Protein Kinase R Modulates c-Fos and c-Jun Signaling to Promote Proliferation of Hepatocellular Carcinoma with Hepatitis C Virus Infection

Double-stranded RNA-activated protein kinase R (PKR) is known to be upregulated by hepatitis C virus (HCV) and overexpressed in hepatocellular carcinoma (HCC). However, the precise roles of PKR in HCC with HCV infection remain unclear. Two HCV replicating cell lines (JFH-1 and H77s), generated by transfection of Huh7.5.1 cells, were used for experiments reported here. PKR expression was modulated with siRNA and a PKR expression plasmid, and cancer-related genes were assessed by real-time PCR and Western blotting; cell lines were further analyzed using a proliferation assay. Modulation of genes by PKR was also assessed in 34 human HCC specimens. Parallel changes in c-Fos and c-Jun gene expression with PKR were observed. Levels of phosphorylated c-Fos and c-Jun were upregulated by an increase of PKR, and were related to levels of phosphorylated JNK1 and Erk1/2. DNA binding activities of c-Fos and c-Jun also correlated with PKR expression, and cell proliferation was dependent on PKR-modulated c-Fos and c-Jun expression. Coordinate expression of c-Jun and PKR was confirmed in human HCC specimens with HCV infection. PKR upregulated c-Fos and c-Jun activities through activation of Erk1/2 and JNK1, respectively. These modulations resulted in HCC cell proliferation with HCV infection. These findings suggest that PKR-related proliferation pathways could be an attractive therapeutic target

Effects of Motilin Receptor Agonists and Ghrelin in Human <i>motilin receptor</i> Transgenic Mice

Gastrointestinal motility is regulated by neural factors and humoral factors. Both motilin and ghrelin improve gastrointestinal motility, but many issues remain unclear. We prepared human motilin receptor transgenic (Tg) mice and performed experiments evaluating the effects of motilin, erythromycin (EM), and ghrelin. EM and ghrelin promoted gastric emptying (GE) when administered either peripherally or centrally to Tg mice. Atropine (a muscarinic receptor antagonist) counteracted GE induced by centrally administered EM, but not that induced by peripherally administered EM. The administration of EM in this model promoted the effect of mosapride (a selective serotonin 5-hydroxytryptamine 4 (5-HT4) receptor agonist), and improved loperamide (a &#956;-opioid receptor agonist)-induced gastroparesis. The level of acyl-ghrelin was significantly attenuated by EM administration. Thus, we have established an animal model appropriate for the evaluation of motilin receptor agonists. These data and the model are expected to facilitate the identification of novel compounds with clinical potential for relieving symptoms of dyspepsia and gastroparesis

Characterization of the gastric motility response to human motilin and erythromycin in human motilin receptor-expressing transgenic mice.

Motilin is a gastrointestinal peptide hormone that stimulates gastrointestinal motility. Motilin is produced primarily in the duodenum and jejunum. Motilin receptors (MTLRs) are G protein-coupled receptors that may represent a clinically useful pharmacological target as they can be activated by erythromycin. The functions of motilin are highly species-dependent and remain poorly understood. As a functional motilin system is absent in rodents such as rats and mice, these species are not commonly used for basic studies. In this study, we examine the usefulness of human MTLR-overexpressing transgenic (hMTLR-Tg) mice by identifying the mechanisms of the gastric motor response to human motilin and erythromycin. The distribution of hMTLR was examined immunohistochemically in male wild-type (WT) and hMTLR-Tg mice. The contractile response of gastric strips was measured isometrically in an organ bath, while gastric emptying was determined using phenol red. hMTLR expression was abundant in the gastric smooth muscle layer. Interestingly, higher levels of hMTLR expression were observed in the myenteric plexus of hMTLR-Tg mice but not WT mice. hMTLR was not co-localized with vesicular acetylcholine transporter, a marker of cholinergic neurons in the myenteric plexus. Treatment with human motilin and erythromycin caused concentration-dependent contraction of gastric strips obtained from hMTLR-Tg mice but not from WT mice. The contractile response to human motilin and erythromycin in hMTLR-Tg mice was affected by neither atropine nor tetrodotoxin and was totally absent in Ca2+-free conditions. Furthermore, intraperitoneal injection of erythromycin significantly promoted gastric emptying in hMTLR-Tg mice but not in WT mice. Human motilin and erythromycin stimulate gastric smooth muscle contraction in hMTLR-Tg mice. This action is mediated by direct contraction of smooth muscle via the influx of extracellular Ca2+. Thus, hMTLR-Tg mice may be useful for the evaluation of MTLR agonists as gastric prokinetic agents

Characterization of Liver Monocytic Myeloid-Derived Suppressor Cells and Their Role in a Murine Model of Non-Alcoholic Fatty Liver Disease.

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of T cell immunity in tumors and inflammatory diseases. They are identified by surface expression of CD11b+Gr1+ in mice, and CD11b+Gr1+ cells accumulate in the livers of obese mice. However, many myeloid cells share these CD11b+Gr1+ markers. Accordingly, the aim of this study was to identify the authentic phenotype of MDSCs and investigate their functions in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were divided into 2 diet groups: a normal control group and high-fat group to induce NAFLD. We demonstrated that monocytic CD11b+Gr1dim cells could be further divided into 2 populations based on side scatter (SSC) during flow cytometry. We found that SSClowCD11b+Gr1dim cells accumulated in the livers of NAFLD mice over time, and that these cells were recruited by the chemokine CCL2 and its receptor CCR2 and might expand in the liver via macrophage colony-stimulating factor stimulation. Furthermore, SSClowCD11b+Gr1dim cells had a strong suppressive ability on T cells; this effect was not observed for SSChighCD11b+Gr1dim cells, and was dependent on nitric oxide production by inducible nitric oxide synthase. Our findings demonstrate that SSClowCD11b+Gr1dim cells represent authentic MDSCs in NAFLD livers, and might serve an important negative feedback function in liver inflammation