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Heterogenous Nuclear Ribonucleoprotein F Modulates Angiotensinogen Gene Expression in Rat Kidney Proximal Tubular Cells

Chih-Chang Wei^*, Deng-Fu Guo^*, Shao-Ling Zhang, Julie R. Ingelfinger and John S.D. Chan^*

^* Centre de recherche, Centre Hospitalier de l’Université de Montréal (CHUM)-Hôtel-Dieu, Pavillon Masson, Montreal, Quebec, Canada; and Harvard Medical School, Massachusetts General Hospital, Pediatric Nephrology Unit, Boston, Massachusetts

Address correspondence to: Dr. John S.D. Chan, Centre de recherche, Centre Hospitalier de l’Université de Montréal (CHUM), Hôtel-Dieu Pavillon Masson, 3850 Saint Urbain Street, Montreal, Quebec, Canada H2W 1T8. Phone: 514-890-8000 ext. 15080; Fax: 514-412-7204; E-mail: john.chan{at}umontreal.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
An insulin-responsive element (IRE) in the rat angiotensinogen (ANG) gene promoter that binds to two nuclear proteins with apparent molecular weights of 48 and 70 kD was identified previously from rat immortalized renal proximal tubular cells (IRPTC). The present studies aimed to identify and clone the 48-kD nuclear protein and to define its action on ANG gene expression. Nuclear proteins were isolated from IRPTC and subjected to two-dimensional electrophoresis. The 48-kD nuclear protein was detected by Southwestern blotting and subsequently identified by mass spectrometry, revealing that it was identical to 46-kD heterogeneous nuclear ribonucleoprotein F (hnRNP F), a nuclear protein that binds to TATA-binding protein and associates with RNA polymerase II and also interacts with nuclear cap-binding complex. The hnRNP F cDNA was cloned from IRPTC by reverse transcriptase–PCR. Bacterially expressed recombinant hnRNP F bound to the rat ANG-IRE, as revealed by gel mobility shift assay. The addition of polyclonal antibodies against hnRNP F yielded a supershift in gel mobility. Transient transfer of sense and antisense hnRNP F cDNA in IRPTC inhibited and enhanced ANG gene expression, respectively. High glucose stimulated and insulin inhibited hnRNP F expression in IRPTC. Expression studies indicated that hnRNP F is present in the kidney, testis, liver, lung, and brain but not in the spleen. In conclusion, these studies demonstrate that hnRNP F binds to rANG-IRE and modulates renal ANG gene expression, implicating that dysregulation of hnRNP F might affect renin-angiotensin system activation and, subsequently, kidney injury in diabetes.

	Introduction

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Insulin regulates the expression of >100 genes by affecting the transcription, mRNA stability, or mRNA translation (1,2). The underlying mechanisms of insulin regulation of gene transcription and mRNA stability are poorly understood. One major obstacle that is hampering the progress at the transcriptional level is that there is no consensus insulin response element (IRE) that can account for the regulation of all insulin-responsive genes. This is in contrast with consensus-responsive elements that have been described for other hormones, e.g., the steroid hormone receptors. However, some genes whose transcription is inhibited by insulin seem to share a common IRE (T[G/A]TTT[T/G][G/T]) core sequence, including phosphoenolpyruvate carboxykinase, IGF-binding protein-1, tyrosine aminotransferase, glucose-6-phosphatase, apolipoprotein C III, and aspartate aminotransferase (3–8). Transacting factors that interact with the above common IRE have been tentatively identified, but none has been shown directly to mediate an insulin response. Moreover, other genes contain a well-defined IRE, but its sequence is substantially different from the common IRE (2); thus, it has been postulated that no single common transacting factor could be associated with all IRE of different genes.

Angiotensinogen (ANG) is a glycoprotein that consists of 452 amino acid residues with an apparent molecular weight of 62 to 65 kD (9,10). ANG is principally expressed in the liver and is the sole substrate in the renin-angiotensin system (RAS). In addition to the well-characterized systemic RAS, the presence of a local intrarenal RAS has now been generally accepted (11,12). The mRNA and protein for all RAS components, including ANG, renin, angiotensin-converting enzyme (ACE), and angiotensin II (Ang II) receptors (AT₁R and AT₂R subtypes), are expressed in murine and rat immortalized renal proximal tubular cells (IRPTC) (13–20). Several studies have reported that Ang II levels, ANG, and renin mRNA expression are elevated in early diabetic kidney (16,21,22), suggesting that hyperglycemia and/or augmented intrarenal Ang II may be directly or indirectly responsible for renal proximal tubular hypertrophy and tubulointerstitial fibrosis in diabetes.

