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:

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

Sequencing of Lp-PLA2-encoding PLA2G7 gene in 2000 Europeans reveals several rare loss-of-function mutations.

Elevated plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA2) activity have been shown to be associated with increased risk of coronary heart disease and an inhibitor of this enzyme is under development for the treatment of that condition. A Val279Phe null allele in this gene, that may influence patient eligibility for treatment, is relatively common in East Asians but has not been observed in Europeans. We investigated the existence and functional effects of low frequency alleles in a Western European population by re-sequencing the exons of PLA2G7 in 2000 samples. In all, 19 non-synonymous single-nucleotide polymorphisms (nsSNPs) were found, 14 in fewer than four subjects (minor allele frequency <0.1%). Lp-PLA2 activity was significantly lower in rare nsSNP carriers compared with non-carriers (167.8±63.2 vs 204.6±41.8, P=0.01) and seven variants had enzyme activities consistent with a null allele. The cumulative frequency of these null alleles was 0.25%, so <1 in 10,000 Europeans would be expected to be homozygous, and thus not potentially benefit from treatment with an Lp-PLA2 inhibitor.

Loss of egg yolk genes in mammals and the origin of lactation and placentation

Embryonic development in nonmammalian vertebrates depends entirely on nutritional reserves that are predominantly derived from vitellogenin proteins and stored in egg yolk. Mammals have evolved new resources, such as lactation and placentation, to nourish their developing and early offspring. However, the evolutionary timing and molecular events associated with this major phenotypic transition are not known. By means of sensitive comparative genomics analyses and evolutionary simulations, we here show that the three ancestral vitellogenin-encoding genes were progressively lost during mammalian evolution (until around 30-70 million years ago, Mya) in all but the egg-laying monotremes, which have retained a functional vitellogenin gene. Our analyses also provide evidence that the major milk resource genes, caseins, which have similar functional properties as vitellogenins, appeared in the common mammalian ancestor approximately 200-310 Mya. Together, our data are compatible with the hypothesis that the emergence of lactation in the common mammalian ancestor and the development of placentation in eutherian and marsupial mammals allowed for the gradual loss of yolk-dependent nourishment during mammalian evolution

A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss.

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

Epigenetic alterations and constitutive Wnt signaling are early events in the progression from Barrett's esophagus to esophageal adenocarcinoma

SUMMARY Barrett's esophagus (BE) is an acquired condition in which the normal squamous epithelium in the distal esophagus is replaced by a metaplastic columnar epithelium, as a complication of chronic gastroesophageal reflux. The clinical significance of this disease is its associated predisposition to esophageal adenocarcinoma (EAC). EAC is a highly lethal disease. Better understanding of the pathogenesis of columnar metaplasia and its progression to cancer might allow the identification of biomarkers that can be used for early diagnosis, which will improve the patient survival. In this study, an improved protocol for methylation-sensitive single-strand conformation analysis, which is used to analyze promoter methylation, is proposed and a methylation-sensitive dot blot assay is described, which allows a rapid, easy, and sensitive detection of promoter methylation. Both methods were applied to study the methylation pattern of the APC promoter in histologically normal appearing gastric mucosa. The APC promoter showed monoallelic methylation, and because the methylated allele differed between the different gastric cell types, this corresponded to allelic exclusion. The APC methylation pattern was frequently altered in noimal gastric mucosa associated with neoplastic lesions, indicating that changes in the pattern of promoter methylation might precede the development of neoplasia, without accompanying histological manifestations. An epigenetic profile of 10 genes important in EAC was obtained in this study; 5 promoter genes (APC, TIMP3, TERT, CDKN2A and SFRP1) were found to be hypermethylated in the tumors. Furthermore, the promoter of APC, TIMP3 and TERT was frequently methylated in BE samples from EAC patients, but rarely in BE samples that did not progress to EAC. These three biomarkers might therefore be considered as potential predictive markers for increased EAC risk. Analysis of Wnt pathway alterations indicated that WNT2 ligand is overexpressed as early as the low-grade dysplastic stage and downregulation by promoter methylation of the SFRP1 gene occurrs already in the metaplastic lesions. Moreover, loss of APC expression is not the only factor involved in the activation of the Wnt pathway. These results indicate that a variety of biologic, mostly epigenetic events occurs very early in the carcinogenesis of BE. This new information might lead to improved early diagnosis of EAC and thus open the way to a possible application of these biomarkers in the prediction of increased EAC risk progression. RESUME L'oesophage de Barrett est une lésion métaplasique définie par le remplacement de la muqueuse malpighienne du bas oesophage par une muqueuse cylindrique glandulaire, suite à une agression chronique par du reflux gastro-esophagien. La plus importante signification clinique de cette maladie est sa prédisposition au développement d'un adénocarcinome. Le pronostic de l'adénocarcinome sur oesophage de Barrett est sombre. Seule une meilleure compréhension de la pathogenèse de l'épithélium métaplasique et de sa progression néoplasique permettrait l'identification de biomarqueurs pouvant être utilisés pour un diagnostic précoce ; la survie du patient serait ainsi augmentée. Dans cette étude, un protocole amélioré pour l'analyse de la méthylation par conformation simple brin est proposé. De plus, une technique d'analyse par dot blot permettant une détection rapide, facile et sensible de la méthylation d'un promoteur est décrite. Les deux méthodes ont été appliquées à l'étude de la méthylation du promoteur du gène APC dans des muqueuses gastriques histologiquement normales. Le promoteur APC a montré une méthylation monoallélique et, parce que les allèles méthylés différaient entre les différents types de cellules gastriques, celle-ci correspondait à une méthylation allélique exclusive. La méthylation d'APC a été trouvée fréquemment altérée dans la muqueuse gastrique normale associée à des lésions néoplasiques. Ceci indique que des changements dans la méthylation d'un promoteur peuvent précéder le développement d'une tumeur, et cela sans modification histologique. Un profil épigénétique des adénocarcinomes sur oesophage de Barrett a été obtenu dans cette étude. Cinq promoteurs (APC, TIMP3, TERT, CDKN2A et SFRP1) ont été trouvés hyperméthylés dans les tumeurs. Les promoteurs d'APC, TIMP3 et TERT étaient fréquemment méthylés dans l'épithélium métaplasique proche d'un adénocarcinome et rarement dans l'épithélium sans évolution néoplasique. Ces trois biomarkers pourraient par conséquent être considérés comme marqueur prédicatif d'un risque accru de développer une tumeur. L'analyse des altérations de la voie Wnt a montré que WNT2 est surexprimé déjà dans des dysplasies de bas-grade et que la dérégulation de SFRP1 par méthylation de son promoteur intervenait dans les lésions métaplasiques. Une perte d'expression d'APC n'est pas le seul facteur impliqué dans l'activation de cette voie. Ces résultats montrent qu'une grande diversité d'événements biologiques, principalement épigénétiques, surviennent très tôt lors de la carcinogenèse de l'oesophage de Barrett. Ces nouveaux éléments pourraient améliorer le diagnostic précoce et rendre possible l'application de ces biomarqueurs dans la prédiction d'un risque accru de développer un adénocarcinome sur un oesophage de Barrett.

