Hyun-Ock Pae

Mitogen-Activated Protein Kinases and Reactive Oxygen Species: How Can ROS Activate MAPK Pathways?

Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that play the major role in signal transduction from the cell surface to the nucleus. MAPKs, which consist of growth factor-regulated extracellular signal-related kinases (ERKs), and the stress-activated MAPKs, c-jun NH2-terminal kinases (JNKs) and p38 MAPKs, are part of a three-kinase signaling module composed of the MAPK, an MAPK kinase (MAP2K) and an MAPK kinase (MAP3K). MAP3Ks phosphorylate MAP2Ks, which in turn activate MAPKs. MAPK phosphatases (MKPs), which recognize the TXY amino acid motif present in MAPKs, dephosphorylate and deactivate MAPKs. MAPK pathways are known to be influenced not only by receptor ligand interactions, but also by different stressors placed on the cell. One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by reactive oxygen species (ROS). Generally, increased ROS production in a cell leads to the activation of ERKs, JNKs, or p38 MAPKs, but the mechanisms by which ROS can activate these kinases are unclear. Oxidative modifications of MAPK signaling proteins and inactivation and/or degradation of MKPs may provide the plausible mechanisms for activation of MAPK pathways by ROS, which will be reviewed in this paper.

Carbon Monoxide Produced by Heme Oxygenase-1 Suppresses T Cell Proliferation via Inhibition of IL-2 Production

Heme oxygenase-1 (HO-1) catabolizes heme into CO, biliverdin, and free iron and serves as a protective enzyme by virtue of its anti-inflammatory, antiapoptotic, and antiproliferative actions. Previously, we have demonstrated that human CD4+ T cells express HO-1 and that HO-1-overexpressing Jurkat T cells tend to display lower proliferative response. The aim of this study is to elucidate the mechanism(s) by which HO-1 can mediate its antiproliferative effect on CD4+ T cells. Among the three HO-1 byproducts, only CO showed suppressive effect on T cell proliferation in response to anti-CD3 plus anti-CD28 Abs, mimicking the antiproliferative action of HO-1. CO blocked the cell cycle entry of T cells, which was independent of the guanylate cyclase/cGMP pathway. CO also suppressed the secretion of IL-2, and this suppressive effect of CO on IL-2 secretion mediated the antiproliferative action of CO. CO selectively inhibited the extracellular signal-regulated kinase pathway, which could explain the suppressive effects of CO on T cell proliferation and IL-2 secretion. Based on these findings, we suggest that HO-1/CO suppresses T cell proliferation and IL-2 secretion, possibly via its inhibition of extracellular signal-regulated kinase activation.

Reactive Oxygen Species in the Activation of MAP Kinases

There are three well-defined subgroups of mitogen-activated protein kinases (MAPKs): the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 MAPKs. Three subgroups of MAPKs are involved in both cell growth and cell death, and the tight regulation of these pathways, therefore, is paramount in determining cell fate. MAPK pathways have been shown to be activated not only by receptor ligand interactions but also by different stressors placed on the cell. MAPK phosphatases (MKPs) dephosphorylate and deactivate MAPKs. Reactive oxygen species (ROS), such as hydrogen peroxide, have been reported to activate ERKs, JNKs, and p38 MAPKs, but the mechanisms by which ROS can activate these kinases are unclear. Oxidative modifications of MAPK signaling proteins and inactivation and/or degradation of MKPs may provide the plausible mechanisms for activation of MAPK pathways by ROS, which will be reviewed in this chapter.

Heme oxygenase in the regulation of vascular biology: from molecular mechanisms to therapeutic opportunities.

