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Departments of Pathology and Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B family member, activates transcription of osmocompensatory
genes in response to extracellular hyperosmotic stress. However, the
function of NFAT5 under isosmotic conditions present in vivo remains
unknown. Here we demonstrate that NFAT5 is necessary for optimal T cell
development in vivo and allows for optimal cell growth ex vivo under
conditions associated with osmotic stress. Transgenic mice expressing
an inhibitory form of NFAT5 in developing and mature T cells exhibited
a 30% reduction in thymic cellularity evenly distributed among thymic
subsets, consistent with the uniform expression and nuclear
localization of NFAT5 in each subset. This was associated with a 25%
reduction in peripheral CD4+ T cells and a 50% reduction
in CD8+ T cells. While transgenic T cells exhibited no
impairment in cell growth or cytokine production under normal culture
conditions, impaired cell growth was observed under both hyperosmotic
conditions and isosmotic conditions associated with osmotic stress.
Transgenic thymocytes also demonstrated increased sensitivity to
osmotic stress. Consistent with this, the system A amino acid
transporter gene ATA2 exhibited NFAT5 dependence under hypertonic
conditions but not in response to amino acid deprivation. Expression of
the TNF-
gene, a putative NFAT5 target, was not altered in
transgenic T cells. These results not only demonstrate an
osmoprotective function for NFAT5 in primary cells but also show that
NFAT5 is necessary for optimal thymic development in vivo, suggesting
that developing thymocytes within the thymic microenvironment are
subject to an osmotic stress that is effectively countered by
NFAT5-dependent responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B proteins, NFATc proteins, and the most
recently identified member, NFAT5 (also designated TonEBP, NFATL1,
OREBP). NF-B proteins (which include Rel-A, NF-B1, NF-B2,
c-Rel, and Rel-B) are activated during both innate and adaptive immune
responses and in response to certain cellular stresses
(1, 2, 3). NF-B-dependent transcriptional responses
regulate cytokine expression, cell survival, and cell proliferation
(2, 4, 5). NFATc proteins (consisting of NFATc1 through
NFATc4) represent a distinct group of rel proteins defined by a common
regulatory domain that is the target of the
calcium/calmodulin-dependent protein phosphatase calcineurin
(6). Calcium-dependent activation of NFATc proteins
regulates transcriptional programs involved in lymphocyte development,
activation, and differentiation (6), as well as
development of the heart, the vasculature, and potentially the nervous
system (7, 8). NFAT5, however, does not contain a
calcineurin-dependent regulatory domain and precedes NFATc proteins in
evolutionary development (9). In marked contrast to
NF-B and NFATc proteins, the biologic function of NFAT5 remains
poorly understood.
The NFAT5 transcription factor was originally identified as tonicity
enhancer binding protein (TonEBP) based on its ability to bind to
tonicity response elements present in the upstream regulatory region of
genes that are induced upon hypertonic stress (10). Known
NFAT5 target genes encode proteins that function to increase the
intracellular concentration of compatible (i.e., organic) osmolytes and
thus compensate for increased extracellular tonicity. These include
aldose reductase (AR3;
Ref. 11), the sodium/myoinositol transporter
(12), and the betaine/
-aminobutyric acid transporter
(13). Although NFAT5 exhibits variable nucleocytoplasmic
localization in different cell lines, hyperosmotic stress induces clear
nuclear translocation (10, 14). Additionally,
NFAT5-dependent reporter gene expression is markedly induced by
hypertonicity (10, 15). These observations have led to the
hypothesis that NFAT5 functions physiologically within the kidney to
counteract the potentially deleterious consequences of the hypertonic
environment of the renal medulla (16). In support of this
hypothesis, NFAT5 protein was observed to be localized to the nucleus
of renal medullary epithelial cells of rats in which the osmolarity of
the renal medulla was elevated by dehydration. Moreover, this nuclear
localization correlated with increased expression of the
sodium/myoinositol transporter gene (17). NFAT5 thus
appears to play an important role in allowing cells to compensate for
hyperosmotic stress under tissue culture conditions or within the
hyperosmotic environment of the renal medulla. However, direct
functional data to support this hypothesis remains lacking.
The possibility that NFAT5 subserves broader or perhaps alternative
functions in vivo, however, is suggested by several observations.
First, NFAT5 RNA expression is not limited to the kidney, but is in
fact ubiquitous (10, 15, 18). Analysis of protein
expression further indicates that NFAT5 is expressed in essentially all
tissues of the developing embryo, with particularly high levels of
expression in the brain and heart (19). In adult tissues
detectable NFAT5 protein is limited to the thymus and mitogen-activated
primary T lymphocytes (15). Despite the apparent lack of
expression of NFAT5 protein in most tissues, NFAT5 is readily
detectable by Western blot analysis in most transformed cell lines
(Refs. 10, 15 , and 18 and our
unpublished observations). NFAT5 protein expression thus appears to
correlate with cell growth. Second, the extremes of hypertonicity
present within the renal medulla do not appear to exist in any other
tissue under physiologic conditions. Third, NFAT5-dependent
transcription can be induced not only by hypertonicity but also by
TCR-dependent signaling events (15). Fourth, in addition
to osmoregulatory genes, NFAT5 has been demonstrated to play a role in
the expression of cytokine genes such as TNF- and lymphotoxin 
in
response to a combination of hypertonic and mitogenic signals
(20). And fifth, a novel Drosophila rel domain
protein designated MESR1, which is more similar to NFAT5 than to any
other NFAT or rel family member, was identified in an overexpression
screen and shown to be capable of suppressing RAS-dependent signaling
(21). This gene was also identified in an overexpression
screen for axon guidance/synaptogenesis phenotypes (22).
Together these observations suggest that the function of NFAT5 may not
be limited to regulating transcription in response to hypertonic
stress, as occurs in the renal medulla. Rather, NFAT5 may function more
broadly and under isotonic conditions to regulate transcriptional
responses that permit optimal cell growth and function.
