BBA - Molecular Cell Research (v.1843, #3)
Editorial Board (i).
Oxidative stress impairs multiple regulatory events to drive persistent cytokine-stimulated STAT3 phosphorylation by Ivan H.W. Ng; Yvonne Y.C. Yeap; Lynette S.R. Ong; David A. Jans; Marie A. Bogoyevitch (483-494).
Although cytokine-driven STAT3 phosphorylation and activation are often transient, persistent activation of STAT3 is a hallmark of a range of pathologies and underpins altered transcriptional responses. As triggers in disease frequently include combined increases in inflammatory cytokine and reactive oxygen species levels, we report here how oxidative stress impacts on cytokine-driven STAT3 signal transduction events. In the model system of murine embryonic fibroblasts (MEFs), combined treatment with the interleukin-6 family cytokine Leukemia Inhibitory Factor (LIF) and hydrogen peroxide (H2O2) drove persistent STAT3 phosphorylation whereas STAT3 phosphorylation increased only transiently in response to LIF alone and was not increased by H2O2 alone. Surprisingly, increases in transcript levels of the direct STAT3 gene target SOCS3 were delayed during the combined LIF + H2O2 treatment, leading us to probe the impact of oxidative stress on STAT3 regulatory events. Indeed, LIF + H2O2 prolonged JAK activation, delayed STAT3 nuclear localisation, and caused relocalisation of nuclear STAT3 phosphatase TC-PTP (TC45) to the cytoplasm. In exploring the nuclear import/export pathways, we observed disruption of nuclear/cytoplasmic distributions of Ran and importin-α3 in cells exposed to H2O2 and the resultant reduced nuclear trafficking of classical importin-α/β-dependent protein cargoes. CRM1-mediated nuclear export persisted despite the oxidative stress insult, with sustained STAT3 Y705 phosphorylation enhancing STAT3 nuclear residency. Our studies thus reveal for the first time the striking impact of oxidative stress to sustain STAT3 phosphorylation and nuclear retention following disruption of multiple regulatory events, with significant implications for STAT3 function.
Keywords: STAT3; Oxidative stress; Cytokines; Nuclear transport;
Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells by Christina Patlaka; Heike Becker; Maria Norgård; Staffan Paulie; Annica Nordvall-Bodell; Pernilla Lång; Göran Andersson (495-507).
Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors.
Keywords: Tartrate-resistant acid phosphatase; Acp5; Adipocyte; Mesenchymal stem cells; Endocytosis; Osteoblast;
Ionizing radiation-inducible miR-494 promotes glioma cell invasion through EGFR stabilization by targeting p190B RhoGAP by Seo-Young Kwak; Ji-Sook Yang; Bu-Yeon Kim; In Hwa Bae; Young-Hoon Han (508-516).
MicroRNAs (miRNAs) play an important role in various stages of tumor progression. miR-494, which we had previously identified as a miRNA induced by ionizing radiation (IR) in the glioma cell line U-251, was observed to enhance invasion of U-251 cells by activating MMP-2. The miR-494-induced invasive potential was accompanied by, and dependent on, epidermal growth factor receptor (EGFR) upregulation and the activation of its downstream signaling constituents, Akt and ERK. The upregulation of EGFR by miR-494 involved the suppression of lysosomal protein turnover. Among the putative target proteins tested, p190B RhoGAP (p190B) was downregulated by miR-494, and its reduced expression was responsible for the increase in EGFR expression. A reporter assay using a luciferase construct containing p190B 3′-untranslated region (3′UTR) confirmed that p190B is a direct target of miR-494. Downregulation of p190B by small interfering RNA (siRNA) transfection closely mimicked the outcomes of miR-494 transfection, and showed increased EGFR expression, MMP-2 secretion, and invasion. Ectopic expression of p190B suppressed the miR-494-induced EGFR upregulation and invasion promotion, thereby suggesting that p190B depletion is critical for the invasion-promoting action of miR-494. Collectively, our results suggest a novel function for miR-494 and its potential application as a target to control invasiveness in cancer therapy.Display Omitted
Keywords: MicroRNA; Invasion; miR-494; EGFR; p190B RhoGAP;
Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms by Hande Ozgen; Nicoletta Kahya; Jenny C. de Jonge; Graham S.T. Smith; George Harauz; Dick Hoekstra; Wia Baron (517-530).
