Serine/threonine protein kinase SGK1 in glucocorticoid-dependent
transdifferentiation of pancreatic acinar cells to hepatocytes
Karen Wallace, Quan Long, Emma A. Fairhall, Keith A. Charlton and Matthew C. Wright
Journal of Cell Science 124, 1602
© 2011. Published by The Company of Biologists Ltd
There was an error published in J. Cell Sci. 124, 405-413. The acknowledgements paragraph should read as follows:
Supported by a grant from Newcastle University. K.W. was supported by a BBSRC PhD Studentship award. E.A.F. is currently a recipient
of an MRC ITTP Studentship. The technical assistance of Dr Trevor Booth with confocal microscopy is gratefully acknowledged. The
expertise and assistance of Trevor R. Jackson is acknowledged. The SGK1F isoform was originally cloned by Ben Hall [Ben Hall, The
identification and characterisation of novel serum and glucocorticoid regulated kinase 1 (SGK1) isoforms in human skin, PhD Thesis,
Newcastle University, 2007]. See also NCBI accession number FM205710.
The authors apologise for this mistake.
Transdifferentiation is a form of metaplasia and describes the
irreversible switching of cellular differentiation in fully
differentiated cells (Burke and Tosh, 2005; Wallace et al., 2010a).
Administration of glucocorticoid to rats results in the appearance
of hepatocytes in the pancreas (Wallace et al., 2009). In mice with
elevated levels of circulating glucocorticoid, extensive areas of the
exocrine (acinar) pancreas express hepatocyte markers, suggestive
of widespread acinar transdifferentiation to hepatocytes (Wallace
et al., 2010b). This pathological response of acinar cells to elevated
glucocorticoid in vivo also occurs to a limited degree in primary
cultures of rat acinar cells (Lardon et al., 2004) and efficiently in
the AR42J-B-13 (B-13) rat pancreatic exocrine cell line (Shen et
al., 2000; Marek et al., 2003), the latter providing a model to study
the mechanism(s) involved.
Previous work has shown that transdifferentiation of B-13 cells
into hepatocyte-like cells (defined as B-13/H cells) is dependent on
induction of the CCAAT enhancer binding protein- (C/EBP-)
(Shen et al., 2000). More recently, we have shown that a transient
repression of WNT3a expression, WNT signalling and Tcf/Lef
transcriptional activity in B-13 cells is an upstream event to
induction of C/EBP- (Wallace et al., 2010c). Thus, siRNA
knockdown of -catenin – the intracellular messenger for WNT
signalling (Hoppler and Kavanagh, 2007) – substituted for
glucocorticoid and led to an induction of C/EBP- expression and
transdifferentiation (Wallace et al., 2010c). Overexpression of a
dominant-negative -catenin protein blocked glucocorticoid-
dependent transdifferentiation (Wallace et al., 2010c).
However, precisely how glucocorticoids interact with the WNT
signalling pathway in B-13 cells remains obscure. It is known that
some nuclear receptors – the super-family of receptor proteins that
includes the glucocorticoid receptor (GR) – interact with
components of the WNT signalling pathway (Mulholland et al.,
2005). However, there are only a small number of investigations
examining interactions of glucocorticoids with WNT signalling,
and interactions appear to depend on the cell type examined. Thus,
glucocorticoid exposure reduces the expression of -catenin in
pituitary cells (Spangler and Delidow, 1998), whereas it has no
effect in osteoblasts (Smith et al., 2002).
The serum- and glucocorticoid-regulated kinases (SGKs) belong
to a family of related serine/threonine kinases including protein
kinase A, protein kinase G and protein kinase C (AGC kinases)
(Firestone et al., 2003; Pearce et al., 2010). The SGK subfamily
consists of three genes, SGK1, SGK2 and SGK3 (Pearce et al.,
2010). SGK1 has been shown to be involved in the regulation of
a number of ion channels, with sodium re-absorption in the kidney
the best studied (for a review, see Lang et al., 2006). In the kidney,
activation of the mineralocorticoid receptor results in the
transcriptional induction of SGK1 expression. Phosphorylation of
SGK1 through active phosphoinositide 3-kinase (PI3K) and mTorC
signalling then renders SGK1 functionally active as a kinase (Pearce
et al., 2010). This results in phosphorylation of the ubiquitin ligase
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-. 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 -catenin, and recombinant SGK1
was shown to directly phosphorylate purified -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 -
catenin by SGK1C represents the mechanism of crosstalk between glucocorticoid and WNT signalling pathways.
