Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin.

7.6 MB·English

by  Ortells, M. Carmen

4368–4384 Nucleic Acids Research, 2012, Vol. 40, No. 10 Published online 28 January 2012
doi:10.1093/nar/gks038
Transcriptional regulation of gene expression
during osmotic stress responses by the
mammalian target of rapamycin
M. Carmen Ortells1, Beatriz Morancho1, Katherine Drews-Elger1, Benoit Viollet2,3,4,
Keith R. Laderoute5, Cristina Lo´pez-Rodrı´guez1 and Jose Aramburu1,*
1Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain,
2INSERM, U1016, Institut Cochin, 75014 Paris, 3CNRS, UMR8104, 75014 Paris, 4Universite´ Paris Descartes,
75014 Paris, France and 5SRI International, Menlo Park, CA 94025, USA
Received April 18, 2011; Revised and Accepted January 10, 2012
ABSTRACT INTRODUCTION
Although stress can suppress growth and prolifer- The mammalian target of rapamycin (mTOR) Ser/Thr
ation, cells can induce adaptive responses that kinase belongs to the family of PI3-kinase-related
kinases (PIKK) and is a central regulator of cell growth
allow them to maintain these functions under
and proliferation due to its ability to activate the biosyn-
stress. While numerous studies have focused on
thesis of proteins, nucleic acids and lipids in response to
the inhibitory effects of stress on cell growth, less
growth-promoting signals (1). The pool of mTOR mol-
is known onhow growth-promoting pathways influ-
ecules is distributed into two main protein complexes
ence stress responses. We have approached this
with distinct functions, mTORC1 and mTORC2, which
question by analyzing the effect of mammalian are defined by specific accessory proteins, such as raptor
target of rapamycin (mTOR), a central growth con- in mTORC1 (2,3), and rictor and Sin1 in mTORC2 (4,5).
troller, on the osmotic stress response. Our results mTORC1 is activated by growth factors, nutrients and
showedthatmammalian cellsexposed tomoderate energy. The mTORC1 complex has substantial influence
hypertonicity maintained active mTOR, which was in growth and proliferation due to its ability to regulate
required to sustain their cell size and proliferative the translation of diverse proteins involved in ribosome
biogenesis and cell cycle control (1), by activating the
capacity. Moreover, mTOR regulated the induction
ribosomal S6 subunit kinases (S6K) 1 and 2 (6), and
of diverse osmostress response genes, including
inactivating the translation repressor 4E-BP1 (7).
targets of the tonicity-responsive transcription
mTORC1 also influences, by direct and indirect mechan-
factor NFAT5 as well as NFAT5-independent
isms, the expression of genes involved in the control of
genes. Genes sensitive to mTOR-included regula-
metabolism, ribosomal biogenesis, growth and prolifer-
tors of stress responses, growth and proliferation. ation (8–12). mTORC2 was first described as a regulator
Among them, we identified REDD1 and REDD2, of the actin cytoskeleton (4,13), but also regulates cell
which had been previously characterized as mTOR growth, differentiation and proliferation (14), at least in
inhibitorsinotherstresscontexts.Weobservedthat part by enhancing the activity of mTORC1 via activation
mTOR facilitated transcription-permissive condi- and stabilization of Akt (15,16). mTORC1and mTORC2
tions for several osmoresponsive genes by differintheirsensitivitytorapamycin,abacterialproduct
thatbindstotheintracellularchaperoneFKBP12toform
enhancing histone H4 acetylation and the recruit-
a complex capable of binding with high affinity and spe-
ment of RNA polymerase II. Altogether, these
cificity to the FRB domain of mTOR. This domain is ac-
results reveal a previously unappreciated role of
cessible to the rapamycin-FKBP12 complex when mTOR
mTOR in regulating transcriptional mechanisms
isaloneorforming partofmTORC1. Rapamycinrapidly
that control gene expression during cellular stress
suppresses the activity of mTORC1 towards many,
responses. although not all, of its substrates (17,18), but does not
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
Present addresses:
Beatriz Morancho, Vall d’Hebron Institute of Oncology, Vall d’Hebron University Hospital, 08035 Barcelona, Spain.
Katherine Drews-Elger, Braman Family Breast Cancer Institute, University of Miami Sylvester Comprehensive Cancer Center, Miami,
FL 33136, USA.
(cid:2)TheAuthor(s)2012.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
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NucleicAcidsResearch,2012,Vol.40,No.10 4369
generally inhibit mTORC2 (19). However, it prevents the and can maintain their proliferative capacity under pro-
formation of new mTORC2 complexes, which in the long longed hypertonic conditions (40,44–48). Mammalian
term can lead to the inhibition of mTORC2-dependent cells react to osmostress by activating the transcription
functions (19). factor NFAT5/TonEBP, which contains a Rel-like
Besides its responsiveness to growth regulatory condi- DNA-binding domain homologous to those of NF-kB
tions, mTOR is also sensitive to diverse stressors, such as and the calcineurin-dependent NFATc proteins (49–51).
hypoxia, DNA damage, oxidative stress and osmotic NFAT5 controls the expression of numerous osmo-
stress (20). DNA damage induces the p53-dependent ex- protective gene products, including chaperones of the
pression ofSestrins 1 and 2 (21), and hypoxia induces the Hsp family (52,53), enzymes involved in osmolyte synthe-
HIF-1a-mediated expression of REDD1 and REDD2 sis, and osmolyte transporters (49,54–56), and regulates
(22), all of which can activate TSC2 (20,21). TSC2 has the adaptation to hypertonicity in cell types as diverse as
GTPase activity and inhibits mTORC1 by converting its renal medullary cells (46), embryonic fibroblasts (46,47),
activator Rheb-GTP to Rheb-GDP (23). Energy stress in neurons (39) or lymphocytes (40,44,47). NFAT5 defi-
general causes the increase in the intracellular [AMP]/ ciency in the mouse causes a severe renal dysfunction
[ATP] ratio and engages the AMP-activated kinase (46) and T-cell immunodeficiency associated with the in-
AMPK, which phosphorylates and activates TSC2 (24). ability of NFAT5-deficient cells to adapt to hypertonic
AMPK can also inhibit mTORC1 directly by stress conditions in vivo (40,47).
