Activation Of Stress-activated Protein Kinase Research Articles

Study on the relationship between cadmium chloride-induced adrenocortical cell of guinea pig apoptosis and stress-activated protein kinase activity

Heavy metals are important environmental pollutants that affect many cellular functions. The signaling pathways that lead to apoptosis of adrenocortical cells following exposure to cadmium chloride (CdCl 2) have not yet been fully clarified. We developed a primary culture of guinea pig adrenocortical cells and treated it with various concentrations of CdCl 2 for different time periods. The apoptosis induced by CdCl 2 was detected by fluorescein isothiocyanate-labeled annexin V and propidium iodide staining using a flow cytometer. Stress-activated protein kinase (SAPK) activities were measured by immunoprecipitation and chemiluminescence assay. After 2 h of treatment, the apoptotic cell rate increased in a dose-dependent manner. The difference between the high-dose group and the control group was significant ( P<0.01). The apoptosis was also found to increase in a time-dependent manner in cells treated with 50 μmol/L CdCl 2. Among the various treatment period groups, the difference between more than 1 h-treated groups and the control group was significant ( P<0.01). The SAPK activity increased with an increase in CdCl 2 dose after 5 min of exposure. However, after 15 min of exposure to CdCl 2, the SAPK activity decreased with an increase in exposure time. Our findings suggest that the SAPK signaling pathway might play an important role in CdCl 2-induced apoptosis of adrenocortical cells.

Cigarette smoke condensate promotes cell proliferation through disturbance in cellular redox homeostasis of transformed lung epithelial type-II cells

Present study was initiated to evaluate the effects of cigarette smoke condensate (CSC) on the cellular changes at molecular levels in non-small lung carcinoma cells (A549). Cigarette smoke condensate at low concentration (0.1 μg/ml) induced cancer cell proliferation, DNA synthesis, reduced glutathione (GSH) levels and intercellular adhesion molecule-1 (ICAM-1) expression without any significant change in reactive oxygen species (ROS) and superoxide radicals (SOR) production. The increased levels of GSH and ICAM-1 due to increased γ-glutamylcysteine synthetase (γ-GCS) activity and transcriptional activation of ICAM-1 gene respectively might be via activation of p38 mitogen activated protein kinase (p38 MAPK). The induction of ICAM-1 expression and cell proliferation reflect the tumor promoting activity of low CSC concentration. On the other hand, high CSC concentration (50 μg/ml), which is doubtful to be achieved in the lungs even in the chain smokers, induced killing effects on cancer cells by increasing apoptosis, ROS and SOR production, inducing cell cycle arrest, and increased ICAM-1 levels. These changes were found to be associated with altered GSH/GSSG ratio which shifted the redox balance towards more oxidizing equivalent followed by activation of p38 MAPK and stress-activated protein kinase (SAPK) involved in signaling cascade and finally transcriptional activation of γ-GCS and ICAM-1 genes. These changes were found to be p38 and SAPK dependent.

Hydrogen peroxide induces the production of tumor necrosis factor-α in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.

Activation of endothelial cells to pathological status by down-regulation of connexin43

We investigated the effects of connexin43 (Cx43) down-regulation on endothelial function. We used two different sequences of Cx43-specific small interference RNA (siRNA) to reduce de novo synthesis of Cx43 in human aortic endothelial cells and then examined the expression profiles, proliferation activity and viability, and angiogenic potential. The involvement of mitogen-activated protein kinase signalling pathways was analysed. In parallel, the effect of inhibition of gap-junctional communication by connexin-mimetic peptides was evaluated. During the down-regulation of Cx43 by the siRNA, the cells exhibited impaired gap-junctional communication, proliferation, viability, and angiogenic potential. In addition, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor were up-regulated. Furthermore, c-jun N-terminal kinase (JNK) and its downstream target c-jun were activated, while caspase-3, p38, and extracellular signal-regulated kinase remained unchanged. Inhibition of JNK by SP600125 blocked the siRNA-induced increased expression of PAI-1 and partially recovered the impaired angiogenic potential. Short-term inhibition of Cx43 channels by connexin-mimetic peptides did not activate JNK. Down-regulation of Cx43 inhibits gap-junctional communication and activates endothelial cells to pathological status, as characterized by up-regulation of coagulatory molecules and impairment of proliferation, viability, and angiogenesis. The processes are associated with activation of JNK signalling pathways and rectified by inhibition of the activation. These results suggest that inadequate expression of Cx43 per se impairs endothelial function by the activation of stress-activated protein kinase.

