A properly functioning immune system is dependent on programmed cell death at virtually every stage of lymphocyte development and activity. This review addresses the phenomenon of activation-induced cell death (AICD) in T lymphocytes, in which activation through the T-cell receptor results in apoptosis. AICD can occur in a cell-autonomous manner and is influenced by the nature of the initial T-cell activation events. It plays essential roles in both central and peripheral deletion events involved in tolerance and homeostasis, although it is likely that different forms of AICD proceed via different mechanisms. For example, while AICD in peripheral T cells is often caused by the induction of expression of the death ligand, Fas ligand (CD95 ligand, FasL), it does not appear to be involved in AICD in thymocytes. This and other mechanisms of AICD are discussed. One emerging model that may complement other forms of AICD involves the inducible expression of FasL by nonlymphoid tissues in response to activated T lymphocytes. Induction of nonlymphoid FasL in this manner may serve as a sensing mechanism for immune cell infiltration, which contributes to peripheral deletion.

The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its anti-apoptotic effect.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

The role of specialized follicular helper T (T(FH)) cells in the germinal center has become well recognized, but it is less clear how effector T cells govern the extrafollicular response, the dominant pathway of high-affinity, isotype-switched autoantibody production in the MRL/MpJ-Fas(lpr) (MRL(lpr)) mouse model of lupus. MRL(lpr) mice lacking the Icos gene have impaired extrafollicular differentiation of immunoglobulin (Ig) G(+) plasma cells accompanied by defects in CXC chemokine receptor (CXCR) 4 expression, interleukin (IL) 21 secretion, and B cell helper function in CD4 T cells. These phenotypes reflect the selective loss of a population of T cells marked by down-regulation of P-selectin glycoprotein ligand 1 (PSGL-1; also known as CD162). PSGL-1(lo) T cells from MRL(lpr) mice express CXCR4, localize to extrafollicular sites, and uniquely mediate IgG production through IL-21 and CD40L. In other autoimmune strains, PSGL-1(lo) T cells are also abundant but may exhibit either a follicular or extrafollicular phenotype. Our findings define an anatomically distinct extrafollicular population of cells that regulates plasma cell differentiation in chronic autoimmunity, indicating that specialized humoral effector T cells akin to T(FH) cells can occur outside the follicle.

OBJECTIVE

To detect and quantify choroidal neovascularization (CNV) in patients with age-related macular degeneration (AMD) using optical coherence tomography (OCT) angiography.

METHODS

Observational, cross-sectional study.

METHODS

A total of 5 normal subjects and 5 subjects with neovascular AMD were included.

METHODS

A total of 5 eyes with neovascular AMD and 5 normal age-matched controls were scanned by a high-speed (100 000 A-scans/seconds) 1050-nm wavelength swept-source OCT. The macular angiography scan covered a 3 × 3-mm area and comprised 200 × 200 × 8 A-scans acquired in 3.5 seconds. Flow was detected using the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm. Motion artifacts were removed by 3-dimensional (3D) orthogonal registration and merging of 4 scans. The 3D angiography was segmented into 3 layers: inner retina (to show retinal vasculature), outer retina (to identify CNV), and choroid. En face maximum projection was used to obtain 2-dimensional angiograms from the 3 layers. The CNV area and flow index were computed from the en face OCT angiogram of the outer retinal layer. Flow (decorrelation) and structural data were combined in composite color angiograms for both en face and cross-sectional views.

METHODS

The CNV angiogram, CNV area, and CNV flow index.

RESULTS

En face OCT angiograms of CNV showed sizes and locations that were confirmed by fluorescein angiography (FA). Optical coherence tomography angiography provided more distinct vascular network patterns that were less obscured by subretinal hemorrhage. The en face angiograms also showed areas of reduced choroidal flow adjacent to the CNV in all cases and significantly reduced retinal flow in 1 case. Cross-sectional angiograms were used to visualize CNV location relative to the retinal pigment epithelium and Bruch's layer and classify type I and type II CNV. A feeder vessel could be identified in 1 case. Higher flow indexes were associated with larger CNV and type II CNV.

