Altered lipid raft composition and defective cell death signal transduction in glycosylphosphatidylinositol anchor-deficient PIG-A mutant cells.
Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal disorder of haematopoietic stem cells caused by somatic PIGA mutations, resulting in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-AP). Because GPI-AP associate with lipid rafts (LR), lack of GPI-AP on PNH cells may result in alterations in LR-dependent signalling. Conversely, PNH cells are a suitable model for investigating LR biology. LR from paired, wild-type GPI(+), and mutant GPI(-) cell lines (K562 and TF1) were isolated and analysed; GPI(-) LR contained important anti-apoptotic proteins, not found in LR from GPI(+) cells. When methyl-beta-cyclodextrin (MbetaCD) was utilized to probe for functional differences between normal and GPI(-) LR, increased levels of phospho-p38 mitogen-activated protein kinase (MAPK), and phospho-p65 nuclear factor NF-kappaB were found in control and GPI(-) cells respectively. Subsequent experiments addressing the inhibition of phosphoinositide-3-kinase (PI3K) suggest that the PI3K/AKT pathway may be responsible for the resistance of K562 GPI(-)cells to negative effects of MbetaCD. In addition, transduction of tumour necrosis factor-alpha (TNF-alpha) signals in a LR-dependent fashion increased induction of p38 MAPK in GPI(+) and increased pro-survival NF-kappaB levels in K562 GPI(-) cells. Therefore, we suggest that the altered LR-dependent signalling in PNH-like cells may induce different responses to pro-inflammatory cytokines from those observed in cells with intact GPI-AP.
Experimental Haematology and Haematopoiesis Section, Taussig Cancer Centre, Cleveland Clinic, Cleveland, OH 44195, USA.
Hadrian
H
Szpurka
Andrew E
AE
Schade
Anna M
AM
Jankowska
Jaroslaw P
JP
Maciejewski
D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D017261:Glycosylphosphatidylinositols / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000473:pathology; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
D016207:Cytokines; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D019869:Phosphatidylinositol 3-Kinases