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First published online August 3, 2006
Journal of Experimental Biology 209, 3257-3265 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02352
Regulation of osmotic stress transcription factor 1 (Ostf1) in tilapia (Oreochromis mossambicus) gill epithelium during salinity stress
Physiological Genomics Group, Department of Animal Science, University of California, Davis, One Shields Avenue, Meyer Hall, Davis, CA 95616, USA
* Author for correspondence (e-mail: dkueltz{at}ucdavis.edu)
Accepted 30 May 2006
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Summary |
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We conclude that Ostf1 induction in gill epithelial cells of tilapia exposed to salinity stress (1) is independent of cortisol or other systemic factors; (2) depends on hypertonicity as the signal; and (3) is based on transient mRNA stabilization. Moreover, our data on primary cell cultures show that systemic signals are necessary to prevent sustained transcriptional induction of Ostf1 during hyperosmotic stress, indicating feedback regulation and a high degree of complexity of osmosensing and signaling networks in euryhaline fishes.
Key words: osmoregulation, teleost, stress signaling, salinity adaptation, gill epithelial cell, tilapia, Oreochromis mossambicus
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). In the gill
epithelium numerous ion and water transporters are regulated at the mRNA level
by salinity. Examples of this regulation include subunits of the
Na+/K+-ATPase and
Na+/K+/2Cl- cotransporter
(Tipsmark et al., 2002), urea
transporter (Mistry et al.,
2001a), taurine transporter
(Takeuchi et al., 2000) and
aquaporin 3 water channel (Cutler and
Cramb, 2002). In addition, other mRNAs such as 14-3-3 and C-type
lectin are also regulated by osmolality in fish gills
(Kültz et al., 2001;
Mistry et al., 2001b). These
responses could potentially be regulated by increases in the synthesis rate of
the corresponding mRNAs and/or by mRNA stabilization mechanisms. Based on
reports on mechanisms of osmotic regulation in mammalian cells it is likely
that variations in mRNA abundance of these transporters result from
transcriptional regulation (Burg et al.,
1996).
Knowledge of transcriptome regulation during salinity adaptation of
euryhaline fishes is still far from comprehensive but recent work in this
field could shed light on mechanisms of osmosensory signal transduction. We
demonstrated recently that Ostf1 (osmotic stress transcription factor 1) is
early and transiently upregulated in gill epithelial cells from tilapia
exposed to hyperosmotic stress at both the mRNA and protein levels
(Fiol and Kültz, 2005).
The function of this protein is not yet discerned, but, based on protein
structure similarity studies, Ostf1 could be a transcriptional regulator.
Tilapia Ostf1 contains DNA binding motifs such as Q-rich regions, as well as
the signature motif of the TSC-22/GILZ/DSIPI protein family. Several members
of this protein family have been characterized as transcriptional regulators
(Mittelstadt and Ashwell,
2001; Shibanuma et al.,
1992).
Tilapia Ostf1 has the highest sequence similarity to the mammalian
glucocorticoid induced leucine zipper (GILZ)
(Fiol and Kültz, 2005),
suggesting that Ostf1 could also respond to glucocorticoids. In accordance
with that notion, plasma cortisol levels increase with hyperosmotic stress
(reviewed by Evans et al.,
2005). Cortisol, referred to as the `seawater-adapting' hormone,
is involved in the upregulation of several hyperosmotic stress responsive
genes (Mommsen et al., 1999).
It is therefore possible that cortisol represents a signal for the induction
of Ostf1 during hyperosmotic stress. Thus, we conducted the present study to
discern the signals and mechanisms involved in the induction of Ostf1 in
tilapia gill epithelial cells during salinity stress.
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Materials and methods |
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).
Gill epithelium was utilized for subsequent RNA or protein isolation.
Cell cultures
Experiments performed with epithelial gill cell primary cultures (EGCPC)
were carried out using epithelial gill tissue scraped off the cartilage and
dispersed into <1 mm3 tissue pieces. FW acclimated fish were
used as a source of EGCPC, which were seeded in plastic tissue culture dishes.
Cells were cultured in minimum essential medium (MEM)-Eagle's medium (ATCC,
Manassa, VA, USA) at 25°C and atmospheric oxygen and CO2.