phosphorylating raptor (25). The decrease in mTOR With regard to mTOR and osmostress, it had been
activity in cells exposed to stress serves to slow down or described that elevated hypertonicity (600–900mOsm/kg)
arrestgrowthandproliferation-promotingprocesseswhen inactivated the mTOR pathway (57–59), although some
cells face conditions that can damage critical components had reported a transient stimulation of S6K1 activity by
suchasDNA.TheimportanceofinhibitingmTORduring hypertonic shock (60). A possible mechanism for mTOR
stress is illustrated by the finding that TSC2(cid:2)/(cid:2) cells inhibition during osmostress might involve AMPK, since
cannotshutdownmTORwhenexposedtoglucosedepriv- this kinase can be activated by elevated hypertonicity
ation or DNA damage and exhibit mTOR-dependent ac- (61,62). However, intense hypertonic stress might not ac-
cumulation of p53 and cell death (26,27). The capacity of curately reflect conditions in which cells would mount an
mTORtoinfluencestressresponsesisalsoevidencedbyits adaptive response, since acute elevation of tonicity
ability to increase the translation and activity of the >600mOsm/kg can induce cell death in hours (45), and
hypoxia-inducible factors HIF-1a and HIF-2a (28,29). impair the osmoprotective function of NFAT5 (47). On
These observations raise the question of whether growth the other hand, we and others had observed that the
signaling pathways are merely less active under stress or activity of NFAT5 in response to more moderate
may also regulate the quality and magnitude of stress re- osmostress (500mOsm/kg) was enhanced in cells with
sponses.Wehaveaddressedthisquestionbyanalyzingthe active PI3-kinase (63) or stimulated with mitogens (64),
effect of mTOR in the response of mammalian cells to which suggests that growth-promoting signals might
hypertonic stress. haveapositive effectonsurvival responsestoosmostress.
The effects of hypertonic stress on mammalian cells In this regard, earlier work had shown that at least in
depend on its intensity and duration. In mammals, some organisms, such as yeast, TOR promoted cell
normal tonicity in most tissues is (cid:3)300mOsm/kg, corres- survival to salt stress (65,66).
ponding to a concentration of extracellular sodium plus Inviewoftheseobservations,weaskedwhethermTOR
potassium ions of (cid:3)150mM. An exception is the renal was active in mammalian cells exposed to moderate
medulla, where local tonicity can reach 600–1700mOsm/ osmostress conditions, and if so, whether it contributed
kg. However, this elevated tonicity is not entirely caused to the induction of an adaptive gene expression
by sodium ions, but is in part contributed by a high con- program. We report that hypertonicity conditions of
centration of urea, which actually helps renal medullary 500mOsm/kg do not inactivate mTORC1 nor mTORC2
cells to tolerate hypertonicity (30). Hypertonicity levels in mouse embryo fibroblasts (MEFs). Moreover, mTOR
of (cid:3)400mOsm/kg and higher can also occur in other activity was required for cell growth and proliferation
sites, such as the skin and respiratory and gastrointes- under osmostress. By using pharmacological mTOR in-
tinal mucosa, as result of hypernatremia caused by hibitors and mRNA microarray analysis of MEFs,
dietary and ambient conditions (31–33). In addition, we found that mTOR regulated the expression of
systemic hypernatremia with plasma tonicities of osmostress-sensitive genes, which comprised regulators
360–430mOsm/kg, which would affect multiple tissues, of stress responses, cell survival and proliferation, and
can result from severe dehydration and osmoregulatory included NFAT5-dependent and -independent targets, re-
disorders(34–40).Studieshaveshownthatelevatedhyper- vealing an influence of mTOR on genes with different
tonicity (>600mOsm/kg) can cause double-strand DNA transcriptional requirements. Among them, we identified
breaks (41) and induce a genotoxic stress response (30), REDD1, previously characterized as an mTOR inhibitor
with activation of ATM and the checkpoint effectors p53 in other stress contexts. Suppression of REDD1 affected
and Chk2 (42,43). However, cells exposed to moderate theexpressionofvariousgenesinducedbyosmoticstress,
osmostress conditions of 400–500mOsm/kg, which are suggesting a regulatory role in the osmostress response.
more likely to reflect physiopathological settings, exhibit Our results also showed that mTOR enhanced the acetyl-
only a transient genotoxic stress-like response and revers- ation of histone H4 at the promoters and transcribed
ible cell cycle arrest without evident DNA damage (44), regions of osmostress-induced genes, and the recruitment
4370 NucleicAcidsResearch,2012,Vol.40,No.10
ofRNApolymeraseII,whichsuggeststhatitcanregulate (200mM final concentration) was used to induce
the transcription of stress response genes by influencing osmotic stress.
their chromatin configuration and RNA polymerase
function. These results reveal a previously unappreciated Real-time quantitative PCR (RT–qPCR)
role of mTOR in regulating transcriptional mechanisms
Total RNA was isolated using the High Pure RNA isola-
that control gene expression during cellular stress
tion kit (Cat. 11828665001, Roche) following manufac-
responses.
turer’s instructions. 1–2mg of total RNA were
retrotranscribed to cDNA using SuperScript III reverse
transcriptase and random primers (Invitrogen). For
MATERIALS AND METHODS
real-time quantitative PCR, LightCycler 480 SYBR
Cells Green I Master Mix (Cat. 11608521, Roche) and a
LightCycler480 system apparatus (Roche) were used fol-
The human embryonic kidney cell line HEK293 was
lowing the instructions provided by the manufacturers.
obtained from the American Type Culture Collection
Samples were normalized to L32 mRNA levels using the
(ATCC). HEK293T cells were kindly provided by Josep
LightCycler 480 SW 1.5 software (Roche).