3β-Hydroxysteroid-Δ24 Reductase Is a Hydrogen Peroxide Scavenger, Protecting Cells from Oxidative Stress-Induced Apoptosis

3beta-Hydroxysteroid-Delta24 reductase (DHCR24) is an endoplasmic reticulum-resident, multifunctional enzyme that possesses antiapoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H(2)O(2)) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24(-/-) mouse embryonic fibroblasts). H(2)O(2) exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase-1 and stress-activated protein kinases, such as p38 MAPK and c-Jun N-terminal kinase. Complementation of the mouse embryonic fibroblasts by adenovirus expressing DHCR24 attenuated the H(2)O(2)-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H(2)O(2) was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such antiapoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced endoplasmic reticulum stress by eliminating ROS. Finally, using in vitro-synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H(2)O(2)-scavenging activity, whereas the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H(2)O(2), thereby protecting cells from oxidative stress-induced apoptosis.

Overexpression of the dual-specificity phosphatase MKP-4/DUSP-9 protects against stress-induced insulin resistance

Insulin resistance, a hallmark of type 2 diabetes and obesity, is associated with increased activity of MAP and stress-activated protein (SAP) kinases, which results in decreased insulin signaling. Our goal was to investigate the role of MAP kinase phosphatase-4 (MKP-4) in modulating this process. We found that MKP-4 expression is up-regulated during adipocyte and myocyte differentiation in vitro and up-regulated during fasting in white adipose tissue in vivo. Overexpression of MKP-4 in 3T3-L1 cells inhibited ERK and JNK phosphorylation and, to a lesser extent, p38MAPK phosphorylation. As a result, the phosphorylation of IRS-1 serine 307 induced by anisomycin was abolished, leading to a sensitization of insulin signaling with recovery of insulin-stimulated IRS-1 tyrosine phosphorylation, IRS-1 docking with phosphatidylinositol 3-kinase, and Akt phosphorylation. MKP-4 also reversed the effect of TNF-alpha to inhibit insulin signaling; alter IL-6, Glut1 and Glut4 expression; and inhibit insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Overexpression of MKP-4 in the liver of ob/ob mice decreased ERK and JNK phosphorylation, leading to a reduction in fed and fasted glycemia, improved glucose intolerance, decreased expression of gluconeogenic and lipogenic genes, and reduced hepatic steatosis. Thus, MKP-4 has a protective effect against the development of insulin resistance through its ability to dephosphorylate and inactivate crucial mediators of stress-induced insulin resistance, such as ERK and JNK, and increasing MKP-4 activity might provide a therapy for insulin-resistant disorders.

Parkin protects against tyrosinase‐mediated dopamine neurotoxicity by suppressing stress‐activated protein kinase pathways

Parkinson's disease (PD) motor symptoms are caused by degeneration of nigrostriatal dopaminergic (DAergic) neurons. The most common causes of hereditary PD are mutations in the PARKIN gene. The ubiquitin ligase parkin has been shown to mediate neuroprotection in cell culture and in vivo, but the molecular mechanisms are not well understood. We investigated the effects of parkin in a human SH-SY5Y neuroblastoma cell culture model of PD, in which transcriptional induction of the enzyme tyrosinase causes a neurotoxic overproduction of cellular DA and its oxidative metabolites. Tyrosinase induction caused formation of reactive oxygen species in the cytosol and mitochondria, and neurotoxicity via activation of apoptotic stress-activated protein kinases and caspase 3. Stable transfection of wild-type parkin suppressed tyrosinase-induced apoptosis, and PD-associated mutations abolished the neuroprotective effect of parkin. Expression of wild-type parkin did not affect reactive oxygen species production, but attenuated the tyrosinase-induced activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as their cognate mitogen-activated protein kinase kinases. PD-associated mutations differentially affected the anti-apoptotic signaling of parkin. Thus, parkin contributes to DAergic neuroprotection by suppression of apoptotic stress-activated protein kinase pathways.

Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086–9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe.

Use of Hyperosmolar Stress to Measure Stress-Activated Protein Kinase Activation and Function in Human HTR Cells and Mouse Trophoblast Stem Cells

Embryo growth is inversely correlated with hyperosmolar stress-induced stress-activated protein kinase/jun kinase (SAPK/JNK) induction. To examine whether stress has similar effects in stem cells derived from the embryo, the authors test trophoblast stem cells. The stress response of human placental and mouse trophoblast stem cell lines are tested here. Peak phosphorylated SAPK/JNK was induced by 400 mM sorbitol at 0.5 hours. At this dose, there is an SAPK/JNK-dependent decrease in mitogenic, phosphorylated cMyc at 0.5 hours preceding an SAPK/JNK-dependent decrease in cell cycle entrance at 24 hours. At 0.5 hours, SAPK/JNK decreases terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis at sorbitol doses from 50 mM to 400 mM and induces phosphorylated cJun prior to an SAPK/JNK-dependent, approximate 8-fold increase in apoptosis by 24 hours at 400 mM. SAPK/JNK phosphorylation peaked at 0.5 to 4 hours and largely subsided by 12 hours. Thus, total SAPK/JNK exists before stress and mediates rapid, homeostatic molecular responses that become biologic consequences after phosphorylated SAPK/JNK ends. This suggests continuity in the homeostatic mechanisms and functions of SAPK/JNK in placental lineage cells during implantation, in which SAPK/JNK is completely responsible for cell cycle arrest and largely responsible for apoptosis.

Transactivator of transcription–tagged cell cycle and apoptosis regulatory protein-1 peptides suppress the growth of human breast cancer cells in vitro and in vivo

Deregulated signaling by the epidermal growth factor receptor family of proteins is encountered in human malignancies including breast cancer. Cell cycle and apoptosis-regulatory protein-1 (CARP-1), a novel, perinuclear phosphoprotein, is a regulator of apoptosis signaling by epidermal growth factor receptors. CARP-1 expression is diminished in human breast cancers, and correlates inversely with human breast cancer grades which could be attributed to increased methylation. The expression of CARP-1, on the other hand, interferes with the ability of human breast cancer cells to invade through the matrigel-coated membranes, to form colonies in the soft agar, and to grow as s.c. tumors in severe combined immunodeficiency (SCID) mice. To test whether CARP-1 is a suppressor of human breast cancer growth, we generated transactivator of transcription (TAT)-tagged CARP-1 peptides. Treatment of human breast cancer cells with affinity purified, TAT-CARP-1 1-198, 197-454, and 896-1150 peptides caused inhibition of human breast cancer cell proliferation and elevated apoptosis. In contrast, TAT-tagged enhanced green fluorescent protein or CARP-1 (1-198(Y192/F)) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1-198(Y192/F)), involves the activation of p38 stress-activated protein kinase and caspase-9. Moreover, administration of TAT-CARP-1 (1-198), but not TAT-tagged enhanced green fluorescent protein or TAT-CARP-1 (1-198(Y192/F)), inhibits growth of human breast cancer cell-derived tumor xenografts in SCID mice. We conclude that CARP-1 is a suppressor of human breast cancer growth, and its expression is diminished in tumors, in part, by methylation-dependent silencing.

Signaling Cascades Triggered by Bacterial Metabolic End Products during Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

