Shin Maeda

IKK-beta links inflammation to obesity-induced insulin resistance.

Inflammation may underlie the metabolic disorders of insulin resistance and type 2 diabetes. IκB kinase β (IKK-β, encoded by Ikbkb) is a central coordinator of inflammatory responses through activation of NF-κB. To understand the role of IKK-β in insulin resistance, we used mice lacking this enzyme in hepatocytes (IkbkbΔhep) or myeloid cells (IkbkbΔmye). IkbkbΔhep mice retain liver insulin responsiveness, but develop insulin resistance in muscle and fat in response to high fat diet, obesity or aging. In contrast, IkbkbΔmye mice retain global insulin sensitivity and are protected from insulin resistance. Thus, IKK-β acts locally in liver and systemically in myeloid cells, where NF-κB activation induces inflammatory mediators that cause insulin resistance. These findings demonstrate the importance of liver cell IKK-β in hepatic insulin resistance and the central role of myeloid cells in development of systemic insulin resistance. We suggest that inhibition of IKK-β, especially in myeloid cells, may be used to treat insulin resistance.

Reactive Oxygen Species Promote TNFα-Induced Death and Sustained JNK Activation by Inhibiting MAP Kinase Phosphatases

TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.

IKKβ Couples Hepatocyte Death to Cytokine-Driven Compensatory Proliferation that Promotes Chemical Hepatocarcinogenesis

IkappaB kinase beta (IKKbeta), required for NF-kappaB activation, links chronic inflammation with carcinogenesis. We investigated whether IKKbeta is involved in chemically induced liver cancer, a model not involving overt inflammation. Surprisingly, mice lacking IKKbeta only in hepatocytes (Ikkbeta(Deltahep) mice) exhibited a marked increase in hepatocarcinogenesis caused by diethylnitrosamine (DEN). This correlated with enhanced reactive oxygen species (ROS) production, increased JNK activation, and hepatocyte death, giving rise to augmented compensatory proliferation of surviving hepatocytes. Brief oral administration of an antioxidant around the time of DEN exposure blocked prolonged JNK activation and compensatory proliferation and prevented excessive DEN-induced carcinogenesis in Ikkbeta(Deltahep) mice. Decreased hepatocarcinogenesis was also found in mice lacking IKKbeta in both hepatocytes and hematopoietic-derived Kupffer cells. These mice exhibited reduced hepatocyte regeneration and diminished induction of hepatomitogens, which were unaltered in Ikkbeta(Deltahep) mice. IKKbeta, therefore, orchestrates inflammatory crosstalk between hepatocytes and hematopoietic-derived cells that promotes chemical hepatocarcinogenesis.

The E3 Ubiquitin Ligase Itch Couples JNK Activation to TNFα-induced Cell Death by Inducing c-FLIPL Turnover

The proinflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNFalpha treatment is determined by the balance between NF-kappaB and Jun kinase (JNK) signaling; NF-kappaB promotes survival, whereas JNK enhances cell death. Critically, identity of a JNK substrate that promotes TNFalpha-induced apoptosis has been outstanding. Here we show that TNFalpha-mediated JNK activation accelerates turnover of the NF-kappaB-induced antiapoptotic protein c-FLIP, an inhibitor of caspase-8. This is not due to direct c-FLIP phosphorylation but depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. JNK1 or Itch deficiency or treatment with a JNK inhibitor renders mice resistant in three distinct models of TNFalpha-induced acute liver failure, and cells from these mice do not display inducible c-FLIP(L) ubiquitination and degradation. Thus, JNK antagonizes NF-kappaB during TNFalpha signaling by promoting the proteasomal elimination of c-FLIP(L).

A large noncoding RNA is a marker for murine hepatocellular carcinomas and a spectrum of human carcinomas

Tumor markers can facilitate understanding molecular cell biology of neoplasia and provide potential targets for the diagnosis and insight for intervention. We here identify a novel murine gene, hepcarcin (hcn), encoding a 7-kb mRNA-like transcript. The gene appears to be the murine ortholog of the human alpha gene, that is, MALAT-1. The gene and homologs lack credible open reading frames, consistent with a highly conserved large noncoding RNA (ncRNA). In all nodules of procarcinogen-induced murine hepatocellular carcinomas (HCCs) and human HCCs, expression was markedly elevated compared to the uninvolved liver. Quantitative analyses indicated a 6–7-fold increased RNA level in HCCs versus uninvolved liver, advancing this as a molecule of interest. This ncRNA was overexpressed in all five non-hepatic human carcinomas analysed, consistent with a potential marker for neoplastic cells and potential participant in the molecular cell biology of neoplasia.

The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4

Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens. Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns—including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA—and in turn activate effector functions, including anti-apoptotic signalling pathways. Certain pathogens, however, such as Salmonella spp., Shigellae spp. and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis. We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase. We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4. Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis. The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.