Key words: Dexamethasone, Stem cell, Reprogramming, LY294002, Liver, Pancreas
Accepted 28 September 2010
Journal of Cell Science 124, 405-413
© 2011. Published by The Company of Biologists Ltd
Serine/threonine protein kinase SGK1 in
glucocorticoid-dependent transdifferentiation of
pancreatic acinar cells to hepatocytes
Karen Wallace, Quan Long, Emma A. Fairhall, Keith A. Charlton and Matthew C. Wright*
Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, NE2 4HH, UK
*Author for correspondence (email@example.com)
Journal of Cell Science
Nedd4-2, a block in epithelial Na+channel (ENaC) ubiquitylation,
a reduction in ENaC endocytosis and increased renal tubular
epithelial Na+transport into the cell (Lang et al., 2006). SGK1
might also increase expression of ENaCs (Boyd and Naray-Fejes-
Toth, 2005). The predominant steroidal regulator of SGK1
expression in the kidney is the mineralocorticoid. Although
glucocorticoids activate the mineralocorticoid receptor, rapid
oxidation by HSD11B2 in the kidney ensures that the
mineralocorticoid receptor primarily responds to mineralocorticoid
and not glucocorticoid levels (Seckl and Walker, 2001).
Despite a clear functional role for SGK1 in the kidney, SGK1 is
also constitutively expressed in a wide range of other tissues.
Expression is also upregulated by a diverse range of factors in
addition to glucocorticoids and mineralocorticoids, including 1,25
dihidroxyvitamin D3 (Akutsu et al., 2001), TGF (Waldegger et
al., 1999), PDGF (Mizuno and Nishida, 2001), PPAR activators
(Hong et al., 2003) and osmotic stress (Waldegger et al., 1997).
Placement of SGK1 in a functional context in other tissues is
therefore problematical. Indeed, Sgk1–/–mice show no overt
phenotype. Only when these mice are placed on a low NaCl diet
does a lack of SGK1 have an effect, which is restricted to salt
wasting (Wulff et al., 2002; Fejes-Tóth et al., 2008). The most
notable feature of SGK1 that sets it apart from other AGC kinases
is the rapid and marked change in expression levels seen with
SGK1 after exposure of cells to transcriptional regulators (Firestone
et al., 2003). Because many of the regulators of SGK1 expression
are associated with stressful cellular conditions, it is likely that
SGK1 has an ancillary role in fine-tuning kinase-dependent control
under stress conditions. SGK2 and SGK3 are closely related to
SGK1 in terms of amino acid sequence, but are not inducible by
glucocorticoid or serum (Kobayashi et al., 1999).
The data in this paper demonstrate that transdifferentiation of B-
13 cells is dependent on an interaction of glucocorticoid with the
GR – and not the closely related mineralocorticoid receptor (MR)
– and induction of SGK1. SGK1 was actively phosphorylated in
B-13 cells by a PI3K-dependent mechanism and we show that
SGK1 phosphorylates -catenin in vitro. These data therefore
suggest that B-13 cell transdifferentiation to B-13/H cells in
response to glucocorticoid is dependent on SGK1 induction, and
its crosstalk with the WNT signalling pathway is through direct
phosphorylation of -catenin by SGK1.
B-13 transdifferentiation is dependent on interaction with
the glucocorticoid receptor and not the mineralocorticoid
Glucocorticoids activate both the GR and the MR (Arriza et al.,
1987). The presence and activity of 11-hydroxysteroid
dehydrogenase (HSD11B) enzymes 1 and 2 determine the extent
to which glucocorticoids remain active GR and MR ligands (Seckl
and Walker, 2001). Expression and activity of HSD11B2 ensures
that glucocorticoids do not activate the MR in selected tissues
(such as the kidney) (Fig. 1A). Fig. 1B confirms that B-13 cells
expressed the GR and expressed barely detectable levels of MR.
