title: Pancreatic B-13 Cell Trans-Differentiation to Hepatocytes Is Dependent on Epigenetic-Regulated Changes in Gene Expression.
abstract: 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 in vitro.
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.
authors.forename: Emma A
authors.initials: EA
authors.lastname: Fairhall
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Charles
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.
authors.forename: Philip M E
authors.initials: PM
authors.lastname: Probert
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.|School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: Wallace
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Gibb
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.
authors.forename: Chandni
authors.initials: C
authors.lastname: Ravindan
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Soloman
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Wright
mesh_terms: D000818:Animals; D002460:Cell Line; D054338:Cell Transdifferentiation / Q000187:drug effects / Q000235:genetics*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008297:Male; D009928:Organ Specificity / Q000235:genetics; D010179:Pancreas / Q000166:cytology* / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D005938:Glucocorticoids; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; C411470:glucocorticoid receptor alpha