In the present study the biologic function of NFAT5 in vivo was evaluated through the use of transgenic mice that express a dominant inhibitory form of NFAT5. Given previous studies demonstrating expression of the NFAT5 protein in thymus and activated T cells (15), transgene expression was directed to thymus and mature peripheral T lymphocytes. Mice that express the transgene exhibited impaired thymic development and reduced numbers of mature T cells in spleen and lymph nodes, consistent with the constitutive nuclear localization of NFAT5 observed in thymocytes as well as both quiescent and activated T cells. Although transgenic T cells exhibited normal ex vivo proliferative responses and cytokine production under standard cell culture conditions, medium depletion and amino acid limitation resulted in impaired cell growth and reduced viability as compared with control mice. These results suggest that NFAT5 is not only important for optimal cell growth in vivo, but within the context of an isosmotic environment NFAT5 appears to play a role in optimally coordinating transcriptional programs in response to osmotic stresses to which cells are normally exposed in vivo during cell growth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A DNA fragment encoding the murine NFAT5 DNA binding domain
(DBD; aa 267–543) was amplified by PCR from a murine NFAT5 cDNA clone
(kindly provided by C. Antos and E. Olson (University of Texas
Southwestern Medical Center, Dallas, TX); GenBank accession no.
AF162853), transferred to an intermediate subcloning vector (pJT501t)
to introduce a 3' influenza hemagglutinin (HA) epitope tag, and
subsequently inserted into the EcoRI site of the CD2
transgene vector (23), resulting in the transgene vector
CD2 mDBD. The region spanning the murine NFAT5 DBD insert was verified
by DNA sequencing (DNA Sequencing Shared Resource, University of
California-San Diego Cancer Center). The transgene DNA fragment was
separated from vector DNA by centrifugation through a 10–40% sucrose
gradient. Fractions containing the 
12-kb transgene fragment were
pooled and exhaustively dialyzed against injection buffer (7.5 mM Tris
(pH 7.4), 0.15 mM EDTA). Pronuclear injections of transgene DNA were
performed by the University of California-San Diego Cancer Center
Transgenic Mouse and Gene Targeting Core using CB6F1 hybrid embryos.
Transgene-positive founders were identified by PCR from tail DNA using
transgene-specific primers (5'-GCATAAGAGTCAAAGAAGTCCC and
5'-CTCCTTTCACTGAACAGCTATGC). Three founder lines (CD2DBD2, CD2DBD10,
CD2DBD17) were generated and transgene expression was verified by
Western blotting using the HA epitope-specific mAb HA.11 (1:4000;
CRP, Denver, PA). The highest expressing transgenic lines (CD2DBD2 and
CD2DBD10) were used in the present studies and gave similar results.
All experiments were performed using transgenic mice backcrossed onto
the C57BL/6 background at least five generations, with control mice
consisting of transgene-negative littermates. Animals were maintained
in accordance with University of California-San Diego animal care
guidelines.
Cell preparation and culture
Splenocyte single cell suspensions were purified by density gradient centrifugation (Lympholyte M; Cedarlane Laboratories, Hornby, Ontario, Canada). CD4+ and CD8+ T lymphocytes were isolated to >95% purity by positive MACS using Ab-conjugated MicroBeads (Miltenyi Biotec, Auburn, CA) as per the manufacturer’s instructions. Con A blasts were generated from splenocyte single cell suspensions. Cells were plated in complete medium as previously described (15) with 2.5 µg/ml Con A (Calbiochem, La Jolla, CA) and 40 U/ml IL-2 (Roche, Basel, Switzerland) at 106 cells per milliliter for 48 h. Cells were washed, resuspended, and cultured as indicated. Isolated T cells were activated in tissue culture dishes precoated with anti-CD3 Ab (145-2C11; BD PharMingen, San Diego, CA) or as previously described (15) and supplemented with 2.5 µg/ml anti-CD28 (37.51; BD PharMingen) and 40 U/ml IL-2. Jurkat E6 cell lines were cultured as described previously (15). SV40 transformed murine ear fibroblasts (kindly provided by B. A. Hamilton, University of California at San Diego, La Jolla, CA) were cultured in DMEM containing high glucose supplemented with 10% heat-inactivated FBS, 100 U penicillin, and 100 µg/ml streptomycin. Hyperosmotic conditions were maintained by incubation in complete medium containing 0.22-µm filter-sterilized 100 mM sucrose (Fisher, Pittsburgh, PA), 50–200 mM D(+)raffinose (Sigma- Aldrich, St. Louis, MO), or 100 mM sodium chloride (Sigma-Aldrich) as indicated. Lymphocyte apoptosis was induced by incubation in complete medium containing 1 µM dexamethasone (Calbiochem) or 250 ng/ml anti-fas Ab (Jo2; BD PharMingen) with 1 mg/ml protein G (Sigma-Aldrich) as indicated. Amino acid deprivation culture conditions were maintained by washing cells twice with Earl’s balanced salt solution (EBSS) with Ca2+ and Mg2+ supplemented with 10% heat-inactivated, dialyzed FBS. Cell culture was then continued in the same EBSS with or without the addition of minimum essential and nonessential amino acids as indicated. For additional stimulation, cells were incubated with complete medium containing 1 µM ionomycin (Calbiochem) and/or 10 ng/ml PMA (Calbiochem) unless otherwise indicated. Osmolarity of the culture medium was measured using a Wescor 5500 vapor pressure osmometer (Wescor, Logan, UT). Jurkat cell transfections and reporter gene analyses were performed as previously described (15).
Generation of stable Jurkat cell lines
A full-length human NFAT5 cDNA clone containing two tandem C-terminal HA epitope tags (pSH320) was digested with BamHI. The resulting 4828-bp fragment was ligated into pBJ5-neo (a mammalian expression vector containing the neomycin resistance marker) to generate p5FL-neo. pJT501 was digested with NotI and EcoRI. The resulting 894-bp fragment was ligated into pBJ-neo to generate p5DBD-neo. Jurkat E6 cells (American Type Culture Collection, Manassas, VA) were electroporated as previously described (15) with 20 µg of linearized (AtaI) pBJ-neo (control), p5FL-neo, or p5DBD-neo plasmid DNA. Cells were plated into 96-well plates by limiting dilution and cultured in complete medium containing 800 µg/ml G418. Three independent clones of the pBJ-neo control were maintained. Three independent clones for each of the NFAT5 constructs were identified based on Western analysis of expression of the HA epitope-tagged expression cassette. Cells were removed from G418 selection at least 48 h before experiments.