Heme oxygenases (HOs) are the rate-limiting enzymes in the catabolism of heme into biliverdin, free iron, and carbon monoxide. Two genetically distinct isoforms of HO have been characterized: an inducible form, HO-1, and a constitutively expressed form, HO-2. HO-1 is a kind of stress protein, and thus regarded as a sensitive and reliable indicator of cellular oxidative stress. The HO system acts as potent antioxidants, protects endothelial cells from apoptosis, is involved in regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in angiogenesis and vasculogenesis. Endothelial integrity and activity are thought to occupy the central position in the pathogenesis of cardiovascular diseases. Cardiovascular disease risk conditions converge in the contribution to oxidative stress. The oxidative stress leads to endothelial and vascular smooth muscle cell dysfunction with increases in vessel tone, cell growth, and gene expression that create a pro-thrombotic/pro-inflammatory environment. Subsequent formation, progression, and obstruction of atherosclerotic plaque may result in myocardial infarction, stroke, and cardiovascular death. This background provides the rationale for exploring the potential therapeutic role for HO system in the amelioration of vascular inflammation and prevention of adverse cardiovascular outcomes.

Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, is antiapoptotic in a variety of models of cellular injury, but the precise mechanisms remain to be established. In human umbilical vein endothelial cells, exogenous CO activated Nrf2 through the phosphorylation of protein kinase R–like endoplasmic reticulum kinase (PERK), resulting in HO-1 expression. CO-induced activation of PERK was followed by the phosphorylation of eukaryotic translation initiation factor 2&agr; and the expression of activating transcription factor 4. However, CO fails to induce X-box binding protein-1 expression and activating transcription factor 6 cleavage. CO had no significant effect on synthesis of endoplasmic reticulum (ER) chaperone proteins such as the 78-kDa glucose-regulated proteins 78 and 94. Instead, CO prevented X-box binding protein 1 expression and activating transcription factor 6 cleavage induced by ER-stress inducers such as thapsigargin, tunicamycin and homocysteine. CO also prevented endothelial apoptosis triggered by these ER inducers through suppression of C/EBP homologous protein expression, which was associated with its activation of p38 mitogen-activated protein kinase. Similarly, endogenous CO produced from endothelial HO-1 induced by either exogenous CO or a pharmacological inducer was also cytoprotective against ER stress through C/EBP homologous protein suppression. Our findings suggest that CO renders endothelial cells resistant to ER stress not only by downregulating C/EBP homologous protein expression via p38 mitogen-activated protein kinase activation but also by upregulating Nrf2-dependent HO-1 expression via PERK activation. Thus, the HO-1/CO system might be potential therapeutics in vascular diseases associated with ER stress.

Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells.

The rhizomes of Cyperus rotundus (C. rotundus) have been used in oriental traditional medicines for the treatment of stomach and bowel disorders, and inflammatory diseases. Nitric oxide (NO) and superoxide (O2-) are important mediators in the pathogenesis of inflammatory diseases. This study was undertaken to address whether the metanol (MeOH) extract of rhizomes of C. rotundus could modulate NO and O2- productions by murine macrophage cell line, RAW 264.7 cells. The MeOH extract of rhizomes of C. rotundus showed the inhibition of NO production in a dose-dependent manner by RAW 264.7 cells stimulated with interferon-gamma plus lipopolysaccharide. The inhibition of NO production by the extract was due to the suppression of iNOS protein, as well as iNOS mRNA expression, determined by Western and Northern blotting analyses, respectively. In addition, the MeOH extract suppressed the production of O2- by phorbol ester-stimulated RAW 264.7 cells in dose- and time-dependent manners. Collectively, these results suggest that the MeOH extract of rhizomes of C. rotundus could be developed as anti-inflammatory candidate for the treatment of inflammatory diseases mediated by overproduction of NO and O2-.

Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer.

