Importance of Sphingosine Kinase 1 in MDCK cells proliferation and survival.

LEOCATA NIETO F; PESCIO L; SALAMA F; STERIN SPEZIALE N

Pinamar, Buenos Aires, Argentina

Congreso; XLI Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2005

SAIB

We have previously demonstrated that 24hrs treatment of subconfluent-MDCK cells with 25ìM tDHS (D,L-threodihydrosphingosine), an inhibitor of Sphingosine Kinase 1 (SK1), reduced cell number (32.5% of control) and viability (16.3%). Incubation with 32P and 14C-palmitic-acid, showed a reduction in sphingosine- 1-phosphate (S1P) production (26% of radiolabelling SLs vs. 51% in the control) and Ceramide (Cer) accumulation (39% vs. 26%), respectively. In order to determine if the effect of tDHS treatment is due to S1P synthesis decrease or to Cer accumulation we carried out two strategies: 1)Inhibition of Cer synthesis by treatment with 50ìM Fumonisine B1 (FB1), together with tDHS: this treatment promoted an attenuation in tDHS effect raising cell number and viability (65.7% and 53%) and decreasing Cer accumulation (11% of radiolabelling SLs). 2)Knockdown of SK1 expression, by transfecting MDCK cells with a SK1-specific siRNA: the treatment induced a)A reduction in SK1 expression (43% of control). b)Cell number reduction (66% of control) but with out loosing cell viability (97%). c)Reduction in total radiolabelled SLs and accumulation of radiolabelled sphingosine, in spite of no reduction in the percentage of incorporation of radioactive mark to S1P was obtained. Taken together these results indicate that pharmacological inhibition of SK1 with tDHS disturbed the sphingolipids-rheostat by accumulating of Cer and decreasing S1P production, affecting cell proliferation and viability. By other side, physiological block of SK1 by the specific siRNA, provoked a less unbalance in the rheostat, with out accumulation of Cer and affecting only cell proliferation.