Characterization of cholesterol homeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts reveals a Niemann-Pick disease type C-like phenotype with enhanced lysosomal Ca2+ storage

Hans Vienken,Dieter Lütjohann,Agnes Rudowski,Josef Pfeilschifter,Gerhild Van Echten-Deckert,Dagmar Meyer Zu Heringdorf,Katja Grabau,Nathalie Mabrouki,Nina Schömel,Ralf Frederik Claas

doi:10.1038/srep43575

Abstract

Sphingosine-1-phosphate (S1P) lyase irreversibly cleaves S1P, thereby catalysing the ultimate step of sphingolipid degradation. We show here that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1−/−-MEFs), in which S1P and sphingosine accumulate, have features of Niemann-Pick disease type C (NPC) cells. In the presence of serum, overall cholesterol content was elevated in Sgpl1−/−-MEFs, due to upregulation of the LDL receptor and enhanced cholesterol uptake. Despite this, activation of sterol regulatory element-binding protein-2 was increased in Sgpl1−/−-MEFs, indicating a local lack of cholesterol at the ER. Indeed, free cholesterol was retained in NPC1-containing vesicles, which is a hallmark of NPC. Furthermore, upregulation of amyloid precursor protein in Sgpl1−/−-MEFs was mimicked by an NPC1 inhibitor in Sgpl1+/+-MEFs and reduced by overexpression of NPC1. Lysosomal pH was not altered by S1P lyase deficiency, similar to NPC. Interestingly, lysosomal Ca2+ content and bafilomycin A1-induced [Ca2+]i increases were enhanced in Sgpl1−/−-MEFs, contrary to NPC. These results show that both a primary defect in cholesterol trafficking and S1P lyase deficiency cause overlapping phenotypic alterations, and challenge the present view on the role of sphingosine in lysosomal Ca2+ homeostasis.

Highlights

Sphingosine-1-phosphate (S1P) lyase catalyses the irreversible cleavage of S1P, which plays an important role in regulation of crucial cellular functions such as cell growth, survival, adhesion and migration[1,2,3]
We show here that in S1P lyase-deficient MEFs, free cholesterol is trapped in NPC1-expressing vesicles, leading to activation of sterol regulatory element binding protein-2 (SREBP-2), upregulation of the low-density lipoprotein (LDL) receptor and enhanced cholesterol uptake in the presence of serum
Release of [3H]cholesterol in the presence of bovine serum albumin (BSA) and apolipoprotein A-I (ApoA-I) as acceptors was not altered in S1P lyase-deficient MEFs (Fig. 1B)

Summary

Introduction

Sphingosine-1-phosphate (S1P) lyase catalyses the irreversible cleavage of S1P, which plays an important role in regulation of crucial cellular functions such as cell growth, survival, adhesion and migration[1,2,3]. A thorough study on lipid profiles in liver and plasma of S1P lyase-deficient mice revealed that aside from sphingolipids, total and free cholesterol, cholesterol esters, phospholipids, and triacylglycerol were strongly elevated in the serum of Sgpl1−/−-mice. Both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol were elevated[16]. We show here that in S1P lyase-deficient MEFs, free cholesterol is trapped in NPC1-expressing vesicles, leading to activation of sterol regulatory element binding protein-2 (SREBP-2), upregulation of the LDL receptor and enhanced cholesterol uptake in the presence of serum. These results shed new light on the interplay between S1P/sphingosine and cholesterol, and on the regulation of lysosomal Ca2+ homeostasis

Methods

Results

Conclusion