Isosmotic medium had an osmolality of 300 mOsmol kg-1 and
hyperosmotic media were prepared by the addition of the appropriate amount of
NaCl to isosmotic medium yielding a medium osmolality of 600, 900 or 1100
mOsmol kg-1 of H2O. When indicated, choline chloride,
sodium gluconate, mannitol or glycerol was added instead of NaCl for
hyperosmotic media preparation. Final osmolality of all media was verified
with a micro-osmometer (Model 3300, Advanced Instruments, Norwood, MA, USA).
Dexamethasone (DEX; D-4902, Sigma, St Louis, MO, USA) was added to a final
concentration of 1 µmol l-1 when indicated. Controls with a
vehicle (ethanol) were always run in parallel.
RNA isolation
Total RNA from gill epithelial cells was extracted using Trizol reagent
(Invitrogen, Carlsbad, CA, USA) as specified by the manufacturer. RNA was
treated with DNase (Turbo DNA free, Ambion, Austin, TX, USA). Purity and
quality was confirmed and quantity determined by measuring absorbance of the
samples at 260 and 280 nm with a Beckman DU520 spectrophotometer and by
denaturing electrophoresis.
DNA synthesis and real-time qPCR
RNA (2 µg) was reverse-transcribed using Superscript III first strand
synthesis reagents (Invitrogen) with a random hexamer:oligo(dT) mix (1:1) as
primers. Abundance of selected transcripts was quantified by quantitative
real-time PCR (qPCR) with a PRISM 7500 real-time thermal cycler (Applied
Biosystems, Foster City, CA, USA). Reactions were performed in duplicate using
20 µl reaction volumes with SYBR Green PCR Master Mix (Applied Biosystems)
or Full Velocity SYBR Green Mix QPCR Master Mix (Stratagene), and 30 pmol of
each primer. PCR conditions were 50°C for 2 min and 95°C for 10 min,
followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were
collected at 60°C. In the osmotic stress experiments, ß-actin
expression was the choice for normalization based on previous experiments
(Fiol and Kültz, 2005;
Fiol et al., 2006). In the
mRNA decay experiments normalization was done using 18S rRNA levels, based on
its high stability and higher reliability of obtaining remaining mRNA values.
Relative expression ratios (referred to also as mRNA fold induction or mRNA
relative abundance) were calculated using the method of Pfaffl
(Pfaffl, 2001):
 |
where E=10(-1/slope), with the slope referring to the standard curve of the target genes (in Fig. 4D,E) or the normalizer gene (not shown, determined analogously E =1.90 for ß-actin and E =1.94 for 18S).
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PCR and sequencing
PCR was performed using the Advantage HF-2 PCR kit (Clontech, Mountain
View, CA, USA) in a MasterCycler (Eppendorf, Westbury, NY, USA) using the
following cycling parameters: 94°C for 1 min, 35 cycles of 94°C for 30
s, 60°C for 30 s, 72°C for 1 min, and then 72°C for 5 min. PCR
products were extracted from agarose gels using GeneClean system (Q-BIO gene,
Irvine, CA, USA) and then double-pass sequenced on an ABI 3730 automated DNA
sequencer.
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Statistical analyses for identifying significant differences from the control
were performed using REST software (Pfaffl
et al., 2002). REST uses the pair-wise fixed reallocation
randomization test to calculate the significance of results. Differences at
P<0.001 were considered significant. The REST software used for
statistical analyses represents an established method for analyzing real-time
PCR results directly. In addition, significant differences of relative
increases between mature Ostf1 and hnOstf1 were assessed using unpaired
t-test carried out with SigmaPlot 9.0 (Systat). Significance
threshold was set at P<0.05 and data are presented as mean
± standard error of mean (s.e.m.). |
Results |
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Considering the participation of cortisol in SW adaptation in fish, as well
as sequence similarity between Ostf1 and the mammalian glucocorticoid
responsive GILZ protein, we decided to test glucocorticoid involvement in the
Ostf1 induction pathway. The synthetic glucocorticoid dexamethasone (DEX) was
used in the following experiment as a potent cortisol receptor agonist. The
concentration of DEX was optimized based on previous reports of its use as a
glucocorticoid receptor agonist in fish gill cells
(McCormick and Bern, 1989) and
other tissues [e.g. rat hepatocytes
(Wehner and Tinel, 1998)]
under hypertonic stress. When media were supplemented with 1 µmol
l-1 DEX, the induction pattern was the same as that of the controls
(Fig. 1A), and were essentially
identical to the vehicle treatments (not shown). Whereas no change was
observed in isosmotic medium, Ostf1 mRNA was 13±3-fold induced at 600
mOsm kg-1, and three- to fourfold increased at 900 and 1100 mOsm
kg-1 when DEX was added to the medium
(Fig. 1A). Thus, glucocorticoid
receptor activation is not responsible for hyperosmotic OSTF1 up-regulation
and did not significantly alter the hyperosmotic induction of Ostf1 in gill
epithelial cells.