Treatment with the glucocorticoid dexamethasone (DEX) resulted
in transdifferentiation of B-13 cells to B-13/H cells, as judged by
the expression of liver markers CYP2E1 and albumin (Fig. 1C)
and to a marked change in morphology (see later). The GR
antagonist mifepristone (RU486) prevented transdifferentiation
(and morphological changes – data not shown), whereas the MR
antagonist spironolactone had no effect (Fig. 1C), suggesting the
effects of DEX on transdifferentiation are mediated entirely through
the GR. This is supported by a failure of the natural MR agonist
aldosterone (ALD) to promote transdifferentiation (Fig. 1C).
Fludrocortisone (FLUD) is a mixed GR and MR agonist and Fig.
1C demonstrates that only RU486 – and not spironolactone –
406Journal of Cell Science 124 (3)
Fig. 1. Transdifferentiation of B-13 cells into B-
13/H cells is dependent on glucocorticoid
interaction with the glucocorticoid receptor, not
the mineralocorticoid receptor. (A)schematic
diagram of glucocorticoid metabolism and
interaction with the GR and MR. In aldosterone-
selective tissues such as the kidney and placenta,
11-hydroxysteroid dehydrogenase 2 (HSD11B2)
oxidises glucocorticoids to an inactive keto form,
reducing glucocorticoid activation of the MR. In
tissues such as the liver, 11-hydroxysteroid
dehydrogenase 1 (HSD11B1) promotes high
concentrations of active glucocorticoid, ensuring
these tissue are responsive to circulating
glucocorticoid levels. (B)RT-PCR analysis of the
expression of the indicated transcript using primers
listed in Table 1. (C)Western blot showing the
expression of the indicated liver markers plus -
actin as loading control. B-13 cells were cultured
with the indicated additions made from 1000-fold
ethanol stocks. Control cells received 0.1% (v/v)
ethanol. Medium and additions were renewed every
2–3 days and cells were harvested for western
blotting analysis (30g total protein/lane) after 14
days of treatment. FLUD, fludrocortisone; ALD,
aldosterone. All data are typical of at least three
Journal of Cell Science
blocked transdifferentiation (confirming that MR does not have a
redundant role in transdifferentiation when GR activity is inhibited).
SGK1 is induced by DEX treatment in B-13 cells
Microarray data has shown that the transdifferentiation of B-13
cells into B-13/H cells is a co-ordinated change in gene expression
that results in a qualitative and quantitative expression that is
similar to that of primary hepatocytes (Wallace et al., 2009). Further
interrogation of this microarray data indicated that there was a
marked (>100-fold) increase in Sgk1 mRNA transcripts in B-13/H
cells, contrasting with few changes in the expression levels of
mRNAs encoding many components of the WNT and PI3K
signalling pathways (supplementary material Fig. S1), which
converge at glycogen synthase kinase 3 (GSK3) phosphorylation
and potentially regulate -catenin levels in B-13 cells (see later).
The induction of all known rat Sgk1 isoforms (for sequence
comparison, see supplementary material Fig. S2) in B-13/H cells,
with the Sgk1c mRNA isoform induced from undetectable levels
in B-13 cells is shown in Fig. 2A. In the Tg(Crh) mouse model of
Cushing’s disease, which results in widespread expression of
hepatocyte-specific gene expression in the acinar pancreas by 21
weeks of age (Wallace et al., 2010b), Sgk1c was also induced from
undetectable levels in the pancreas (Fig. 2B). Induction of Sgk1
transcripts in B-13/H and Tg(Crh) also resulted in an induction of
SGK1 proteins (Fig. 2C). Induction of Sgk1c mRNA and SGK1
protein occurs in B-13 cells before induction of C/EBP-,
hepatocyte marker gene expression and conversion to B-13/H cells
(Fig. 2D,E). The possibility arises therefore, that SGK1 induction
could be a crucial upstream event that controls B-13
transdifferentiation to B-13/H cells.
SGK1 expression promotes B-13 transdifferentiation to
A specific chemical inhibitor for SGK1 is not currently available.
An inhibition of SGK1 expression was therefore used to determine
whether SGK1 has a role in transdifferentiation. An siRNA designed
to promote the degradation of mRNA encoding C/EBP- inhibited
DEX-dependent C/EBP- induction and transdifferentiation (Fig.
