Tyrosine Phosphorylation Of CD28 Research Articles

CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes

AbstractLipopolysaccharide (LPS) is a major gramnegative bacterial component that stimulates innate immune response and also induces B-lymphocyte activation. Recent studies have revealed that common molecular patterns of microorganisms such as LPS are recognized by toll-like receptors (TLRs). B cells have 2 known TLRs that mediate LPS signaling, TLR4 and RP105 (CD180). While TLR4 is expressed on immune cells of various types, RP105 is preferentially expressed on mature B cells. Here we demonstrate that CD19 plays a major role in regulating signal transduction through RP105. Anti-RP105 ligation induced normal proliferation of B cells from mice deficient for MyD88, an adaptor protein that mediates most TLR pathways. By contrast, the loss of CD19 resulted in modest B-cell proliferation against anti-RP105 stimulation as well as LPS stimulation. LPS induced tyrosine phosphorylation of CD19, which was RP105-dependent but TLR4-independent. CD19 formed a complex with Lyn and Vav following RP105 ligation, and CD19 expression was required for optimal Lyn activation and Vav phosphorylation. Consistently, B cells deficient for CD19 exhibited specific defect in the activation of c-Jun N-terminal kinases following RP105 ligation and LPS stimulation. In contrast, CD19 and phosphatidylinositol 3-kinase independently regulated intracellular calcium mobilization induced by anti-RP105 stimulation. Thus, signaling through the B-cell–specific LPS receptor RP105 is uniquely regulated by the B-cell–specific signaling component, Lyn/CD19/Vav complex.

Free Fatty Acids Shift Insulin-induced Hepatocyte Proliferation towards CD95-dependent Apoptosis

Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an activation of the epidermal growth factor receptor (EGFR) and subsequent cell proliferation (1). Free fatty acids (FFAs) are known to induce lipoapoptosis in liver cells in a c-Jun-NH2-terminal kinase (JNK)-dependent, but death receptor-independent way (2). As non-alcoholic steatohepatitis (NASH) is associated with hyperinsulinemia and increased FFA-blood levels, the interplay between insulin and FFA was studied with regard to hepatocyte proliferation and apoptosis in isolated rat and mouse hepatocytes. Saturated long chain FFAs induced apoptosis and JNK activation in primary rat hepatocytes, but did not activate the CD95 (Fas, APO-1) system, whereas insulin triggered EGFR activation and hepatocyte proliferation. Coadministration of insulin and FFAs, however, abolished hepatocyte proliferation and triggered CD95-dependent apoptosis due to a JNK-dependent association of the activated EGFR with CD95, subsequent CD95 tyrosine phosphorylation and formation of the death-inducing signaling complex (DISC). JNK inhibition restored the proliferative insulin effect in presence of FFAs and prevented EGFR/CD95 association, CD95 tyrosine phosphorylation and DISC formation. Likewise, in presence of FFAs insulin increased apoptosis in hepatocytes from wild type but not from Alb-Cre-FAS(fl/fl) mice, which lack functional CD95. It is concluded that FFAs can shift insulin-induced hepatocyte proliferation toward hepatocyte apoptosis by triggering a JNK signal, which allows activated EGFR to associate with CD95 and to trigger CD95-dependent apoptosis. Such phenomena may contribute to the pathogenesis of NASH.

