Osmotic Stress Transcription Factor Research Articles

Cloning and expression of Na +/K +-ATPase and osmotic stress transcription factor 1 mRNA in black porgy, Acanthopagrus schlegeli during osmotic stress

We cloned complementary DNA (cDNA) encoding the Na +/K +-ATPase (NKA) and the osmotic stress transcription factor 1 (OSTF1) from the kidney and gill, respectively, of the black porgy, Acanthopagrus schlegeli. Black porgy NKA full-length cDNA consists of 3078 base pairs (bp) and encodes a protein of 1025 amino acids; OSTF1 partial cDNA consists of 201 bp. To investigate the osmoregulatory ability of black porgy when black porgy were transferred to freshwater (FW), we examined the expression of NKA and OSTF1 mRNA in osmoregulatory organs, i.e., gill, kidney and intestine, using quantitative polymerase chain reaction (QPCR). To determine the hypoosmotic stressor specificity of the induction of NKA and OSTF1, black porgy were exposed to 30°C water temperature for 24 h. In the gill, NKA mRNA was 4.2 times higher in FW, its expression in the kidney was 5.7 times higher in 10‰ seawater (10‰ SW) than in SW. In contrast, OSTF1 mRNA in the gill was 3.7 times higher in FW than in SW. The expression of heat shock protein 90 (HSP90) mRNA occurred not only during transfer to FW, but also in high-temperature water in all tested tissues, although the mRNA levels were not significantly different. Plasma osmolality level was decreased and cortisol level was increased when the fish were transferred from SW to FW. These results suggest that NKA and OSTF1 genes play important roles in hormonal regulation in osmoregulatory organs and that these genes are specific to hypoosmotic stress, improving the hyperosmoregulatory ability of black porgy in hypoosmotic environments.

Specific TSC22 domain transcripts are hypertonically induced and alternatively spliced to protect mouse kidney cells during osmotic stress

We recently cloned a novel osmotic stress transcription factor 1 (OSTF1) from gills of euryhaline tilapia (Oreochromis mossambicus) and demonstrated that acute hyperosmotic stress transiently increases OSTF1 mRNA and protein abundance [Fiol DF, Kültz D (2005) Proc Natl Acad Sci USA102, 927-932]. In this study, a genome-wide search was conducted to identify nine distinct mouse transforming growth factor (TGF)-beta-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 mechanism 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 mOsmol x kg(-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.

Regulation of osmotic stress transcription factor 1 (Ostf1) in tilapia(Oreochromis mossambicus) gill epithelium during salinity stress

Mechanisms of induction of osmotic stress transcription factor 1 (Ostf1) were analyzed in gill epithelium of tilapia exposed to salinity stress. Experiments with primary cultures of gill epithelial cells revealed that hyperosmotic Ostf1 induction was independent of systemic factors. In addition, the synthetic glucocorticoid receptor agonist dexamethasone did not affect Ostf1 levels, arguing against cortisol being the signal for Ostf1 induction during hyperosmotic stress. Exposure of primary gill cell cultures to a hyperosmotic agent that is cell permeable and non-hypertonic (glycerol) did not trigger Ostf1 induction. However, when gill cells were exposed to hypertonicity (either in the form of NaCl or other forms) Ostf1 was rapidly and significantly induced. Analysis of hnRNA and mRNA levels revealed that Ostf1 upregulation in gill cells of intact fish and primary cultures of gill epithelial cells was mediated by transient mRNA stabilization. In addition to the initial transient mRNA stabilization a subsequent transcriptional induction of Ostf1 was observed. In cultured gill cells increase in Ostf1 mRNA synthesis was stable and very potent, whereas in gill cells of intact fish this increase was transient. This observation suggests positive feedback by Ostf1 or one of its targets and negative feedback by systemic factors on Ostf1 transcription. 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.

TILAPIA'S HYPEROSMOTIC TRANSCRIPTION FACTORS

Salt-tolerant fish like tilapia, Orechromis mossambicus, revert from actively absorbing salt in freshwater to secreting salt across their gill epithelium in seawater. This requires a fast remodelling of the cellular gill epithelium upon transfer from freshwater to seawater and is accompanied by changes in mRNA levels of several ATPases, ion transporters and water channels. Given what we know about mammalian osmotic regulation, it seems reasonable that transcriptional regulation plays a role in inducing these changes. So far, only one transcription factor (TonEBP) is known to be involved in vertebrate osmoregulation. Diego Fiol and colleagues from the University of California Davis now have evidence for two novel transcription factors that are specifically induced in response to hyperosmotic stress in fish.Fiol's team reasoned that any transcriptional changes observed after they moved tilapia from freshwater to seawater would reflect a response to help the fish cope with increased saltiness. They got their first glimpse of the transcription factors that potentially mediate this response when they screened a cDNA library obtained by subtracting cDNA of a freshwater fish from cDNA of a fish that the authors had exposed to seawater for four hours. Among the induced transcripts they found two novel transcription factors. One of these they called osmotic stress transcription factor 1 (OSTF1), and to find some clues regarding its function they compared its gene sequence to other vertebrate gene sequences. They found that OSTF1 was similar to a splice variant of a glucocorticoid-induced leucine zipper (GILZ) transcription factor that responds to glucocorticoid treatment. This suggests that cortisol, a glucocorticoid hormone that plays a major role in the acclimation of fish to high salinities, may have a similar effect on OSTF1. The second novel factor was a homologue of TFIIB, a general transcription factor found in vertebrates. Tilapia TFIIB showed high sequence identity (89.6%) with other vertebrate TFIIBs and, thus, is likely to be very similar in function to these TFIIB homologues. Importantly, the team found that OSTF1 and TFIIB possess similar phosphorylation sites for stress-signalling kinases, indicating that these two novel factors mediate stress responses and appear to be affected by similar stress signals.But were these two transcription factors really induced by hyperosmotic stress caused by a move from freshwater to seawater? The authors used quantitative PCR to follow the time course of mRNA expression before and after they transferred fish from freshwater to seawater, and found that OSTF1 and TFIIB levels significantly increased between one and four hours after the transfer. So these two factors are indeed co-induced when saltiness increases. Since there are many stress-induced genes, the authors took great care to show that these transcription factors were specifically linked to osmotic stress. Exposure to oxidative stress (H2O2) and heat-shock led to strong induction of heat-shock protein70, a general stress protein, but did not affect OSTF1 and TFIIB levels. So OSTF1 and TFIIB are not part of the general stress response, but are induced specifically by hyperosmotic stress.Although the exact roles of these two novel transcription factors and the genes they affect need to be elucidated, homologies and sites of post-translational regulation provide insights into their possible functions. Their rapid induction suggests a role in the transduction of the osmosensory signal to the acclimation response to hyperosmotic conditions. Since TFIIB is known to interact with other transcription factors, its co-induction with OSTF1 in tilapia gill cells may indicate that these two transcription factors interact during osmotic stress.

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

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.