Resveratrol strongly enhances the retinoic acid-induced superoxide generating activity via up-regulation of gp91-phox gene expression in U937 cells

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Highlights

  • Resveratrol effectively enhanced the RA-induced O2-generating activity.

  • Resveratrol strongly enhanced gene expression of gp91-phox.

  • Resveratrol caused remarkable accumulation of cytochrome b558 and p47-phox proteins.

  • Resveratrol promoted histone acetylation around the promoter of gp91-phox gene.

  • Resveratrol enhances the O2-generating activity via transactivation of gp91-phox.

Abstract

The membrane bound cytochrome b558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O2)-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, dramatically activates the O2-generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and resveratrol, the O2-generating activity increased more than 5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and resveratrol strongly enhanced transcription of the gp91-phox compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and resveratrol caused remarkable accumulation of protein levels of gp91-phox (to 4-fold), p22-phox (to 5-fold) and p47-phox (to 4-fold) compared with those of the RA-treatment alone. In addition, ChIP assay suggested that resveratrol participates in enhancing the gene expression of gp91-phox via promoting acetylation of Lys-9 residues and Lys-14 residues of histone H3 within chromatin around the promoter regions of the gene. These results suggested that resveratrol strongly enhances the RA-induced O2-generating activity via up-regulation of gp91-phox gene expression in U937 cells.

Introduction

Upon bacterial infection, phagocytes (e.g. neutrophil, monocyte and macrophage) rapidly phagocytose the invading bacteria and generate superoxide anion (O2) [1]. At the initial reaction, the O2-generating system, a membrane-associated protein complex, generates O2, which is released inside phagosomes or outside the cells. O2 is used for the precursor of the stronger reactive oxygen species such as hydrogen peroxide, hydroxyl radical and hypochlorite which are used to kill the invading bacteria. Five specific protein factors (membrane proteins p22-phox and gp91-phox (the small and large subunits of cytochrome b558), cytosolic proteins p40-phox, p47-phox and p67-phox) and small G-protein Rac are essential for the O2-generating system [2]. The system is dormant in resting phagocytes, but is activated during phagocytosis to carry an electron from NADPH to molecular oxygen resulting in O2 generation. The importance of this system is emphasized by a genetic disorder known as chronic granulomatous disease (CGD), where phagocytes cannot generate O2 upon stimulation. In particular, it is well known that the four genes encoding p22-phox, gp91-phox, p47-phox and p67-phox are responsible for CGD [3]. Since the O2-generating system does not act in undifferentiated phagocytes including leukemia cells, this system is regarded as one of the differentiation indexes in phagocytes [4].

Human leukemia cell lines have been frequently used as models to study not only differentiation of leukocytes but also therapy of leukemia [5]. U937 cells, one of the human monoblastic leukemic cell lines, lack the O2-generating activity [6]. Upon stimulation by various agents: phorbol esters, dimethylsulfoxide, interferon-γ, tumor necrosis factor, vitamin D3 (VD3) and all-trans retinoic acid (RA), U937 cells can be differentiated to macrophage-like cells and produce the O2 [5], [6], [7]. Among them, RA has been used to improve treatment for leukemia including acute promyelocytic leukemia [8]. In fact, leukemia therapy using RA has been improved stepwise in combination with cytotoxic chemotherapy [9]. In addition, more effective and safer phytochemicals are expected as modifiers in leukemia therapy. In recent year, we reported that curcumin, a phenolic compound responsible for the yellow color of turmeric, dramatically enhances retinoic acid-induced superoxide generating activity via accumulation of p47-phox and p67-phox proteins in U937 cells [10].

Resveratrol (3, 5, 4′-trihydroxyl-trans-stilbene) is a bioactive polyphenolic compound belonging to stilbenoids. Since resveratrol targets numerous molecules, including transcription factors, tumor suppressors, cell cycle regulators, apoptotic regulators and enzymes, it has several prophylactic and therapeutic effects such as anti-inflammatory, -oxidant, -cancer, -leukemia, -diabetic and -neurodegenerative effects [11], [12], [13], [14], [15], [16], [17]. Interestingly, it has been reported that resveratrol is an inducer of differentiation of human leukemia cells [18]. However, little is known regarding mechanisms of up-regulation of the O2-generating activity by resveratrol during RA-induced differentiation of leukocytes. In this study, we show that resveratrol strongly enhances the RA-induced O2-generating activity via up-regulation of gp91-phox gene expression in U937 cells.

Section snippets

Materials

Oxyresveratrol, piceatannol, pinostilbene, pterostilbene, resveratrol, rhapontigenin, trans-stilbene (Tokyo Chemical Industry, Tokyo, Japan), pinosylvin (Cosmo Bio, Tokyo, Japan), phorbol 12-myristate 13-acetate (PMA) (Calbiochem, Darmstadt, Germany), RA, luminol (Sigma, St Louis, MO, USA), PMSF (Wako, Osaka, Japan) and Diogenes (National Diagnostics, Atlanta, GA, USA) were obtained. Monoclonal anti-gp91-phox antibody, monoclonal anti-p47-phox antibody, monoclonal anti-p67-phox antibody (BD

Resveratrol dramatically enhances the RA-induced O2-generating activity in U937 cells

First, we examined effects of RA and stilbenoids on the O2-generating activity in U937 cells (Fig. 1A). Untreated U937 cells generated a negligible level of O2 when stimulated with PMA. In contrast, the O2-generating activity was dramatically induced in the presence of RA. While resveratrol and pinostilbene slightly induced the O2-generating activity, pinosylvin and piceatannol showed no effect on the activity. Other stilbenoids (oxyresveratrol, pterostilbene, rhapontigenin and

Acknowledgements

We thank H. Madhyastha and R. Madhyastha for editorial reading of the manuscript. This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 16K00895, to H. K. and No. 15K09671, to F. K.). This work was also partly supported by the Grant for Joint Research Project of The Institute of Medical Science, The University of Tokyo (No. 2016-S301, to H. K.).

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