Mechanisms of tauroursodeoxycholate-mediated inhibition of apoptosis

The hydrophilic bile acid ursodeoxycholic acid (UDCA), which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay in the treatment of cholestatic liver disease. TUDC prevents bile acid-induced cholestasis and induces choleresis. Its beneficial effects involve an increased hepatobiliary secretion as well as cytoprotective and antiapoptotic effects. The choleretic effect of TUDC largely depends on a rapid targeting and insertion of intracellularly stored transport ATPases into the canalicular membrane, such as the bile salt export pump (Bsep) and multidrug resistance protein-2 (Mrp2) [1]. It was recently shown that the TUDC-induced signaling involves phosphorylation of the epidermal growth factor receptor (EGFR) at tyrosine 845 and 1173, but not Tyr1045. Tyr845, a known Src kinase target, triggers an activating autophosphorylation at Tyr1173 of the EGFR [2]. Furthermore, c-Src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K) and Ras/Raf are activated within minutes by TUDC, which triggers downstream dual activation of extracellular signal-regulated kinases (Erks) and p38 mitogen-activated protein kinase (p38MAPK) [1-4]. Recent data indicate that integrins are a long-searched sensor for TUDC in the liver, because TUDC interacts with α5s1 integrins in the liver resulting in a conformational change with activation of s1 integrin and the initiation of downstream integrin signaling. In contrast to the previously reported s1 integrin activation in response to hypoosmolarity or insulin, TUDC-induced integrin activation occurs inside the hepatocyte and requires TUDC uptake via the basolateral Na+/taurocholate cotransporting polypeptide (Ntcp) [2]. However, other mechanisms of TUDC action may contribute to its choleretic effect under pathological cholestatic conditions. Hepatocyte apoptosis can be triggered by the retention of hydrophobic bile acids in hepatocytes by activation of ligand-independent death receptor pathways in vivo and in vitro. Toxic bile acids such as taurolithocholyl-3 sulfate (TLCS) and glycochenodeoxycholate (GCDC) have previously been shown to induce apoptosis in a CD95-dependent apoptosis, involving activation of the acidic sphingomyelinase, increased ceramide formation and activation of the protein kinase Cζ (PKCζ) followed by an oxidative stress response via serine phosphorylation of p47phox and NADPH oxidase activation. Putative inhibition of phosphatase activities by reactive oxygen species (ROS) triggers an activation of the Src family kinase Yes and activation of the c-Jun-N-terminal kinase-1/-2 (JNK). The activated Yes kinase associates with the EGFR resulting in a Yes-mediated EGFR transactivation. Activated EGFR then associates in a JNK-dependent way with CD95 and catalyzes CD95 tyrosine phosphorylation, which triggers CD95 membrane trafficking, death-inducing signaling complex (DISC) formation, i.e. association of Fas-associated death domain (FADD) and caspase 8 to the death receptor, followed by caspase 8-dependent mitochondrial amplification of pro-apoptotic signaling, which finally leads to apoptotic cell death [5]. Bile salt-induced apoptosis can be antagonized by TUDC. This cytoprotective effect of TUDC results from an inhibition of JNK activation and CD95 trafficking to the cell surface and activation of caspases 8 and 9 [6]. However, the molecular basis of TUDC-sensing with regard to anti-apoptosis is not yet settled, but may involve a TUDC-mediated inhibition of bile salt-induced phosphorylation of the MAP kinase kinase 4/7 (MKK4/7) upstream of JNK activation Furthermore, similar to cAMP, TUDC induced a protein kinase A (PKA)-sensitive serine/threonine phosphorylation of the CD95 in presence of GCDC, which was identified as an internalization signal for CD95. Inhibition of hepatocyte apoptosis by cAMP involves both PKA-dependent serine/threonine phosphorylation of the CD95 and inhibition of EGFR activation, which results in an inhibition of CD95 tyrosine phosphorylation, CD95 membrane targeting, and DISC formation. mRNA analyses show that the dual specificity MAP kinase phosphatase MKP-1, a transcriptionally regulated immediate early gene-encoded enzyme, is upregulated in response to TUDC after stimulation with GCDC for up to 60 min, which was already shown for cAMP [7]. Preliminary data suggest that TUDC-induced integrin activation is involved in mediating its anti-apoptotic effect.

Prosurvival and Immunosuppressive Functions of CD28 in the Myeloma Tumor Microenvironment

Abstract 473Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable for most patients. Myeloma cells are the transformed counterpart of the normal, bone marrow-resident long lived plasma cells (LLPC) that can survive for years to decades and are also responsible for long-term production of protective antibody titers. Critical interactions between MM and their bone marrow stromal cells (BMSC), that are important for their long term survival and chemotherapy resistance (in MM) identifies potential therapeutic targets, and are often the target for many IMIDs (thalidomide, lenalidomide). However, the specific molecular and cellular components of these interactions remain poorly characterized.These interactions directly transduce pro-survival signals to the myeloma cells as well as induce niche production of supportive soluble factors, the prototypic example being MM induction of stromal IL-6 - a key pro-MM survival cytokine. Despite this importance, the specific molecular and cellular components involved in these interactions remain poorly characterized. We have previously shown that CD28 expressed on human myeloma cells directly transduces a survival signal to MM cells upon binding its CD80/CD86 ligands on conventional (myeloid) dendritic cells (DC), and that DC preferentially co-localize with myeloma cells in the patient bone marrow niche. In our previous presentation at ASH (2010), we showed that myeloma cells interact with DCs to produce immunosuppressive factors such as IDO, and protects myeloma cells against cell death via a CD28-B7 mediated interaction. We now show that DC-IDO not only suppresses T-cell proliferation in invitro assays, but also contributes to the immunosuppressive milieu by inducing naïve T-cells to form T-regs (Fig 1). We hypothesize that while IDO activity by itself can suppress T-cell proliferation and induce T-cell growth arrest and apoptosis, the generation of T-regs by these immunosuppressive DCs (previous studies have shown a close association of T-regs with myeloma cells) form two facets of an immunosuppressive defense that myeloma cells mount against the body’s anti-myeloma immune response. [Display omitted] As we have shown in our earlier presentations at ASH, on the myeloma side, the activation of CD28 induces pro-survival responses that can be extinguished by blocking CD28-B7 interactions between myeloma cells and DCs. Now we show that CD28 activation is accompanied by rapid tyrosine phosphorylation of CD28, association of p85 (PI3K), activation of Vav-1 and increase in CD28 associated tyrosine kinase activity, as shown by immunoprecipitation, western and kinase activity assays. Our data suggests a role for SLP76 downstream of Vav1 in CD28 mediated survival of myeloma cells. Immunoassays with protein extracts from myeloma cells that were previously co-cultured with DCs and isolated using positive magnetic selection show a decrease in BLIMP1 expression (Fig 2) that correlates with published data by other groups that indicates that DC mediated increase in myeloma clonogenicity/tumorigenicity is accompanied by increases in BCL6 (a negative regulator of BLIMP1). Blocking CD28-B7 interactions between myeloma and dendritic cells reversed this decrease in BLIMP1 expression. The implications of this to myeloma survival is currently under study in our lab. [Display omitted] In summary, we propose that CD28 expressed by myeloma cells serves as a central molecular bridge within a complex and integrated cellular and soluble factor microenvironment necessary for MM cell survival. CD28 directly delivers a pro-survival signal to the myeloma cell, and by ligating CD80/CD86 on conventional DC backsignals to these stromal cells to elicit immunosuppressive enzyme IDO and inducing immunosuppressive T-regulatory cells. Although undoubtedly incomplete, this model begins to point to novel therapeutic targets for the treatment of multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

