Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91 phox ) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91 phox /NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca 2؉ . However, the significance of Ca 2؉ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca 2؉ ionophore that induces Ca 2؉ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca 2؉ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca 2؉ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.Photosynthetic plants have developed various mechanisms to cope with oxidative stress, such as the production of antioxidants and enzymes that scavenge reactive oxygen species (ROS).3 Plants are also equipped with mechanisms for producing ROS in response to internal and external stimuli. ROS production is induced during many physiological processes, including stress responses, cell growth, hormonal responses, stomatal closure, and disease resistance (see Refs. 1-4 and references therein).ROS production is induced in plants in response to recognition of pathogenic signals, such as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) or elicitors. Elicitor-induced ROS production is preceded by a rapid increase in the cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) (5-7) and is inhibited both by Ca 2ϩ chelators such as EGTA and BAPTA, and by Ca 2ϩ channel blockers such as La 3ϩ (6,8). The overexpression of rice two-pore channel 1 (OsTPC1), which is a putative voltage-gated Ca 2ϩ channel, enhanced elicitor-induced ROS production (9). Elicitor-induced ROS production is also inhibited by diphenylene iodonium (DPI), which is known to inhibit NADPH oxidase activity (6, 10). NADPH oxidase activity in the microsomal membrane fraction from tomato and tobacco was activated by adding Ca 2ϩ in vitro (11), suggesting that elicitor-induced ROS production by plant NADPH oxidase might be dependent on Ca 2ϩ . In mammalian phagocytes, ROS production is mediated by the NADPH-dependent phagocytic oxidase (phox) complex, which consists of the catalytic subunit gp91 phox /NADPH oxidase (NOX) 2, together with the regulatory subunits p22 phox , p40 phox , p47 phox , p67 phox , and the small GTP-binding protein Rac (12). In...

Recent articles have reported an association between fatty liver disease and systemic insulin resistance in humans, but the causal relationship remains unclear. The liver may contribute to muscle insulin resistance by releasing secretory proteins called hepatokines. Here we demonstrate that leukocyte cell–derived chemotaxin 2 (LECT2), an energy-sensing hepatokine, is a link between obesity and skeletal muscle insulin resistance. Circulating LECT2 positively correlated with the severity of both obesity and insulin resistance in humans. LECT2 expression was negatively regulated by starvation-sensing kinase adenosine monophosphate-activated protein kinase in H4IIEC hepatocytes. Genetic deletion of LECT2 in mice increased insulin sensitivity in the skeletal muscle. Treatment with recombinant LECT2 protein impaired insulin signaling via phosphorylation of Jun NH2-terminal kinase in C2C12 myocytes. These results demonstrate the involvement of LECT2 in glucose metabolism and suggest that LECT2 may be a therapeutic target for obesity-associated insulin resistance.

While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal ®broblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and signi®cantly shortened the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27 Kip1 . These results suggest that the loss of substrate attachment induces the cell cycle arrest through the upregulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27 Kip1 degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Its pathogenesis is frequently linked to liver inflammation. Gain-of-function mutations in the gene encoding β-catenin are frequent genetic modifications found in human HCCs. Thus, we investigated whether inflammation was a component of β-catenin-induced tumorigenesis using genetically modified mouse models that recapitulated the stages of initiation and progression of this tumoral process. Oncogenic β-catenin signaling was found to induce an inflammatory program in hepatocytes that involved direct transcriptional control by β-catenin and activation of the NF-κB pathway. This led to a specific inflammatory response, the intensity of which determined the degree of tumor aggressiveness. The chemokine-like chemotactic factor leukocyte cell-derived chemotaxin 2 (LECT2) and invariant NKT (iNKT) cells were identified as key interconnected effectors of liver β-catenin-induced inflammation. In genetic deletion models lacking the gene encoding LECT2 or iNKT cells, hepatic β-catenin signaling triggered the formation of highly malignant HCCs with lung metastasis. Thus, our results identify inflammation as a key player in β-catenin-induced liver tumorigenesis. We provide strong evidence that, by activating pro-and antiinflammatory mediators, β-catenin signaling produces an inflammatory microenvironment that has an impact on tumoral development. Our data are consistent with the fact that most β-catenin-activated HCCs are of better prognosis.

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