In conditions of limited chromophore supply rods entrap 11-cis-retinal leading to loss of cone function and cell death.

RPE65 is a retinoid isomerase required for the production of 11-cis-retinal, the chromophore of both cone and rod visual pigments. We recently established an R91W knock-in mouse strain as homologous animal model for patients afflicted by this mutation in RPE65. These mice have impaired vision and can only synthesize minute amounts of 11-cis-retinal. Here, we investigated the consequences of this chromophore insufficiency on cone function and pathophysiology. We found that the R91W mutation caused cone opsin mislocalization and progressive geographic cone atrophy. Remnant visual function was mostly mediated by rods. Ablation of rod opsin corrected the localization of cone opsin and improved cone retinal function. Thus, our analyses indicate that under conditions of limited chromophore supply rods and cones compete for 11-cis-retinal that derives from regeneration pathway(s) which are reliant on RPE65. Due to their higher number and the instability of cone opsin, rods are privileged under this condition while cones suffer chromophore deficiency and degenerate. These findings reinforce the notion that in patients any effective gene therapy with RPE65 needs to target the cone-rich macula directly to locally restore the cones' chromophore supply outside the reach of rods.

Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice.

Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1(-/-) mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1(-/-) mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1(-/-) mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1(-/-) mice were not protected from intrahepatic accumulation of diacylglyerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload.

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Dysfunction in endoplasmic reticulum-mitochondria crosstalk underlies SIGMAR1 loss of function mediated motor neuron degeneration.

Mutations in Sigma 1 receptor (SIGMAR1) have been previously identified in patients with amyotrophic lateral sclerosis and disruption of Sigmar1 in mouse leads to locomotor deficits. However, cellular mechanisms underlying motor phenotypes in human and mouse with disturbed SIGMAR1 function have not been described so far. Here we used a combination of in vivo and in vitro approaches to investigate the role of SIGMAR1 in motor neuron biology. Characterization of Sigmar1(-/-) mice revealed that affected animals display locomotor deficits associated with muscle weakness, axonal degeneration and motor neuron loss. Using primary motor neuron cultures, we observed that pharmacological or genetic inactivation of SIGMAR1 led to motor neuron axonal degeneration followed by cell death. Disruption of SIGMAR1 function in motor neurons disturbed endoplasmic reticulum-mitochondria contacts, affected intracellular calcium signalling and was accompanied by activation of endoplasmic reticulum stress and defects in mitochondrial dynamics and transport. These defects were not observed in cultured sensory neurons, highlighting the exacerbated sensitivity of motor neurons to SIGMAR1 function. Interestingly, the inhibition of mitochondrial fission was sufficient to induce mitochondria axonal transport defects as well as axonal degeneration similar to the changes observed after SIGMAR1 inactivation or loss. Intracellular calcium scavenging and endoplasmic reticulum stress inhibition were able to restore mitochondrial function and consequently prevent motor neuron degeneration. These results uncover the cellular mechanisms underlying motor neuron degeneration mediated by loss of SIGMAR1 function and provide therapeutically relevant insight into motor neuronal diseases.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.