Targeting Human B-Precursor Acute Lymphoblastic Leukemia Cells with Recombinant Human CD19 Ligand

AbstractAbstract 599CD19 is a 95-kDa B-lineage restricted receptor molecule that functions as a key regulator of transmembrane signals in both B-cells and B-cell precursors. Here we report the cloning and characterization of a novel high-mobility group (HMG)-box protein as the membrane-associated natural ligand of human CD19 receptor (CD19-L) on human immature as well as mature lymphoid cells. We cloned the gene encoding CD19-L from a human thymus cDNA library by yeast two-hybrid screening using the cDNA encoding human CD19 extracellular domain (AA 1 to 273) (CD19ECD) fused to the GAL4 DNA binding domain as the bait plasmid. The cDNA for the surface membrane-associated CD19-L protein is 2290-bp in length encoding a 487-aa protein with a predicted molecular mass of 54-kDa. The comparison of the amino acid sequence of CD19-L protein with reported sequences in Genebank Database revealed that CD19-L is a new member of the HMG-box protein family. CD19-L contains two leucine-rich hydrophobic nuclear export signal (NES) motifs associated with unconventional ER- and Golgi-independent transport of nuclear/cytoplasmic secretory proteins to the surface membrane. Expression of the CD19-L gene expression is limited to the lymphocyte compartment within the human lymphohematopoietic system and particularly abundant in T-lineage cells. CD19-L displays abundant expression on immature double-positive (DP) thymocytes as well as leukemic T-cell precursors from T-lineage ALL patients corresponding to immature double-negative (DN) pro-thymocyte and DP cortico-thymocyte stages of human T-cell ontogeny. CD19-L is also expressed on B-lineage lymphoid cells at all stages of human B-cell ontogeny, including fetal liver derived biphenotypic CD2+CD19+ pro-B/T cells, pro-B cells, pre-B cells and mature B-cells. Soluble recombinant human CD19-L protein produced in a baculovirus expression system exhibited exquisite specificity for the extracellular domain of CD19 and had profound effects on apoptosis-related signaling and gene expression in CD19+ human leukemia cells. Engagement of CD19 co-receptor on B-lineage ALL cells with soluble CD19-L protein perturbed CD19-associated signaling network and triggered tyrosine phosphorylation of CD19 in a time-dependent fashion with peak phosphorylation occurring within 1–5 min. CD19-phosphorylation was associated with rapid and transient activation of SYK tyrosine kinase. Treatment of B-lineage ALL cells with 100 ng/mL CD19-L for 24 h corrupted the regulation of gene expression and altered the expression levels of 13 genes directly involved in regulation of apoptosis. We next examined the ability of CD19-L to induce apoptosis in leukemic B-cell precursors from chemotherapy-resistant CD19-positive human B-lineage ALL cell lines NALM-6 (pre-B ALL), RS4;11 (MLL-AF4+ Pro-B ALL), and ALL-1 (BCR-ABL+ Pre-pre-B ALL). CD19-L (but not control proteins CD19ECD or CD19ICD) caused apoptosis in each of these 3 ALL cell lines. As CD19-L specifically targets CD19ECD, excess CD19ECD protein was able to compete with surface CD19 receptor for CD19-L binding and thereby prevent CD19-L induced apoptosis. Excess CD19 intracellular domain protein (CD19ICD) that was included as a negative control did not affect CD19-L induced apoptosis. CD19-L was also capable of causing apoptosis in chemotherapy-resistant primary leukemic cells from relapsed CD19+ B-lineage ALL patients. This collection of experimental data provides compelling evidence that CD19-L is a potent biotherapeutic new agent candidate against CD19+ lymphoid malignancies. The identification of human CD19-L may lead to therapeutic innovation for B-lineage ALL as well as other B-lineage lymphoid malignancies by providing an effective alternative to CD19-directed monoclonal antibody-based biotherapeutic agents that have encountered several limitations in clinical settings. Disclosures:No relevant conflicts of interest to declare.

