Limiting B-13 cell exposure to glucocorticoid for 6 hours results in a near quantitatively identical trans-differentiation to the B-13/H phenotype.

<p><b>A</b>, Gr transcriptional activity in B-13 cells after the indicated period of time of treatment with DEX and/or RU486. “6 hours DEX treatment” refers to cells pulse treated with DEX for 6 hours prior to washing as outlined in methods section. Cells were transfected as outlined in methods section with GRE4-pGL4.28 and RL-TK prior to measurement of luciferase and renilla activities. Data are the mean and standard deviation of 3 separate transfections in the same experiment, typical of 3 separate experiments. *Significantly different (two tailed) normalised luciferase reporter gene activity versus control vehicle treated cells; $significantly different (two tailed) normalised luciferase reporter gene activity versus DEX only treated cells, P > 0.05. <b>B–D</b>, Expression levels of the indicated 18S rRNA normalised transcript relative to the levels present in B-13 cells (open bars) 14 days after continuous DEX treatment (grey bars) or limited 6 hours DEX treatment (hashed bars), data are the mean and standard deviation of at least 3 separate cell samples. <b>E</b>, Photomicrographs of B-13 cells 14 days after continuous DEX treatment or limited 6 hours DEX treatment. <b>F</b>, Western blot for the indicated protein in B-13 cells 14 days after continuous DEX treatment or limited 6 hours DEX treatment.</p

Quantitative RT-PCR determination of transcripts in B-13, B-13/H cells and comparison to rat liver and pancreas levels.

<p>Time course for the expression of amylase mRNA (<b>A</b>) or Cebp-α (-■-), Cebp- β (-●-), albumin (-▲-) and Cyp2e1 (-♦) mRNAs (<b>B</b>) in B-13 cells after treatment with 10nM DEX. Data are mean and standard deviation of three independent experiments. B-13 cells treated with 0.1% (v/v) ethanol (i.e. vehicle control) had minimal effect on all transcripts and are not shown for clarity. After 3–4 days, B-13 cells unexposed to DEX required sub-culturing since they continued to proliferate to avoid cell death. Therefore, they were not appropriate controls for B-13 cells treated with DEX after this time since DEX treatment resulted in a cessation of proliferation and sub-culture was not required. <b>C—E,</b> Expression levels of the indicated 18S rRNA-normalised transcript, relative to the levels present in B-13 cells, data are the mean and standard deviation of at least 3 separate cell samples/rat tissue samples.</p

Glucocorticoid treatment results in a pulse of DNA methylation that is dependent on the Gr and is essential for B-13 cell trans-differentiation to B-13/H cells.

<p><b>A</b>, genomic DNA methylation in B-13 cells treated as indicated [DEX (6 hrs), B-13 cells treated with a pulse 6 hour treatment]. <b>B</b>, RT-PCR analysis for the expression of the indicated transcript in the indicated cell and tissue samples. <b>C</b>, qRT-PCR for Mecp2 mRNA in the indicated time point, expressed relative to the levels present in B-13 cells. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of 3 separate experiments. <b>D</b>, genomic DNA methylation in B-13 cells at the peak of methylation after DEX treatment (12 hours) with treatments as indicated. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of 3 separate experiments. ^*Significantly different (two tailed) DNA methylation versus control vehicle treated cells; ^$significantly different (two tailed) DNA methylation versus DEX only treated cells, P > 0.05. <b>E</b>, Western blot for the indicated proteins in B-13 cells treated for 14 days as indicated, results typical of at least 3 separate determinations. <b>F</b>, Photomicrographs of B-13 cells 7 days after continuous DEX treatment with or without the indicated epigenetic inhibitors or vehicle control (cells were pre-treated with these inhibitors 2 hours before first exposure to DEX). Medium was changed as outlined in methods section with DEX and epigenetic inhibitor treatments renewed at each media change (results typical of at least 8 separate experiments.</p

Pancreatic B-13 Cell Trans-Differentiation to Hepatocytes Is Dependent on Epigenetic-Regulated Changes in Gene Expression

<div><p>The proliferative B-13 pancreatic cell line is unique in its ability to generate functional hepatocyte-like (B-13/H) cells in response to exposure to glucocorticoid. In these studies, quantitatively comparable hepatic levels of liver-specific and liver-enriched transcription factor and hepatocyte defining mRNA transcripts were expressed after 10–14 days continuous treatment with glucocorticoid. This conversion in phenotype was associated with increased Gr-α mRNA expression and translation of a functional N-terminally truncated variant protein that localized to the nucleus in B-13/H cells. A short (6 hours) pulse exposure to glucocorticoid was also sufficient to transiently activate the Gr and irreversibly drive near identical conversion to B-13/H cells. Examination of epigenetic-related mechanisms demonstrated that B-13 DNA was rapidly methylated and de-methylated over the initial 2 days in response to both continuous or pulse exposure with glucocorticoid. DNA methylation and glucocorticoid-dependent conversion to an hepatic B-13/H phenotype was blocked by the methylation inhibitor, 5-azacytidine. Conversion to an hepatic B-13/H phenotype was also blocked by histone deacetylase inhibitors. Previous experiments have identified N-terminal Sgk1 variant proteins as pivotal to the mechanism(s) associated with pancreatic–hepatic differentiation. Both continuous and pulse exposure to DEX was sufficient to result in a near-similar robust transcriptional increase in Sgk1c mRNA expression from undetectable levels in B-13 cells. Notably, expression of Sgk1c mRNA remained constitutive 14 days later; including after pulse exposure to glucocorticoid and this induction was inhibited by 5-azacytidine or by histone deacetylase inhibitors. These data therefore suggest that exposing B-13 cells to glucocorticoid results in a Gr-dependent pulse in DNA methylation and likely other epigenetic changes such as histone modifications that leads to constitutive expression of Sgk1c and irreversible reprogramming of B-13 cells into B-13/H cells. Understanding and application of these mechanism(s) may enhance the functionality of stem cell-derived hepatocytes generated <i>in vitro</i>.</p></div

Primers used for RT-PCR and qRT-PCR.

