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

Wallace, Karen; Quan, Long; Fairhall, Emma A.; Charlton, Keith A.; Wright, Matthew

doi:10.1242/jcs.090084

Cited by 8 publications

(34 citation statements)

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“…Figure 2A Previous work has identified that SGK1 is markedly up-regulated in B-13 cells when treated with glucocorticoid, most notably an alternatively transcribed variant 3 (also termed variant C). 4 Transfecting B-13 cells with expression vectors encoding the different human SGK1 variants demonstrated that SGK1 v3 (and an unassigned human-specific F variant) directed B-13 cells into hepatocytes without glucocorticoid treatment, demonstrating a pivotal role for specific SGK1 transcript induction in the trans-differentiation response. 4 Figure 2D demonstrates that transcripts for SGK1 v3 and SGK1 variant F were detectable in NP08 and NP10 pancreata, with the highest To establish whether adult human pancreatic acinar cells are capable of trans-differentiation to hepatocyte-like cells in vitro, acinar cells were isolated and cultured with or without the addition of 10µM dexamethasone (DEX), a synthetic glucocorticoid.…”

Section: Adult Human Acinar Cells Express Hepatocyte Markers In Vivo mentioning

confidence: 99%

“…4 Transfecting B-13 cells with expression vectors encoding the different human SGK1 variants demonstrated that SGK1 v3 (and an unassigned human-specific F variant) directed B-13 cells into hepatocytes without glucocorticoid treatment, demonstrating a pivotal role for specific SGK1 transcript induction in the trans-differentiation response. 4 Figure 2D demonstrates that transcripts for SGK1 v3 and SGK1 variant F were detectable in NP08 and NP10 pancreata, with the highest To establish whether adult human pancreatic acinar cells are capable of trans-differentiation to hepatocyte-like cells in vitro, acinar cells were isolated and cultured with or without the addition of 10µM dexamethasone (DEX), a synthetic glucocorticoid. Figure 4A indicates that cells from patient NP8 expressed a range of hepatic markers at the mRNA level (CYP2E1, CPSI, albumin, CYP3A4) in response to DEX treatment, with no expression detected in cells cultured in basal media.…”

Section: Adult Human Acinar Cells Express Hepatocyte Markers In Vivo mentioning

confidence: 99%

“…The B-13 rat pancreatic cell line is an expandable progenitor-like cell with the capacity to generate an unlimited supply of functional rat hepatocyte-like cells (termed B-13/H cells) in vitro, without the need for animal donors. [1][2][3][4][5][6] The cell line appears to be unique in that it differentiates into B-13/H cells that are both qualitatively and quantitatively comparable to freshly isolated hepatocytes. [1][2][3][4][5][6] Furthermore, B-13 cells undergo a change to B-13/H cells in response to the addition of a single glucocorticoid hormone, providing a simple and highly reproducible system for hepatocyte generation.…”

Section: Introductionmentioning

confidence: 99%

“…We sought to investigate whether the trans-differentiation of acinar cells to hepatocytes is a physiologically feasible outcome in normal cells, both in vitro and in vivo. In a series of papers, we have shown that the B-13 to B-13/H trans-differentiation is regulated by Wnt signalling and serine/threonine protein kinase (SGK1) induction 4,5 and is related to a pathophysiological response of the rodent pancreas to glucocorticoid. 7,8 B-13 cells appear to be hyper-responsive to glucocorticoid with respect to hepatic differentiation, but the response occurs in normal rodent pancreas acinar cells as a result of physiologically abnormally high concentrations of glucocorticoid, both in vitro and in vivo.…”

Section: Introductionmentioning

confidence: 99%

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Adult human exocrine pancreas differentiation to hepatocytes – potential source of a human hepatocyte progenitor for use in toxicology research

et al. 2013

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Adult human exocrine pancreas differentiation to hepatocytes -potential source of a human hepatocyte progenitor for use in toxicology research. Toxicology Research 2013, 2(1), 80-87. Fairhall et al In vitro hepatocyte generation 3Reduction 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-differntiating 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, transdifferentiation 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 glucocorticoidthat of a differentiation into hepatocytelike 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.