3A), as predicted from previous work which has shown that
overexpression of C/EBP in B-13 cells substitutes for the effects
Role for SGK1 in transdifferentiation
Fig. 2. Transdifferentiation of B-13 cells into B-
13/H cells is associated with an induction of
SGK1 gene expression. (A)RT-PCR analysis of
the indicated transcript using primers as outlined
in Table 1 (see also supplementary material Fig.
S2 illustrating 5? region alignment of alternative
transcribed Sgk1 cDNA sequences and positions
of selective upstream primers). (B)RT-PCR
analysis of the indicated mouse mRNA transcripts
in pairs of age-matched wild-type (W/T) and
Tg(Crh) female mouse liver, pancreas and kidney
tissues. (C)Western blot of SGK1 expression
(30g total protein/lane). (D)Time course for the
induction of the indicated transcripts after
treatment with DEX for the indicated number of
days. (E)Time course for the induction of the
indicated protein after treatment with DEX for the
indicated number of days. All data are typical of at
least three separate experiments.
Journal of Cell Science
of DEX and promotes transdifferentiation (Shen et al., 2000).
Transfection with Sgk1 siRNA similarly inhibited DEX-dependent
SGK1 induction and transdifferentiation (Fig. 3B) whereas an
siRNA designed to knock down expression of the low-affinity
glucocorticoid binding protein (LAGS), used as a control, had no
effect (Fig. 3C).
To further test the hypothesis that SGK1 induction is involved
in transdifferentiation, B-13 cells were transfected with a series of
human SGK1 expression constructs (SGK1 genes are highly
homologous between species; rat and human SGK1A isoforms
show 97% identical amino acid homology with only three
functional amino acid differences over the entire 431 amino acids;
see supplementary material Fig. S3). Fig. 4A demonstrates that
expression of either the human SGK1C (orthologous to the C form
in rat) or SGK1F (which has no orthologue in the rat) isoforms
alone resulted in a transdifferentiation of B-13 cells. Fig. 4B
demonstrates that SGK1C and SGK1F also significantly inhibited
the transcriptional activity of Tcf/Lef to levels similar to those
obtained with DEX treatment, in contrast to the SGK1A and
To establish whether the kinase function of SGK1C and SGK1F
is required for promoting B-13 transdifferentiation, mutant variants
of each protein were generated. Work by others has demonstrated
that phosphorylation of SGK1A at Ser422 by mTorC2 (Garcia-
Martinez and Alessi, 2008) enables 3-phosphoinositide-dependent
408 Journal of Cell Science 124 (3)
Fig. 3. siRNA knockdown of SGK1 expression inhibits DEX-dependent
transdifferentiation of B-13 cells to B-13/H cells. B-13 cells cultured in six-
well plates were transfected with 4g of siRNA designed to inhibit the
expression of (A) C/EBP-, (B) SGK1 or (C) LAGS. After 7 days, cells were
harvested and analysed by western blotting for the indicated protein. All data
are typical of at least three separate experiments.
Fig. 4. Expression of human SGK1C or SGK1F isoforms in B-13 cells
promotes the transdifferentiation of B-13 cells to B-13/H cells. B-13 cells
cultured in six-well plates were transfected with 2g of the indicated plasmid
construct [plus 2g of either Topflash (T) or Fopflash (F) combined with
0.02g of transfection control Renilla reporter vector] or treated with 10 nM
DEX for 14 days to generate B-13/H cells. 14 days after transfection, cells
were harvested and analysed by western blotting (A,C) or analysed for
expression of luciferase activities (B,D). KD, kinase-dead. *P<0.05, compared
with Topflash/RL-TK-transfected (i.e. no expression construct) B-13 cells
using ANOVA (two-tailed).
Journal of Cell Science
protein kinase-1 (PDK1, activated via the PI3K pathway) to
phosphorylate the T-loop (Biondi et al., 2001), which results in a
functional kinase (Pearce et al., 2010). However, when these
residues were mutated in SGK1 isoforms to alanines, the proteins
were unstable when expressed in B-13 cells and expression could
not be reliably and repeatedly confirmed (data not shown). Mutating
the lysine residue in SGK1A at residue 127 impedes binding of
ATP and also results in a kinase-dead (KD) protein (Park et al.,
1999). SGK1 isoforms were therefore generated with this residue
mutated to a methionine.
