Effect of the cPKCγ-Ng Signaling System on Rapid Eye Movement Sleep Deprivation-Induced Learning and Memory Impairment in Rats

Objective: Rapid eye movement sleep deprivation (REM-SD) can cause a decline in learning and memory and lead to changes in behavior. Therefore, REM sleep plays a key role in processes that govern learning and memory. However, the mechanism underlying REM-SD-induced learning and memory impairment is unclear and the underlying molecular signaling still needs to be identified. In the present study, we investigated the role of the cPKCγ-Ng signaling pathway in REM-SD-induced learning and memory impairment.Method: Sixty male rats were divided into Control, REM-SD, REM-SD+cPKCγ activator PMA, REM-SD+cPKCγ inhibitor H-7, and sleep revival (SR) groups. The Morris water maze was used to assess spatial learning and memory. Western blot analysis was used to detect cPKCγ total protein expression and membrane translocation levels, and Ng total protein expression and phosphorylation levels.Results: The REM-SD group performed worse on the Morris water maze test than the control group. Western blot analysis showed that cPKCγ membrane translocation and Ng phosphorylation levels were significantly lower in the REM-SD group. SR following REM-SD restored learning and memory ability, cPKCγ transmembrane translocation, and Ng phosphorylation levels, but not to levels observed before REM-SD. PMA and H-7 significantly improved/disrupted task ability as well as cPKCγ transmembrane translocation and Ng phosphorylation levels in REM-SD rats.Conclusion: The REM-SD induced learning and memory impairment in rats and may be associated with the cPKCγ-Ng signaling pathway. Specifically, activation of the cPKCγ-Ng signaling pathway may protect against REM-SD.

Functional Analysis of the Promoter Regions of Two Apoptosis-Related Genes (Bcl-2 and Cycs) and Their Regulation by Zn in Yellow Catfish

B-cell lymphoma 2 (Bcl-2) and cytochrome c (Cycs) are two important proteins relevant to cellular apoptosis. In this study, we characterized the functions of the promoter regions of two apoptosis-related genes, Bcl-2 and Cycs, in yellow catfish Pelteobagrus fulvidraco. We obtained a 1989 bp Bcl-2 promoter and an 1830 bp Cycs promoter and predicted several key transcription factor binding sites (TFBSs) on the promoters, such as Kruppel-like factor 4 (KLF4), signal transducer and activator of transcription factor 3 (STAT3), forkhead box O (FOXO), metal-responsive element (MRE) and hepatocyte nuclear factor 1α (HNF-1α). Zinc (Zn) increased the activities of the Bcl-2 promoter but decreased the activities of the Cycs promoter. Metal-responsive transcription factor 1 (MTF-1) and HNF-1α directly bound with Bcl-2 and Cycs promoters, and they positively regulated the activity of the Bcl-2 promoter but negatively regulated the activity of the Cycs promoter. Zn promoted the binding ability of HNF-1α to the Bcl-2 promoter but decreased its binding ability to the Cycs promoter. However, Zn had no significant effect on the binding capability of MTF-1 to the regions of Bcl-2 and Cycs promoters. Zn upregulated the mRNA and total protein expression of Bcl-2 but downregulated the mRNA and total protein expression of Cycs. At the same time, Annexin V–FITC/PI staining showed that Zn significantly reduced the apoptosis of primary hepatocytes. For the first time, our study provides evidence for the MRE and HNF-1α response elements on the Bcl-2 and Cycs promoters, offering new insight into the mechanism by which Zn affects apoptosis in vertebrates.

Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

Macroautophagy is a conserved degradative process for maintaining cellular homeostasis and plays a key role in aging and various human disorders. The microtubule-associated protein 1A/1B light chain 3B (MAP1LC3B or LC3B) is commonly analyzed as a key marker for autophagosomes and as a proxy for autophagic flux. Three paralogues of the LC3 gene exist in humans: LC3A, LC3B and LC3C. The molecular function, regulation and cellular localization of LC3A and LC3C have not been investigated frequently, even if a similar function to that described for LC3B appears likely. Here, we have selectively decapacitated LC3B by three separate strategies in primary human fibroblasts and analyzed the evoked effects on LC3A, LC3B and LC3C in terms of their cellular distribution and co-localization with p62, a ubiquitin and autophagy receptor. First, treatment with pharmacological sirtuin 1 (SIRT1) inhibitors to prevent the translocation of LC3B from the nucleus into the cytosol induced an increase in cytosolic LC3C, a heightened co-localization of LC3C with p62, and an increase LC3C-dependent autophagic flux as assessed by protein lipidation. Cytosolic LC3A, however, was moderately reduced, but also more co-localized with p62. Second, siRNA-based knock-down of SIRT1 broadly reproduced these findings and increased the co-localization of LC3A and particularly LC3C with p62 in presumed autophagosomes. These effects resembled the effects of pharmacological sirtuin inhibition under normal and starvation conditions. Third, siRNA-based knock-down of total LC3B in cytosol and nucleus also induced a redistribution of LC3C as if to replace LC3B in the nucleus, but only moderately affected LC3A. Total protein expression of LC3A, LC3B, LC3C, GABARAP and GABARAP-L1 following LC3B decapacitation was unaltered. Our data indicate that nuclear trapping and other causes of LC3B functional loss in the cytosol are buffered by LC3A and actively compensated by LC3C, but not by GABARAPs. The biological relevance of the potential functional compensation of LC3B decapacitation by LC3C and LC3A warrants further study.

Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination: links between altered multifunction of enolase and neurodegenerative diseases

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ϵ-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.

The antioxidant effects of garlic saponins protect PC12 cells from hypoxia-induced damage

Hypoxia frequently occurs under several different cellular circumstances. Excess reactive oxygen species that are induced by hypoxia may result in cell injury and dysfunction. Recently, garlic has been found to possess some biological and pharmacological activities. The present study examined the effects of garlic saponins (GSP) on the survival of differentiated PC12 (dPC12) cells and the oxidative–antioxidant system. dPC12 cells were exposed to 2 % O2 in order to establish a neuronal insult model. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay and lactate dehydrogenase (LDH) release assay. The expression of selected genes (catalase (CAT), p65 and neuron-specific class III β-tubulin) was evaluated by real-time PCR and immunoblot assays. CAT activity, malondialdehyde (MDA) and 8-hydroxy-deoxyguanosine (8-OH-dG) concentrations were also determined. The data showed that hypoxia dramatically damaged dPC12 cells, while treatment with approximately 5 × 10− 2–10 ng/ml GSP improved cell viability, decreased LDH leakage and caused the cells to maintain neuronal-like characteristics in hypoxia. The production of MDA and 8-OH-dG was attenuated by GSP. CAT activity in dPC12 cells pretreated with GSP was higher than that of the hypoxic control. Moreover, GSP up-regulated CAT expression and decreased the total protein expression as well as the nuclear expression of p65 in hypoxic cells. These data indicate that GSP has antioxidant properties that can protect dPC12 cells from hypoxia-induced damage, which may be related to the up-regulation of CAT expression and activity as well as a decrease in the expression and nucleus distribution of p65 through effects on redox-sensitive signalling pathways.

A Novel AHI-1-BCR-ABL-JAK2 Interaction Complex Mediates Cellular Resistance to Tyrosine Kinase Inhibitors in CML .

Abstract 38 The molecular signature of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene originating in a multipotent hematopoietic stem cell. The BCR-ABL oncoprotein (p210BCR-ABL) has constitutively elevated tyrosine kinase activity that perturbs several signalling cascades, including the PI3K/AKT, JAK2/STAT5, NF-kB, and RAS/MAPK pathways. The current first line treatment for CML is the tyrosine kinase inhibitor imatinib mesylate (IM) that induces clinical remission in most chronic phase CML patients. However, early relapses and IM-resistant disease have emerged and are frequently associated with mutations in the BCR-ABL kinase domain. Our recent studies indicate that CML stem cells are less responsive to IM and other tyrosine kinase inhibitors and are critical target population for IM resistance. It is therefore critical to identify other therapies that target CML stem cells to prevent acquisition of resistance. One candidate target is AHI-1 (Abelson helper integration site 1), a recently discovered oncogene that is deregulated in primary leukemic stem cells from CML patients. AHI-1 contains several domains indicative of signalling functions, including an SH3 and a WD40-repeat domain. We have recently identified a novel AHI-1-BCR-ABL-JAK2 interaction complex that modulates BCR-ABL transforming activity both in vitro and in vivo and play a key role in the IM response/resistance of primary CML stem/progenitor cells. To investigate AHI-1's involvement in mediating this cellular resistance to IM and to test the comparative ability of new ABL and JAK2 inhibitors to inhibit this complex in CML cells, AHI-1 was either stably overexpressed in K562 cells by transduction of EF1a-AHI-1-IRES-YFP lentivirus or suppressed in K562 cells using RNA interference. Interestingly, an increase in cellular proliferation and colony formation and a decrease in apoptosis were observed in the presence of 1, 5 and 10 uM of IM when AHI-1 was overexpressed. Survival of these cells was similar to IM resistant K562 cells, which are highly resistant to IM in vitro and display higher AHI-1 protein expression than parental K562 cells. Suppression of AHI-1 had the opposite effect, with cells displaying heightened sensitivity to IM at concentrations as low as 1 uM. Phosphorylation and total protein expression levels of several proteins known to be involved in BCR-ABL signalling, including JAK2, STAT5, MAPK, SRC, AKT and NF-kB (P105, P50, and P65 subunits), were quantified by Western blot analysis. Elevated phosphorylation and total protein expression levels of several of these proteins were observed when AHI-1 was overexpresessed, in particular in the JAK2/STAT5 pathway and especially in the presence of Interleukin 3. Due to the strong effects AHI-1 had on this signalling cascade, we next inhibited JAK2 activity using a selective JAK2 inhibitor, TG101209, that is highly effective against the V617F mutation and inhibits JAK2 and STAT5 activities in polycythemia vera progenitor cells. AHI-1 overexpressing cells showed reduced proliferation and colony formation when treated with IM and TG101209 in combination compared to either IM or TG101209 alone. Interestingly, treatment with IM (5 uM) or dasatinib (150 nM, DA) in combination with TG101209 (100 nM) resulted in greater inhibition (81% and 85%) of CD34+ CML stem/progenitor cells from IM nonresponders (n=4), compared to the same cells treated with a combination of IM and DA (∼60%, p<0.05), as measured by colony-forming cell assays. CFSE tracking analysis of cell division in these cells further demonstrated additive antiproliferative activity as a result of combined ABL and JAK2 inhibitors. These results suggest that targeting both BCR-ABL and JAK2 activities may be a potential therapeutic option for IM resistant patients. Disclosures: No relevant conflicts of interest to declare.

