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

Abstract

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-β. 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 β-catenin, and recombinant SGK1 was shown to directly phosphorylate purified β-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 β-catenin by SGK1C represents the mechanism of crosstalk between glucocorticoid and WNT signalling pathways.

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

“…Previous experience with rat B-13 cells has demonstrated that the hepatocyte-like B-13/H cells derived therefrom show good comparability with primary rat hepatocytes with respect to several cytochrome P450s and sensitivities to drug/chemical toxicity [8][9][10][12][13][14]. Exposing HPAC cells to glucocorticoid had a variety of effects also observed when B-13 cells were treated with glucocorticoid, including an inhibition in proliferation, phenotypic changes and low level expression 7 of a variety of hepatocyte-specific transcripts (e.g.…”

“…RNA and protein extracts were prepared and RT-PCR performed as previously outlined [13,14] using primers described in Table 1. Western blotting, immunocytochemistry and immunohistochemistry were performed as described [13,14].…”

“…Differentiation is triggered by exposure to glucocorticoid which -via the glucocorticoid receptor (GR) -induces serine threonine kinase 1, β-catenin phosphorylation, transient wnt signalling repression, epigenetic changes and induction of hepatic transcription factors Cebpα, Cebpβ, Hnf1α 4 and Hnf4α [8,10,13,14]. Recently, the human pancreatic adenocarcinoma cell line (HPAC) phenotype was shown to be suppressed by glucocorticoid exposure and to undergo a qualitatively similar expression of genes associated with hepatocytes (although the levels were relatively low compared to intact human liver [15]).…”

B-13 progenitor-derived hepatocytes (B-13/H cells) model lipid dysregulation in response to drugs and chemicals

“…Furthermore, B-13 cells do not require an extensive array of recombinant growth factors and/or other signalling inhibitor treatments to direct them to become hepatocytes. Exposure to a single glucocorticoid hormone is all that is required ( Shen et al, 2000 , Wallace et al, 2011 ). Critically, in contrast to stem cell-derived hepatocyte-like cells which generally only form a foetal level of hepatic differentiation, B-13/H cells express many adult hepatocyte genes at adult hepatocyte levels ( Marek et al, 2003 , Fairhall et al, 2013a , Fairhall et al, 2016 ).…”