CONCLUSIONS

Optical coherence tomography angiography provides depth-resolved information and detailed images of CNV in neovascular AMD. Quantitative information regarding CNV flow and area can be obtained. Further studies are needed to assess the role of quantitative OCT angiography in the evaluation and treatment of neovascular AMD.

Local radiation of tumor masses is an established modality for the therapy of a range of human tumors. It has recently been recognized that doses of radiation, lower than or equal to those that cause direct cytolysis, may alter the phenotype of target tissue by up-regulating gene products that may make tumor cells more susceptible to T-cell-mediated immune attack. Previously, we demonstrated that radiation increased Fas (CD95) gene expression in carcinoembryonic antigen (CEA)-expressing murine tumor cells, which consequently enhanced their susceptibility to CEA-specific CTL-mediated killing. The present study was designed to determine whether these phenomena also occur with human tumor cells. Here, 23 human carcinoma cell lines (12 colon, 7 lung, and 4 prostate) were examined for their response to nonlytic doses of radiation (10 or 20 Gy). Seventy-two hours postirradiation, changes in surface expression of Fas (CD95), as well as expression of other surface molecules involved in T-cell-mediated immune attack such as intercellular adhesion molecule 1, mucin-1, CEA, and MHC class I, were examined. Twenty-one of the 23 (91%) cell lines up-regulated one or more of these surface molecules postirradiation. Furthermore, five of five irradiated CEA(+)/A2(+) colon tumor cells lines demonstrated significantly enhanced killing by CEA-specific HLA-A2-restricted CD8(+) CTLs compared with nonirradiated counterparts. We then used microarray analysis to broaden the scope of observed changes in gene expression after radiation and found that many additional genes had been modulated. These up-regulated gene products may additionally enhance the tumor cells' susceptibility to T-cell-mediated immune attack or serve as additional targets for immunotherapy. Overall, the results of this study suggest that nonlethal doses of radiation can be used to make human tumors more amenable to immune system recognition and attack and form the rational basis for the combinatorial use of cancer vaccines and local tumor irradiation.

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca-/- cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.

Inherited factors play a major role in the predisposition to nonalcoholic fatty liver disease (NAFLD), and the rs738409 C->>G polymorphism of PNPLA3/adiponutrin, encoding for the isoleucine-to-methionine substitution at residue 148 (I148M) protein variant, has recently been recognized as a major determinant of liver fat content. However, the effect of the rs738409 polymorphism on the severity of liver fibrosis in patients with NAFLD is still unknown. In this study, we considered 253 Italian patients, 179 healthy controls, and 71 family trios with an affected child with NAFLD. Analyses were replicated in 321 patients from the United Kingdom. The rs738409 polymorphism was determined by TaqMan assays. Liver histology was scored according to Kleiner et al. Hepatic expression of genes regulating liver damage was assessed by real-time polymerase chain reaction in 52 patients. The rs738409 GG genotype was more prevalent in patients than in controls (14% versus 3%, adjusted odds ratio [OR] = 3.29, 95% confidence interval [CI] = 1.8-6.9), and in the family study, the G allele was overtransmitted to affected children (P = 0.001). In Italian and United Kingdom patients, adiponutrin genotype influenced alanine aminotransferase levels and the severity of steatosis. Adiponutrin genotype was associated with the expression of genes involved in the steatosis-related liver damage, including the proapoptotic molecule Fas ligand. In the whole series combined, adiponutrin genotype was associated with steatosis grade >1 (OR = 1.35, 95% CI = 1.04-1.76), nonalcoholic steatohepatitis (OR = 1.5, 95% CI = 1.12-2.04), and fibrosis stage >1 (OR = 1.5, 95% CI = 1.09-2.12), independent of age, body mass index, and diabetes. Adiponutrin genotype demonstrated a dose effect with heterozygote risk intermediate between CC and GG homozygotes.

