Cytoprotection against ischemia-induced DNA cleavages and cell injuries in the rat liver by pro-vitamin C via hydrolytic conversion into ascorbate

To search a regimen for prevention of post-ischemic reperfusional (I/R) injuries, I/R in the liver was induced by 30-min clamping and subsequent unfastening of the portal vein of a rat, which underwent previous intravenous administration with ascorbic acid (Asc) of 1 mg/kg or the autooxidation-resistant pro-vitamin C, 2-O-alpha-D-glucosylated Asc (Asc2G) or 2-O-phosphorylated Asc (Asc2P) of 1 mg Asc equivalent/kg from the viewpoint of utilization of antioxidants that can promptly scavenge I/R-derived reactive oxygen species. The administration with Asc, Asc2P or Asc2G prevented some features of hepatic I/R injuries such as release of hepatic marker enzymes GOT and GPT into the blood vessel, cellular degenerative symptoms including vacuolation and cell fragmentation, and nuclear DNA strand cleavage as detected by TUNEL staining. The preventive effects on I/R injuries were in the order: Asc2G > Asc2P >> Asc. This order of preventive degrees of three anti-oxidants is partly attributable to proper efficiency of conversion to vitamin C and stability in blood stream; Asc2P was moderately converted to a free monoanion form of Asc in human serum, but, in rat serum, so efficiently converted to Asc as to undergo the resultant oxidative decomposition before reaching the liver, whereas Asc2G underwent scarce conversion to Asc in human serum but moderate conversion in rat serum, suggesting that Asc2P might be less cytoprotective against I/R injury than Asc2G in the rat liver in a way different from the human liver. In contrast Asc was so susceptible to autooxidation as to be rapidly decomposed in either rat or human serum. The concentrations of ascorbyl radicals (AscR) in serum were unchanged during I/R for sham-operated rats, but appreciably diminished time-dependently for I/R-operated rats as shown by ESR spectra. A marked increase in serum AscR occurred in rats receiving Asc, Asc2G or Asc2P, but it was time-dependently restored down to the pre-ischemic level of AscR in I/R-operated rats more rapidly than in sham-operated rats. Thus, hepatic I/R injuries were shown to be prevented more markedly by Asc2G or Asc2P than by Asc, which is attributable to efficiencies of both vitamin C conversion and subsequent AscR retention.     

Cytoprotection by pro-vitamin C against ischemic injuries in perfused rat heart together with differential activation of MAP kinase family

The cardiac muscle cells are known to be killed by ischemia-reperfusion (I/R) treatment that produce reactive oxygen species (ROS). We analyzed the function of the autooxidation-resistant pro-vitamin C, 2-O-alpha-D-glucosylated derivative (Asc2G) of ascorbic acid (Asc), in protecting against I/R injury of the heart in rat. The serum release of the intracellular enzyme CPK due to I/R injury decreased upon injection with Asc2G. Out of the mitogen-activated protein (MAP) kinase family members, MAP kinase and JNK underwent the down-regulation in contrast to up-regulation of p38 compared with the I/R-treated control in the absence of Asc2G. These data suggest important roles for differential activation of the MAP kinase family in cytoprotection against I/R injury by Asc2G.     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in Cycling Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.     

Cytoprotection of vascular endotheliocytes by phosphorylated ascorbate through suppression of oxidative stress that is generated immediately after post-anoxic reoxygenation or with alkylhydroperoxides

Vascular endotheliocytes BAE-2 underwent the gradually proceeding cell death until 48 h after reoxygenation (Reox) following 3 h anoxia (Anox), but protected by pre-Anox administration with L-ascorbic acid (Asc)-2-O-phosphate (Asc2P), an autooxidation-resistant Asc derivative, but not by Asc itself. This cytoprotection with Asc2P was achieved in a glucose (Glc)-lacking buffer more advantageously than in a Glc-containing buffer where less efficiency had been demonstrated for Asc entry into BAE-2 cells than in a Glc-lacking buffer. Superoxide anion radicals were detected explosively in the extracellular space at 2-5 min after Reox following the Anox treatment of HUVE endotheliocytes, and were thereafter retained at levels as high as approximately one-half of the maximum level until 60 min after Reox, as shown by cytochrome c reduction assay. Superoxide anions at 3 and 60 min after Reox were suppressed by pre-Anox administration with Asc2P, but not with Asc or dehydro-Asc, and were not suppressed by post-Anox administration with Asc2P; the cytoprotection may need the intracellular accumulation of the ROS-scavenging effector Asc that is converted from Asc2P until 3 min after Reox. The ROS-generator tert-butylhydroperoxide (t-BuOOH) also induced both the diminished cell viability and nuclear DNA strand cleavages of BAE-2 endotheliocytes, which were also protected dose-dependently with Asc2P. The cytoprotection was attributed to reduction of intracellular ROS including hydroperoxide and hydrogen peroxide with Asc2P as shown by fluorometry with the redox indicator CDCFH-DA. Thus Anox/Reox-induced cell death can be prevented by Asc2P that suppresses ROS-generation immediately after Reox following Anox more efficiently in the intracellular sphere rather than in the extracellular space.     

