression of tyrosine hydroxylase and reduced dopamine production in the brain. Moreover, the level of phosphorylated calmodulin kinase II (CaMKII) and glycogen synthase kinase 3 α/β (GSK3α/β) was higher in sEH-/- mice than in WT mice. Collectively, these findings suggest that sEH is a key player in neurite outgrowth of neurons, OB development in the brain, and the development of anxiety-like behavior, by regulating the CaMKII-GSK3α/β signaling pathway.

Accumulating evidence suggests AKT1 and DRD2-AKT-GSK3 signaling involvement in schizophrenia. AKT1 activity is also required for lithium, a GSK3 inhibitor, to modulate mood-related behaviors. Notably, GSK3 inhibitor significantly alleviates behavioral deficits in Akt1^-/- female mice, whereas typical/atypical antipsychotics have no effect. In agreement with adjunctive therapy with lithium in treating schizophrenia, our data mining indicated that the average utilization rates of lithium in the Taiwan National Health Insurance Research Database from 2002 to 2013 are 10.9% and 6.63% in inpatients and outpatients with schizophrenia, respectively. Given that lithium is commonly used in clinical practice, it is of great interest to evaluate the effect of lithium on alleviating Akt1-related deficits. Taking advantage of Akt1^+/- mice to mimic genetic deficiency in patients, behavioral impairments were replicated in female Akt1^+/- mice but were alleviated by subchronic lithium treatment for 13 days. Lithium also effectively alleviated the observed reduction in phosphorylated GSK3α/β expression in the brains of Akt1^+/- mice. Furthermore, inhibition of Akt expression using an Akt1/2 inhibitor significantly reduced neurite length in P19 cells and primary hippocampal cell cultures, which was also ameliorated by lithium. Collectively, our findings implied the therapeutic potential of lithium and the importance of the AKT1-GSK3 signaling pathway.

Urotensin II (UII) is a neural hormone that induces cardiac hypertrophy and may be involved in the pathogenesis of cardiac remodeling and heart failure. Hypertrophy has been linked to histone deacetylase 5 (HDAC5) phosphorylation and nuclear factor κB (NF-κB) translocation, both of which are predominantly mediated by G protein-coupled receptor kinase 5 (GRK5). In the present study, we found that UII rapidly and strongly stimulated nuclear export of HDAC5 and nuclear import of NF-κB in H9c2 cells overexpressing the urotensin II receptor (H9c2UT). Hence, we hypothesized that GRK5 and its signaling pathway may play a role in UII-mediated cellular hypertrophy. H9c2UT cells were transduced with a GRK5 small hairpin RNA interference recombinant lentivirus, resulting in the down-regulation of GRK5. Under UII stimulation, reduced levels of GRK5 in H9c2UT cells led to suppression of UII-mediated HDAC5 phosphorylation and activation of the NF-κB signaling pathway. In contrast, UII-mediated activations of ERK1/2 and GSK3α/β were not affected by down-regulation of GRK5. In a cellular hypertrophy assay, down-regulation of GRK5 significantly suppressed UII-mediated hypertrophy of H9c2UT cells. Furthermore, UII-mediated cellular hypertrophy was inhibited by amlexanox, a selective GRK5 inhibitor, in H9c2UT cells and neonatal cardiomyocytes. Our results suggest that GRK5 may be involved in a UII-mediated hypertrophic response via activation of NF-κB and HDAC5 at least in part by ERK1/2 and GSK3α/β-independent pathways.

In mammals, motility and fertilizing ability of spermatozoa develop during their passage through the epididymis. After ejaculation, sperm undergo capacitation and hyperactivation in the female reproductive tract - a motility transition that is required for sperm penetration of the egg. Both epididymal initiation of sperm motility and hyperactivation are essential for male fertility. Motility initiation in the epididymis and sperm hyperactivation involve changes in metabolism, cAMP (cyclic adenosine mono-phosphate), calcium and pH acting through protein kinases and phosphatases. Despite this knowledge, we still do not understand, in biochemical terms, how sperm acquire motility in the epididymis and how motility is altered in the female reproductive tract. Recent data show that the sperm specific protein phosphatase PP1γ2, glycogen synthase kinase 3 (GSK3), and the calcium regulated phosphatase calcineurin (PP2B), are involved in epididymal sperm maturation. The protein phosphatase PP1γ2 is present only in testis and sperm in mammals. PP1γ2 has a isoform-specific requirement for normal function of mammalian sperm. Sperm PP1γ2 is regulated by three proteins - inhibitor 2, inhibitor 3 and SDS22. Changes in phosphorylation of these three inhibitors and their binding to PP1γ2 are involved in initiation and activation of sperm motility. The inhibitors are phosphorylated by protein kinases, one of which is GSK3. The isoform GSK3α is essential for epididymal sperm maturation and fertility. Calcium levels dramatically decrease during sperm maturation and initiation of motility suggesting that the calcium activated sperm phosphatase (PP2B) activity also decreases. Loss of PP2B results in male infertility due to impaired sperm maturation in the epididymis. Thus the three signaling enzymes PP1γ2, GSK3, and PP2B along with the documented PKA (protein kinase A) have key roles in sperm maturation and hyperactivation. Significantly, all these four signaling enzymes are present as specific isoforms only in placental mammals, a testimony to their essential roles in the unique aspects of sperm function in mammals. These findings should lead to a better biochemical understanding of the basis of male infertility and should lead to novel approaches to a male contraception and managed reproduction.