CONCLUSIONS

In patients with NAFLD, adiponutrin rs738409 C->>G genotype, encoding for I148M, is associated with the severity of steatosis and fibrosis and the presence of nonalcoholic steatohepatitis.

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

BACKGROUND

Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response.

RESULTS

Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2.

CONCLUSIONS

We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.

In order to examine the effect of HTLV-I proviral load on the pathogenesis of HAM/TSP, we measured the HTLV-I proviral load in peripheral blood mononuclear cells (PBMC) from a large number of HAM/TSP patients and asymptomatic HTLV-I carriers. To measure the proviral load, we used an accurate and reproducible quantitative PCR method using a dual-labeled fluorogenic probe (ABI PRISM 7700 Sequence Detection System). The mean +/- standard error of mean (s.e.m.) HTLV-I proviral copy number per 1 x 10(4) PBMC was 798 +/- 51 (median 544) in 202 HAM/TSP patients; 120 +/- 17 (median 34) in 200 non HAM-related (general) asymptomatic HTLV-I carriers (RC); and 496 +/- 82 (median 321) in 43 asymptomatic HTLV-I carriers genetically related to HAM/TSP patients (FA). The prevalence of HAM/TSP rises exponentially with log (proviral load) once the proviral load exceeds 1% PBMC. The HTLV-I proviral load of female patients with HAM/TSP was significantly higher than that of male patients, however there was no significant difference in proviral load between sexes in RC. There was a significant correlation between the proviral load and the concentration of neopterin in CSF of HAM/TSP patients. These results indicate that the HTLV-I proviral load in PBMC may be related to the inflammatory process in the spinal cord lesion. The increased proviral load in FA suggests the existence of genetic factors contributing to the replication of HTLV-I in vivo.

Studying rare human genetic diseases often leads to a better understanding of normal cellular functions. Fanconi anemia (FA), for example, has elucidated a novel DNA repair mechanism required for maintaining genomic stability and preventing cancer. The FA pathway, an essential tumor-suppressive pathway, is required for protecting the human genome from a specific type of DNA damage; namely, DNA interstrand cross-links (ICLs). In this review, we discuss the recent progress in the study of the FA pathway, such as the identification of new FANCM-binding partners and the identification of RAD51C and FAN1 (Fanconi-associated nuclease 1) as new FA pathway-related proteins. We also focus on the role of the FA pathway as a potential regulator of DNA repair choices in response to double-strand breaks, and its novel functions during the mitotic phase of the cell cycle.

Mitochondria trigger apoptosis by releasing caspase activators, including cytochrome c (cytC). Here we show, using a pH-sensitive green fluorescent protein (GFP), that mitochondria-dependent apoptotic stimuli (such as Bax, staurosporine and ultraviolet irradiation) induce rapid, Bcl-2-inhibitable mitochondrial alkalinization and cytosol acidification, followed by cytC release, caspase activation and mitochondrial swelling and depolarization. These events are not induced by mitochondria-independent apoptotic stimuli, such as Fas. Activation of cytosolic caspases by cytC in vitro is minimal at neutral pH, but maximal at acidic pH, indicating that mitochondria-induced acidification of the cytosol may be important for caspase activation; this finding is supported by results obtained from cells using protonophores. Cytosol acidification and cytC release are suppressed by oligomycin, a FoF1-ATPase/H +-pump inhibitor, but not by caspase inhibitors. Ectopic expression of Bax in wild-type, but not FoF1/H+-pump-deficient, yeast cells similarly results in mitochondrial matrix alkalinization, cytosol acidification and cell death. These findings indicate that mitochondria-mediated alteration of intracellular pH may be an early event that regulates caspase activation in the mitochondrial pathway for apoptosis.