The kinase function of SGK1C and F isoforms is essential for
promoting the transdifferentiation of B-13 cells to B-13/H cells
because KD mutant SGK1C and SGK1F proteins failed to inhibit
Tcf/Lef transcriptional function or induce expression of CYP2E1
or albumin (Fig. 4C,D) in contrast to the wild-type equivalents
The PI3K pathway has an ancillary role in B-13
transdifferentiation to B-13/H cells
The PI3K pathway is activated by a range of growth factors and
has been shown to lead to the phosphorylation of SGK1 in the T
loop (at Thr256 of the wild-type protein) as part of its modification
to an active kinase (Biondi et al., 2001). To further determine
whether endogenous SGK1 phosphorylation and kinase activity is
required for B-13 transdifferentiation, cells were treated with the
PI3K inhibitor LY294002.
LY294002 treatment alone did not promote transdifferentiation
to B-13/H cells because there was no change in morphology of the
cells (Fig. 5A) or expression of hepatic markers, in contrast to
DEX treatment (Fig. 5B). LY294002 might have had a weak
glucocorticoid effect because there was a small induction of SGK1
and a less potent reduction in total -catenin levels compared with
DEX, but this did not result in any overt change in B-13 cell
differentiation. However, LY294002 treatment inhibited the
phosphorylation of SGK1 induced by DEX at both activating
positions, reduced the levels of phosphorylated -catenin and
inhibited the degree to which B-13 cells expressed liver markers
by ~50% (Fig. 5B), indicating that SGK1 phospho-activation
correlates with B-13 transdifferentiation to B-13/H cells.
SGK1C and SGK1F phosphorylate -catenin
Previous work has demonstrated that -catenin phosphorylation,
which targets the protein for degradation (Hoppler and Kavanagh,
2007), and reductions in the levels of -catenin are early upstream
Role for SGK1 in transdifferentiation
Fig. 5. Inhibition of PI3K signalling inhibits
SGK1 phosphorylation and B-13
transdifferentiation. (A)B-13 cells were
treated with or without DEX (10 nM) or
LY294002 (5M). After 14 days, cells
appeared typically as shown. (B)Cells
harvested and analysed for the indicated
protein by western blotting. CPS, carbamoyl
phosphate synthase I. Data are typical of at
least three independent experiments.
(C)Schematic diagram illustrating overlap
between the WNT and PI3K pathways.
Journal of Cell Science
events to C/EBP- induction and B-13 transdifferentiation to B-
13/H cells (Wallace et al., 2010c). It was therefore hypothesised
that expression of SGK1C and SGK1F or induction of endogenous
SGK1C resulted in phosphorylation of -catenin. Thus, B-13 cells
were transfected with SGK1C, SGK1F or SGK1A (as a control)
and the effects on -catenin examined. Expression of both SGK1C
and SGK1F resulted in phosphorylation of -catenin and reduced
levels of total -catenin, whereas the SGK1A isoform had no
effect (Fig. 6A).
To determine whether SGK1 could directly phosphorylate -
catenin (rather than activate other kinases that mediate the
phosphorylation of -catenin), purified recombinant SGK1 was
incubated with purified recombinant -catenin and phosphorylation
was examined by western blotting. -catenin phosphorylation only
occurred when SGK1 and ATP were present (Fig. 6B).
