Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalk between SA-dependent immunity and phosphate-starvation responses.

KEY MESSAGE: Selective Arabidopsis thaliana inositol phosphate kinase functions modulate response amplitudes in innate immunity by balancing signalling adjustments with phosphate homeostasis networks. Pyrophosphorylation of InsP6 generates InsP7 and/or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. As bona fide co-factors for several phytohormone networks, InsP7/InsP8 modulate key developmental processes. With requirements in transducing jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 exemplifies a versatile metabolite for crosstalks between different cellular pathways during diverse stress exposures. Here we show that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2) implicated in InsP8 biosynthesis, suppress salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, constitutive activation of defenses lead to enhanced resistance against the Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that upregulated SA-signaling sectors potentiate increased expression of several phosphate-starvation inducible (PSI)-genes, previously known in these mutants. In reciprocation, upregulated PSI-genes moderate expression amplitudes of defense-associated markers. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators in defense-phosphate homeostasis and in reprogramming stress-appropriate response intensities.

Effects of triptolide on the sphingosine kinase - Sphingosine-1-phosphate signaling pathway in colitis-associated colon cancer.

BACKGROUNDS: Triptolide (TP) exhibits effective activity against colon cancer in multiple preclinical models, but the mechanisms underlying the observed effects are not fully understood. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in the regulation of colon cancer progression. The aim of this study was to investigate the effect of TP on the sphingosine kinase (SPHK)-S1P signaling pathway in colitis-associated colon cancer. METHODS: An azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model and the THP-1 cell line were used to evaluate the therapeutic effects and mechanisms of TP in colitis-associated colon cancer (CACC). Various molecular cell biology experiments, including Western blotting, real-time PCR and immunofluorescence, were used to obtain relevant experimental data. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was also established to detect the levels of S1P in tissue and plasma. RESULTS: In the AOM/DSS mouse model, TP treatment induced a dose-dependent decrease in tumor incidence and inhibited macrophage recruitment and M2 polarization in the tumors. TP also efficiently decreased the S1P levels and SPHK1/S1PR1/S1PR2 expression and significantly inhibited activation of the S1P-mediated phosphorylation of ERK protein in macrophages. CONCLUSIONS: The results indicated that TP might influence the recruitment and polarization of tumor-associated macrophages by suppressing the SPHK-S1P signaling pathway.

Discovery and Differential Processing of HLA Class II-Restricted Minor Histocompatibility Antigen LB-PIP4K2A-1S and Its Allelic Variant by Asparagine Endopeptidase.

Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML.

Emerging Connections of S1P-Metabolizing Enzymes with Host Defense and Immunity During Virus Infections.

The sphingosine 1-phosphate (S1P) metabolic pathway is a dynamic regulator of multiple cellular and disease processes. Identification of the immune regulatory role of the sphingosine analog FTY720 led to the development of the first oral therapy for the treatment of an autoimmune disease, multiple sclerosis. Furthermore, inhibitors of sphingosine kinase (SphK), which mediate S1P synthesis, are being evaluated as a therapeutic option for the treatment of cancer. In conjunction with these captivating discoveries, S1P and S1P-metabolizing enzymes have been revealed to display vital functions during virus infections. For example, S1P lyase, which is known for metabolizing S1P, inhibits influenza virus replication by promoting antiviral type I interferon innate immune responses. In addition, both isoforms of sphingosine kinase have been shown to regulate the replication or pathogenicity of many viruses. Pro- or antiviral activities of S1P-metabolizing enzymes appear to be dependent on diverse virus-host interactions and viral pathogenesis. This review places an emphasis on summarizing the functions of S1P-metabolizing enzymes during virus infections and discusses the opportunities for designing pioneering antiviral drugs by targeting these host enzymes.

Acylglycerol Kinase Maintains Metabolic State and Immune Responses of CD8+ T Cells.

CD8+ T cell expansions and functions rely on glycolysis, but the mechanisms underlying CD8+ T cell glycolytic metabolism remain elusive. Here, we show that acylglycerol kinase (AGK) is required for the establishment and maintenance of CD8+ T cell metabolic and functional fitness. AGK deficiency dampens CD8+ T cell antitumor functions in vivo and perturbs CD8+ T cell proliferation in vitro. Activation of phosphatidylinositol-3-OH kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling, which mediates elevated CD8+ T cell glycolysis, is tightly dependent on AGK kinase activity. Mechanistically, T cell antigen receptor (TCR)- and CD28-stimulated recruitment of PTEN to the plasma membrane facilitates AGK-PTEN interaction and AGK-triggered PTEN phosphorylation, thereby restricting PTEN phosphatase activity in CD8+ T cells. Collectively, these results demonstrate that AGK maintains CD8+ T cell metabolic and functional state by restraining PTEN activity and highlight a critical role for AGK in CD8+ T cell metabolic programming and effector function.