OBJECTIVE

Diffusion tensor imaging (DTI) may be a useful index of microstructural changes implicated in diffuse axonal injury (DAI) linked to persistent postconcussive symptoms, especially in mild traumatic brain injury (TBI), for which conventional MR imaging techniques may lack sensitivity. We hypothesized that for mild TBI, DTI measures of DAI would correlate with impairments in reaction time, whereas the number of focal lesions on conventional 3T MR imaging would not.

METHODS

Thirty-four adult patients with mild TBI with persistent symptoms were assessed for DAI by quantifying traumatic microhemorrhages detected on a conventional set of T2*-weighted gradient-echo images and by DTI measures of fractional anisotropy (FA) within a set of a priori regions of interest. FA values 2.5 SDs below the region average, based on a group of 26 healthy control adults, were coded as exhibiting DAI.

RESULTS

DTI measures revealed several predominant regions of damage including the anterior corona radiata (41% of the patients), uncinate fasciculus (29%), genu of the corpus callosum (21%), inferior longitudinal fasciculus (21%), and cingulum bundle (18%). The number of damaged white matter structures as quantified by DTI was significantly correlated with mean reaction time on a simple cognitive task (r = 0.49, P = .012). In contradistinction, the number of traumatic microhemorrhages was uncorrelated with reaction time (r = -0.08, P = .71).

CONCLUSIONS

Microstructural white matter lesions detected by DTI correlate with persistent cognitive deficits in mild TBI, even in populations in which conventional measures do not. DTI measures may thus contribute additional diagnostic information related to DAI.

We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.

The CD95 (APO-1/Fas) system is an important mediator of T-cell cytotoxicity. We investigated this system in 22 hepatocellular carcinomas (HCCs) from patients. All HCCs had partially or completely lost the expression of the CD95 receptor constitutively expressed by normal liver cells and might thus evade CD95-mediated killing. We also considered a new mechanism of immune evasion, namely, the active destruction of T-lymphocytes by tumor cells expressing CD95 ligand (CD95L). CD95L messenger RNA and protein could be detected in the HCCs. In coculture experiments, HepG2 hepatoblastoma cells, expressing CD95L mRNA after treatment with cytostatic drugs, killed CD95+ Jurkat lymphocytes. Our data suggest that tumor cells can evade immune attack by down-regulation of the CD95 receptor and killing of lymphocytes through expression of CD95L.

Both insulin resistance and hyperinsulinemia have been reported to be independent risk factors for cardiovascular diseases. However, little is known regarding insulin signaling in the vascular tissues in insulin-resistant states. In this report, insulin signaling on the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways were compared in vascular tissues of lean and obese Zucker (fa/fa) rats in both ex vivo and in vivo studies. Ex vivo, insulin-stimulated tyrosine phosphorylation of insulin receptor beta subunits (IRbeta) in the aorta and microvessels of obese rats was significantly decreased compared with lean rats, although the protein levels of IRbeta in the 2 groups were not different. Insulin-induced tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and their protein levels were decreased in the aorta of obese rats compared with lean rats. The association of p85 subunit to the IRS proteins and the IRS-associated PI 3-kinase activities stimulated by insulin in the aorta of obese rats were significantly decreased compared with the lean rats. In addition, insulin-stimulated serine phosphorylation of Akt, a downstream kinase of PI 3-kinase pathway, was also reduced significantly in isolated microvessels from obese rats compared with the lean rats. In euglycemic clamp studies, insulin infusion greatly increased tyrosine phosphorylation of IRbeta- and IRS-2-associated PI 3-kinase activity in the aorta of lean rats, but only slight increases were observed in obese rats. In contrast, insulin stimulated tyrosine phosphorylation of MAP kinase (ERK-1/2) equally in isolated microvessels of lean and obese rats, although basal tyrosine phosphorylation of ERK-1/2 was higher in the obese rats. To our knowledge, these data provided the first direct measurements of insulin signaling in the vascular tissues, and documented a selective resistance to PI 3-kinase (but not to MAP kinase pathway) in the vascular tissues of obese Zucker rats.