The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage.
Keywords: Oligodendrocyte; MBP; Nucleocytoplasmic shuttling; Proliferation;
Energy adaptive response during parthanatos is enhanced by PD98059 and involves mitochondrial function but not autophagy induction by Chen-Tsung Huang; Duen-Yi Huang; Chaur-Jong Hu; Dean Wu; Wan-Wan Lin (531-543).
Parthanatos is a programmed necrotic demise characteristic of ATP (adenosine triphosphate) consumption due to NAD+ (nicotinamide adenine dinucleotide) depletion by poly(ADP-ribose) polymerase 1 (PARP1)-dependent poly(ADP-ribosyl)ation on target proteins. However, how the bioenergetics is adaptively regulated during parthanatos, especially under the condition of macroautophagy deficiency, remains poorly characterized. Here, we demonstrated that the parthanatic inducer N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) triggered ATP depletion followed by recovery in mouse embryonic fibroblasts (MEFs). Notably, Atg5−/− MEFs showed great susceptibility to MNNG with disabled ATP-producing capacity. Moreover, the differential energy-adaptive responses in wild-type (WT) and Atg5−/− MEFs were unequivocally worsened by inhibition of AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and mitochondrial activity. Importantly, Atg5−/− MEFs disclosed diminished SIRT1 and mitochondrial activity essential to the energy restoration during parthanatos. Strikingly, however, parthanatos cannot be exasperated by bafilomycin A1 and MNNG neither provokes microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and p62 elimination, suggesting that parthanatos does not induce autophagic flux. Intriguingly, we reported unexpectedly that PD98059, even at low concentration insufficient to inhibit MEK, can promote mitochondrial activity and facilitate energy-restoring process during parthanatos, without modulating DNA damage responses as evidenced by PARP1 activity, p53 expression, and γH2AX (H2A histone family, member X (H2AX), phosphorylated on Serine 139) induction. Therefore, we propose that Atg5 deficiency confers an infirmity to overcome the energy crisis during parthanatos and further underscore the deficits in mitochondrial quality control, but not incapability of autophagy induction, that explain the vulnerability in Atg5-deficient cells. Collectively, our results provide a comprehensive energy perspective for an improved treatment to alleviate parthanatos-related tissue necrosis and disease progression and also provide a future direction for drug development on the basis of PD98059 as an efficacious compound against parthanatos.Display Omitted
Keywords: Parthanatos; ATP consumption; AMPK; SIRT1; Mitochondrion; PD98059;
Analysis of α3 GlyR single particle tracking in the cell membrane by Kristof Notelaers; Susana Rocha; Rik Paesen; Nick Smisdom; Ben De Clercq; Jochen C. Meier; Jean-Michel Rigo; Johan Hofkens; Marcel Ameloot (544-553).
Single particle tracking (SPT) of transmembrane receptors in the plasma membrane often reveals heterogeneous diffusion. A thorough interpretation of the displacements requires an extensive analysis suited for discrimination of different motion types present in the data. Here the diffusion pattern of the homomeric α3-containing glycine receptor (GlyR) is analyzed in the membrane of HEK 293 cells. More specifically, the influence of the α3 RNA splice variants α3K and α3L on lateral membrane diffusion of the receptor is revealed in detail. Using a combination of ensemble and local SPT analysis, free and anomalous diffusion parameters are determined. The GlyR α3 free diffusion coefficient is found to be 0.13 ± 0.01 μm2/s and both receptor variants display confined motion. The confinement probability level and residence time are significantly elevated for the α3L variant compared to the α3K variant. Furthermore, for the α3L GlyR, the presence of directed motion was also established, with a velocity matching that of saltatory vesicular transport. These findings reveal that α3 GlyRs are prone to different types of anomalous diffusion and reinforce the role of RNA splicing in determining lateral membrane trafficking.
