Excessive hepatomegaly of mice with hepatocyte-targeted elimination of integrin linked kinase following treatment with 1,4-bis [2-(3,5-dichaloropyridyloxy)] benzene

Abstract

TCBOPOP (1,4-bis [2-(3,5-dichaloropyridyloxy)] benzene) an agonist of the constitutive androstane receptor (CAR), produces rapid hepatocyte hyperplasia and hepatomegaly in the absence of hepatic injury. In this study we demonstrate that integrin-linked kinase (ILK), which is involved in transmission of the extracellular matrix (ECM) signaling by way of integrin receptors, plays an important role in regulating TCPOBOP-induced proliferation of hepatocytes and hepatomegaly. Hepatocyte-specific ILK knockout mice (ILK/liver-/- mice) and wildtype mice (WT) were given a single dose of TCPOBOP (3 mg/kg) by oral gavage. Mice were sacrificed at days 1, 2, 5, and 7 after TCPOBOP administration. WT mice showed maximum proliferation on days 1 and 2, which came back to baseline levels by days 5 and 7 after TCPOBOP administration. The ILK/liver-/- mice, on the other hand, showed a prolonged and a sustained proliferative response as evident by an increased number of proliferative cell nuclear antigen assay (PCNA)-positive cells even at days 5 and 7 after TCPOBOP administration. At day 7 the WT mice showed close to a 2.5-fold increase in liver weight, whereas the ILK/liver-/- mice showed a 3.7-fold increase in liver weight. The prolonged proliferative response in the ILK/liver-/- mice seems to be due to sustained induction of CAR leading to sustained induction of c-Myc, which is known to be a key mediator of TCPOPOP-CAR induced direct liver hyperplasia.

Conclusion: The data indicate that ECM-mediated signaling by way of ILK is essential for adjustment of final liver size and proper termination of TCPOBOP-induced proliferation of hepatocytes.

Fig 1

Liver enlargement after TCPOBOP administration. A) Liver size 7 day after administration of TCPOBOP B) Liver to body weight ratios of WT and ILK/Liver−/− mice at day 0, 1, 2 5 and 7 days after TCPOBOP administration. Each data point is the mean ± SE from more than three measurements per point.

Fig 2

Cell proliferation kinetics after TCPOBOP administration (A) Hepatocyte proliferation was quantitated by counting the PCNA positive cells. PCNA positive cells were counted in low-power fields (200×) in 3 sections from 3 different ILK/Liver −/− or WT livers. Each data point is the mean ± SE from more than three measurements per point. (B) Representative photomicrographs of PCNA stained liver section of ILK/Liver−/− and WT mice at day 1, and 7 after TCPOBOP administration. (C) Western Blot analysis of PCNA. Pooled liver samples from at least 3 mice were used for protein analysis.

Fig 3

A) Gel shift assay for CAR. Pooled liver samples from at least 3 mice were used for preparing nuclear samples. P represents probe only, C represent cold probe (B) mRNA levels of CAR before and after TCPOBOP administration. Pooled liver samples from at least 3 mice were used for preparing RNA. (C) Real time PCR for UGT1A1. Levels were normalized relative to expression of cyclophillin in each sample. Fold change in gene expression was calculated by using the 2(−ΔΔCt) method.

Fig 4

(A) Protein levels of various mitogenic and mitoinhibitory molecules. Pooled liver samples from at least 3 mice were used for preparing nuclear, cytoplasmic and total cell lysates. (B) HGF protein and mRNA levels after TCPOBOP administration.

Fig 5

A) Protein levels of c-myc after TCPOBOP administration. B) mRNA levels of c-myc after TCPOBOP administration. C) Gel shift assay for c-myc after TCPOBOP administration. C represents cold competition (20 fold more as compared to biotinalated probe). 24 h sample for the WT and 24 h sample for the KO were used for cold competition D) Representative photomicrographs of Immunohistochemistry for c-myc in WT and ILK/liver−/− mice at day 1 and 7 after TCPOBOP administration. Arrows indicate nuclear localization of c-myc. NOTE: Pooled liver samples from at least 3 mice were used for preparing RNA and nuclear extracts. (E) FoxM1 mRNA level after TCPOBOP administration.