Omentin, a novel adipocytokine inhibits TNF-induced vascular inflammation in human endothelial cells

Omentin is a recently identified adipocytokine with insulin-sensitizing effect. While lack of omentin may be related to the pathogenesis of obesity-related cardiovascular diseases, its effect in vasculature is largely unknown. We examined effects of omentin on vascular endothelial inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured vascular endothelia cells. The cyclic guanosine monophosphate (cGMP) content was measured using enzyme immunoassay. Treatment of human umbilical vein endothelial cells with omentin (300 ng/ml, 20 min) induced phosphorylation of 5′-AMP-activated protein kinase (AMPK) (Thr 172) and endothelial nitric oxide (NO) synthase (eNOS) (Ser 1177). Consistently, omentin increased the cGMP level. Pretreatment with omentin (300 ng/ml, 30 min) significantly inhibited the phosphorylation of JNK as well as expression of cyclooxygenase (COX)-2 by TNF-α (5 ng/ml, 20 min–24 h). An inhibitor of JNK significantly inhibited the TNF-α-induced COX-2 expression. Inhibitory effect of omentin on TNF-α-induced COX-2 was reversed by a NOS inhibitor. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced COX-2 expression in vascular endothelial cells. Omentin inhibits COX-2 induction via preventing the JNK activation presumably through activation of AMPK/eNOS/NO pathways.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.     

Arabidopsis RPT2a encoding the 26S proteasome subunit is required for various aspects of root meristem maintenance, and regulates gametogenesis redundantly with its homolog, RPT2b

The 26S proteasome plays fundamental roles in the degradation of short-lived regulatory proteins, thereby controlling diverse cellular processes. In Arabidopsis, the essential RPT2 subunit is encoded by two highly homologous genes: RPT2a and RPT2b. Currently, only RPT2a has been reported to regulate various developmental processes, including the maintenance of the root apical meristem (RAM), although the roles of RPT2a in the RAM are still obscure. Here, we analyzed the cell type-specific requirement for RPT2a. When RPT2a was expressed locally in the rpt2a mutant, pleiotropic defects in the RAM, such as cell death and distorted cellular organization, were rescued differently, suggesting that RPT2a regulates various specific activities, which converge to maintain the RAM. On the other hand, the homologous RPT2b was also expressed in meristems, and the expression of RPT2b protein under the control of the RPT2a promoter complemented the rpt2a RAM defects, although the rpt2b mutant showed no obvious defect in all developmental aspects we examined. These results show that RPT2b might work in the RAM, but is dispensable for RAM maintenance in the presence of RPT2a. In contrast, the rpt2a rpt2b double mutant was lethal in male and female gametophytes, suggesting that RPT2a and RPT2b are redundantly required for gametogenesis. Furthermore, we showed that similar meristematic and gametophytic defects were caused by mutations in other subunit genes, RPT5a and RPT5b, suggesting that proper activity of the proteasome, not an RPT2-specific function, is required. Taken together, our results suggest that RPT2a and RPT2b contribute differently to the proteasome activity required for each developmental context.     

Dual functional significance of calcineurin homologous protein 1 binding to Na(+)/H(+) exchanger isoform 1

Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na(+)/H(+) exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246-C254, 2007). However, Pang et al. (J Biol Chem 276: 17367-17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.     

Decreased plasma granulysin and increased interferon-gamma concentrations in patients with newly diagnosed and relapsed tuberculosis

Granulysin and interferon-gamma (IFN-γ) have broad antimicrobial activity which controls Mycobacterium tuberculosis (M. tuberculosis) infection. Circulating granulysin and IFN-γ concentrations were measured and correlated with clinical disease in Thai patients with newly diagnosed, relapsed and chronic tuberculosis (TB). Compared to controls, patients with newly diagnosed, relapsed and chronic TB had lower circulating granulysin concentrations, these differences being significant only in newly diagnosed and relapsed TB (P < 0.001 and 0.004, respectively). Granulysin concentrations in patients with newly diagnosed and relapsed TB were significantly lower than in those with chronic TB (P= 0.003 and P= 0.022, respectively). In contrast, significantly higher circulating IFN-γ concentrations were found in patients with newly diagnosed and relapsed TB compared to controls (P < 0.001). The IFN-γ concentrations in newly diagnosed and relapsed patients were not significantly different from those of patients with chronic TB. However, in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from patients with newly diagnosed, relapsed and chronic TB with purified protein derivative (PPD) or heat killed M. tuberculosis (H37Ra) enhanced production of granulysin by PBMCs. In vitro, stimulation of PBMCs of newly diagnosed TB patients with PPD produced greater amounts of IFN-γ than did controls, while those stimulated with H37Ra did not. The results demonstrate that patients with active pulmonary TB have low circulating granulysin but high IFN-γ concentrations, suggesting possible roles in host defense against M. tuberculosis for these agents.     

Physiological stress exacerbates murine colitis by enhancing proinflammatory cytokine expression that is dependent on IL-18

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18(-/-) mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18(-/-) mice did not aggravate colitis compared with that in the IL-18(-/-) group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.     

Oxidative stress-induced posttranslational modification of TRPV1 expressed in esophageal epithelial cells

Human esophageal epithelium is continuously exposed to physical stimuli or to gastric acid that sometimes causes inflammation of the mucosa. Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive, Ca(2+)-selective ion channel activated by capsaicin, heat, and protons. It has been reported that activation of TRPV1 expressed in esophageal mucosa is involved in gastroesophageal reflux disease (GERD) or in nonerosive GERD symptoms. In this study, we examined the expression and function of TRPV1 in the human esophageal epithelial cell line Het1A, focusing in particular on the role of oxidative stress. Interleukin-8 (IL-8) secreted by Het1A cells upon stimulation by capsaicin or acid with/without 4-hydroxy-2-nonenal (HNE) was measured by ELISA. Following capsaicin stimulation, the intracellular production of reactive oxygen species (ROS) was determined using a redox-sensitive fluorogenic probe, and ROS- and HNE-modified proteins were determined by Western blotting using biotinylated cysteine and anti-HNE antibody, respectively. HNE modification of TRPV1 proteins was further investigated by immunoprecipitation after treatment with synthetic HNE. Capsaicin and acid induced IL-8 production in Het1A cells, and this production was diminished by antagonists of TRPV1. Capsaicin also significantly increased the production of intracellular ROS and ROS- or HNE-modified proteins in Het1A cells. Moreover, IL-8 production in capsaicin-stimulated Het1A cells was enhanced by synthetic HNE treatment. Immunoprecipitation studies revealed that TRPV1 was modified by HNE in synthetic HNE-stimulated Het1A cells. We concluded that TRPV1 functions in chemokine production in esophageal epithelial cells, and this function may be regulated by ROS via posttranslational modification of TRPV1.