Targeting the CD28-B7 Pro-Survival Pathway In Multiple Myeloma

AbstractAbstract 132Multiple myeloma (MM), an incurable neoplasia of terminally differentiated plasma cells, are critically dependent on their interactions with bone marrow stromal cells (BMSC) for essential survival signals, growth and immunosuppressive factors. Very little is known about the specific BM cell type or the molecular elements in these interactions, an understanding of which could provide novel targets that could be interdicted to enhance conventional chemotherapy. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma tumoral expansion. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with BM cells that express B7 (CD80/CD86) such as dendritic cells (DCs, that are known to be closely associated with MM cells in the BM) or with CD86+ MM cells themselves. We had previously shown (ASH2008, #I-769) that blocking CD28-CD86 interactions between myeloma cells with high affinity B7 ligand CTLA4Ig (Abatacept®) sensitized myeloma cells to chemotherapy. Now we show that myeloma cells co-cultured with myeloid DCs in vitro derive both direct and indirect survival signals from DCs, and this can be partially blocked by commercially available reagents. Our data show that flow cytometric analysis of mononuclear cells (MNC) from BM aspirates of myeloma patients with increased CD138+ plasma cell populations (9-58%), show an increased CD11b+ (myeloid) population (20-37%) as well, which is in contrast to healthy transplant donor controls (12-15% CD11b+, 4–6% CD138+). Moreover, a larger fraction (11-47%) of the myeloma CD138+ plasma cells expressed CD28 compared to healthy control (3.3-7.7%). Also, when we analyzed gene expression datasets (NCBI #GSE5900 and GSE4204) from plasma cells (PC) of normal donors, monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SM) and newly diagnosed multiple myeloma (MM), we found a progressive increase in patients showing CD28 expression with increasing severity of disease (normal<MGUS<SM<MM) (Fig 1A). When we sorted the highest scoring MM group (n=538) into 8 genetic subgroups as defined earlier, CD28 expression was found to peak in the MF subgroup (typically associated with poor survival in myeloma patients) (Zhan et al. 2006, Blood 108, pp. 2020) relative to total population (p<0.0001) (Fig 1B). Antibody mediated activation of MM-CD28 over 48 hrs increased viability of myeloma cell line MM.1S cultured under serum starvation (3.7%) or with drugs ATO (1.9%), melphalan (18%) or dexamethasone (3.3%) to 66%, 21%, 33% and 11% respectively. Viability of MM.1S cells or primary CD138+ plasma cells (isolated from myeloma BM aspirates) cultured under serum starvation was enhanced >3 fold (p<0.001) when co-cultured with monocyte derived DCs, and in MM.1S this was partially reversed when either MM-CD28 or DC-B7 was blocked (Fig 2). Similar protection of MM.1S was also observed against a gradient of dexamethasone or melphalan. CD28 activation was accompanied by rapid tyrosine phosphorylation of CD28, association of p85 (PI3K), activation of Vav-1 and increase in CD28 associated tyrosine kinase activity, as shown by immunoprecipitation, western and kinase activity assays. We had previously shown that MM-CD28 interaction drive DC production of pro-survival factor IL-6 and immunosuppressive factor IDO via DC-B7 “backsignaling” (ASH2008 #I-769). Now we show that MM induced DC production of IL-6 (8 ng/ml) was partially inhibited in presence of CD28 blocking αCD28(Fab) fragments (3 ng/ml) or with protein kinase C (PKC) inhibitor Bisindolylmaleimide-I (2.1ng/ml). Activity of the immunosuppressive enzyme IDO in these co-cultures was completely inhibited in the presence of a novel IDO inhibitor from Incyte corporation, and this helped partially reverse IDO mediated suppression of T-cell proliferation in proliferation assays using co-culture supernatants. In conclusion, our data characterizes CD28-B7 pathway and DCs in the BM as vital for myeloma survival and also as possible targets to include in future strategies in the treatment of myeloma. [Display omitted] [Display omitted] Disclosures:Boise:University of Chicago: Patents & Royalties.

Sorafenib Activates CD95 and Promotes Autophagy and Cell Death via Src Family Kinases in Gastrointestinal Tumor Cells

Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95; the present studies have determined how sorafenib and vorinostat individually contribute to CD95 activation. Sorafenib (3-6 micromol/L) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib-induced (3 micromol/L) CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 micromol/L), or sorafenib (3 micromol/L) and vorinostat (500 nmol/L) treatment promoted higher levels of CD95 phosphorylation which correlated with DISC formation, receptor surface localization, and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to localize plasma membrane or induce autophagy. Knockdown/knockout of Src family kinases abolished sorafenib-induced CD95 tyrosine phosphorylation, DISC formation, and the induction of cell death and autophagy. Knockdown of platelet-ived growth factor receptor-beta enhanced Src Y416 and CD95 tyrosine phosphorylation, which correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased reactive oxygen species levels, and in a delayed NF kappa B-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95; however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by approximately 50%. Thus, sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation, whereas vorinostat contributes to CD95 activation via the initial facilitation of reactive oxygen species generation and subsequently of FAS-L expression.

