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Genetics and Development |
1 Promoter for Treatment of Progressive Renal Diseases
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Departments of * Internal Medicine and Advanced Medicine and Advanced Research Institute of the Sciences and Humanities, Nihon University, Tokyo Japan; Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan; and || Gentier Biosystems Incorporation, Tokyo, Japan
Address correspondence to: Dr. Noboru Fukuda, Department of Internal Medicine, Nihon University School of Medicine, Ooyaguchi-kami 30-1, Itabashi-ku, Tokyo 173-8610, Japan. Phone: +81-3-3972-8111; Fax: +81-3-3972-8666; fukudan{at}med.nihon-u.ac.jp
Received for publication June 22, 2005. Accepted for publication November 2, 2005.
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Abstract |
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1 was designed as a gene-silencing agent for progressive renal diseases, and the distribution and the effects of this polyamide on renal injury were examined in Dahl-salt sensitive (Dahl-S) rats. For identification of transcription factor binding elements for activation of the rat TGF-1 gene, recombinant TGF-1 reporter plasmids were transfected into HEK-293 cells, and promoter activity was measured. Py-Im polyamide was designed to the activator protein-1 binding site of the rat TGF-1 promoter. This Py-Im polyamide showed strong, fast, and specific binding to the target DNA in gel mobility shift and Biacore assays. Py-Im polyamide significantly inhibited TGF-1 promoter activity and expression of TGF-1 mRNA and protein in rat mesangial cells. Intravenously administered fluorescein-labeled polyamide distributed to the kidney of rats. Py-Im polyamide significantly inhibited expression of TGF-1 mRNA and protein in the renal cortex of Dahl-S rats and reduced the increase in urinary protein and albumin in Dahl-S rats independent of changes in blood pressure. These results indicate that Py-Im polyamide that targets TGF-1 will be a novel gene-silencing agent for the TGF-1–associated diseases, including progressive renal diseases. |
Introduction |
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Pyrrole-imidazole (Py-Im) polyamides first were identified from duocarmycin A and distamycin A, which recognize and bind DNA with sequence specificities and are small synthetic molecules that are composed of the aromatic rings of N-methylpyrrole and N-methylimidazole amino acids (1–3). Synthetic Py-Im polyamides bind to specific nucleotide sequences in the minor groove of double-helical DNA with high affinity and block binding of specific proteins. Therefore, Py-Im polyamides may be useful tools in molecular biology medicine. Py-Im polyamides are resistant to nucleases and do not require particular delivery systems (4). Various types of sequence-specific DNA-binding Py-Im polyamides have been developed to regulate gene expression by targeting the promoter regions of enhancer and transcription factor binding elements (5). Binding of enhancers to the major and minor grooves of DNA can be inhibited by the minor groove-binding Py-Im polyamides (6). DNA recognition by Py-Im polyamides depends on a code of side-by-side pairing of Py and Im in the minor groove; pairing of Im opposite Py (Im/Py) targets a G-C base pair, and Py/Im targets a C-G base pair. Py/Py targets either a T-A or an A-T base pair (3). The binding constants and specificity of Py-Im polyamides are comparable to those of transcription factors. Dickinson et al. (7) reported the genes of the HIV were silenced by Py-Im polyamides to their regulatory sequences.