Keywords: Glycine receptor; Alpha3 subunit; RNA splicing; Single particle tracking; Confined motion; Directed motion;
Glucose-induced dissociation of glucokinase from its regulatory protein in the nucleus of hepatocytes prior to nuclear export by Martin Tobias Kaminski; Julia Schultz; Rica Waterstradt; Markus Tiedge; Sigurd Lenzen; Simone Baltrusch (554-564).
The glucose phosphorylating enzyme glucokinase regulates glucose metabolism in the liver. Glucokinase activity is modulated by a liver-specific competitive inhibitor, the glucokinase regulatory protein (GRP), which mediates sequestration of glucokinase to the nucleus at low glucose concentrations. However, the mechanism of glucokinase nuclear export is not fully understood. In this study we investigated the dynamics of glucose-dependent interaction and translocation of glucokinase and GRP in primary hepatocytes using fluorescence resonance energy transfer, selective photoconversion and fluorescence recovery after photobleaching. The formation of the glucokinase:GRP complex in the nucleus of primary hepatocytes at 5 mmol/l glucose was significantly reduced after a 2 h incubation at 20 mmol/l glucose. The GRP was predominantly localized in the nucleus, but a mobile fraction moved between the nucleus and the cytoplasm. The glucose concentration only marginally affected GRP shuttling. In contrast, the nuclear export rate of glucokinase was significantly higher at 20 than at 5 mmol/l glucose. Thus, glucose was proven to be the driving-force for nuclear export of glucokinase in hepatocytes. Using the FLII12Pglu-700μ-δ6 glucose nanosensor it could be shown that in hepatocytes the kinetics of nuclear glucose influx, metabolism or efflux were significantly faster compared to insulin-secreting cells. The rapid equilibration kinetics of glucose flux into the nucleus facilitates dissociation of the glucokinase:GRP complex and also nuclear glucose metabolism by free glucokinase enzyme. In conclusion, we could show that a rise of glucose in the nucleus of hepatocytes releases active glucokinase from the glucokinase:GRP complex and promotes the subsequent nuclear export of glucokinase.
Keywords: Glucokinase; Glucokinase regulatory protein; Hepatocyte; Pancreatic beta cell; Fluorescence microscopy; FLII12Pglu-700μ-δ6;
The 58-kDa microspherule protein (MSP58) represses human telomerase reverse transcriptase (hTERT) gene expression and cell proliferation by interacting with telomerase transcriptional element-interacting factor (TEIF) by Che-Chia Hsu; Chang-Han Chen; Tsung-I Hsu; Jan-Jong Hung; Jiunn-Liang Ko; Bo Zhang; Yi-Chao Lee; Han-Ku Chen; Wen-Chang Chang; Ding-Yen Lin (565-579).
58-kDa microspherule protein (MSP58) plays an important role in a variety of cellular processes including transcriptional regulation, cell proliferation and oncogenic transformation. Currently, the mechanisms underlying the oncogenic effect of MSP58 are not fully understood. The human telomerase reverse transcriptase (hTERT) gene, which encodes an essential component for telomerase activity that is involved in cellular immortalization and transformation, is strictly regulated at the gene transcription level. Our previous study revealed a novel function of MSP58 in cellular senescence. Here we identify telomerase transcriptional element-interacting factor (TEIF) as a novel MSP58-interacting protein and determine the effect of MSP58 on hTERT transcription. This study thus provides evidence showing MSP58 to be a negative regulator of hTERT expression and telomerase activity. Luciferase reporter assays indicated that MSP58 could suppress the transcription of hTERT promoter. Additionally, stable overexpression of MSP58 protein in HT1080 and 293T cells decreased both endogenous hTERT expression and telomerase activity. Conversely, their upregulation was induced by MSP58 silencing. Chromatin immunoprecipitation assays showed that MSP58 binds to the hTERT proximal promoter. Furthermore, overexpression of MSP58 inhibited TEIF-mediated hTERT transactivation, telomerase activation, and cell proliferation promotion. The inhibitory effect of MSP58 occurred through inhibition of TEIF binding to DNA. Ultimately, the HT1080-implanted xenograft mouse model confirmed these cellular effects. Together, our findings provide new insights into both the biological function of MSP58 and the regulation of telomerase/hTERT expression.
