J Am Soc Nephrol 14: 283–288, 2003 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.

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

TonEBP (tonicity-responsive enhancer binding protein) is a accumulation of urea (UT-A), protection of cells from the high transcriptional activator of the Rel family that includes NF-␬B concentration of urea (HSP70). In cultured cells, TonEBP is and NFAT. The Rel family proteins share unique structural stimulated by an increase in ambient tonicity in temporal features in the DNA binding domains. The DNA binding correlation with the upregulation of the target genes (7). Like- domains of TonEBP and NF-␬B form dimers despite minimal wise in the kidney medulla, changes in the activity of TonEBP identity in amino acid sequences (1), and the dimerization is in response to water loading or thirsting leads to corresponding required for DNA binding (2,3). Ironically, the DNA binding changes in the expression of SMIT, BGT1, and AR expression domain of TonEBP shares 43% of amino acids with those of (8). Thus, hypertonicity in the renal medulla is an important NFAT isoforms that do not dimerize (4). TonEBP is also called local signal for maintenance of differentiation or tissue-specific NFAT5 on the basis of this amino acid similarity (5). gene expression, and TonEBP is a key player in this. TonEBP was first cloned as a candidate transcriptional ac- TonEBP is widely expressed during early development and tivator that binds specifically to the DNA sequence called in adulthood: TonEBP is abundantly expressed in brain, heart, tonicity-responsive enhancer (TonE) (4). On the basis of a liver, and many other organs (9). Mature T cells express high variety of analyses, including reporter gene expression, in levels of TonEBP (10), which stimulates certain cytokine vitro, and sometimes in vivo binding to the TonE sites up- genes including TNF-␣ (2). In epithelial cells, TonEBP is ␣ ␤ stream of the promoters, TonE has been proposed to be the induced by the 6 4 integrin and is required for carcinoma regulatory element that mediates stimulation of several genes invasion (11). Function of TonEBP in other organs is not in response to hypertonicity (see reference 6 for a recent review understood. To study the function of TonEBP in the kidney and references therein). These genes include the sodium/myo- medulla or other organs and tissues, we sought to develop tools inositol cotransporter (SMIT), sodium/chloride/betaine con- to knock out expression of TonEBP. Here we report develop- transporter (BGT1), aldose reductase (AR), vasopressin-regu- ment of RNA interference that silences TonEBP expression. lated urea transporter (UT-A), and heat shock protein 70 (HSP70–2). Expression of all of these genes is much higher in Materials and Methods the hypertonic renal medulla than the cortex, and they are Preparation of siRNA Duplexes essential for the function of the renal medulla: accumulation of 23-nucleotide single-stranded RNAs shown in Figure 1 were chem- compatible osmolytes to protect cells from the deleterious ically synthesized and purified using a commercial source (Dharma- effects of hypertonicity (SMIT, BGT1, AR), counter-current con Research, Lafayette, CO). For annealing of siRNA, 20 ␮M single strands were incubated in annealing buffer (100 mM potassium ace- tate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90°C followed by1hat37°C. More than 95% was annealed Received September 16, 2002. Accepted October 18, 2002. Correspondence to Dr. H. Moo Kwon, Nephrology, N3W143, University of on the basis of analysis using a 15% polyacrylamide gel in 40 mM Maryland, 22 South Greene St., Baltimore, MD 21201. Phone: 410-706-4382; Tris-acetate, 1 mM EDTA, and 30 mM NaCl. Fax: 410-706-4314; E-mail: [email protected] 1046-6673/1402-0283 Cell Culture Journal of the American Society of Nephrology HeLa cells in passages 5 to 10 were grown at 37°C in Dulbecco Copyright © 2003 by the American Society of Nephrology modified Eagle’s medium (Life Technologies, Rockville, MD) sup- DOI: 10.1097/01.ASN.0000045050.19544.B2 plemented with 10% fetal bovine serum, 2 mM glutamine, 100 ␮g/ml 284 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 283–288, 2003

