Regulation of MRP2/ABCC2 and BSEP/ABCB11 expression in sandwich cultured human and rat hepatocytes exposed to inflammatory cytokines TNF-{alpha}, IL-6, and IL-1{beta}

Abstract

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-α, IL-6, or IL-1β. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1β. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-α and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-α or IL-1β. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.

FIGURE 1.

Diagram of MRP2/ABCC2 (upper panel)- and BSEP/ABCB11 (lower panel)-selective tryptic peptide for LC-MS/MS quantification. MSD, membrane-spanning domain; NBD, nucleotide binding domain; SRM, selected reaction monitoring; MRM, multiple reaction monitoring.

FIGURE 2.

Changes of MRP2/ABCC2 and BSEP/ABCB11 mRNA expression in SC human hepatocytes exposed to TNF-α, IL-6, and IL-1β. The mRNA expression was normalized to GAPDH. The data represent the relative -fold of mRNA expression of MRP2/ABCC2 (A) or BSEP/ABCB11 (B) to control cultures; data represent the mean ± S.D. (n = 3). *, statistically significant (p ≤ 0.05 by ANOVA).

FIGURE 3.

Changes of MRP2/ABCC2 and BSEP/ABCB11 protein expression in SC human hepatocytes exposed to TNF-α, IL-6, and IL-1β. The membrane protein fraction was prepared from SC human hepatocytes from 2–3 wells of a 24-well plate and subjected to LC-MS/MS SRM analysis for absolute quantification of MRP2/ABCC2 (A) or BSEP/ABCB11 (B) protein. The data represent the mean ± S.D. (n = 3). *, statistically significant (p ≤ 0.05 by ANOVA). SRM, selected reaction monitoring.

FIGURE 4.

Changes of Mrp2 and Bsep/Abcb11 mRNA expression in SC rat hepatocytes exposed to TNF-α, IL-6, and IL-1β. The mRNA expression was normalized to GAPDH. The data represents the relative -fold of mRNA expression of Mrp2 (A) or Bsep/Abcb11 (B) to control cultures, mean ± S.D. (n = 3). *, statistically significant (p ≤ 0.05 by ANOVA).

FIGURE 5.

Changes of Mrp2/Abcc2 and Bsep/Abcb11 protein expression in SC rat hepatocytes exposed to TNF-α, IL-6, and IL-1β. The membrane protein fraction was prepared from SC human hepatocytes from 2–3 wells of a 24-well plate and subjected to LC-MS/MS SRM analysis for absolute quantification of Mrp2/Abcc2 (A) or Bsep (B) protein. The data represent the mean ± S.D. (n = 3). *, statistically significant (p ≤ 0.05 by ANOVA).

FIGURE 6.

Proinflammatory cytokine level in plasma of LPS-treated rats. Plasma was collected 6 h post-LPS treatment. Pooled plasma was subjected to cytokine measurements using ELISA kits. The data represent the average of two measurements.

FIGURE 7.

Changes of Mrp2/Abcc2 and Bsep/Abcb11 mRNA expression in LPS-treated rats. The total mRNA was extracted from liver of LPS-treated rats. Mrp2/Abcc2 and Bsep/Abcb11 mRNA expression was normalized to GAPDH. The data represent the relative -fold of mRNA expression of Mrp2/Abcc2 (A) or Bsep (B) to control rats, mean ± S.D. (n = 3). *, statistically significant (p ≤ 0.05 by ANOVA).

FIGURE 8.

Changes of Mrp2 and Bsep protein in the liver of LPS-treated rats. The membrane protein fraction was prepared from liver tissues of LPS-treated rats and was subjected to LC-MS/MS SRM analysis for absolute quantification of Mrp2/Abcc2 (A) or Bsep (B) protein. The data represent the mean ± S.D. (n = 3). *, statistically significant (p ≤ 0.05 by ANOVA).