Keywords: 58-kDa microspherule protein; Telomerase; Human telomerase reverse transcriptase; Telomerase transcriptional element-interacting factor;
Regulation of Hsf4b nuclear translocation and transcription activity by phosphorylation at threonine 472 by Jun Zhang; Zengyi Ma; Jiyan Wang; Shulian Li; Yaqin Zhang; Yuelin Wang; Mingli Wang; Xiaoli Feng; Xiang Liu; Guangchao Liu; Qiang Lou; Xiukun Cui; Yuanfang Ma; Zheng Dong; Yan-zhong Hu (580-589).
Hsf4b, a key regulator of postnatal lens development, is subjected to posttranslational modifications including phosphorylation. However, the phosphorylation sites in Hsf4b and their biological effects on the transcription activity of Hsf4b are poorly understood. Here we examined 17 potential phosphorylation residues in Hsf4b with alanine-scanning assays and found that a T472A mutation diminished Hsf4b-mediated expression of Hsp25 and αB-crystallin. In contrast, the phosphomimetic mutation of T472D enhanced their expression. Further investigation demonstrated that Hsf4b could interact with nuclear-transporter importin β-1 and Hsc70 via amino acids 246–320 and 320–493, respectively. T472A mutation reduced Hsf4b's interaction with importin β-1, while enhancing its interaction with Hsc70, resulting in Hsf4b cytosolic re-localization, protein instability and transcription activity attenuation. At the upstream, MEK6 was found to interact with Hsf4b and enhance Hsf4b's nuclear translocation and transcription activity, probably by phosphorylation at sites such as T472. Taken together, our results suggest that phosphorylation of Hsf4b at T472 by protein kinases such as MEK6 regulates Hsf4b interaction with the importin β-1-Hsc70 complex, resulting in blockade of its nuclear translocation and transcriptional activity of Hsf4b.
Keywords: Hsf4b; T472; Phosphorylation; Importin β-1; Hsc70; MEK6;
Epigenetic regulation of microRNA-128a expression contributes to the apoptosis-resistance of human T-cell leukaemia Jurkat cells by modulating expression of Fas-associated protein with death domain (FADD) by Nami Yamada; Shunsuke Noguchi; Minami Kumazaki; Haruka Shinohara; Kohei Miki; Tomoki Naoe; Yukihiro Akao (590-602).
Increased expression of miR-128a is often observed in acute lymphoblastic leukaemia (ALL) compared with its expression in acute myeloid leukaemia (AML). The objective of this study was to investigate the role of miR-128a, especially that in the Fas-signalling pathway, in T-cell leukaemia cells. The role of miR-128a in Fas-mediated apoptosis was examined by using Fas-activating antibody (CH-11)-susceptible Jurkat cells and -resistant Jurkat/R cells. Whereas ectopic expression of miR-128a conferred Fas-resistance on Jurkat cells by directly targeting Fas-associated protein with death domain (FADD), antagonizing miR-128a expression sensitized Jurkat/R cells to the Fas-mediated apoptosis through derepression of FADD expression. Myeloid leukaemia HL60 and K562 cells were also CH-11-resistant, sharing a similar resistant mechanism with Jurkat/R cells. Furthermore, CH-11 induced demethylation of the promoter region of miR-128a with resultant up-regulation of miR-128a expression in Jurkat/R cells, which was shown to be a mechanism for the resistance of Jurkat/R cells to Fas-mediated apoptosis. Our results indicate that the induction of miR-128a expression by DNA demethylation is a novel mechanism of resistance to Fas-mediated apoptosis.
