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