The effects of MK-801 on the phosphorylation of Ser338-c-Raf-MEK-ERK pathway in the rat frontal cortex

MK-801 induces psychotomimetic behavioural changes in animals. ERKs play an important role in the pathogenesis of schizophrenia and in the action of antipsychotics and psychotomimetics. We observed phosphorylation of ERK-signalling-pathway-associated molecules in the rat frontal cortex and their association with rat behaviour after MK-801 administration. After injecting 0.25-1 mg/kg MK-801, ERK phosphorylation decreased compared to vehicle treatment, and rats showed increased locomotion. After 2 mg/kg treatment, ERK phosphorylation increased and rat motility started to decrease. After treating with 4-8 mg/kg, ERK phosphorylation once again decreased and rats showed hypomotility and ataxia. ERK phosphorylation levels were maintained from 15 min to 90 min after 1 or 2 mg/kg treatment. Ser338-c-Raf and MEK phosphorylation showed similar dose-dependent and temporal patterns to those of ERK. Taken together, Ser338-c-Raf-MEK-ERK phosphorylation by MK-801 in the rat frontal cortex showed a specific pattern and may be associated with behavioural changes induced by MK-801

cAMP signaling inhibits radiation-induced ATM phosphorylation leading to the augmentation of apoptosis in human lung cancer cells

Background: The ataxia–telangiectasia mutated (ATM) protein kinase plays a central role in coordinating the cellular response to radiation-induced DNA damage. cAMP signaling regulates various cellular responses including metabolism and gene expression. This study aimed to investigate the mechanism through which cAMP signaling regulates ATM activation and cellular responses to ionizing radiation in lung cancer cells. Methods: Lung cancer cells were transfected with constitutively active stimulatory G protein (GαsQL), and irradiated with γ-rays. The phosphorylation of ATM and protein phosphatase 2A was analyzed by western blotting, and apoptosis was assessed by western blotting, flow cytometry, and TUNNEL staining. The promoter activity of NF-κB was determined by dual luciferase reporter assay. BALB/c mice were treated with forskolin to assess the effect in the lung tissue. Results: Transient expression of GαsQL significantly inhibited radiation-induced ATM phosphorylation in H1299 human lung cancer cells. Treatment with okadaic acid or knock down of PP2A B56δ subunit abolished the inhibitory effect of Gαs on radiation-induced ATM phosphorylation. Expression of GαsQL increased phosphorylation of the B56δ and PP2A activity, and inhibition of PKA blocked Gαs-induced PP2A activation. GαsQL enhanced radiation-induced cleavage of caspase-3 and PARP and increased the number of early apoptotic cells. The radiation-induced apoptosis was increased by inhibition of NF-κB using PDTC or inhibition of ATM using KU55933 or siRNA against ATM. Pretreatment of BALB/c mice with forskolin stimulated phosphorylation of PP2A B56δ, inhibited the activation of ATM and NF-κB, and augmented radiation-induced apoptosis in the lung tissue. GαsQL expression decreased the nuclear levels of the p50 and p65 subunits and NF-κB-dependent activity after γ-ray irradiation in H1299 cells. Pretreatment with prostaglandin E2 or isoproterenol increased B56δ phosphorylation, decreased radiation-induced ATM phosphorylation and increased apoptosis. Conclusions: cAMP signaling inhibits radiation-induced ATM activation by PKA-dependent activation of PP2A, and this signaling mechanism augments radiation-induced apoptosis by reducing ATM-dependent activation of NF-κB in lung cancer cells.This study was supported by a National Research Foundation (NRF) grant funded by the Korea government (MEST) (No. 2007–2001258), by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (2012R1A1A2044374), and a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (0720540).Peer Reviewe

Increased phosphorylation of Ser473-Akt, Ser9-GSK-3beta and Ser133-CREB in the rat frontal cortex after MK-801 intraperitoneal injection

GSK-3beta is regarded as playing an important part in the pathogenesis of schizophrenia and the action of psychotomimetic agents. We observed phosphorylation of molecules associated with the GSK-3beta signalling pathway in the rat brain after MK-801 injection, which induces a schizophrenia-like state in humans. Ser9-GSK-3beta phosphorylation was increased after injection of 1 mg/kg MK-801 in the rat frontal cortex but not in the hippocampus or cerebellum. This increase peaked at 30 min and was maintained until 90 min after injection. The phosphorylation showed a dose-dependent increase up to 1 mg/kg MK-801, followed by a decrease at higher dosage. Furthermore, phosphorylation of Ser473-Akt and Ser133-CREB showed similar temporal, dose-dependent and regionally specific patterns with those of Ser9-GSK-3beta. However, phosphorylation of Dvl and Ser33-beta-catenin was not affected by MK-801. These results suggest that GSK-3beta phosphorylation by MK-801 may be associated with the Akt-GSK-3beta pathway rather than with the Wnt-Dvl-GSK3beta pathway

Potential role and mechanism of IFN-gamma inducible protein-10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in rheumatoid arthritis