The present studies explore the role of polymicrobial infection in the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) and analyze signaling pathways activated upon this induction. We hypothesized that activation of the cellular stress-activated mitogen-activated protein kinase (MAPK) p38 pathway would play a key role in the bacterium-mediated disruption of viral latency similar to that of previously reported results obtained with other inducers of gammaherpesvirus lytic replication. KSHV within infected BCBL-1 cells was induced to replicate following exposure to metabolic end products from gram-negative or -positive bacteria that were then simultaneously exposed to specific inhibitors of signal transduction pathways. We have determined that bacterium-mediated induction of lytic KSHV infection is significantly reduced by the inhibition of the p38 MAPK pathway. In contrast, inhibition of the phosphatidylinositol 3-kinase pathway did not impair induction of lytic replication or p38 phosphorylation. Protein kinase C, though activated, was not the major pathway used for bacterium-induced viral reactivation. Furthermore, hyperacetylation of histones 3 and 4 was detected. Collectively, our results show that metabolic end products from these pathogens induce lytic replication of KSHV in BCBL-1 cells primarily via the activation of a stress-activated MAPK pathway. Importantly, we demonstrate for the first time a mechanism by which polymicrobial bacterial infections result in KSHV reactivation and pathogenesis.

Toll-like Receptor-7 Tolerizes Malignant B Cells and Enhances Killing by Cytotoxic Agents

Chronic activation through Toll-like receptors (TLR) occurs in a number of pathologic settings, but has not been studied to the same extent as primary activation. TLR7, expressed by B cells and some dendritic cells, recognizes molecular patterns associated with viruses that can be mimicked by synthetic imidazoquinolines. In response to primary stimulation with the imidazoquinoline, S28690, human mononuclear cells produced tumor necrosis factor-alpha, but were unable to do so upon restimulation with S28690. This state of "tolerization" lasted at least 5 days. Using chronic lymphocytic leukemia B cells as a model to facilitate biochemical analysis, the tolerized state was found to be associated with altered receptor components, including down-regulated expression of TLR7 mRNA and decreased levels of interleukin-1 receptor-associated kinase 1. Tolerization was characterized by a transcriptionally regulated block in stress-activated protein kinase and nuclear factor kappaB activation, with relatively preserved activation of extracellular signal-regulated kinase (ERK). Tolerized chronic lymphocytic leukemia cells were found to be more sensitive to cytotoxic chemotherapeutic agents, in part through altered stress-activated protein kinase signaling pathways. This property of the TLR7-tolerized state may potentially be exploited in the treatment of B cell cancers.

Role of apoptotic signaling pathways in regulation of inflammatory responses to ricin in primary murine macrophages

Because of its lethal effects, ease of preparation, and ability to be delivered by aerosolization, ricin has been developed as a lethal weapon by various terrorist groups. When introduced into the pulmonary system of rodents, ricin causes pathological changes in the lung that are known to occur in acute respiratory distress syndrome (ARDS). Early response cytokines such as TNF-α and IL-1 are known to play a critical role in the pathogenesis of ARDS. Ricin induces the release of these pro-inflammatory cytokines and the transcriptional activation of the genes that encode them in vitro and in vivo. Macrophages, considered to act as upstream regulators of inflammatory cascades, may play a central role in the pathogenesis and the development of ricin-induced ARDS because of their ability to make and secrete pro-inflammatory cytokines. Exposure of primary macrophages to ricin in vitro led to activation of stress-activated protein kinases, increased expression of pro-inflammatory mRNA transcripts, subsequent increase in the synthesis and secretion of TNF-α, and apoptotic cell death. Interestingly, macrophages required the engagement of the apoptotic cascade for the maximal synthesis and release of some pro-inflammatory mediators. This work identifies a cross talk between the apoptotic and inflammatory signaling pathways induced by ricin in primary macrophages.

Fractionated and acute irradiation induced signaling in a murine tumor

The effect of fractionated doses of Co(60) gamma-irradiation (2 Gy per fraction over 5 days), as is delivered in cancer radiotherapy, was compared with acute doses of 10 and 2 Gy, in a serially transplanted mouse fibrosarcoma grown in Swiss mice. The aspects that were studied included the three major mitogen-activated protein (MAP) kinases, namely p44 MAP kinase, p38 MAP kinase, and stress-activated protein (SAP) kinase, which are known to be involved in determining the cell fate following exposure to ionizing radiation. The response of dual specificity phosphatase PAC1 which is involved in the dephosphorylation of MAP kinases was also looked at. There were significant differences in the response to different dose regimens for all the factors studied. Fractionated irradiation elicited an adaptive response with a sustained activation over 7 days of prosurvival p44 MAP kinase which was balanced by the increased activation of proapoptotic p54 SAP kinase up to 1 day post-irradiation, whereas, phosphorylated p38 MAP kinase showed a decrease at most time points. PAC1 was induced following fractionated irradiation and may be acting as a feed back regulator of p44 MAP kinase. The activation of SAP kinase after fractionated irradiation may be a stress response, whereas, constitutively activated p44 MAP kinase may play an important role in the induction of radioresistance during fractionated radiotherapy of cancer and may serve as a promising target for specific inhibitors to enhance the efficacy of radiotherapy.

