Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP.

The accumulation of compatible osmolytes during osmotic stress is observed in virtually all organisms. In mammals, the hypertonicity-induced expression of osmolyte transporters and synthetic enzymes is conferred by the presence of upstream tonicity-responsive enhancer (TonE) sequences. Recently, we described the cloning and initial characterization of TonE-binding protein (TonEBP), a transcription factor that translocates to the nucleus and associates with TonE sequences in a tonicity-dependent manner. We now report that hypertonicity induces an increase in TonEBP phosphorylation that temporally correlates with increased nuclear localization of the molecule. TonEBP phosphorylation is not affected by a number of kinase inhibitors, including the p38 inhibitor SB-203580. In addition, in vitro binding assays show that the association of TonEBP with TonE sequences is not affected by phosphorylation. Thus TonEBP phosphorylation is an early step in the response of cells to hypertonicity and may be required for nuclear import or retention.

Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity.

Tonicity-responsive enhancer binding protein (TonEBP) regulates transcription of tonicity responsive genes such as the sodium-myo-inositol cotransporter (SMIT), the sodium-chloride-betaine cotransporter (BGT1), and aldose reductase (AR). To characterize signals that activate TonEBP in Madin-Darby canine kidney (MDCK) cells, the abundance and nuclear distribution of TonEBP were studied after the osmolality of the culture medium was changed. Hypertonicity but not hyperosmolality is effective in activation of TonEBP as expected. Surprisingly, exposure to hypotonic medium leads to a dramatic downregulation of TonEBP both in abundance and nuclear distribution, indicating that under isotonic conditions, TonEBP is at a low-level activated state and can respond to both increase and decrease in tonicity. Additional experiments suggest that cellular ionic strength is the signal that initiates regulation of TonEBP. The increase in abundance of TonEBP is mediated by an increase in mRNA abundance and a parallel increase in synthesis of TonEBP. The stability of TonEBP mRNA is not affected by hypertonicity indicating that transcription plays a major role in the induction of TonEBP by hypertonicity.

Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity.

Hypertonicity (most often present as high salinity) is stressful to the cells of virtually all organisms. Cells survive in a hypertonic environment by increasing the transcription of genes whose products catalyze cellular accumulation of compatible osmolytes. In mammals, the kidney medulla is normally hypertonic because of the urinary concentrating mechanism. Cellular accumulation of compatible osmolytes in the renal medulla is catalyzed by the sodium/myo-inositol cotransporter (SMIT), the sodium/chloride/betaine cotransporter, and aldose reductase (synthesis of sorbitol). The importance of compatible osmolytes is underscored by the necrotic injury of the renal medulla and subsequent renal failure that results from the inhibition of SMIT in vivo by administration of a specific inhibitor. Tonicity-responsive enhancers (TonE) play a key role in hypertonicity-induced transcriptional stimulation of SMIT, sodium/chloride/betaine cotransporter, and aldose reductase. We report the cDNA cloning of human TonE binding protein (TonEBP), a transcription factor that stimulates transcription through its binding to TonE sequences via a Rel-like DNA binding domain. Western blot and immunohistochemical analyses of cells cultured in hypertonic medium reveal that exposure to hypertonicity elicits slow activation of TonEBP, which is the result of an increase in TonEBP amount and translocation to the nucleus.

Tyrosine kinase inhibitors and immunosuppressants perturb the myo-inositol but not the betaine cotransporter in isotonic and hypertonic MDCK cells.

BACKGROUND: The sodium/myo-inositol cotransporter (SMIT) and the betaine cotransporter (BGT1) are essential for the accumulation of myo-inositol and betaine, and hence cell survival in a hypertonic environment. The underlying molecular mechanism involves an increase in transcription of the SMIT and BGT1 genes through binding of a trans-acting factor to enhancer elements in the 5' flanking region of both genes, resulting in increased mRNA abundance and increased activity of the cotransporters. Current evidence regarding transcriptional and post-transcriptional regulation indicates that both cotransporters are regulated in parallel. METHODS: To investigate the signal transduction of hypertonic stress, we examined the effect of tyrosine kinase inhibitors and immunosuppressants on the hypertonicity-induced activity of the two cotransporters in Madin-Darby canine kidney (MDCK) cells. RESULTS: None of the agents studied affected BGT1 activity in isotonic or hypertonic conditions. Treatment of MDCK cells with genistein, a tyrosine kinase inhibitor, increased SMIT activity in hypertonic but not isotonic conditions. The stimulation of SMIT by genistein was accompanied by a parallel increase in mRNA abundance. In contrast, treating cells with tyrphostin A23, another tyrosine kinase inhibitor, or cyclosporine A, an immunosuppressant, inhibited SMIT activity in hypertonic cells. FK506, another immunosuppressant, increased SMIT activity, but only in isotonic conditions. CONCLUSIONS: These results provide the first evidence of divergent regulatory pathways modulating SMIT and BGT activity.