Bile Acid-induced Epidermal Growth Factor Receptor Activation in Quiescent Rat Hepatic Stellate Cells Can Trigger Both Proliferation and Apoptosis

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47(phox) and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Czeta, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H2O2), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.

CD95 activation in the liver: Ion fluxes and oxidative signaling

Apoptosis is characterized by typical features as cell shrinkage, nuclear condensation, DNA fragmentation, and apoptotic body formation. Whereas some signs of apoptosis are cell type-and death signal-dependent, apoptotic cell volume decrease is an early and ubiquitous event and little is known about the signalling events, which are localized upstream of the plasma membrane transport steps leading to apoptotic cell volume decrease and the proapoptotic events, which are induced by osmolyte loss and cell shrinkage. Ion fluxes and oxidative signaling were recently shown to play an important role in signal transduction with respect to apoptotic cell death within the liver, as a ceramide-dependent activation of the NADPH oxidase was identified as the source of reactive oxygen species generation in rat hepatocytes upon treatment with CD95 ligand, hydrophobic bile salts or hyperosmolarity. The NADPH oxidase-derived ROS signal then allows via Yes, JNK, and EGFR activation for CD95 tyrosine phosphorylation as a prerequisite for CD95 targeting to the plasma membrane and formation of the death inducing signalling complex. Other covalent modifications such as CD95-tyrosine-nitration or CD95-serine/threonine-phosphorylation can interfere with the CD95 activation process. The findings not only provide a mechanistic explanation for the high susceptibility of dehydrated cells for apoptosis, but also give insight into the role of ion fluxes and oxidative signaling with respect to apoptotic cell death within the liver.

CD95 tyrosine phosphorylation is required for CD95 oligomerization

Proapoptotic stimuli, such as CD95 ligand and hydrophobic bile acids induce an epidermal growth factor receptor (EGFR)-catalyzed tyrosine phosphorylation of CD95-death receptor in hepatocytes, as a prerequisite for CD95-translocation to the plasma membrane, formation of the death-inducing signalling complex and execution of apoptotic cell death. However, the molecular role played by CD95 tyrosine phosphorylation remained unclear. The present study shows that CD95-tyrosine phosphorylation is required for CD95-oligomerization. Fluorescence resonance energy transfer (FRET)-analysis in Huh7 hepatoma cells, which were cotransfected with CD95-YFP/CD95-CFP revealed that stimulation of these cells with CD95 ligand, proapoptotic bile acids or hyperosmolarity resulted within 30 min in an intracellular FRET-signal, suggestive for CD95/CD95-oligomerization. After 120 min the FRET-signal was detected in the plasma membrane, indicating translocation of the CD95/CD95-oligomer to the plasma membrane. CD95/CD95-oligomerization was abolished in presence of AG1478 or a JNK-inhibitory peptide, i.e. maneuvers known to prevent EGFR-catalyzed CD95-tyrosine phosphorylation. Transfection studies with YFP/CFP-coupled CD95-mutants, which contain tyrosine/phenylalanine-exchanges in positions 232 and 291 (CD95(Y232,291F)), revealed that at least one tyrosine (Y(232,291))-phosphorylated CD95 is required for CD95/CD95-oligomerization. FRET-studies in mouse embryonic fibroblasts, which in contrast to Huh7 express endogenous CD95, revealed that EGF, but not CD95L induced EGFR-homomerization, whereas CD95 ligand, but not EGF resulted in EGFR/CD95-heteromerization. These findings suggest that EGFR-catalyzed CD95-tyrosine phosphorylation is involved in the CD95/CD95-oligomerization process, which is induced by proapoptotic stimuli and is required for apoptosis induction.

