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J Am Soc Nephrol 14:283-288, 2003
© 2003 American Society of Nephrology

Silencing of TonEBP/NFAT5 Transcriptional Activator by RNA Interference

Ki Young Na, Seung Kyoon Woo, Sang Do Lee and H. Moo Kwon

Division of Nephrology, Johns Hopkins University, Baltimore, Maryland.

Correspondence to Dr. H. Moo Kwon, Nephrology, N3W143, University of Maryland, 22 South Greene St., Baltimore, MD 21201. Phone: 410-706-4382; Fax: 410-706-4314;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. TonEBP is a transcriptional activator that is expressed throughout development in many tissues and cell types. In the kidney medulla, TonEBP appears to be an important local regulator of differentiation by virtue of stimulating several genes. To study the function of TonEBP, two small interfering RNA (siRNA) duplexes were developed that reduced TonEBP expression effectively via RNA interference. The silencing lasted only 3 d after introduction of the TonEBP-siRNA’s. As expected, TonEBP-driven reporter gene expression and expression of the sodium/myo-inositol cotransproter (SMIT), aldose reductase (AR) and heat shock protein 70 (HSP70) mRNA were significantly decreased in cells where TonEBP expression was silenced. These data provide direct evidence that the SMIT, AR, and HSP70 genes are targets of TonEBP, although the potential role of other proteins, such as accessory proteins, cannot be excluded. The TonEBP-siRNA is an effective tool that should prove useful in the investigation of loss-of-function relationship in cells. E-mail: mookwon@hotmail.com


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TonEBP (tonicity-responsive enhancer binding protein) is a transcriptional activator of the Rel family that includes NF-{kappa}B and NFAT. The Rel family proteins share unique structural features in the DNA binding domains. The DNA binding domains of TonEBP and NF-{kappa}B form dimers despite minimal identity in amino acid sequences (1), and the dimerization is required for DNA binding (2,3). Ironically, the DNA binding domain of TonEBP shares 43% of amino acids with those of NFAT isoforms that do not dimerize (4). TonEBP is also called NFAT5 on the basis of this amino acid similarity (5).

TonEBP was first cloned as a candidate transcriptional activator that binds specifically to the DNA sequence called tonicity-responsive enhancer (TonE) (4). On the basis of a variety of analyses, including reporter gene expression, in vitro, and sometimes in vivo binding to the TonE sites upstream of the promoters, TonE has been proposed to be the regulatory element that mediates stimulation of several genes in response to hypertonicity (see reference 6 for a recent review and references therein). These genes include the sodium/myo-inositol cotransporter (SMIT), sodium/chloride/betaine contransporter (BGT1), aldose reductase (AR), vasopressin-regulated urea transporter (UT-A), and heat shock protein 70 (HSP70–2). Expression of all of these genes is much higher in the hypertonic renal medulla than the cortex, and they are essential for the function of the renal medulla: accumulation of compatible osmolytes to protect cells from the deleterious effects of hypertonicity (SMIT, BGT1, AR), counter-current accumulation of urea (UT-A), protection of cells from the high concentration of urea (HSP70). In cultured cells, TonEBP is stimulated by an increase in ambient tonicity in temporal correlation with the upregulation of the target genes (7). Likewise in the kidney medulla, changes in the activity of TonEBP in response to water loading or thirsting leads to corresponding changes in the expression of SMIT, BGT1, and AR expression (8). Thus, hypertonicity in the renal medulla is an important local signal for maintenance of differentiation or tissue-specific gene expression, and TonEBP is a key player in this.

TonEBP is widely expressed during early development and in adulthood: TonEBP is abundantly expressed in brain, heart, liver, and many other organs (9). Mature T cells express high levels of TonEBP (10), which stimulates certain cytokine genes including TNF-{alpha} (2). In epithelial cells, TonEBP is induced by the {alpha}6{beta}4 integrin and is required for carcinoma invasion (11). Function of TonEBP in other organs is not understood. To study the function of TonEBP in the kidney medulla or other organs and tissues, we sought to develop tools to knock out expression of TonEBP. Here we report development of RNA interference that silences TonEBP expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of siRNA Duplexes
23-nucleotide single-stranded RNAs shown in Figure 1 were chemically synthesized and purified using a commercial source (Dharmacon Research, Lafayette, CO). For annealing of siRNA, 20 µM single strands were incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90°C followed by 1 h at 37°C. More than 95% was annealed on the basis of analysis using a 15% polyacrylamide gel in 40 mM Tris-acetate, 1 mM EDTA, and 30 mM NaCl.



