Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells.

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

PPARβ/δ activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells.

Owing to its high fat content, the classical Western diet has a range of adverse effects on the heart, including enhanced inflammation, hypertrophy, and contractile dysfunction. Proinflammatory factors secreted by cardiac cells, which are under the transcriptional control of nuclear factor-κB (NF-κB), may contribute to heart failure and dilated cardiomyopathy. The underlying mechanisms are complex, since they are linked to systemic metabolic abnormalities and changes in cardiomyocyte phenotype. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolism and are capable of limiting myocardial inflammation and hypertrophy via inhibition of NF-κB. Since PPARβ/δ is the most prevalent PPAR isoform in the heart, we analyzed the effects of the PPARβ/δ agonist GW501516 on inflammatory parameters. A high-fat diet induced the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6, and enhanced the activity of NF-κB in the heart of mice. GW501516 abrogated this enhanced proinflammatory profile. Similar results were obtained when human cardiac AC16 cells exposed to palmitate were coincubated with GW501516. PPARβ/δ activation by GW501516 enhanced the physical interaction between PPARβ/δ and p65, which suggests that this mechanism may also interfere NF-κB transactivation capacity in the heart. GW501516-induced PPARβ/δ activation can attenuate the inflammatory response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed a high-fat diet. This is relevant, especially taking into account that PPARβ/δ has been postulated as a potential target in the treatment of obesity and the insulin resistance state.

Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression.

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis.

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.

Cell death-induced activation of epidermal growth factor receptor in keratinocytes: implications for restricting epidermal damage in dermatitis.

Recent findings have implicated Fas/Fas ligand (FasL) in mediating the death of keratinocytes in spongiotic lesions. We asked whether dying keratinocytes could potentially initiate a protective response of the skin to limit the destruction of the epidermis in the spongiotic areas. In addition to apoptosis, treatment of keratinocyte cultures in vitro with FasL triggers a profound phoshorylation of the epidermal growth factor receptor (EGFR) and of its downstream effectors ERK and protein kinase B (PKB/Akt). Using a variety of inhibitors and blocking antibodies, we demonstrated that: (i) apoptosis is required for the generation of the signal(s) leading to the activation of EGFR, ERK, and Akt; (ii) the activation of EGFR, ERK, and Akt by FasL is indeed mediated by its bona fide receptor Fas; (iii) the activation of EGFR is essential for the subsequent activation of ERK and Akt; and (iv) apoptotic keratinocytes secrete soluble EGFR ligands (including amphiregulin) that are processed from membrane-bound proligand forms by metalloproteinase(s). Our findings demonstrate a potential mechanism for the restriction and repair of spongiotic damage in eczemas.

Increased vulnerability to kainic acid-induced epileptic seizures in mice underexpressing the scaffold protein Islet-Brain 1/JIP-1.

Islet-Brain 1, also known as JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein mainly involved in the regulation of the pro-apoptotic signalling cascade mediated by c-Jun-N-terminal kinase (JNK). IB1/JIP-1 organizes JNK and upstream kinases in a complex that facilitates JNK activation. However, overexpression of IB1/JIP-1 in neurons in vitro has been reported to result in inhibition of JNK activation and protection against cellular stress and apoptosis. The occurrence and the functional significance of stress-induced modulations of IB1/JIP-1 levels in vivo are not known. We investigated the regulation of IB1/JIP-1 in mouse hippocampus after systemic administration of kainic acid (KA), in wild-type mice as well as in mice hemizygous for the gene MAPK8IP1, encoding for IB1/JIP-1. We show here that IB1/JIP-1 is upregulated transiently in the hippocampus of normal mice, reaching a peak 8 h after seizure induction. Heterozygous mutant mice underexpressing IB1/JIP-1 showed a higher vulnerability to the epileptogenic properties of KA, whereas hippocampal IB1/JIP-1 levels remained unchanged after seizure induction. Subsequently, an increasing activation of JNK in the 8 h following seizure induction was observed in IB1/JIP-1 haploinsufficient mice, which also underwent more severe excitotoxic lesions in hippocampal CA3, as assessed histologically 3 days after KA administration. Taken together, these data indicate that IB1/JIP-1 in hippocampus participates in the regulation of the neuronal response to excitotoxic stress in a level-dependent fashion.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

Loss of insulin synthesis and beta cell survival induced by oxidized LDL require activation of reticulum endoplasmic stress: a mechanism countered by HDL

