Activation of p38 Mitogen-Activated Protein Kinase Is Required for Death Receptor–Independent Caspase-8 Activation and Cell Death in Response to Sphingosine Chang-Hwan Yoon, 1 Min-Jung Kim, 1 Moon-Taek Park, 1 Joo-Yun Byun, 1 Young-Hyun Choi, 2 Hwan-Soo Yoo, 3 Yong-Moon Lee, 3 Jin-Won Hyun, 4 and Su-Jae Lee 1 1 Department of Chemistry, Hanyang University, Seoul, Korea; 2 Department of Oriental Medicine, Dougeui University College of Oriental Medicine, Busan, Korea; 3 Department of Biochemistry, College of Pharmacy, Chungbuk National University, Chongju, Korea; and 4 College of Medicine and Applied Radiological Science Research Institute, Cheju National University, Jeju-si, Korea Abstract Sphingosine induces activation of multiple signaling pathways that play critical roles in controlling cell death. However, the precise molecular mechanism of cell death induced by sphingosine remains to be clarified. In this study, we show that sphingosine induces death receptor–independent caspase-8 activation and apoptotic cell death via p38 mitogen-activated protein kinase (MAPK) activation and that suppression of the MAPK/extracellular signal–regulated kinase (ERK) kinase/ERK pathway by protein phosphatase 2A (PP2A) is required for p38 MAPK activation. Treatment of cells with sphingosine induced suppression of ERK and activation of p38 MAPK. Inhibition of p38 MAPK led to the marked suppression of death receptor–independent caspase-8 activation and subsequent cell death induced by sphingosine. Interestingly, pretreatment with phorbol 12-myristate 13-acetate or transfection of MAPK/ERK kinase/ERK resulting in ERK activation completely attenuated sphingosine-induced p38 MAPK activation. PP2A activity was additionally elevated on sphingosine treatment. Small interfering RNA targeting of PP2A effectively attenuated sphingosine-induced p38 MAPK activation through restoration of ERK activity, suggesting PP2A-mediated opposing regulation of ERK andp38MAPK.Ourfindingsclearlyimplythatactivation of p38 MAPK promotes death receptor–independent activation of caspase-8 and apoptotic cell death pathways,thusprovidinganovelcellularmechanismfor the anticancer activity of sphingolipid metabolites. (Mol Cancer Res 2009;7(2):361–70) Introduction Sphingolipid metabolites, such as ceramide, sphingosine, and sphingosine 1-phosphate (S1P), are key regulators of diverse cellular processes, including apoptosis, cell prolifera- tion, differentiation, and inflammation (1). Considerable attention has been focused on ceramide as a potential endogenous mediator of apoptosis in response to cytokines, antigens, anticancer drugs, or environmental stress (2, 3). Indeed, these diverse exogenous stimuli rapidly enhance intracellular levels of ceramide through sphingomyelinase- mediated hydrolysis of sphingomyelin. Ceramide is further metabolized by ceramidase to generate sphingosine during the early stages of apoptosis. Moreover, sphingosine itself is capable of triggering apoptosis when added exogenously to a variety of leukemic cells or solid cancer cell lines (4). These findings have led to the proposal that sphingosine plays a key role in apoptosis signaling. Accumulating evidence suggests a role of mitogen-activated protein kinase (MAPK) in sphingosine-induced apoptosis. For instance, sphingosine treatment results in complete inhibition of extracellular signal–regulated kinase (ERK) activity in leuke- mic and solid cancer cells (5-7), indicating that suppression of this pathway is required for sphingosine-induced apoptosis. Moreover, sphingosine is suggested to function as a mediator of stress responses, leading to the activation of c-jun NH 2 -terminal kinase (JNK) or p38 MAPK (8-11). However, the molecular mechanism by which apoptotic cell death occurs in response to sphingosine has been widely explored but not precisely deciphered. A close relationship exists between the ERK pathway and the p38 MAPK or JNK pathway in a variety of eukaryotic cells (8-11). The concomitant inactivation of survival signals may be necessary for JNK- and p38 MAPK–mediated cell death (11). Interestingly, deprivation of neurotrophic factors or UV irradiation not only activates stress kinase cascades but also leads to dramatic inhibition of the ERK pathway (12). In addition, ERK is required for survival signaling in response to cellular growth factors (13). Furthermore, overexpression of Received 2/5/08; revised 10/5/08; accepted 10/13/08; published OnlineFirst 3/10/09. Grant support: Korea Science and Engineering Foundation (KOSEF) and Ministry of Education, Science and Technology (MEST), Korean government, through its National Nuclear Technology Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article is available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). C-H. Yoon, M-J. Kim, and M-T. Park contributed equally to this work. Requests for reprints: Su-Jae Lee, Laboratory of Molecular Biochemistry, Department of Chemistry, Hanyang University, 17 Haengdang-Dong, Seongdong- Ku, Seoul 133-791, Korea. Phone: 82-2-2220-2557; Fax: 82-2-2299-0762. E-mail: sj0420@hanyang.ac.kr Copyright D 2009 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-08-0069 Mol Cancer Res 2009;7(3). March 2009 361 on July 3, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst March 10, 2009; DOI: 10.1158/1541-7786.MCR-08-0069