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 re
Trang 1induced 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.
Trang 2mechanisms 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, nothtransform-ing
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
Trang 3alignment 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).
Trang 4Regulation 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,
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 l M aldosterone (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).
Trang 533-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
55¼ TSC22D2-2 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)
Trang 6Response of the four TSC22D2 variants to
hyperosmolality and aldosterone
We quantified abundances of individual TSC22D2
transcripts by quantitative PCR using the PCR primers
depicted in Fig 4A and Table 1 All four TSC22D2 variants are expressed at comparable levels in mouse kidney papilla, medulla, and cortex (data not shown)
To analyze the regulation of the four TSC22D2 vari-ants in response to hyperosmolality and aldosterone
C
D
E
A
B
Fig 4 Detection and characterization of alternative TSC22D2 transcripts (A) Align-ment of TSC22D2 (ENSMUST00000029383) with TSC22D2 transcripts predicted by
GENSCAN and GENOMEWISE ⁄ GENEWISE and genomic DNA (chromosome 3) Positions of PCR primers designed to differentiate between splice variants are indicated by arrows below the schematic representation
of genomic DNA (B) Products of PCR amplification using splice variant-specific TSC22D2 PCR primers (C) Nucleotide sequence of the PCR products amplified by the A–D primer pair In-frame stop and start codons are over-lined in gray and black, respectively (D) Schematic representation
of the exon–intron structure of all identified TSC22D2 transcripts Positions of PCR prim-ers designed to amplify individual splice vari-ants are indicated by arrowheads (E) Partial deduced amino acid sequence of the exon 2 region of all identified TSC22D2 transcripts The TSC22D domain is boxed Regions encoded by exon 2A and exon 2B are prin-ted in bold Splice variant-specific potential phosphorylation sites are underlined Aster-isk indicates the presence of an in-frame stop codon in the corresponding mRNA.
Trang 7we exposed mIMCD3 cells to either of those stimuli
alone and to a combination of both All four
TSC22D2 variants are transiently upregulated by
hyperosmotic stress (Fig 5) The highest degree
of hyperosmotic upregulation was observed for
TSC22D2-4 Aldosterone with or without
hyperosmol-ality did not significantly affect the abundance of any
individual TSC22D2 variant, consistent with the results
obtained when all TSC22D2 variants were quantified together (Figs 3, 5)
Regulation of TSC22D2 variants by hyperosmolality
To identify the signal responsible for hyperosmotic upregulation of TSC22D2 we exposed mIMCD3 cells for 5 h to hyperosmotic media (550 mOsmolÆkg)1) pre-pared by addition of NaCl, choline chloride, sodium gluconate, mannitol, urea or glycerol TSC22D2-4 was always upregulated by hypertonic media (choline chlor-ide, sodium gluconate, mannitol, NaCl) independent of the presence of Na+ or Cl– in such media (Fig 6) In contrast, hyperosmolality due to nonhypertonic gly-cerol or urea did not alter TSC22D2-4 levels (Fig 6) Similar results were obtained for the other three TSC22D2 variants (data not shown) These data dem-onstrate that neither Na+nor Cl–nor hyperosmolality per se represent the signal for upregulation of
Table 2 Sequences corresponding to 3¢ acceptor and 5¢ donor
exon ⁄ intron boundaries in TSC22D2 transcripts.
5¢-Donor
EXON ⁄ intron
3¢-Acceptor intron⁄ EXON Exon 1 AGACAG ⁄ gtatgtaca gtctcacag ⁄ GAATCC Exon 2 A
Exon 1 AGACAG ⁄ gtatgtaca .ctttgctag ⁄ AATTTT Exon 2B
Exon 1 AGACAG ⁄ gtatgtaca .tttttccag⁄ TGCATC Exon 3
Exon 2 A GGATAG ⁄ gtatgatta .tttttccag ⁄ TGCATC Exon 3
Exon 2B AAATTG ⁄ gtaagactt .tttttccag ⁄ TGCATC Exon 3
Exon 3 GCAATG ⁄ gtaagtagg .tcttcacag ⁄ GATCTG Exon 4
Fig 5 Response of TSC22D2 alternative transcripts to hyperosmotic stress 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 l M aldosterone (triangles), or to both hyper-osmolality and aldosterone simultaneously (open circles) Each panel shows the time course response for a particular TSC22D2 alternative transcript as 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).