In previous studies, we showed that high glucose stimulates and insulin inhibits ANG gene expression and cell hypertrophy in IRPTC (23–31). Moreover, we identified a putative IRE motif that contains nucleotides N-878 to N-864 (5'-CCT TCC CGC CCT TCA-3') upstream of the transcription start site of the rat ANG gene promoter (32). This ANG-IRE binds to two major nuclear proteins with apparent molecular weights of approximately 48 and 70 kD from IRPTC, as revealed by Southwestern blotting (31). It seems that high glucose and insulin enhanced and inhibited the expression of 48- and 70-kD nuclear protein expression in IRPTC, respectively. These data suggest that 48- and 70-kD nuclear protein might mediate the effect of high glucose and insulin on ANG gene expression in IRPTC.

The present studies aimed to identify and clone the 48-kD nuclear protein and to investigate its action on ANG gene expression. We identified the 48-kD nuclear protein as 46-kD heterogeneous nuclear ribonucleoprotein F (hnRNP F) by two-dimensional (2-D) electrophoresis and mass spectrometry. We demonstrated that hnRNP F binds to ANG-IRE and modulates ANG gene expression in IRPTC. Finally, high glucose stimulated and insulin inhibited hnRNP F expression in IRPTC. These studies demonstrate that 46-kD hnRNP F is one of the IRE-binding proteins (BP) that binds to the rat ANG gene promoter and modulates ANG gene expression in IRPTC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
D(+)-Glucose, D-mannitol, and insulin were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). -[32P-ATP] (3000 Ci/mol) and D-threo-[1,2 ¹⁴C]chloramphenicol were obtained from Amersham-Pharmacia Biotech (Baie d’Urfé, QC, Canada). Rabbit polyclonal antiserum raised against the full-length human hnRNP F and antiserum predominantly recognizing hnRNP F (CTARRYIGIVKQAGLER corresponding to amino acids 215 to 230 of human hnRNP F [33]) were a gift from Dr. Taka-aki Tamura (Chiba University, Chiba, Japan) and Dr. Christine Milcarek (University of Pittsburgh School of Medicine, Pittsburgh, PA), respectively. The characteristics of these antisera have been reported (34,35). The bacterial expression vector pGex 4T-3 and mammalian expression vector pcDNA 3.1 were purchased from Amersham-Pharmacia Biotech and InVitrogen (Burlington, ON, Canada), respectively. Restriction modified enzymes were acquired from InVitrogen., Amersham-Pharmacia Biotech, or Roche Diagnostics (Laval, QC, Canada).

Oligonucleotides for rat ANG-IRE N-882 to N-855 (5'-CCT CCC TTC CCG CCC TTC ACT TTC TAG T-3') (32), mutants of ANG N-882 to N-885 (M1, 5'-CCT CCC TTC CAT TAC TTC ACT TTC TAG T-3'; M2, 5'-CCT CCC TTA AAT AAG ACC ACT TTC TAG T-3'; M3, 5'-CCT CCC TTC CCT TCC TTC ACT TTC TAG T-3'; M4, 5'-CCT CCC TTC CCT CCC TTC ACT TTC TAG T-3'), rANG-IRE motif N-878 to N-864 (5'-CCT TCC CGC CCT TCA-3'), concatameric rANG-IRE motif (3 X N-878 to N-864, 5'-CCT TCC CGC CCT TCA CCT TCC CGC CCT TCA CCT TCC CGC CCT TCA-3'), IRE of human glyceraldehyde-3-phosphate dehydrogenase gene (hGAPDH-IRE; N-473 to N-477, 5'-CCA ACT TTC CCG CCT CTC AGC CTT TGA A-3') (36), and IRE of rat glucagon gene (N-267 to N-242, 5'-AGT TTT CAC GCC TGA CTG AGA TTG A-3') (37) were synthesized by InVitrogen. The oligonucleotide that contains the consensus SP1-binding site (5'-TCG CCC CGC CCC CGA TCG AAT-3') (38) was purchased from Promega (Fisher-Scientific, Montreal, QC, Canada).

The plasmid that contains the concatameric rANG-IRE motif DNA was constructed by inserting the double-stranded concatameric rANG-IRE motif oligonucleotide with the Not-1 enzyme restriction site added on both termini into the polyclonal site of pcDNA 3.1 by conventional method. The double-stranded concatameric rANG-IRE motif DNA fragment then was incised from the plasmid and used for labeling as probe.

