Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect.
Previous studies demonstrated that Kit activation confers radioprotection. However, the mechanism by which Kit signaling interferes with cellular response to ionizing radiation (IR) has not been firmly established. Based on the role of the sphingomyelin (SM) cycle apoptotic pathway in IR-induced apoptosis, we hypothesized that one of the Kit signaling components might inhibit IR-induced ceramide production or ceramide-induced apoptosis. Results show that, in both Ba/F3 and 32D murine cell lines transfected with wild-type c-kit, stem cell factor (SCF) stimulation resulted in a significant reduction of IR-induced apoptosis and cytotoxicity, whereas DNA repair remained unaffected. Moreover, SCF stimulation inhibited IR-induced neutral sphingomyelinase (N-SMase) stimulation and ceramide production. The SCF inhibitory effect on SM cycle was not influenced by wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor. The SCF protective effect was maintained in 32D-KitYF719 cells in which the PI3K/Akt signaling pathway is abolished due to mutation in Kit docking site for PI3K. In contrast, phospholipase C gamma (PLC gamma) inhibition by U73122 totally restored IR-induced N-SMase stimulation, ceramide production, and apoptosis in Kit-activated cells. Moreover, SCF did not protect 32D-KitYF728 cells (lacking a functional docking site for PLC gamma 1), from IR-induced SM cycle. Finally, SCF-induced radioprotection of human CD34(+) bone marrow cells was also inhibited by U73122. Altogether, these results suggest that SCF radioprotection is due to PLC gamma 1-dependent negative regulation of IR-induced N-SMase stimulation. Beyond the scope of Kit-expressing cells, it suggests that PLC gamma 1 status could greatly influence the post-DNA damage cellular response to IR, and perhaps, to other genotoxic agents.
Maddens
Stéphane
S
Institut Nationale de la Santé et de la Recherche Médicale E9910, Institut Claudius Régaud, Toulouse, France.
Charruyer
Alexandra
A
Plo
Isabelle
I
Dubreuil
Patrice
P
Berger
Stuart
S
Salles
Bernard
B
Laurent
Guy
G
Jaffrézou
Jean-Pierre
JP
D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D001402:B-Lymphocytes / Q000378:metabolism; D016923:Cell Death / Q000528:radiation effects; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D003037:Cobalt Radioisotopes; D004260:DNA Repair / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007211:Indoles; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D011837:Radiation-Protective Agents; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D019089:Stem Cell Factor / Q000494:pharmacology / Q000502:physiology*; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002518:Ceramides; D003037:Cobalt Radioisotopes; D004791:Enzyme Inhibitors; D004963:Estrenes; D007211:Indoles; D007527:Isoenzymes; D011760:Pyrrolidinones; D011837:Radiation-Protective Agents; D013109:Sphingomyelins; D019089:Stem Cell Factor; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C007293:DAPI; D019869:Phosphatidylinositol 3-Kinases; D019009:Proto-Oncogene Proteins c-kit; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; D051966:Phospholipase C gamma