Specific TSC22 domain transcripts are hypertonically
induced and alternatively spliced to protect mouse kidney
cells during osmotic stress
Diego F. Fiol, Sally K. Mak* and Dietmar Ku
¨ltz
Physiological Genomics Group, Department of Animal Science, University of California, Davis, CA, USA
In the mammalian kidney, the papilla is routinely
exposed to severe hyperosmolality and to large changes
in interstitial osmolality. These stressful conditions are
a prerequisite for operation of the urinary concentra-
ting mechanism and maintenance of systemic salt
and water balance. Thus, renal papillary (and outer
medullary) cells have special mechanisms to adapt to
variable and severe hyperosmolality. Cellular adapta-
tion to hyperosmotic stress is controlled via a complex
array of cellular signaling mechanisms that modify
gene and protein expression and protein function
to promote osmoprotection [1]. Such signaling
Keywords
aldosterone; hyperosmotic stress;
hypertonicity; kidney; mIMCD3 cells
Correspondence
D. Ku
¨ltz, Physiological Genomics Group,
Department of Animal Science, University of
California, Davis, One Shields Avenue,
Davis, CA 95616, USA
Fax: +1 530 752 0175
Tel: +1 530 752 2991
E-mail: dkueltz@ucdavis.edu
*Present address
The Parkinson’s Institute, Sunnyvale, CA,
USA
(Received 28 July 2006, revised 23 October
2006, accepted 3 November 2006)
doi:10.1111/j.1742-4658.2006.05569.x
We recently cloned a novel osmotic stress transcription factor 1 (OSTF1)
from gills of euryhaline tilapia (Oreochromis mossambicus) and demonstra-
ted that acute hyperosmotic stress transiently increases OSTF1 mRNA
and protein abundance [Fiol DF, Ku
¨ltz D (2005) Proc Natl Acad Sci USA
102, 927–932]. In this study, a genome-wide search was conducted to iden-
tify nine distinct mouse transforming growth factor (TGF)-b-stimulated
clone 22 domain (TSC22D) transcripts, including glucocorticoid-induced
leucine zipper (GILZ), that are orthologs of OSTF1. These nine TSC22D
transcripts are encoded at four loci on chromosomes 14 (TSC22D1, two
splice variants), 3 (TSC22D2, four splice variants), X (TSC22D3, two
splice variants), and 5 (TSC22D4). All nine mouse TSC22D transcripts are
expressed in renal cortex, medulla and papilla, and in the mIMCD3 cell
line. The two TSC22D3 transcripts (including GILZ) are upregulated by
aldosterone but not by hyperosmolality in mIMCD3 cells. In contrast,
TSC22D4 is stably upregulated by hyperosmolality in mIMCD3 cells and
increased in renal papilla compared with cortex. Moreover, all four
TSC22D2 transcripts are transiently upregulated by hyperosmolality and
resemble tilapia OSTF1 in this regard. All TSC22D2 transcripts depend
on hypertonicity as the signal for their upregulation and are unresponsive
to increases in cell-permeable osmolytes. mRNA stabilization is the mech-
anism for TSC22D2 upregulation by hyperosmolality. Overexpression of
TSC22D2–4 in mIMCD3 cells confers protection towards osmotic stress,
as evidenced by a 2.7-fold increase in cell survival after 3 days at
600 mOsmolÆkg
)1
. Based on variable responsiveness to aldosterone and
hyperosmolality in kidney cells we conclude that mouse TSC22D genes
have diverse physiological functions. TSC22D2 and TSC22D4 are involved
in adaptation of renal cells to hypertonicity suggesting that they represent
important elements of osmosensory signal transduction in mouse kidney
cells.
Abbreviations
GILZ, glucocorticoid-induced leucine zipper; OSTF1, osmotic stress transcription factor 1; TGF, transforming growth factor; TonEBP, tonicity-
response element binding protein.