Keywords: miR-128a; T-cell leukaemia; FADD; Apoptosis resistance; DNA methylation;
Nitric oxide-matrix metaloproteinase-9 interactions: Biological and pharmacological significance by Shane O'Sullivan; Carlos Medina; Mark Ledwidge; Marek W. Radomski; John F. Gilmer (603-617).
Nitric oxide (NO) and matrix metalloproteinase 9 (MMP-9) levels are found to increase in inflammation states and in cancer, and their levels may be reciprocally modulated. Understanding interactions between NO and MMP-9 is of biological and pharmacological relevance and may prove crucial in designing new therapeutics. The reciprocal interaction between NO and MMP-9 have been studied for nearly twenty years but to our knowledge, are yet to be the subject of a review. This review provides a summary of published data regarding the complex and sometimes contradictory effects of NO on MMP-9. We also analyse molecular mechanisms modulating and mediating NO-MMP-9 interactions. Finally, a potential therapeutic relevance of these interactions is presented.
Keywords: Matrix metalloproteinase-9; MMP-9; Nitric oxide; Expression; Activation; Regulation;
Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer by Ting Ting Sun; Yan Wang; Hong Cheng; Xiao Hu Zhang; Juan Juan Xiang; Jie Ting Zhang; Siu Bun Sydney Yu; Tracey Amanda Martin; Lin Ye; Lai Ling Tsang; Wen Guo Jiang; Xiaohua Jiang; Hsiao Chang Chan (618-628).
How mutations or dysfunction of CFTR may increase the risk of malignancies in various tissues remains an open question. Here we report the interaction between CFTR and an adherens junction molecule, AF-6/afadin, and its involvement in the development of colon cancer. We have found that CFTR and AF-6/afadin are co-localized at the cell–cell contacts and physically interact with each other in colon cancer cell lines. Knockdown of CFTR results in reduced epithelial tightness and enhanced malignancies, with increased degradation and reduced stability of AF-6/afadin protein. The enhanced invasive phenotype of CFTR-knockdown cells can be completely reversed by either AF-6/afadin over-expression or ERK inhibitor, indicating the involvement of AF-6/MAPK pathway. More interestingly, the expression levels of CFTR and AF-6/afadin are significantly downregulated in human colon cancer tissues and lower expression of CFTR and/or AF-6/afadin is correlated with poor prognosis of colon cancer patients. The present study has revealed a previously unrecognized interaction between CFTR and AF-6/afadin that is involved in the pathogenesis of colon cancer and indicated the potential of the two as novel markers of metastasis and prognostic predictors for human colon cancer.
Keywords: CFTR; AF-6/afadin; Adherens junction; Colon cancer; Metastasis;
Cellular iron homeostasis mediated by the Mrs4–Ccc1–Smf3 pathway is essential for mitochondrial function, morphogenesis and virulence in Candida albicans by Ning Xu; Yijie Dong; Xinxin Cheng; Qilin Yu; Kefan Qian; Jiwei Mao; Chang Jia; Xiaohui Ding; Bing Zhang; Yulu Chen; Biao Zhang; Laijun Xing; Mingchun Li (629-639).
Iron bioavailability is crucial for mitochondrial metabolism and biosynthesis. Dysregulation of cellular iron homeostasis affects multiple aspects of mitochondrial physiology and cellular processes. However, the intracellular iron trafficking pathway in Candida albicans remains unclear. In this study, we characterized the Mrs4–Ccc1–Smf3 pathway, and demonstrated its important role in maintaining cellular iron levels. Double deletion of vacuolar iron exporter SMF3 and mitochondrial iron transporter MRS4 further elevated cellular iron levels in comparison with the single MRS4 deletion. However, deletion of vacuolar iron importer CCC1 in the mrs4∆/∆ mutant restored cellular iron homeostasis to normal wild-type levels, and also normalized most of the defective phenotypes in response to various environmental stresses. Our results also suggested that both Mrs4 and Ccc1 contributed to the maintenance of mitochondrial function. The mrs4∆/∆ and mrs4∆/∆smf3∆/∆ mutants exhibited an obvious decrease in aconitase activities and mitochondrial membrane potential, whereas deletion of CCC1 in the mrs4∆/∆ mutant effectively rescued these defects. Furthermore, we also found that the Mrs4–Ccc1–Smf3 pathway was indispensable for cell-wall stability, antifungal drug tolerance, filamentous growth and virulence, supporting the novel viewpoint that mitochondria might be the promising target for better antifungal therapies. Interestingly, the addition of exogenous iron failed to rescue the defects on non-fermentable carbon sources or hyphae-inducing medium, indicating that the defects in mitochondrial respiration and filamentous development might result from the disturbance of cellular iron homeostasis rather than environmental iron deprivation. Taken together, our results propose the Mrs4–Ccc1–Smf3 pathway as a potentially attractive target for antifungal drug development.
