Inhibition of SK1 stimulates sphingolipids de novo synthesis enhancing ceramida accumulation.

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

Rosario , Santa Fe, Argentina

Congreso; XLII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2006

SAIB

We reported that Sphingosine Kinase 1 (SK1) inhibition with D,L,-threo-dihydrosphingosine (tDHS), in subconfluent MDCK cultures, causes cell number and viability decrease (33% and 16%). Incubation with 14C-palmitic acid (PA) shows an increase of de novo synthesized Ceramide (Cer) (39% of labeling sphingolipids (SLs) vs. 26% in control). Since Cer synthesis inhibition with Fumonisine B1 attenuates the apoptotic effect, we concluded that such an effect is due to Cer accumulation. On the other hand, reduction of SK1 expression by using SK1-siRNA, also decreases both cell number and viability (60% and 85%) and raises Cer synthesis (43% vs. 35%). Labeling assays with 14C-PA show that both tDHS and siRNA increase total 14C-PASLs by 60% and 43%, respectively. To evaluate if the increase in total SLs synthesis raises Cer production thus contributing with SK1 inhibition effect on cell viability, we studied serine-palmitoyl transferase (SPT), which catalyzes the rate-limiting step of de novo synthesis, by treating MDCK cells with SPT inhibitor, Lcicloserine, plus tDHS. We found attenuation in tDHS effect: a raise in cell number and viability and a decrease in Cer production. These results indicate that SK1 inhibition abrogates a negative control on SLs de novo synthesis (possibly on SPT activity) thus enhancing Cer production, which could not be clearing by its conversion to Sphingosine-1P.