Transcription of the sodium/myo-inositol cotransporter gene is regulated by multiple tonicity-responsive enhancers spread over 50 kilobase pairs in the 5'-flanking region.

The sodium/myo-inositol cotransporter is a plasma membrane protein responsible for concentrative cellular accumulation of myo-inositol in a variety of tissues. When cells in kidney and brain are exposed to a hyperosmolar salt condition (hypertonicity) due to the operation of urinary concentration mechanism and pathological conditions, respectively, they survive the stress of hypertonicity by raising the cellular concentration of myo-inositol. Transcription of the sodium/myo-inositol cotransporter gene is markedly stimulated in response to hypertonicity, leading to an increase in the activity of the cotransporter, which in turn drives the osmoprotective accumulation of myo-inositol. To understand the molecular mechanisms by which hypertonicity stimulates transcription, we analyzed the 5'-flanking region of the cotransporter gene for cis-acting regulatory sequences. We identified five tonicity-responsive enhancers that are scattered over 50 kilobase pairs. All the enhancers are variations of the same type of enhancer interacting with the transcription factor named tonicity-responsive enhancer binding protein. In vivo methylation experiments demonstrated that exposure of cells to hypertonicity increases the binding of tonicity-responsive enhancer binding protein to the enhancer sites, indicating that all of these enhancers are involved in the transcriptional stimulation. We conclude that the sodium/myo-inositol cotransporter gene is regulated by a large region (approximately 50 kilobase pairs) upstream of the gene.

Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity.

We have previously identified a tonicity-responsive enhancer (TonE) in the promoter region of the canine BGT1 gene. TonE mediates hypertonicity-induced stimulation of transcription. Here, we characterize TonE and TonE binding proteins (TonEBPs) to provide a biochemical basis for cloning of the TonEBPs. Mutational analysis applied to both hypertonicity-induced stimulation of transcription and TonEBP binding reveals that TonE is 11 base pairs in length, with the consensus sequence of (C/T)GGAAnnn(C/T)n(C/T). Activity of the TonEBPs increases in response to hypertonicity with a time course similar to that of transcription of the BGT1 gene. Studies with inhibitors indicate that translation, but not transcription, is required for activation of the TonEBPs. Phosphorylation is required for the stimulation of transcription but not for activation of DNA binding by the TonEBPs. In vivo methylation by dimethyl sulfate reveals that the TonE site of the BGT1 gene is protected with a time course like that of activity of the TonEBPs and activation of transcription. Ultraviolet cross-linking indicates that the TonEBPs share a DNA binding subunit of 200 kDa.

Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia.

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin-deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.

Integrin phosphorylation is modulated during the differentiation of F-9 teratocarcinoma stem cells.

The retinoic acid-induced differentiation of F-9 teratocarcinoma cells in monolayer culture is accompanied by the accumulation of fibrillar fibronectin deposits, the appearance of a highly structured actin cytoskeleton, and the redistribution of integrin to apparent sites of substrate contact. We have studied the 140-kD fibronectin receptor during this process and report that although the integrin molecule is present in equivalent amounts before and after differentiation, the level of integrin phosphorylation decreases dramatically as the cells differentiate. This loss of phosphorylation coincides temporally with the observed changes in actin, fibronectin, and integrin organization. The phosphorylation state of integrin thus may mediate developmentally regulated cell-matrix interactions.

Altered accumulations of fibronectin are not dependent on fibronectin modifications during the differentiation of F-9 teratocarcinoma stem cells.

F-9 teratocarcinoma stem cells differentiate into parietal endoderm when monolayer cultures are treated with retinoic acid. This change in phenotype is accompanied by increased accumulation and altered organization of fibronectin deposits. Although both stem cells and treated cells synthesize and accumulate fibronectin, only the treated cells deposit a fibrillar array of the protein. We have monitored the accumulation of fibronectin in nontreated and treated F-9 cells with indirect immunofluorescence and have biochemically analyzed the fibronectin synthesized by each cell type with one- and two-dimensional acrylamide gels and peptide maps. Our data suggest that no differences exist between these fibronectins to account for the observed changes in accumulation. Thus, another mechanism may regulate the organization of matrix deposition.