Trang 8TSC22D2 Instead, hypertonicity is the stimulus that
triggers TSC22D2 upregulation
mRNA stabilization of TSC22D2 during
hyperosmotic stress
Next, we analyzed the mechanism of TSC22D2
upreg-ulation in response to hyperosmotic stress
Transcrip-tion in mIMCD3 cells was completely blocked by a
1 h preincubation in 10 lm actinomycin D Even 5 lm actinomycin was sufficient to effectively block tran-scription in mIMCD3 cells (Fig S2) Cells were then exposed to hyperosmotic stress, aldosterone, and control conditions (isosmotic medium) The half-life
of TSC22D2-4 was 2.8 ± 0.2 h for controls and aldosterone treatment but increased to > 20 h as a consequence of hyperosmolality (Fig 7) TSC22D2-1⁄ 2 and TSC22D2-3 responded similarly, with half-lives increasing from 2.8 ± 0.3 to > 10 h and 2.3 ± 0.3 to
> 15 h, respectively, in response to hyperosmolality (data not shown) These results indicate that mRNA stabilization is the mechanism responsible for hyper-osmotic upregulation of TSC22D2 transcripts
Osmoprotection of mIMCD3 cells by overexpression of TSC22D2-4
To evaluate whether TSC22D2 upregulation protects cells from hyperosmotic stress we generated stably transfected mIMCD3 cells that overexpress
TSC22D2-4 We first generated a mIMCD3 cell line with a Flp recombinase target site stably integrated into the genome (mIMCD3FRT cells; Fig S1) to generate a good control for future experiments with stably selec-ted cells We then cotransfecselec-ted V5-epitope-tagged TSC22D2-4 in pcDNA5FRT vector together with a Flp recombinase expression vector to insert
TSC22D2-4 into the FRT site in exchange for the LacZ gene The transgenic TSC22D2-4 cell line expressed 100-fold higher levels of TSC22D2-4 compared with mIMCD3FRT control cells (Fig 8A) A single protein with the expected molecular mass (17 kDa) was detec-ted in TSC22D2-4 cells using V5 antibody (Fig 8B) The transgenic TSC22D2-4 cells showed signifi-cantly greater hyperosmotic stress tolerance than mIMCD3FRT control cells We incubated these two cell lines for 24 h under hyperosmotic stress conditions that lead to a high frequency of apoptosis in wild-type mIMC3 cells (600–650 mOsmÆkg)1) [19] Under these conditions, TSC22D2-4 cells had a significantly improved phenotype (Fig 9A) and cell numbers were significantly higher compared with mIMCD3FRT con-trol cells (Fig 9B), indicating that high levels of TSC22D2-4 protect cells during hyperosmotic stress
Discussion
Mammals have four loci encoding at least nine TSC22D transcripts
We have identified four loci in the mouse genome that encode nine homalogs of the tilapia osmotic stress
Fig 6 Response of TSC22D2-4 to different hyperosmotic media in
mIMCD3 cells Osmolarity was increased from 300 to 550 mOsm
with the addition of the indicated compounds After 5 h, cells were
collected and TSC22D2-4 mRNA levels were determined by
quanti-tative PCR Results represent means ± SEM for three independent
experiments Asterisks indicate significant differences with respect
to isosmotic controls (P < 0.05).
Fig 7 Stability of TSC22D2-4 transcript mIMCD3 cells were
prein-cubated for 1 h with 10 lgÆmL)1actinomycin D in isosmotic
med-ium (300 mOsmolÆkg)1) Treatments were initiated at time zero
when cells were exposed to hyperosmolality by increasing medium
osmolality to 550 mOsmÆkg)1by addition of NaCl (black circles), to
1 l M aldosterone (black triangles), or isosmotic control conditions
(open circles) mRNA levels were determined by quantitative
real-time PCR and normalized to L32 mRNA Results are depicted as
means ± SEM for three independent experiments Asterisks
indi-cate significantly different values with respect to the value at time
zero (P < 0.05).