Stress-induced Inactivation of the c-Myb Transcription Factor through Conjugation of SUMO-2/3 Proteins

Post-translational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play an important role in regulation of the stability and the transcriptional activity of c-Myb. Conjugation of small ubiquitin-like modifier type 1 (SUMO-1) to lysines in the negative regulatory domain strongly suppresses its transcriptional activity. Here we report conjugation of two other members of the SUMO protein family, SUMO-2 and SUMO-3, and provide evidence that this post-translational modification negatively affects transcriptional activity of c-Myb. Conjugation of SUMO-2/3 proteins is strongly enhanced by several different cellular stresses and occurs primarily on two lysines, Lys(523) and Lys(499). These lysines are in the negative regulatory domain of c-Myb and also serve as acceptor sites for SUMO-1. Stress-induced SUMO-2/3 conjugation is very rapid and independent of activation of stress-activated protein kinases of the SAPK and JNK families. PIAS-3 protein was identified as a new c-Myb-specific SUMO-E3 ligase that both catalyzes conjugation of SUMO-2/3 proteins to c-Myb and exerts a negative effect on c-Myb-induced reporter gene activation. Interestingly, co-expression of a SPRING finger mutant of PIAS-3 significantly suppresses SUMOylation of c-Myb under stress. These results argue that PIAS-3 SUMO-E3 ligase plays a critical role in stress-induced conjugation of SUMO-2/3 to c-Myb. We also detected stress-induced conjugation of SUMO-2/3 to c-Myb in hematopoietic cells at the levels of endogenously expressed proteins. Furthermore, according to the negative role of SUMO conjugation on c-Myb capacity, we have observed rapid stress-induced down-regulation of the targets genes c-myc and bcl-2 of c-Myb. Our findings demonstrate that SUMO-2/3 proteins conjugate to c-Myb and negatively regulate its activity in cells under stress.

Trypanosoma cruzi-Mediated IFN-γ-Inducible Nitric Oxide Output in Macrophages Is Regulated byiNOSmRNA Stability

Although the effects of activated macrophages (Muphi) on the intracellular parasite Trypanosoma cruzi are well documented, little is known about how host-Muphi functions are affected by this pathogen before activation. This study is aimed at assessing the capacity of T. cruzi infection to modulate J77.4 murine Muphi NO generation following IFN-gamma stimulation, and identifying mechanisms regulating this modulation. Results show that parasite infection potentiates Muphi to produce inducible NO synthase (iNOS) mRNA and protein as well as NO following IFN-gamma stimulation above IFN-gamma alone controls. This potentiation occurs through the concomitant activation of NF-kappaB, ERK1/ERK2 MAPK, and stress-activated protein kinase signaling pathways. Activation of the JAK/STAT pathway by IFN-gamma then leads to STAT1alpha translocation and the transcription of a stable iNOS mRNA species. A decreased rate of iNOS mRNA degradation results in elevated levels of iNOS protein and NO production. Maximal iNOS expression is likely achieved through NF-kappaB activation by T. cruzi, whereas iNOS mRNA stability results from ERK1/ERK2 MAPK and stress-activated protein kinase activation by the infection. Taken together, our data show that T. cruzi-infected Muphi NO generation is controlled at both pre- and posttranscriptional levels and relies on signaling pathway cross-talk. This is the first report of a parasite pathogen capable of heightening host mRNA stability.