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 ␮g/ml aprotinin, 5 ␮M leupeptin, 1 mM phenylmethylsulfonylfluoride, and 10 ␮M 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 for1hin20mMTris-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 ϫ 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 Figure 1. Small interfering RNA (siRNA) duplexes targeted for min in 0.5% Triton X-100 in Tris-buffered saline. The cells were then human TonEBP. (A) The TonEBP mRNA is schematically shown. incubated for 30 min in a 1:400 dilution of the TonEBP antiserum in Numbered boxes represent exons from which the transcript is made PBS at room temperature. TonEBP was visualized by incubation in a (9). Exons 2 to 4 are alternatively spliced (see text for details). 1:400 dilution of Alexa 568-conjugated goat anti-rabbit antiserum Translation starts used by various TonEBP isoforms are indicated (Molecular Probes, Eugene, OR) in PBS containing 3% BSA. The with arrowheads above exons 1 and 5. Translation stop at exon 15 is coverslips were mounted in Prolong antifade (Molecular Probes) for marked with downward arrowhead. Regions targeted by siRNA du- observation. plexes 279R, 569R, and 1469R are indicated below. (B) The sense (top) and antisense (bottom) sequences of 279R, 569R, and inv569R Northern Blot Analyses that have the inverted sequence of 569R. 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 streptomycin, and 100 units/ml penicillin. Hypertonic medium was nitrocellulose membrane. Membranes were hybridized overnight with made by addition of 100 mM NaCl to the control isotonic medium. 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, Transfection and Luciferase Assays X01677). After washing under stringent conditions (60°Cin75mM The day before transfection, HeLa cells were seeded at 50 to 70% NaCl, 7.5 mM Na-citrate, and 0.1% SDS), radioactivity was detected confluence in 6-well plates. Transfection of siRNA was carried out using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). 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 ϭ 2) Results showed that dual transfection was more effective in silencing TonEBP Design of TonEBP-siRNA Duplexes than single transfection. For analysis of reporter gene expression, 0.3 To silence the expression of TonEBP by RNA interference, ␮g of TonE-driven Photinus luciferase reporter plasmid or 0.1 ␮gof we designed small interfering RNA (siRNA) duplexes. Wide- ␤-actin promotor driven Photinus luciferase reporter (12) was trans- spread alternative splicing in exons 2, 3, and 4 of the TonEBP fected into cells in each well using Lipofectamine 2000 (Life Tech- gene produces five different transcripts that encode four dif- nologies) 24 h after the transfection of siRNA. Each construct was ferent polypeptides; the start codon for three such polypeptides transfected along with 40 ng of pRL-TK, where the thymidine kinase is in exon 1, and these polypeptides are in frame with the start promotor drove expression of Renilla luciferase. After transfection, codon in exon 5 from which translation of the shortest polypep- the cells were maintained in isotonic medium for 24 h and then tide starts (9). The siRNA duplexes 279R and 569R are tar- switched to hypertonic medium or maintained in isotonic medium for geted to the two start codons as shown in Figure 1: the codons 16 h. Activity of the Photinus and Renilla luciferase in extracts of the transfected cells were determined using a commercial kit, Dual- start at nucleotides 279 and 569 of the TonEBP cDNA (Gen- Luciferase Reporter Assay System (Promega, Madison, WI). For each Bank accession number AF089824), respectively. As a control, sample, activity of the Photinus luciferase is divided by the activity of inverted sequence of 569R (inv569R) was used. The siRNA the Renilla luciferase to correct for transfection efficiency. The cor- duplex 1469R was targeted to the nucleotides 1469 to 1489 of rected TonE-driven luciferase activity was expressed relative to that TonEBP cDNA corresponding to exons 7 and 8. Each of the of the ␤-actin promoter-driven luciferase as described previously (12). siRNA duplexes has 21 bp with symmetric U di-nucleotide J Am Soc Nephrol 14: 283–288, 2003 RNAi of TonEBP 285

3'-overhangs as described by Elbashir et al. (13). Each of the tion of 279R or 569R reduced the abundance of TonEBP both siRNA duplexes 279R, 569R, and 1469R targets all the splice in isotonic and hypertonic conditions (Figure 2A) without variant transcripts because the targeted sequence is not alter- grossly changing nucleocytoplasmic distribution of TonEBP natively spliced. (Figure 2B), demonstrating that the siRNA silenced TonEBP expression. These effects were specific to TonEBP in that Characterization of TonEBP-siRNA Duplexes expression of HSC70 was not affected. On the other hand, We chose HeLa cells to use the TonEBP-siRNA duplexes inv569R did not affect the abundance of TonEBP, indicating because they are transfected with high efficiency, typically that the silencing of TonEBP by 279R and 569R was due to over 80% (not shown). When HeLa cells were switched to RNA interference. In addition, 1469R did not affect the abun- hypertonic medium, the abundance of TonEBP increased some dance of TonEBP (not shown), demonstrating that not all fourfold (Figure 2A) in 16 h as in MDCK cells (7). Introduc- sequence-matched siRNA was effective in RNA interference. 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 abun- dance 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 silenc- ing. 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.

Silencing TonEBP Reduces TonE-Driven Transcription RNA interference of TonEBP provides a unique opportunity to examine cellular function of TonEBP because direct inhib- itor of TonEBP is not known. To examine TonE-driven tran- scription, a TonE-driven luciferase reporter construct was transfected into HeLa cells after transfection of siRNA du-

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 Figure 3. Dose-dependence of TonEBP silencing. HeLa cells were probed for TonEBP and HSC70. Positions of molecular weight mark- transfected as in Figure 2 with different concentrations of 569R as ers are shown at right. (B) Immunocytochemical detection of TonEBP indicated or 400 nM of inv569R (control). Cells were treated with in HeLa cells treated as in panel A, except that the cells were grown hypertonic medium for 16 h before harvest. Immunoblot analyses of on glass coverslips. Representatives of four independent experiments TonEBP and HSC70 were performed. A representative of three inde- are shown in panels A and B. pendent experiments is shown. 286 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 283–288, 2003

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.

plexes. 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 hyper- tonicity. When transfected with 569R, luciferase expression was decreased by 40% in isotonic conditions (P Ͻ 0.05, t test) and by 80% in hypertonic conditions (P Ͻ 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 Ͻ 0.05) and an 85% reduction in hy- pertonic conditions (P Ͻ 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 lu- ciferase expression in isotonic and hypertonic conditions, pro- viding the first direct link in vivo between TonEBP and TonE- driven transcription.