Cell growth assays
Cell proliferation assays based on [3H]thymidine incorporation were performed by activating isolated CD8+ T cells in triplicate in 96-well culture plates (105 cells per well) with plate-bound anti-CD3 plus anti-CD28. Cells were pulsed with 1 µCi of [3H]thymidine (Amersham, Arlington Heights, IL) for 12 h before termination of the culture and harvested onto glass filter mats using a 96-well harvester (Tomtec, Hamden, CT). Incorporated [3H]thymidine was counted using a MicroBeta TriLux scintillation counter (PerkinElmer Life Sciences, Wellesley, MA). To measure cell growth, 106 isolated CD8+ T cells were activated in triplicate in 2-ml cultures in a 12-well culture dish, or 0.2 x 106/ml Jurkat E6 cells were cultured in triplicate in 1-ml cultures in a 24-well culture dish. At the indicated times cells were counted and viability was assessed by flow cytometric determination of propidium iodide (PI; Calbiochem) exclusion. The number of viable cells was calculated by multiplying total cell number by the percentage of cells that were PI negative. Similar results were obtained by trypan blue (Life Technologies, Gaithersburg, MD) exclusion. In vivo bromodeoxyuridine (BrdU) incorporation was performed with 4- to 6-wk-old mice. Mice were injected i.p. with 1 µM BrdU (BD PharMingen) in PBS. One hour later thymus and spleen were harvested and cells were fixed in ice-cold 70% ethanol for 30 min at -20°C. Cells were then processed for staining with a FITC-conjugated anti-BrdU Ab (BD PharMingen) according to the manufacturer’s instructions. Finally, cells were resuspended in PBS containing 10 µg/ml PI and cell cycle distribution (DNA vs BrdU) was analyzed by flow cytometry.
Immunofluorescence
Fibroblasts were seeded on coverslips in 12-well tissue culture dishes at 50% confluence and allowed to grow to at least 70% confluence. Thymocyte samples were prepared by rapid isolation and dispersal of single cell suspensions through a 70-µm filter into ice-cold PBS. Thymic subsets (CD4 and CD8 double negative, double positive, and single positive) were isolated by fluorescence cell sorting using CD4-FITC and CD8-PE marker Abs (BD PharMingen). CD4+ T cells were isolated and activated as described. Thymocytes and CD4+ T cells were cytospun (5 min at 600 rpm; Cytospin-3; Thermo Shandon, Pittsburgh, PA) onto poly-L-lysine-coated slides (Sigma-Aldrich). Attached cells were fixed in 4% formaldehyde for 10 min and washed three times in PBS. Fixed cells were blocked and permeabilized by incubation in PBS containing 5% donkey serum and 0.2% Triton X-100 for 30 min. Incubation with 1:1000 rabbit polyclonal anti-NFAT5 Ab (15) was performed for 1 h in the same blocking/permeabilization solution diluted 1/1 in PBS. Cells were then washed five times in PBS and incubated for 1 h with a Cy3-conjugated anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1/500 in PBS with 2% donkey serum. Cells were then washed three times and stained with DAPI (Sigma-Aldrich) for 5 min. All incubations were performed at room temperature. Deconvolution images were obtained through the University of California-San Diego Cancer Center Digital Imaging Shared Resource. Images were captured with a DeltaVision deconvolution microscopy system (Applied Precision, Issaquah, WA). Individual images were derived from 10–20 optical sections obtained at 0.2-µm increments. The data sets were deconvolved and analyzed using SoftWorx software (Applied Precision) on a Silicon Graphics Octane workstation. For accurate quantitation of NFAT5 expression in lymphocytes, exposure times and Cy3 signal scaling were kept constant between images as indicated, and fluorescence intensity values were obtained from nondeconvolved images using the Data Inspector program in SoftWorx. NFAT5 expression was quantitated by determining Cy3 fluorescence intensity per cell as a mean of at least 30 cells in a representative x20 field. Similar results were obtained by determining mean Cy3 fluorescence intensity within 10–20 uniformly staining regions within a single cell (x100).
RNA analysis
RNA was isolated from Con A blasts using TRIzol (Invitrogen, San
Diego, CA), and 15 µg of total RNA per sample was size-fractionated
on a 1% formaldehyde-agarose gel. Radiolabeled cDNA fragments used as
probes consisted of the following: a 1267-bp
EcoRI-XhoI fragment from a murine AR cDNA
expressed sequence tag (EST) clone (GenBank accession no.
AA791571; American Type Culture Collection), an 850-bp
EcoRI-NotI fragment from a murine ATA2 cDNA EST
clone (GenBank accession no. AA764095; Research Genetics, Huntsville,
AL), a 1110-bp EcoRI-NotI fragment from a murine
TNF- cDNA EST clone (GenBank accession no. AA920586; Research
Genetics), and a murine GAPDH fragment (Ambion, Austin, TX).
Hybridizations and high stringency washes were performed using standard
procedures. RNA levels were quantitated by phosphorescence using a
Storm Phosphoimaging system and ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). Fold induction values were calculated after
normalization to the GAPDH signal.
Flow cytometry
mAbs used for flow cytometric analyses were obtained from BD
PharMingen and include anti-CD4 (GK1.5), anti-CD8 (53-6.7),
anti-B220 (RA3-6B2), anti-CD25 (7D4), anti-CD44 (IM7),
anti-CD69 (H1.2F3), anti-IL-2 (JES6-5H4), anti-IFN-
(XMG1.2), anti-IL-4 (11B11), and anti-BrdU (3D4). Flow
cytometry was performed on a FACScan flow cytometer using CellQuest
software (BD Biosciences, Palo Alto, CA).
