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Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia ( Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyper-osmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (p < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (p<0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO induced activation of the MIB pathway.
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Investigation of IMPA1.1 regulation in euryhaline fishes using genetic manipulations of cell cultures
Aquatic animals existing in variable salinity environments must possess mechanisms to maintain cellular osmotic balance essential for function. When facing hyper-osmotic stress, some cells can produce biochemically inert compatible organic osmolytes, such as myoinositol, to combat the osmotic gradient across the cell membrane. Myo-inositol is commonly utilized in this manner across widely diverse taxa. The final enzyme in the myo-inositol synthesis pathway is IMPA1.1 which has been shown to be highly upregulated in multiple tissues in several fish species in response to hyperosmotic challenge including Oreochromis mossambicus, a teleost fish tolerant of extreme hypersaline conditions. When the regulatory region of IMPA1.1 from this species was cloned upstream of multiple reporter genes and transfected into cell cultures, high reporter activity was observed in cells of both this species and a distantly related species, suggesting high conservation of regulatory elements and utilization of a common hyperosmotic response signaling pathway. Current work includes further genetic manipulations of this pathway in cell culture models to decipher its components from cellular osmosensing leading to differential regulation of relevant genes. Supported by NSF award 1656371 and BARD award IS-4800-15 R.
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- Award ID(s):
- 1656371
- PAR ID:
- 10139305
- Date Published:
- Journal Name:
- ICBF Proceeedings
- Volume:
- 13
- Issue:
- Calgary
- Page Range / eLocation ID:
- 72
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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