Establishing the Relationship between mRNA Expression and Protein Abundance for Seven Transporters in Human Small Intestine: A Pilot Study

Background: Previous meta-analyses have described the regional-specific intestinal expression of transporters in humans based on a combination of mRNA and protein expression data from relative quantification methods for incorporation into mechanistic PBPK models [1]. Quantitative proteomic techniques allow the relationship between mRNA and protein abundance to be defined, and while it is suggested that these factors can be poorly correlated [2], specific data from human intestine is limited. Aim: To assess the relationship between relative mRNA expression and protein abundances for seven transporters expressed in human jejunum by a targeted proteomic technique.Methods: Human jejunum (n=4) was harvested under informed consent from donors undergoing elective surgery at Salford Royal Hospital. The mRNA (gene) and protein expression (in crude membranes) of seven transporters; P-gp, BCRP, MRP2, OATP2B1, HPT1, OST-α, OST-β was quantified by RT-PCR (mRNA) against 2 housekeeper proteins (GAPDH and Villin) and by the Quantification conCATemer (QconCAT) LC-MS/MS targeted proteomics technique. Results: mRNA and protein expression levels for seven transporters in human jejunum were quantified. Relative mRNA expression was reference gene-dependent; however the between sample precision of expression was systematically higher using villin. The rank order of mRNA broadly follows that of protein with HPT1, BCRP, P-gp and OST-β consistently demonstrating the highest expression. HPT1, P-gp and OST-α mRNA-protein expression were highly correlated (r2 > 0.70), BCRP and OST-β were moderately correlated (r2 > 0.40) and no correlation (r2 <0.40) existed for MRP2 and OATP2B1.Conclusion: The initial findings of investigations into the relationship between transporter relative mRNA expression and protein abundance in human jejunum are reported and suggest the mRNA-protein expression relationship is transporter-dependent. Clearly more data are required; however given the discrepancies in mRNA-protein expression, caution is advised if using mRNA jejunal transporter expression in PBPK models.References: 1.Harwood MD, Neuhoff S, Carlson GL, Warhurst G and Rostami-Hodjegan A (2013) Absolute abundance and function of intestinal drug transporters: a prerequisite for fully mechanistic in vitro-in vivo extrapolation of oral drug absorption. Biopharm Drug Dispos 34:2-28. 2. Vogel C, Marcotte EM 2012. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet 13(4):227-232.