Ceramide induces p38 MAPK-dependent apoptosis and Bax translocation via inhibition of Akt in HL-60 cells

doi:10.1016/j.canlet.2007.10.030

Cancer Letters

Volume 260, Issues 1–2, 18 February 2008, Pages 88-95

https://doi.org/10.1016/j.canlet.2007.10.030 Get rights and content

Abstract

Ceramide induces apoptosis through caspase activation, cytochrome c release, and Bax translocation in HL-60 cells. However, the upstream signal transduction pathways that induce Bax translocation during ceramide-mediated apoptosis have not been well defined yet. In this study, the activation of p38 mitogen-activated protein kinase (MAPK) was found to be critical for the induction of apoptosis and subcellular redistribution of Bax. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a dominant-negative p38 MAPK attenuated DNA fragmentation, caspase-3 activation, and Bax translocation in response to ceramide. Overexpression of Akt also led to suppression of Bax translocation to mitochondria during ceramide-induced apoptosis in HL-60 cells. We also provide evidence for cross-talk between p38 MAPK and Akt pathways. Expression of myr-Akt or inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 had no effect on p38 MAPK activation by ceramide as assessed by phosphorylation, while inhibition of p38 MAPK by a pharmacological inhibitor or a dominant-negative p38 inhibited Akt dephosphorylation in response to ceramide, suggesting that ceramide-induced p38 MAPK activation negatively regulates the Akt pathway.

Introduction

Ceramide is a sphingolipid second messenger that is involved in mediating a variety of cell functions including growth arrest, differentiation and apoptosis [1], [2], [3]. A number of diverse apoptosis-inducing agents such as tumor necrosis factor-α, chemotherapeutic agents, Fas ligation, irradiation, and corticosteroids generate ceramide by hydrolysis of membrane sphingomyelin by sphingomyelinases or by de novo synthesis [3], [4], [5], [6], [7]. Moreover, exogenous cell-permeable ceramide and endogenous ceramide generated by sphigomyelinase activation specifically induce apoptosis in many different cell types, suggesting that ceramide can act as a mediator of apoptosis.

Ceramide activates a number of signaling cascades including mitogen-activated protein kinases. Ceramide has also been shown to activate proapototic mechanisms by activating ceramide-activated phosphatases and ceramide-activated kinases, which in turn regulate PKC, Akt, and Bcl-2 family proteins [3], [8], [9]. It is well established that mitochondria are important in the control of apoptosis [10], [11], [12], and several studies have addressed the involvement of mitochondrial dysfunction in ceramide-mediated apoptosis. The regulation of mitochondrial membrane integrity and the release of cytochrome c from mitochondria are important processes during ceramide-mediated apoptosis [13], [14], [15]. Members of the antiapoptotic Bcl-2 family are key regulators of mitochondrial-dependent apoptosis. Of these, Bax is a critical regulator of the ceramide-induced apoptotic pathway, upstream of cytochrome c release. Ceramide induces mitochondrial translocation of Bax upon the induction of apoptosis in HL-60 cells [14]. Furthermore, ceramide-induced apoptosis is sensitized in Bax-transfected cells and inhibited by loss of Bax [16]. A recent study suggested that TNFα-mediated mitochondrial generation of ceramide is involved in translocation of Bax to mitochondria and subsequent release of cytochrome c and cell death [17], suggesting a role for the mitochondrial ceramide pool in the mitochondrial pathway of apoptosis.

Although Bax translocation plays a critical role in ceramide-induced apoptosis, the upstream signal transduction pathways that induce Bax translocation during ceramide-mediated apoptosis have not been well defined yet. Therefore, we investigated the regulation of Bax translocation in ceramide-induced apoptosis. The p38 mitogen-activated protein kinase (MAPK) is known as a potential upstream regulator of Bax [18]. p38 MAPK is activated by a variety of cellular stresses including ultraviolet light, hyperosmolarity, heat shock, and proinflammatory cytokines, and acts at early step prior to dysfunction of mitochondria and caspase activation [19], [20], [21]. Translocation of Bax can be also modulated by the PI3K-Akt pathway, which is one of the important pathways that are critical in cell survival [22], [23]. Akt is known to inhibit apoptosis by inactivating proapoptotic proteins such as Bad, procaspase-9, and forkhead and by activating antiapoptotic proteins such as NFκB and cyclic adenosine monophosphate (cAMP)-response element binding protein [24], [25], [26].

Recent studies have shown that ceramide-induced apoptosis is associated with activation of stress-activated protein kinase p38 MAPK [13], [27], and inactivation of Akt kinase pathway [28], [29]. Therefore, we asked whether p38 MAPK and Akt kinase are involved in the regulation of ceramide-induced mitochondrial Bax translocation. We found that activation of p38 MAPK is critical for the induction of apoptosis and Bax subcellular redistribution, and also demonstrated that Akt is capable of suppressing Bax translocation to mitochondria during ceramide-induced apoptosis in HL-60 cells. Furthermore, the present data showed that inactivation of p38 MAPK inhibits ceramide-induced dephosphorylation of Akt, indicating p38MAPK functions as an upstream regulator of Akt.

Section snippets

Reagents and antibodies

Fetal bovine serum was purchased from WEL GENE (Daegu, Korea), and the ECL kit was from Amersham Pharmacia. C₆-ceramide, SB203580, and LY294002 were from Sigma Chemical Co. (St. Louis, MO, USA), and caspase-3 substrate from Biomol (Plymouth Meeting, PA, USA). Antibodies to cytochrome c and PI3 kinase (p85, p110α) were from Cell Signaling Technology (Beverly, MA, USA), and antibodies to p38 MAPK, phospho-p38, Bax, Akt, pAkt (Ser473), and HRP-conjugated secondary antibody were from Santa Cruz

p38 MAPK is involved in ceramide-induced apoptosis in HL-60 cells

Previously, we reported that ceramide induced apoptosis through caspase activation, cytochrome c release, and Bax translocation in HL-60 cells [14]. Since there is an evidence that p38 MAPK acts at an early step prior to mitochondrial dysfunction and caspase activation [18], we analyzed the involvement of p38 MAPK activation in ceramide-mediated apoptosis to elucidate the signaling events that mediate these events. Consistent with previous results in other cell lines [13], [15], [27], treatment

Discussion

Previous studies have implicated p38 MAPK and Akt as regulators of mitochondria-dependent apoptosis. Because ceramide modulates the activity of p38 MAPK and Akt in various cell systems, we hypothesized that these signaling pathways may be involved in ceramide-induced mitochondria-dependent apoptosis. In this study, we provided evidence for the role of p38 MAPK in ceramide-induced apoptosis in HL-60 cells. The present study showed that ceramide induced apoptosis, caspase-3 activation, Bax

Acknowledgements

This work was supported by Grants, from the Basic Research Program of the Korea Science and Engineering Foundation, R01-2003-000-10855-0, and the Strategic Research Program of Chung-Ang University.

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Ceramide induces p38 MAPK-dependent apoptosis and Bax translocation via inhibition of Akt in HL-60 cells

Abstract

Introduction

Section snippets

Reagents and antibodies

p38 MAPK is involved in ceramide-induced apoptosis in HL-60 cells

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Cell Biol.

Mol. Cell. Neurosci.

FEBS Lett.

Exp. Cell Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neurosci. Lett.

J. Biol. Chem.

J. Biol. Chem.

Programmed cell death by ceramide

Science

Functions of ceramide in coordinating cellular responses to stress

Science

Daunorubicin induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis

EMBO J.

Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis

EMBO J.