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First published online May 30, 2008
Journal of Experimental Biology 211, 1964-1968 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017368
The cloning of eel osmotic stress transcription factor and the regulation of its expression in primary gill cell culture
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong
* Author for correspondence (e-mail: ckcwong{at}hkbu.edu.hk)
Accepted 31 March 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: chloride cell, organic osmolyte, osmosensor, pavement cell, Ostf, transcription factor
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Lang et al., 1998a). It is
crucial for many cellular functions, including ion transport, cell migration,
cell growth and cell death (Haussinger,
1996; Haussinger,
1998; Lang et al.,
1998b). This regulatory process is particularly important in gill
epithelia of euryhaline fish, as their gill cells are in direct contact with
waters of different salinity. Although the physiological functions of fish
gills have been studied extensively (Evans
et al., 2005), very little is known about how gill cells sense and
tolerate a large osmotic fluctuation. Major gaps exist in our understanding of
how gill cells detect volume perturbations and transmit signals to activate
volume regulatory mechanisms. In 2005, Kultz's group cloned two putative
transcriptional regulators in gills of tilapia: the osmotic stress
transcription factor 1 (Ostf1) and the basal transcription factor IIB (TFIIB),
which are classified as the `early hyperosmotically up-regulated proteins'
(Fiol and Kultz 2005;
Fiol et al., 2006). The
regulation of Ostf1 mRNA expression in tilapia gills and primary gill cell
culture was demonstrated to be activated by hypertonic treatment
(Fiol and Kultz 2005;
Fiol et al., 2006). Recently,
the partial cDNA sequence of Ostf was cloned in black porgy
(Choi and An, 2008). However,
unlike the tilapia model, the expression of Ostf1 mRNA in the gills of black
porgy was found to be activated only under hypo-osmotic stress. Until now,
Ostf has only been cloned in two species of fish. In addition, the mechanism
of regulation of Ostf expression has not been fully elucidated. In order to
study the underlying mechanism in the regulation of its expression in
branchial ion transporting cells, it would be necessary to clone the gene from
a truly euryhaline fish. Further study in this area warrants our understanding
of the evolutionary role as well as the functions of Ostf or related factors
in osmoregulation. In the first part of the present study, we aimed to clone the Ostf cDNA from gills of Japanese eels. The expression profiles of Ostf mRNA upon freshwater-to-seawater transfer, and vice versa, were measured in PercollTM-gradient-isolated pavement cells (PVCs) and chloride cells (CCs). Additionally, we investigated the regulation of Ostf expression using primary gill cell culture. Our data indicate that the basal expression level of Ostf is significantly higher in gill CCs than PVCs. Hyperosmotic acclimation significantly activated Ostf expression in CCs. Hypo-osmotic adaptation, however, had no obvious effect on Ostf mRNA levels. Using primary cell culture, we demonstrated that Ostf gene activation is influenced by an increase in intracellular ionic strength and affected by the integrity of the cytoskeleton.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The
identity of the isolated CCs was confirmed by mitochondria staining
(Tse et al., 2006). For
real-time PCR analysis, the isolated gill cells were dissolved in TRIzol®
Reagent (Invitrogen, Carlsbad, CA, USA) for total RNA extraction.
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Primary gill cell culture
The culture was established using gill filaments obtained from
freshwater-adapted eels. Single-seeded preparations were used, as described in
our previous study (Tse et al.,
2007). After tryptic digestion of the gill cells, the cell
suspension was then filtered and washed. The cells were resuspended in
Leibovitz's L-15 medium (Gibco; Invitrogen) supplemented with 5% fetal bovine
serum (FBS; HyClone®; Perbio Sciences, Logan, UT, USA), 1%
penicillin/streptomycin, 1% gentamycin (Gibco; Invitrogen) and seeded at a
density of 2x106 cells cm–2 onto
collagen-coated culture plates (Iwaki, Chiba, Japan). The cells were incubated
at 22°C in a growth chamber with humidified air. One day after seeding,
each well was rinsed with PBS to remove mucous and unattached cells. The cells
were then exposed to hypertonic stress and/or drug treatment. Hypertonic
stress was induced by either the addition of (1) membrane-nonpermeable solute
(NaCl; Sigma, St Louis, MO, USA) or (2) membrane-permeable solute (urea;
Sigma) to the medium, making its osmolarity 500 mOsmol l–1.
In addition, in some experiments the cells were cotreated with actinomycin D
(Act D, 1 µmol l–1) (Calbiochem, Darmstadt, Germany) to
determine if the effect was transcriptionally dependent. To test if the gene
induction by hypertonicity can be modulated by organic osmolytes, the cultured
cells were exposed to media containing high NaCl (final osmolarity of 500
mOsmol l–1) and, at the same time, with or without 5 mmol
l–1 inositol, betaine or taurine (Sigma). To test the
involvement of cytoskeleton in Ostf activation, the hypertonic-exposed cells
were co-treated with colchicine (100 µmol l–1;
Calbiochem). After 6 h of incubation, total RNA was extracted for the
measurement of Ostf and GAPDH mRNA levels.
