TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity

Abstract

While hyperosmolality of the kidney medulla is essential for urinary concentration, it imposes a great deal of stress. Cells in the renal medulla adapt to the stress of hypertonicity (hyperosmotic salt) by accumulating organic osmolytes. Tonicity-responsive enhancer (TonE) binding protein (TonEBP) (or NFAT5) stimulates transcription of transporters and a synthetic enzyme for the cellular accumulation of organic osmolytes. We found that dominant-negative TonEBP reduced expression of HSP70 as well as the transporters and enzyme. Near the major histocompatibility complex class III locus, there are three HSP70 genes named HSP70-1, HSP70-2, and HSC70t. While HSP70-1 and HSP70-2 were heat inducible, only HSP70-2 was induced by hypertonicity. In the 5' flanking region of the HSP70-2 gene, there are three sites for TonEBP binding. In cells transfected with a reporter plasmid containing this region, expression of luciferase was markedly stimulated in response to hypertonicity. Coexpression of the dominant-negative TonEBP reduced the luciferase expression. Mutating all three sites in the reporter plasmid led to a complete loss of induction by hypertonicity. Thus, TonEBP rather than heat shock factor stimulates transcription of the HSP70-2 gene in response to hypertonicity. We conclude that TonEBP is a master regulator of the renal medulla for cellular protection against high osmolality via organic osmolytes and molecular chaperones.

FIG. 1.

Expression of HSP70 and TonEBP in mIMCD cells. Confluent mIMCD cells were treated with hypertonic medium (lane HT; made by addition of 100 mM NaCl), were heat shocked (lane HS; 42°C), or were untreated (C) for 4 h. Then, 5 μg of total RNA was used for Northern blot detection of HSP70 mRNA (top) and 40 μg of protein of cell lysate was used for immunoblot detection of HSP70, HSC70, and TonEBP.

FIG. 2.

Effects of DN-TonEBP on mRNA expression. MDCK cells stably transfected with pcDNA3.1 (lanes V) or pcDNA3.1 driving expression of DN-TonEBP (lanes R6, a clonal line) were cultured overnight in control isotonic (lanes C) or hypertonic medium (lanes HT). (A) Cell lysate containing 40 μg of protein was used in each lane for immunoblot analysis of TonEBP. Bands representing TonEBP and DN-TonEBP are indicated on the right. (B) Five micrograms of RNA was used for Northern blot detection of mRNA for SMIT, BGT1, AR, HSP70, COX-2, αB-crystallin, and glyceraldehyde 3-phosphate dehydrogenase.

FIG. 3.

RPA to detect expression of specific HSP70 isoforms. (A) Genomic structure near the mouse major histocompatibility complex class III region is shown. The rectangles and vertical bar represent exons. HSC70t consists of two exons while HSP70-1 and HSP70-2 each consists of one exon. Bent arrows show the direction of transcription. The region that contains TonE sequences (TonEs) is indicated with a bar. (B) RNA samples of Fig. 1 were analyzed to detect isoform-specific transcripts as indicated.

FIG. 4.

TonE's found upstream of the HSP70-2 gene and their binding to TonEBP. (A) Sequences and locations (relative to the start codon of the HSP70-2 gene) of four sequences (TonEA to TonED) that fit the TonE consensus. (B) EMSA was performed using 0.5 nM (each) ³²P-labeled hTonE, TonEA, TonEB, TonEC, and TonED. Nuclear extracts were isolated from confluent MDCK cells treated with isotonic (lane C) or hypertonic medium (lanes HT) overnight. In some lanes, 50 nM hTonE (lane WT [TGGAAAATTAC]) or inactive mutant hTonE (lane m [TGGAtAATTAC]) was added as a competitor. TonEBP antiserum (lane IM) or preimmune serum (lane PI) was used to confirm the presence of TonEBP in the bands. (C) In order to compare their affinity to TonEBP, 50 nM hTonE (lane WT), mutant hTonE (lane m), TonEA (lane A), TonEB (lane B), TonEC (lane C), or TonED (lane D) was used to compete with 0.5 nM ³²P-hTonE for TonEBP binding.

FIG. 5.

Activity of HSP70-2 promoter in response to hypertonicity and heat shock. A 4.1-kb genomic DNA fragment covering nucleotides −4,158 to −43 upstream of the start codon was cloned into a luciferase reporter vector. mIMCD cells were transfected with the reporter plasmid along with pcDNA3.1 (vector) or pcDNA3.1 driving expression of DN-TonEBP or TonEBP as indicated. The transfected cells were treated with hypertonic medium or heat shock. The activity of luciferase is shown relative to the vector control. Data are the mean ± standard deviation (n = 4). Note that the scale for the heat shock-treated group is shown on the right.

FIG. 6.

Role of TonE in activity of HSP70-2 promoter. TonEA, TonEB, and TonED in the reporter plasmid described in Fig. 5 was inactivated by site-directed mutagenesis in various combinations as shown. Mutated TonE's are indicated by filled ovals. Relative luciferase activity in mIMCD cells cultured in isotonic and hypertonic medium is shown. Data are mean ± standard deviation (n = 3).