Endosomal Acidification and Activation of NADPH Oxidase Isoforms Are Upstream Events in Hyperosmolarity-induced Hepatocyte Apoptosis

Hyperosmotic exposure of rat hepatocytes induced a rapid oxidative-stress(ROS) response as an upstream signal for proapoptotic CD95 activation. This study shows that hyperosmotic ROS formation involves a rapid ceramide- and protein kinase Czeta (PKCzeta)-dependent serine phosphorylation of p47phox and subsequent activation of NADPH oxidase isoforms. Hyperosmotic p47phox phosphorylation and ROS formation were sensitive to inhibition of sphingomyelinases and were strongly blunted after knockdown of acidic sphingomyelinase (ASM) or of p47phox protein. Hyperosmolarity induced a rapid bafilomycin- and 4,4 '-diisothiocyanostilbene-2,2 '-disulfonic acid disodium salt (DIDS)-sensitive acidification of a vesicular compartment, which was accessible to endocytosed fluorescein isothiocyanate-dextran and colocalized with ASM, PKCzeta, and the NADPH oxidase isoform Nox 2 (gp91phox). Bafilomycin and DIDS prevented the hyperosmolarity-induced increase in ceramide formation, p47phox phosphorylation, and ROS formation. As shown recently (Reinehr, R., Becker, S., Höngen, A., and Häussinger, D. (2004) J. Biol. Chem. 279, 23977-23987), hyperosmolarity induced a Yes-dependent activation of JNK and the epidermal growth factor receptor (EGFR), followed by EGFR-CD95 association, EGFR-catalyzed CD95-tyrosine phosphorylation, and translocation of the EGFR-CD95 complex to the plasma membrane, where formation of the deathinducing signaling complex occurs. These proapoptotic responses were not only sensitive to inhibitors of sphingomyelinase, PKCzeta, or NADPH oxidases but also to ASM knockdown, bafilomycin, and DIDS, i.e. maneuvers largely preventing hyperosmolarity-induced endosomal acidification and/or ceramide formation. In hepatocytes from p47phox knock-out mice, hyperosmolarity failed to activate the CD95 system. The data suggest that hyperosmolarity induces endosomal acidification as an important upstream event for CD95 activation through stimulation of ASM-dependent ceramide formation and activation of NADPH oxidase isoforms.

Hyperosmotic activation of the CD95 death receptor system

Apoptosis is characterized by cell shrinkage, nuclear condensation, DNA fragmentation and apoptotic body formation. These features distinguish apoptosis from other types of cell death, such as necrosis. Whereas some signs of apoptosis, such as externalization of phosphatidylserine, altered mitochondrial function or activation of caspases are cell type- and death signal-dependent, apoptotic cell volume decrease (AVD) is an early and ubiquitous event and little is known about the signalling events, which are localized upstream of the plasma membrane transport steps leading to AVD and the proapoptotic events, which are induced by osmolyte loss and cell shrinkage. In hepatocytes hyperosmotic shrinkage sensitizes the cells towards CD95 ligand-induced apoptosis by activating the CD95 system. This complex process with a NADPH oxidase-derived reactive oxygen species signal as an important upstream event, allows via Yes, JNK and epidermal growth factor-receptor activation for CD95 tyrosine phosphorylation as a prerequisite for CD95 targeting to the plasma membrane and formation of the death inducing signalling complex. Other covalent modifications such as CD95-tyrosine-nitration or CD95-serine/threonine-phosphorylation can interfere with the CD95 activation process. The findings not only provide a mechanistic explanation for the high susceptibility of dehydrated cells for apoptosis, but also give insight into the role of AVD.

Mechanisms of immune suppression by interleukin‐10 and transforming growth factor‐β: the role of T regulatory cells

Specific immune suppression and induction of tolerance are essential processes in the regulation and circumvention of immune defence. The balance between allergen-specific type 1 regulatory (Tr1) cells and T helper (Th) 2 cells appears to be decisive in the development of allergy. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals. In contrast, there is a high frequency of allergen-specific interleukin-4 (IL-4)-secreting T cells in allergic individuals. Allergen-specific immunotherapy can induce specific Tr1 cells that abolish allergen-induced proliferation of Th1 and Th2 cells, as well as their cytokine production. Tr1 cells utilize multiple suppressor mechanisms, such as IL-10 and transforming growth factor-beta (TGF-beta) as secreted cytokines and various surface molecules, such as cytotoxic T-lymphocyte antigen 4 and programmed death-1. IL-10 only inhibits T cells stimulated by low numbers of triggered T-cell receptors, which depend on CD28 costimulation. IL-10 inhibits CD28 tyrosine phosphorylation, preventing the binding of phosphatidylinositol 3-kinase p85 and consequently inhibiting the CD28 signalling pathway. In addition, IL-10 and TGF-beta secreted by Tr1 cells skew the antibody production from immunoglobulin E (IgE) towards the non-inflammatory isotypes IgG4 and IgA, respectively. Induction of antigen-specific Tr1 cells can thus re-direct an inappropriate immune response against allergens or auto-antigens using a broad range of suppressor mechanisms.