A number of studies have identified TGF-1 as a critical factor in kidney diseases such as glomerulosclerosis (8) and mesangioproliferative glomerulonephritis (9,10). TGF-1 stimulates the proliferation of mesangial cells and the production of extracellular matrix (11,12). TGF-1 has been reported to induce Thy-1–associated nephritis (13), diabetic nephropathy (14), and interstitial nephritis associated with obstructive nephropathy (15) in rats. TGF-1 was reported recently to induce epithelial-mesenchymal transformation in renal tissue, which plays a critical role in the pathogenesis of nephritis (16). Thus, TGF-1 induces nephrosclerosis and interstitial nephropathy by its effects on mesangial cell growth, extracellular matrix formation, and epithelial-mesenchymal transformation. TGF-1 is expressed highly in various animal models of progressive renal disease such as hypertensive renal sclerosis, diabetic nephropathy, and focal renal sclerosis (17–20). Dahl salt-sensitive (Dahl-S) rats show glomerulosclerosis, which is known to be associated with TGF-1 (17). To develop a novel gene-silencing agent for the treatment of progressive renal diseases, we designed and synthesized a Py-Im polyamide targeting TGF-1 and examined its distribution and effects on TGF-1 expression and renal injury in Dahl-S rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthesis of Polyamide Targeting Rat TGF-1
Polyamide targeting rat TGF-1 (polyamide) was designed to span the boundary of the activator protein-1 (AP-1) binding site (–2303 to –2297) of the TGF-1 promoter (Figure 1A). Numbering refers to the start of the open reading frame as +1 (21). Mismatch polyamide (mismatch) was designed not to bind transcription binding sites of the promoter. Polyamides were synthesized according to previously described methods (22) (Figure 1B).
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Kinetics of binding of polyamide and mismatch to target sequence were assessed by surface plasmon resonance technique with a molecular interaction model. Biotin double-strand DNA was immobilized to streptavidin-functionalized sensorchip SA (Biacore, Uppsala, Sweden). Measurements of binding curves of polyamide or mismatch to the DNA and data processing were performed on a Biacore X (Biacore) according to recommended protocols.
Construction and Activity of Rat TGF-1 Promoter Plasmid and Effect of Polyamide on Promoter Activity
Rat TGF-1 promoter cloned into the KpnI site of the pGL3-basic vector was a gift from Dr. Kyoung Lim (Ajou University School of Medicine, Suwon, Korea). The promoter deletion mutant (–2676 to 72) was cleaved with KpnI and BpuI102I and self-ligated to the promoter-pGL3-basic vector fragment with DNA blunting kit (TaKaRa; Otsu, Shiga, Japan). Other deletion mutants were generated by cleaving with KpnI and SacI, XhoI, PvuII, Van91I, NsiI, BstPI, SacII, or Bsu36I to obtain promoter terminating at –2424, –2153, –1715, –1474, –1327, –1104, –742, and –513 to 72.
HEK-293 cells were seeded onto 24-well plates and grown in DMEM (Sigma-Aldrich, St. Louis, MO) with 20% calf serum. At 70 to 90% confluence, a mixture of reporter plasmid (1 µg/well) and phRG-TK vector (0.01 µg/well; Promega) as an internal control was used to transfect cells with Lipofectamine2000 (Invitrogen, Carlsbad, CA) as described previously (23) and then changed to flesh medium in the presence or absence 1.0 mM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich). Cells were incubated for an additional 24 h and scraped into 100 µl of cold lysis buffer (PBS [pH 7.4] and 1 mM PMSF). Luciferase activity was measured with a Dual-luciferase reporter assay system (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) (24).
For evaluation of the effect of polyamide on promoter activity, HEK-293 cells were transfected with rat TGF-1 promoter plasmid and incubated with 0.1 or 1.0 µM polyamide or 1.0 µM mismatch in the presence of 1.0 µM PMA for 24 h. Luciferase activity then was measured.
Distribution of Fluorescein-Labeled Polyamide In Vitro and In Vivo
Glomeruli were isolated from the kidneys of 4-wk-old Wistar-Kyoto rats (SHR Corp., Funabashi, Chiba, Japan) by a graded-sieve technique as described previously (25). Mesangial cells were isolated from explants of whole glomeruli according to the differential growth capacities of glomerular epithelial and mesangial cells.
Mesangial cells were seeded onto plates and grown in RPMI-1640 medium (Sigma-Aldrich) with 10% FCS. After 24 h, cells were incubated with 2 µM fluorescein-labeled polyamide in RPMI-1640 medium for 2 h. Cells were washed, and fresh RPMI-1640 was added. After 22 h, cells were viewed at x200 under live cell conditions and then fixed in 4% paraformaldehyde for 10 min. Nuclei were stained with Hoechst 33342 (Invitrogen) and viewed again.