Previous studies have shown that glycogen synthase kinase 3β (GSK3β) suppression is a potential strategy for human acute myeloid leukemia (AML) therapy. However, the cytotoxic mechanism associated with GSK3β suppression remains unresolved. Thus, the underlying mechanism of N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418)-elicited GSK3β suppression in the induction of AML U937 and HL-60 cell death was investigated in this study. Our study revealed that AR-A014418-induced MCL1 downregulation remarkably elicited apoptosis of U937 cells. Furthermore, the AR-A014418 treatment increased p38 MAPK phosphorylation and decreased the phosphorylated Akt and ERK levels. Activation of p38 MAPK subsequently evoked autophagic degradation of 4EBP1, while Akt inactivation suppressed mTOR-mediated 4EBP1 phosphorylation. Furthermore, AR-A014418-elicited ERK inactivation inhibited Mnk1-mediated eIF4E phosphorylation, which inhibited MCL1 mRNA translation in U937 cells. In contrast to GSK3α, GSK3β downregulation recapitulated the effect of AR-A014418 in U937 cells. Transfection of constitutively active GSK3β or cotransfection of constitutively activated MEK1 and Akt suppressed AR-A014418-induced MCL1 downregulation. Moreover, AR-A014418 sensitized U937 cells to ABT-263 (BCL2/BCL2L1 inhibitor) cytotoxicity owing to MCL1 suppression. Collectively, these results indicate that AR-A014418-induced GSK3β suppression inhibits ERK-Mnk1-eIF4E axis-modulated de novo MCL1 protein synthesis and thereby results in U937 cell apoptosis. Our findings also indicate a similar pathway underlying AR-A014418-induced death in human AML HL-60 cells.

Keywords: 4EBP1 downregulation; GSK3β suppression; MCL1 downregulation; eIF4E dephosphorylation; leukemia.

Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)-defined as PAX7-expressing cells with myogenic potential-can provide an abundant source of donor material for muscle stem cell therapy. As in vitro myogenesis is decoupled from in vivo timing and 3D-embryo structure, it is important to characterize what stage or type of muscle is modeled in culture. Here, gene expression profiling is analyzed in hESCs over a 50 day skeletal myogenesis protocol and compared to datasets of other hESC-derived skeletal muscle and adult murine satellite cells. Furthermore, day 2 cultures differentiated with high or lower concentrations of CHIR99021, a GSK3A/GSK3B inhibitor, were contrasted. Expression profiling of the 50 day time course identified successively expressed gene subsets involved in mesoderm/paraxial mesoderm induction, somitogenesis, and skeletal muscle commitment/formation which could be regulated by a putative cascade of transcription factors. Initiating differentiation with higher CHIR99021 concentrations significantly increased expression of MSGN1 and TGFB-superfamily genes, notably NODAL, resulting in enhanced paraxial mesoderm and reduced ectoderm/neuronal gene expression. Comparison to adult satellite cells revealed that genes expressed in 50-day cultures correlated better with those expressed by quiescent or early activated satellite cells, which have the greatest therapeutic potential. Day 50 cultures were similar to other hESC-derived skeletal muscle and both expressed known and novel SMP surface proteins. Overall, a putative cascade of transcription factors has been identified which regulates four stages of myogenesis. Subsets of these factors were upregulated by high CHIR99021 or their binding sites were significantly over-represented during SMP activation, ranging from quiescent to late-activated stages. This analysis serves as a resource to further study the progression of in vitro skeletal myogenesis and could be mined to identify novel markers of pluripotent-derived SMPs or regulatory transcription/growth factors. Finally, 50-day hESC-derived SMPs appear similar to quiescent/early activated satellite cells, suggesting they possess therapeutic potential.