The data in this paper demonstrate that the transdifferentiation of
B-13 cells into B-13/H cells is glucocorticoid dependent, mediated
via the glucocorticoid receptor and dependent on an induction of
SGK1 expression. siRNA-mediated knockdown of SGK1 and
overexpression of SGK1 inhibited and promoted (without the
requirement for glucocorticoid) transdifferentiation, respectively,
unequivocally demonstrating a regulatory role for SGK1 in the
response. Our data also suggest that the mechanism is likely to
involve crosstalk with the WNT signalling pathway, which has
previously been shown to regulate B-13 cell transdifferentiation
(Wallace et al., 2010c), because overexpression of SGK1 inhibited
transcriptional activity of distal WNT signalling, an effect that was
absent when the SGK1 kinase function was blocked. The
transdifferentiation of pancreatic acinar cells to hepatocyte-like
410Journal of Cell Science 124 (3)
Fig. 6. SGK1 can directly phosphorylate -catenin. (A)B-13 cells
cultured in six-well plates were transfected with 2g of the indicated
plasmid construct or treated with 10 nM DEX for 14 days to generate B-
13/H cells. After 14 days, cells were harvested and analysed for the indicated
protein. (B)In vitro phosphorylation of -catenin. Western blot for the
indicated protein after incubation. Results presented are typical of three
Table 1. DNA oligonucleotide sequences employed in RT-PCR or PCR genotyping
Oligo ID5?-3? sequenceComments
57Will amplify rat NM_012576.2 nuclear receptor subfamily 3, group C,
member 1 (Nr3c1) cDNA sequence of 396 bp.
Will amplify rat NM_013131.1 nuclear receptor subfamily 3, group C,
member 2 (Nr3c2) cDNA sequence of 503 bp.
56 Will amplify rat NM_017080.2 hydroxysteroid 11- dehydrogenase 1
cDNA sequence of 209 bp.
55 Will amplify rat NM_017081.1 hydroxysteroid 11- dehydrogenase 2
cDNA sequence of 364 bp.
50 Will amplify rat (NM_017008), human (NM_002046) or mouse
(NM_008084) glyceraldehyde 3-phosphate dehydrogenase cDNA
sequence of 243bp.
60Will amplify rat (NM_019232) Sgk1a cDNA sequence of 128 bp (see
Supplementary material Fig. S2).
60Will amplify rat Sgk1b cDNA sequence of 131 bp (see Supplementary
material Fig. S2).
60 Will amplify rat Sgk1c cDNA sequence of 143 bp (see Supplementary
material Fig. S2).
55 Will amplify mouse Sgk1a (NM_011361.3) cDNA sequence of 309 bp.
55Will amplify mouse Sgk1b (NM_001161850.2) cDNA sequence of 284 bp.
55Will amplify mouse Sgk1c (NM_001161849.2) cDNA sequence of 317 bp.
aAnnealing temperature (°C) and 35 PCR cycles.
Journal of Cell Science
cells in primary culture (Shen et al., 2000; Lardon et al., 2004;
Sumitran-Holgersson et al., 2009) and in vivo (Wallace et al.,
2009; Wallace et al., 2010c) is therefore likely to be a pathological
response to stressful conditions that could be mediated, in part, by
an induction of SGK1.
Interestingly, only selective variants of SGK1 were effective
regulators of B-13 transdifferentiation. Expression of wild-type
SGK1A had no effect. This suggests that the data are unlikely to
be associated with a non-specific effect, such as the overexpression
of a kinase. The various SGK1 forms differ only in their N-
terminal sequences, suggesting that the ability to promote
transdifferentiation is dependent on a unique interaction of the N-
terminus (in SGK1C and SGK1F) with an unknown factor(s).
Because SGK1 is capable of phosphorylating the intracellular
WNT signalling mediator -catenin, it is likely that the N-terminal
sequence in these isoforms are crucial (in a cellular context) for
effective -catenin phosphorylation, perhaps by providing a direct
interaction with -catenin. There is however, no significant
similarity between the N terminal sequences of SGK1C and SGK1F
(data not shown). Despite this lack of N terminal similarity, both
proteins required an intact functional kinase function for effective
promotion of B-13 transdifferentiation to B-13/H cells.
The crucial role of SGK1 in transdifferentiation is also supported
by the requirement for PI3K signalling. This would be expected to
inhibit transdifferentiation of B-13 cells in the absence of
glucocorticoid exposure through
phosphorylation and inactivation of GSK3 kinase activity and
therefore the accumulation of -catenin (Liu et al., 2009) (see also
Fig. 5C). However, the PI3K pathway also phosphorylates and
activates SGK1 by PDK1 (Biondi et al., 2001; Alessi, 2001; Mora
et al., 2004). Addition of the PI3K inhibitor LY294002 inhibited
DEX-dependent transdifferentiation. This also suggests that induction
of SGK1 is crucial for transdifferentiation, either to overcome the
effects of the PI3K pathway on GSK3, or to generate an isoform of
SGK1 specific for this function (such as the rat SGK1C orthologue,
which is induced from undetectable levels by DEX treatment).