IKKβ Is Required for Prevention of Apoptosis Mediated by Cell-Bound but Not by Circulating TNFα

Abstract IκB kinase β (IKKβ) is required for NF-κB activation and suppression of TNFα-mediated liver apoptosis. To investigate how IKKβ suppresses apoptosis, we generated hepatocyte-specific Ikkβ knockout mice, Ikkβ Δhep , which exhibit little residual NF- κB activity but are healthy with normal liver function. Unexpectedly, Ikkβ Δhep mice are slightly more sensitive than controls to LPS-induced liver apoptosis but are highly susceptible to liver destruction following concanavalin A (ConA)-induced T cell activation. Unlike LPS, a potent inducer of circulating TNFα, ConA exerts cytotoxic effects through cell-bound TNFα, which activates type 1 and 2 TNF receptors (TNFR). While TNFR2 does not contribute to NF-κB activation, it is important for ConA-induced JNK activation, which is augmented by the absence of IKKβ. Using JNK-deficient mice we show that JNK is required for ConA-induced liver damage. Thus, the antiapoptotic function of IKKβ, which is most critical in situations that involve cell-bound TNFα, is mediated partially through attenuation of JNK activity.

Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan

Background—VacA and CagA proteins have been reported to be major virulence factors of Helicobacter pylori. However, antibodies against these proteins are frequently found in the sera of Japanese patients regardless of their gastroduodenal status. Aim—To evaluate the expression of VacA and CagA proteins by H pylori strains isolated in Japan. Methods—By using specific antibodies raised against recombinant VacA and CagA proteins, the expression of VacA and CagA was evaluated in 68 H pylori strains isolated from Japanese patients; a vacuolating assay and genotyping of thevacA gene were also used in the evaluation. The results were analysed in relation to the gastroduodenal diseases of the hosts. Results—VacA and CagA proteins were expressed in 59/68 (87%) and in 61/68 (90%) isolates respectively. The vacuolating assay was positive in 57/68 (84%) isolates, indicating that most immunologically VacA positive strains produced active cytotoxin. The prevalence of infection with strains expressing CagA and positive for vacuolating activity (Type I) was very high, 54/68 (79%), irrespective of the gastroduodenal status of the host. Conclusion—Most H pylori isolates in Japan are positive for vacuolating cytotoxin and CagA, and thus these virulence factors cannot be used as markers to discern the risk of developing serious gastroduodenal pathologies in the hosts. However, the high prevalence of infection with strains positive for vacuolating cytotoxin and CagA may contribute to the characteristics of H pylori infection in Japan.

IκB kinase (IKK)β, but not IKKα, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss

Transcription factor, nuclear factor κB (NF-κB), is required for osteoclast formation in vivo and mice lacking both of the NF-κB p50 and p52 proteins are osteopetrotic. Here we address the relative roles of the two catalytic subunits of the IκB kinase (IKK) complex that mediate NF-κB activation, IKKα and IKKβ, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKKβ subunit as a transducer of signals from receptor activator of NF-κB (RANK) to NF-κB. Although IKKα is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. However, IKKβ is required for osteoclastogenesis in vitro and in vivo. IKKβ also protects osteoclasts and their progenitors from tumor necrosis factor α–induced apoptosis, and its loss in hematopoietic cells prevents inflammation-induced bone loss.

IκB kinase-α acts in the epidermis to control skeletal and craniofacial morphogenesis

IκB kinase-α (IKK-α) exhibits protein-kinase-dependent and -independent functions. Its kinase activity is required for lymphoid organogenesis and mammary gland development, whereas a kinase-independent activity is required for epidermal keratinocyte differentiation. In addition to failed epidermal differentiation, IKK-α-deficient mice exhibit abnormal skeletal and craniofacial morphogenesis. As similar defects are not exhibited by mice that experience systemic inhibition of NF-κB, we postulated that the morphogenetic defects in IKK-α-deficient mice are not caused by reduced NF-κB activity but instead are due to failed epidermal differentiation that disrupts proper epidermal–mesodermal interactions. We tested this hypothesis by introducing an epidermal-specific Ikka (also known as Chuk) transgene into IKK-α-deficient mice. Mice lacking IKK-α in all cell types including bone and cartilage, but not in basal epidermal keratinocytes, exhibit normal epidermal differentiation and skeletal morphology. Thus, epidermal differentiation is required for proper morphogenesis of mesodermally derived skeletal elements. One way by which IKK-α controls skeletal and craniofacial morphogenesis is by repressing expression of fibroblast growth factor (FGF) family members, such as FGF8, whose expression is specifically elevated in the limb bud ectoderm of IKK-α-deficient mice.