Insulin-like growth factor 2 (IGF2) mRNA binding protein 2 (IMP2) was selectively deleted from adult mouse muscle; two phenotypes were observed: decreased accrual of skeletal muscle mass after weaning and reduced wheel-running activity but normal forced treadmill performance. Reduced wheel running occurs when mice are fed a high-fat diet but is normalized when mice consume standard chow. The two phenotypes are due to altered output from different IMP2 client mRNAs. The reduced fiber size of IMP2-deficient muscle is attributable, in part, to diminished autocrine Igf2 production; basal tyrosine phosphorylation of the insulin and IGF1 receptors is diminished, and Akt1 activation is selectively reduced. Gsk3α is disinhibited, and S536-phosphorylated ε subunit of eukaryotic initiation factor 2B [eIF2Bε(S536)] is hyperphosphorylated. Protein synthesis is reduced despite unaltered mTOR complex 1 activity. The diet-dependent reduction in voluntary exercise is likely due to altered muscle metabolism, as contractile function is normal. IMP2-deficient muscle exhibits reduced fatty acid oxidation, due to a reduced abundance of mRNA of peroxisome proliferator-activated receptor α (PPARα), an IMP2 client, and PPARα protein. IMP2-deficient muscle fibers treated with a mitochondrial uncoupler to increase electron flux, as occurs with exercise, exhibit reduced oxygen consumption from fatty acids, with higher oxygen consumption from glucose. The greater dependence on muscle glucose metabolism during increased oxygen demand may promote central fatigue and thereby diminish voluntary activity.

Cobalt protoporphyrin (CoPP) is a potent HO-1 inducer and generally known to be an antioxidant in various cell types. Little is known about the CoPP-induced cyclooxygenase-2 (COX-2) expression and its downstream signaling in microglial cells. In current study, CoPP caused concentration- and time-dependent increases in COX-2 expression in microglial cells. Furthermore, activation of apoptosis signal-regulating kinase (ASK) 1/MAP kinase involved in CoPP-induced COX-2 expression in microglia. CoPP also induced P2X7 receptor activation, and treatment of P2X7 inhibitors effectively reduced CoPP-induced COX-2 expression. Protein inhibitor of activated STAT (PIAS) 1 is reported to be involved in modulating anti-inflammatory response through negative regulation of transcription factors. Interestingly, treatment with CoPP markedly induced PIAS1 degradation which is regulated by PI3K, Akt, and glycogen synthase kinase 3α/β (GSK3α/β) signaling pathways. These results suggest that CoPP induces COX-2 expression through activating P2X7 receptors and ASK1/MAP kinases as well as PIAS1 degradation signaling pathways. Our study provides a new insight into the regulatory effect of CoPP on neuroinflammation in microglial cells.

Proteomic analyses are useful for understanding the metabolic pathways governing embryo development. This study investigated the presence of enzymes involved in glycolysis and glycogenesis in in vitro-produced bovine embryos at five developmental stages leading up to blastocyst formation. The enzymes examined were: (1) glycolytic: hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase mutase 1/2 (PKM-1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (2) glycogenic: glycogen synthase kinase-3 isoforms α/ β (GSK-3α/β). Glucose transporter-1 (GLUT-1) was also analysed. The developmental stages examined were: (1) 2-4-cell, (2) 5-8-cell, (3) 16-cell, (4) morula and (5) expanded blastocyst. The enzymes HK-I, PFK-1, PKM-1/2, GAPDH and GLUT-1 were differentially expressed throughout all stages (P<0.05). GSK-3α and β were also differentially expressed from the 2-4-cell to the expanded blastocyst stage (P<0.05) and GLUT-1 was identified throughout. The general trend was that the abundance of PFK1, GAPDH and PKM-1/2 decreased whereas HK-I, phospho-GSK3α (P-GSK3α) and P-GSK3β levels increased as the embryo advanced. In contrast, GLUT-1 expression peaked at the 16-cell stage. These data combined suggest that in vitro bovine embryo metabolism switches from being glycolytic-centric to glycogenic-centric around the 16-cell stage, the developmental window also characterised by embryonic genome activation.

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3β, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3β is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. Both GSK3 isoforms, present in sperm from several species including human, are suggested to play a role in epididymal initiation of sperm motility. Using genetic approaches, we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3 isoforms are expressed at high levels during the onset of spermatogenesis. Conditional knockout of GSK3α, but not GSK3β, in developing testicular germ cells in mice results in male infertility. Mice lacking one allele each of GSK3α and GSK3β are fertile. Despite overlapping expression and localization in differentiating spermatids, GSK3β does not substitute for GSK3α. Loss of GSK3α impairs sperm hexokinase activity resulting in low ATP levels. Net adenine nucleotide levels in caudal sperm lacking GSK3α resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1γ2 with its protein interactors occurring during epididymal sperm maturation is impaired in sperm lacking GSK3α. The isoform-specific requirement for GSK3α is likely due to its specific binding partners in the sperm principal piece. Testis and sperm are unique in their specific requirement of GSK3α for normal function and male fertility.