Real-time PCR analysis
Purified sample RNA with an
A260/A280 ratio of 1.8–2.0 was
used. Briefly, 0.5 µg of total cellular RNA was reversed transcribed
(iScript; BioRad). PCR reactions were conducted with the iCycler iQ real-time
PCR detection system using iQTM SYBR® Green Supermix (Bio-Rad).
Primers for Ostf (TCCGCCAGCTCCTTGATTTG-forward,
AGCAGGCAATGGATCTTGTGAA-reverse) (Tse et
al., 2007) and GAPDH (GCGCCAGCCAGAACATCATC-forward,
CGTTAAGCTCGGGGATGACC-reverse) (GenBank accession no. AB075021) were used. The
PCR products were cloned into pCRII TOPO® (Invitrogen) and subjected to
dideoxy sequencing for verification. The copy number of the transcripts for
each sample was calculated in reference to the parallel amplifications of
known concentrations of the respective cloned PCR fragments. Standard curves
were constructed and the amplification efficiencies were approximately
0.9–0.95. The occurrence of primer-dimers and secondary products was
inspected using melting curve analysis. Our data indicated that the
amplification was specific. There was only one PCR product amplified for each
individual set of primers. Control amplification was done either without
reverse transcriptase or without RNA.
Statistical analysis
All data are presented as means ± s.e.m. Statistical significance is
tested by Student's t-test. Groups were considered significantly
different if P<0.05.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Differential expression of Ostf mRNA in gill PVCs and CCs after transfer
To investigate whether Ostf is differentially regulated in freshwater PVCs
and CCs, we isolated the cells by PercollTM gradient centrifugation and
measured Ostf mRNA level using real-time PCR assay. In freshwater-adapted
eels, gill CCs expressed a significantly higher (
4 times) level of the
transcript than that of the PVCs (Fig.
2A). To examine the effect of hypotonic and hypertonic stress on
gill Ostf expression levels, freshwater-to-seawater and seawater-to-freshwater
transfer experiments were conducted. In the freshwater-to-seawater transfer
experiment, Ostf transcript levels in CCs and PVCs increased significantly by
4- and 0.8-folds, respectively, at 0.25 days post-transfer, after which they
decreased back to basal levels (Fig.
2B). After transfer of the fish from seawater to freshwater, there
was no obvious change in Ostf expression levels in either CCs or PVCs (data
not shown). No significant change in Ostf transcript level was observed in the
freshwater-to-freshwater and seawater-to-seawater transfer experiments (data
not shown).
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Attenuation of Ostf mRNA expression by organic osmolytes and colchicine
It is well known that cells respond to volume perturbations by modulating
the transport of inorganic and organic osmolytes in the processes of
regulatory volume change (Lang et al.,
1998a; Strange,
2004; Wehner et al.,
2003). The alteration in the composition of intracellular organic
osmolytes is important to relieve volume perturbation. We tested this
hypothesis by investigating whether an accumulation of organic osmolytes can
attenuate the hypertonic stress-induced Ostf expression. In this regard, Ostf
mRNA was measured in cells exposed to hypertonic medium (NaCl, 500 mOsmol
l–1) with or without the addition of 5 mmol
l–1 of inositol, betaine or taurine. Our data demonstrated
that the hypertonicity-induced Ostf mRNA expression was attenuated by the
addition of inositol, betaine or taurine in the culture medium
(Fig. 4A).
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In our previous study, we demonstrated that hypertonic treatment (NaCl, 500
mOsmol l–1) of the gill cells caused cell shrinkage, probably
by affecting membrane tension (Tse et al.,
2007). Thus, it would be interesting to know if the induction of
the Ostf transcript is related to cytoskeleton organization. The treatment of
the hypertonic-treated cells (NaCl, 500 mOsmol l–1) with
colchicine (an inhibitor to microtubule polymerization) abolished the
induction of Ostf expression (Fig.
4B).
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DISCUSSION |
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|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). In the present study, we attempted to address
this issue by cloning Ostf cDNA in gills of Japanese eels and determining its
expression profile in the two major types of ion-transporting gill cells (i.e.
CC and PVC) of acclimating fish. Furthermore, using the primary gill cell
culture model, we deciphered the possible mechanisms in the regulation of Ostf
expression.