Water-soluble chitosan oligomer (WSCO) has been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we have shown that WSCO is able to inhibit proliferation of human leukemia HL-60 cells and induce these cells to differentiate. Treatment with WSCO for 4 days resulted in a concentration-dependent reduction in HL-60 cell growth as measured by cell counting and MTT assay. This effect was accompanied by a marked increase in the proportion of G(0)/G(1) cells as measured by flow cytometry. WSCO also induced differentiation of the cells as measured by phorbol ester-dependent reduction of NBT, morphological changes as examined by Wright-Giemsa staining and expression of CD11b but not of CD14 as analysed by flow cytometry, indicating differentiation of HL-60 cells toward granulocyte-like cells. A combination of low dose of WSCO with all-trans retinoic acid, a differentiating agent toward granulocyte-like cells, exhibited a synergistic effect on the differentiation. In addition, treatment of HL-60 cells with WSCO for 6 or 8 days resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis and quantitatively by Annexin V technique using flow cytometry. Collectively, there is a potential for WSCO in the treatment of myeloid leukemia.

Synergistic cooperation between water-soluble chitosan oligomers and interferon-γ for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages

The effects of water-soluble chitosan oligomers (WSCO) on the synthesis of nitric oxide (NO) by murine peritoneal macrophages and on macrophage-mediated cytotoxicity towards murine fibrosarcoma Meth A cells were investigated. WSCO alone had no effect on NO synthesis and killing of tumor cells. However, treatment of macrophages with a combination of WSCO and interferon-gamma (IFN-gamma) synergically increased NO synthesis and enhanced killing of tumor cells. The synergism between IFN-gamma and WSCO in NO synthesis and tumoricidal activity was mainly dependent on increased secretion of tumor necrosis factor-alpha by WSCO.

Methanol extract of Cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing NF-κB activation

Cordyceps pruinosa has been used in traditional folk medicine to treat numerous diseases. The molecular mechanism of C. pruinosa pharmacological and biochemical actions of macrophages in inflammation has not been clearly elucidated. We examined how the methanol extract of C. pruinosa regulates production of IL-1beta, TNF-alpha, nitric oxide (NO), and prostaglandin E(2) (PGE(2)) in vitro and in vivo. The extract inhibits these inflammatory mediators in LPS-stimulated murine macrophage cell line RAW264.7 and primary macrophages, by suppressing gene expression of IL-1beta, TNF-alpha, inducible nitric oxide synthase, and cyclooxygenase-2. Moreover, the extract suppresses the nuclear transcription factor NF-kappaB activation in LPS-stimulated RAW264.7 cells. Administration of the extract significantly decreases the plasma levels of these inflammatory mediators in LPS-injected mice. These results suggest that the C. pruinosa methanol extract suppresses inflammation through suppression of NF-kappaB-dependent inflammatory gene expression, suggesting that the C. pruinosa extract may be beneficial for treatment of endotoxin shock or sepsis.

Differential expressions of heme oxygenase-1 gene in CD25- and CD25+ subsets of human CD4+ T cells.

Growing evidence suggests that the immunomodulatory heme oxygenase-1 (HO-1) may have an important role in regulating T-cell responses. In this study, we investigated whether CD4(+)CD25(-) and CD4(+)CD25(+) T cells of human CD4(+) subpopulation could differentially express HO-1. Our results obtained from qualitative reverse transcriptase-polymerase chain reaction and quantitative flow cytometry analyses revealed that the CD4(+)CD25(+) T cells constitutively express HO-1 and that T cell stimulation with plate-bound anti-CD3 in combination with soluble anti-CD28 not only induced HO-1 gene expression in the CD4(+)CD25(-) T cells but also up-regulated HO-1 gene expression in the CD4(+)CD25(+) T cells. Our further studies showed that CD28 signal alone was enough to induce HO-1 expression and CD3 signal, of which signal alone did not induce HO-1 expression, was required at least for full HO-1 expression in both CD25(-) and CD25(+) subsets of human CD4(+) T cells. In addition, transfection of human Jurkat T cells with HO-1 suppressed the cellular proliferation, and this effect was reversed by zinc protoporphyrin, a specific HO competitive inhibitor. Taken together, we have first reported that human CD4(+)CD25(+) regulatory T cells constitutively express HO-1 and that HO-1 inhibits Jurkat T cell proliferation.