There might be targets of SGK1 in B-13 cells other than -
catenin that are involved in the transdifferentiation response because
many of the targets for SGK1 phosphorylation are shared with
other AGC kinase members (Lang et al., 2006). One potential
target is forkhead box O1 which is the target for SGK1 in pre-
adipocytes and is reported to regulate differentiation to fat cells (Di
Petro et al., 2010). Another target might be N-myc downstream
regulated genes (NDRGs), which are unique targets of SGK1
(Murray et al., 2004). Indeed, NDRG isoform 4A2 has recently
been implicated in rat pancreatic duct cell differentiation (Wang
and Hill, 2009).
This paper therefore shows that SGK1 is implicated in the
transdifferentiation of B-13 cells into B-13/H hepatocyte-like cells
and adds knowledge to the changes that occur in cells under
pathophysiological conditions. These data also give an insight into
potential ways to manipulate cell differentiation that might be of
particular use in the drive to generate hepatocytes in vitro for a wide
range of uses, from toxicological screening to bioartificial livers.
Materials and Methods
Cell isolation and culture
B-13 cells were routinely cultured in Dulbecco’s modified Eagle Medium (DMEM)
supplemented with 10% (v/v) fetal calf serum, 80 g/ml penicillin and 80 g/ml
streptomycin as previously outlined and under which conditions the cells remained
proliferative and phenotypically stable (Marek et al., 2003; Wallace et al., 2010c).
Treatment with 10 nM DEX results in the transdifferentiation to hepatocyte-like cells
which is maximal (75–85% of all cells) after 14 days, whereupon these cells are
referred to as B-13/H cells/cultures (Wallace et al., 2010c). Rat hepatocytes were
isolated from adult male 250–300 g body weight Sprague–Dawley rats by collagenase
perfusion (Wright et al., 2001) with additional tissues harvested and snap frozen
Tg(Crh) transgenic mice overexpressing a rat corticotrophin releasing factor transgene
that results in chronic elevated circulating glucocorticoid and Cushing’s disease
(Stenzel-Poore et al., 1992; Wallace et al., 2010b) were obtained from the Jackson
Role for SGK1 in transdifferentiation
Table 2. Plasmid DNA constructs employed
Construct Vector Details
Topflash Topflash-luciferase Construct containing two sets (with the second set in the reverse orientation) of three copies of the TCF-binding
site upstream of a thymidine kinase promoter and luciferase open reading frame.
Fopflash Fopflash-luciferase An identical vector to Topflash, but with mutated TCF response elements.
Renilla pRL-TK Construct containing a renilla gene under control of a thymidine kinase minimal promoter, purchased from
Promega (Southampton, UK).
hSGK1A pFLAG-CMV2 Wild-type SGK1. Construct encodes full-length 2–431 amino acid protein.
hSGK1C pFLAG-CMV2 Altered N terminus to wild type. Construct encodes full-length 2–459 amino acid protein.
hSGK1D pFLAG-CMV2 Altered N terminus to wild type. Construct encodes full-length 2–526 amino acid protein.
hSGK1F pFLAG-CMV2 Altered N terminus to wild type. Construct encodes full-length 2–421 amino acid protein.
hSGK1A KD mutant pFLAG-CMV2 SGK1A-K127M. SGK1A sequence mutated at residue 127. Protein with no kinase activity (Park et al., 1999).
hSGK1C KD mutant pFLAG-CMV2 SGK1C-K155M. SGK1C sequence mutated at residue 155, equivalent to residue at 127 in SGK1A and therefore
predicted to have no kinase activity.
hSGK1D KD mutant pFLAG-CMV2 SGK1D-K222M. SGK1D sequence mutated at residue 222, equivalent to residue at 127 in SGK1A and
therefore predicted to have no kinase activity.
hSGK1F KD mutant pFLAG-CMV2 SGK1F-K117M. SGK1F sequence mutated at residue 117, equivalent to residue at 127 in SGK1A and therefore
predicted to have no kinase activity.