Statistical analyses
All data represent the mean of independent experiments. Error values represent the SEM calculated from a minimum of three experiments. Student’s t test was performed to determine significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NFAT5 protein localizes to both the cytoplasm and nucleus in
tissue culture cell lines. Exposure to hypertonic culture conditions
induces marked cytoplasmic clearing and concentration of NFAT5 in the
nucleus concomitant with an overall increase in NFAT5 staining
(10, 14, 18, 19, 20, 24). In previous studies NFAT5 RNA was
shown to be ubiquitously expressed, while NFAT5 protein was readily
detectable only in thymus or in activated T lymphocytes
(15). To investigate the potential role of NFAT5 in T cell
development and activation, the nucleocytoplasmic distribution of NFAT5
protein in primary thymocytes and CD4+ T cells
was determined by deconvolution immunofluorescence microscopy using an
antiserum raised to the DBD of NFAT5 (15). As a control
for NFAT5 staining, primary murine dermal fibroblasts were cultured
under isotonic or hypertonic conditions for 2 h. As expected
(24), primary skin fibroblasts exhibited constitutive
nuclear NFAT5 staining under isotonic culture conditions as well as
diffuse cytoplasmic staining. Exposure to hypertonic culture conditions
resulted in the complete elimination of NFAT5 from the cytoplasm (Fig. 1
, A vs B), thus
validating the specificity of the antisera for immunohistochemical
localization of NFAT5. Primary murine thymocytes were isolated and flow
sorted into CD4 and CD8 double-negative, double-positive, and
single-positive subsets. Cells of each thymic subset exhibited equally
strong, multifocal nuclear staining for NFAT5, with little or no
detectable cytoplasmic staining, indicating that NFAT5 is not
differentially expressed or regulated during thymic development (Fig. 1, E–H). Interestingly, there was a clear
inverse relationship between NFAT5 staining and the DAPI nuclear
counterstain. Given that DAPI most strongly stains areas of condensed,
heterochromatic DNA, this result suggests that NFAT5 is potentially
localized to areas of more accessible, transcriptionally active
euchromatic DNA. Staining of quiescent CD4+ T
cells demonstrated low but detectable NFAT5 staining in the nucleus in
a pattern similar to that seen in thymocytes, as well as detectable
cytoplasmic staining (Fig. 1C). Although prior Western
analysis of NFAT5 protein expression in quiescent T cells demonstrated
no detectable immunoreactivity (15), immunofluorescence
microscopy represents a more sensitive method of detection.
CD4+ T cell blasts activated with anti-CD3
plus anti-CD28 exhibited significantly increased NFAT5 staining
that was localized to the nucleus with no detectable cytoplasmic
staining (Fig. 1D). Quantitation of NFAT5 expression
observed by immunofluorescence microscopy demonstrates that NFAT5 is
induced in activated T cells to a level similar to that present in
thymocytes (Fig. 1, inset), consistent with results from
Western analysis of thymus and peripheral T cells (15).
The nuclear localization of NFAT5 in thymocytes as well as quiescent
and activated T cells and the marked induction of NFAT5 protein
expression upon T cell activation suggest that NFAT5 is potentially
active during both thymic development and T cell activation.
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In an effort to further define the physiologic function of NFAT5
in vivo, expression of an inhibitory (i.e., DN) form of NFAT5 was
directed to thymus and mature peripheral T cells using the human CD2
transgene expression vector (23). The DN NFAT5 transgene
consists of the isolated DBD (Fig. 2A), which has been shown
previously to significantly inhibit endogenous NFAT5 transcriptional
activity in cell-based transient transfection assays (10, 15), most likely by interfering with NFAT5 dimerization
(20, 25). The specificity of the DN NFAT5 was further
validated in additional reporter gene assays which demonstrated that,
while expression of DN NFAT5 inhibited inducible transcription mediated
by NFAT5 binding sites, there was no inhibition of transcription
mediated by NFATc, NF-B, or AP1 DNA binding sites (Fig. 2B). Western analysis of several independent DN NFAT5
transgenic lines demonstrated strong expression of the DN NFAT5
transgene in both thymus and spleen (Fig. 2C). To verify
that transgenic expression of the NFAT5 DBD results in the functional
inhibition of the endogenous NFAT5 protein, inducible expression of the
AR gene upon hypertonic stress was analyzed in nontransgenic vs
transgenic mice. AR is an NFAT5 target gene that is induced upon
hyperosmotic stimulation (26, 27, 28). Previous studies
demonstrated that expression of the NFAT5 DBD is capable of inhibiting
the hyperosmotic induction of the endogenous AR gene in cell lines
(28). As expected, hyperosmotic induction of AR mRNA in
primary T lymphocytes was reduced by 40 ± 5.2%
(n = 3) in cells expressing the DN NFAT5 transgene as
compared with nontransgenic controls after normalization to the level
of GAPDH mRNA expression (Fig. 2, D and E). This
level of inhibition mediated by overexpression of the NFAT5 DBD is
similar to that observed in transfected cell lines
(28).
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A), consistent with the role
of NFAT5 in regulating transcription of osmoprotective genes. The
specificity of the observed increased sensitivity of DN NFAT5
transgenic cells to hypertonic stress-dependent cell death was
established by investigating the sensitivity of these cells to either
growth factor withdrawal or fas-dependent cell death. While DN NFAT5
transgenic cells exhibited increased sensitivity to hypertonic stress,
the decrease in viability associated with either growth factor
withdrawal or the induction of apoptosis by cross-linking fas was
identical in both transgenic and nontransgenic cells (Fig. 3B). The sensitivity of thymocytes to hypertonic stress was
also determined. Thymocytes were placed into culture containing varying
concentrations of raffinose, and cell viability was assessed 12 h
later. Under these conditions thymocytes from DN NFAT5 transgenic mice
also exhibited increased sensitivity to hypertonic stress (Fig. 3C). However, induction of cell death by either incubation
with dexamethasone or anti-fas Abs showed no difference comparing
transgenic and nontransgenic cells (Fig. 3D). These results
indicate that the DN NFAT5 transgene specifically inhibits endogenous
NFAT5 function and further demonstrate the osmoprotective function of
NFAT5 in primary cells under ex vivo hypertonic tissue culture
conditions. Although NFAT5 has been demonstrated to regulate the
expression of genes such as AR that mediate the induction of compatible
osmolytes in response to hyperosmotic stress, these results represent
the first functional data that demonstrate a role for NFAT5 in
protecting cells from cell death induced by hyperosmotic stress.
