Role of Human Sphingosine-1-phosphate Phosphatase 1 in the Regulation of Intra- and Extracellular Sphingosine-1-phosphate Levels and Cell Viability

Sphingosine-1-phosphate (S1P) is a highly bioactive lipid that exerts numerous biological effects both intracellularly as a second messenger and extracellularly by binding to its G-protein-coupled receptors of the endothelial differentiation gene family (S1P receptors-(1–5)). Intracellularly, at lea...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2003-09, Vol.278 (36), p.34541-34547
Main Authors: Johnson, Korey R., Johnson, Kristy Y., Becker, Kevin P., Bielawski, Jacek, Mao, Cungui, Obeid, Lina M.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Blotting, Northern
Cell Division
Cell Line
Cell Survival
Cloning, Molecular
Coloring Agents - pharmacology
DNA, Complementary - metabolism
Endoplasmic Reticulum - metabolism
Gene Expression Regulation, Enzymologic
Humans
Lysophospholipids
Membrane Proteins
Mice
Microscopy, Confocal
Microscopy, Fluorescence
Models, Biological
Molecular Sequence Data
Phosphoric Monoester Hydrolases - chemistry
Protein Isoforms
Protein Structure, Tertiary
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger - metabolism
RNA, Small Interfering - metabolism
Sequence Homology, Amino Acid
Signal Transduction
Sphingolipids - chemistry
Sphingolipids - metabolism
Sphingosine - analogs & derivatives
Sphingosine - chemistry
Sphingosine - metabolism
Tetrazolium Salts - pharmacology
Thiazoles - pharmacology
Time Factors
Tissue Distribution
Tumor Cells, Cultured
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Sphingosine-1-phosphate (S1P) is a highly bioactive lipid that exerts numerous biological effects both intracellularly as a second messenger and extracellularly by binding to its G-protein-coupled receptors of the endothelial differentiation gene family (S1P receptors-(1–5)). Intracellularly, at least two enzymes, sphingosine kinase and S1P phosphatase, regulate the activity of S1P by governing the phosphorylation status of S1P. To study the regulation of S1P levels, we cloned the human isoform of S1P phosphatase 1 (hSPPase1). The hSPPase1 has 78% homology to the mouse SPPase at the amino acid level with 6–8 possible transmembrane domains. Confocal microscopy revealed green fluorescent protein-tagged hSPPase1, expressed in either MCF7 or HEK293 cells, co-localized to endoplasmic reticulum with calreticulin. According to Northern blot analysis, hSPPase1 is expressed in most tissues, with the strongest levels found in the highly vascular tissues of placenta and kidney. Transient overexpression of hSPPase1 exhibited a 2-fold increase in phosphatase activity against S1P and dihydro-S1P, indicating that the expressed protein was functional. Small interfering RNA (siRNA) knockdown of endogenous hSPPase1 drastically reduced hSPPase1 mRNA levels, as confirmed by reverse transcription PCR, and resulted in an overall 25% reduction of in vitro phosphatase activity in the membrane fractions. Sphingolipid mass measurements in hSPPase1 siRNA knockdown cells revealed a 2-fold increase of S1P levels and concomitant decrease in sphingosine. In vivo labeling of hSPPase1 siRNA-treated cells showed accumulation of S1P within cells, as well as significantly increased secretion of S1P into the media, indicating that hSPPase1 regulates secreted S1P. In addition, siRNA-induced knockdown of hSPPase1 endowed resistance to tumor necrosis factor-α and the chemotherapeutic agent daunorubicin. Collectively, these data suggest that regulation of hSPPase1 with the resultant changes in cellular and secreted S1P could have important implications to cell proliferation, angiogenesis, and apoptosis.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M301741200