Keywords: The Mrs4–Ccc1–Smf3 pathway; Mitochondrial function; Filamentous development; Virulence; Candida albicans;
Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kB-dependent signaling by Paola Castri; Yang-ja Lee; Todd Ponzio; Dragan Maric; Maria Spatz; Joliet Bembry; John Hallenbeck (640-651).
Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24 kDa (PARP-124) and an 89 kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regard to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of INOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of “ischemia”.Green areas (left and right) indicate events that have been investigated in this study. The formation of the multiprotein complex between PARP-1 and NF-kB (p65+p50) is a known phenomenon in the literature (see Refs. [11,20,57]). The orange area in the middle represents a hypothetical mechanism that might explain the differential expression of downstream effectors of NF-kB and cell viability.Display Omitted
Keywords: PARP-1; ARTD1; Brain ischemia; OGD; NF-kB;
Heterodimerization of Mouse Orexin type 2 receptor variants and the effects on signal transduction by Chunmei Wang; Yanyou Pan; Rumin Zhang; Bo Bai; Jing Chen; Harpal S. Randeva (652-663).
Orexin-A and Orexin-B play important roles in many physiological processes in which Orexins orchestrate diverse downstream effects via two G-protein coupled receptors: Orexin1R and Orexin2R. Two alternative C-terminus splice variants of the mouse Orexin receptors mOX2αR and mOX2βR have recently been identified. This study explored the possibility of heterodimerization between mOX2αR and mOX2βR, and investigated novel signal transduction characteristics after stimulation. The dimerization of mOX2αR and mOX2βR was confirmed by BRET and co-immunoprecipitation assays. Meanwhile, in HEK293 cells, co-expression of mOX2αR and mOX2βR resulted in a strengthened increase in activation of ERK1/2, with maximal activation at 5 min and 100 nM. Furthermore, heterodimerization also elicits stronger intracellular Ca2 + elevation after Orexin(s) stimulation, followed by a slower decline in intracellular Ca2 + to a steady endpoint. Protein Kinase C Inhibitor significantly inhibited these downstream effects. In addition, the cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase and the T-lymphocyte activation of nuclear factor-responsive element reporter activity were significantly up-regulated after Orexin(s) stimulation. Besides, Orexin-A/-B induced a significantly higher rate of HEK293 cell proliferation in cells co-expressing mOX2αR/mOX2βR compared to the control group. Taken together, we provide conclusive evidence that mOX2αR can form a functional heterodimer with mOX2βR and this leads to increased PKC and decreased protein kinase A activity by ERK signal pathway leading to a significant increase in cell proliferation. The nature of this signaling pathway has significant implications for the role of Orexin in the regulation of physiological processes including the homeostasis of feeding.Display Omitted
Keywords: Hypocretin (Orexin); GPCR; mOX2αR; mOX2βR; Heterodimerization; BRET;
Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA by Ji-Sun Kim; Ji-Hui Seo; Sa-Ouk Kang (664-674).
Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA¯) encoding γ-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA¯ cells, the expression of a growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA¯ cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA¯ cells (YakAOE/gcsA¯) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components that act downstream in the YakA signaling pathway.
Keywords: Glutathione; YakA; Transition from growth to development; Dictyostelium discoideum;