Inhibition of S1P degradation rescues 661W cells from oxidative stress

Abstract

 

Purpose

 

Sphingolipids are a broad class of molecules with the double role of cell membranes components and intra/extra cellular signal mediators, controlling proliferation, differentiation, stress survival and apoptosis. Ceramide (Cer) is the core of most complex sphingolipids and from Cer deacylation sphingosine and sphingosine-1-phosphate (S1P) are obtained. This latter exerts a pro-survival and proliferative activity, as opposed to Cer promoting cell cycle arrest and apoptosis. Retinal degeneration and in particular Retinitis Pigmentosa (RP) have been associated to Cer accumulation and cell death induction. In a murine model of human RP (rd10 mutant mice), we demonstrated that inhibition of Cer synthesis and accumulation in retina rescue from photoreceptors age related apoptosis (Strettoi et al, PNAS 2009). Our aim is targeting sphingolipid metabolites to reduce oxidative damage in retinal photoreceptor cell colture.

 

Methods

 

Murine 661W cone-like cell line were treated with 75µM 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI), an inhibitor of S1PLyase, for 2 hours; next, cells were treated with 1mM H₂O₂ for different times. Cell proliferation was determined by MTT assay after 24 hours. ERK 1/2 and AKT/PKB phosphorylation was evaluated by Western blotting with specific antibodies at 12 hours.

 

Results

 

We show that enhanced stability of S1P, obtained through THI administration, reduces inhibitory H₂O₂ effect on proliferation (Fig.1). We demonstrate that THI reverses H₂O₂ -induced ERK1/2 de-phosphorylation and Akt phosphorylation on Ser473 (Fig.2).

 

Conclusions

 

We conclude that S1P stabilization can be considered a therapeutic target to photoreceptor survival in oxidative stress conditions.

 

 

Fig.1: THI effect on 661W cells stressed with H₂O₂ for 24 hours.

 

 

Fig.2: THI effect on Akt and ERKs phosphorylation, in 661W cells, stressed with H₂O₂ for 12 hours.

 

Keywords: 695 retinal degenerations: cell biology • 449 cell survival • 648 photoreceptors