FEBS Journal 274 (2007) 109–124 ª2006 University of California Journal compilation ª2006 FEBS 109
mechanisms stimulate accumulation of the compatible
organic osmolytes glycine-betaine, myo-inositol, tau-
rine, sorbitol, and glycerophosphorylcholine [2–4].
Accumulation of glycine-betaine, inositol, and sorbitol
is transcriptionally regulated and depends, at least in
part, on the transcription factor tonicity-response ele-
ment binding protein (TonEBP) [5]. TonEBP also acti-
vates additional genes that are important for osmotic
stress adaptation, including HSP70 and UT-A urea
transporter [6,7]. In addition to the TonEBP pathway,
hyperosmolality activates a very complex network of
intracellular signaling pathways in renal medullary
cells, including MAP kinase pathways [8], the p53
pathway [9], DNA-dependent protein kinases [10], and
protein kinase A-dependent pathways [11]. Thus, the
response of mammalian kidney cells to hyperosmotic
stress is highly complex and involves many different
pathways and elements. Proper understanding of the
cellular hyperosmotic stress response enabling compu-
tational modeling of this response is highly desirable
because it would open avenues for manipulating stress-
resistance networks of cells in states of renal disease
and disorders of water and electrolyte balance. How-
ever, better knowledge about key elements of osmo-
sensory signal transduction pathways and their
interactions within osmotic stress signaling networks is
required before in silico models that correctly reflect
and predict cellular responses to osmotic stress can be
devised.
We recently cloned a novel immediate early gene
osmotic stress transcription factor 1 (OSTF1) that is
involved in the cellular osmotic stress response of gill
cells of euryhaline tilapia [12]. In this fish, OSTF1
mRNA and protein levels rapidly and transiently
increase in response to hyperosmotic stress, peaking at
2 and 4 h, respectively. The rapid and transient activa-
tion kinetics is characteristic of immediate early genes.
OSTF1 belongs to the TSC22D family of leucine zip-
per proteins that are thought to be transcription fac-
tors in mammalian cells. In mouse tissues, TSC22D
genes are regulated by glucocorticoids and transform-
ing growth factor b(TGF-b) [13,14]. However, nothing
is known about the osmotic regulation of any mouse
TSC22D isoform. In addition, a systematic genome-
wide analysis of mouse TSC22D gene products, identi-
fying all family members, is lacking.
In this study, we identified nine murine TSC22D
transcripts and investigated their regulation by hyper-
osmolality and aldosterone, which is a mineralocorti-
coid hormone important for modulation of the urinary
concentrating mechanism. Moreover, TSC22D2 was
identified as the closest functional mouse ortholog of
tilapia OSTF1 and the mechanism and physiological
significance of hyperosmotic upregulation of this gene
was analyzed.
Results
Identification of TSC22D family members in the
mouse genome
We recently cloned tilapia OSTF1 and showed that it
is a rapidly induced osmotic stress transcription factor
[12]. To identify possible functional homologs of tila-
pia OSTF1 in mammals, we carried out an exhaustive
search of the complete annotated mouse genome using
the ENSEMBL database (http://www.ensembl.org)
[15]. This search yielded six gene products with expec-
tation values ranging from 6.1e-69 to 3.2e-21. These
proteins are the products of transcripts encoded at
four different loci (Table 1). In order to avoid ambi-
guity, we follow the recently updated and unified
MGD nomenclature guidelines for TSC22D proteins
in this study (Mouse Genome Informatics) [16].
TSC22D1-1 and TSC22D1-2 are splice variants that
are located on chromosome 14, TSC22D2 is located
on chromosome 3, TSC22D3-1 and TSC22D3-2 are
splice variants that are located on chromosome X, and
TSC22D4 is located on chromosome 5 (Table 1).