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  		Figure 1. Small interfering RNA (siRNA) duplexes targeted for human TonEBP. (A) The TonEBP mRNA is schematically shown. Numbered boxes represent exons from which the transcript is made (9). Exons 2 to 4 are alternatively spliced (see text for details). Translation starts used by various TonEBP isoforms are indicated with arrowheads above exons 1 and 5. Translation stop at exon 15 is marked with downward arrowhead. Regions targeted by siRNA duplexes 279R, 569R, and 1469R are indicated below. (B) The sense (top) and antisense (bottom) sequences of 279R, 569R, and inv569R that have the inverted sequence of 569R.

 
Cell Culture
HeLa cells in passages 5 to 10 were grown at 37°C in Dulbecco modified Eagle’s medium (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin. Hypertonic medium was made by addition of 100 mM NaCl to the control isotonic medium.

Transfection and Luciferase Assays
The day before transfection, HeLa cells were seeded at 50 to 70% confluence in 6-well plates. Transfection of siRNA was carried out using Oligofectamine (Life Technologies) with 10 to 1000 nM siRNA duplex in final culture medium. Cells were transfected twice with a 16-h interval and analyzed 2 to 14 d later. Preliminary results (n = 2) showed that dual transfection was more effective in silencing TonEBP than single transfection. For analysis of reporter gene expression, 0.3 µg of TonE-driven Photinus luciferase reporter plasmid or 0.1 µg of {beta}-actin promotor driven Photinus luciferase reporter (12) was transfected into cells in each well using Lipofectamine 2000 (Life Technologies) 24 h after the transfection of siRNA. Each construct was transfected along with 40 ng of pRL-TK, where the thymidine kinase promotor drove expression of Renilla luciferase. After transfection, the cells were maintained in isotonic medium for 24 h and then switched to hypertonic medium or maintained in isotonic medium for 16 h. Activity of the Photinus and Renilla luciferase in extracts of the transfected cells were determined using a commercial kit, Dual-Luciferase Reporter Assay System (Promega, Madison, WI). For each sample, activity of the Photinus luciferase is divided by the activity of the Renilla luciferase to correct for transfection efficiency. The corrected TonE-driven luciferase activity was expressed relative to that of the {beta}-actin promoter-driven luciferase as described previously (12).

Immunoblot Analyses
Cells were lysed for 30 min at 4°C in a lysis buffer (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) with freshly added protease inhibitors: 0.2 µg/ml aprotinin, 5 µM leupeptin, 1 mM phenylmethylsulfonylfluoride, and 10 µM E64. The cell lysate was cleared by centrifugation, separated on a 6% SDS-polyacrylamide gel, and blotted onto a nitrocellulose membrane. To detect a specific protein, the blots were incubated with an antiserum/antibody at a 2000-fold diution for 1 h in 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat milk. Monoclonal antibody for HSC70 was obtained from Stressgene (Victoria, British Columbia, Canada), and polyclonal antiserum against TonEBP was described previously (4). The blots were then incubated with a secondary antibody conjugated with alkaline phosphatase and visualized using a commercial substrate for alkaline phosphatase (Sigma Chemical, St Louis, MO).

Immunocytochemical Analyses
HeLa cells were seeded on 18 x 18–mm glass coverlips and transfected with the siRNA as described above. The cells were fixed for 15 min in 3% paraformaldehyde in PBS and permeablized for 15 min in 0.5% Triton X-100 in Tris-buffered saline. The cells were then incubated for 30 min in a 1:400 dilution of the TonEBP antiserum in PBS at room temperature. TonEBP was visualized by incubation in a 1:400 dilution of Alexa 568-conjugated goat anti-rabbit antiserum (Molecular Probes, Eugene, OR) in PBS containing 3% BSA. The coverslips were mounted in Prolong antifade (Molecular Probes) for observation.

