Ceramide Kinase Regulates the Production of Tumor Necrosis Factor α (TNFα) via Inhibition of TNFα-converting Enzyme

Nadia F Lamour,Dayanjan S Wijesinghe,Jennifer A Mietla,Katherine E Ward,Robert V Stahelin,Charles E Chalfant

doi:10.1074/jbc.m111.310169

Nadia F Lamour, Dayanjan S Wijesinghe + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m111.310169

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Abstract

Tumor necrosis factor α (TNFα) is a well known cytokine involved in systemic and acute inflammation. In this study, we demonstrate that ceramide 1-phosphate (C1P) produced by ceramide kinase (CERK) is a negative regulator of LPS-induced TNFα secretion. Specifically, bone marrow-derived macrophages isolated from CERK knock-out mice (CERK(-/-)) generated higher levels of TNFα than the wild-type mice (CERK(+/+)) in response to LPS. An increase in basal TNFα secretion was also observed in CERK(-/-) murine embryonic fibroblasts, which was rescued by re-expression of wild-type CERK. This effect was due to increased secretion and not transcription. The secretion of TNFα is regulated by TNFα-converting enzyme (TACE also known as ADAM17), and importantly, the activity of TACE was higher in cell extracts from CERK(-/-) as compared with wild type. In vitro analysis also demonstrated that C1P is a potent inhibitor of this enzyme, in stark contrast to ceramide and sphingosine 1-phosphate. Furthermore, TACE specifically bound C1P with high affinity. Finally, several putative C1P-binding sites were identified via homology throughout the protein sequence of TACE. These results indicate that C1P produced by CERK has a negative effect on the processing/secretion of TNFα via modulation of TACE activity.

Highlights

Pro-TNF␣ is transformed into the active/soluble form through proteolysis by TNF␣-converting enzyme (TACE)
Ex Vivo Cells from CERKϪ/Ϫ Mice Secrete Increased Levels of TNF␣—Compounds structurally related to ceramide 1-phosphate (C1P) have been shown previously to block TNF␣ production in cells challenged with LPS in culture and in vivo in septic shock models (54 –58)
Our laboratory found that ceramide kinase (CERK) and its product C1P play a role in the suppression of TNF␣ production

Summary

Background

Pro-TNF␣ is transformed into the active/soluble form through proteolysis by TNF␣-converting enzyme (TACE). We demonstrate that ceramide 1-phosphate (C1P) produced by ceramide kinase (CERK) is a negative regulator of LPS-induced TNF␣ secretion. Several putative C1Pbinding sites were identified via homology throughout the protein sequence of TACE These results indicate that C1P produced by CERK has a negative effect on the processing/secretion of TNF␣ via modulation of TACE activity. Genetic mouse models lacking key molecules involved in TLR4-mediated signaling are protected from LPSinduced septic shock [8], and one key molecule produced in response to LPS is the proinflammatory/tissue-damaging cytokine, tumor necrosis factor ␣ (TNF␣) [9]. We find that genetic ablation of the enzyme, ceramide kinase (CERK), leads to a significant increase in TNF␣ production in response to LPS. This study demonstrates that the loss of CERK leads to an increase in TACE activity, and TACE is directly and inhibited by the product of CERK, ceramide 1-phosphate (C1P)

EXPERIMENTAL PROCEDURES

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DISCUSSION