SMS Knockdown And Overexpression:Impact On Drug Transporters In Caco-2

Sphingomyelin synthase (SMS) is the last enzyme involved in the biosynthesis of sphingomyelin (SM) in mammals.lt contains two isoforms (SMS1 and SMS2). SMS1 is resided at cis-, medial-Golgi, while SMS2 locates at the plasma membrane. SMS activity closely relates to plasma membrane structures as well as the expression of membrane proteins.Drug transporters, a special family of membrane proteins,are recognized as an important determinant for drug metabolism and theirabnormalexpression are related to the occurrence of some diseases. Whether SMSinflunce the expression and function of drug transporter.and what is the molecular mechanismbehind the potential effect are interesting questions. We chose human colon cancer cell (Caco-2) as model, using gene knockdown and overexpression technology, with RT-PCR, Western blot and HPLC analysis methods to to investigate the role of SMS in the regulation of drug transporters.Small interfering RNA (siRNA) targeting human SMS1 and SMS2 were transfected intoCaco-2 cells, which obviously downregulated the expression of P-gp and MRP2 rather than other drug transporters, consequentlydecreased their function significantly. Meanwhile, Caco-2 cell line that stably overexpresses human SMS1 was constructed. SMS1 overexpression causedobvious increase both in the expression of P-gp and MRP2, and the expression of Pregnane X receptor(PXR) which is nuclear receptor transcription factor of P-gp and MPR2.The possible involvement of signaling pathways such as janus protein tyrosine kinase 2 (JAK-2) pathway and extracellular signal regulated kinases (ERK) pathwayin the regulation of SMS on transporters was explored.We found thatthephosphorylation levels of JAK-2 and ERK were evidently increasedin SMS1 overexpression Caco-2 cells.p-ERK, p-JAK-2 may act on hPXR to increase the expression of P-gp and MRP2.Furthermore, SMS1 overexpression also causedthe change in the expression of ezrin and moesin, which weremembers of ERM protein family and were considered to be involved in the regulation of drug transporters.These results indicate that SMS have a significant effect on the expression and function of drug transporters, especially P-gp and MRP2.