Sphingosine kinase 2 is a negative regulator of inflammatory macrophage activation.

Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2-/- mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.

From APOBEC to ZAP: Diverse mechanisms used by cellular restriction factors to inhibit virus infections.

Antiviral restriction factors are cellular proteins that inhibit the entry, replication, or spread of viruses. These proteins are critical components of the innate immune system and function to limit the severity and host range of virus infections. Here we review the current knowledge on the mechanisms of action of several restriction factors that affect multiple viruses at distinct stages of their life cycles. For example, APOBEC3G deaminates cytosines to hypermutate reverse transcribed viral DNA; IFITM3 alters membranes to inhibit virus membrane fusion; MXA/B oligomerize on viral protein complexes to inhibit virus replication; SAMHD1 decreases dNTP intracellular concentrations to prevent reverse transcription of retrovirus genomes; tetherin prevents release of budding virions from cells; Viperin catalyzes formation of a nucleoside analogue that inhibits viral RNA polymerases; and ZAP binds virus RNAs to target them for degradation. We also discuss countermeasures employed by specific viruses against these restriction factors, and mention secondary functions of several of these factors in modulating immune responses. These important examples highlight the diverse strategies cells have evolved to combat virus infections.

A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.

Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Natural T-cell ligands that are created by genetic variants can be transferred between cells by extracellular vesicles.

CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen-presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II-negative infected or transformed cells need to be transferred to surrounding antigen-presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T-cell epitope, but rather on characteristics of the full-length protein. This mechanism of (neo)antigen transfer from HLA class II-negative cells to surrounding antigen-presenting cells may play a crucial role in induction of anti-tumor immunity.

Fas/S1P1 crosstalk via NF-κB activation in osteoclasts controls subchondral bone remodeling in murine TMJ arthritis.

Enhanced turnover of subchondral trabecular bone is a hallmark of rheumatoid arthritis (RA) and it results from an imbalance between bone resorption and bone formation activities. To investigate the formation and activation of osteoclasts which mediate bone resorption, a Fas-deficient MRL/lpr mouse model which spontaneously develops autoimmune arthritis and exhibits decreased bone mass was studied. Various assays were performed on subchondral trabecular bone of the temporomandibular joint (TMJ) from MRL/lpr mice and MRL+/+ mice. Initially, greater osteoclast production was observed in vitro from bone marrow macrophages obtained from MRL/lpr mice due to enhanced phosphorylation of NF-κB, as well as Akt and MAPK, to receptor activator of nuclear factor-κB ligand (RANKL). Expression of sphingosine 1-phosphate receptor 1 (S1P1) was also significantly upregulated in the condylar cartilage. S1P1 was found to be required for S1P-induced migration of osteoclast precursor cells and downstream signaling via Rac1. When SN50, a synthetic NF-κB-inhibitory peptide, was applied to the MRL/lpr mice, subchondral trabecular bone loss was reduced and both production of osteoclastogenesis markers and sphingosine kinase (Sphk) 1/S1P1 signaling were reduced. Thus, the present results suggest that Fas/S1P1 signaling via activation of NF-κB in osteoclast precursor cells is a key factor in the pathogenesis of RA in the TMJ.

Phosphatidylinositol 4-Phosphate 5-Kinase ß Controls Recruitment of Lipid Rafts into the Immunological Synapse.

Phosphatidylinositol 4,5-biphosphate (PIP2) is critical for T lymphocyte activation serving as a substrate for the generation of second messengers and the remodeling of actin cytoskeleton necessary for the clustering of lipid rafts, TCR, and costimulatory receptors toward the T:APC interface. Spatiotemporal analysis of PIP2 synthesis in T lymphocytes suggested that distinct isoforms of the main PIP2-generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), play a differential role on the basis of their distinct localization. In this study, we analyze the contribution of PIP5Kß to T cell activation and show that CD28 induces the recruitment of PIP5Kß to the immunological synapse, where it regulates filamin A and lipid raft accumulation, as well as T cell activation, in a nonredundant manner. Finally, we found that Vav1 and the C-terminal 83 aa of PIP5Kß are pivotal for the PIP5Kß regulatory functions in response to CD28 stimulation.

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLß2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue.

Apigenin attenuates heart injury in lipopolysaccharide-induced endotoxemic model by suppressing sphingosine kinase 1/sphingosine 1-phosphate signaling pathway.