Lluı´s Parra (Vall d’Hebron Institute of Oncology).
AMPKa1/a2 double knockout MEFs were kindly
Microarray experiments and analysis
provided by Benoit Viollet (INSERM, Institut Cochin,
CNRS and Universite´ Paris Descartes) and Tomi NIH3T3-immortalized wild-type and NFAT5-deficient
Makela (Institute of Biotechnology, University of MEFs (46) from passages 30–35 were plated (175000
Helsinki). Briefly, MEFs were obtained from 12.5-day cells in 60-mm dishes) in isotonic medium and 2 days
mouse embryos from Prkaa1(AMPKa1)(cid:2)/(cid:2), Prkaa2 later were either left untreated or treated for 8h with
(AMPKa2)-floxed mice (AMPKa1(cid:2)/(cid:2), AMPKa2fl/fl) of 500mOsm/kg. When indicated, the mTOR catalytic in-
mixed background (Bl6/CD1) (67). Deletion of hibitor Torin1 was added 1h before hypertonicity
AMPKa2 was done by infecting the cells in vitro with (500mOsm/kg) treatment. Cells were lysed in RLT
Adeno-Cre, and cells were immortalized at passage 2 buffer (300ml, RNeasy system, QIAGEN Cat. 74104)
with a carboxy-terminal fragmentof p53 (68,69). Wild- and total RNA was isolated using the manufacturer
type-AMPK MEFs were immortalized by the same pro- protocol. The RNA integrity was assessed using Agilent
cedure. Wild-type and NFAT5-deficient MEFs were 2100 Bioanalyzer (Agilent), and only samples with high
prepared from 13.5-day littermate embryos (129sv back- integrity[RNAintegritynumber(RIN)>7.5]weresubse-
ground) using the NIH3T3 protocol to obtain spontan- quently used in microarray experiments. Amplification,
eously immortalized cells (46). HEK293 cells and MEFs labeling and hybridizations were performed according to
were maintained in Dulbecco’s modified Eagle’s Medium protocolsfromAmbionandAffymetrix.Briefly,250ngof
(DMEM) (Gibco) supplemented with 10% heat- total RNA were amplified using the Ambion(cid:3) WT
inactivated fetal bovine serum, 4mM L-glutamine Expression Kit (Ambion/Applied Biosystems), labeled
(Gibco), 1mM sodium pyruvate (Gibco) and 50mM using the WT Terminal Labeling Kit (Affymetrix Inc),
b-mercaptoethanol (Gibco). Splenocytes from 8- to and then hybridized to Mouse Gene 1.0 ST Array
12-weeks-old wild-type and Nfat5(cid:2)/(cid:2) mice (46) were (Affymetrix) in a GeneChip(cid:3) Hybridization Oven 640.
isolated by density gradient sedimentation with Washing and scanning were performed using the
Hybridization Wash and Stain Kit and the GeneChip(cid:3)
Lymphoprep (Axis-Shield PoC AS) and stimulated with
System of Affymetrix (GeneChip(cid:3) Fluidics Station 450
2.5mg/ml concanavalin A (Sigma-Aldrich) plus 25ng/ml
and GeneChip(cid:3) Scanner 3000 7G). Three independent
ofIL-2(Chiron)during24hinculturemediumcontaining
microarray hybridizations were performed for each ex-
DMEM supplemented with 10% heat-inactivated fetal
perimental condition: cells maintained at 300mOsm/kg
bovine serum, 2mM L-glutamine, 1mM sodium
or exposed to 500mOsm/kg (8h) without or with
pyruvate, 50mM b-mercaptoethanol, penicillin–strepto-
100nM Torin1 in the case of wild-type MEFs, or
mycin and non-essential amino acids (Gibco) and then
300mOsm/kg and 500mOsm/kg for Nfat5(cid:2)/(cid:2) MEFs.
cultured under isotonic or hypertonic conditions as
Microarray data analysis was performed as follows: after
indicated in the figure legend.
quality control of raw data, it was background-corrected,
quantile-normalizedandsummarizedtoagenelevelusing
Hypertonic stress
the robust multi-chip average (RMA) (70) obtaining a
The osmolarity of the culture medium was measured in a total of 28853 transcript clusters, excluding controls,
VAPRO 5520 vapor pressure osmometer (Wescor). which roughly correspond to genes. Core annotations
Culture medium with supplements had an osmolality of (version NetAffx 30, human genome 18) were used to
330mOsm/kg, and was adjusted to 300mOsm/kg by summarize data into transcript clusters. Linear Models
adding 10% sterile H O (Milli-Q Biocel A10, Millipore). for Microarray (LIMMA) (71), a moderated t-statistics
2
Media were made hypertonic by adding NaCl from a model, was used for detecting differentially expressed
sterile 4M stock solution in water. Over an isotonic genes between the conditions in study. Correction for
baseline of 300mOsm/kg, addition of 50mM NaCl multiplecomparisonswasperformedusingfalsediscovery
raised the osmolarity to 400mOsm/kg and 100mM rate. Genes with an adjusted P-value < 0.05 or with a
NaCl to 500mOsm/kg. In some experiments, sorbitol P<0.01 for those comparisons with few results after
NucleicAcidsResearch,2012,Vol.40,No.10 4371
adjusting P-values were selected as significant. eightcyclesof30sonand30soffonhighpowerforcells
Hierarchical cluster analysis was also performed to stimulated for (cid:6)4h and six cycles for cells stimulated 8h,
analyze how data aggregated and linear model for regres- to obtain DNA fragments between 500 and 1000bp, and
sion purposes. All data analysis was performed in R centrifuged 10min at 18000(cid:5)g to remove insoluble
(version 2.11.1) with packages aroma.affymetrix, debris. Supernatants were collected and 10% of each
Biobase, Affymetrix, LIMMA and genefilter. The micro- sample was separated to use as a measure of chromatin
array data have been deposited in NCBI’s Gene input for normalization. The rest of the sample was
Expression Omnibus and are accessible through GEO diluted 10 times in ChIP dilution buffer (16.7mM Tris–
Series accession number GSE27485.