Northern Blot Analyses
RNA was isolated using Trizol reagent (Life Technologies). Five micrograms of RNA from each sample was separated on an 1% agarose gel containing 2.2 M formaldehyde and transferred onto a nitrocellulose membrane. Membranes were hybridized overnight with radiolabeled cDNA probes: human AR (GenBank accession number J05474), canine SMIT (M85068), human HSP70 (M11717), mouse calcyclin (X66449), human cyclooxygenase 2 (COX2, M90100), and human glyceraldehydes 3-phosphate dehydrogenase (G3PDH, X01677). After washing under stringent conditions (60°C in 75 mM NaCl, 7.5 mM Na-citrate, and 0.1% SDS), radioactivity was detected using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Design of TonEBP-siRNA Duplexes
To silence the expression of TonEBP by RNA interference, we designed small interfering RNA (siRNA) duplexes. Widespread alternative splicing in exons 2, 3, and 4 of the TonEBP gene produces five different transcripts that encode four different polypeptides; the start codon for three such polypeptides is in exon 1, and these polypeptides are in frame with the start codon in exon 5 from which translation of the shortest polypeptide starts (9). The siRNA duplexes 279R and 569R are targeted to the two start codons as shown in Figure 1: the codons start at nucleotides 279 and 569 of the TonEBP cDNA (GenBank accession number AF089824), respectively. As a control, inverted sequence of 569R (inv569R) was used. The siRNA duplex 1469R was targeted to the nucleotides 1469 to 1489 of TonEBP cDNA corresponding to exons 7 and 8. Each of the siRNA duplexes has 21 bp with symmetric U di-nucleotide 3'-overhangs as described by Elbashir et al. (13). Each of the siRNA duplexes 279R, 569R, and 1469R targets all the splice variant transcripts because the targeted sequence is not alternatively spliced.

Characterization of TonEBP-siRNA Duplexes
We chose HeLa cells to use the TonEBP-siRNA duplexes because they are transfected with high efficiency, typically over 80% (not shown). When HeLa cells were switched to hypertonic medium, the abundance of TonEBP increased some fourfold (Figure 2A) in 16 h as in MDCK cells (7). Introduction of 279R or 569R reduced the abundance of TonEBP both in isotonic and hypertonic conditions (Figure 2A) without grossly changing nucleocytoplasmic distribution of TonEBP (Figure 2B), demonstrating that the siRNA silenced TonEBP expression. These effects were specific to TonEBP in that expression of HSC70 was not affected. On the other hand, inv569R did not affect the abundance of TonEBP, indicating that the silencing of TonEBP by 279R and 569R was due to RNA interference. In addition, 1469R did not affect the abundance of TonEBP (not shown), demonstrating that not all sequence-matched siRNA was effective in RNA interference.



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  		Figure 2. Silencing of TonEBP in HeLa cells. (A) Immunoblots of transfected HeLa cells under isotonic or hypertonic condition. The cells were transfected with buffer (B) or 400 nM of TonEBP-siRNA duplex 279R, 569R, or inv569R (inv) as described in Materials and Methods and analyzed 65 h later. They were treated with isotonic or hypertonic medium for 16 h before analysis. Immunoblots were probed for TonEBP and HSC70. Positions of molecular weight markers are shown at right. (B) Immunocytochemical detection of TonEBP in HeLa cells treated as in panel A, except that the cells were grown on glass coverslips. Representatives of four independent experiments are shown in panels A and B.