A time course experiment was performed in order to evaluate the dynamic
nature of the response. EPCGC were exposed to either isosmotic medium or 600
mOsm kg-1 hyperosmotic medium with the addition of NaCl. Ostf1 mRNA
was increased at comparable levels at 2, 4 and 8 h
(Fig. 1B). Interestingly, this
sustained increase differed from the time course previously obtained with
whole fish exposed to hyperosmotic stress where the induction was transient,
peaking at 2 h and reaching background levels after 6-8 h
(Fiol and Kültz,
2005).
Ostf1 is up-regulated by hypertonic stress
Our results presented above show that gill epithelial cells are able to
sense hyperosmolality independently of systemic stimuli such as cortisol and
respond by increasing the Ostf1 mRNA steady state level. In order to assess
the nature of the stimulatory signal more closely, different hyperosmotic
media were tested for their ability to trigger Ostf1 upregulation. EGCPC were
exposed for 2 h to acute hyperosmotic stress by adding either choline
chloride, sodium gluconate, glycerol or mannitol to increase the osmolality
from 300 mOsm kg-1 (isosmotic medium) to 600 mOsm
kg-1.
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Analysis of the mechanism of Ostf1 mRNA upregulation
Determination of intronic Ostf1 sequence and validation of qPCR quantification
In principle, steady-state Ostf1 mRNA levels can be upregulated either
through an increase in the transcription rate or through a mRNA stabilization
mechanism decreasing the degradation rate of the mRNA. In order to assess
which of these mechanisms applies we decided to analyze Ostf1 mRNA synthesis
rate during hyperosmotic stress.
We analyzed the effect of hyperosmotic stress on the transcription rate of
the Ostf1 gene using the assay of Elferink and Reiners, Jr
(Elferink and Reiners, Jr,
1996), which measures the quantity of nascent, unspliced
transcript (hnRNA) from a particular gene using quantitative real-time RT-PCR,
representing a surrogate measure of the gene transcription rate.
Knowledge of the sequence of unspliced mRNA is required to carry out this
assay. Because tilapia Ostf1 nucleotide sequence was published as cDNA and the
tilapia Ostf1 genomic sequence was unavailable, we carried out a homology
analysis between tilapia Ostf1 cDNA and genomic sequences of Danio rerio,
Fugu rubripes and Tetraodon nigroviridis. Based on the structure
of the orthologous loci we were able to determine possible exon-exon
boundaries in the tilapia Ostf1 cDNA. Subsequent PCR-based cloning yielded an
intronic Ostf1 sequence, not present in the mature mRNA (and cDNA). All three
analyzed genomes predicted a transcript with an exon composition that matched
the tilapia Ostf1 cDNA 3' region. On the other hand, sequences
homologous to the tilapia Ostf1 cDNA 5' region were predicted by ENSEMBL
as part of different transcripts. In these three genomes, sequences homologous
to the 3' part of tilapia Ostf1 cDNA were located quite distantly
(
20 000 bp in Fugu and Tetraodon and 50 000 bp in
Danio) from the regions that are homologous to the 5' part of
tilapia Ostf1 cDNA.
Having identified the conserved sequence features among the genomes
compared, we were able to predict that tilapia genomic Ostf1 constitutes at
least three exons encompassing the entire ORF
(Fig. 3A). The lengths of the
introns located between the predicted exons were relatively conserved in the
three fish genomes. Intron 1/2 was very long in every case (20 000 bp in
Tetraodon and Fugu and 50 000 bp Danio) but intron 2/3
was shorter (403, 425 and 1079 bp in Tetraodon, Fugu and
Danio, respectively). Exon 2 showed a high degree of sequence
conservation, with a length of 52 bp in all three species, firmly supporting
the prediction of tilapia Ostf1 exon-exon boundaries.