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To determine whether inhibition of NFAT5 function in vivo
influences thymic development, the cellularity of the thymus and spleen
of nontransgenic and transgenic mice was compared. Thymuses from
transgenic mice exhibited a 30% reduction in cellularity as compared
with nontransgenic mice, while transgenic spleen exhibited a 15%
decrease in overall cellularity (Fig. 4A). To determine whether the
reduction in splenic cellularity reflected a reduction in the number of
mature peripheral T cells, the percentages of
CD4+ and CD8+ T cells
present in both spleen and peripheral lymph node were determined by
flow cytometry. Spleen from DN NFAT5 transgenic mice exhibited a 50%
reduction in the percentage of CD8+ T cells and a
25% reduction in the percentage of CD4+ T cells
(Fig. 4B). Given the observed frequencies of
CD4+ and CD8+ cells in the
spleen, the overall reduction in splenic cellularity observed in the
transgenic mice is accounted for by a reduction in the absolute number
of CD4+ and CD8+ T cells. A
reduction in the percentage of CD8+ T cells was
also observed in cells obtained from peripheral lymph nodes (Fig. 4C). To determine whether the reduction in T cell population
functionally affected the ability of DN NFAT5 transgenic mice to elicit
an Ag-specific immune response in vivo, nontransgenic and transgenic
mice were immunized intradermally with plasmid DNA encoding
-galactosidase in a CMV-based expression vector (29, 30). As expected, DN NFAT5 transgenic mice developed impaired
Ag-specific Ab and cytotoxic T cell responses that correlated with a
reduction in the frequency of Ag-specific precursor cells as determined
by IFN- ELISPOT and with the reduced frequency of
CD4+ and CD8+ T cells in
the transgenic mice (data not shown). These results indicate that
inhibition of NFAT5 function in vivo results in impaired thymic
development associated with peripheral T cell lymphopenia, which, not
surprisingly, gives rise to impaired immune responses. The reduced
number of peripheral T cells is likely secondary to the observed
impairment in thymic development. However, peripheral T cell expansion
via homeostatic mechanisms regulating Ag-independent T cell
proliferation may also occur independent of thymic influence.
Therefore, these results do not exclude the possibility that NFAT5 may
play a role in regulating peripheral T cell homeostasis independent of
a role in regulating thymic development.
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D). These results
indicate that the observed reduction in overall size (i.e.,
cellularity) of thymus from DN NFAT5 transgenic mice is equally
distributed among all thymic subsets and therefore is not the result of
inhibition of a specific thymic developmental transition. To determine
whether the observed reduction in thymic cellularity was due to a
reduction in the rate of cell proliferation in the thymus, the
percentage of thymocytes in S phase of the cell cycle was determined by
measuring BrdU incorporation in vivo. One hour after injection of BrdU,
thymocytes were isolated and the frequency of BrdU-positive cells was
determined by flow cytometry. Essentially identical percentages of
BrdU-positive cells were observed in nontransgenic (8.0 ± 0.2%,
n = 6) and transgenic (8.2 ± 0.5%,
n = 6) thymuses. This result indicates that there was
no significant difference in the proliferative rate of thymocytes that
might account for the reduction in overall thymic cellularity. In
contrast, parallel analysis of splenocytes by in vivo BrdU
incorporation demonstrated a significant 70% reduction in
BrdU-positive cells present in the spleen of transgenic animals
(2.1 ± 0.5%, n = 3) as compared with
nontransgenic controls (6.0 ± 0.4%, n = 3). This
reduction in the percentage of transgenic splenocytes in S phase likely
reflects the observed reduction in the number of T cells in the
spleen. Normal function of DN NFAT5 transgenic T cells under non-stress culture conditions
As a broad assay of T cell function, the proliferative responses
of purified splenic CD8+ T cells from transgenic
and nontransgenic littermate control mice were quantitated by tritiated
thymidine incorporation, which reflects the number of cells in S phase
of the cell cycle. Cells were stimulated with plate-bound anti-CD3
plus anti-CD28 in the presence of excess exogenous IL-2 and
subjected to 12-h pulses of [3H]thymidine at
varying times after stimulation. Culture medium was replenished 2 days
after stimulation. Both transgenic and nontransgenic T cells exhibited
similar proliferative responses (Fig. 5A). Moreover, both control
and transgenic T cells exhibited essentially identical cell growth over
the course of a 6-day culture period based on direct quantitation of
cell number (data not shown). As an additional measure of T cell
activation, intracellular cytokine analyses were performed. Splenocyte
preparations were stimulated with Con A and analyzed for intracellular
cytokine production 48 h later. The number of IL-2- and
IFN--positive cells, as a percentage of either
CD4+ or CD8+ cells, was the
same when comparing transgene-negative vs transgene-positive T cells
(Fig. 5B). The stimulation conditions used in these
experiments preferentially allowed differentiation and expansion of
IFN--producing Th1 cells rather than IL-4-producing Th2 cells.
However, under conditions that induce differentiation of Th2 cells,
IL-4 production by transgene-negative and transgene-positive cells also
showed no significant difference (data not shown). In addition, there
were no differences in TCR expression, as measured by CD3
expression, or in the activation-dependent inducible expression of CD25
and CD69, which represent cell surface markers of T cell activation
(data not shown). Together these results indicate that under standard
ex vivo cell culture conditions, inhibition of NFAT5 function has no
significant effect on T cell growth or effector function (i.e.,
cytokine production).
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As described above, DN NFAT5 transgenic T cells and thymocytes
exhibit reduced viability upon exposure to hypertonic culture
conditions as compared with nontransgenic cells. However, the control
nontransgenic cells also exhibited significant cell death (Fig. 3, A and C), reflecting the highly toxic and
nonphysiologic nature of this culture condition. The rapid, essentially
instantaneous transition from isotonic to hypertonic culture conditions
represents a stress that is unlikely to occur in vivo. Moreover, the
rapid transition to a hypertonic environment does not allow sufficient
time to induce compensatory mechanisms, thus resulting in significant
cell death. To further investigate the growth response of DN NFAT5
transgenic T cells under hypertonic stress conditions, cells were
cultured under conditions of medium depletion in which tissue culture
medium is not replenished over time. Continuous culture without medium
exchange results in gradually increasing osmolarity of the medium over
time in culture (see Fig. 6E).
