Identification of a novel transport-independent function of PiT1/SLC20A1 in the regulation of TNF-induced apoptosis.
PiT1/SLC20A1 is a sodium-dependent P(i) transporter expressed by most mammalian cells. Interestingly, PiT1 transcription has been shown to be up-regulated by the tumor necrosis factor α (TNF), and we have now investigated the possible involvement of PiT1 in TNF-induced apoptosis. We show that PiT1-depleted cells are more sensitive to the proapoptotic activity of TNF (i.e. when the antiapoptotic NFκB pathway is inactivated). These observations were made in the human HeLa cancer cell line either transiently or stably depleted in PiT1 by RNA interference and in immortalized mouse embryonic fibroblasts isolated from PiT1 knock-out embryos. Depletion of the closely related family member PiT2 had no effect on TNF-induced apoptosis, showing that this effect was specific to PiT1. The increased sensitivity of PiT1-depleted cells was evident regardless of the presence or absence of extracellular P(i), suggesting that a defect in P(i) uptake was not involved in the observed phenotype. Importantly, we show that the re-expression of a P(i) uptake mutant of PiT1 in PiT1(-/-) mouse embryonic fibroblasts delays apoptosis as efficiently as the WT protein, showing that this function of PiT1 is unrelated to its transport activity. Caspase-8 is more activated in PiT1-depleted cells, and our data reveal that the sustained activation of the MAPK JNK is up-regulated in response to TNF. JNK activity is actually involved in PiT1-depleted cell death because specific JNK inhibitors delay apoptosis.
Salaün
Christine
C
Growth and Signaling Research Center, INSERM U845, F-75015 Paris, France. christine.salaun@inserm.fr
Leroy
Christine
C
Rousseau
Alice
A
Boitez
Valérie
V
Beck
Laurent
L
Friedlander
Gérard
G
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*
D047428:Protein Kinase Inhibitors; C493586:SLC20A1 protein, human; C493587:Slc20a1 protein, mouse; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D014409:Tumor Necrosis Factor-alpha; D048670:MAP Kinase Kinase 4; C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8
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