Although two of these proteins have been previously
described as TSC-22 (TSC22D1-2) and glucocorticoid-
induced leucine zipper (GILZ) (TSC22D3-2), the other
four have not been characterized or only referred to as
TSC22-like or GILZ-like proteins. Multiple sequence
Table 1. Mouse OSTF1-like predicted transcripts. aa, amino acid; nt, nucleotide.
Transcript Name
Chromosome
location Accession EMBL ENSEMBL
Length
(aa) (nt)
OSTF1 homology
Score E-value
TSC22D1-1 14 band D3 AF201285 ENSMUST00000048371 1057 4581 298 2.5e-26
TSC22D1-2 TSC-22 14 band D3 L25785 ENSMUST00000022587 143 1670 299 1.0e-27
TSC22D2 3 band D BC058221 ENSMUST00000029383 167 2002 256 3.7e-23
TSC22D3-1 X band F1 AF201289 ENSMUST00000033807 201 1377 688 6.1e-69
TSC22D3-2 GILZ X band F1 AF024519 ENSMUST00000055738 137 1972 324 2.3e-30
TSC22D4 THG1 5 band G1 AF315352 ENSMUST00000049554 387 2672 240 3.2e-21
Osmotic regulation of TSC22D in kidney cells D. F. Fiol et al.
110 FEBS Journal 274 (2007) 109–124 ª2006 University of California Journal compilation ª2006 FEBS
alignment shows that the six mouse proteins and
tilapia OSTF1 share a conserved region of 70 amino
acids, which comprises the TSC22D family signature
motif and a leucine-zipper domain. The N- and C-ter-
mini are least conserved in all proteins. In particular,
N-termini are highly heterogeneous, accounting for
variability in total protein lengths ranging from 124 to
1057 amino acids (Table 1, Fig. 1). The protein with
the highest overall sequence similarity to tilapia
OSTF1 is TSC22D3-1, based on highest degree of con-
servation of the N-terminus (Fig. 1).
Expression of TSC22D family members in kidney
mouse and mIMCD3 cells
We analyzed the expression of the six mouse TSC22D
transcripts in kidney to learn whether any of them
functionally resembles tilapia OSTF1. Levels of expres-
sion of the six transcripts were determined by quantita-
tive PCR in three regions of the kidney that are
characterized by increasing interstitial osmolality in the
order from cortex (lowest) to medulla (intermediate) to
papilla (highest). All six transcripts are expressed in all
three regions of the kidney. Renal TSC22D2 is most
abundant being expressed at levels that are between
one and two orders of magnitude lower than that of
the highly abundant ribosomal protein L32 (Fig. 2).
The level of expression of TSC22D1-2 and TSC22D2
is similar in cortex, medulla, and papilla (Fig. 2).
However, TSC22D3-1, TSC22D3-2, and TSC22D4 are
significantly more abundant in papilla, whereas
TSC22D1-1 is more abundant in cortex. The data
suggest that hyperosmolality could potentially be
responsible for altering the expression of four TSC22D
transcripts. The level of expression of all six transcripts
was also determined in mIMCD3 cells. All six tran-
scripts are expressed in mIMCD3 cells and expression
levels are similar to those in mouse kidney medulla
in vivo (data not shown). Therefore, mIMCD3 cells are
a good model for evaluating mechanisms of regulation
of the mouse TSC22D transcripts.
B
A
Fig. 1. Schematic structure (A) and multiple sequence alignment of the TSC22D motif (B) of tilapia OSTF1 and mouse TSC22D family mem-
bers identified by a genome-wide search. Large gray cylinders correspond to the conserved TSC22 ⁄leucine zipper motif. Smaller white cylin-
ders represent local regions of high homology. Residues shaded in darker tones correspond to higher level of homology in the alignment.
Fig. 2. Relative expression levels of mouse TSC22D transcripts in
kidney papilla, medulla and cortex.Expression levels of TSC22D
transcripts were determined by quantitative PCR. C, cortex; M,
medulla; P, papilla. Results are depicted as means ± SEM of three
independent experiments. Significant differences between kidney
regions are indicated by asterisks (P<0.05).