Rearrangement of RET proto-oncogene is the major event in the etiopathogenesis of papillary thyroid carcinoma (PTC). We report a high prevalence of BRAF(V599E) mutation in sporadic PTC and in PTC-derived cell lines. The BRAF(V599E) mutation was detected in 23 of 50 PTC (46%) and in three of four PTC-derived cell lines. The prevalence of the BRAF(V599E) mutation in PTC is the highest reported to date in human carcinomas, being only exceeded by melanoma. PTC with RET/PTC rearrangement as well as the TPC-1 cell line (the only one harboring RET/PTC rearrangement) did not show the BRAF(V599E) mutation. BRAF(V599E) mutation was not detected in any of 23 nodular goiters, 51 follicular adenomas and 18 follicular carcinomas. A distinct mutation in BRAF (codon K600E) was detected in a follicular adenoma. Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC. BRAF(V599E) mutation did not coexist with alterations in any of the RAS genes in any of the tumors. These results suggest that BRAF(V599E) mutation is frequent in the etiopathogenesis of PTC. The BRAF(V599E) mutation appears to be an alternative event to RET/PTC rearrangement rather than to RAS mutations, which are rare in PTC. BRAF(V599E) may represent an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation.

The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro-interleukin-1β (pro-IL-1β) into secreted IL-1β, is induced by endogenous and exogenous danger signals. Lipopolysaccharide (LPS), a toll-like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals. Using mouse models of methionine choline-deficient (MCD) diet-induced NASH and high-fat diet-induced NASH, we found up-regulation of the inflammasome [including NACHT, LRR, and PYD domains-containing protein 3 (NALP3; cryopyrin), apoptosis-associated speck-like CARD-domain containing protein, pannexin-1, and pro-caspase-1] at the messenger RNA (mRNA) level increased caspase-1 activity, and mature IL-1β protein levels in mice with steatohepatitis in comparison with control livers. There was no inflammasome activation in mice with only steatosis. The MCD diet sensitized mice to LPS-induced increases in NALP3, pannexin-1, IL-1β mRNA, and mature IL-1β protein levels in the liver. We demonstrate for the first time that inflammasome activation occurs in isolated hepatocytes in steatohepatitis. Our novel data show that the saturated fatty acid (FA) palmitic acid (PA) activates the inflammasome and induces sensitization to LPS-induced IL-1β release in hepatocytes. Furthermore, PA triggers the release of danger signals from hepatocytes in a caspase-dependent manner. These hepatocyte-derived danger signals, in turn, activate inflammasome, IL-1β, and tumor necrosis factor α release in liver mononuclear cells.

CONCLUSIONS

Our novel findings indicate that saturated FAs represent an endogenous danger in the form of a first hit, up-regulate the inflammasome in NASH, and induce sensitization to a second hit with LPS for IL-β release in hepatocytes. Furthermore, hepatocytes exposed to saturated FAs release danger signals that trigger inflammasome activation in immune cells. Thus, hepatocytes play a key role in orchestrating tissue responses to danger signals in NASH.

Epidemiological studies in Greenland Eskimos led to the hypothesis that marine oils rich in n-3 fatty acids (also referred to as omega (omega)-3 fatty acids) are hypolipidemic and ultimately antiatherogenic. Metabolically controlled trials in which large amounts of fish oil were fed to normal volunteers and hyperlipidemic patients showed that these fatty acids (FAs) are effective at lowering plasma cholesterol and triglyceride levels. Although more recent trials using smaller, more practical doses of fish oil supplements have confirmed the hypotriglyceridemic effect, they have shown little effect on total cholesterol levels; hypertriglyceridemic patients have even experienced increases in low density lipoprotein cholesterol (LDL-C) levels of 10-20% while taking n-3 FA supplements. Discrepancies among fish oil studies regarding the effects of n-3 FAs on LDL-C levels may be understood by noting that, in the majority of studies reporting reductions in LDL-C levels, saturated fat intake was lowered when switching from the control diet to the fish oil diet. When fish oil is fed and saturated fat intake is constant, LDL-C levels either do not change or may increase. Levels of high density lipoprotein cholesterol have been found to increase slightly (about 5-10%) with fish oil intake. Plasma apolipoprotein levels change in concert with their associated lipoprotein cholesterol levels. Although the decrease in triglyceride levels appears to result from an inhibition in hepatic triglyceride synthesis, the mechanisms leading to the increases in LDL and HDL have not been determined. Finally, fatty fish or linolenic acid may serve as alternative sources of long-chain n-3 FAs, but further studies will be needed to document their hypolipidemic and/or antiatherogenic effects.

