Rapid hyperosmotic coinduction of two tilapia (Oreochromis mossambicus) transcription factors in gill cells

Abstract

Gills of euryhaline teleosts are excellent models for studying osmotic-stress adaptation because they directly contact the aquatic environment and are an important effector tissue during osmotic stress. We acclimated tilapia (Oreochromis mossambicus) from fresh water (FW) to seawater (SW); performed suppression subtractive hybridization of gill mRNAs; and identified two transcription factors, osmotic stress transcription factor 1 (OSTF1) and the tilapia homolog of transcription factor II B (TFIIB), that are rapidly and transiently induced during hyperosmotic stress. mRNA levels increase 6-fold for OSTF1 and 4-fold for TFIIB, and they reach maxima 2 h after SW transfer. Protein levels increase 7.5-fold for OSTF1 and 9-fold for TFIIB, and they reach maxima 4 h after SW transfer. Induction of OSTF1 and TFIIB increases gradually with increasing salinity. Induction of OSTF1 and TFIIB is specific for osmotic stress and absent during oxidative stress (1 mM H2O2) or heat shock (+10 degrees C). Bioinformatic analysis of OSTF1 reveals that it is a transcription factor of the TGF-beta-stimulated clone 22/GILZ family. Because some mammalian homologs are strongly induced by glucocorticoids, OSTF1 may represent the molecular link between the SW hormone cortisol and transcriptional regulation of ion transport and cell differentiation in teleost gills. Coinduction of OSTF1 and TFIIB may serve to recruit TFIIB preferentially to OSTF1 target genes during hyperosmotic stress and compensate for reduced rates of transcription resulting from salt-induced chromatin compaction. We conclude that OSTF1 and TFIIB are critical elements of osmosensory signal transduction in euryhaline teleosts that mediate osmotic adaptation by means of transcriptional regulation.

Fig. 1.

Isolation of OSTF1 and TFIIB as transcriptionally induced clones identified by suppression subtractive hybridization (SSH). SSH clones 2 (A) and 10 (B) were fully extended by using PCR-based methods. (C) Semiquantitative RT-PCR from samples of FW fish (control) and fish acclimated to SW for 4 h.

Fig. 3.

Time-course analysis of hyperosmotic induction of OSTF1 and TFIIB protein abundance. OSTF1 (A) and TFIIB (B) protein levels were determined by Western blot analysis and quantified by densitometry. (Inset) A typical Western blot image. Experiments were performed with n = 4. Data are given as means ± SEM. ^*, P < 0.05; ^**, P < 0.01, compared with control sample (0 h, FW).

Fig. 2.

Time-course analysis of hyperosmotic induction of OSTF1 and TFIIB mRNA. OSTF1 (A) and TFIIB (B) transcript abundance was analyzed by using quantitative RT-PCR. Experiments were performed with n = 4. Data are given as means ± SEM. ^*, P < 0.05; ^**, P < 0.01, compared with control sample (0 h, FW).

Fig. 4.

Analysis of OSTF1, TFIIB, and Hsp70 mRNA expression during hyperosmotic stress. OSTF1 (A), TFIIB (B), and Hsp70 (C) transcript abundances of fish exposed to different salinities for 2 h were quantified by using quantitative RT-PCR. Experiments were performed with n = 6. Data are given as means ± SEM. ^*, P < 0.05; ^**, P < 0.01, compared with control sample (FW).

Fig. 5.

Analysis of OSTF1, TFIIB, and Hsp70 mRNA expression during oxidative stress (1 mM H₂O₂) and heat shock (+10°C). OSTF1 (A), TFIIB (B), and Hsp70 (C) transcript levels of fish exposed to oxidative stress or heat shock for 2 h were measured by using quantitative RT-PCR. Experiments were performed with n = 6. Data are shown as means ± SEM. ^*, P < 0.05; ^**, P < 0.01, compared with control sample (FW, 26°C).

Fig. 6.

Phylogenetic trees for OSTF1 and TFIIB. (A) Multiple-sequence alignment of tilapia OSTF1 and homologs from mouse (Mus musculus, AAG41222.1), human (Homo sapiens, BAC03934.1), zebrafish (Danio rerio, AAH56586.1), frog (Xenopus laevis, AAH43841.1), chicken (Gallus gallus, BAA11565.1), mosquito (Anopheles gambiae, EAA14371.1), fruit fly (Drosophila melanogaster,XP_395024.1), and nematode (Caenorhabditis elegans, NP_510086). (B) Multiple-sequence alignment of tilpia OSTF1 and human homologs GILZ (AAG12456), THG-1 (CAB43491), KIAA0669 protein (AAH44643), chr3 TSC-22 related protein (AAG41224), TSC-22 (AAG53077), KIAA1994 protein (AB082525.1), and chr X TSC-22 related protein (BAC03934.1). (C) Multiple-sequence alignment of tilapia TFIIB and TFIIB homologs from zebrafish (NP_955991), human (NP_001505), mouse (AAH16637.1), frog (CAA44668), mosquito (XP_310128), fruit fly (NP_476888), nematode (AAG24202), yeast (Schizosaccharomyces pombe, CAB11044), plant (Arabidopsis thaliana (AAF02810), and archaebacteria (Halobacterium sp. NRC-1, AAG18850). GenBank accession numbers are given in parentheses above.

Fig. 7.

Putative phosphorylation sites, motifs, and interaction domains in OSTF1 (A) and TFIIB (B). The N terminus is shown on the left, and the C terminus is shown on the right. For methods of analysis, please refer to the text.