Pertussis toxin promotes macrophage survival through inhibition of acid sphingomyelinase and activation of the phosphoinositide 3-kinase/protein kinase B pathway

doi:10.1016/j.cellsig.2007.04.001

Cellular Signalling

Volume 19, Issue 8, August 2007, Pages 1772-1783

https://doi.org/10.1016/j.cellsig.2007.04.001 Get rights and content

Abstract

Apoptosis is an important mechanism involved in regulating the number of macrophages present at sites of inflammation. Several lines of evidence indicate that blocking macrophage apoptosis can increase atherosclerosis. We previously reported that oxidized LDL can inhibit apoptosis in cultured bone marrow-derived macrophages. We used pertussis toxin (PTX) to test whether G protein coupled receptors are activated by oxLDL. PTX is a bacterial toxin that inhibits G_i activation by ADP-ribosylating the α subunit of G_i, preventing the subunit from interacting with receptors. Unexpectedly, we found that PTX by itself selectively blocks macrophage apoptosis in a dose-dependent manner. PTX acts in part by inhibiting acid sphingomyelinase activity which in turn prevents generation of ceramide, which is required for macrophage apoptosis. A G_i activator peptide, mastoparan, increased ceramide levels in macrophage and induced apoptosis, but pre-treatment with PTX partially overrode mastoparan-induced apoptosis. The anti-apoptotic effect of PTX was found to require ADP-ribosylation. PTX failed to prevent A-SMase activation or apoptosis in macrophages lacking TLR4. The anti-apoptotic effect of PTX involved the same signaling pathways as those of oxidized LDL, in that both inhibited acid sphingomyelinase, and activated the phosphoinositide 3 kinase (PI3K)/protein kinase B (PKB) pathway which leads to nuclear localization of the transcription factor NFκB and up-regulation of Bcl-X_L. These results indicate that G_i proteins, TLR4, A-SMase and the PI3K/PKB pathway are crucial components for regulation of macrophage apoptosis.

Introduction

In previous studies using mouse peritoneal macrophages we showed that oxidized low density lipoproteins (oxLDL), or lysophosphatidylcholine (LPC), stimulated the activity of phospholipase D (PLD). This enzyme activity generates phosphatidic acid (PA) from glycerophospholipids. PA is a potent mitogenic agent and second messenger that has been implicated in various pathophysiological processes, including atherosclerosis [1]. We found that PLD activation by oxLDL or LPC was inhibited by pertussis toxin, abbreviated PTX [2], [3]. PTX is a protein that is secreted by Bordetella pertussis and consists of five different subunits, designated S1 to S5. There are two S4 subunits, and so the complete toxin molecule is a hexamer [4]. PTX consists of an A subunit that carries the biologic activity and a B subunit that binds the complex to the cell membrane [4]. The S1 subunit constitutes the A protomer and the B oligomer is formed by the remaining five subunits [4]. PTX is a member of the family of ADP-ribosylating bacterial toxins. The S1 subunit of PTX ADP-ribosylates Cys352 of G_i. This modification of cysteine prevents G protein heterotrimers from interacting with receptors to block their coupling and activation. PTX has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in cell signaling processes. The inhibitory effect of PTX on PLD activation by oxLDL or LPC suggested that G_i proteins were involved in this process.

Some of the effects of PTX can be explained through actions other than G-protein inhibition. For example, PTX has been shown to use Toll-like receptor (TLR) 4 signaling to mediate some of its pathologic effects. It facilitates the break-down of the blood–brain barrier and is widely used as an adjuvant in experimental autoimmune encephalomyelitis [5]. TLRs are expressed on several cell types, including cells of the immune system [6]. One action of TLR4 signaling results in translocation of NF-kB, which induces transcription of a variety of genes, including those for pro-inflammatory cytokines. Therefore, some of the effects of PTX can be explained through actions other than G-protein inhibition.

In the present study, we show that PTX inhibits acid sphingomyelinase activation and the resulting accumulation of ceramides in bone marrow-derived macrophages. This is associated with inhibition of apoptosis that normally results from withdrawal of M-CSF from these cells. PTX caused phosphorylation of protein kinase B (PKB), activation of the transcription factor NFκB and up-regulation of Bcl-X_L, an anti-apoptotic Bcl-2 protein. These are the same mechanisms involved in the inhibition of apoptosis by oxLDL [7].

Section snippets

Reagents

Pertussis toxin (PTX), PTX B-oligomer, propidium iodide and protease inhibitor cocktail and RPMI 1640 medium were purchased from Sigma-Aldrich. Annexin V-FITC conjugated antibody was obtained from BD Pharmingen. Caspases FLICA kit was from Immunochemistry Technologies. C2-ceramide and C2-dihydroceramide were purchased from Avanti Polar Lipids. ³H-Palmitate and [N-methyl-¹⁴C] bovine sphingomyelin were obtained from Mandel Scientific. Mastoparan, mastoparan 17, and all the kinase inhibitors were

Pertussis toxin can selectively protect macrophages from apoptosis induced by cytokine withdrawal

We have previously shown that BMDM undergo apoptosis if incubated in the absence of M-CSF for 24 h or more, and that this is blocked by incubation with oxLDL [7]. To test if a G-protein-coupled membrane receptor(s) might be involved in oxLDL mediated cell survival, the G-protein inhibitor PTX, was used. Unexpectedly, we found that control incubations done with PTX in the absence of cytokine or oxLDL also promoted cell viability in a dose-dependent manner in macrophages (Fig. 1A). The

Discussion

There are several reports in the literature showing that some of the biological effects of oxLDL are sensitive to PTX. Whitman et al. demonstrated that uptake of acetylated LDL by macrophages is regulated by PTX-sensitive G proteins [34]. It was also reported that oxLDL-induced macrophage proliferation is mediated through a PTX-sensitive G-protein-coupled receptor [35] while another group documented that oxLDL induced cytotoxicity in vascular smooth muscle cells is mediated through

Acknowledgements

We would like to thank C. Chingkoe for technical assistance. This study was supported by grants from the Heart and Stroke Foundation of British Columbia and Yukon (Canada), and BFU2006-13689/BFI from Ministerio de Educacion y Ciencia (Madrid, Spain).

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¹: These authors have contributed equally to this work.

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Pertussis toxin promotes macrophage survival through inhibition of acid sphingomyelinase and activation of the phosphoinositide 3-kinase/protein kinase B pathway

Abstract

Introduction

Section snippets

Reagents

Pertussis toxin can selectively protect macrophages from apoptosis induced by cytokine withdrawal

Discussion

Acknowledgements

Biochim. Biophys. Acta

J. Lipid Res.

Trends Immunol.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Biol. Chem.

Blood

Exp. Hematol.

Semin. Cell Dev. Biol.

J. Lipid Res.

FEBS Lett.

FEBS Lett.

J. Lipid Res.

Atherosclerosis

J. Lipid Res.

J. Lipid Res.

Exp. Hematol.

Biochem. Biophys. Res. Commun.

Blood

J. Biol. Chem.