D. F. Fiol et al. Osmotic regulation of TSC22D in kidney cells
FEBS Journal 274 (2007) 109–124 ª2006 University of California Journal compilation ª2006 FEBS 111
Regulation of TSC22D transcripts in mIMCD3
cells by hyperosmotic stress and aldosterone
The responsiveness of TSC22D transcripts to hyper-
osmotic stress and ⁄or aldosterone treatment was
determined in mIMCD3 cells in 24-h time course
experiments. Acute hypertonicity increases the expres-
sion of TSC22D2, TSC22D4 and TSC22D3-2. Of
interest, TSC22D2 is elevated early and transiently,
showing increases of 2.6- and 3.1-fold at 4 and 6 h of
treatment, respectively, and returning to baseline levels
within 12 h. In contrast, TSC22D3-2 and TSC22D4
show a slower but more stable upregulation, increasing
three- and sixfold, respectively, after 24 h of treatment
(Fig. 3). These results are in agreement with higher
levels of TSC22D3-2 and TSC22D4 in renal papilla
in vivo (see previous paragraph, Fig. 2). Aldosterone
induces a rapid increase in TSC22D3-2 (4-fold at 1 h,
AB
CD
EF
Fig. 3. Response of TSC22D transcripts to hyperosmotic stress and aldosterone in mIMCD3 cells.Cells were exposed to hyperosmolality by
increasing medium osmolality from 300 to 550 mOsm by addition of NaCl (filled circles), to 1 lMaldosterone (triangles), or to both hyper-
osmolality and aldosterone simultaneously (open circles). Each panel shows the time course response for a particular transcript determined
by quantitative PCR. Results are depicted as means ± SEM for three independent experiments. Asterisks indicate significantly differences
with respect to the value at time zero (P<0.05).
Osmotic regulation of TSC22D in kidney cells D. F. Fiol et al.
112 FEBS Journal 274 (2007) 109–124 ª2006 University of California Journal compilation ª2006 FEBS
33-fold at 12 h, 10-fold at 24 h) and TSC22D3-1 (five-
fold at 4–6 h hours) (Fig. 3). Of interest, a combina-
tion of hyperosmotic stress and aldosterone does
not potentiate the transient increase in TSC22D3-2
(Fig. 3). By contrast, hyperosmotic stress and aldoster-
one in combination prevent transient short-term effects
and offset each other. Taken together, the data on
osmotic regulation of TSC22D transcripts implicate
TSC22D2 as the closest functional homolog of tilapia
OSTF1.
Identification of alternative TSC22D2 transcripts
Because of its similar osmotic regulation compared
with tilapia OSTF1 we investigated mouse TSC22D2
in more depth. Two additional alternative transcripts
encoding splice variants of TSC22D2 protein were
identified that differed from the original cDNA
ENSMUST00000029383 (TSC22D2-1; Fig. 1, Table 1).
These two additional cDNAs (GENSCAN000000732
55 ¼TSC22D2-2 and ENSMUSESTG00000010047 ¼
TSC22D2-3) were predicted using the Ensembl data-
base and gene prediction software genscan and
genomewise ⁄genewise.genscan is a bioinformatic
tool that predicts gene loci and their exon ⁄intron
composition based on the genomic DNA sequence
[17]. genomewise ⁄genewise gene-prediction software
assembles cDNA sequences based on the analysis and
integration of EST data [18]. Taking advantage of
information provided by these two complementary
approaches we thoroughly examined the TSC22D2
gene for alternative splicing events. Alignment of the
three identified TSC22D2 splice variants against the
genomic TSC22D2 sequence revealed differences in
exon composition. Two splice variants (TSC22D2-1 ⁄2)
consist of three exons, whereas the third splice variant
(TSC22D2-3) has four exons as a result of inclusion of
an extra 72 bp exon in the second position (Fig. 4A).
