Hypertonicity-induced binding of the transcription factor TonEBP͞ OREBP to its cognate DNA element, ORE͞TonE, is associated with increased transcription of several osmotically regulated genes. Previously, it was found that hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP͞OREBP and, more slowly, increases TonEBP͞OREBP abundance. Also, the C terminus of TonEBP͞OREBP was found to contain a transactivation domain (TAD). We have now tested for tonicity dependence of the TAD activity of the 983 C-terminal amino acids of TonEBP͞OREBP. HepG2 cells were cotransfected with a reporter construct and one of several TAD expression vector constructs. The reporter construct contained GAL4 DNA binding elements, a minimal promoter, and the Photinus luciferase gene. TAD expression vectors generate chimeras comprised of the GAL4 DNA binding domain fused to (i) the 983 C-terminal amino acids of TonEBP͞OREBP, (ii) 17 glutamine residues, (iii) the TAD of c-Jun, or (iv) no TAD. All TAD-containing chimeras were functional at normal extracellular osmolality (300 mosmol͞kg), but the activity only of the chimera containing the 983 C-terminal amino acids of TonEBP͞OREBP varied with extracellular NaCl concentration, decreasing by >80% at 200 mosmol͞kg and increasing 8-fold at 500 mosmol͞kg. The chimera containing the 983 C-terminal amino acids of TonEBP͞OREBP was constitutively localized to the nucleus and showed tonicity-dependent posttranslational modification consistent with phosphorylation. The activity at 500 mosmol͞kg was reduced by herbimycin, a tyrosine kinase inhibitor and by 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole, a protein kinase CK2 inhibitor. Thus, the 983 C-terminal amino acids of TonEBP͞OREBP contain a TAD that is regulated osmotically, apparently by tonicity-dependent phosphorylation.

Organisms, almost universally, adapt to hyperosmotic stress through increased accumulation of organic osmolytes but the molecular mechanisms have only begun to be addressed. Among mammalian tissues, renal medullary cells are uniquely exposed to extreme hyperosmotic stress. Sorbitol, synthesized through aldose reductase, is a predominant osmolyte induced under hyperosmotic conditions in renal cells. Using a rabbit renal cell line, we originally demonstrated that hyperosmotic stress induces transcription of the aldose reductase gene. Recently, we cloned the rabbit aldose reductase gene, characterized its structure, and found the first evidence of an osmotic response region in a eukaryotic gene. Now, we have progressively subdivided this 3221-base pair (bp) region into discrete fragments in reporter gene constructs. Thereby, we have functionally defined the smallest sequence able to confer hyperosmotic response on a downstream gene independent of other putative cis-elements, that is, a minimal essential osmotic response element (ORE). The sequence of the ORE is CGGAAAATCAC(C) (bp ؊1105/؊1094). A 17-bp fragment (؊1108/؊1092) containing the ORE used as a probe in electrophoretic mobility shift assays suggests hyperosmotic induction of a slowly migrating band. Isolation of trans-acting factor(s) and characterization of their interaction with the ORE should elucidate the basic mechanisms for regulation of gene expression by hyperosmotic stress.

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