Autoimmune regulator (AIRE) gene is expressed in human activated CD4+ T-cells and regulated by mitogen-activated protein kinase pathway

The autoimmune regulator (AIRE) gene is a gene responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Here we show that AIRE is expressed in human peripheral CD4-positive T-cells, and most highly in antigen-and interleukin 2-stimulated T (IL-2T) cells. Mitogen-activated protein kinases (MAPKs), including MAPK kinase (MEK) 1/2 and p38 MAPK, were phosphorylated in IL-2T cells and the expression of the AIRE gene was inhibited by a specific p38 MAPK inhibitor (SB203580), thereby indicating that AIRE gene expression is controlled by the MAPK pathway in IL-2T cells. These data suggested the possible significance of the AIRE gene in the peripheral immune system.     

Preventive effects of phosphorylated ascorbate on ultraviolet-B induced apoptotic cell death and DNA strand cleavage through enrichment of intracellular vitamin C in skin epidermal keratinocytes

Mortality of mouse keratinocytes Pam212 that were irradiated with ultraviolet-B (UVB) was shown to be repressed by pre-irradiated administration with L-ascorbic acid (Asc) or more markedly with Asc-2-O-phosphate (Asc2P), but not with dehydroascorbic acid (DehAsc) or Asc-2-O-alpha-glucoside (Asc2G), although not repressed by post-irradiated administration. The cytoprotection by Asc2P was restricted against UVB below 5-20 mJ/cm2, and exhibited markedly by administration for a period over 2 h, which may be caused by intracellular Asc that was accumulated via dephosphorylation of Asc2P and was increased, 6-24 h after, to levels above twice as abundant as those of Asc-administration. Pre-irradiated Asc2P-administration slightly repressed a DNA ladder-like electrophoretic pattern for UVB-irradiated keratinocytes, containing the histone-bound DNA fragments as shown by ELISA assay, and appreciably repressed the DNA-3'OH cleavage terminals as shown by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain. Thus, prevention of UVB-induced cell death by Asc2P was shown to occur concurrently with inhibition of DNA cleavages and enrichment of intracellular Asc.     

Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein

We have recently reported that Trypanosoma cruzi infection protects cardiomyocytes against apoptosis induced by growth factor deprivation. Cruzipain, a major parasite antigen, reproduced this survival effect by a Bcl-2-dependent mechanism. In this study, we have investigated the molecular mechanisms of cruzipain-induced cardiomyocyte protection. Neonatal BALB/c mouse cardiac myocytes were cultured under minimum serum conditions in the presence of cruzipain or T. cruzi (Tulahuen strain). Some cultures were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 or specific inhibitors of the mitogen-activated protein kinase (MAPK) family members such as the mitogen-activated protein kinase kinase (MEK1) inhibitor PD098059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 MAPK inhibitor SB203580. Inhibition of PI3K and MEK1 but not JNK or p38 MAPK increased the apoptotic rate of cardiomyocytes treated with cruzipain. Phosphorylation of Akt, a major target of PI3K, and ERK1/2, MEK1-targets, was achieved at 15 min and 5 min, respectively. In parallel, these kinases were strongly phosphorylated by T. cruzi infection. In cultures treated with cruzipain, cleavage of caspase 3 was considerably diminished after serum starvation; Bcl-2 overexpression was inhibited by PD098059 but not by Ly294002, whereas Bad phosphorylation and Bcl-xL expression were increased and differentially modulated by both inhibitors. The results suggest that cruzipain exerts its anti-apoptotic property in cardiac myocytes at least by PI3K/Akt and MEK1/ERK1/2 signaling pathways. We further identified a differential modulation of Bcl-2 family members by these two signaling pathways.     