 
We characterized the RNA interference of 569R further. The silencing of TonEBP was dependent on the concentration of 569R employed in transfection (Figure 3). The silencing reached maximum of 70 to 90% reduction in TonEBP abundance at 400 nM. Other investigators also noted that siRNA did not eliminate targeted proteins completely (13,14). We are aware of two factors that contribute to the incomplete silencing. First, transfection efficiency in HeLa cells is typically about 90%; i.e., 10% of cells are not transfected and therefore not subjected to RNA interference. Second, the half-life of TonEBP is 10 h (7). Thus, even after a complete absence of TonEBP mRNA for 2 d, 5% of TonEBP should remain. The silencing was also time-dependent. As shown in Figure 4, silencing was evident 2 and 3 d after transfection. However, by 7 d, the silencing disappeared. This observation is in line with the observation that RNAi in the HeLa-derived Magi-CCR5 cell line is maximal 60 h after transfection (14). We conclude that RNA interference of TonEBP expression in HeLa cells is transient and dependent on the concentration of TonEBP-siRNA duplex.



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  		Figure 3. Dose-dependence of TonEBP silencing. HeLa cells were transfected as in Figure 2 with different concentrations of 569R as indicated or 400 nM of inv569R (control). Cells were treated with hypertonic medium for 16 h before harvest. Immunoblot analyses of TonEBP and HSC70 were performed. A representative of three independent experiments is shown.

 


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  		Figure 4. Time-dependence of TonEBP silencing. HeLa cells were transfected with 400 nM inv569R (inv) or 569R, and immunoblot analyses were performed 2, 3, 7, or 14 d later. Cells were treated with isotonic (I) or hypertonic medium (H) for 16 h before immunoblot analyses. A representative of three independent experiments is shown.

 
Silencing TonEBP Reduces TonE-Driven Transcription
RNA interference of TonEBP provides a unique opportunity to examine cellular function of TonEBP because direct inhibitor of TonEBP is not known. To examine TonE-driven transcription, a TonE-driven luciferase reporter construct was transfected into HeLa cells after transfection of siRNA duplexes. In HeLa cells transfected with inv569R (Figure 5, A and B) or no siRNA (not shown), TonE-driven luciferase expression increased more than 20-fold in response to hypertonicity. When transfected with 569R, luciferase expression was decreased by 40% in isotonic conditions (P < 0.05, t test) and by 80% in hypertonic conditions (P < 0.001) (Figure 5A). There was a 58% reduction in the fold-induction of luciferase expression in response to hypertonicity. Likewise, transfection of 279R leads to a 60% reduction in luciferase expression in isotonic conditions (P < 0.05) and an 85% reduction in hypertonic conditions (P < 0.005) (Figure 5B). There was a 56% reduction in the fold-induction of luciferase expression in response to hypertonicity. Thus, silencing of TonEBP by RNA interference leads to a dramatic reduction in TonE-driven luciferase expression in isotonic and hypertonic conditions, providing the first direct link in vivo between TonEBP and TonE-driven transcription.



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  		Figure 5. Silencing of TonEBP reduces TonE-driven transcription. HeLa cells were transfected with 400 nM of inv569R (inv), 569R (A), or 279R (B). Twenty-four hours later, the cells were transfected with the hTonE-GL3 reporter construct and cultured in isotonic or hypertonic medium for 16 h. Relative luciferase activity was measured as described in Materials and Methods. Mean ± SD, n = 4.

 
Silencing TonEBP Reduces Expression of TonEBP Target Genes
HeLa cells express only the sodium/myo-inositol cotransporter (SMIT), aldose reductase (AR), and heat shock protein 70 (HSP70) among the known TonEBP target genes. In isotonic conditions, the abundance of mRNA for these genes was very low. The abundance of these mRNA decreased by silencing of TonEBP (Figure 6), but the decreases did not reach statistical significance (n = 3 or 4). In response to hypertonicity, the mRNA abundance increased clearly. RNA interference of TonEBP led to 45 to 55% decrease in the abundance of mRNA of these genes in hypertonic conditions when corrected for RNA loading using the abundance of G3PDH mRNA (P < 0.05 for SMIT, AR, and HSP70; n = 3 or 4). These data provide direct evidence that TonEBP stimulates the transcription of the SMIT, AR, and HSP70 genes.



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  		Figure 6. Effects of TonEBP silencing on mRNA expression. Cells were transfected and treated as described in Figure 2. Northern analyses were performed to detect mRNA for AR, SMIT, HSP70, calcyclin, and G3PDH. A representative of three or four experiments is shown for each gene.