As previously demonstrated (Fig. 5A), at early time points
after activation of isolated CD8+ T cells both
control and transgenic cells proliferated equally. However, at later
time points in extended culture under conditions of medium depletion
the DN NFAT5 transgenic T cells showed a markedly reduced rate of
incorporation of [3H]thymidine (Fig. 6A). Moreover, direct quantitation of cell expansion after
stimulation demonstrated a reduction in cell numbers at later time
points (Fig. 6B), consistent with results from the
proliferation assay. Thus, DN NFAT5 transgenic T cells exhibited
impaired cell growth under conditions of medium depletion that was not
observed in control mice. Interestingly, in parallel experiments
transgenic cells exhibited no increased sensitivity to a reduction in
pH or even serum deprivation (data not shown), suggesting that the
sensitivity to medium depletion may be due specifically to an increase
in the osmolarity of the medium.
|
, C and
D). Measurement of the osmolarity of the medium over the
culture period demonstrated a 23% increase in osmolarity (Fig. 6E). These results are consistent with the observed
impairment in cell growth of primary T cells expressing the DN NFAT5
transgene under conditions of medium depletion, and further demonstrate
the growth advantage conferred by overexpression of NFAT5. Similar to
results with primary T cells, differences in growth/survival of the
stably transfected cell lines were not apparent at early time points in
the culture but were detectable only later (i.e., day 3 or later).
Together these results clearly demonstrate that NFAT5 functions to
permit optimal cell growth under suboptimal growth conditions
associated with medium depletion. Impaired growth of DN NFAT5 transgenic T cells in response to amino acid deprivation
Amino acid deprivation under isotonic conditions has been
demonstrated to induce a decrease in cell volume and an increase in
intracellular potassium, thus representing an osmotic stress condition
(24, 31, 32). Of particular interest, amino acid
deprivation also induced NFAT5 nuclear localization (24).
To investigate whether the impaired growth of DN NFAT5 transgenic T
cells observed under conditions of medium depletion could also be
manifest by amino acid deprivation, T cells from transgenic and
littermate control mice were subject to culture in medium containing
varying concentrations of amino acids. As expected, culture in medium
containing no amino acids resulted in essentially no proliferation as
measured by [3H]thymidine uptake. However, at
suboptimal concentrations of amino acids, DN NFAT5 transgenic T cells
exhibited a greater inhibition of proliferation compared with T cells
from littermate control mice (Fig. 7A).
|
B). These
results demonstrate that inhibition of endogenous NFAT5 activity
resulting from expression of the DN NFAT5 transgene results in an
increased sensitivity of thymocytes to conditions of limiting
amino acids. Thus, osmotic stress induced not only by exposure to a
hypertonic environment (e.g., addition of raffinose), but also by amino
acid limitation under isotonic conditions (24, 31, 32),
results in impaired cell viability in cells from DN NFAT5 transgenic
mice as compared with nontransgenic controls.
Analysis of potential NFAT5 target genes: ATA2 and TNF-
To further investigate the impaired viability of cells from DN
NFAT5 transgenic mice upon exposure to either hypertonic conditions or
amino acid deprivation, the expression of AR mRNA was compared with
that of the system A transporter ATA2 (Fig. 8, A and B). The
system A amino acid transport system is a sodium-dependent transport
system for neutral amino acids that exhibits osmosensitivity
(32). The induction of the ATA2 gene by hypertonicity
(33, 34) and the nuclear localization of NFAT5 in response
to amino acid deprivation (24) suggests that NFAT5 may
play a role in regulating transcription of this gene. Exposure of
nontransgenic T cell blasts to either hypertonicity or amino acid
deprivation resulted in a similar induction of AR mRNA. The magnitude
of induction is less than that observed previously (Fig. 2, D and E) because the duration of the induction
was shortened (6 vs 16 h) to optimize for the induction of ATA2.
In DN NFAT5 transgenic T cells the induction of AR mRNA by either
hypertonicity or amino acid deprivation was reduced relative to
nontransgenic cells, consistent with a role for NFAT5 in the
transcriptional regulation of the AR gene in response to hyperosmotic
stress. ATA2 mRNA was also induced by both hypertonicity and amino acid
deprivation, although the magnitude of induction by amino acid
deprivation was significantly greater. However, while the induction of
the ATA2 gene by hypertonicity was inhibited in the DN NFAT5 transgenic
cells, induction by amino acid deprivation was not inhibited. These
results indicate that, in contrast to the AR gene in which NFAT5 plays
a role in the induction in response to either amino acid deprivation or
hypertonicity, the ATA2 gene exhibits NFAT5 dependence only in the
context of a hypertonic stimulus, suggesting that amino acid
deprivation activates transcriptional programs independent of NFAT5
that are capable of regulating the ATA2 gene.
|
gene in response to a combined stimulus of
hypertonicity and PMA. Specifically, stimulation of primary T cell
blasts with PMA plus 100 mM NaCl resulted in the induction of TNF-
protein expression (20). To determine whether TNF- gene
expression was altered in DN NFAT5 transgenic mice, T cell blasts were
rested for 16 h, then stimulated with ionomycin plus PMA or 100 mM
NaCl plus PMA for 8 h and RNA was isolated for Northern analysis.
The overall yield of RNA from cells stimulated with 100 mM NaCl plus
PMA was reduced 55% as compared with all other samples, reflecting
the toxicity of the hypertonic stimulus. Analysis of TNF- gene
expression, normalized to the level of GAPDH mRNA expressed,
demonstrated that ionomycin plus PMA induced TNF- mRNA expression
equally in both transgenic and nontransgenic cells. Moreover,
stimulation with 100 mM NaCl plus PMA resulted in a significant
inhibition of TNF- mRNA in both transgenic and nontransgenic cells
(Fig. 8, C and D). These results indicate that
the TNF- gene is not induced by hypertonicity. Moreover, NFAT5 does
not appear to play a role in the regulation of TNF- mRNA
expression. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biologic function of NFAT5 in vivo
Inhibition of NFAT5 function in thymocytes and mature peripheral T
cells in vivo through the development of transgenic mice expressing an
inhibitory or DN NFAT5 transgene demonstrated a significant reduction
both in thymic cellularity and in the number of mature peripheral T
cells, particularly CD8+ T cells. These results
imply a role for NFAT5 in T cell development and clearly demonstrate
that NFAT5 functions in vivo under normal physiologic conditions not
associated with overt hypertonic stress. These results are consistent
with the observed constitutive nuclear localization of NFAT5 in
thymocytes and quiescent or activated T cells (Fig. 1), as well as the
previously demonstrated induction of NFAT5 protein and transcriptional
activity upon T cell activation under isotonic conditions
(15). Although the present study restricted the inhibition
of NFAT5 function in vivo to thymus and mature T cells, inhibition of
NFAT5 in other cell types may also result in similarly impaired cell
growth or development given the ubiquitous nature of NFAT5 expression
(15, 19).
