Serine/threonine protein kinase SGK1 in glucocorticoid-dependent transdifferentiation of pancreatic acinar cells to hepatocytes
Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne NE24HH, UK.Journal of Cell Science (Impact Factor: 5.43). 02/2011; 124(Pt 3):405-13. DOI: 10.1242/jcs.077503
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.
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ABSTRACT: Serum-and-glucocorticoid-inducible-kinase-1 (SGK1) is under regulation of several hormones, mediators and cell stressors. More specifically, SGK1 expression is particularly sensitive to glucocorticoids, mineralocorticoids, and TGFβ. Moreover, SGK1 expression is exquisitely sensitive to hypertonicity, hyperglycemia, and ischemia. SGK1 is activated by insulin and growth factors via phosphatidylinositol-3-kinase, 3-phosphoinositide dependent-kinase PDK1, and mTOR. SGK1 up-regulates the Na+/K+-ATPase, a variety of carriers (e.g. NCC, NKCC, NHE1, NHE3, SGLT1, several amino acid transporters) and many ion channels (e.g. ENaC, SCN5A, TRPV4-6, Orai1/STIM1, ROMK, KCNE1/KCNQ1, GluR6, CFTR). SGK1 further up-regulates a number of enzymes (e.g. glycogen-synthase-kinase-3, ubiquitin-ligase Nedd4-2), and transcription factors (e.g. forkhead-transcription-factor FOXO3a, β-catenin, nuclear-factor-kappa-B NFκB). SGK1 sensitive functions contribute to regulation of epithelial transport, excitability, degranulation, matrix protein deposition, coagulation, platelet aggregation, migration, cell proliferation, and apoptosis. Apparently, SGK1 is not required for housekeeping functions, as the phenotype of SGK1 knockout mice is mild. However, excessive SGK1 expression and activity participates in the pathophysiology of several disorders, including hypertension, obesity, diabetes, thrombosis, stroke, inflammation, autoimmune disease, fibrosis, and tumor growth. A SGK1 gene variant (prevalence ~3-5% prevalence in Caucasians, ~10% in Africans) predisposes to hypertension, stroke, obesity, and type 2 diabetes. Moreover, excessive salt intake and/or excessive release of glucocorticoids, mineralocorticoids, and TGFβ up-regulates SGK1 expression thus predisposing to SGK1-related diseases.Hormones (Athens, Greece) 04/2012; 12(2):160-71. DOI:10.14310/horm.2002.1401 · 1.20 Impact Factor
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ABSTRACT: Reduction in the use of animals in toxicology is an important goal despite the continued need to assess drug and chemical safety in man. However, a limitation to in vitro screening for drug and chemical toxicity is the lack of available human hepatocytes and the difficulties associated with generating fully functional hepatocytes from stem cells. Previously, we have shown that a rat pancreatic acinar cell line is capable of trans-differentiating into fully functional hepatocyte-like cells in response to glucocorticoid via a serine/threonine protein kinase mechanism alone. Here we demonstrated that differentiation only occurs with glucocorticoids, not other steroids. We also investigated the potential of human pancreatic cells to undergo the same process. Analysis of adult human pancreata at the level of mRNA, protein and by immunohistochemical staining demonstrated that long term systemic exposure to glucocorticoid therapy resulted in differentiation of exocrine tissue to hepatocyte-like tissue. Glucocorticoid treatment of human pancreatic acinar cells in culture also resulted in trans-differentiation to hepatocyte-like cells. Both in vivo and in vitro, trans-differentiation of pancreas cells to hepatocytes was associated with an induction of SGK1 variant transcripts that have been previously shown to drive B-13 differentiation to hepatocytes. Adult exocrine human pancreas therefore responds in a similar qualitative fashion to that previously observed in rodents exposed to elevated glucocorticoid – that of a differentiation into hepatocyte-like cells. Understanding the enhanced response of B-13 cells to glucocorticoid and engineering this response in a replicating human acinar cell could generate an unlimited supply of functional human hepatocytes in vitro that could be useful in a variety of applications, including screening drugs and chemicals for hepatic metabolism and toxicity.12/2012; 2(1):80-87. DOI:10.1039/C2TX20061A
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ABSTRACT: The rat pancreatic “B-13” acinar cell is a stable progenitor cell line that differentiates into hepatocyte-like cells (B-13/H cells) in 2D un-coated plastic culture with simple culture media in response to glucocorticoid exposure. Examination of cytochrome P450 indicated that the expression of a range of genes were similar to freshly isolated hepatocytes and that these gene products were functional on the basis of spectrophotometrically-detectable reduced carbon-monoxide haemoprotein and metabolism of several drugs. Since normal hepatocytes readily de-differentiate under similar conditions, we hypothesized that B-13 cells have undergone a variety of alterations that stabilise a progenitor phenotype and restrict differentiation to hepatocytes only (which if capitulated in human cells, could generate a readily accessible supply of functional human hepatocytes in vitro). To examine this hypothesis, the B-13 karyotype; pluripotency-inducing transcription factor expression and forced over-expression of these factors in B-13 cells were examined. B-13 cells were also injected into NOD/SCID mice and engraftment and differentiation assessed by RT-PCR, Western blotting, immunohistochemistry and fluorescent in situ hybridization (FISH). B-13 cells expressed four pluripotency-inducing transcription factors c-Myc, Klf4, Oct4 and Sox2 with only c-Myc expression maintained after glucocorticoid treatment. Over-expression of the pluripotency-inducing transcription factors blocked B-13/H formation in response to glucocorticoid. Injection of B-13 cells into NOD/SCID mice resulted in their engraftment to the pancreas and liver, with restricted differentiation to hepatocytes in the liver. The cells did not engraft to any other tissues examined. The ability of B-13 cells to specifically generate functional hepatocytes in vitro in response to glucocorticoid is therefore associated with genetic rearrangements that may facilitate expression of genes associated with plasticity (without leading to pluripotency), which are repressed by glucocorticoid treatment.Toxicology Research 01/2013; 2(6):308 - 320. DOI:10.1039/C3TX50030F · 3.98 Impact Factor
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