 
Using cDNA microarray analysis and differential selection of transcripts, we identified a number of genes for which transcription was increased in response to hypertonicity (reference 15 and unpublished observation). A few of them are expressed in HeLa cells. Cyclooxygenase 2 (COX2) mRNA was expressed in HeLa cells, and its expression increased fivefold in response to hypertonicity. The abundance of COX2 mRNA was not affected by silencing of TonEBP (not shown), indicating that TonEBP does not regulate the COX2 gene. This is consistent with our earlier finding that the dominant negative form of TonEBP does not affect expression of COX2 in MDCK cells (16). Another hypertonicity-induced gene expressed in HeLa cells was calcyclin (17). The mRNA abundance of calcyclin increased twofold by hypertonicity but was not affected by the silencing of TonEBP (Figure 6). Thus, TonEBP does not regulate the calcyclin gene either.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results collectively demonstrate that we developed two siRNA duplexes that silenced TonEBP expression via RNA interference. The silencing was more effective in hypertonic conditions, reducing the abundance of TonEBP up to 90%. On the other hand, the silencing was transient, lasting only 3 d, as has been reported by other investigators (14). The transitory nature of RNA interference in mammalian cells limits its use in certain cases, but it can be beneficial as explained below.

TonEBP was identified on the basis of its specific binding to the TonE sites in vitro (4). Investigating the role of TonEBP in cellular context in vivo has been hampered by the lack of cell lines that do not express TonEBP and the lack of inhibitors that act directly on TonEBP. In transient transfection assays, expression of a dominant negative form of TonEBP (DN-TonEBP) leads to inhibition of TonE-driven expression of luciferase from a plasmid co-transfected with the expression vector for DN-TonEBP (4). When DN-TonEBP was stably expressed, expression of SMIT and some other tonicity-regulated genes was inhibited in certain colonies (6), but the inhibition invariably disappeared as cells continued proliferation (not shown). In fact, most colonies expressing DN-TonEBP did not display reduced transcription of SMIT or AR, raising doubts about the cellular function of TonEBP in regulating the tonicity-responsive genes. Because generation of cells stably expressing a protein after transfection takes time to allow massive proliferation, we suspect that in time cells adapt to and overcome the inhibitory effects of DN-TonEBP.

The results in this study clearly demonstrate that expression of the SMIT, AR, and HSP70 mRNA is lowered when TonEBP is silenced by RNA interference. This provides the first undisputable loss-of-function evidence that TonEBP is required for transcription of the SMIT, AR, and HSP70 genes, although potential role of an accessory protein cannot be precluded. On the basis of our experience with stable expression of DN-TonEBP (see above), we suspect that cells will adapt to prolonged silencing of TonEBP in time. Because of high efficiency in transfection, we were able to effectively silence TonEBP acutely and examine its effect on gene expression in HeLa cells. Thus, RNA interference provided a tool to effectively knock out TonEBP transiently so that the role of TonEBP can be studied without the complications of cellular adaptation. RNA interference may also be useful therapeutically when temporary downregulation of a gene is required.

TonEBP is widely expressed in a variety of organs (10,9) in addition to the kidney in which TonEBP expression varies tremendously among segments of tubules and cell types (8). Loss-of-function test should be useful in investigating the role of TonEBP in a given cell type or tissue. RNAi described here provides a potential tool to downregulate TonEBP expression in a tissue-specific manner. Effective methods to deliver sufficient amount of siRNA duplexes to the target cells with high efficiency need to be developed.


