prostaglandin-d2 and verlukast

15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a representative J-series cyclopentenone prostaglandin bearing an electrophilic α,β-unsaturated carbonyl group. In the present study, treatment of human breast cancer MCF-7 cells with 15d-PGJ(2) caused the up-regulation of the glutamate cysteine ligase catalytic (GCLC) subunit, the rate-limiting enzyme in glutathione (GSH) synthesis. 15d-PGJ(2) treatment caused nuclear translocation and transactivation of Nrf2, a redox-sensitive transcription factor responsible for induced expression of antioxidant and other cytoprotective genes. siRNA knockdown of Nrf2 abrogated 15d-PGJ(2)-induced GCLC expression. Following 15d-PGJ(2) treatment, the intracellular GSH level was initially diminished but eventually enhanced even above the basal level. The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) abolished the 15d-PGJ2-induced Nrf2 activation and GCLC expression. Pharmacologic inhibition or siRNA knockdown of Akt, the target of phosphoinositide 3-kinase (PI3-K), attenuated 15d-PGJ(2)-induced Nrf2 activation and GCLC expression, and NAC treatment inhibited phosphorylation of Akt, and subsequently Nrf2 activation and GCLC upregulation. 9,10-Dihydro-15-PGJ2, a nonelectrophilic analogue of 15d-PGJ(2) that lacks the ability to form a conjugate with GSH, failed to induce activation of Akt and Nrf2 as well as ROS generation. These findings, taken all together, suggest that intracellular accumulation of ROS formed as a consequence of initial depletion of GSH can activate Akt, which in turn induces Nrf2 activation and subsequently the expression of GCLC, leading to the restoration of GSH. Interestingly, the extracellular GSH level was increased, concomitantly with the depletion of the intracellular GSH following 15d-PGJ(2) treatment. However, 15d-PGJ(2) was unable to influence both intra- and extra-cellular GSH levels when multidrug resistance-associated protein 1 (MRP1), the efflux pump for GSH conjugates, was blocked by its antagonist, MK571. Moreover, 15d-PGJ(2)-induced GCLC expression was attenuated by the MK571 and also by siRNA knockdown of MRP1, suggesting that MRP1 contributes to 15d-PGJ(2)-mediated up-regulation of GCLC by pumping out the 15d-PGJ(2)-GSH conjugate. It is speculated that 15d-PGJ(2), once effluxed through MRP, liberates from the GSH conjugate, and the free 15d-PGJ(2) re-enters the cell and forms the GSH conjugate again. In conclusion, MRP1 mediates Nrf2-dependent up-regulation of GCLC in

Topics: Acetylcysteine; Breast Neoplasms; Cell Line, Tumor; Female; Glutamate-Cysteine Ligase; Glutathione; Humans; Multidrug Resistance-Associated Proteins; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinases; Propionates; Prostaglandin D2; Proto-Oncogene Proteins c-akt; Quinolines; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Signal Transduction; Up-Regulation

We previously reported that interleukin (IL)-4 upregulates the expression of leukotriene C(4) synthase (LTC(4)S) by human cord blood--derived mast cells (hMCs), augments their high-affinity Fc receptor for IgE (Fc(epsilon)RI)-dependent generation of eicosanoids and cytokines, and induces a calcium flux in response to cysteinyl leukotrienes (cys-LTs) and uridine diphosphate (UDP) that is blocked by cys-LT receptor antagonists. We speculated that this IL-4-dependent, receptor-mediated response to the cys-LTs and UDP might induce cytokine generation by hMCs without concomitant exocytosis. Unlike hMCs maintained in cytoprotective stem cell factor (SCF) alone, hMCs primed for 5 d with IL-4 responded to UDP (1microM), LTC(4) (100 nM), and LTD(4) (100 nM) by producing IL-5, tumor necrosis factor (TNF)-alpha, and especially large quantities of macrophage inflammatory protein (MIP)-1beta de novo at 6 h, preceded by the induced expression of the corresponding mRNAs. Cys-LT- and UDP-mediated cytokine production by the primed hMCs occurred without histamine release or PGD(2) generation and was inhibited by the CysLT1 receptor antagonist MK571. Additionally, pretreatment of hMCs with MK571 or with the cys-LT biosynthetic inhibitor MK886 decreased IL-5 and TNF-alpha production in response to IgE receptor cross-linkage, implying a positive feedback by endogenously produced cys-LTs. Cys-LTs and UDP thus orchestrate a novel, IL-4-regulated, non-IgE-dependent hMC activation for cytokine gene induction that could be initiated by microbes, cellular injury, or neurogenic or inflammatory signals; and this pathobiologic event would not be recognized in tissue studies where hMC activation is classically defined by exocytosis.

Topics: Cysteine; Cytokines; DNA-Binding Proteins; Exocytosis; Interleukin-4; Leukotriene Antagonists; Leukotrienes; MAP Kinase Signaling System; Mast Cells; NFATC Transcription Factors; Nuclear Proteins; Propionates; Prostaglandin D2; Quinolines; Receptors, IgE; Transcription Factors; Uridine Diphosphate