Trang 9transcription factor OSTF1 All four genes belong to
the TSC22D family of leucine zipper proteins that
form homo- and heterodimers with other family
mem-bers Four TSC22D isoforms have previously been
des-cribed: TSC22D1-2 (TSC-22), TSC22D3-2 (GILZ),
TSC22D3-1 and TSC22D4
TSC22D1-2 was first isolated based on rapid and
transient transcriptional induction by TGF-b1 [13] It
also increases in response to anticancer drugs,
prog-esterone, and growth inhibitors [20] and has been
implied in mechanisms of tumorigensis
TSC22D3-2 was identified as a protein that is
induced following the treatment of thymocytes with
dexamethasone [14] Its mRNA increases threefold as
early as 30 min and by more than 10-fold within 4 h
of aldosterone exposure in principal cells of the renal
cortical collecting duct [21] In contrast, it is
downreg-ulated by estrogen in MCF-7 human breast cancer
cells [22] GILZ interacts with NF-jB and Raf and
inhibits AP-1, FoxO3, and Raf-mediated apoptotic
pathways [23–25] This protein mediates aldosterone
actions by stimulation of epithelial sodium
trans-port in kidney [26]
TSC22D3-1 was identified in porcine brain as a
77 kDa protein that shares immunoreactivity with the sequence-unrelated nonamer neuropeptide DSIP [27]
It was later found to be the most highly glucocorti-coid-induced cDNA among over 9000 tested in a cDNA gene chip array in human peripheral blood mononuclear cells [28]
TSC22D4 was identified in humans as a protein cap-able of forming heterodimers with TSC-22 [20] Its mouse homolog is involved in pituitary organogenesis [29]
In this study we identified five additional TSC22D transcripts that are encoded by genes located on chro-mosomes 3 (TSC22D2-1, TSC22D2-2, TSC22D2-3, TSC22D2-4) and 14 (TSC22D1-1) Although some of these novel transcripts have been previously described
in the context of high-throughput cDNA sequencing projects [30,31] their functions are unknown However, based on their sequence similarity to known TSC22D proteins they may be transcription factors that are involved in the regulation of cell proliferation, apop-tosis, and stress response pathways
A
B
Fig 9 TSC22D2-4 confers increased tolerance to hyperosmotic stress in mIMCD3 cells (A) Representative images of transfected and control (FRT) cells after exposure to 600 and 650 mOsm for
24 h (B) Count of viable transfected and control (FRT) cells after exposure to isoosmotic (300 mOsm) or hyperosmotic (600 mOsm) media for 72 h Asterisks indicate significant differences (P < 0.05) Results represent means ± SEM for three independent experiments.
A
B
Fig 8 Overexpression of TSC22D2-4 in mIMCD3 cells (A)
Deter-mination of expression levels of endogenous and transfected
TSC22D2-4 by quantitative real-time PCR Abundance is expressed
relative to L32 content Error bars are too small to be visible on the
logarithmic scale that is depicted Asterisks indicate significant
dif-ferences (P < 0.05) Results represent means ± SEM for three
independent experiments (B) Identification of transfected
TSC22D2-4 ⁄ V5-His-tagged fusion protein expression by western
blot using V5 antibody.