HCl pH 8.1, 0.01% SDS, 1.1% Triton X-100, 1.2mM
EDTA, 167mM NaCl and protease inhibitor cocktail
Cell viability, cell-size determination and
set III, EDTA free) for immunoprecipitation. Samples
proliferation assays
were precleared with protein A-sepharose beads
Flowcytometry wasdonewithaBDLSRflowcytometer (Amersham, cat. 17-0780-01) that had been pre-adsorbed
(BD Biosciences). For viability and cell-cycle analysis, with 57mg/ml salmon sperm DNA (Roche, cat.
cells were labeled with 5mg/ml of the DNA dye Hoechst 11467140001) and 0.1mg/ml of bovine serum albumin
33342 (SIGMA) in suspension in cytometry tubes for 1h (BSA) (New England Biolabs) by rocking for 1h at 4(cid:4)C.
at 37(cid:4)C in a water bath. Viability was determined by After removing the preclearing beads, the specific
forward and side scatter parameters (FSC/SSC) in the antibodies were added to the lysates and incubated over-
total population of cells. Non-viable cells were readily night at 4(cid:4)C in rotation. Protein A-Sepharose beads
identified by their distinct position in the FSC/SSC
pre-adsorbed with 60mg/ml salmon sperm DNA and
plots.Cellsize(FSCparameter)wasanalyzedinthepopu-
0.1mg/ml of BSA were then added, incubated for 3–4h at
lation of cells in G1 phase, after staining with the DNA 4(cid:4)C, and then washed once with low salt wash buffer
dye Hoechst 33342. Cell proliferation was analyzed
(20mM Tris–HCl pH 8.1, 0.1% SDS, 1% Triton X-100,
in CFDA-SE-labeling experiments. Briefly, MEFs were
2mM EDTA, 150mM NaCl), once with high salt wash
labeled with 5mM carboxyfluorescein diacetate
buffer (20mM Tris–HCl pH 8.1, 0.1% SDS, 1% Triton
succinimidyl ester (CFDA-SE, Molecular Probes,
X-100, 2mM EDTA, 500mM NaCl), once with LiCl
Invitrogen) at Day 0 and then analyzed at 48 and 72h
immune complex wash buffer (10mM Tris–HCl pH 8.1,
after labeling. The decrease in CFDA-SE fluorescence in-
250mMLiCl,1%NP40,1%sodiumdeoxycholate,1mM
tensity in cells, which was proportional to the number of
EDTA) and twice with 1(cid:5) TE (10mM Tris–HCl pH 8.1
celldivisions,wasanalyzedbytwo-colorflowcytometryin
and 1mM EDTA). To elute the DNA, beads were
the population of live cells, identified by staining with
incubated with 200ml elution buffer (1% SDS and
Hoechst 33342 and excluding cells with a sub-G0/G1
DNA content. 100mM NaHCO3) for 20min at room temperature with
shaking. To reverse the crosslinking, samples were
RNA interference assays incubated overnight at 65(cid:4)C with 1mg RNase per sample
(Roche, cat. 11119915001) and 200mM NaCl final
ON-TARGETplus SMARTpool small interfering RNA
concentration. DNA was purified using the QIAGEN
(siRNA) pools were purchased from Dharmacon:
PCR purification system (Cat. 28104). DNA was then
nontargeting scramble (D-001810-10), mouse Ddit4/
subjected to real time quantitative PCR (RT–qPCR)
REDD1 (L-056656-01) and mouse Ddit4l/REDD2
with the primers described in Supplementary Methods.
(L-056952-00). siRNA (40nM) was transfected using
Immunoprecipitated DNA from each sample was
Lipofectamine 2000 (Invitrogen) in a six-well plate
normalized to its respective chromatin input.
format following the manufacturer’s protocol.
Chromatin immunoprecipitation
Statistical analysis
Two days before stimulation 0.4(cid:5)106 MEFs (wild-type-
The statistical significance of the experimental data was
AMPK) were plated in 10-cm diameter dishes and 1 day
determined with paired Student’s t-test.
before applying the stimulus the medium was replaced by
fresh isotonic one. Cells were treated during the indicated
timeswith500mOsm/kgwithorwithoutTorin1(100nM)
Reagents, antibodies and primers, western blots, luciferase
pretreatment. Cells were fixed with 1% formaldehyde for
reporter assays and fluorescence microscopy
10min at room temperature and with continuous agita-
tion.Formaldehydewasthenquenchedwithglycine(final The detailed list of reagents (AICAR, rapamycin, Torin1
concentration of 125mM) for 5min. After washing the and general chemicals), antibodies (western blot, chroma-
plates twice with cold PBS and once with cold PBS + tin immunoprecipitation and immunofluorescence) and
PMSF, cells were lysed in SDS lysis buffer [50mM Tris– primers used for mRNA analysis and chromatin
HCl pH 8.1, 1% SDS, 10mM EDTA and protease in- immunoprecipitation is provided in Supplementary
hibitor cocktail set III, EDTA-free (Cat. 539134, ‘MaterialsandMethods’section.Likewise,thedescription
Calbiochem)] for 5–10min and then stored at (cid:2)80(cid:4)C for ofwesternblotting, luciferase reporterassaysand fluores-
at least 24h. Lysates were sonicated in 1.5ml polypropyl- cence microscopy experiments are also included in
enetubeswithabathsonicator(DiagenodeBioruptor)for Supplementary ‘Materials and Methods’ section.
