A Novel Role for Tm7sf2 Gene in Regulating TNFα Expression

We have explored the role of Tm7sf2 gene, which codifies for 3β-hydroxysterol Δ14-reductase, an endoplasmic reticulum resident protein, in the sensitivity to endoplasmic reticulum stress and in the resulting inflammatory response. We used mouse embryonic fibroblasts, derived from Tm7sf2^+/+ and Tm7sf2^−/− mice, to determine the in vitro effects of thapsigargin on NF-κB activation. Our results show that the Tm7sf2 gene controls the launch of the unfolded protein response and presides an anti-inflammatory loop thus its absence correlates with NF-κB activation and TNFα up-regulation. Our data also show that Tm7sf2 gene regulates liver X receptor activation and its absence inhibits LXR signalling. By expressing the hTm7sf2 gene in KO MEFs and observing a reduced NF-κB activation, we have confirmed that Tm7sf2 gene is linked to NF-κB activation. Finally we used genetically modified mice in an in vivo model of ER stress and of inflammation. Our results show a significant increase in renal TNFα expression after tunicamycin exposure and in the oedematogenic response in Tm7sf2^−/− mice. In conclusion, we have shown that the Tm7sf2 gene, to date involved only in cholesterol biosynthesis, also controls an anti-inflammatory loop thereby confirming the existence of cross talk between metabolic pathways and inflammatory response.     

Low-pH-induced apoptosis: role of endoplasmic reticulum stress-induced calcium permeability and mitochondria-dependent signaling

The acidic microenvironment around tumor cells is a major determinant in cancer growth, metabolism, and metastasis. However, its role in cancer physiology is still not clearly understood. In the present investigation, an attempt has been made to explore the effect of acidic environment on physiology of cancer cells. Exposure of Raji cells to extracellular acidic environment was associated with enhanced cytosolic calcium level and endoplasmic reticulum stress response. X-box binding protein 1 (XBP1) splicing, CCAAT/enhancer-binding protein homologous protein (CHOP), and glucose-regulated protein 78 kDa (GRP78) upregulation suggested endoplasmic reticulum stress generation. On the other hand, real-time-based upregulation of Bax gene expression and flow cytometric analysis of cytochrome c release as well as enhanced active caspase-3 further confirmed mitochondrion-mediated events leading to induction of apoptosis. The expression of TP53 and p21 was upregulated. These observations collectively strongly suggest that both endoplasmic reticulum stress-mediated calcium release and Bax targeting might be altering mitochondrion membrane potential which in turn could induce secondary apoptotic signals; subsequently, endoplasmic reticulum stress can also lead to nuclear localization of Nuclear factor-κB (NF-κB) which in turn favors p53 mediated apoptotic signals.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0568-6) contains supplementary material, which is available to authorized users.     

A Combined “Omics” Approach Identifies N-Myc Interactor as a Novel Cytokine-induced Regulator of IRE1α Protein and c-Jun N-terminal Kinase in Pancreatic Beta Cells

Type 1 diabetes is an autoimmune disease with a strong inflammatory component. The cytokines interleukin-1β and interferon-γ contribute to beta cell apoptosis in type 1 diabetes. These cytokines induce endoplasmic reticulum stress and the unfolded protein response (UPR), contributing to the loss of beta cells. IRE1α, one of the UPR mediators, triggers insulin degradation and inflammation in beta cells and is critical for the transition from “physiological” to “pathological” UPR. The mechanisms regulating inositol-requiring protein 1α (IRE1α) activation and its signaling for beta cell “adaptation,” “stress response,” or “apoptosis” remain to be clarified. To address these questions, we combined mammalian protein-protein interaction trap-based IRE1α interactome and functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines to identify novel cytokine-induced regulators of IRE1α. Based on this approach, we identified N-Myc interactor (NMI) as an IRE1α-interacting/modulator protein in rodent and human pancreatic beta cells. An increased expression of NMI was detected in islets from nonobese diabetic mice with insulitis and in rodent or human beta cells exposed in vitro to the pro-inflammatory cytokines interleukin-1β and interferon-γ. Detailed mechanistic studies demonstrated that NMI negatively modulates IRE1α-dependent activation of JNK and apoptosis in rodent and human pancreatic beta cells. In conclusion, by using a combined omics approach, we identified NMI induction as a novel negative feedback mechanism that decreases IRE1α-dependent activation of JNK and apoptosis in cytokine-exposed beta cells.     