Silencing TonEBP Reduces Expression of TonEBP Target Genes HeLa cells express only the sodium/myo-inositol cotrans- Figure 5. Silencing of TonEBP reduces TonE-driven transcription. porter (SMIT), aldose reductase (AR), and heat shock protein HeLa cells were transfected with 400 nM of inv569R (inv), 569R (A), 70 (HSP70) among the known TonEBP target genes. In iso- or 279R (B). Twenty-four hours later, the cells were transfected with tonic conditions, the abundance of mRNA for these genes was the hTonE-GL3 reporter construct and cultured in isotonic or hyper- very low. The abundance of these mRNA decreased by silenc- tonic medium for 16 h. Relative luciferase activity was measured as Ϯ ϭ ing of TonEBP (Figure 6), but the decreases did not reach described in Materials and Methods. Mean SD, n 4. statistical significance (n ϭ 3 or 4). In response to hypertonic- ity, the mRNA abundance increased clearly. RNA interference of TonEBP led to 45 to 55% decrease in the abundance of expressed in HeLa cells. Cyclooxygenase 2 (COX2) mRNA mRNA of these genes in hypertonic conditions when corrected was expressed in HeLa cells, and its expression increased for RNA loading using the abundance of G3PDH mRNA (P Ͻ fivefold in response to hypertonicity. The abundance of COX2 0.05 for SMIT, AR, and HSP70; n ϭ 3 or 4). These data mRNA was not affected by silencing of TonEBP (not shown), provide direct evidence that TonEBP stimulates the transcrip- indicating that TonEBP does not regulate the COX2 gene. This tion of the SMIT, AR, and HSP70 genes. is consistent with our earlier finding that the dominant negative Using cDNA microarray analysis and differential selection form of TonEBP does not affect expression of COX2 in of transcripts, we identified a number of genes for which MDCK cells (16). Another hypertonicity-induced gene ex- transcription was increased in response to hypertonicity (ref- pressed in HeLa cells was calcyclin (17). The mRNA abun- erence 15 and unpublished observation). A few of them are dance of calcyclin increased twofold by hypertonicity but was J Am Soc Nephrol 14: 283Ð288, 2003 RNAi of TonEBP 287

lines that do not express TonEBP and the lack of inhibitors that act directly on TonEBP. In transient transfection assays, ex- pression 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-regu- lated genes was inhibited in certain colonies (6), but the inhi- bition 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 undis- putable 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 pro- longed silencing of TonEBP in time. Because of high effi- ciency 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 effec- tively 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 therapeuti- cally 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). Figure 6. Effects of TonEBP silencing on mRNA expression. Cells Loss-of-function test should be useful in investigating the role were transfected and treated as described in Figure 2. Northern anal- of TonEBP in a given cell type or tissue. RNAi described here yses were performed to detect mRNA for AR, SMIT, HSP70, calcy- provides a potential tool to downregulate TonEBP expression clin, and G3PDH. A representative of three or four experiments is in a tissue-specific manner. Effective methods to deliver suf- shown for each gene. ficient amount of siRNA duplexes to the target cells with high efficiency need to be developed. not affected by the silencing of TonEBP (Figure 6). Thus, TonEBP does not regulate the calcyclin gene either. Acknowledgments This work was supported by National Institutes of Health Grant Discussion RO1 DK42479 and American Heart Association Grant-In-Aid The results collectively demonstrate that we developed two 0150368N. SDL was supported by the National Kidney Foundation siRNA duplexes that silenced TonEBP expression via RNA Fellowship. 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 References 1. Stroud JC, Lo´pez-Rodrõ«guez C, Rao A, Chen L: Structure of a has been reported by other investigators (14). The transitory TonEBP-DNA complex reveals DNA encircled by a transcrip- nature of RNA interference in mammalian cells limits its use in tion factor. Nature Struct Biol 9: 90Ð94, 2002 certain cases, but it can be beneficial as explained below. 2. Lo«pez-Rodrõ«guez C, Aramburu J, Jin L, Rakeman AS, Michino TonEBP was identified on the basis of its specific binding to M, Rao A: Bridging the NFAT and NF␬B families: NFAT5 the TonE sites in vitro (4). Investigating the role of TonEBP in dimerization regulates cytokine gene transcription in response to cellular context in vivo has been hampered by the lack of cell osmotic stress. Immunity 15: 47Ð58, 2001 288 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 283Ð288, 2003

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