4372 NucleicAcidsResearch,2012,Vol.40,No.10
RESULTS dephosphorylation of the mTORC1 substrate 4E-BP1
(Figure 1A), whereas rapamycin had a partial effect, in
mTOR remains active under sustained osmotic stress and
agreement with previous publications (17,18).
regulates the induction of osmostress response genes
Since it had been reported that AMPK can be transi-
AsafirstapproximationtoanalyzewhethermTORcould ently activated by intense osmotic stress conditions
influence the osmotic stress response in mammalian cells, (>600mOsm/kg) (61,62), we tested whether the lower
we first tested whether this pathway was active during osmostress levels used in our assays activated AMPK.
hypertonic stress conditions of 500mOsm/kg, that were Our results showed that hypertonic conditions of
strong enough to induce a robust osmoadaptive gene ex- 500mOsm/kg had minimal or no effect on the
pression response but not so high that they would LKB1-mediated phosphorylation of AMPKa in T172, in
suppress cell growth and proliferative capacity. Analysis contrast with the strong phosphorylation induced by the
of diagnostic substrates downstream mTORC1 AMPK activator AICAR (Supplementary Figure S3A).
(phosphorylated-S235/236 in the ribosomal subunit S6, Experiments using AMPKa1/a2-double knockout MEFs
and phosphorylation-dependent electrophoretic mobility (Supplementary Figure S3B) showed that wild-type and
shift of 4E-BP1) and mTORC2 (phosphorylated S473 of AMPKa-null cells exhibited similar inducibility and
Akt) showed that although osmostress caused a partial rapamycin sensitivity of osmostress response genes
inhibition of both mTOR complexes in MEFs, they (Supplementary Figure S3C), indicating that AMPK
retained substantial activity under hypertonic stress does not have a strong effect on mTOR activity and ex-
(Figure 1A and Supplementary Figure S1A). The induc- pression of osmoregulatory genes under moderate
tionofNFAT5observedinthesameexperimentservedas osmostress conditions.
an indicator of the ongoing osmostress response. This ex-
perimentalsoshowedthatrapamycinspecificallyinhibited
Identification of mTOR-regulated genes in the osmotic
mTORC1, but not mTORC2, in MEFs exposed to
stress response
osmotic stress, whereas both mTOR complexes were effi-
ciently inhibited by Torin1, an ATP-competitive mTOR The results above showed that mTOR was active in
inhibitor whose mechanism of action is unrelated to that proliferatingcellsexposedtophysiopathologicosmostress
ofrapamycin(18)(Figure1A).Wethentestedtheeffectof conditions and contributed to enhance the expression of
rapamycin and Torin1 on the expression of the endogen- several osmoprotective genes. In order to get a broader
ous mRNAs of several osmoresponsive genes in response picture of genes whose expression was regulated by
to hypertonic stress (500mOsm/kg) induced by either osmostress and sensitive to mTOR, we used RNA micro-
hypernatremia or sorbitol (Figure 1A). Comparison of array analysis in MEFs. RNA from three independently
both inhibitors on the induction of Akr1b3 (aldose reduc- performed experiments was analyzed with the Affymetrix
tase), Aqp1 (aquaporin 1), Hspa1b (Hsp70.1) and Slc5a3 Mouse Gene 1.0 ST array. The experiments included
(sodium/myoinositol cotransporter, SMIT) showed that a parallel analysis of osmoresponsive genes in
Torin1 was moderately stronger than rapamycin NFAT5-deficient MEFs to identify NFAT5-dependent
(Figure 1B). Relative mRNA levels for the osmostress- genes. Cells were exposed to hypertonic stress for 8h,
induced genes were obtained after normalization to L32 since our previous experiments (Figure 1C) had shown
mRNA, which we had previously compared with another that this time point was sufficient for robust induction
usual normalization control, Gapdh, and confirmed as in- of osmotic stress response genes, but not so long that
sensitive to mTOR inhibitors (Supplementary Figure cells had fully adjusted to a hypertonic environment (48)
S1B). Experiments also showed that rapamycin and nor to the prolonged inhibition of mTOR (12). For these
Torin1 caused a stronger inhibition of gene expression at assays, we used Torin1 instead of rapamycin because it
latertimepoints(Figure1C),inparallelwiththeprogres- had a stronger effect in downregulating osmostress
sive inactivation of mTOR (Figure 1A). Analysis of inde- response genes and we considered that it would allow a
pendent cell types yielded similar results, as we observed clearer identification of mTOR-regulated genes when
thatinductionofHspa1b,Slc5a3andSlc6a6(sodiumand comparing independent samples in the microarray
chloride-dependenttaurinetransporter,TauT)byosmotic analysis. The results revealed that Torin1 significantly
stress (400mOsm/kg) in mitogen-activated splenocytes affected the expression of numerous genes in cells
was also sensitive to rapamycin (Supplementary Figure exposed to osmotic stress (Figure 2A). We identified 107
S2A), and that rapamycin caused a significant inhibition genesthatwereinducedbyosmoticstress(cid:7)2timeswitha
of the osmostress-induced, NFAT5-activated ORE-Luc P<0.01, of which 24 (22%) were repressed by Torin1 by
reporter activity in the human embryonic kidney cell line atleast35%(SupplementaryTableS1).Thisanalysisalso
HEK293 and MEFs (Supplementary Figure S2B). The showedthatanother74geneswererepressedby(cid:7)50%by
finding that mTORC2 was active in rapamycin-treated osmotic stress, and the inhibition of eight of them (11%)
MEFs exposed to osmostress indicated that the was attenuated by Torin1 (Supplementary Table S2). We
downregulation of gene expression caused by rapamycin detected few mRNAs that were consistently inhibited by
wasduetotheinhibitionofmTORC1,andsuggestedthat Torin1 at 8h, and none upregulated, in non-stressed cells
the greater potency of Torin1 likely reflected its ability to (Supplementary Table S3). This modest effect likely re-
inhibit mTORC1-dependent functions better than flected that inhibition of mTOR for the last 9h in cells
rapamycin (18). This interpretation was also supported that until then had been growing actively had a limited
by the observation that Torin1 caused a complete impact on the representation of preexisting mRNAs.