Regulation of CCAAT/Enhancer-binding Protein Homologous Protein (CHOP) Expression by Interleukin-1β in Pancreatic β Cells

Apoptosis contributes to immune-mediated pancreatic β cell destruction in type I diabetes. Exposure of β cells to interleukin-1β (IL-1β) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in β cells treated with IL-1β. In addition, prolonged exposure to high glucose further increases IL-1β-triggered CHOP expression. IL-1β also causes increased expression of C/EBP-β and a reduction of MafA, NFATc2, and Pdx-1 expression in β cells. Inhibition of the NF-κB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects β cells from IL-1β-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of β cells.     

The Endoplasmic Reticulum-based Acetyltransferases,ATase1 and ATase2, Associate with the Oligosaccharyltransferase to Acetylate Correctly Folded Polypeptides

The endoplasmic reticulum (ER) has two membrane-bound acetyltransferases responsible for the endoluminal N^ϵ-lysine acetylation of ER-transiting and -resident proteins. Mutations that impair the ER-based acetylation machinery are associated with developmental defects and a familial form of spastic paraplegia. Deficient ER acetylation in the mouse leads to defects of the immune and nervous system. Here, we report that both ATase1 and ATase2 form homo- and heterodimers and associate with members of the oligosaccharyltransferase (OST) complex. In contrast to the OST, the ATases only modify correctly folded polypetides. Collectively, our studies suggest that one of the functions of the ATases is to work in concert with the OST and “select” correctly folded from unfolded/misfolded transiting polypeptides.     

The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the “K loop,” which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.     

Fyn Activation of mTORC1 Stimulates the IRE1α-JNK Pathway,Leading to Cell Death

We previously reported that the skeletal muscle-specific overexpression of Fyn in mice resulted in a severe muscle wasting phenotype despite the activation of mTORC1 signaling. To investigate the bases for the loss of muscle fiber mass, we examined the relationship between Fyn activation of mTORC1, JNK, and endoplasmic reticulum stress. Overexpression of Fyn in skeletal muscle in vivo and in HEK293T cells in culture resulted in the activation of IRE1α and JNK, leading to increased cell death. Fyn synergized with the general endoplasmic reticulum stress inducer thapsigargin, resulting in the activation of IRE1α and further accelerated cell death. Moreover, inhibition of mTORC1 with rapamycin suppressed IRE1α activation and JNK phosphorylation, resulting in protecting cells against Fyn- and thapsigargin-induced cell death. Moreover, rapamycin treatment in vivo reduced the skeletal muscle IRE1α activation in the Fyn-overexpressing transgenic mice. Together, these data demonstrate the presence of a Fyn-induced endoplasmic reticulum stress that occurred, at least in part, through the activation of mTORC1, as well as subsequent activation of the IRE1α-JNK pathway driving cell death.     

Interactions between NF-κB and SP3 Connect Inflammatory Signaling with Reduced FGF-10 Expression

Inflammation inhibits normal lung morphogenesis in preterm infants. Soluble inflammatory mediators present in the lungs of patients developing bronchopulmonary dysplasia disrupt expression of multiple genes critical for development. However, the mechanisms linking innate immune signaling and developmental programs are not clear. NF-κB activation inhibits expression of the critical morphogen FGF-10. Here, we show that interactions between the RELA subunit of NF-κB and SP3 suppress SP1-mediated FGF-10 expression. SP3 co-expression reduced SP1-mediated Fgf-10 promoter activity, suggesting antagonistic interactions between SP1 and SP3. Chromatin immunoprecipitation of LPS-treated primary mouse fetal lung mesenchymal cells detected increased interactions between SP3, RELA, and the Fgf-10 promoter. Expression of a constitutively active IκB kinase β mutant not only decreased Fgf-10 promoter activity but also increased RELA-SP3 nuclear interactions. Expression of a dominant-negative IκB, which blocks NF-κB nuclear translocation, prevented inhibition of FGF-10 by SP3. The inhibitory functions of SP3 required sequences located in the N-terminal region of the protein. These data suggested that inhibition of FGF-10 by inflammatory signaling involves the NF-κB-dependent interactions between RELA, SP3, and the Fgf-10 promoter. NF-κB activation may therefore lead to reduced gene expression by recruiting inhibitory factors to specific gene promoters following exposure to inflammatory stimuli.     

