Lentiviral siRNA silencing of sphingosine-1-phosphate receptors S1P1 and S1P2 in smooth muscle

Sphingosine-1-phosphate regulates diverse biological processes through five receptor types, S1P1-5. Two or more S1P receptors are usually co-expressed on target cells. We have previously shown that smooth muscle cells of the gut co-express S1P1 and S1P2 receptors that could mediate distinct functions. In the absence of selective agonists and antagonists, we developed siRNA constructs to silence each receptor separately. The constructs were based on identical sequences in several mammalian species. A lentiviral vector-based system was used to deliver siRNA into HEK293T cells and smooth muscle cells. One S1P1 and two S1P2 siRNA constructs specifically inhibited ectopic expression of S1P1 and S1P2 receptors, respectively, as determined by immunocytochemistry and Western blot, and endogenous expression of S1P1 and S1P2 receptors in smooth muscle cells, as determined by RT-PCR. Measurement of PLC-β and Rho kinase activities, which mediate initial and sustained muscle contraction, confirmed receptor silencing and showed that contraction is mediated exclusively by S1P2 receptors.