Arachidonic acid induces Fas and FasL upregulation in human leukemia U937 cells via Ca2+/ROS-mediated suppression of ERK/c-Fos pathway and activation of p38 MAPK/ATF-2 pathway

doi:10.1016/j.toxlet.2009.08.016

Toxicology Letters

Volume 191, Issues 2–3, 15 December 2009, Pages 140-148

https://doi.org/10.1016/j.toxlet.2009.08.016 Get rights and content

Abstract

Arachidonic acid (AA)-induced apoptotic death of human leukemia U937 cells was characteristic of increase in intracellular Ca²⁺ concentration ([Ca²⁺]i), ROS generation, ERK inactivation, p38 MPAK activation, degradation of procaspase-8 and production of truncated Bid (tBid). Moreover, AA treatment upregulated Fas/FasL protein expression and transcription of Fas/FasL mRNA. Downregulation of FADD blocked AA-induced procaspase-8 degradation and rescued viability of AA-treated cells. BAPTA-AM (Ca²⁺ chelator) pretreatment abolished AA-induced ROS generation, while N-acetylcystein (NAC, ROS scavenger) was unable to alter AA-elicited [Ca²⁺]i increase. Pretreatment with BAPTA-AM or NAC abrogated p38 MAPK activation and restored ERK activation. Suppression of p38 MAPK or transfection of constitutively active MEK1 abolished AA-induced Fas and FasL upregulation. AA treatment repressed ERK-mediated c-Fos phosphorylation but evoked p38 MAPK-mediated ATF-2 phosphorylation. Knockdown of c-Fos and ATF-2 by siRNA reflected that c-Fos counteracted the effect of ATF-2 on Fas/FasL upregulation. Taken together, our data indicate that Fas/FasL upregulation in AA-treated U937 cells is elicited by Ca²⁺/ROS-mediated suppression of ERK/c-Fos pathway and activation of p38 MAPK/ATF-2, and suggest that autocrine Fas-mediated apoptotoic mechanism is involved in AA-induced cell death.

Introduction

The enzymes phospholipase A₂ (PLA₂) hydrolyze fatty acids at the sn-2 position of phospholipids, which generates free acids and lysophospholipids are involved in several cell functions including the processing of pro-inflammatory mediators, cell proliferation, and apoptosis (Kini, 1997, Triggiani et al., 2000, Taketo and Sonoshita, 2002). Our recent studies showed that Naja naja atra PLA₂ elicited Fas and FasL upregulation in human leukemia U937 cells and induced apoptotic death of human leukemia U937 cells (Liu et al., 2009). In view of the inability of catalytically inactive PLA₂ to upregulate Fas/FasL protein expression in U937 cells, PLA₂-induced Fas/FasL upregulation was thus suggested to be related to the catalytic activity of PLA₂. Noticeably, apoptosis is ultimately executed by caspases that operate both through the receptor-mediated pathway containing members of the tumor necrosis factor (TNF) family (e.g. TNF-R1, Fas, TRAIL-R1 and TRAIL-R2) and the mitochondrial-mediated pathway involving cytochrome c release from the mitochondria (Kroemer et al., 1998). The abnormalities in the apoptotic response are related to the development of drug resistance in leukemia (Wang, 2003, Testa and Riccioni, 2007). Thus, understanding the mechanism of the phospholipid hydrolytic products responsible for upregulating Fas/FasL in PLA₂-treated U937 cells may implicate a new paradigm for leukemia therapy.

Arachidonic acid (AA), a biologically active polyunsaturated fatty acid, is released from membrane phospholipids endogenously upon agonist-induced activation of PLA₂ or upon cellular activation by a wide array of external stimuli (Gijon and Leslie, 1997). Upon release, AA is rapidly converted into a number of metabolites with enhanced or altered biological activity (Pompeia et al., 2003a, Pompeia et al., 2003b). Although many effects of AA on cell function are mediated by its conversion into eicosanoids (Rizzo, 2002), several studies have shown that AA per se is involved in the regulation of a number of signal transduction pathways (Rizzo and Carlo-Stella, 1996, Rizzo et al., 1999, Rao et al., 1994). AA-derived prostaglandins, leukotrienenes and lipoxins are identified to be involved in inflammation, cancer, autoimmunity and allergic diseases (Harizi et al., 2008). The functions of infiltrating neutrophiles and other leukocytes are not only directly affected by AA but also its metabolites generated by lipoxygenases and cyclooxygenases (Hii and Ferrante, 2007). Moreover, AA physiologically modulates the ion current of voltage-gated calcium channels (Roberts-Crowley et al., 2009). Alternatively, a number of studies suggest a role for AA, either released endogenously by PLA₂ or presented exogenously to cells, in apoptosis of hematopoietic cells (Finstad et al., 1994, Seatter et al., 1997). Although AA influences growth and survival of hematopoietic cells in a cell-type specific manner (Finstad et al., 1998), it is found that leukemic cells are more sensitive to the antiproliferative effect of AA than normal cells (Rizzo et al., 1999).

