Effect of oncostatin M on uridine diphosphate-5'-glucuronosyltransferase 1A1 through cross talk with constitutive androstane receptor

Abstract

Hyperbilirubinemia remains a common condition in neonates. The constitutive androstane receptor (CAR) is an orphan nuclear receptor that has been shown to participate in the activation of the uridine diphosphate-5'-glucuronosyltransferase 1A1 (UGT1A1) gene, which plays an important role in bilirubin clearance. Oncostatin M (OSM), a member of the IL-6 family, is involved in the maturation of fetal hepatocytes. We have demonstrated that low OSM levels are a potential indicator of neonatal jaundice and the need for phototherapy. In this study we examined the effects of OSM on CAR-mediated signaling to investigate its potential role in neonatal jaundice via the CAR-UGT1A1 pathway. We observed that OSM positively augmented the CAR and UGT1A1 expressions and CAR-mediated signaling in vivo and in vitro, through cross talk between the nuclear CAR receptor and the plasma membrane OSM receptor, via the MAPK cascade. These data suggest that OSM might play a role in bilirubin metabolism via the CAR-UGT1A1 pathway.

Figure 1

Effects of OSM on CAR and UGT1A1 expressions. HepG2 cells were treated with ethanol or 10⁻³ m PB, 10⁻⁶ m CITCO, or 10⁻⁵ m RIF with or without 10⁻⁶ m OSM for 36 h. A, Total RNA was obtained from HepG2 cells and analyzed for the mRNA expression of CAR, UGT1A1, and CYP3A4 using real-time quantitative PCR. The mRNA levels were normalized to β-actin mRNA and expressed as fold induction over control. B, Whole-cell extracts were prepared as described in Materials and Methods. CAR, UGT1A1, and β-actin protein levels were determined by Western blotting using anti-CAR, -UGT1A1, and -β-actin antibodies. Relative protein levels are expressed by taking the control values obtained from the ethanol-treated cells as one. C, Total RNA was obtained from HepG2 cells and analyzed for the mRNA expressions of UGT1A1 using real-time quantitative PCR. The mRNA levels were normalized to β-actin mRNA and expressed as fold induction over control. The results represent the mean ± sd of triplicate determinations (*, P < 0.01 compared with ethanol-treated cells; **, P < 0.01 compared with non-OSM-treated cells).

Figure 2

Effects of OSM on CAR-mediated transcription. HepG2 cells were cotransfected with 1 μg of a reporter gene construct, (NR1)³-tk-CAT or tk-CAT vector with or without pCMX-CAR expression vector. The cells were treated with ethanol vehicle or 10⁻³ m PB or 10⁻⁶ m CITCO or 10⁻⁶ m progesterone, with or without 10⁻⁶ m OSM for 36 h. The amount of CAT was determined using a CAT ELISA kit, according to the manufacturer’s instructions. The results represent the mean ± sd of triplicate determinations (*, P < 0.01 compared with ethanol-treated cells; **, P < 0.01 compared with the cells without OSM treatment).

Figure 3

Effects of CAR siRNA on UGT1A1 expression and CAR-mediated transcription in the presence of CAR ligands. A, HepG2 cells were transfected with CAR or control siRNA. Whole-cell extracts were prepared as described in Materials and Methods. CAR protein levels were determined by Western blotting using anti-CAR antibodies. Total RNA was also obtained from HepG2 cells and analyzed for the expression of CAR mRNAs using RT-PCR. PCR products were separated on 3% Nu-Sieve agarose gels and visualized by ethidium bromide staining. B, HepG2 cells were transfected with CAR or control siRNAs and treated with ethanol vehicle or 10⁻³ m PB or 10⁻⁶ m CITCO with or without 10⁻⁶ m OSM for 36 h. Whole-cell extracts were prepared and CAR, UGT1A1, and β-actin protein levels were determined by Western blotting. Relative protein levels are expressed by taking the control values obtained from the ethanol-treated cells as one. Each bar represents the mean ± sd from three independent experiments.

Figure 4

MAPK inhibitor abolishes the effects of OSM on CAR-UGT1A1 pathway. A, Schema of CAR transduction cascade and inhibitor. B, HepG2 cells were treated with ethanol or 10⁻³ m PB or 10⁻⁶ m CITCO, with or without 10⁻⁶ m OSM, PD98059, or STAT inhibitor for 36 h. Whole-cell extracts were prepared as described in Materials and Methods. CAR, UGT1A1, and β-actin protein levels were determined by Western blotting using anti-CAR, -UGT1A1, and -β-actin antibodies. Relative protein levels are expressed by taking the control values obtained from the ethanol-treated cells as one. C, HepG2 cells were cotransfected with 1 μg of a reporter gene construct, (NR1)³-tk-CAT, or tk-CAT vector. The cells were treated with ethanol or 10⁻³ m PB or CITCO, with or without 10⁻⁶ m OSM, PD98059, or STAT inhibitor for 36 h. The amount of CAT was determined using a CAT ELISA kit, according to the manufacturer’s instructions. The results represent the mean ± sd of triplicate determinations (*, P < 0.01 compared with ethanol-treated cells; **, P < 0.01 compared with PB or CITCO-treated cells without OSM).

Figure 5

Effects of OSM on CAR and UGT1A1 expression in neonatal mice in vivo. Neonatal ICR mice (3 d old) were administered PB (100 mg/kg), TCPOBOP (0.3 mg/kg), or ethanol, with or without OSM (0.1 mg/kg) or PD98059 (1 mg/kg) via ip injection every 24 h. The animals were killed under ether anesthesia 72 h after the injection, and the livers were removed, immediately frozen, and stored at −70 C. A, The mRNA expression of CAR, UGT1A1, and CYP2B10 in the livers of mice after 72-h exposure to TCPOBOP or ethanol, with or without OSM or PD98059, was analyzed using real-time quantitative PCR. The mRNA levels were normalized to β-actin mRNA and expressed as fold induction over control. B, CAR, UGT1A1, and β-actin protein expression in the livers of mice after 72-h exposure to PB, TCPOBOP, or ethanol, with or without OSM, was determined by Western blotting using anti-CAR, -UGT1A1, and -β-actin antibodies. Relative protein levels are expressed by taking the control values obtained from the ethanol-treated mouse as one. C, CAR, UGT1A1, and β-actin protein expression in the livers of mice after 72-h exposure to TCPOBOP or ethanol, with or without OSM or PD98059, were determined by Western blotting using anti-CAR, -UGT1A1, and -β-actin antibodies. Relative protein levels are expressed by taking the control values obtained from the ethanol-treated mouse as one. Five neonatal mice were analyzed for each treatment group. The results represent the mean ± sd of triplicate determinations (*, P < 0.01 compared with ethanol treatment without OSM; **, P < 0.01 compared with PB or TCPOBOP treatment without OSM).