M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity

Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.     

Macrophage inflammatory protein-1α induces osteoclast formation by activation of the MEK/ERK/c-Fos pathway and inhibition of the p38MAPK/IRF-3/IFN-β pathway

Multiple myeloma (MM) is a bone disease that affects many individuals. It was recently reported that macrophage inflammatory protein (MIP)-1α is constitutively secreted by MM cells. MIP-1α causes bone destruction through the formation of osteoclasts (OCs). However, the molecular mechanism underlying MIP-1α-induced OC formation is not well understood. In the present study, we attempted to clarify the mechanism whereby MIP-1α induces OC formation in a mouse macrophage-like cell line comprising C7 cells. We found that MIP-1α augmented OC formation in a concentration-dependent manner; moreover, it inhibited IFN-β and ISGF3γ mRNA expression, and IFN-β secretion. MIP-1α increased the expressions of phosphorylated ERK1/2 and c-Fos and decreased those of phosphorylated p38MAPK and IRF-3. We found that the MEK1/2 inhibitor U0126 inhibited OC formation by suppressing the MEK/ERK/c-Fos pathway. SB203580 induced OC formation by upregulating c-fos mRNA expression, and SB203580 was found to inhibit IFN-β and IRF-3 mRNA expressions. The results indicate that MIP-1α induces OC formation by activating and inhibiting the MEK/ERK/c-Fos and p38MAPK/IRF-3 pathways, respectively, and suppressing IFN-β expression. These findings may be useful in the development of an OC inhibitor that targets intracellular signaling factors.     

Involvement of Mos-MEK-MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.     

MAPK signaling in H9c2 cardiomyoblasts exposed to cholesterol secoaldehyde – Role of hydrogen peroxide

3β-Hydroxy-5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known to be formed in ozone- and singlet oxygen-mediated oxidations of cholesterol, has been detected in the atherosclerotic plaque and in the brain of patients suffering from Alzheimer’s disease and Lewy body dementia. Previously, we have shown that, in H9c2 cardiomyoblasts, ChSeco induces oxidative stress followed by apoptosis involving both intrinsic and extrinsic signaling pathways. In the present study, we investigated the nature of reactive oxygen species (ROS) and its associated redox signaling in H9c2 cells upon treatment with ChSeco. Both catalase and deferoxamine, which lowered intracellular ROS, were found to alleviate the ChSeco-induced cytotoxicity. ChSeco-treated H9c2 cells showed a significant decrease in the intracellular catalase activity, suggesting the involvement of H₂O₂ in the associated cytotoxicity. Additionally, in ChSeco-exposed cells, there was a marked increase in lipid peroxidation and pre-treatment with SB 203580 (p38 MAPK inhibitor) and MEK1/2 inhibitor (ERK1/2 and JNK inhibitor) rendered protection against the cytotoxicity. An early increase in the expression of p-SAPK/JNK or delayed p38 MAPK did not alter ATF-2 but decreased c-Jun expression in these cells. Overall, these findings are consistent with MAPK signaling resulting from increased cellular H₂O₂ in ChSeco-induced cytotoxicity in cardiomyoblasts.     

Cytoprotection by bcl-2 gene transfer against ischemic liver injuries together with repressed lipid peroxidation and increased ascorbic acid in livers and serum

The maximum gene exhibition was shown to be achieved at 48 h after transfection with human bcl-2 (hbcl-2) genes built in an SV40 early promoter-based plasmid vector and HVJ-liposome for cultured rat hepatocytes. The similar procedure of hbcl-2 transfection was therefore conducted for livers in rats via the portal vein, and after 48 h followed by post-ischemic reperfusion (I/R) operation for some hepatic lobes. The I/R-induced hepatic injuries were in situ observed as both cell morphological degeneration and cellular DNA strand cleavages around capillary vessels of the ischemic liver lobes as detected by HE stain and TUNEL assay, and were biochemically observed as release of two hepatic marker enzymes AST and ALT into serum. All the I/R-induced injuries examined were appreciably repressed for rats transfected with hbcl-2; hbcl-2 was expressed in hepatocytes around the capillaries of ischemic regions such as the median lobe and the left lobe, but scarcely around those of non-ischemic regions. Thus cytoprotection against I/R-induced injuries may be attributed to the I/R-promoted expression of transferred hbcl-2 genes. The possibility was examined firstly by methylphenylindole method, which showed that I/R-enhanced lipid peroxidation in the reference vector-transfected livers were markedly repressed in the hbcl-2-transfected livers. Contents of ascorbic acid (Asc) in serum and livers of hbcl-2-transfected rats were enriched, unexpectedly, versus those of non-transfected rats, and were as abundant as 1.90-fold and 1.95- to 2.60-fold versus those in the pre-ischemic state, respectively. After I/R, an immediate decline in serum Asc occurred in hbcl-2-transfectants, and was followed by prompt restoration up to the pre-ischemic Asc levels in contrast to the unaltered lower Asc levels in non-transfectants except a transient delayed increase. Hepatic Asc contents were also diminished appreciably at the initial stage after I/R in the ischemic lobes of hbcl-2-transfectants, which however retained more abundant Asc versus non-transfectants especially at the initial I/R stage when scavenging of the oxidative stress should be most necessary for cytoprotection. The results showed a close correlation between cytoprotection by exogenously transferred hbcl-2 and repressive effects on the lipid peroxidation associated with Asc consumption or redistribution.     