Bile Salt–Induced Apoptosis Involves NADPH Oxidase Isoform Activation

Hydrophobic bile salts trigger a rapid oxidative stress response as an upstream event of CD95 activation and hepatocyte apoptosis. The underlying mechanisms were studied by Western blot, immunocytochemistry, protein knockdown, and fluorescence resonance energy transfer microscopy in rat hepatocytes and human hepatoma cell line 7 (Huh7). The rapid oxidative stress formation in response to taurolithocholate-3-sulfate (TLCS) was inhibited by diphenyleneiodonium, apocynin, and neopterin, suggestive for the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. TLCS induced a rapid serine phosphorylation of the regulatory subunit p47phox, which was sensitive to inhibition of sphingomyelinase and protein kinase Czeta (PKCzeta). Inhibitors of p47phox phosphorylation and p47phox protein knockdown abolished the TLCS-induced oxidative stress response and blunted subsequent CD95 activation. Consequences of TLCS-induced oxidative stress were c-Jun-N-terminal kinase activation and Yes-dependent activation of the epidermal growth factor receptor (EGFR), followed by EGFR-catalyzed CD95 tyrosine phosphorylation, formation of the death-inducing signaling complex, and execution of apoptosis. As shown by fluorescence resonance energy transfer experiments in Huh7 cells, TLCS induced a c-Jun-N-terminal kinase-dependent EGFR/CD95 association in the cytosol and trafficking of this protein complex to the plasma membrane. Inhibition of EGFR tyrosine kinase activity by AG1478 allowed for cytosolic EGFR/CD95 association, but prevented targeting of the EGFR/CD95 complex to the plasma membrane. Both processes, and TLCS-induced Yes and EGFR activation, were sensitive to inhibition of sphingomyelinase, PKCzeta, or NADPH oxidases. The data suggest that hydrophobic bile salts activate NADPH oxidase isoforms with the resulting oxidative stress response triggering activation of the CD95 system and apoptosis.

Involvement of NADPH Oxidase Isoforms and Src Family Kinases in CD95-dependent Hepatocyte Apoptosis

CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47phox. gp91phox (Nox 2) and p47phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47phox, which was sensitive to inhibitors of sphingomyelinase and protein kinase Czeta (PKCzeta). These inhibitors and p47phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKCzeta are upstream events of the CD95L-induced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N-acetylcysteine, inhibitors of NADPH oxidase, PKCzeta, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr845 and Tyr1173 but not at Tyr1045. Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKCzeta, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47phox, Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKCzeta-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation.

Fluorescence resonance energy transfer analysis of proapoptotic CD95–EGF receptor interactions in Huh7 cells†

Hyperosmolarity- and CD95 ligand (CD95L)-induced interactions between CD95 (Fas/APO-1) and the epidermal growth factor receptor (EGFR) involve EGFR-catalyzed CD95 tyrosine phosphorylation. Such interactions were studied by means of fluorescence resonance energy transfer (FRET) and CD95 receptor mutagenesis in Huh7 hepatoma cells. In cells cotransfected with EGFR-cyan fluorescent protein and CD95-yellow fluorescent protein, FRET studies showed a rapid, hyperosmolarity-induced, c-Jun-N-terminal kinase-dependent CD95-EGFR association in the cytosol with subsequent microtubule-dependent translocation of the protein complex to the plasma membrane. Inhibition of EGFR tyrosine kinase activity by AG1478 and cyclic adenosine monophosphate had no effect on hyperosmotic CD95-EGFR association in the cytosol but prevented CD95 tyrosine phosphorylation, targeting of the protein complex to the plasma membrane, and formation of the death-inducing signaling complex (DISC). The requirement of EGFR-mediated CD95 tyrosine phosphorylation for hyperosmotic and CD95L-induced CD95 membrane targeting and DISC formation was also shown in CD95 mutagenesis experiments. CD95 mutants with tyrosine-phenylalanine exchanges at positions 232 and 291 failed to translocate to the plasma membrane and to recruit Fas-associated death domain and caspase 8, although these mutants still associated with the EGFR in the cytosol in response to hyperosmolarity and CD95L. Cells transfected with these mutants were also resistant to CD95L-induced apoptosis. Single mutations of tyrosine 91, 232, and 291 failed to inhibit CD95 membrane targeting, DISC formation, or CD95L-induced apoptosis. In conclusion, we identify EGFR-CD95 interaction and phosphorylation of critical CD95 tyrosine residues as important early events in hyperosmotic and CD95L-induced CD95 activation and apoptosis induction.

NADPH Oxidase-Dependent Reactive Oxygen Species are Important to the Early Stage of CD95 Engagement in Hepatocytes *

Abstract CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91 phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47 phox . gp91 phox (Nox 2) and p47 phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47 phox , which was sensitive to inhibitors of sphingomyelinase and protein kinase Cζ (PKCζ). These inhibitors and p47 phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKCζ are upstream events of the CD95Linduced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N -acetylcysteine, inhibitors of NADPH oxidase, PKCζ, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr845 and Tyr1173 but not at Tyr1045. Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKCζ, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47 phox , Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKCζ-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation. (Hepatology 2005;42:956–958.)