NucleicAcidsResearch,2012,Vol.40,No.10 4373
Figure 1. Effect of rapamycin and Torin1 on the induction of osmostress response genes. (A) MEFs immortalized with a p53-carboxy-terminal
fragment (immortalized MEFs) were cultured in isotonic (300mOsm/kg) or hypertonic medium (500mOsm/kg, upon addition of 100mM NaCl)
withoutorwithrapamycin(50nM)orTorin1(100nM).NFAT5,phospho-S6(Ser235/236),S6,phospho-AKT(Ser473),AKT,4E-BP1andb-actin
(loading control) were detected by Western blotting. One representative experiment is shown (other experiments are shown in Supplementary
Figure S1). (B) RNA was isolated from immortalized MEFs cultured in isotonic (300mOsm/kg) medium or medium made hypertonic
(500mOsm/kg) by addition of 100mM NaCl or 200mM sorbitol, during 8h without or with 50nM rapamycin (Ra) or 100nM Torin1 (T1).
mRNA abundance for Akr1b3, Aqp1, Hspa1b and Slc5a3 normalized to L32 mRNA is represented relative to hypertonic conditions (100%).
Bars represent the mean±SEM of five independent experiments (*P<0.05). (C) RNA was isolated from immortalized MEFs cultured in
isotonic (300mOsm/kg) or hypertonic medium (500mOsm/kg, upon addition of 100mM NaCl) without or with rapamycin (50nM) or Torin1
(100nM).mRNAabundanceforAkr1b3andSlc5a3normalizedtoL32mRNAisrepresentedrelativetothe8-htimepoint(100%).Barsrepresent
the mean±SEM of four independent experiments (*P<0.05).
However, the combination of osmotic stress and Torin1 of them. Therefore, although a majority of the genes
downregulated the expression of 12 genes, and increased strongly induced by osmostress were NFAT5-regulated,
theexpression of2,thatwere notsignificantly affectedby there was no association between the dependence on
either osmotic stress or Torin1 alone (Supplementary NFAT5 and sensitivity to mTOR inhibition.
Table S4). These results indicated that mTOR regulated WevalidatedbyRT–qPCRasampleof14geneschosen
the induction and repression of a selective pattern of bytheirrobustinductionbyosmostress inthemicroarray
osmostress responsive genes. Comparison of wild-type experiments, and also illustrative of Torin1-sensitive and
and NFAT5-deficient MEFs showed that 74% (79 of -insensitive, as well as NFAT5-dependent and -independ-
107) of the genes induced by osmostress were regulated ent genes (Figure 2B). This analysis confirmed the
by NFAT5, of which 17 (22%) were Torin1-sensitive sensitivity to Torin1 of the NFAT5-dependent genes
(Supplementary Table S1). Of 28 NFAT5-independent Ddit4, Ddit4l, Slc1a3 and the NFAT5-independent
genes, Torin1 inhibited the induction of 7 (25%) Amd1, Bpgm and Slc19a2 (Figure 2B). Also consistent
4374 NucleicAcidsResearch,2012,Vol.40,No.10
Figure 2. Identificationofosmostress-regulatedgenesandtheirsensitivitytomTORinhibition.(A)Scatterplotinwhichthegraydotsrepresentthe
expressionpatternoftheentiredatasetandthebluecrossesthegeneswhoseinductionbyosmostresswasrepressedbyTorin1orwhoserepressionby
osmostresswasattenuatedbyTorin1.Thepanelsintherightshowtheheatmapsofthegeneprobesfoundtoberegulatedbyosmostressandtheir
sensitivity to Torin1. Expression levels correspond to the log2 of the mean of three independent samples per condition (center panel) and for each
individualsample(rightpanel).Thebrightnessofred(induced)andgreen(downregulated)representsthemagnitudeofthechangeintheexpression
ofeachgene.GenenamesandvaluesareprovidedinSupplementaryTablesS1–S4.(B)RT–qPCRvalidationofTorin1sensitivityofosmoresponsive
genesidentifiedinthemicroarrayanalysis.RNAfromthethreeindividualsamplesusedforthemicroarrayanalysisplusoneadditionalexperiment
were analyzed by RT–qPCR for the expression of the indicated genes and normalized to L32. Samples correspond to wild-type MEFs cultured in
isotonic (I, white bars) (300mOsm/kg) or hypertonic (H, black bars) medium (500mOsm/kg) during 8h without or with Torin1 (100nM). RNA
levels are represented relative to the amount of mRNA in hypertonic conditions, which was given an arbitrary value of 100. Bars represent the
mean±SEM of four independent experiments (*P<0.05).
NucleicAcidsResearch,2012,Vol.40,No.10 4375
withthemicroarraydata,Torin1hadminimalornoeffect and proliferation capacity in cells exposed to osmostress.
on the induction of the NFAT5-dependent Hist1h1d and For this approach, MEFs were labeled with the fluores-
Kcnj2, and the NFAT5-independent genes Fosl1, Itga3, cent dye CFDA-SE and then cultured for 48 or 72h in
Prl2c3/c5 and Slc39a10 (Figure 2B). We observed that isotonicorhypertonicmediumwithorwithoutrapamycin
Akr1b3, Aqp1 and Mrps6 had not scored as or Torin1. As cells proliferate, the concentration of
Torin1-sensitive with sufficient statistical significance in CFDA-SE per cell decreases as the initial label is
the microarray, but showed to be inhibited by Torin1 distributed to daughter cells in successive rounds of
when analyzed by RT–qPCR. This result likely reflects division. Besides CFDA-SE fluorescence, we monitored
differences in sensitivity between both techniques, and the overall viability and cell size by flow cytometry.