Introduction IFN-gamma inducible protein-10 (CXCL10), a member of the CXC chemokine family, and its receptor CXCR3 contribute to the recruitment of T cells from the blood stream into the inflamed joints and have a crucial role in perpetuating inflammation in rheumatoid arthritis (RA) synovial joints. Recently we showed the role of CXCL10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in an animal model of RA and suggested the contribution to osteoclastogenesis. We tested the effects of CXCL10 on the expression of RANKL in RA synoviocytes and T cells, and we investigated which subunit of CXCR3 contributes to RANKL expression by CXCL10. Methods Synoviocytes derived from RA patients were kept in culture for 24 hours in the presence or absence of TNF-α. CXCL10 expression was measured by reverse transcriptase polymerase chain reaction (RT-PCR) of cultured synoviocytes. Expression of RANKL was measured by RT-PCR and western blot in cultured synoviocytes with or without CXCL10 and also measured in Jurkat/Hut 78 T cells and CD4+ T cells in the presence of CXCL10 or dexamethasone. CXCL10 induced RANKL expression in Jurkat T cells was tested upon the pertussis toxin (PTX), an inhibitor of Gi subunit of G protein coupled receptor (GPCR). The synthetic siRNA for Gαi2 was used to knock down gene expression of respective proteins. Results CXCL10 expression in RA synoviocytes was increased by TNF-α. CXCL10 slightly increased RANKL expression in RA synoviocytes, but markedly increased RANKL expression in Jurkat/Hut 78 T cell or CD4+ T cell. CXCL10 augmented the expression of RANKL by 62.6%, and PTX inhibited both basal level of RANKL (from 37.4 ± 16.0 to 18.9 ± 13.0%) and CXCL10-induced RANKL expression in Jurkat T cells (from 100% to 48.6 ± 27.3%). Knock down of Gαi2 by siRNA transfection, which suppressed the basal level of RANKL (from 61.8 ± 17.9% to 31.1 ± 15.9%) and CXCL10-induced RANKL expression (from 100% to 53.1 ± 27.1%) in Jurkat T cells, is consistent with PTX, which inhibited RANKL expression. Conclusions CXCL10 increased RANKL expression in CD4+ T cells and it was mediated by Gαi subunits of CXCR3. These results indicate that CXCL10 may have a potential role in osteoclastogenesis of RA synovial tissue and subsequent joint erosion

The effects of repeated administrations of MK-801 on ERK and GSK-3beta signalling pathways in the rat frontal cortex

Repeated administrations of NMDA receptor antagonists induce behavioural changes which resemble the symptoms of schizophrenia in animals. ERK and GSK-3beta associated signalling pathways have been implicated in the pathogenesis of psychosis and in the action mechanisms of various psychotropic agents. Here, we observed the phosphorylations of ERK and GSK-3beta and related molecules in the rat frontal cortex after repeated intraperitoneal injections of MK-801, over periods of 1, 5, and 10 d. Repeated treatment with 0.5, 1, and 2 mg/kg MK-801 increased the phosphorylation levels of the MEK-ERK-p90RSK and Akt-GSK-3beta pathways and concomitantly and significantly increased CREB phosphorylation in the rat frontal cortex. However, single MK-801 treatment did not induce these significant changes. In addition, the immunoreactivities of HSP72, Bax, and PARP were not altered, which suggests that neuronal damage may not occur in the rat frontal cortex in response to chronic MK-801 treatment. These findings suggest that chronic exposure to MK-801 may induce pro-survival and anti-apoptotic activity without significant neuronal damage in the rat frontal cortex. Moreover, this adaptive change might be associated with the psychotomimetic action of MK-801

Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells

Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the a subunit of Gs (G alpha sQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked G alpha sQL-stimulated Bak reporter luciferase activity. Expression of G alpha sQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Gas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Gas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.Seo M, 2009, BIOCHEM BIOPH RES CO, V381, P153, DOI 10.1016/j.bbrc.2009.01.188Decker RH, 2008, SEMIN RESP CRIT CARE, V29, P285, DOI 10.1055/s-2008-1076748Taylor RC, 2008, NAT REV MOL CELL BIO, V9, P231, DOI 10.1038/nrm2312Kim SY, 2008, J BIOL CHEM, V283, P1350, DOI 10.1074/jbc.M702344200Youle RJ, 2008, NAT REV MOL CELL BIO, V9, P47, DOI 10.1038/nrm2308Kim SY, 2007, EXP MOL MED, V39, P583Wang W, 2007, CLIN CANCER RES, V13, P4891, DOI 10.1158/1078-0432.CCR-07-0416Siu YT, 2007, FEBS J, V274, P3224, DOI 10.1111/j.1742-4658.2007.05884.xSugimoto Y, 2007, J BIOL CHEM, V282, P11613, DOI 10.1074/jbc.R600038200Adams JM, 2007, ONCOGENE, V26, P1324, DOI 10.1038/sj.onc.1210220POTTI A, 2006, EXPERT OPIN BIOL TH, V6, P709Zhang XM, 2005, P NATL ACAD SCI USA, V102, P4459, DOI 10.1073/pnas.0501076102Yano T, 2003, KIDNEY INT, V64, P2052Lewerenz J, 2003, J NEUROCHEM, V87, P522, DOI 10.1046/j.1471-4159.2003.02019.xChen M, 2002, APOPTOSIS, V7, P313Baliga BC, 2002, HEMATOL ONCOL, V20, P63, DOI 10.1002/hon.685Wei MC, 2001, SCIENCE, V292, P727Lizcano JM, 2000, BIOCHEM J, V349, P547Gu CH, 2000, J BIOL CHEM, V275, P20726Greenlee RT, 2000, CA-CANCER J CLIN, V50, P7Geng YJ, 1999, CIRC RES, V84, P34Daaka Y, 1997, P NATL ACAD SCI USA, V94, P2180CHITTENDEN T, 1995, NATURE, V374, P733GONG JP, 1993, CANCER RES, V53, P5096CLAPHAM DE, 1993, NATURE, V365, P403CONKLIN BR, 1993, CELL, V73, P631BROWN AM, 1991, ADV EXP MED BIOL, V308, P119REID BJ, 1987, GASTROENTEROLOGY, V93, P1