Involvement of the Src family kinase yes in bile salt-induced apoptosis

Hydrophobic bile acids induce CD95 (Fas, APO-1)-dependent hepatocyte apoptosis, which involves epidermal growth factor receptor (EGFR)-catalyzed CD95 tyrosine phosphorylation. The mechanisms underlying bile salt-induced EGFR activation remain unclear. Bile acid-induced EGFR activation was studied in 24-hour cultured rat hepatocytes and perfused rat liver. The proapoptotic bile salts taurolithocholate-3-sulfate (TLCS), glycochenodesoxycholate (GCDC) and taurochenodeoxycholate (TCDC), but not taurocholate (TC), activate within 1 minute the Src kinase family member Yes, followed by an association of Yes with EGFR and subsequent EGFR activation. EGFR phosphorylation by TLCS involves tyrosines 845 and 1173 but not 1045. Yes/EGFR association and EGFR activation were sensitive to inhibition by SU6656 but not by PP-2. cAMP had no effect on TLCS and GCDC-induced Yes activation but induced Ser/Thr phosphorylation of Yes and prevented Yes/EGFR association and subsequent EGFR activation. Both SU6656 and cAMP had no effect on bile salt-induced c-Jun N-terminal kinase activation, but blocked bile salt-induced CD95 tyrosine phosphorylation, membrane trafficking of CD95, formation of the death-inducing signaling complex, and apoptosis. In 4-day cultured hepatocytes, knockdown of either Yes or EGFR strongly attenuated bile salt-induced CD95 activation and apoptosis. The data identify the Src kinase Yes as an upstream target of proapoptotic bile acids, which triggers EGFR activation, subsequent CD95 tyrosine phosphorylation, and apoptosis. The antiapoptotic effect of cAMP involves a protein kinase A-dependent inhibition of Yes/EGFR association, thereby preventing EGFR activation, which is required for CD95 activation.

Translocation of CD28 to lipid rafts and costimulation of IL-2.

Stimulation of the CD28 costimulatory receptor can lead to an increased surface lipid raft expression in T lymphocytes. Here, we demonstrate that CD28 itself is recruited to lipid rafts in both Jurkat and peripheral blood T lymphocytes. This recruitment of CD28 is triggered by engagement with either anti-CD28 mAbs or a natural ligand of CD28, B7.2 (CD86). All detectable tyrosine-phosphorylated CD28 is in the lipid raft fractions, as is all of the CD28 associated with phosphatidylinositol 3-kinase, which is recruited to CD28 by tyrosine phosphorylation. Targeting the CD28 cytoplasmic domain to lipid rafts results in its tyrosine phosphorylation, indicating that tyrosine phosphorylation of CD28 may occur after translocation to lipid rafts. Studies with Jurkat cells deficient in Lck and CD45 demonstrate that movement of CD28 into lipid rafts does not require Lck and CD45 and can occur despite reduction of CD28 tyrosine phosphorylation to below the levels of detection. Analysis of murine CD28 mutants reveals a correlation between translocation to lipid rafts and costimulation of IL-2 production. Taken together with the known importance of lipid rafts in T cell activation, these observations suggest that translocation to lipid rafts may play an important role in CD28 signaling.

The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95.

Hyperosmotic exposure of rat hepatocytes triggers epidermal growth factor receptor (EGFR) activation, which results in an activation of the CD95 system and sensitizes the cells toward apoptosis (Reinehr, R., Schliess, F., and Haüssinger, D. (2003) FASEB J. 17, 731-733). The mechanisms underlying the hyperosmotic EGFR activation were studied. Hyperosmotic exposure (405 mosm) resulted in a rapid activation of the Src kinase family members Yes, Fyn, and Lck. Hyperosmotic Yes, but not Fyn activation, was antioxidant-sensitive and was followed by a rapid Yes/EGFR association. PP-2 abolished the hyperosmotic activation of Fyn and Lck but not activation of Yes and EGFR and their association. However, these latter processes were prevented in the presence of SU6656. SU6656 and antioxidants, but not PP-2 and AG1478, also inhibited the hyperosmotic JNK activation. Cyclic AMP had no effect on hyperosmotic Yes and JNK activation but prevented EGFR/Yes association and EGFR activation in an H89-sensitive way. When the hyperosmolarity-induced Yes-EGFR protein complex started to disappear after 30 min, an association between EGFR and CD95 became apparent, which was followed by CD95 tyrosine phosphorylation and activation. SU6656 but not PP-2 also inhibited EGFR/CD95 association, CD95 tyrosine phosphorylation, CD95 membrane trafficking, and death-inducing signaling complex (DISC) formation. EGFR knockdown had no effect on hyperosmotic Yes activation but prevented CD95 tyrosine phosphorylation, membrane targeting, and DISC formation. Hyperosmotic EGFR and CD95 activation was also largely blunted following Yes knockdown. The data suggest that hyperosmotic signaling triggers an oxidative stress-dependent Yes activation, which is followed by JNK and EGFR activation and subsequent activation of the CD95 system. However, the functional relevance of hyperosmolarity-induced Fyn and Lck activation remains to be elucidated.