Fanconi anemia (FA) is a multigenic autosomal recessive cancer susceptibility syndrome. The FA pathway regulates the monoubiquitination of FANCD2 and the assembly of damage-associated FANCD2 nuclear foci. How FANCD2 monoubiquitination is coupled to the DNA-damage response has remained undetermined. Here, we demonstrate that the ATR checkpoint kinase and RPA1 are required for efficient FANCD2 monoubiquitination. Deficiency of ATR function, either in Seckel syndrome, which clinically resembles Fanconi anemia, or by siRNA silencing, results in the formation of radial chromosomes in response to the DNA cross-linker, mitomycin C (MMC), thus mimicking the chromosome instability of FA cells.

Expression of BAX, without another death stimulus, proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1 beta-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family), as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However, the fall in mitochondrial membrane potential, production of reactive oxygen species, cytoplasmic vacuolation, and plasma membrane permeability that are downstream of BAX still occurred. Thus, BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases.

c-Jun N-terminal kinases (JNKs) participate in cellular responses to mitogenic stimuli, environmental stresses, and apoptotic agents. The mechanisms by which JNK integrates with other signaling pathways and regulates the diverse cellular events are unclear. We found JNK, but not p38-mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase 2, to be persistently activated in apoptosis induced by gamma radiation, UV-C, and anti-Fas treatment. Direct correlation was found between JNK activation and apoptosis induced by UV-C and gamma radiation; however, JNK induction and apoptosis induced by Fas signaling were not well correlated. Overexpression of activated JNK1 caused cell death in transfected cells, and the expression of a dominant-negative mutant of MAPK kinase 1 or JNK1 (but not a dominant-negative mutant of p38-MAPK or c-Raf) prevented the UV-C- and gamma radiation-induced cell death. The inductions of JNK in T-cell activation and apoptosis were distinguished by the different activation patterns, transient versus persistent, respectively. Co-treatment with a tyrosine phosphatase inhibitor (sodium orthovanadate) and T-cell activation signals (phorbol 12-myristate 13-acetate plus ionomycin) prolonged JNK induction, followed by T-cell apoptosis. Our data revealed the requirement of the JNK pathway in radiation-induced apoptosis and implicated the importance of the duration of JNK activation in determining the cell fates.

Death receptors have been recently identified as a subgroup of the TNF-receptor superfamily with a predominant function in induction of apoptosis. The receptors are characterized by an intracellular region, called the death domain, which is required for the transmission of the cytotoxic signal. Currently, five different such death receptors are known including tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2. The signaling pathways by which these receptors induce apoptosis are rather similar. Ligand binding induces receptor oligomerization, followed by the recruitment of an adaptor protein to the death domain through homophilic interaction. The adaptor protein then binds a proximal caspase, thereby connecting receptor signaling to the apoptotic effector machinery. In addition, further pathways have been linked to death receptor-mediated apoptosis, such as sphingomyelinases, JNK kinases and oxidative stress. These pro-apoptotic signals are counteracted by several mechanisms which inhibit apoptosis at different levels. This review summarizes the current and rapidly expanding knowledge about the biological functions of death receptors and the mechanisms to trigger or to counteract cell death.