Sepsis is a cluster of heterogeneous syndromes associated with progressive endotoxemic developments, ultimately leading to damage of multiple organs, including the heart. This study is to investigate the effects of apigenin on heart injury in lipopolysaccharide-induced endotoxemic rat model. Normal Wistar rats were randomly divided into four groups: control group, LPS group (15 mg/kg), LPS plus apigenin groups with different apigenin doses (50 mg/kg, 100 mg/kg). Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) were measured after the rats were sacrificed. SphK1/S1P signaling pathway proteins, cleaved caspase-3, cleaved caspase-9, Bax and Bcl-2 in heart were measured by Western blot. In vitro, we evaluated the protective effect of apigenin on rat embryonic heart-derived myogenic cell line H9c2 induced by LPS. Apigenin decreased serum levels of CK-MB, LDH, TNF-α, IL-6, IL-1ß. SphK1/S1P signaling pathway proteins, cleaved caspase-3, cleaved caspase-9, Bax in heart were found inhibited and Bcl-2 increased in the apigenin groups in vivo. In addition, apigenin inhibited intracellular calcium, the MAPK pathway and SphK1/S1P signaling pathway in vitro. Apigenin exerts pronounced cardioprotection in rats subjected to LPS likely through suppressing myocardial apoptosis and inflammation by inhibiting the SphK1/S1P signaling pathway.

The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases.

Macrophages switch to an anti-inflammatory, 'regulatory'-like phenotype characterized by the production of high levels of interleukin (IL)-10 and low levels of pro-inflammatory cytokines to promote the resolution of inflammation. A potential therapeutic strategy for the treatment of chronic inflammatory diseases would be to administer drugs that could induce the formation of 'regulatory'-like macrophages at sites of inflammation. In the present study, we demonstrate that the clinically approved cancer drugs bosutinib and dasatinib induce several hallmark features of 'regulatory'-like macrophages. Treatment of macrophages with bosutinib or dasatinib elevates the production of IL-10 while suppressing the production of IL-6, IL-12p40 and tumour necrosis factor α (TNFα) in response to Toll-like receptor (TLR) stimulation. Moreover, macrophages treated with bosutinib or dasatinib express higher levels of markers of 'regulatory'-like macrophages including LIGHT, SPHK1 and arginase 1. Bosutinib and dasatinib were originally developed as inhibitors of the protein tyrosine kinases Bcr-Abl and Src but we show that, surprisingly, the effects of bosutinib and dasatinib on macrophage polarization are the result of the inhibition of the salt-inducible kinases. Consistent with the present finding, bosutinib and dasatinib induce the dephosphorylation of CREB-regulated transcription co-activator 3 (CRTC3) and its nuclear translocation where it induces a cAMP-response-element-binding protein (CREB)-dependent gene transcription programme including that of IL-10. Importantly, these effects of bosutinib and dasatinib on IL-10 gene expression are lost in macrophages expressing a drug-resistant mutant of salt-inducible kinase 2 (SIK2). In conclusion, our study identifies the salt-inducible kinases as major targets of bosutinib and dasatinib that mediate the effects of these drugs on the innate immune system and provides novel mechanistic insights into the anti-inflammatory properties of these drugs.

A potent Brucella abortus 2308 Δery live vaccine allows for the differentiation between natural and vaccinated infection.

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. However, the current Brucella abortus vaccines (S19 and RB51) are deficient; they can cause abortion in pregnant animals. Moreover, when the vaccine S19 is used, tests cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent vaccine is needed. A Brucella abortus 2308 ery promoter mutant (Δery) was constructed to overcome these drawbacks. The growth of the Δery mutant was significantly attenuated in macrophages and mice and induced high protective immunity in mice. Moreover, Δery induced an anti-Brucella-specific IgG (immunoglobulin G) response and stimulated the expression of interferon-gamma (INF-γ) and interleukin-4 (IL-4). Furthermore, the expression of EryA antigen allowed for the serological differentiation between natural and vaccinated infection in mice. These results indicate that the Δery mutant is a potential attenuated live vaccine candidate against virulent Brucella abortus 2308 (S2308) infection.

The role of sphingosine 1-phosphate in inflammation and cancer.

The enzymes that catalyze formation of the bioactive sphingolipid, sphingosine 1-phosphate, sphingosine kinase 1 and 2, are predictive markers in inflammatory diseases and cancer as evidenced by data from patients, knockout mice and the use of available molecular and chemical inhibitors. Thus, there is a compelling case for therapeutic targeting of sphingosine kinase. In addition, there are several examples of functional interaction between sphingosine 1-phosphate receptors and sphingosine kinase 1 that can drive malicious amplification loops that promote cancer cell growth. These novel aspects of sphingosine 1-phosphate pathobiology are reviewed herein.

Ceramide kinase regulates TNFα-stimulated NADPH oxidase activity and eicosanoid biosynthesis in neuroblastoma cells.