Elevated circulating concentrations in modified LDL-cholesterol particles (e.g. oxidised LDL) and low levels in HDL increase not only the risk for diabetic patients to develop cardiovascular diseases but also may contribute to development and progression of diabetes by directly having adverse effects on β-cells. Chronic exposure of β-cells to 2 mM human oxidised LDL-cholesterol (oxLDL) increases the rate of apoptosis, reduce insulin biosynthesis and the secretory capacity of the cells in response to nutrients. In line with the protective role, HDL efficiently antagonised the harmful effects of ox- LDL, suggesting that low levels of HDL would be inefficient to protect β-cells against oxLDL attack in patients. Activation of endoplasmic reticulum (ER) stress is pointed out to contribute to β-cell dysfunction elicited by environmental stressors. In this study we investigated whether activation of ER stress is required for oxLDL to mediate detrimental effects on β-cells and we tested the potential antagonist properties of HDL: The mouse MIN6 insulin-secreting cells were cultured with 2 mM of LDL-cholesterol preparation (native or in vitro oxidized) in the presence or absence of 1 mM of HDL-cholesterol or the ER stress inhibitor 4-phenylbutyrate (4-PBA): Prolonged exposure of MIN6 cells to 2 mM oxLDL-cholesterol for 48 hours led to an increase in expression of ER stress markers such as ATF4, CHOP and p58 and stimulated the splicing of XBP-1 whereas, induction of these markers was not observable in the cells cultured with native LDL. Treatment of the cells with the 4-PBA chemical chaperone molecule efficiently blocked activation of the ER stress markers induced by oxLDL. The latter mediates β-cell dysfunction and apoptosis by diminishing the expression of islet brain 1 (IB1) and Bcl2. The levels of these two proteins were preserved in the cells that were co-treated with oxLDL and the 4-PBA. Consistent with this result we found that blockade of ER stress activation alleviated the loss of insulin synthesis and abolished apoptosis evoked by oxLDL. However incubation of the cells with 4-PBA did not prevent impairment of insulin secretion elicited by oxLDL, indicating that ER stress is not responsible for the oxLDL-mediated defect of insulin secretion. Co-incubation of the cells with HDL mimicked the effects of 4-PBA on the expression of IB1 and Blc2 and thereby counteracted oxLDL attacks on insulin synthesis and cell survivals. We found that HDL efficiently inhibited activation of the ER stress mediated by oxLDL: These data highlight the contribution of the ER stress in the defects of insulin synthesis and cell survivals induced by oxLDL and emphasize the potent role of HDL to counter activation of the oxLDL-mediated ER-stress activation:

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Single nucleotide polymorphisms, apoptosis, and the development of severe late adverse effects after radiotherapy.

PURPOSE: Evidence has accumulated in recent years suggestive of a genetic basis for a susceptibility to the development of radiation injury after cancer radiotherapy. The purpose of this study was to assess whether patients with severe radiation-induced sequelae (RIS; i.e., National Cancer Institute/CTCv3.0 grade, > or =3) display both a low capacity of radiation-induced CD8 lymphocyte apoptosis (RILA) in vitro and possess certain single nucleotide polymorphisms (SNP) located in candidate genes associated with the response of cells to radiation. EXPERIMENTAL DESIGN: DNA was isolated from blood samples obtained from patients (n = 399) included in the Swiss prospective study evaluating the predictive effect of in vitro RILA and RIS. SNPs in the ATM, SOD2, XRCC1, XRCC3, TGFB1, and RAD21 genes were screened in patients who experienced severe RIS (group A, n = 16) and control subjects who did not manifest any evidence of RIS (group B, n = 18). RESULTS: Overall, 13 and 21 patients were found to possess a total of <4 and > or =4 SNPs in the candidate genes. The median (range) RILA in group A was 9.4% (5.3-16.5) and 94% (95% confidence interval, 70-100) of the patients (15 of 16) had > or =4 SNPs. In group B, median (range) RILA was 25.7% (20.2-43.2) and 33% (95% confidence interval, 13-59) of patients (6 of 18) had > or =4 SNPs (P < 0.001). CONCLUSIONS: The results of this study suggest that patients with severe RIS possess 4 or more SNPs in candidate genes and low radiation-induced CD8 lymphocyte apoptosis in vitro.

Human high-density lipoprotein particles prevent activation of the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreatic beta cells.

AIMS/HYPOTHESIS: We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. MATERIALS AND METHODS: Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. CONCLUSIONS/INTERPRETATION: These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.