Based on the exon-exon boundary sites determined, we designed specific PCR
primers flanking the exon2/exon3 boundary to amplify intron 2/3. PCR primers
were designed based on the cDNA sequence plus the addition of two nucleotides
of the 3' acceptor and 5' donor consensus sequences
(5'-G/gt-ag/G-3') in order to avoid amplification of the more
abundant mature mRNA (Fig. 3B).
A single PCR amplification product of 700 bp was obtained and further
purified and sequenced (Fig.
3C).
A schematic structure of Ostf1 unspliced mRNA showing the exonic and
intronic regions identified by this analysis, is depicted in
Fig. 4A. The intron 2/3
sequence was then used for the design of specific Ostf1 hnRNA qPCR primers.
Validation of Ostf1 mRNA and hnRNA qPCR primers is shown in
Fig. 4B-E. One specific band
was detected in agarose gel electrophoresis of the PCR products, and a single
peak was observed in the melting curve analysis of the qPCR for each reaction
(Fig. 4B,C). No bands were
detected in the minus RT samples (Fig.
4B), indicating the absence of genomic DNA contamination and hence
validating the suitability of the method. Standard curves produced with serial
dilutions of the cDNA templates are also shown
(Fig. 4D,E). Different samples
were used in each standard curve for the preparation of serial dilutions in
order to overcome the relatively lower content of hnRNA compared to the mature
mRNA (
Ct 5.18±0.45, N=46). Ostf1 mRNA-specific primers
are, as depicted in Fig. 4A,
able to also amplify the hnRNA. However, given the much higher content of the
mature RNA no corrections of the data were made because the error was not
significant in the context of the biological and experimental variation (a
Ct value of 5.18 implies a 36-fold difference in absolute amount
between Ostf1 mature mRNA and hnRNA, amounting to an error of only 2.8%).
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) the synthesis rate of a specific mRNA can be estimated
through the quantification of the steady state of the immature hnRNA. Thus, we
quantified hnOstf1 and Ostf1 mRNA levels using the validated specific qPCR
primers in gill epithelial cells of fish exposed to hyperosmotic stress in a
time course experiment. Significant differences between total mRNA and hnRNA
increases were detected only after 2 h of exposure to hyperosmotic stress
(Fig. 5A). Whereas total mRNA
increased about fivefold, hnRNA only increased less than threefold. At later
time-points, similar ratios of both kinds of transcripts relative to controls
were detected. These results suggest that two mechanisms are responsible for
Ostf1 mRNA increase. First, mRNA stabilization occurs very rapidly and early
(within 2 h of hyperosmotic stress). Second, this is followed by an increase
in the mRNA synthesis rate (from 2 to 6 h), perhaps as a result of
autoregulatory feedback.
When a similar analysis was done in EGCPC equivalent results were obtained
(Fig. 5B). Differences between
total mRNA and hnRNA were observed only after 2 h of exposure. At this time,
total mRNA increased sixfold whereas hnRNA increased only twofold. At 4 and 8
h, comparable increases for both total mRNA and hnRNA were found. Results
depict again a mRNA stabilization event early on (2 h) followed by an
increase in mRNA synthesis rate (Fig.
5B). Whereas timing of mRNA stabilization was identical in intact
fish and isolated cells, the effect of hyperosmolality was more pronounced and
sustained in cultured cells, suggesting negative feedback by systemic factors
on Ostf1 transcription.
Analysis of mRNA stability of Ostf1
In order to further validate the mRNA stabilization component of the Ostf1
induction mechanism, we analyzed the response of Ostf1 mRNA to hyperosmotic
stress in EGCPC incubated with the RNA synthesis inhibitor actinomycin D. In
cells kept under isosmotic conditions, a decline in Ostf1 mRNA amount was
observed, which is due to its degradation over time
(Fig. 6). Based on the time
course of mRNA degradation in the presence of actinomycin D a half-life
shorter than 30 min was estimated for Ostf1 mRNA, which is indicative of a
typical immediate early gene. When EGCPC were exposed to hypertonic stress,
Ostf1 degradation rate was significantly decreased. After the initial 30 min
during which the values were the same as in isosmotic controls, Ostf1 mRNA
levels remained significantly higher compared to isosmotic controls
(Fig. 6). These results confirm
that mRNA stabilization is involved in the rapid upregulation of Ostf1 mRNA
during hyperosmotic stress.