   Acknowledgments
 
This work was supported by National Institutes of Health Grant RO1 DK42479 and American Heart Association Grant-In-Aid 0150368N. SDL was supported by the National Kidney Foundation Fellowship.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stroud JC, López-Rodríguez C, Rao A, Chen L: Structure of a TonEBP-DNA complex reveals DNA encircled by a transcription factor. Nature Struct Biol 9: 90–94, 2002[CrossRef][Medline]
  2. López-Rodríguez C, Aramburu J, Jin L, Rakeman AS, Michino M, Rao A: Bridging the NFAT and NF{kappa}B families: NFAT5 dimerization regulates cytokine gene transcription in response to osmotic stress. Immunity 15: 47–58, 2001[CrossRef][Medline]
  3. Lee SD, Woo SK, Kwon HM: Dimerization is required for phosphorylation and DNA binding of TonEBP/NFAT5. Biochem Biophys Res Comm 294: 968–975, 2002[CrossRef][Medline]
  4. Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM: Tonicity-responsive enhancer binding protein, a Rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci USA 92: 2538–2542, 1999
  5. López-Rodríguez C, Aramburu J, Rakeman A, Rao A: NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun. Proc Natl Acad Sci USA 92: 7214–7219, 1999
  6. Woo SK, Lee SD, Kwon HM: TonEBP transcriptional activator in the cellular response to increased osmolality. Pflüger Arch Eur J Physiol 444: 579–585, 2002[CrossRef][Medline]
  7. Woo SK, Dahl SC, Handler JS, Kwon HM: Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity. Am J Physiol Renal Physiol 278: F1006–F1012, 2000[Abstract/Free Full Text]
  8. Cha JH, Woo SK, Han KH, Kim YH, Handler JS, Kim J, Kwon HM: Hydration status affects nuclear distribution of transcription factor tonicity responsive enhancer binding protein in rat kidney. J Am Soc Neprhol 12: 2221–2230, 2001[Abstract/Free Full Text]
  9. Maouyo D, Kim JY, Lee SD, Wo Y, Woo SK, Kwon HM: Mouse TonEBP-NFAT5: expression in early development and alternative splicing. Am J Physiol Renal Physiol 282: F802–F809, 2002[Abstract/Free Full Text]
  10. Trama J, Lu Q, Hawley RG, Ho SN: The NFAT-related protein NFATL1 (TonEBP/NFAT5) is induced upon T cell activation in a calcineurin-dependent manner. J Immunol 165: 4884–4894, 2000[Abstract/Free Full Text]
  11. Jauliac S, López-Rodríguez C, Shaw LM, Brown LF, Rao A, Toker A: The role of NFAT transcription factors in integrin-mediated carcinoma invasion. Nature Cell Biol 4: 540–544, 2002[CrossRef][Medline]
  12. Miyakawa H, Woo SK, Chen CP, Dahl SC, Handler JS, Kwon HM: Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity. Am J Physiol Renal Physiol 274: F753–F761, 1998[Abstract/Free Full Text]
  13. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498, 2001[CrossRef][Medline]
  14. Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA: siRNA-directed inhibition of HIV-1 infection. Nature Medicine 8: 681–686, 2002[CrossRef][Medline]
  15. Nahm O, Woo SK, Handler JS, Kwon HM: Involvement of multiple kinase pathways in stimulation of gene transcription by hypertonicity. Am J Physiol Cell Physiol 282: C49–C58, 2002[Abstract/Free Full Text]
  16. Woo SK, Lee SD, Na KY, Park WK, Kwon HM: TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity. Mol Cell Biol 22: 5753–5760, 2002[Abstract/Free Full Text]
  17. Courtois-Coutry N, Le Moellic C, Boulkroun S, Fay M, Cluzeaud F, Escoubet B, Farman N, Blot-Chabaud M: Calcyclin is an early vasopressin-induced gene in the renal collecting duct: Role in the long term regulation of ion transport. J Biol Chem 277: 25728–25734, 2002[Abstract/Free Full Text]
Received for publication September 16, 2002. Accepted for publication October 18, 2002.




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J. H. Esensten, A. V. Tsytsykova, C. Lopez-Rodriguez, F. A. Ligeiro, A. Rao, and A. E. Goldfeld
NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone
Nucleic Acids Res., July 12, 2005; 33(12): 3845 - 3854.
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 Am. J. Physiol. Renal Physiol.Home page
K.-H. Han, S. K. Woo, W.-Y. Kim, S.-H. Park, J.-H. Cha, J. Kim, and H. M. Kwon
Maturation of TonEBP expression in developing rat kidney
Am J Physiol Renal Physiol, November 1, 2004; 287(5): F878 - F885.
[Abstract] [Full Text] [PDF]

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