IL-6 and IL-10 Anti-Inflammatory Activity Links Exercise to Hypothalamic Insulin and Leptin Sensitivity through IKKβ and ER Stress Inhibition

Overnutrition caused by overeating is associated with insulin and leptin resistance through IKKβ activation and endoplasmic reticulum (ER) stress in the hypothalamus. Here we show that physical exercise suppresses hyperphagia and associated hypothalamic IKKβ/NF-κB activation by a mechanism dependent upon the pro-inflammatory cytokine interleukin (IL)-6. The disruption of hypothalamic-specific IL-6 action blocked the beneficial effects of exercise on the re-balance of food intake and insulin and leptin resistance. This molecular mechanism, mediated by physical activity, involves the anti-inflammatory protein IL-10, a core inhibitor of IKKβ/NF-κB signaling and ER stress. We report that exercise and recombinant IL-6 requires IL-10 expression to suppress hyperphagia-related obesity. Moreover, in contrast to control mice, exercise failed to reverse the pharmacological activation of IKKβ and ER stress in C3H/HeJ mice deficient in hypothalamic IL-6 and IL-10 signaling. Hence, inflammatory signaling in the hypothalamus links beneficial physiological effects of exercise to the central action of insulin and leptin.     

Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting RAGE Signaling in Diabetic Atherosclerosis

A naturally occurring bile acid, ursodeoxycholic acid (UDCA), is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, the detailed action mechanisms of UDCA in atherosclerosis are not fully understood. In this study, we demonstrated whether UDCA exerts anti-atherogenic activity in diabetic atherosclerosis by targeting ER stress and “receptor for advanced glycation endproduct” (RAGE) signaling. UDCA markedly reduced ER stress, RAGE expression, and pro-inflammatory responses [including NF-κB activation and reactive oxygen species (ROS) production] induced in endothelial cells (ECs) by high glucose (HG). In particular, UDCA inhibited HG-induced ROS production by increasing the Nrf2 level. In macrophages, UDCA also blocked HG-induced RAGE and pro-inflammatory cytokine expression and inhibited foam cell formation via upregulation of the ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1. In the diabetic mouse model, UDCA inhibited atheromatous plaque formation by decreasing ER stress, and the levels of RAGE and adhesion molecules. In conclusion, UDCA exerts an anti-atherogenic activity in diabetic atherosclerosis by targeting both ER stress and RAGE signaling. Our work implicates UDCA as a potential therapeutic agent for prevention or treatment of diabetic atherosclerosis.     

Bindarit: An anti-inflammatory small molecule that modulates the NFκB pathway

The activation of nuclear factor (NF)κB pathway and its transducing signaling cascade has been associated with the pathogenesis of many inflammatory diseases. The central role that IκBα and p65 phosphorylation play in regulating NFκB signalling in response to inflammatory stimuli made these proteins attractive targets for therapeutic strategies. Although several chemical classes of NFκB inhibitors have been identified, it is only for a few of those that a safety assessment based on a comprehensive understanding of their pharmacologic mechanism of action has been reported. Here, we describe the specific anti-inflammatory effect of bindarit, an indazolic derivative that has been proven to have anti-inflammatory activity in a variety of models of inflammatory diseases, including lupus nephritis, arthritis and pancreatitis. The therapeutic effects of bindarit have been associated with its ability to selectively interfere with monocyte recruitment and the “early inflammatory response,” although its specific molecular mechanisms have remained ill-defined. For this purpose, we investigated the effect of bindarit on the LPS-induced production of inflammatory cytokines (MCP-1 and MCPs, IL-12β/p40, IL-6 and IL-8/KC) in both a mouse leukaemic monocyte-macrophage cell line and bone marrow-derived macrophages (BMDM). Bindarit inhibits the LPS-induced MCP-1 and IL-12β/p40 expression without affecting other analyzed cytokines. The effect of bindarit is mediated by the downregulation of the classical NFκB pathway, involving a reduction of IκBα and p65 phosphorylation, a reduced activation of NFκB dimers and a subsequently reduced nuclear translocation and DNA binding. Bindarit showed a specific inhibitory effect on the p65 and p65/p50 induced MCP-1 promoter activation, with no effect on other tested activated promoters. We conclude that bindarit acts on a specific subpopulation of NFκB isoforms and selects its targets wihtin the whole NFκB inflammatory pathway. These findings pave the way for future applications of bindarit as modulator of the inflammatory response.Key words: inflammation, NFκB, MCP-1, IL-12β/p40, macrophages, lipopolysaccharide, bindarit