Analysis of transgenic thymocytes and T cells ex vivo demonstrated that
inhibition of NFAT5 resulted in increased sensitivity to hyperosmotic
stress as measured by cell viability, thus directly demonstrating the
osmoprotective function of NFAT5 in primary cells. The observed lack of
any enhanced sensitivity of transgenic T cells to other mechanisms of
inducing cell death (Fig. 3, B and D) further
substantiates the conclusion that NFAT5 specifically regulates
osmoprotective transcriptional responses. Transgenic T cells exhibited
no impairment in growth responses or cytokine production under normal
cell culture conditions (Fig. 5), further supporting the conclusion
that NFAT5 functions specifically to allow cells to optimally
compensate for hyperosmotic conditions. To further investigate the
osmoprotective function of NFAT5, two alternative means of inducing an
osmotic stress were used. Culture under conditions of medium depletion
results in a very slow increase in extracellular osmolarity, in
contrast to rapid exposure of cells to hypertonic medium, which is
particularly toxic to lymphoid cells (Fig. 3A and Ref.
36). The gradual increase in extracellular osmolarity that
takes place upon medium depletion (Fig. 6E) allows for the
optimal induction of adaptive compensatory mechanisms that may require
hours to be fully activated, such as adaptive transcriptional responses
potentially mediated by NFAT5. Consistent with this result, stable
overexpression of NFAT5 in the Jurkat T cell line resulted in enhanced
cell growth and viability under conditions of medium depletion, while
expression of DN NFAT5 impaired cell growth and viability (Fig. 6, C and D).
An alternative means of inducing an osmotic stress under isotonic
conditions uses amino acid deprivation. Amino acid limitation induces a
reduction in cell volume due to loss of intracellular amino acids,
which function as compatible organic osmolytes (24, 31).
Amino acid limitation thus induces osmocompensatory mechanisms similar
to those induced by exposing cells to a hypertonic environment, but it
does so by loss of intracellular osmolytes under isotonic conditions
rather than loss of intracellular water under hypertonic conditions
(24, 31). Interestingly, transgenic T cells exhibited
impaired cell growth upon both medium depletion and amino acid
deprivation (Figs. 6, A and B, and
7A). These results indicate that NFAT5 functions to allow
the cell to compensate for suboptimal growth conditions associated with
osmotic stress induced by either increased extracellular osmolarity
(medium depletion) or decreased intracellular osmolarity under isotonic
conditions (amino acid limitation). Thus, the osmoprotective function
of NFAT5 in ex vivo cultures, particularly in response to osmotic
stress induced not only by extracellular hyperosmolarity but also by
intracellular osmolyte loss under isotonic conditions, suggests that
the observed impairment in T cell development in vivo under physiologic
isosmotic conditions results from inhibition of the osmoprotective
function of NFAT5.
Exposure of cells to hyperosmotic stress results in osmotic outflow of water and a concomitant reduction in cell volume, resulting in an increase in intracellular ionic strength. Cell survival requires the homeostatic maintenance of intracellular conditions within an appropriate range that is compatible with normal metabolic and biochemical function within the cell. While most cell types are capable of rapidly activating various ionic transport mechanisms to induce a regulatory volume increase in response to hyperosmotic stress (37, 38), lymphoid cell lines as well as primary thymocytes preferentially undergo apoptosis (36). The reduced thymic cellularity observed in the DN NFAT5 transgenic mice, as well as the increased sensitivity of both thymocytes and mature T cells to hyperosmolar stress, may thus reflect a greater dependence on NFAT5-dependent adaptive mechanisms to compensate for osmotic stress. Moreover, the extremely high density of cells within the thymus, combined with abundant apoptosis associated with thymic selection and the associated release of macromolecules that may increase extracellular osmolarity, may create a microenvironment in which NFAT5-dependent osmocompensatory mechanisms are critical. The importance of the thymic microenvironment in revealing a function for NFAT5 in vivo is highlighted by the lack of any NFAT5-specific phenotype upon culture of thymocytes or mature T cells ex vivo under normal tissue culture conditions. However, the sensitivity of thymocytes and mature T cells ex vivo to conditions associated with hyperosmotic stress provides further support for the notion that cells within the thymic microenvironment are subject to osmotic stress conditions that are optimally compensated for by NFAT5-dependent transcriptional responses.
Cell growth and physiologic osmotic stress
The present studies specifically investigate the function of NFAT5 in developing and mature T cells. However, the ubiquitous expression of NFAT5 mRNA (10, 15, 18), and in particular the enhanced expression of NFAT5 protein in actively proliferating cells (15, 19), suggests that the demands of cell growth may impose an osmotic stress that is relieved through transcription of NFAT5 target genes in all cell types. As described above, exposure of cells to a hypertonic environment results in a decrease in cell volume that is associated with an increase in intracellular ionic strength. NFAT5-dependent transcription of genes that increase the intracellular concentration of compatible organic osmolytes provides a means to increase cell volume, thereby restoring cell volume and intracellular ionic strength to a steady state that is optimal for normal cell function. However, with the exception of cells of the renal medulla, mammalian cells exist in an isosmotic environment.
Under isosmotic conditions, a reduction or loss of intracellular osmolytes may also lead to a functional reduction in cell volume and an increase in ionic strength that could compromise normal cellular metabolic and biochemical function. The rapid synthesis of macromolecules (e.g., proteins, nucleic acids) from low m.w. precursors (e.g., amino acids, nucleotides) that takes place concomitantly with the induction of cell growth represents one mechanism by which the concentration of organic as well as ionic osmolytes within the cell may be effectively reduced under isosmotic conditions (37, 38). The biosynthetic demands of cell growth under isosmotic conditions may induce an osmotic stress that could be deleterious to optimal cell growth without a mechanism to compensate for the osmotic disequilibrium. Thus, the impaired thymic development observed in DN NFAT5 transgenic mice, as well as the impaired cell growth under conditions of osmotic stress, may reflect the inability of cells that are defective in NFAT5 function to completely compensate for osmotic stress associated with cell growth.
