Elsevier

Biochemical Pharmacology

Volume 67, Issue 7, 1 April 2004, Pages 1259-1267
Biochemical Pharmacology

The biphasic effects of cyclopentenone prostaglandins, prostaglandin J2 and 15-deoxy-Δ12,14-prostaglandin J2 on proliferation and apoptosis in rat basophilic leukemia (RBL-2H3) cells

https://doi.org/10.1016/j.bcp.2003.10.037Get rights and content

Abstract

Mast cells produce chemical mediators, including histamine and arachidonate metabolites such as prostaglandin D2 (PGD2) after antigen stimulation. Cyclopentenone prostaglandins of the J series, prostaglandin J2 (PGJ2) and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), are thought to be derivatives of PGD2. In this study, the biphasic effects of the PGJ2 and 15d-PGJ2 on proliferation and apoptosis in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mast cells, were examined. At low concentrations, 1 or 3 μM PGJ2 and 15d-PGJ2 induced cell proliferation, respectively. At high concentrations (10–30 μM) both the inhibition of viability and decrease in histamine content in RBL-2H3 cells were dose dependent. These effects were independent of the nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ), since troglitazone, an agonist of PPARγ did not cause any effects in RBL-2H3 cells. Cell death induced by PGJ2 and 15d-PGJ2 was the result of apoptotic processes, since RBL-2H3 cells treated with 30 μM of the prostaglandins had condensed nuclei, DNA fragmentation and increase in activities of caspase-3 and -9. Moreover, PGJ2 or 15d-PGJ2-induced apoptotic effects were prevented by the caspase inhibitor, z-VAD-fmk. In conclusion, the PGJ2 or 15d-PGJ2-induced apoptosis in RBL-2H3 cells occurs mainly via mitochondrial pathways instead of by PPARγ-dependent mechanisms.

Introduction

Mast cells are regarded as critical effector cells in the inflammatory reaction that underlies immediate hypersensitivity-mediated clinical conditions such as bronchial asthma, pollinosis and atopic dermatitis. After activation by cross-linking of FcεRI IgE receptors, mast cells initiate the production and secretion of chemical mediators, such as histamine, lipid-derived PGs and leukotrienes, as well as several types of cytokines. The soluble mediators and cytokines recruit cells to the site of inflammation, resulting in an inflammatory response [1].

PGD2 is a major cyclooxygenase product in various kinds of cells, including mast cells [2], [3]. PGD2 has potent pharmacological actions such as bronchoconstriction, peripheral vasodilation, inhibition of platelet aggregation and neuromodulation, so on [4], [5]. Two different types of PGD2 synthesizing enzymes, PGD synthetase (PGDS) have been characterized, purified and cloned by Urade et al. [6]. One type of PGDS is a glutathione requiring enzyme, hematopoietic PGDS that is present in mast cells. PGD2 is readily dehydrated to the cyclopentenone prostaglandins of the J series (PGJ2 series), such as PGJ2 and 15d-PGJ2, via an albumin-independent mechanism [7], [8]. The PGJ2 series display several unique characteristics associated with cell proliferation and apoptosis. Specifically, a cytotoxic effect of Δ12-PGJ2 is readily induced after translocation, accumulation in nuclei, and binding to nuclear proteins [9]. In addition the electrophilic carbons in cyclopentenone rings of the 15d-PGJ2 can directly conjugate with a thiol residue and to modify the function of proteins [10]. Furthermore, 15d-PGJ2 is a natural ligand of the nuclear receptor, PPARγ. The mechanisms involved in PPARγ-dependent [11], [12] and PPARγ-independent cytotoxic effects [13] are unknown.

Because so little is known about the biological significance of the PGJ2 series in mast cells [14], [15], [16], we have studied the cytotoxic effects of the PGJ2 and 15d-PGJ2 in mast cells.

Apoptosis is the physiological process of cell death that occurs in all multicellular organisms. In mast cells, apoptosis is also a key process in the regulation of inflammation and proliferation. It is also responsible for maintaining a constant cell number in tissues under normal conditions. Mast cell hyperplasia occurs in several clinical conditions; for example, host responses to parasites and the chronic inflammatory conditions that are present during either Crohn’s disease or rheumatoid arthritis [17]. In contrast, mast cells also express Fas antigen and undergo apoptosis by direct activation of Fas antigen [18]. In many cases, apoptotic pathways are mediated via the p53 tumor suppressor protein [19]. The involvement of p53 in initiating apoptosis results in the up-regulation of proapoptotic members of the bcl-family, such as bax, in mitochondria [20]. The role of survival/apoptosis-regulated genes in mast cells activation is now being investigated [17].

In this study, the exogenous PGJ2 and 15d-PGJ2 both induced bifunctional effects on RBL-2H3 cells. Specifically, proliferation occurred at the lower concentrations and apoptosis was observed at the higher concentrations. These concentration-dependent bifunctional effects were independent of PPARγ and DP-receptor. And the apoptotic effects were mainly mediated by mitochondrial pathways, not involved in p53 accumulation.

Section snippets

Assay of cell proliferation and viability

The rat mast cell line RBL-2H3 was kindly supplied from Dr. Michael A. Beaven (National Institutes of Health). The incorporation of 5-bromo-2′-deoxyuridine (BrdU) in place of thymidine is carried out to monitor cell proliferation. Briefly, RBL-2H3 cells (2×104 cells/200 μL/well) were cultured with BrdU in 96-well culture plate for 24 hr at 37°, and BrdU assay was carried out according to manufacture’s protocol. The optical density was then measured on a microplate reader with a wavelength of 450 

Biphasic effects of PGJ2 and 15d-PGJ2 on proliferation of RBL-2H3 cells

The incorporation of BrdU in place of thymidine is measured to monitor cell proliferation. Cell proliferation was enhanced by PGJ2 at 1 μM (1.23-fold compared with the control) and by 15d-PGJ2 at 3 μM (1.27-fold), showing that the potency of cell proliferative effect induced by 15d-PGJ2 was slightly less than that of PGJ2 (Fig. 1). Incubation of RBL-2H3 cells with high concentrations of either PGJ2 or 15d-PGJ2 caused significant cytotoxic effects. Cell proliferation was suppressed by 48.4±9.6%,

Discussion

Mast cells play key roles in allergy and inflammatory responses via synthesis and release of inflammatory mediators. Some of the mediators are preformed and stored in secretory granules. Others such as the PGs, which are arachidonic acid metabolites, are generated de novo. PGJ2 and 15d-PGJ2, dehydration of PGD2, are known as cytotoxic inducer [7]. They also have been identified as ligands for PPARγ and regulate adipocyte differentiation and inflammatory response in macrophages [12].

In this

Acknowledgements

We are grateful to Dr. Takashi Inui (Tsu City College) for helpful suggestions and Dr. Yo Sato (RIKEN Harima Institute) for assistance with the TUNEL assay, as well as critical suggestions. We also thank Dr. Yasushi Fujitani (Takeda Chemical Industry, Ltd.) for critical suggestions, and Prof. Junya Tanaka (Ehime University, Dept. Physiology) for assistance with the fluorescence staining assay.

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