HNF4alpha expression amplifies the glucocorticoid-induced conversion of a human pancreatic cell line to an hepatocyte-like cell.

The pancreas and liver are closely related developmentally and trans-differentiation of cells from one tissue into the cells of the other has been documented to occur after injury or exposure to selected growth factors or glucocorticoid hormones. To generate a readily-expandable source of human hepatocyte-like (H-13) cells, the human pancreatic adenocarcinoma cell (HPAC) line was stably transfected with a construct encoding the variant 2 hepatocyte nuclear factor 4 α (HNF4α) using a piggyBac vector and transient expression of a transposase. Through induction of transgene HNF4α regulated via an upstream glucocorticoid response element in combination with existing modulating effects of glucocorticoid, H-13 cells were converted into quantitatively similar hepatocyte-like (H-13/H) cells based on expression of a variety of hepatocyte proteins. H-13/H cells also demonstrated the ability to store glycogen and lipids. These data provide proof of concept that regulated expression of genes associated with hepatocyte phenotype could be used to generate quantitatively functional human hepatocyte-like cells using a readily expandable cell source and simple culture protocol. This approach would have utility in Toxicology and Hepatology research.

Glucocorticoid-induced pancreatic-hepatic trans-differentiation in a human cell line in vitro.

The rodent pancreatic AR42J-B13 (B-13) cell line differentiates into non-replicative hepatocyte-like cells in response to glucocorticoid mediated via the glucocorticoid receptor (GR). The aims of this study were to identify a human cell line that responds similarly and investigate the mechanisms underpinning any alteration in differentiation. Exposing the human pancreatic adenocarcinoma (HPAC) cell line to 1-10 µM concentrations of dexamethasone (DEX) resulted an inhibition of proliferation, suppressed carcinoembryonic antigen expression, limited expression of pancreatic acinar and hepatic gene expression and significant induction of the constitutively-expressed hepatic CYP3A5 mRNA transcript. These changes were associated with a pulse of genomic DNA methylation and suppressed notch signalling activity. HPAC cells expressed high levels of GR transcript in contrast to other nuclear receptors - such as the glucocorticoid-activated pregnane X receptor (PXR) - and GR transcriptional function was activated by DEX in HPAC cells. Expression of selected hepatocyte transcripts in response to DEX was blocked by co-treatment with the GR antagonist RU486. These data indicate that the HPAC response to glucocorticoid exposure includes an inhibition in proliferation, alterations in notch signalling and a limited change in the expression of genes associated with an acinar and hepatic phenotype. This is the first demonstration of a human cell responding to similarly to the rodent B-13 cell regarding formation of hepatocyte-like cells in response to glucocorticoid. Identifying and modulating the ablating factor(s) may enhance the hepatocyte-like forming capacity of HPAC cells after exposure to glucocorticoid and generate an unlimited in vitro supply of human hepatocytes for toxicology studies and a variety of clinical applications.

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

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.

Serine/threonine protein kinase SGK1 in glucocorticoid-dependent transdifferentiation of pancreatic acinar cells to hepatocytes.

Elevated glucocorticoid levels result in the transdifferentiation of pancreatic acinar cells into hepatocytes through a process that requires a transient repression of WNT signalling upstream of the induction of C/EBP-ß. However, the mechanism by which glucocorticoid interacts with WNT signalling is unknown. A screen of microarray data showed that the serine/threonine protein kinase SGK1 (serum- and glucocorticoid-regulated kinase 1) was markedly induced in the model B-13 pancreatic rat acinar cell line after glucocorticoid treatment (which converts them into hepatocyte-like 'B-13/H' cells) and this was confirmed at the level of mRNA (notably an alternatively transcribed SGK1C form) and protein. Knockdown of SGK1 using an siRNA designed to target all variant transcripts inhibited glucocorticoid-dependent transdifferentiation, whereas overexpression of the human C isoform (and also the human SGK1F isoform, for which no orthologue in the rat has been identified) alone - but not the wild-type A form - inhibited distal WNT signalling Tcf/Lef transcription factor activity, and converted B-13 cells into B-13/H cells. These effects were lost when the kinase functions of SGK1C and SGK1F were mutated. Inhibition of SGK1 kinase activity also inhibited glucocorticoid-dependent transdifferentiation. Expression of SGK1C and SGK1F resulted in the appearance of phosphorylated ß-catenin, and recombinant SGK1 was shown to directly phosphorylate purified ß-catenin in vitro in an ATP-dependent reaction. These data therefore demonstrate a crucial role for SGK1 induction in B-13 cell transdifferentiation to B-13/H hepatocytes and suggest that direct phosphorylation of ß-catenin by SGK1C represents the mechanism of crosstalk between glucocorticoid and WNT signalling pathways.

AR42J-B-13 cell: an expandable progenitor to generate an unlimited supply of functional hepatocytes.

Hepatocytes are the preparation of choice for Toxicological research in vitro. However, despite the fact that hepatocytes proliferate in vivo during liver regeneration, they are resistant to proliferation in vitro, do not tolerate sub-culture and tend to enter a de-differentiation program that results in a loss of hepatic function. These limitations have resulted in the search for expandable rodent and human cells capable of being directed to differentiate into functional hepatocytes. Research with stem cells suggests that it may be possible to provide the research community with hepatocytes in vitro although to date, significant challenges remain, notably generating a sufficiently pure population of hepatocytes with a quantitative functionality comparable with hepatocytes. This paper reviews work with the AR42J-B-13 (B-13) cell line. The B-13 cell was cloned from the rodent AR42J pancreatic cell line, express genes associated with pancreatic acinar cells and readily proliferates in simple culture media. When exposed to glucocorticoid, 75-85% of the cells trans-differentiate into hepatocyte-like (B-13/H) cells functioning at a level quantitatively similar to freshly isolated rat hepatocytes (with the remaining cells retaining the B-13 phenotype). Trans-differentiation of pancreatic acinar cells also appears to occur in vivo in rats treated with glucocorticoid; in mice with elevated circulating glucocorticoid and in humans treated for long periods with glucocorticoid. The B-13 response to glucocorticoid therefore appears to be related to a real pathophysiological response of a pancreatic cell to glucocorticoid. An understanding of how this process occurs and if it can be generated or engineered in human cells would result in a cell line with the ability to generate an unlimited supply of functional human hepatocytes in a cost effective manner.