Five milligrams of fluorescein-labeled polyamide was injected into Wistar rats (250 g body wt) via the tail vein. After 24 h, the kidneys, aorta, heart, and brain were removed, and frozen specimens were made and viewed at x200. Subsequently, 50 to 100 mg of each tissue was homogenized in DMSO (Sigma-Aldrich). Each sample was centrifuged and freeze-dried. The precipitate was reconstituted with 50 µl of N,N-dimethylformamide (Sigma-Aldrich). Urine was also collected for 24 h in metabolic cages. Fluorescein-labeled polyamide in 20 µl of N,N-dimethylformamide and 20 µl of urinary samples were subjected to HPLC (0.01% CHO2NH4/CH3CN 0 to 100% linear gradient from 0 to 30 min through a Chemcobond 5-ODS-H column [Chemco Scientific, Osaka, Japan]). Fluorescence was measured with a fluorescence detector (FP-2020 Plus; Nihon Bunko, Tokyo, Japan).
In Vivo Experimental Design
Seven-week-old male Dahl-S rats (CLEA Japan, Tokyo, Japan) were used in this study. Rats were divided into two groups and were fed 0.3% NaCl (low-salt [LS]) or 8% NaCl (high-salt [HS]) diet (both from Oriental Yeast, Tokyo, Japan) ad libitum for 2 wk. One milligram of polyamide or mismatch was dissolved in 100 µl of DMSO plus 100 µl of H2O and injected via the tail veins of HS rats every 2 d for 2 wk (total 7 mg of polyamide per rat). Control rats received injections of 100 µl of DMSO plus 100 µl of H2O (Figure 2). Systolic BP (SBP) was measured by the tail-cuff method. Urine was collected in metabolic cages for 24 h every week. Urinary protein and albumin excretion were determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).
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Real-time quantitative PCR was performed with cDNA diluted four times and TaqMan Universal Master Mix and an ABI 7500 sequence detector (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Assay-on-Demand primers and probes (TGF-1, Rn00572010-m1; connective tissue growth factor [CTGF], Rn00573960-g1; collagen type 1 
1, Rn00801649-g1; fibronectin, Rn00569575-m1 and TaqMan Rodent GAPDH control reagents) were purchased from Applied Biosystems. Real-time PCR data were analyzed with a standard curve. Correlation coefficients for the standard curves all were >0.90.
Measurement of TGF-1 Protein
TGF-1 protein levels in urine and conditioned medium were determined by enzyme immunoassay (TGF-1 Emax ImmunoAssay System; Promega) as described previously (26). Mesangial cells were treated with 0.1 or 1.0 µM polyamide or 1.0 µM mismatch in the presence of 1.0 µM PMA for 24 h. Conditioned medium was collected and diluted with TGF-1 sample buffer. Because this assay detects only active TGF-1 protein, each sample was acidified to convert latent TGF-1 to the active form. Urine samples from Dahl-S rats that were fed an HS diet for 2 wk were collected for 24 h and diluted 1:1000 in sample buffer.
TGF-1 Immunofluorescence
Unstained 3-µm-thick paraffin sections of renal cortex were deparaffinized, hydrated, and heated for antigen unmasking. Sections were blocked with Serum Blocking Reagent G (R&D Systems, Minneapolis, MN) for 15 min. The slides then were incubated with diluted primary antibody (mAb to TGF-1; R&D Systems) for 3 h at room temperature, washed in PBS, and incubated with fluorescein-conjugated polyclonal goat anti-mouse antibody (Dako, Carpinteria, CA) for 30 min at room temperature. After being washed in PBS, sections were incubated with Hoechst 33342 and viewed at x200.
Statistical Analyses
Values are reported as mean ± SEM. A t test was used for unpaired data. Two-way ANOVA and Duncan multiple range tests were also used. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 Promoter Analysis1 gene, a rat TGF-1 promoter-pGL3-basic luciferase reporter chimeric plasmid was created (Figure 3). The start site of the rat 2.5-kb TGF-1 transcript (892 nucleotides upstream of ATG) (24) is at a position homologous to that of human TGF-1 (858 and 852 nucleotides upstream of ATG) (27) and mouse (856 nucleotides upstream of ATG) (28). A comparison of this conserved region with that of other species revealed 91.4% overall identity with the mouse sequence and 81.7% identity with the human sequence (29).