흰쥐 간 IMP 탈수소 효소의 활성과 안정도에 영향을 주는 인자

IMP dehydrogenase is the key enzyme in the biosynthesis of guanine nucleotides, and is known to have close relationship with many biological phenomena including malignant transformation. Its inhibitors have been utilized in attempt to interrupt the growth of cancer cells and viruses. This experiment was performed to define the conditions for assay of IMPO activity and puriftcation of the enzyme by elucidating the effect of various factors on its activity and stability. The enzyme was prepared from partially hepatectomized rat liver by treatment with ammonium sulfate and OEAE-celluiose chromatography. The enzyme exhibited maxium activity at pH 7.0 and sharp decrease in its activity below and above pH 7.0. The enzyme required free sulfhydryl groups such as mercaptoethanol and dithioerythritol for its full activity, and mercaptoethanol inhibited 1MPO activity at the concentration above 10 mM. Among the monovalent cations tested, both K+ and Na + manifested activating effect in contrast with the inhibitory effect of Li+, and the activating effect of sodium was only about half the effect of potassium. All the divalent cations such as Mg2 '. Ca2 +, Cu2 +, Zn2 + exhibited strong inhibitory effect on IMPO activity. EOTA was added to the buffers to inhibit various phosphatases and also to remove any trace divalent actions, and did not exert any inhibitory effect on IMPO activity. Glycerol added to purification buffers to mimic intracellular environment manifested slightly inhibitory effect at 10% concentration, but the ethanol showed relatively strong inhibition. As the duration of dialysis increased, the IMPD activity was increased proportionally until 24 hours. And ammonium sulfate used for salting out IMPD also showed strong inhibitory effect, therefore complete removal of the salt are required before assaying the activity. When IMPD preparation was stored at ~60'C. ~20C. 4°C, and 25°C for 7 days, only the preparation stored at ~ 60"C retained most of the initial activity, and that stored at 25°C lost most of its activity in 24 hours. Thiols such as dithioerythritol and mercaptoethanol exhibited stabilizing effect on IMPD stored at 4('C, and K+, glycerol, and EDTA did not show any protective effect Change of Tris buffer (pH 7.4) buffer neither to phosphate nor to pH 8.0 improved the stability of IMPD

Influences of Nonmyristoylated alpha Subunit of Gi2 on Adenylate Cyclase Signaling Pathway in COS-7 Cells

To investigate the functional significance of myristoylation of inhibitory GlP binding protein IY. subunit, the rat eDNA encoding GiZIY. was mutated at the glycine-2 modified normally by myristoylation. The glycine was replaced with alanine or deleted to construct G2A GiZiY. and (~2-9)GzIY. mutants, respectively. The mutant and wild type GiZiY. cDNAs were expressed transiently in COS-7 cells, and the intracellular localization of the proteins were analyzed by SDS-PAGE and immunoblot. Wild type Gi2IY. protein was localized mainly on the particulate fraction, but the nonmyristoylated G2A- and (~2-9)GiZIY. proteins were localized to the cytosol. These results confirmed that myristoylation of GzIY. protein is required for its membrane binding as well as for GillY. and GOiY.. The basal level of cAMP in the COS cells transfected with Gz·iY. cDNAs ranged from 5.8 to 31 pmol/mq-proteins. When cells were treated with 10 liM isoproterenoi, the cAMP level increased by 8- to 20 fold from the basal state, but the levels in COS cells expressing nonmyristoylated mutants were lower than that of control. Forskolin-stimulated accumulation of cAMP was increased in all the cells, and it also decreased in cells expressing nonmyristoylated GizCi by 15% to 45% from the control. There was no significant alteration in the immunoreactivity of GsiY. quantitated from the immunoblot. These results suggested that the nonmyristoylated GzIY. may decrease the adenylate cyclase activity. It might be possible that there is some crosstalk between the expression of Gu« and that of adenylate cyclase