A neutral sphingomyelinase resides in sphingolipid-enriched microdomains and is inhibited by the caveolin-scaffolding domain: potential implications in tumour necrosis factor signalling

Sphingomyelinases hydrolyse sphingomyelin to ceramide, a process involved in signal-transduction routes leading to apoptosis and various other cellular responses. In the present study, we investigated the sphingomyelinase content of caveolae, invaginated plasma-membrane microdomains that contain a variety of signalling molecules. These structures are highly enriched in sphingomyelin as well as in ceramide, which suggests that metabolism of these lipids might, to some extent, occur locally. By cell fractionation, we demonstrate that, in addition to a previously reported minute amount of acidic sphingomyelinase activity, a substantial amount of neutral sphingomyelinase activity resides in caveolae of human skin fibroblasts. This caveolar neutral sphingomyelinase activity was also detected in Niemann–Pick disease type A fibroblasts, which are completely devoid of functional acidic sphingomyelinase. Neutral (but not acidic) sphingomyelinase activity was specifically inhibited by a peptide that corresponds to the scaffolding domain of caveolin, which suggests a direct molecular interaction between the two proteins. In addition, this finding implies a cytosolic orientation of the caveolar neutral sphingomyelinase. Interestingly, stimulation of fibroblasts with tumour necrosis factor α (TNFα) resulted in a partial shift of its p55 receptor to caveolin-enriched membrane fractions and the appearance of caveolin-sensitive neutral sphingomyelinase activity in the non-caveolar fractions. These results suggest that (part of) the presently identified caveolar neutral sphingomyelinase activity is involved in TNFα signalling.

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Neutral sphingomyelinase 2 (nSMase2) is the primary neutral sphingomyelinase isoform activated by tumour necrosis factor-α in MCF-7 cells

Biochemical Journal ◽

10.1042/bj20101752 ◽

2011 ◽

Vol 435 (2) ◽

pp. 381-390 ◽

Cited By ~ 29

Author(s):

Christopher J. Clarke ◽

Emily A. Cloessner ◽

Patrick L. Roddy ◽

Yusuf A. Hannun

Keyword(s):

Tumour Necrosis Factor ◽

Cell Lines ◽

Tumour Necrosis ◽

Tumour Necrosis Factor Α ◽

Neutral Sphingomyelinase ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor ◽

Mcf 7

Activation of N-SMase (neutral sphingomyelinase) is an established part of the response of cytokines such as TNF (tumour necrosis factor)-α. However, it remains unclear which of the currently cloned N-SMase isoforms (nSMase1, nSMase2 and nSMase3) are responsible for this activity. In MCF-7 cells, we found that TNF-α induces late, but not early, increases in N-SMase activity, and that nSMase2 is the primary isoform activated, most likely through post-transcriptional mechanisms. Surprisingly, overexpression of tagged or untagged nSMase3 in multiple cell lines had no significant effect on in vitro N-SMase activity. Moreover, only overexpression of nSMase2, but not nSMase1 or nSMase3, had significant effects on cellular sphingolipid levels, increasing ceramide and decreasing sphingomyelin. Additionally, only siRNA (small interfering RNA) knockdown of nSMase1 significantly decreased basal in vitro N-SMase activity of MCF-7 cells, whereas nSMase2 but not nSMase3 siRNA inhibited TNF-α-induced activity. Taken together, these results identify nSMase2 as the major TNF-α-responsive N-SMase in MCF-7 cells. Moreover, the results suggest that nSMase3 may not possess in vitro N-SMase activity and does not affect cellular sphingolipid levels in the cell lines evaluated. On the other hand, nSMase1 contributes to in vitro N-SMase activity, but does not affect cellular sphingolipids much.

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Substrate specificity and inducibility of TACE (tumour necrosis factor α-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity

Biochemical Society Symposium ◽

10.1042/bss0700039 ◽

2003 ◽

Vol 70 ◽

pp. 39-52 ◽

Cited By ~ 54

Author(s):

Roy A. Black ◽

John R. Doedens ◽

Rajeev Mahimkar ◽

Richard Johnson ◽

Lin Guo ◽

...

Keyword(s):

Substrate Specificity ◽

Recent Work ◽

Tumour Necrosis Factor ◽

Tumour Necrosis ◽

Transforming Growth Factor ◽

Intrinsic Activity ◽

Converting Enzyme ◽

Tumour Necrosis Factor Α ◽

Factor Α ◽

Necrosis Factor

Tumour necrosis factor α (TNFα)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor α, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.

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