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We searched for transcription factor binding sites between –2424 and –2153 with TFSEACH (http://mbs.cbrc.jp/research/db/TFSEARCH.html) (30). This region contained an AP-1 binding site (–2303 to –2297). Polyamide was designed to bind to this region (Figure 1A).
Binding and Specificity of Polyamide to Double-Stranded DNA and Inhibition of AP-1 Complex Binding
Gel mobility shift and Biacore assays allow for the determination of the binding affinity and specificity of polyamides for target DNA. Polyamide bound the appropriate 21-bp double-stranded DNA but did not bind the 2-bp mutated DNA, whereas mismatch did not show binding to appropriate DNA (Figure 4A). As shown in Figure 4B, a single mobility band was observed when the DNA were incubated with AP-1 or treatment with mismatch. Polyamide inhibited AP-1 binding to target DNA.
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1 Promoter Activity and Expression of TGF-11 promoter plasmid. Polyamide was significantly and in a concentration-dependent manner decreased luciferase activity stimulated by PMA (Figure 5A). In mesangial cells, polyamide decreased expression of TGF-1 mRNA (Figure 5B) and protein (Figure 5C) stimulated by PMA in a concentration-dependent manner. Mismatch did not affect luciferase activity or expression of TGF-1 mRNA and protein in response to PMA.
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HPLC analysis of fluorescein-labeled polyamide in urine, kidney, aorta, heart, and brain is shown in Figure 6C. Fluorescein-labeled polyamide was clearly detected in urine, kidney, and aorta but not in heart or brain.
Effect of Polyamide on Urinary Protein and TGF-1 Expression in Dahl-S Rats
Intravenous administration of polyamide or mismatch did not affect the body weights of Dahl-S rats (Figure 7A). SBP was remarkably increased in HS rats compared with LS rats. Polyamide or mismatch did not affect SBP (Figure 7B). Both urinary protein and albumin were significantly increased in HS rats compared with levels in LS rats. Treatment with polyamide significantly reduced the increased urinary protein and albumin in HS rats. Mismatch did not affect urinary protein or albumin levels (Figure 7, C and D).
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1, CTGF, collagen type 1 1, and fibronectin mRNA in the renal cortex of Dahl-S rats are shown in Figure 8, A through D. Expression of these mRNA was significantly higher in HS rats than in LS rats. Treatment with polyamide significantly decreased expression of these mRNA in HS rats. Mismatch did not affect expression of these mRNA.
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1 protein in Dahl-S rats is shown in Figure 8E. Urinary excretion of TGF-1 protein was significantly higher in HS rats than in LS rats. Treatment with polyamide significantly decreased urinary excretion of TGF-1 protein in HS rats. Mismatch did not affect urinary excretion of TGF-1 protein in HS rats.
Immunofluorescence of TGF-1 in Renal Cortex
Immunofluorescence of TGF-1 in the renal cortex of Dahl-S rats is shown in Figure 9. TGF-1 staining in glomeruli and nephrotubuli in HS rats was stronger than that in LS rats. Treatment with polyamide considerably reduced TGF-1 staining in glomeruli and nephrotubuli in HS rats. Mismatch did not affect TGF-1 staining.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To design a polyamide that targets rat TGF-1, we analyzed the rat TGF-1 promoter structure and PMA-stimulated activity in promoter deletion mutants. Positive-regulatory elements that were stimulated by PMA were found at bases –2424 to –2153, which contain an AP-1 binding site. AP-1 elements respond to AP-1 transcription factors such as Jun homodimers or Fos/Jun heterodimers, PMA, angiotensin II, and v-Src. Stimulation of TGF-1 promoter activity occurs by binding to the AP-1 element (33,34). Polyamide targeting TGF-1 was designed not to cover AP-1 consensus sequences but spans the boundary of AP-1 binding site with the intention of obtaining specificity to the promoter. This polyamide showed strong, fast, and specific binding to the target DNA in gel mobility shift and Biacore assays. Fluorescein-labeled polyamide distributed sufficiently in the kidney without any delivery systems and localized in the nuclei of cultured cells for long periods of time. Best et al. (35,36) reported that fluorescein-polyamide conjugates exhibit good nuclear uptake in a wide variety of cell lines. In our in vivo experiments, polyamide administered intravenously was delivered sufficiently to the kidney and aorta and was localized to nuclei. Thus, this polyamide bound to target DNA in vitro and in vivo. HPLC analysis also confirmed that the presence of polyamide administered intravenously was in the urine, kidney, aorta, liver, and lung. Polyamide was not delivered in heart and brain. It is possible that the differentiated cardiac tissue does not express TGF-1 in normal heart, and this polyamide does not pass through the blood-brain barrier.