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Section: Adult Human Acinar Cells Express Hepatocyte Markers In Vivo mentioning

confidence: 99%

Section: Adult Human Acinar Cells Express Hepatocyte Markers In Vivo mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Adult human exocrine pancreas differentiation to hepatocytes – potential source of a human hepatocyte progenitor for use in toxicology research

et al. 2013

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“…This laboratory and others have therefore investigated a number of signaling pathways involved in B-13/H generation in part, to understand how stem cell-derived hepatic phenotype might be amplified to normal in vivo levels. In this respect, roles for the glucocorticoid receptor (Gr) [ 1 ], serine/threonine protein kinase 1 (Sgk1) [ 12 ], Wnt signalling [ 8 ] and induction of the liver-enriched transcription factors C/EBPα and C/EBPβ in the trans-differentiation have been identified [ 1 , 7 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors

Halland

Schmidt

Weiß

et al. 2014

ACS Med. Chem. Lett.

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From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure. KEYWORDS: Serum glucocorticoid regulated kinase, WNT signaling, AGC kinase, virtual screening T he serum and glucocorticoid regulated kinase 1 (SGK1) belongs to the serine/threonine kinase family (AGC kinases) and is an important downstream effector in the phosphatidylinositol-3-kinase pathway. SGK1 regulates transport, hormone release, cell proliferation, and apoptosis.1,2 There is a strong body of evidence that dysfunction or dysregulation of SGK1 is involved in many pathological conditions ranging from cancer, hypertension, diabetes, and thrombotic events to neurodegeneration.3−6 Albeit ubiquitously expressed, the transcription is highly regulated and stimulated by physiological events like hyperglycaemia, cell shrinkage, ischemia, glucocorticoids, mineralocorticoids, and insulin as well as by inflammatory mediators including TGFβ. SGK1 regulates several ion channels such as ENaC, KCNE1/KCNQ1, carriers like NCC, NHE3, SGLT1, Na(+)/K(+)-ATPase, and transcription factors including FOXO3a, NF-κβ, and β-catenin.7−11 Recently, it was shown that β-catenin phosphorylation by SGK1 mediates the crosstalk between the corticoid-and WNT-signaling pathways. 7,12,13 Surprisingly, despite the highly relevant, validated biological function of SGK1 only a few inhibitors with appropriate selectivity and potency for the selective interference with SGK1 have been described so far.14−17 Here we report the identification and optimization of highly active and selective SGK1 inhibitors as chemical tools for the further elucidation and validation of the biological role of SGK1. 18Computational screening, particularly 3D ligand-based virtual screening technologies, have emerged as efficient methods for the discovery of novel drug candidates.19−21 Ligand-based virtual screening uses known active molecules to identify new ligands sharing a set of relevant molecular properties such as molecular shape and electrostatics. A limited number of inhibitors of SGK1 have been described in the literature so far including the azaindoles 1 and 2, the hydrazides 3 and 4, and their 3-aminoindazole isosteres 5 and 6 as shown in Figure 1. 22Cocrystal structures of ligands 1 and 2 in the SGK1 enzyme supported a bidentate interaction of the ligand scaffold with the kinase hinge region.14 Using a docking model 23 a bidentate hinge interaction of the para-phenolic substituent was also assumed for 3 and 4 as well as a tridentate interaction for compounds 5 and 6

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

Cited by 8 publications

References 0 publications

Adult human exocrine pancreas differentiation to hepatocytes – potential source of a human hepatocyte progenitor for use in toxicology research

Adult human exocrine pancreas differentiation to hepatocytes – potential source of a human hepatocyte progenitor for use in toxicology research

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

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors

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