Lysophospholipids such as lysophosphatidylcholine (LysoPC), sphingosylphosphorylcholine, lysophosphatidic acid and sphingosine 1-phosphate regulate a wide array of biological processes (Meyer zu Heringdorf and Jokobs, 2007, Matsumoto et al., 2007). LysoPC is recognized as an important cell signaling molecule produced under physiological conditions by the action of PLA₂ on phosphatidylcholine. In addition to a well-known role in atherosclerosis, LysoPC is able to enhance endothelial proliferation and permeability, stimulate adhesion and activation of lymphocytes, initiate chemotaxis of macrophages, impair migration and proliferation in vascular smooth muscle cells and modify platelet aggregation and coagulation pathways (Kougias et al., 2006). Recent studies revealed that LysoPC induces apoptosis of H19-7 hippocampal progenitor cells through upregulation of FasL protein expression (Sun et al., 2009). Obviously, LysoPC displays cytotoxicity in a cell-specific manner.

Noticeably, PLA₂-elicited ROS generation was located at an upstream position responsible for autocrine Fas-mediated death pathway in PLA₂-treated U937 cells (Liu et al., 2009). Given that AA and LysoPC have a capability in inducing ROS generation in neutophils or leukemia cells (Cherny et al., 2001, Pompeia et al., 2003b, Bostan et al., 2003, Muller et al., 2002), the effects of AA and LysoPC on Fas/FasL protein expression of U937 cells were thus investigated in the present study. Compared with other human malignant hematopoietic cell lines, U937 cells had a lower expression of cell surface Fas protein (Kim et al., 2000). Reduction in transcription of FAS gene in variants of U937 cells heavily offers resistance to Fas-mediated apoptosis (Blomberg et al., 2009). Several studies showed that chemotherapeutic drugs and physicochemical stimuli induce apoptosis of U937 cells through upregulation of Fas protein expression (Micheau et al., 1997, Caricchio et al., 1998). Moreover, U937 cells were sensitive to FasL-induced apoptosis (Perez-Cruz et al., 2003). Taken together, this makes U937 cells be a good model in response to autocrine Fas-mediated death pathway. Our data showed that, unlike LysoPC, AA treatment induced Fas/FasL upregulation in U937 cells. Moreover, AA induced increase in intracellular Ca²⁺ concentration and ROS generation, which activated p38 MAPK and inactivated ERK. AA-elicited inactivation of ERK/c-Fos pathway proceeded in concert with AA-evoked p38 MAPK/ATF-2, resulting in upregulation of Fas and FasL in AA-treated cells. The results are presented in this report.

Section snippets

Materials and methods

AA, N-acetylcysteine (NAC), LysoPC, SB202190 (p38 MAPK inhibitor), digitonin, propidium iodide and MTT were purchased from Sigma–Aldrich Inc. BAPTA-AM, Fluo-4 AM, dichlorodihydrofluorescein diacetate (H₂DCFDA), annexin V-FITC kit and rhodamine-123 were the products of Molecular Probes. Anti-caspase-3 and anti-caspase-8 antibodies were obtained from Calbiochem, and anti-β-actin antibodies were the product of Chemicon International Inc. Anti-p38 MAPK, anti-phospho-p38 MAPK, anti-JNK,

Results and discussion

Upon exposure to AA and LysoPC, U937 cells showed a concentration- and time-dependent decrease in cell viability (Fig. 1A). Compared with LysoPC, AA showed a higher cytotoxicity toward U937 cells. Because the dose required for half-maximum inhibition of viability was around 25 μM after AA treatment for 24 h, this single treatment was used for further assessment of cytotoxicity. Fig. 1B shows an increase in Fas and FasL protein expression after AA treatment. Although LysoPC was reported to induce

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grant NSC98-2320-B110-002-MY3 from the National Science Council, ROC (to L.S. Chang), and grant of National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center.

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Arachidonic acid induces Fas and FasL upregulation in human leukemia U937 cells via Ca2+/ROS-mediated suppression of ERK/c-Fos pathway and activation of p38 MAPK/ATF-2 pathway

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Conflict of interest

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

Cell Dev. Biol.

Blood

Trends Mol. Med.

Mol. Cell

Gene

Biochm. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Blood

Prostaglandins Leukot. Essent. Fatty Acids

Blood

Cell Calcium

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Cell. Signal.

Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis

FEBS J.

Phospholipae A2 modulates respiratory burst developed by neutrophils in patients with rheumatoid arthritis

J. Cell. Mol. Med.

Fas/Fas ligand interactions are involved in ultravilolet-B-induced human lymphocyte apoptosis

J. Immunol.

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2-treated human neuroblastoma SK-N-SH cells through ROS- and Ca2+-mediated p38 MAPK activation

J. Cell. Biochem.

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A role for UV-light-induced c-Fos: stimulation of nucleotide excision repair and protection against sustained JNK activation and apoptosis

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Deregulation of the mitochondrial apoptotic machinery and development of molecular targeted drugs in acute myeloid leukemia

Curr. Cancer Drug Targets

Arachidonic acid induces Fas and FasL upregulation in human leukemia U937 cells via Ca²⁺/ROS-mediated suppression of ERK/c-Fos pathway and activation of p38 MAPK/ATF-2 pathway

Phospholipae A₂ modulates respiratory burst developed by neutrophils in patients with rheumatoid arthritis

Upregulation of Fas and FasL in Taiwan cobra phospholipase A₂-treated human neuroblastoma SK-N-SH cells through ROS- and Ca²⁺-mediated p38 MAPK activation