Analysis of the Ostf1 mRNA structure
Messenger RNA stabilization is a mechanism generally associated with the
binding of stabilizing proteins to the 3' UTR of the mRNA
(Guhaniyogi and Brewer, 2001).
Messenger RNAs regulated by this mechanism usually have an extended
3'UTR containing specific sequences known as adenine/uridine-rich
elements (AREs). These elements mediate the rapid turnover of several mRNAs
encoding proteins involved in the regulation of cellular growth and responses
to exogenous agents (Chen and Shyu,
1995). Ostf1 3'UTR is more than 2200 bp long and includes
four Class I AREs, containing the motifs AUUUA or AUUUUA and one Class III
ARE, constituting a U-rich region (Fig.
7). These structural characteristics support a rapid mRNA
stabilization mechanism as observed in the hyperosmotic upregulation of Ostf1
mRNA abundance.
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) and many
genes induced in gill cells in vivo are also induced in EGCPC,
including Hsp70 (Kültz,
1996) and seven novel hyperosmotic stress responsive genes
(Fiol et al., 2006). EGCPC
showed only a substantial increase of Ostf1 mRNA at 600 mOsm whereas 900 and
1100 mOsm kg-1 were close to control. Apical membranes are
generally less permeable than basolateral membranes of cells. Therefore, our
data suggest that osmolalities significantly exceeding those seen at the
basolateral side of gill cells in vivo, overwhelm the adaptive system
of isolated EGCPC and prevent the adaptive increase in Ostf1.
Upregulation of Ostf1 was observed in EGCPC, indicating that gill
epithelial cells respond to the hyperosmotic stress directly at the cellular
level. This capability of gill cells suggests that exogenous systemic factors
such as cortisol are not required for Ostf1 induction during hyperosmotic
stress, despite strong homology of Ostf1 to mammalian glucorticoid-induced
leucine zipper (GILZ) protein. Indeed, further experiments showed that gill
epithelial cells do not induce Ostf1 in response to dexamethasone (a potent
glucocorticoid receptor agonist) confirming that corticosteroids are not
required for hyperosmotic Ostf1 induction. This finding was unexpected since
cortisol is a corticosteroid hormone that is critical for seawater acclimation
and glucocorticoid receptor is induced by hyperosmolality in euryhaline
teleosts (Scott et al., 2004).
It promotes adaptive changes in response to hyperosmotic stress, including
chloride cell differentiation and proliferation in tilapia and salmonid gills
(McCormick, 1990;
Madsen, 1990a;
Madsen, 1990b), and stimulates
branchial Na+/K+-ATPase activity in a variety of fishes
(Madsen et al., 1995;
McCormick, 1995). However,
cortisol proved unable to stimulate other adaptive responses such as active
ion transport across tilapia opercular epithelium
(Foskett et al., 1981).
Exploiting the advantages of the EGCPC model, we analyzed the nature of the
Ostf1 induction signal by testing the effect of different hyperosmotic media.
These experiments demonstrate that hyperosmotic medium per se
(increased glycerol) is insufficient to elicit Ostf1 induction, which occurs
only when hyperosmotic media are prepared with non-permeable solutes. Thus,
hypertonicity is required to elicit Ostf1 induction. Our findings are in
agreement with data on other cell types exposed to hyperosmotic stress. For
instance, in mammalian renal medullary cells hypertonicity is also the signal
for induction of mRNAs of the TonEBP transcription factor and of multiple
genes involved in compatible osmolyte accumulation, and protein and DNA
stabilization (Woo et al.,
2000; Chakravarty et al.,
2002). The molecular nature of the hypertonicity signal is not yet
known. Hypertonicity is known to cause many secondary effects including cell
shrinkage, macro- and micromolecular crowding, changes in the organization of
cell membranes, altered water movements across cell membranes (osmosis), and
stress on the cytoskeleton (Kültz and
Burg, 1998). Such secondary effects are independent of the
particular solutes responsible for hypertonicity, and indeed our results
illustrate that there is no specific sodium or chloride ion requirement for
Ostf1 induction. Therefore, we conclude that one or more of the
above-mentioned secondary effects associated with hypertonicity are
responsible for Ostf1 induction.