Nucleic acid medicines such as antisense DNA, ribozymes, and decoy have been developed as gene-silencing agents. Decoys, in particular, inhibit the binding of target transcription factors in a manner similar to polyamides. However, because these agents are degraded easily by nucleases, they require drug-delivery systems for sufficient distribution into organs. Because polyamides are completely resistant to nucleases and can be delivered into organs without delivery systems, polyamides will be more feasible gene-silencing medicines.
Dahl-S rats are useful models of human progressive renal disease. Dahl-S rats develop severe hypertension, glomerulosclerosis with thickening of capillary artery walls, and interstitial fibrosis in response to salt loading. Tamaki et al. (17) reported increased levels of TGF-1, plasminogen activator inhibitor-1, fibronectin, and collagen type 1 in the renal cortex of Dahl-S rats. In these experiments, expression of TGF-1 mRNA and protein was enhanced in the renal cortex of HS rats. Treatment with polyamide significantly inhibited this increased expression of TGF-1 mRNA and protein. Expression of mRNA for CTGF, a potent growth factor that stimulates the proliferation of mesenchymal cells, including mesangial cells, and induces the production of extracellular matrix downstream of TGF-1 signaling (37–39), was inhibited by polyamide. However, when cell cultures were treated with exogenous TGF-1, polyamide did not block CTGF production. Because the CTGF promoter also has an AP-1 site, this finding further supports the specificity of polyamide for the TGF-1 promoter. Expression of downstream extracellular matrix components such as collagen type 1 and fibronectin were also increased in the renal cortex of HS rats. This, too, was suppressed significantly by treatment with polyamide. In addition, the increased urinary protein and albumin in HS rats was significantly decreased by treatment with polyamide without a reduction in BP. These findings suggest that the polyamide ameliorated the renal damages by inhibition of TGF-1 in organs and extracellular matrix components in the renal cortex of Dahl-S rats.
In our study, although an HS diet did not induce considerable renal damage in Dahl-S rats during a 2-wk period, treatment with polyamide significantly suppressed TGF-1 immunofluorescence in the glomeruli and nephrotubuli of HS rats. Dahly et al. (40) injected anti–TGF- antibody into salt-loaded Dahl-S rats every day for 2 wk and found a significant decrease in BP, proteinuria, and degree of glomerulosclerosis and renal interstitial fibrosis. Polyamide completely inhibited the increases in proteinuria and albuminuria in salt-loaded Dahl-S rats, along with suppression of TGF-1 staining in glomeruli and nephrotubuli. These findings suggest that polyamide may be feasible gene-silencing agent for the treatment of progressive renal diseases.
We believe that polyamides will be important gene-silencing agents in the postgenome era. Polyamides can be readily designed and synthesized to target any gene. In the future, control of gene expression by polyamides may ameliorate diseases that are not treatable with current medicines.
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Acknowledgments |
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Footnotes |
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) or 8% NaCl (high salt; 
) diet. Dahl-S rats that were fed a high-salt diet also received an intravenous injection of 1 mg of polyamide (•) or mismatch polyamide (mismatch; 
) every 2 d (total 7 mg) for 2 wk. *P < 0.05 versus Dahl-S rats that were fed a high-salt diet without polyamide.