suggests that the number of osmoresponsive genes that MEFs exposed to osmotic stress proliferated more
could be affected by mTOR inhibition might be greater slowly than those maintained in isotonic conditions,
than detected in the microarray experiments. Analysis of but showed comparable viability and cell size (Figure 3A
several genes representative of different degrees of sensi- and B). Inhibition of mTOR impaired cell proliferation
tivitytoTorin1,Amd1,Bpgm,Ddit4,Ddit4landMrps6,in and caused a decrease in their size, clearly evident in
an independent MEF line confirmed that they were also Torin1-treated cells, in both isotonic and hypertonic con-
inhibited by rapamycin (Supplementary Figure S4), simi- ditions (Figure 3B). Visual examination of the cells
larly to what we had previously observed with Akr1b3,
indicatednoobvioussignsoftoxicityundertheconditions
Aqp1, Hspa1b and Slc5a3 (Figures 1B and 2B).
tested (Supplementary Figure S5). Altogether, these
Besidesgeneswithalreadyknownosmoprotectivefunc-
results indicate that mTOR was required to sustain
tions, such as Akr1b3, Aqp1, Hspa1b and Slc5a3
growth and proliferative capacity during osmostress.
(38,49,72,73), mTOR regulated the expression of others
We were intrigued by the finding that hypertonic stress
that had not been previously shown to respond to
upregulated the mTOR-dependent expression of REDD1
osmostress, although they had been described in the
and REDD2 mRNAs, two homologous genes whose
context of other stress responses: Ddit4 and Ddit4l in
products had been described to inhibit mTOR in certain
hypoxia, DNA damage and oxidative stress (74), Figf/
contexts,suchashypoxia(74,84).REDD1protein,likeits
VEGF-D (75) in hypoxia, and Slc19a2 in DNA damage
mRNA,wasinducedbyosmoticstressinarapamycinand
(76) (Supplementary Tables S1–S5). Some of the
Torin1-sensitive manner (Figure 4A). We could not asses
Torin1-inhibited genes encoded for positive regulators of
the induction of REDD2 protein due to the lack of
proliferation: Amd1 (77), Figf/VEGF-D (78) and Tacstd2/
antibodies validated in the literature. We focused on
Trop2 (79) (Supplementary Table S1). The set of genes
REDD1 and tested its effect on the activity of mTOR
repressed by osmostress and whose repression was
and induction of osmoresponsive genes. Suppression of
attenuated by Torin1 included Atg10, a regulator of
REDD1 with siRNA did not affect the phosphorylation
autophagosome formation (80), and Gstz1, which partici-
of the mTORC1 substrate S6K1 (Figure 4B), which sug-
pates in cellular responses to oxidative stress (81).
gested that induction of endogenous REDD1 by osmotic
(Supplementary Table S2). Another gene found in this
stress did not inhibit mTOR, in contrast to its inhibitory
analysis was Sesn2, whose basal expression was not
effectdescribedinotherstressresponses.However,lossof
affected by osmostress, but was reduced by Torin1 in
REDD1 in MEFs caused a decrease in the induction of
isotonic and hypertonic conditions (Supplementary
aldose reductase and Hsp70.1 by osmotic stress, without
Table S3). Sesn2 can be induced by DNA damage, and
affecting the induction of REDD2 (Figure 4C). In the
itsproductSestrin2hasbeenshowntoinhibitmTORvia
same set of experiments, downregulation of REDD2
TSC2 (21). Torin1 also enhanced the expression of Pdk4
with siRNA did not cause significant changes in the in-
and Pim1 in cells exposed to osmostress (Supplementary
ductionofosmostressresponsegenesnorintheactivityof
TableS4).Thesegeneshavebeendescribedinthecontext
mTORC1(Figure4BandC).However,sincewehavenot
of cellular responses to starvation or inhibition of growth
confirmed the induction of REDD2 protein, we are
signaling (82,83). The stress-related function of the differ-
ent genes is summarized in Supplementary Table S5. It is cautious about the interpretation of this result regarding
important to note that the expression of several of these the potential lack of effect of endogenous REDD2. Our
genes was affected to a greater or lesser degree, but not microarray analysis showed that inhibition of mTOR in
eliminated, upon inhibiting mTOR (Supplementary cells exposed to osmostress enhanced the expression of
Tables S1–S4), suggesting that this pathway regulated Pdk4 (pyruvate dehydrogenase kinase 4) (Supplementary
their magnitude of induction but was not an absolute Table S4). Since Pdk4 had been recently shown to be re-
requirement. pressed by REDD1 in MEFs (85), we tested whether
The finding that mTOR regulated the expression of a mTOR-mediated induction of REDD1 during osmostress
complex pattern of genes raised the question of how they was involved in the inhibition of Pdk4, and found that
might contribute to cell functions under stress. However, suppression of REDD1 prevented the downregulation of
since the contribution of mTOR to their magnitude of PDK4 by osmotic stress to a similar extent as Torin1
expression varied depending on the particular gene, we (Figure 4C). Altogether, these results indicated that
considered that trying to reproduce the effect of mTOR although the effects of REDD1 were moderate, it could
inhibition by suppressing or overexpressing combinations modulate,bothpositively(aldosereductase,Hsp70.1)and
of genes would be complex. Nonetheless, we tested negatively (PDK4), the expression of several
whether mTOR activity was important to sustain growth osmoresponsive genes.
4376 NucleicAcidsResearch,2012,Vol.40,No.10
Figure 3. EffectofosmoticstressandmTORinhibitorsoncellgrowthandproliferativecapacity.ImmortalizedMEFswerelabeledwithCFDA-SE
and cultured during 48 or 72h in isotonic (300mOsm/kg) or hypertonic medium (500mOsm/kg, upon addition of 100mM NaCl) without or with
rapamycin(50nM)orTorin1(100nM).(A)Cellviability(FSC/SSCdotplots),(B)size(FSCparameter)andrelativeproliferation(proportionalto
dilutionofCFDA-SEsignal)wereanalyzedbyflowcytometry.TheFSCparameterin(B)wasanalyzedincellsinG1,gatedbystainingtheculture
with the DNA dye Hoechst 33342. Flow cytometry graphics are representative of three independent experiments with similar results.
mTOR regulates osmostress-induced changes in chromatin hypertonic stress-induced rapid changes in the chromatin
configuration and RNA pol II recruitment to configuration of the aldose reductase gene (86), we asked
osmoresponsive genes whethermTORcouldregulatetheseprocessesinNFAT5-
dependent and -independent osmostress-responsive genes.