Identification of AHI-1 Involvement in Mediating Cellular Resistance to Imatinib Mesylate in Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disorder arising from the neoplastic transformation of a pluripotent hematopoietic stem cell that acquires a unique BCR-ABL fusion gene. The BCR-ABL oncoprotein displays constitutively elevated tyrosine kinase activity that deregulates cellular proliferation and apoptosis control through effects on several common signal transduction cascades, including the PI3K/AKT, JAK2/STAT5, and NF-kB pathways. The current first line treatment for CML involves administration of the tyrosine kinase inhibitor imatinib mesylate (IM) that has shown promise in treating chronic phase CML patients. However, early relapses and IM-resistant disease have emerged and are frequently associated with mutations in the BCR-ABL kinase domain that affect inhibitor binding. AHI-1 (Abelson helper integration site 1) is a recently discovered oncogene that has been demonstrated to be highly deregulated in a CML cell line (K562) and in primary leukemic stem/progenitor cells from CML patients. AHI-1 contains several unique domains that are indicative of signalling functions, including both an SH3 and a WD40-repeat domain. We have recently demonstrated that overexpression of murine Ahi-1 is able to transform IL-3 dependent Baf3, resulting in cells able to grow in the absence of growth factors. When these transduced cells were injected into sublethally irradiated NOD/SCID immunodeficient mice, the mice developed leukemia, demonstrating the oncogenic properties of Ahi-1. Interestingly, these in vitro and in vivo effects can be enhanced by co-transduction of BCR-ABL in these cells. In addition, a direct interaction between AHI-1 and BCR-ABL at endogenous levels was identified in K562 cells and this protein interaction complex further mediated IM response/resistance in CML stem/progenitor cells. To further investigate AHI-1’s involvement in mediating this cellular resistance to IM, AHI-1 was either stably overexpressed in K562 cells by transduction of EF1a-AHI-1-IRES-YFP lentivirus or was suppressed in K562 cells using a lentiviral-mediated RNA interference approach. Interestingly, overexpression of AHI-1 in K562 cells significantly increased cellular survival in the presence of 1, 5 and 10 uM of IM as measured by a viability assay; survival of these cells was similar to that observed in an IM resistant K562 cell line reported to be highly resistant to IM in vitro. Furthermore, suppression of AHI-1 had the opposite effect, with cells displaying heightened sensitivity to IM at concentrations as low as 1 uM. Phosphorylation and protein expression levels of several proteins known to be involved in BCR-ABL signalling, including JAK2, STAT5, AKT and NF-kB (P105, P50, and P65 subunits), were then quantified by Western blot analysis. Interestingly, elevated phosphorylation and protein expression levels of JAK2, and STAT5 and total protein expression levels NF-kB p105/p55 subunits were observed in both the AHI-1 overexpressing K562 cells and IM resistant K562 cells, while reduced phosphorylation and protein expression of these same proteins was observed in AHI-1 suppressed K562 cells. Differential expression of phosphorylated NF-kB p65 subunit at serine 536 was observed, while total protein expression levels did not significantly differ. Phosphorylated AKT expression levels were only affected in AHI-1 suppressed K562 cells, and total AKT protein expression was not affected in AHI-1 overexpressed or suppressed cells. Interestingly, AHI-1 protein expression was highly elevated at endogenous levels in the IM resistant K562 cells relative to a parental K562 cell line. These findings suggest that AHI-1 may play an important role in mediating cellular resistance to IM through activation of several signalling proteins involved in BCR-ABL signalling pathway, including JAK2 and STAT5.

β-Adrenergic agonists regulate Na-K-ATPase via p70S6k

We have recently reported that the β-adrenergic agonist isoproterenol regulates the alveolar epithelial cell Na-K-ATPase via MAPK/extracellular signal-regulated kinase and rapamycin-sensitive pathways. Here we report that isoproterenol phosphorylated the protein S6 kinase (p70S6k) in alveolar epithelial cells, which was inhibited by both rapamycin and the MEK1/2 inhibitor U-0126. In alveolar epithelial cells transfected with a p70S6k dominant negative construct, isoproterenol did not increase Na-K-ATPase total protein expression, whereas in cells transfected with a rapamycin-resistant mutant, the isoproterenol-mediated increase in Na-K-ATPase was not prevented by rapamycin. Accordingly, we provide here first evidence that isoproterenol regulates Na-K-ATPase via p70S6k in alveolar epithelial cells.