The cloned eel Ostf shows high DNA and amino acid sequence homologies with
those of tilapia (AY679524). The TSC-22 family signature sequence is
identified in the eel Ostf cDNA. In the mammalian system, the
TSC-22-domain-containing transcripts were reported to be important in
protecting mouse kidney cells from osmotic stress
(Fiol et al., 2007). PCR
primers were designed according to the cloned eel Ostf sequence and were used
for the analysis of its expression. In the first part of the study, we
conducted in vivo experiments in which we compared the expression of
Ostf mRNA in the PercollTM-gradient-isolated gill CCs and PVCs of the
fish acclimating in either the freshwater or seawater condition. The
identification of these two cell types was on the basis of the evidence
reported in our previous studies (Tse et
al., 2006; Wong and Chan,
1999). Our data indicated that the basal levels of Ostf transcript
were significantly higher in CCs than in PVCs. In the freshwater-to-seawater
transfer experiments, the Ostf mRNA expression levels in CCs and PVCs
increased significantly after 6h and then decreased to the basal level from
day 1 onwards. This observation indicated that the induction of Ostf is fast
but transient. Comparatively, the induction was more substantial in gill CCs
than in PVCs. In the course of seawater-to-freshwater transfer, there was no
significant change in Ostf mRNA expression measured in either CCs or PVCs.
This is comparable with the results reported in the tilapia model, which
indicated only hypertonic stress-induced Ostf1 expression
(Fiol and Kultz 2005;
Fiol et al., 2006). It is
believed that the modulation in the expression of the ion transporters and
channels is induced by immediate early gene (IEG) transcription factors
(Fiol and Kultz, 2005). When we
compared the time windows of gene induction in gill cells of
seawater-acclimating eels, our previous study demonstrated that most of the
ion transporters increased their expression considerably 24h after transfer to
seawater (Tse et al., 2006).
The early induction of Ostf expression (at 6h of acclimation), which preceded
the expression of ion transporters/channels (at 24h of acclimation), suggests
that Ostf is involved in the regulation of hyperosmotic responses in the gill
cells (e.g. the expression of ion transporters or channels). The data
demonstrate that Ostf was induced by hypertonic stress and its expression may
play an important role in fish hyper-osmoregulation in the seawater
environment.
It is known that a profound alteration in cell volume affects intracellular
ionic strength (macromolecular crowding), cell membrane tension and,
consequently, integrity of the cytoskeleton architecture
(Lang et al., 1998a;
Wehner et al., 2003). Although
a considerable number of studies have been carried out to search for
osmosensing mechanisms in the mammalian system, the conclusive model of the
molecular osmosensors in animal cells has not yet been confirmed. Recently,
our group has reported that there was an activation in the process of
`regulatory volume increase' as well as an increase in Ostf mRNA expression in
gill cells after hypertonic (500mOsmoll–1) treatment
(Tse et al., 2007). Hence,
Ostf expression is thought to be one of the downstream targets in hyperosmotic
responses. In the second part of the present study, we further determined that
Ostf expression is induced in the hypertonic solution prepared using NaCl but
not using urea. This observation indicates that Ostf activation is exerted by
an increase in intracellular ionic strength and is directly coupled to one of
the putative osmosensors, macromolecular crowding
(Lang et al., 1998a;
Wehner et al., 2003). To
further elucidate this possibility, the hypertonic-treated cells were
incubated in media containing organic osmolytes (i.e. betaine, taurine or
inositol). This treatment presumably decreases the intracellular ionic
strength by stimulating the cellular accumulation of organic osmolytes
(Sheikh-Hamad et al., 2000).
Consistently, the incubation abolished the induction of Ostf mRNA expression.
Since the change in intracellular ion strength would affect cell volume and
cytoskeleton architecture (Di Ciano et al.,
2002; Lionetto et al.,
2002; Tse et al.,
2006), we decided to test the effect of colchicine (an inhibitor
of microtubule polymerization) to hypertonicity-induced Ostf expression.
Obviously, colchicine treatment reduced Ostf gene expression. Although
colchicine can inhibit spindle formation during mitosis, this effect on Ostf
expression may not be significant in our study, as the time of drug treatment
in the primary cell culture was relatively short (6 h). Therefore, our
observation supports the notion that Ostf induction is dependent on the
integrity of the cytoskeleton, which is modulated in the process of cell
volume regulation. Using eel intestinal epithelia, Lionetto et al. reported
that colchicine treatment inhibited the hypertonicity-induced short-circuit
current by about 50–80% (Lionetto et
al., 2002). This observation supports the important role of the
cytoskeleton in cellular osmo-responses.
In summary, we are the first to measure the differential expression profiles of Ostf mRNA in gill CCs and PVCs during hyper- and hypo-osmotic adaptation. In addition, we have demonstrated the possible correlation between the expression of Ostf transcript and the two putative osmosensors (macromolecular crowding and cytoskeleton). The studies described here provide a fundamental understanding of the mechanisms of cellular osmoregulation in fish gills.
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Acknowledgments |
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P<0.05 compared
with the isolated freshwater PVCs. The results were obtained from four
independent experiments.
