Stereoselectivity of Induction of the Retinoblastoma Gene Product (pRb) Dephosphorylation by D-erythro-Sphingosine Supports a Role for pRb in Growth Suppression by Sphingosine

Sphingosine has been shown to inhibit cell growth in many cell lines although the mechanism of this effect remains obscure. More recently, D-erythro-sphingosine has been shown to act as an early inducer of dephosphorylation of the retinoblastoma gene product (pRb) in the lymphoblastic leukemia cell line MOLT-4 [Chao, R., Khan, W., & Hannun, Y. A. (1992) J. Biol. Chem., 267, 23459-23462]. In the current study, the role of the natural D-erythro-sphingosine in regulation of cell growth and pRb dephosphorylation was evaluated using chemically synthesized pure isomers of sphingosine. Of the four possible stereoisomers of sphingosine, D-erythro-sphingosine was most active in inducing dephosphorylation of pRb protein with an EC_50% of 0.6 µM whereas its enantiomer L-erythro-sphingosine was 8-fold less potent with an EC_50% of 5 µM. The dose responses for inhibition of cell growth were nearly identical to the EC_50% for pRb dephosphorylation with D-erythro-sphingosine causing 50% inhibition at 0.6 µM whereas L-erythro-sphingosine was 5-6-fold less potent. All of the stereoisomers were taken up by the cells, and the greater potency of D-erythro-sphingosine was not due to differences in cellular uptake. The metabolism of D-erythro-sphingosine was also studied to evaluate the possible role of sphingosine metabolites on regulation of retinoblastoma protein. Evidence is provided against a role for ceramide or sphingosine 1-phosphate as mediators of the effects of sphingosine on pRb dephosphorylation. These results support a specific role for D-erythro-sphingosine in regulation of phosphorylation of pRb and provide evidence for a role of pRb dephosphorylation in mediating the growth inhibitory effects of sphingosine.