Trang 10All nine TSC22D transcripts are expressed in
mouse kidney cells
Expression of all nine TSC22D transcripts was
con-firmed in mouse kidney and in the mIMCD3 cell line
The levels of expression of all nine TSC22D transcripts
in mIMCD3 cells in vitro were comparable with renal
tissue in vivo suggesting that mIMCD3 cells are a useful
model for studying mechanisms of regulation and
func-tions of TSC22D isoforms in mammalian kidney cells
The lack of previous evidence for expression of
sev-eral TSC22D transcripts identified in this study
sug-gests that they may be particularly important for
specific biological functions that are prevalent in renal
cells The multitude of alternatively spliced TSC22D2
gene products could be important for generating
func-tional variability in response to different environmental
cues However, in the case of hyperosmolality all four
splice variants of TSC22D2 are significantly
upregulat-ed even though the magnitude and kinetics of this
up-regulation was somewhat splice variant specific (see
Discussion below) and the structures of the respective
protein products are also different Variable exon 2
usage produces proteins with different N-termini
adja-cent to the conserved TSC22D motif This region is
responsible for transactivation suggesting that TSC22D
variants with truncated N-termini (in particular the
novel TSC22D2-4 variant) may be transcriptional
repressors that sequester other TSC22D family
members [20]
TSC22D2 and TSC22D4 are regulated by
hyperosmolality in kidney cells
In mIMCD3 cells exposed to hyperosmolality
TSC22D2 and TSC22D4 transcripts increase
signifi-cantly but with different kinetics The increase in
TSC22D2 transcripts is transient and closely resembles
that observed previously for tilapia OSTF1 [12] Thus,
despite the higher degree of structural homology of
tilapia OSTF1 with murine TSC22D3-1, the novel
murine TSC22D2 transcripts represent the closest
functional homologs of tilapia OSTF1 The magnitude
and kinetics of hyperosmotic upregulation of TSC22D2
splice variants shows some differences TSC22D2-1 and
TSC22D2-4 responded earlier and more robustly than
TSC22D2-2 and TSC22D2-3
Splice variants of other genes that respond
differen-tially to osmotic stress have been reported before, e.g
for cyclooxygenase 1 in human intestinal epithelial
cells [32] In addition such regulation has been
observed for other types of stress For instance,
Dro-sophila heat shock transcription factor is regulated by
alternative splicing in response to heat⁄ cold stress [33] The splicing factor hSlu7 was reported to alter its sub-cellular distribution and thus modulate alternative spli-cing after UV stress [34] In fact, alternative splispli-cing of pre-mRNA encoding transcription factors represents a common mechanism for generating the complexity and diversity of gene regulation patterns [35–38] This mechanism produces a variety of functionally distinct isoforms from a single gene by use of different combi-nations of splice junctions For example, alternative splicing within the DNA-binding domain of Pax-6 alters DNA-binding specificity of the resulting proteins [39] Alternative splicing of the transactivation domains in Pax-8 [40], the POU homeodomain family protein Pit-1 [41] and the zinc finger transcription fac-tor GATA-5 [42] also results in protein isoforms with different transactivation properties Deletion by spli-cing of the transactivation domain in AML1a [43] and CREB [44] produces proteins with dominant negative activity This may also be the case for TSC22D2 splice variants with a truncated transactivation domain, in particular TSC22D2-4 Thus, alternative splicing of TSC22D2 may confer increased complexity of gene regulation in response to hyperosmotic stress Our data indicate that TSC22D2-4 represents a survival factor for renal cells exposed to hyperosmolality suggesting that it promotes osmotic adaptation programs, poss-ibly by acting as a transcriptional repressor of pro-apoptotic genes
The time course of hyperosmotic induction of the murine TSC22D4 transcript is slower than TSC22D2, more stable, and more closely resembles that observed previously for TonEBP [45], although more transient hyperosmotic activation of TonEBP similar
to that of TSC22D2 has also been reported recently [46] Moreover, significantly higher levels of TSC22D4
in renal papilla vs cortex raise the possibility that this gene is stably upregulated by hyperosmolality not only in vitro but also in vivo Of interest, AP1 (jun, fos) and NF-jB are transcription factors that are regulated by osmotic stress [47–52] and, intriguingly, they are known to interact with TSC22D3-2 (GILZ) [23–25]
TSC22D3 is regulated by aldosterone
in kidney cells Aldosterone is the major corticosteroid hormone regu-lating electrolyte and fluid homeostasis in all verte-brates [53,54] The major action of the hormone on renal Na+transport is localized to the collecting duct Our results show that both TSC22D3 transcripts increase transiently in response to aldosterone