Using two different approaches (calculation of mRNA synthesis rate after
hnRNA quantification, and measurement of mRNA decay after blocking mRNA
synthesis) we found that Ostf1 upregulation in tilapia gill epithelial cells
exposed to hyperosmotic stress is based initially on rapid mRNA stabilization.
Ostf1 mRNA synthesis rate was quantified using a previously validated method
(Elferink and Reiners, Jr,
1996). This method measures mRNA transcription rates via
quantification of the steady state of nascent hnRNA. Transcriptional
activities determined this way were previously validated by equivalent results
obtained with nuclear run-on assays, representing an alternative method of
transcription rate determination. However, compared to nuclear run-on assays,
the method used in our study has the advantages of being more sensitive,
easier to perform, and suitable for a broader range of target tissues compared
with nuclear run-on assay.
In addition, analysis of the Ostf1 3'UTR revealed the presence of
regulatory elements characteristic of mRNAs regulated by mRNA stabilization.
This initial mRNA stabilization is followed by augmentation of the mRNA
synthesis rate. Both mechanisms, mRNA stabilization and transcriptional
induction, are involved in upregulation of other hypertonicity-responsive
genes. For instance, mRNA stabilization in response to hypertonicity is seen
for GADD45 genes (Chakravarty et al.,
2002), TonEBP transcription factor
(Cai et al., 2005) and
aquaporin (Leitch et al.,
2001). Genes induced by transcriptional induction in response to
hypertonicity include aldose reductase, SMIT and BGT1
(Burg et al., 1997).
Stabilization of the mRNA is a regulatory mechanism involved in rapid
responses to various forms of cellular stress, including heat shock
(Andrews et al., 1987), UV
irradiation (Wang et al.,
2000; Westmark et al.,
2005), hypoxia (Levy et al.,
1998) and nutrient deprivation
(Yaman et al., 2002). This
mechanism permits a rapid increase of steady state mRNA levels by slowing
degradation. It is characteristic of inducible transcription factors and other
immediate early genes with high rates of mRNA turnover
(Bakheet et al., 2001). Thus,
stabilization of Ostf1 mRNA during hypertonicity supports a regulatory role of
Ostf1 protein for reorganization of gill epithelium during salinity
stress.
Of interest, we observed significant differences in Ostf1 mRNA upregulation profiles when comparing intact fish with cultured cells. Whereas Ostf1 mRNA increase is transient in intact fish, with a peak at 2 h and decline to normal levels within 6-8 h, cultured cells show a more sustained increase for at least 8 h. This difference suggests that negative feedback by systemic factors prevents sustained increases of Ostf1 mRNA in gill cells in situ, perhaps because sustained increases in Ostf1 may be detrimental to gill function.
We and others have previously used short-term cultures of gill epithelial
cells for physiological and toxicological studies
(Kültz, 1996;
Sandbacka et al., 1999). This
model system has many advantages but it also has the disadvantage that
cultured cells are generally not exposed to osmotic gradients across their
basolateral and apical surfaces. Generation of such gradients in culture is
theoretically possible by growing cells on permeable supports but practically
difficult because epithelial resistance of confluent EGCPC is different from
that of gill epithelium in situ. Many responses in epithelia, such as
ion transport phenomena, are dependent on an osmotic gradient and cell
polarity and may be difficult to reproduce in the EGCPC system. Nevertheless,
our data clearly demonstrate that this is not the case for Ostf1 upregulation,
which is independent of transcellular osmotic gradients and cell polarity.
Thus, it is possible that gill epithelial cells sense hyperosmotic stress
directly via the apical membrane, or indirectly via
subsequent increases in plasma osmolality via the basolateral
membrane. Further experiments are necessary to find out which of these
possible mechanisms applies.
In summary, we conclude that Ostf1 induction in gill epithelial cells of tilapia exposed to salinity stress is independent of cortisol or other systemic factors. Instead, it depends directly on hypertonicity as the primary induction signal and is based on rapid and transient mRNA stabilization followed by transcriptional induction. Systemic signals prevent sustained transcriptional induction of Ostf1 during hyperosmotic stress, indicating negative feedback regulation and possible detrimental effects of Ostf1 on gill function when its levels remain high for an extended period.
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