PPP3R1 Promoter Polymorphism (Allelic Variation) Affects Tacrolimus Treatment Efficacy by Modulating E2F6 Binding...

Tacrolimus is widely used as a first-line immunosuppressant in transplant immunology; however, its clinical application is constrained by the narrow therapeutic index and considerable interindividual variability. In this study, we identified the potential regulatory role of a novel PPP3R1 promoter polymorphism, rs4519508 C > T, in the tacrolimus pharmacodynamic pathway.

Dual-luciferase reporter assays and bioinformatic analysis were applied to assess the impact of allelic variation. Electrophoretic mobility shift assays (EMSA) validated the altered binding of transcription factors. Quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blots were used to determine the immunosuppressive effect of tacrolimus.

Assays revealed that rs4519508 C > T markedly enhanced PPP3R1 promoter activity. EMSA assays validated the binding of E2F6 to rs4519508 C (wild-type) and the binding was significantly weaker to the rs4519508 T (mutant-type). The overexpression of E2F6 significantly reduced the transcriptional activity and expression of PPP3R1 when the rs4519508 site presented as major C allele, an effect that was not observed with the rs4519508 T allele. Furthermore, the downregulation of E2F6 raises the level of downstream immune cytokines inhibited by TAC.

This study proposed that E2F6 suppresses the expression of PPP3R1, while rs4519508 C > T impairs the binding of E2F6, and thus elevates the level of PPP3R1, so that the inhibition of the downstream immune cytokines by TAC is attenuated. Our findings reported the potential regulatory role of a novel polymorphism, PPP3R1 rs4519508 C > T, which may serve as pharmacodynamic-associated pharmacogenetic biomarker indicating individual response variability of tacrolimus, and thus aid the clinical management of transplant immunology.