<p>Primers used for RT-PCR and qRT-PCR.</p

Expression and transcriptional function of Gr in B-13 and B-13/H cells.

<p><b>A,</b> RT-PCR analysis for the expression of rat Gr mRNA transcripts (Gr-α and Gr-β) in the indicated cell and tissue samples. The Gr-β variant uses an alternate acceptor splice site at the 3' terminal exon resulting in a frame-shift and a shorter isoform with a distinct C-terminus compared to the α isoform. Data are typical of at least 4 separate B-13 and B-13/H cell cultures, after 30 cycles. <b>B</b>, Expression levels of the Gr-α mRNA transcripts relative to 18S rRNA transcript levels, and then normalised to B-13 cell. Data are the mean and standard deviation of at least 3 separate cell samples/rat tissue samples. <b>C</b>, Western blot for Gr protein, total cell protein/lane, B-13 and B-13/H 10μg/lane; rat tissue samples, 40μg/lane, typical of at least 4 separate investigations. <b>D</b>, Immunocytochemical staining for Gr (green) in B-13 and B-13/H cells with DNA stained using DAPI (blue), typical of 3 separate investigations. Arrow with circled dotted line indicates position of a cell nucleus, bar = 50μm. <b>E</b>, Gr transcriptional function as determined through transfection of GRE4-pGL4.28 and measurement of luciferase and renilla activities. Data are the mean and standard deviation of 3 separate transfections in the same experiment, typical of 3 separate experiments.</p

Sustained Isoprostane E2 Elevation, Inflammation and Fibrosis after Acute Ischaemia-Reperfusion Injury Are Reduced by Pregnane X Receptor Activation

<div><p>Liver grafts donated after cardiac death are increasingly used to expand the donor pool but are prone to ischaemic-type biliary lesions. The anti-inflammatory effects of the activated pregnane X receptor have previously been shown to be beneficial in a number of inflammatory liver conditions. However, its role in reducing peri-portal inflammation and fibrosis following ischaemia-reperfusion injury has not been investigated. Hepatic injury and its response to pregnane X receptor activation was examined after partial hepatic ischaemia-reperfusion injury induced by surgically clamping the left and middle lobar blood vessels in rats. Molecular and pathological changes in the liver were examined over the following 28 days. Ischaemia-reperfusion injury resulted in transient cholestasis associated with microvillar changes in biliary epithelial cell membranes and hepatocellular injury which resolved within days after reperfusion. However, in contrast to chemically-induced acute liver injuries, this was followed by sustained elevation in isoprostane E2, peri-portal inflammation and fibrosis that remained unresolved in the ischaemic reperfused lobe for at least 28 days after clamping. Administration of pregnenolone-16α-carbonitrile—a rodent-specific pregnane X receptor activator—resulted in significant reductions in cholestasis, hepatic injury, ischaemic lobe isoprostane E2 levels, peri-portal inflammation and fibrosis. Hepatic ischaemia-reperfusion injury therefore results in inflammatory and fibrotic changes that persist well beyond the initial ischaemic insult. Drug-mediated activation of the pregnane X receptor reduced these adverse changes in rats, suggesting that the pregnane X receptor is a viable drug target to reduce ischaemic-type biliary lesions in recipients of liver transplants donated after cardiac death.</p></div

IRI results in a transient cholestasis and altered biliary physiology.

<p><b>(A)</b> Partial hepatic ischaemia model with bile duct isolation (Left). Schematic representation of groups in both studies (Right). (<b>B)</b> Comparison of bile flow rates (normalised to total body weight). (<b>C)</b> Comparison of serum bile acid concentration between the IRI and sham IRI groups. (<b>D)</b> TEM images comparing BEC microvilli morphology during early (day 1) and late (day 28) reperfusion timepoints. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p

PXR activation reduces IRI-induced ductal reactions and the numbers of fibrogenic cells in the liver.

<p><b>(A, C, E)</b> CK-19, α-SMA and vimentin immunohistochemistry respectively between IRI+PCN and IRI+vehicle groups–typical views from the indicated treatment group at the indicated time point after clamp release (scale bar represents 100μm) with (<b>D, E F)</b> corresponding stain quantification, data are the mean and standard deviation from 5 separate animals at each time point and treatment, *Significantly different compared to IRI + vehicle group, p<0.05.</p

IRI results in progressive fibrosis.

<p><b>(A)</b> α-SMA immunohistochemistry—typical views from the indicated treatment group post and time point (upper panels) and quantification of α-SMA immunohistochemistry staining, scale bar represents 100μm. (<b>B)</b> quantification of α-SMA staining. <b>(C)</b> Sirius red staining–typical views from the indicated treatment group post and time point (upper panels) and quantification of Sirius red staining, scale bar represents 100μm. (<b>D</b>) quantification of Sirius red staining. <b>(E-F)</b> qRT-PCR analysis in ischaemic and sham ischaemic lobes. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p