Four alternatively transcribed human SGK1 transcripts are currently described on the NCBI database. Isoform 1 (NM_005627.3) is the wild-type form SGK1A
and three other forms were identified by Simon et al., (Simon et al., 2007): isoform 2 (NM_001143676.1) is SGK1D; isoform 3 (NM_001143677.1) is SGK1C;
isoform 4 (NM_001143678.1) is SGK1B. The SGK1F form sequence has been directly submitted to the NCBI only: account number CAI19718. pFLAG-CMV2
was purchased from Sigma (Poole, UK) – all constructs using this vector encode SGK1 protein fused to an N-terminal flag sequence underlined
Journal of Cell Science
Laboratory (Bar Harbor, ME). Female adult mice were used in all studies (since only
males could be used for successful mating with wild type females). Mice showed
overt clinical signs of Cushing’s syndrome within 15 weeks of age (e.g. hair loss,
obesity, thinning skin). Animals were killed at 21 weeks of age by cervical dislocation
and tissues removed for analyses. Littermate wild-type females were used as controls.
Total RNA was purified from cells using Trizol (Invitrogen, Paisley, UK) and RT-
PCR performed as previously outlined (Haughton et al., 2006). Primer sequences are
given in Table 1.
Western blotting was performed essentially as previously outlined (Marek et al.,
2005) with the following antibodies: rabbit anti-human SGK1 (#S5188, raised to a
synthetic peptide corresponding to the C-terminus of human SGK1A, amino acids
412–431) purchased from Sigma (Poole, UK); anti-SGK1-P422 (sc16745-R) obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies have
previously been detailed (Wallace et al., 2010c). Detection was achieved using
appropriate horseradish-peroxidase-conjugated anti-IgG secondary antibodies and
chemiluminescence (ECL, GE Healthcare, Amersham, UK).
Cells were transfected with siRNAs purchased from Qiagen (Southampton, UK)
using Effectene as previously described (Wallace et al., 2010c) using siRNA designed
to knock down rat C/EBP- (Rn-Cebpb-4/SI01498028); rat SGK1 (Rn-sgk1-
6/SI04717524) or rat low-affinity glucocorticoid binding site (LAGS) also referred
to as progesterone receptor membrane component 1 (Rn-Pgrmc1-2/SI02900023) a
gene that encodes a glucocorticoid and progesterone binding protein of unknown
function (Marek et al., 2009) and used as a control.
Plasmid DNA transfection
Cells were transfected with a range of plasmid DNA constructs (see Table 2) using
Effectene (Qiagen, Southampton, UK) as outlined by the manufacturers. In all cases,
cells were co-transfected with a Renilla expression vector (RL-TK) obtained from
Promega (Southampton, UK) at a ratio of 6:1 (TK plasmid: RL-TK plasmid).
Luciferase and Renilla activities were determined using the Dual-Luc kit (Promega,
Southampton, UK) and a luminometer.
In vitro phosphorylation of -catenin
5 ng purified (?NT60) N-terminal GST-tagged human SGK1 (Stressgen, Victoria,
Canada) was added to 5 g recombinant N-terminal GST-tagged full-length human
-catenin (SignalChem, Richmond, Canada) in a final volume of 30 l containing
6.7 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer, 1.7 mM EGTA,
0.67 mM EDTA, 4.2 mM sodium -glycerophosphate, 6.7 mM MgCl2, 83 M
dithiothreitol and 1.7 M -methyl aspartic acid, pH 7.2. When required, ATP was
added at a final concentration of 50 M. Reactions were initiated by the addition of
-catenin and incubated for 60 minutes at 30°C before heating at 95°C in SDS-
PAGE loading buffer to denature all components before being subjected to SDS-
PAGE and western blotting analysis.
One-way ANOVA was used to test for statistical significance.
Supported by a grant from Newcastle University. K.W. was
supported by a BBSRC PhD Studentship award. E.A.F. is currently a
recipient of an MRC ITTP Studentship. The technical assistance of
Trevor Booth with confocal microscopy is gratefully acknowledged.
The expertise and assistance of Trevor R. Jackson is acknowledged.
Deposited in PMC for release after 6 months.
Supplementary material available online at
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Role for SGK1 in transdifferentiation
Journal of Cell Science