Chester K. Williams

ORE, a Eukaryotic Minimal Essential Osmotic Response Element THE ALDOSE REDUCTASE GENE IN HYPEROSMOTIC STRESS

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.

Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration

Hypertonicity-induced binding of the transcription factor TonEBP/OREBP to its cognate DNA element, ORE/TonE, is associated with increased transcription of several osmotically regulated genes. Previously, it was found that hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP/OREBP and, more slowly, increases TonEBP/OREBP abundance. Also, the C terminus of TonEBP/OREBP 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 TonEBP/OREBP. 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 TonEBP/OREBP, (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 mosmol/kg), but the activity only of the chimera containing the 983 C-terminal amino acids of TonEBP/OREBP varied with extracellular NaCl concentration, decreasing by >80% at 200 mosmol/kg and increasing 8-fold at 500 mosmol/kg. The chimera containing the 983 C-terminal amino acids of TonEBP/OREBP was constitutively localized to the nucleus and showed tonicity-dependent posttranslational modification consistent with phosphorylation. The activity at 500 mosmol/kg was reduced by herbimycin, a tyrosine kinase inhibitor and by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, a protein kinase CK2 inhibitor. Thus, the 983 C-terminal amino acids of TonEBP/OREBP contain a TAD that is regulated osmotically, apparently by tonicity-dependent phosphorylation.

cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/ osmotic response element-binding protein

Hypertonicity-induced increase in activity of the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) protects renal cells by increasing transcription of genes, including those involved in increased accumulation of organic osmolytes. We previously showed that hypertonicity increases transactivating activity of TonEBP/OREBP. Assay with a binary GAL4 transactivation system showed that the 984 C-terminal amino acids of TonEBP/OREBP (amino acids 548–1531) contain a tonicity-dependent transactivation domain (TAD). Also, amino acids 548–1531 undergo tonicity-dependent phosphorylation, and some inhibitors of protein kinases reduce the tonicity-dependent transactivation. In the present studies we examined the role of protein kinase A (PKA). Results: (i) An inhibitor of PKA (H89) reduces tonicity-dependent increases in transactivation, ORE/TonE reporter activity, and induction of aldose reductase and betaine transporter mRNAs. (ii) Overexpression of the catalytic subunit of PKA (PKAc) increases transactivation activity of amino acids 548–1531 and activity of an ORE/TonE reporter. The increases are much greater under isotonic than under hypertonic conditions. (iii) A dominant-negative PKAc reduces activity of an ORE/TonE reporter. (iv) PKAc activity increases with tonicity but cAMP does not. (v) TonEBP/OREBP and PKAc coimmunoprecipitate. (vi) amino acids 872–1271, including N– and C-terminal polyglutamine stretches, demonstrate tonicity-dependent transactivation, albeit less than amino acids 548–1531, and a similar role for PKA. Conclusions: (i) PKA plays an important role in TonEBP/OREBP activation of tonicity-dependent gene expression; (ii) PKA activation of TonEBP/OREBP appears to be cAMP-independent; and (iii) amino acids 872–1271 are sufficient for tonicity-dependent transactivation of TonEBP/OREBP.

Functional consensus for mammalian osmotic response elements

The molecular mechanisms underlying adaptation to hyperosmotic stress through the accumulation of organic osmolytes are largely unknown. Yet, among organisms, this is an almost universal phenomenon. In mammals, the cells of the renal medulla are uniquely exposed to high and variable salt concentrations; in response, renal cells accumulate the osmolyte sorbitol through increased transcription of the aldose reductase (AR) gene. In cloning the rabbit AR gene, we found the first evidence of an osmotic response region in a eukaryotic gene. More recently, we functionally defined a minimal essential osmotic response element (ORE) having the sequence CGGAAAATCAC(C) (bp -1105 to -1094). In the present study, we systematically replaced each base with every other possible nucleotide and tested the resulting sequences individually in reporter gene constructs. Additionally, we categorized hyperosmotic response by electrophoretic mobility shift assays of a 17-bp sequence (-1108 to -1092) containing the native ORE as a probe against which the test constructs would compete for binding. In this manner, binding activity was assessed for the full range of osmotic responses obtained. Thus we have arrived at a functional consensus for the mammalian ORE, NGGAAAWDHMC(N). This finding should accelerate the discovery of genes previously unrecognized as being osmotically regulated.The molecular mechanisms underlying adaptation to hyperosmotic stress through the accumulation of organic osmolytes are largely unknown. Yet, among organisms, this is an almost universal phenomenon. In mammals, the cells of the renal medulla are uniquely exposed to high and variable salt concentrations; in response, renal cells accumulate the osmolyte sorbitol through increased transcription of the aldose reductase (AR) gene. In cloning the rabbit AR gene, we found the first evidence of an osmotic response region in a eukaryotic gene. More recently, we functionally defined a minimal essential osmotic response element (ORE) having the sequence CGGAAAATCAC(C) (bp -1105 to -1094). In the present study, we systematically replaced each base with every other possible nucleotide and tested the resulting sequences individually in reporter gene constructs. Additionally, we categorized hyperosmotic response by electrophoretic mobility shift assays of a 17-bp sequence (-1108 to -1092) containing the native ORE as a probe against which the test constructs would compete for binding. In this manner, binding activity was assessed for the full range of osmotic responses obtained. Thus we have arrived at a functional consensus for the mammalian ORE, NGGAAAWDHMC(N). This finding should accelerate the discovery of genes previously unrecognized as being osmotically regulated.

Activator Protein-1 Contributes to High NaCl-induced Increase in Tonicity-responsive Enhancer/Osmotic Response Element-binding Protein Transactivating Activity

Tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1–4 and NF-κB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-κB·AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE·AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE·AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCl-induced increase of TonEBP/OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE·AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity.

High NaCl- and urea-induced posttranslational modifications that increase glycerophosphocholine by inhibiting GDPD5 phosphodiesterase

Glycerophosphocholine (GPC) is high in cells of the renal inner medulla where high interstitial NaCl and urea power concentration of the urine. GPC protects inner medullary cells against the perturbing effects of high NaCl and urea by stabilizing intracellular macromolecules. Degradation of GPC is catalyzed by the glycerophosphocholine phosphodiesterase activity of glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). We previously found that inhibitory posttranslational modification (PTM) of GDPD5 contributes to high NaCl- and urea-induced increase of GPC. The purpose of the present studies was to identify the PTM(s). We find at least three such PTMs in HEK293 cells: (i) Formation of a disulfide bond between C25 and C571. High NaCl and high urea increase reactive oxygen species (ROS). The ROS increase disulfide bonding between GDPD5-C25 and -C571, which inhibits GDPD5 activity, as supported by the findings that the antioxidant N-acetylcysteine prevents high NaCl- and urea-induced inhibition of GDPD5; GDPD5-C25S/C571S mutation or over expression of peroxiredoxin increases GDPD5 activity; H2O2 inhibits activity of wild type GDPD5, but not of GDPD5-C25S/C571S; and peroxiredoxin is relatively low in the renal inner medulla where GPC is high. (ii) Dephosphorylation of GDPD5-T587. GDPD5 threonine 587 is constitutively phosphorylated. High NaCl and high urea dephosphorylate GDPD5-T587. Mutation of GDPD5-T587 to alanine, which cannot be phosphorylated, decreases GPC-PDE activity of GDPD5. (iii) Alteration at an unknown site mediated by CDK1. Inhibition of CDK1 protein kinase reduces GDE-PDE activity of GDPD5 without altering phosphorylation at T587, and CDK1/5 inhibitor reduces activity of GDPD5- C25S/C571S-T587A.