Next,weanalyzedtheeffectofmTORinhibitionontran-
In agreement with Tong et al. (86), our analysis of the
scriptional mechanisms potentially involved in the induc-
Akr1b3 (aldose reductase) gene showed that hypertonic
tion of osmostress responsive genes. We observed that
stress-induced extensive acetylation of histone H4 at dif-
Torin1 did not inhibit the initial recruitment of NFAT5
ferent regions since the first hour of stimulation, followed
to the Akr1b3 (aldose reductase) enhancer but caused its
by eviction of nucleosomes from the transcription start
partial dissociation from chromatin at later time points
site (TSS) and upstream regions (Figure 5B). Torin1 in-
((cid:7)4h) (Figure 5A). We also observed that Torin1, and
hibited the acetylation of H4 throughout an extended
rapamycin to a lesser extent, reduced the basal constitu-
region of (cid:3)7kb by 4h, but did not affect nucleosome
tive nuclear accumulation of NFAT5 at 8h, but did not
eviction (Figure 5B). We then tested the effect of
impair its translocation induced by osmotic stress
osmostress and Torin1 on H4 acetylation and eviction
(Supplementary Figure S6). These results indicated that in two other genes: the NFAT5-regulated Ddit4l
the activity of NFAT5 was reduced upon prolonged (REDD2), and the NFAT5-independent Bpgm (2,3-
mTOR inhibition. However, seeing that Torin1 inhibited bisphosphoglycerate mutase). The increase in H4 acetyl-
the induction of only a set (22%) of NFAT5-dependent ation induced by osmotic stress in these genes was rather
genes, as well as a similar proportion (25%) of modest in comparison with Akr1b3, but was still
NFAT5-independent ones, we considered that NFAT5 Torin1-sensitive (Supplementary Figure S7). Similarly to
might not be a primary target of mTOR, and wondered what we had observed in the Akr1b3 gene, osmotic stress
whetherTorin1mightaffectothertranscriptionregulatory caused a progressive nucleosome eviction in Ddit4l and
mechanisms. Prompted by a recent work showing that Bpgm that was not affected by Torin1.
NucleicAcidsResearch,2012,Vol.40,No.10 4377
Figure 4. mTOR-sensitive inductionofREDD1 by osmoticstress. (A) Immortalized MEFs orHEK293T cells were cultured during8h inisotonic
(300mOsm/kg) or hypertonic medium (500mOsm/kg, upon addition of 100mM NaCl) without or with rapamycin (50nM) or Torin1 (100nM).
REDD1,phospho-S6K1(Thr389),S6K1andb-actin(loadingcontrol)weredetectedbyWesternblotting.(B)ImmortalizedMEFsweretransfected
with control non-targeting siRNA (scramble) or siRNA specific for REDD1 or REDD2. After 24h, they were cultured during 8h in isotonic
(300mOsm/kg) or hypertonic medium (500mOsm/kg, upon adding 100mM NaCl). (C) Effect of suppressing REDD1 and REDD2 on the
osmostress-mediated induction of REDD1, REDD2, aldose reductase (AR), Hsp70.1, and PDK4. RNA levels for each gene and condition were
normalizedtoL32mRNAandarerepresentedrelativetotheamountofmRNAinhypertonicconditionsincellstransfectedwithcontrolscramble
siRNA,whichwasgivenanarbitraryvalueof100.Barsrepresentthemean±SEMofthreeindependentexperiments(*P<0.05;n.s.notstatistically
significant).
WealsoanalyzedwhetherinhibitionofmTORaffected formsthanfortheAkr1b3gene,butnonethelessthiseffect
the recruitment of RNA polymerase II, a more direct in- was significant and inhibited by either rapamycin or
dicator of transcriptional activity. Osmotic stress induced Torin1 (Figure 6). For Ddit4l, binding of RNA pol II to
a rapid and substantial recruitment of RNA pol II to the its TSS and transcribed regions was minimally increased
TSS and transcribed region (exon 2) of Akr1b3 by osmostress compared to Akr1b3 and Bpgm (Figure 6).
(Supplementary Figure S8). Inhibition of the The modest effect of osmostress in enhancing H4
osmostress-induced RNA pol II recruitment by Torin1 acetylation of Ddit4l and its occupancy by RNA pol II
was detected in 1h, and preceded the partial dissociation suggested that the osmostress-induced mTOR-sensitive
of NFAT5 from the Akr1b3 enhancer, indicating that in- mechanism for upregulating its mRNA could either
hibition of RNA pol IIwas notdue to thedissociation of involve chromatin-regulatory events different from those
NFAT5 from upstream regions. Further analysis showed regulating Akr1b3 and Bpgm, or additional post-
that both rapamycin and Torin1 were significantly effect- transcriptional mechanisms acting on RNA processing
ive at inhibiting the binding of RNA pol II to the TSS of or stability.
Akr1b3 (Figure 6), as well as the recruitment to the TSS
and exon2 of active forms of RNA pol II involved in
transcription initiation and elongation, identified respect-
DISCUSSION
ively by phosphorylated Ser5 and Ser2 in the heptad
repeat of the carboxy-terminal domain (CTD) of its Our study reveals a previously unappreciated capacity of
largest subunit Rbp1 (87). Regarding Bpgm, we observed mTORtoregulate theosmostress response by influencing
a moderate effect of osmostress in enhancing the recruit- the transcription of stress-responsive genes. We used
mentofRNApolIIanditsphosphorylatedSer5andSer2 hypertonic stress conditions (500mOsm/kg) sufficient to