Elsevier

Cellular Signalling

Volume 21, Issue 3, March 2009, Pages 405-412
Cellular Signalling

Ceramide 1-phosphate (C1P) promotes cell migration: Involvement of a specific C1P receptor

https://doi.org/10.1016/j.cellsig.2008.11.003Get rights and content

Abstract

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid that is implicated in the regulation of cell homeostasis and the control of inflammation. It is mitogenic for fibroblasts and macrophages, and has been described as potent inhibitor of apoptosis. Using RAW 264.7 macrophages we have now discovered a new biological activity of C1P: stimulation of cell migration. This novel action can only be observed when C1P is applied exogenously to the cells in culture, and not by increasing the intracellular levels of C1P. This fact led to identify a specific receptor through which C1P stimulates cell migration. The receptor is coupled to Gi proteins and causes phosphorylation of extracellularly regulated kinases 1 and 2, and protein kinase B (also known as Akt) upon ligation with C1P. Inhibition of either of these pathways completely abolished C1P-stimulated macrophage migration. In addition, C1P stimulated the DNA binding activity of nuclear factor kappa B, and blockade of this transcription factor resulted in complete inhibition of macrophage migration. This newly identified receptor could be an important drug target for treatment of illnesses that are associated to inflammatory processes, or to diseases in which cell migration is a major cause of pathology, as it occurs in metastatic tumors.

Introduction

Ceramide is a central molecule in sphingolipid metabolism, and plays critical roles in the regulation of diverse pathophysiological processes [1], [2], [3], [4]. It can be generated by de novo synthesis in the endoplasmic reticulum [3], or through activation of different sphingomyelinases in cell membranes [5], [6]. Specifically, ceramide induces cell cycle arrest and apoptosis [1], [2] in most cell types. A key metabolite derived from ceramide is ceramide 1-phosphate (C1P), which is formed through direct phosphorylation of ceramide by ceramide kinase [7]. C1P was first identified in human leukemia HL-60 cells [8] but its role in cell biology remained unnoticed until we found that it stimulated DNA synthesis and cell division in rat fibroblasts [9]. More recently, we demonstrated that C1P blocks cell death in bone marrow-derived macrophages incubated in the absence of macrophage colony stimulating factor (M-CSF), a condition that renders these cells apoptotic. The mechanism whereby C1P blocked apoptosis involved direct inhibition of acid sphingomyelinase (A-SMase) [10], and activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, also known as Akt) pathway [11].

Macrophages are an important component of both innate and adaptive immunity. They are also involved in a number of chronic diseases characterized by unregulated chronic inflammation, such as autoimmune diseases, atherosclerosis [12], or multiple sclerosis [13], and in tumor progression and metastasis [14]. Macrophage populations in tissues are determined by the rates of recruitment of monocytes from the bloodstream into the tissue, the rates of macrophage proliferation and apoptosis, and the rate of macrophage migration or efflux. Here we show that C1P stimulates macrophage migration through a specific receptor that is coupled to Gi proteins.

Section snippets

Materials

Ceramide 1-phosphate (from bovine brain, containing predominantly stearic and nervonic acids), ceramide (from bovine brain), phosphatidic acid, lysophosphatidic acid, Dulbecco's Modified Eagle's medium (DMEM), A23187, interleukin 1-β GDPβS, GTPγS and pertussis toxin were from Sigma Aldrich. N-Acetyl-d-sphingosine (C2-ceramide), sphingosine 1-phosphate, and VPC 23019 were obtained from Avanti Polar lipids. Fetal bovine serum (FBS) was supplied by Gibco. PD98059, LY290042, SC-524, and caffeic

Results and discussion

Leukocyte chemotaxis toward sites of inflammation or tumorigenesis is primarily mediated by chemokine signaling [16]. Fig. 1A and B shows that exogenous C1P potently stimulated migration of RAW 264.7 macrophages in a time and concentration-dependent manner. By contrast, except for sphingosine 1-phosphate (S1P), whose migration-promoting effect is discussed below, the structurally related lipid mediators ceramide, phosphatidic acid (PA), or lysoPA (LPA) did not stimulate chemotaxis (Fig. 1C).

Acknowledgements

We thank Dr. Urs P. Steinbrecher and Dr. Vincent Duronio (Department of Medicine, University of British Columbia, Vancouver, Canada) for their suggestions and advice. This work was supported by grant BFU2006-13689/BFI from Ministerio de Educación y Ciencia (Madrid, Spain); grant 040732, grant IT-309-07 from the “Departamento de Educación, Universidades e Investigación del Gobierno Vasco”, and grant S-PE07UN15 from “Departamento de Industria, Comercio y Turismo del Gobierno Vasco”

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