The length of the first and last exons is also variable in
the three splice variants of TSC22D2 (Fig. 4A).
We then tested for expression of the newly predicted
TSC22D2 transcripts (TSC22D2-2 ⁄3) in mouse kidney
cells. Specific PCR primer pairs were designed to
amplify TSC22D2-2 (primer pair E–F), TSC22D2-3
(primer pair A–C), and all splice variants (primer pairs
A–B and A–D). We had already used primer pair A–B
for previous quantification of overall TSC22D2 tran-
script abundance as it amplifies all possible splice vari-
ants (Fig. 4A, Table S1). Expression of TSC22D2-2
and TSC22D2-3 was confirmed based on the presence
of RT-PCR products having the expected sizes
(Fig. 4B,lanes A–C and E–F, respectively). In addi-
tion, using the primer pair A–D we detected three
different PCR products of 493, 406 and 334 bp instead
of the two products that we expected based on the pri-
mer design shown in Fig. 4A (amplicon ± exon 2).
Therefore, the three PCR products obtained with
primers A–D were purified, sequenced, and aligned to
each other (Fig. 4C). The sequence of two of these
PCR products matched the predicted sequence for
TSC22D2-1 ⁄2 and TSC22D2-3 (Fig. 4C). These
sequences differed by the presence of the 72-bp exon 2
in TSC22D2-3 as predicted.
Surprisingly, however, an additional unpredicted
fragment was discovered by PCR analysis (TSC22D2-
4, Fig. 4). Sequencing of the corresponding PCR prod-
uct confirmed that TSC22D2-4 represents an entirely
novel splice variant that was not predicted by any of
the bioinformatics methods used in our study nor
reported to exist previously. TSC22D2-4 included an
alternative second exon of 159 bp but lacked the 72 bp
exon 2. Schematic exon ⁄intron structures of all four
TSC22D2 splice variants are compared in Fig. 4D with
emphasis on the two alternative exons 2A (72 bp) and
2B (159 bp), which are not present simultaneously in
any TSC22D2 transcript in mIMCD3 cells (Fig. 4B).
Next, we analyzed the exon ⁄intron regions flanking
TSC22D2 exons 2A and 2B. All of these sequences
match splice donor and acceptor consensus sites very
well (5¢-AG ⁄GT AG ⁄G-3¢) (Table 2). In addition, the
homologous intron ⁄exon regions that flank exons 2A
and 2B in human TSC22D2 are 95% identical to
mouse sequences indicating a high degree of conserva-
tion of these critical areas compared with the overall
much lower homology of TSC22D2 genomic sequence
(< 50%; data not shown). Taken together, these
observations strongly support alternative splicing
events that give rise to TSC22D2 transcripts with dif-
ferent exon 2 sequences.
Protein products for TSC22D2-1 and TSC22D2-2
differ only by variable length of the first and last exons
from each other (Fig. 4A). In contrast, TSC22D2-3
and TSC22D2-4 differ more substantially from the
other TSC22D2 variants because of the presence of an
additional exon (exon 2A ⁄2B) (Fig. 4E). In particular,
TSC22D2-4 differs greatly from the other variants
because it lacks a large portion of the N-terminus due
to the presence of four in-frame stop codons in
exon 2B (Fig. 4C,E). An ATG codon following imme-
diately after the last of these four stop codons may
represent the transcription initiation site for a protein
with a much shorter N-terminus (Fig. 4E). Each of the
four possible TSC22D2 protein products also differs
with respect to the presence of consensus phosphoryla-
tion sites for a number of stress-responsive protein
kinases (Fig. 4E).
D. F. Fiol et al. Osmotic regulation of TSC22D in kidney cells
FEBS Journal 274 (2007) 109–124 ª2006 University of California Journal compilation ª2006 FEBS 113