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-myc gene expression and endothelial cell proliferation.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.     

Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.     

Nuclear mitogen-activated protein kinase activation by protein kinase czeta during reoxygenation after ischemic hypoxia

We examined the upstream kinases for mitogen-activated protein kinase (MAPK) activation during ischemic hypoxia and reoxygenation using H9c2 cells derived from rat cardiomyocytes. Protein kinase C (PKC)zeta, an atypical PKC isoform mainly expressed in rat heart, has been shown to act as an upstream kinase of MAPK during ischemic hypoxia and reoxygenation by analyses with PKC inhibitors, antisense DNA, a dominant negative kinase defective mutant, and constitutively active mutants of PKCzeta. Immunocytochemical observations show PKCzeta staining in the nucleus during ischemic hypoxia and reoxygenation when phosphorylated MAPK is also detected in the nucleus. This nuclear localization of PKCzeta is inhibited by treatment with wortmannin, a phosphoinositide 3-kinase inhibitor that also inhibits MAPK activation in a dose-dependent manner. This is supported by the inhibition of MAPK phosphorylation by another blocker of phosphoinositide 3-kinase, LY294002. An upstream kinase of MAPK, MEK1/2, is significantly phosphorylated 15 min after reoxygenation and observed mainly in the nucleus, whereas it is present in the cytoplasm in serum stimulation. The phosphorylation of MEK is blocked by PKC inhibitors and phosphoinositide 3-kinase inhibitors, as observed in the case of MAPK phosphorylation. These observations indicate that PKCzeta, which is activated by phosphoinositide 3-kinase, induces MAPK activation through MEK in the nucleus during reoxygenation after ischemic hypoxia.     

RAF associates with phosphorylated nuclear BubR1 during endoreduplication induced by JAK inhibition

The role of JAK signaling in cell cycle transit and maintenance of genomic stability was determined in HL-60 human myeloblastic leukemia cells. We have previously reported that a pan-JAK inhibitor caused ERK-dependent endoreduplication. In the current study we find that JAK inhibition caused nuclear re-localization of RAF-1, which could be inhibited by RAF inhibitor GW5074. GW5074 also inhibited JAK inhibitor-induced appearance of nuclear phosphorylated RAF-1(pS621RAF) and MEK, and it inhibited the JAK inhibitor-induced co-immunoprecipitation of nuclear RAF-1 and MEK. JAK inhibition also increased nuclear BubR1 phosphorylation, which was diminished by RAF inhibitor GW5074. RAF-1 and BubR1 in the nucleus co-immunoprecipitated; and GW5074 eliminated this. Furthermore, inhibiting RAF with GW5074 blocked the pan-JAK inhibitor-induced endoreduplication. These data thus show that JAK inhibition causes nuclear re-localization and phosphorylation of RAF and MEK where RAF binds BubR1 with ensuing nuclear RAF-dependent BubR1 phosphorylation. Inhibiting RAF inhibited this and endoreduplication. The results suggest that there is a JAK/RAF/MEK/BubR1 axis that can regulate genomic stability. In this hypothetical model JAK suppresses RAF/MEK phosphorylation and nuclear re-localization, but JAK inhibition induces the phosphorylations and re-localization with association of RAF and phosphorylated BubR1 in the nucleus leading to endoreduplication.Key words: endoreduplication, JAK, genomic instability, MAPK, HL-60 cells