It is, of course, important to note the lack of any impairment in growth of transgenic T cells ex vivo under normal tissue culture conditions, which indicates that the level of osmotic stress inherent in cell growth under normal tissue culture conditions does not require NFAT5-dependent compensation. Rather, it may be the combination of intrinsic osmotic stress associated with cell growth in conjunction with extracellular osmotic stress within the cellular microenvironments found in vivo that gives rise to NFAT5-dependent responses that are necessary for optimal cell growth. Consistent with this, overexpression of the putative Drosophila NFAT5 homolog gives rise to both eye development and synaptogenesis phenotypes that could result from altered cell growth (21, 22).
NFAT5 target genes
The present studies provide direct functional evidence that NFAT5
plays a role in the induction of the AR gene in response to
hyperosmotic stress (Figs. 2 and 8). Regulation of target genes that
control intracellular compatible osmolyte concentrations is consistent
with the observed osmoprotective function of NFAT5 demonstrated in this
study. Amino acids, in addition to their function as critical cell
nutrients, also function as an important class of compatible osmolytes
that are depleted during cell growth as a result of the induction of
protein synthesis. Thus, reduction in intracellular amino acids during
cell growth may contribute to a state of osmotic stress that could,
without compensation, result in impaired cell growth. Among the various
amino acid transporters, function of the transport system A for neutral
amino acids in particular has been shown to be osmosensitive in a
variety of cell systems (32), including rat thymocytes
(39), suggesting that genes encoding the system A
transporter may be regulated by NFAT5 (24). Analysis of
the system A transporter gene ATA2 (Fig. 8, A and
B), which has been shown to be induced by hypertonicity
(33, 34), demonstrated that induction of this gene in
response to extracellular hypertonicity was inhibited in cells from DN
NFAT5 transgenic mice, thus indicating that NFAT5 plays a role (direct
or indirect) in the osmotic regulation of the ATA2 gene. However,
induction of ATA2 mRNA by amino acid deprivation was not only stronger
than the induction observed with a hypertonic stimulus, but it also
showed no inhibition in cells from the DN NFAT5 transgenic mice, in
contrast to the AR gene. These results indicate that the ATA2 amino
acid transporter gene exhibits multiple mechanisms of regulation. Given
that amino acids function not only as compatible osmolytes but also as
essential cell nutrients, an NFAT5-independent means of regulating the
ATA2 gene is not surprising and likely results from activation of as
yet poorly characterized amino acid-sensing pathways
(40).
In addition to genes that regulate intracellular compatible osmolytes,
genes encoding the inflammatory cytokines TNF- and lymphotoxin
have also been identified as putative NFAT5 target genes
(20). In the present studies (Fig. 8, C and
D), comparison of the induction of TNF- mRNA in T cell
blasts from DN NFAT5 transgenic mice and transgene-negative littermate
controls showed no differences, suggesting that NFAT5 is not involved
in the induction of the TNF- gene in primary T lymphocytes.
Moreover, while stimulation with ionomycin plus PMA resulted in the
induction of TNF- mRNA, stimulation with PMA plus 100 mM NaCl
actually resulted in a decrease in TNF- mRNA levels. This result
contradicts results in which an increase in the production of TNF-
protein, as measured by ELISA, was observed in primary T cell blasts
that had been similarly treated (20). Exposure of primary
lymphocytes to hypertonic conditions resulted in significant toxicity,
as reflected by a 55% reduction in the recovery of mRNA from cells
incubated in 100 mM NaCl. Therefore, it is possible that an apparent
increase in TNF- protein production upon hypertonic stimulation
(20) could result from release of intracellular protein as
a result of cell death rather than an induction of TNF- gene
expression. However, the possibility that inhibition of the endogenous
NFAT5 protein by the DN NFAT5 transgene may be insufficient to alter
TNF- gene expression in T cell blasts cannot be excluded. Further
studies will be required to determine whether cytokine genes such as
these represent physiologically relevant targets of NFAT5.
Conclusion
In summary, the results presented here reveal new insight into the biologic function of the NFAT5 transcription factor by not only demonstrating the osmoprotective function of NFAT5 in primary cells but also demonstrating that NFAT5 is necessary for optimal T cell development in vivo. Initially defined as a transcription factor that regulates the transcription of tonicity-responsive genes upon exposure to extracellular hyperosmotic stress, a condition of potentially unique physiologic relevance to cells of the renal medulla, the present data support a model in which extracellular hyperosmolarity in fact mimics osmotic stresses that occur under physiologic, isosmotic conditions, particularly within the cellular microenvironments that exist in vivo. The activation of NFAT5 in response to overt hyperosmotic stress as it occurs in the renal medulla (17) may thus represent a specialized adaptation of a physiologic osmoprotective or cell volume regulatory mechanism that is functional in essentially all cell types in vivo under isosomotic conditions (37, 38). In this context, NFAT5 mediates adaptive cell volume increases that are necessary to reestablish conditions of intracellular ionic strength most compatible with optimal cell function by regulating the transcription of osmoregulatory genes in response to conditions of intracellular osmotic stress. Given the dynamic alterations in cell volume and ionic strength that take place during cell growth, NFAT5 may play a particularly important role in optimally compensating for normal osmotic stresses associated with actively proliferating cells in vivo. In light of the experimentally intractable problem of accurately reproducing normal cellular microenvironments ex vivo under tissue culture conditions, evaluation of this model of the biologic function of NFAT5 will require further genetic manipulation of NFAT5 function in whole animal systems.
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Acknowledgments |
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Footnotes |
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2 Address correspondences and reprint requests to Dr. Steffan N. Ho, Department of Pathology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0644. E-mail address: snho{at}ucsd.edu
3 Abbreviations used in this paper: AR, aldose reductase; HA, hemagglutinin; EBSS, Earl’s balanced salt solution; EST, expressed sequence tag; DN, dominant negative; DBD, DNA binding domain; BrdU, bromodeoxyuridine; PI, propidium iodide.
Received for publication July 22, 2002. Accepted for publication September 13, 2002.
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