Application of multivariate statistical procedures to identify transcription factors that correlate with MRP2, 3, and 4 mRNA in adult human livers

Abstract

Multidrug resistance-associated proteins 2-4 (MRP2-4) are membrane efflux transporters critical for the hepatic clearance of pharmaceuticals and endogenous chemicals. Little is known about the constitutive regulation of MRP2-4 mRNA in normal human liver. The purpose of this study was to identify transcription factors whose expression significantly correlates with MRP2-4 mRNA in human liver specimens. Ninety adult human livers were profiled for mRNA expression of MRP2-4 as well as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPAR alpha) and gamma (gamma), liver X receptor alpha (LXR alpha), farnesoid X receptor (FXR), glucocorticoid receptor (GR), retinoid X receptor alpha (RXR alpha), hepatocyte nuclear factor 1 alpha (HNF1 alpha) and 4 alpha (4 alpha), and nuclear factor E2-related factor 2 (Nrf2) transcription factors. Using linear regression and stepwise selection of partial R(2)-values, CAR, HNF1 alpha, and PPAR alpha mRNA exhibited the greatest correlation with MRP2, 3, and 4, respectively. Interindividual variation in the expression of the identified transcription factors may account for the variability in constitutive mRNA levels of MRP2-4. The multivariate approach presented in this study should aid in predicting signalling pathways that participate either directly or indirectly in regulating hepatic drug disposition.

Figure 1. Frequency distribution of MRP2-4 mRNA in adult human livers

mRNA levels of MRP2-4 transporters are presented as frequency distributions on a logarithmic scale normalized to GAPDH mRNA.

Figure 2. Correlation of MRP2-4 and transcription factor mRNA expression

Comparisons between each MRP gene and a panel of transcription factors were determined by linear regression and presented as R-values. Statistical significance (*) was set at p < 0.005.

Figure 3. MRP:transcription factor interactions

Correlative transcription factors were ranked using stepwise selection and the factors with the highest partial R² values are presented. Partial R² values for each transcription factor are plotted in comparison to the overall model R² on a scale of 0-1.0. Linear regressions for MRP2-CAR, MRP3-HNF1α, and MRP4-PPARα mRNA are shown. Data are presented as mean mRNA expression (normalized to GAPDH mRNA). Confidence intervals (95%) are depicted as dashed lines.

Figure 4. Transcription factor interrelationships and correlation to MRP2-4 mRNA

Correlation coefficients (R-values) obtained from linear regression analysis in Figure 1 are presented for MRP2 (CAR and PXR), MRP3 (HNF1α and RXRα), and MRP4 (PPARα and GR) mRNA. Partial correlation coefficients (R-values) were calculated for each MRP-transcription factor association, controlling for variability in the second transcription factor. The symbol | denotes the partial variable. Statistical significance (*) was set at p < 0.005.