JAB1 Determines the Response of Rheumatoid Arthritis Synovial Fibroblasts to Tumor Necrosis Factor-α

Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-alpha stimulation. Here, we show that JAB1 is a critical regulator of the TNF-alpha-mediated anti-apo-ptosis pathways in RA FLSs. We found that knockdown of JAB1 using small interfering (si)RNA led to restoration of the TNF-alpha-induced apoptosis response, reduction of nuclear factor-kappaB activity, delayed degradation of IkappaB-alpha, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-alpha to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-alpha stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-alpha can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-kappaB and JNK in RA FLSs.

Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7-docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1-Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.

Roles of TNF‐alpha and IL‐1 in mediating the inflammatory responses to ricin in macrophages

Because of its lethal effects, ease of preparation, and ability to be delivered by aerosolization, ricin has been developed as a lethal weapon by various terrorist groups. When introduced into the pulmonary system of rodents, ricin causes pathological changes in the lung that are known to occur in acute respiratory distress syndrome (ARDS). Early response cytokines such as TNF-α and IL-1 are known to play a critical role in the pathogenesis of ARDS. Ricin directly induced the release of these proinflammatory cytokines and the transcriptional activation of the genes that encode them in vitro and in vivo. Macrophages, considered to be an upstream regulator of inflammatory cascades, may play a central role in the pathogenesis and the development of ricin-induced ARDS because of their ability to make and secrete proinflammatory cytokines. Exposure of macrophages to ricin in vitro leads to activation of stress-activated protein kinases, increased expression of TNF-α and IL-1 mRNAs, and subsequent increase in the secretion of TNF-α and IL-1 proteins. We asked whether primary macrophages derived from wild-type mice and mice deficient in TNF-α and IL-1 signaling respond differently in vitro to the proinflammatory effects of ricin. We treated bone marrow-derived macrophages with graded concentrations of ricin. RNA was extracted and subjected to real-time RT-PCR analysis. Macrophages isolated from mutant mice displayed altered expression of mRNAs encoding selected inflammatory genes. These data suggest the importance of TNF-α and IL-1 in modulating ricin-mediated inflammatory responses in macrophages.

1004. c-Jun NH2-Terminal Kinase (JNK)1 and JNK2 Have Distinct Roles in Adenovirus-Mediated Transgene Expression

The c-Jun NH2-terminal kinase (JNK) signaling pathway is induced by various stress stimuli and is implicated in cell death and differentiation. However, the specific function of this pathway depends on the cell type, and the underlying mechanistic basis remains unclear. In the current study, we examined the role of JNK1 and JNK2 in adenovirus-mediated transgene expression. Using mouse embryonic fibroblasts (MEFs) deficient in either Jnk1 (Jnk1-/-) or Jnk2 (Jnk2-/-), we found that adenovirus encoding green fluorescence protein (Ad/GFP) induced much more GFP expression in Jnk2-/- MEFs than that in wild-type cells, while Jnk1-/- MEFs showed less GFP expression. We also confirmed that more GFP protein expression was observed in Ad/GFP-infected Jnk2-/- MEFs than that in wild-type or Jnk1-/- MEFs by immunoblotting. In addition, we demonstrated that adenovirus vector Ad/RGD-GFP that contains an RGD motif in the HI loop of the fiber knob expressing GFP had similar effect on GFP expression in MEFs, indicating that coxsackievirus and adenovirus receptor was not correlated with the difference between Jnk2-/- MEFs and others. Moreover, transfection of human cancer cells with the plasmid dnJNK2 encoding dominan- negative JNK2 mutant resulted in increase of GFP expression induced by Ad/GFP infection. Similarly, transfection with CMV/GFP plasmid that contains CMV promoter resulted in an increase of GFP expression in Jnk2-/- fibroblasts and dnJNK2 transfectant. These results were consitent with previous report showing that activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the CMV promoter. Furthermore, we demonstrated that adenovirus vector Ad/RSV-Luc containing the RSV promoter expressing luciferase had similar effect on transgene expression in Jnk2-/- fibroblasts and dnJNK2 transfectant, indicating that adenovirus-mediated gene expression was regulated promoter independently in our cell system. Thus, JNK1 and JNK2 play different roles in adenovirus-mediated transgene expression in MEFs and human cancer cells, and it might be useful finding for future direction of adenovirus gene therapy. Further studies will be needed to clarify the exact molecular mechanisms by which they do so.