SphK1 is essential for TNFa induced NF-kB activation in human melanoma cells.

ALVAREZ SE; KORDULA T; MILSTIEN S; SPIEGEL S

Richmond, VA USA

Congreso; Massey Cancer Center 2006 Research Retreat; 2006

Sphingosine kinase (SphK) is the key enzyme that catalyzes the synthesis of sphingosine-1-phosphate (S1P), a lipid mediator that regulates a variety of biological processes including cell growth, survival and inflammation. SphK1 is transiently activated in response to a variety of agonists including PDGF, EGF and TNF-a. It has been shown that activation of SphK1 is critical for prevention of apoptosis during TNF-a stimulation in human hepatocytes and endothelial cells. More interestingly, enforced expression of SphK1 increase S1P levels and block MCF-7 cell death induced by anti-cancer drugs and TNF-a. In this study, we show that SphK1 is essential in TNF-a induced NF-kB activation in human melanoma cells. We used matched melanoma cells, one lacking filamin A (M2) that do not respond to TNF-a and one with filamin A (A7) that are fully responsive to TNF-a.  siRNA directed against SphK1 (siSphK1), but not SphK2, markedly reduced TNF-a induced NF-kB binding activity in A7 cells. Moreover, NF-kB driven luciferase activity was also decreased. Basal levels of IkB-a, but not IkB-b, are dramatically reduced in siSphK1 M2 and A7 melanoma cells. Similarly, phosphorylation of IKB-a is also impaired after reduction of SphK1. In A7 cells, stimulation with TNF-a induces a transient increase in IKKab phosphorylation which is greatly reduced by knocking down SphK1. It is very well known that translocation of p65 (RelA) to the nucleus is essential for NF-kB modulation of gene transcription. siSphK1 blocked the TNF-a induced translocation of p65 in A7 cells. In contrast, p50 translocation was not affected. On the other hand, S1P can activate phosphorylation of IKBa and DNA binding activity of NF-kB in both cell lines, but the effect is stronger in M2 cells, indicating that is not dependent of filamin A. Interestingly VPC-23019, a S1P1/S1P3 receptor antagonist, block S1P but not TNF-a induced IKB-a phosphorylation. Taken together, our results suggest an important role of SphK1 in the activation of NF-kB in melanoma cells through the translocation of p65 to the nucleus. Furthermore, our data support a possible role of internal S1P in TNF-a induced NF-kB activation.