A persistent inflammatory reaction is a hallmark of chronic and acute pathologies in the central nervous system (CNS) and greatly exacerbates neuronal degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNFα) plays a pivotal role in the initiation and progression of inflammatory processes provoking oxidative stress, eicosanoid biosynthesis, and the production of bioactive lipids. We established in neuronal cells that TNFα exposure dramatically increased Mg(2+)-dependent neutral sphingomyelinase (nSMase) activity thus generating the bioactive lipid mediator ceramide essential for subsequent NADPH oxidase (NOX) activation and oxidative stress. Since many of the pleiotropic effects of ceramide are attributable to its metabolites, we examined whether ceramide kinase (CerK), converting ceramide to ceramide-1-phosphate, is implicated both in NOX activation and enhanced eicosanoid production in neuronal cells. In the present study, we demonstrated that TNFα exposure of human SH-SY5Y neuroblastoma caused a profound increase in CerK activity. Depleting CerK activity using either siRNA or pharmacology completely negated NOX activation and eicosanoid biosynthesis yet, more importantly, rescued neuronal viability in the presence of TNFα. These findings provided evidence for a critical function of ceramide-1-phospate and thus CerK activity in directly linking sphingolipid metabolism to oxidative stress. This vital role of CerK in CNS inflammation could provide a novel therapeutic approach to intervene with the adverse consequences of a progressive CNS inflammation.

Inositol 1,4,5-trisphosphate 3-kinase B controls survival and prevents anergy in B cells.

Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), its reaction product, play an important role in the control of B lymphocyte fate and function in vivo. In order to investigate the fine mechanisms of Itpkb and Ins(1,3,4,5)P4 action in B cells, we crossed Itpkb(-/-) mice with transgenic mice expressing a 3-83µÎ´ B cell receptor (BCR) specific for membrane-bound MHC-I H2-K(b) and H2-K(k) molecules. On a non-deleting H2-K(d) genetic background, we show that Itpkb is important for the control of Bim protein expression and B cell survival rather than for the control of B cell development from one stage to another. Analyses of cell surface markers expression, proapoptotic Bim protein expression, in vitro survival and in vivo turnover demonstrated that BCR transgenic Itpkb(-/-) B cells exhibit an anergic phenotype with the notable exception of their enhanced antigen-induced calcium signalling. On a deleting H2-K(b) genetic background, we show that Itpkb is not essential for BCR editing or negative selection. These data establish Itpkb as an important regulator of B cell survival and anergy in vivo.

Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-kappaB activation in mouse embryonic stem cells.

SAG (sensitive to apoptosis gene; also known as RBX2 or ROC2) is a dual-function protein with antioxidant activity when acting alone or E3 ligase activity when complexed with other components of SCF (Skp1, cullins, F-box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene-trapping strategy significantly sensitized mouse embryonic stem (ES) cells to radiation, with a sensitizing enhancement rate of 1.5-1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24h after irradiation as well as enhancement of radiation-induced apoptosis. Furthermore, Sag elimination abrogated IkappaBalpha degradation leading to inhibition of NF-kappaB activation. Further detailed analysis revealed that IkappaBalpha is a direct substrate of SAG-SCF(beta-TrCP) E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NF-kappaB.

Heat shock protein 70B' (HSP70B') expression and release in response to human oxidized low density lipoprotein immune complexes in macrophages.

Heat shock proteins (HSPs) have been implicated in the activation and survival of macrophages. This study examined the role of HSP70B', a poorly characterized member of the HSP70 family, in response to oxidatively modified LDL (oxLDL) and immune complexes prepared with human oxLDL and purified human antibodies to oxLDL (oxLDL-IC) in monocytic and macrophage cell lines. Immunoblot analysis of cell lysates and conditioned medium from U937 cells treated with oxLDL alone revealed an increase in intracellular HSP70B' protein levels accompanied by a concomitant increase in HSP70B' extracellular levels. Fluorescence immunohistochemistry and confocal microscopy, however, demonstrated that oxLDL-IC stimulated the release of HSP70B', which co-localized with cell-associated oxLDL-IC. In HSP70B'-green fluorescent protein-transfected mouse RAW 264.7 cells, oxLDL-IC-induced HSP70B' co-localized with membrane-associated oxLDL-IC as well as the lipid moiety of internalized oxLDL-IC. Furthermore, the data demonstrated that HSP70B' is involved in cell survival, and this effect could be mediated by sphingosine kinase 1 (SK1) activation. An examination of regularly implicated cytokines revealed a significant relationship between HSP70B' and the release of the anti-inflammatory cytokine interleukin-10 (IL-10). Small interfering RNA knockdown of HSP70B' resulted in a corresponding decrease in SK1 mRNA levels and SK1 phosphorylation as well as increased release of IL-10. In conclusion, these findings suggest that oxLDL-IC induce the synthesis and release of HSP70B', and once stimulated, HSP70B' binds to the cell-associated and internalized lipid moiety of oxLDL-IC. The data also implicate HSP70B' in key cellular functions, such as regulation of SK1 activity and release of IL-10, which influence macrophage activation and survival.