The expression vectors and plasmid that contains the coding sequence for chloramphenicol acetyltransferase (CAT) without the promoter (pOCAT) or with Rous sarcoma virus enhancer/promoter sequence (pRSV/CAT) fused to the 5' end of the CAT coding sequence, respectively, were a gift from Dr. Joel F. Habener (Massachusetts General Hospital, Boston, MA). Thin-layer chromatography plates were purchased from Fisher Scientific (Montreal, QU, Canada).

Cell Culture
IRPTC at passages 12 to 16 were used in the present studies. The characteristics of IRPTC, which express the mRNA and protein of ANG, renin, ACE, and Ang II receptors, have been described previously (39).

Cellular and Tissue Nuclear Extract Preparation
Nuclear extracts from 10 plates (150 x 20 mm) each of confluent IRPTC that were previously incubated in DMEM with 5 mM glucose and 20 mM D-mannitol, 25 mM glucose, or 25 mM glucose plus insulin (10^–7 M) for 24 h and rat tissues (liver, kidney, testis, lung, brain, and spleen) were prepared according to the method of Henninghausen and Lubon (40) with slight modifications, as we have described elsewhere (31,41).

2-D Electrophoresis
2-D electrophoresis was carried out with the IPGphor Isoelectric Focusing Unit (Amersham-Pharmacia Biotech). For isoelectrofocusing (IEF), precast 13-cm IPG strips (pH 3 to 10, nonlinear, Immobiline DryStrips; Amersham-Pharmacia Biotech) were pre-equilibrated with 750 µg of nuclear extracts in 250 µl of rehydration buffer (8 M urea, 1 M thiourea, 4% CHAPS, 2% IPG buffer, 1% NP-40, 0.1 M dithiothreitol (DTT), and 0.0001% of bromophenol blue [BPB]) for >12 h according to the supplier’s manual. IEF was run for 90 kV-hours at 25°C. After IEF separation, the IPG strips were immediately equilibrated for 15 min with a buffer that contained 50 mM Tris-HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, 2% DTT, and 0.0001% of BPB. Then, the strips were re-equilibrated with another buffer that contained 50 mM Tris-HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, 2% iodoacetamide, and 0.0001% BPB for an additional 15 min. For 2-D separation, the IPG strips were placed above 10% PAGE that contained SDS and electrophoresed. Amersham’s rainbow markers served as molecular weight markers. IRPTC nuclear extracts (100 µg) were run on the same 10% PAGE-SDS as controls. Each sample was divided into two strips for 2-D electrophoresis. One gel was stained with Coomassie Brilliant Blue R-250 (Amresco, Solon, OH) to visualize proteins. The other was electrotransferred to a Hybond C-extra membrane (Amersham-Pharmacia Biotech) for Southwestern blotting.

Southwestern Blotting
Southwestern blotting was performed according to the procedure of Kwast-Welfeld et al. (42) with slight modifications (31,41). Briefly, IRPTC nuclear proteins (200 µg) were resolved on a 4 to 20% PAGE-SDS gradient or on 10% PAGE-SDS (43), then electrotransferred to a Hybond C-extra membrane, which was incubated with 10% (wt/vol) nonfat milk proteins in a binding buffer that contained 10 mM HEPES (pH 7.0), 10 mM MgCl₂, 50 mM NaCl, 0.25 mM EDTA, and 2.5% glycerol (vol/vol) for 24 h at 4°C. The membrane was washed at least twice with binding buffer that contained 0.25% nonfat milk proteins. Subsequently, it was hybridized overnight with ³²P-labeled concatameric ANG-IRE motif DNA (approximately 1.0 to 2.0 pmol; 10⁶ cp/ml) in binding buffer that contained 0.25% nonfat milk proteins and 300 µg/ml nondenatured herring sperm DNA at 4°C. The membrane was finally washed, air-dried, and exposed for autoradiography.

Matrix-Assisted Laser Desorption/Ionization Mass Spectroscopy
Spots on the gel corresponding to positive signals of the Southwestern blot membrane were picked up for matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS). All MALDI-MS analyses were performed at the Quebec Genome Centre (McGill University, Montreal, QC, Canada). Briefly, protein samples first were cleaved by trypsin and then subjected to MALDI-MS. MALDI-MS analysis was conducted at 20 kV accelerating voltage and 23 kV reflecting voltage. For protein identification, peptide mass fingerprints were searched by the Mascot program developed by Matrix Science Ltd. (freely accessible at www.matrixscience.com).

Cloning of hnRNP F
hnRNP F was cloned from IRPTC by conventional reverse transcription–PCR. Sense and antisense primers corresponding to nucleotides N + 1 to N + 19 (5'-ATG ATG CTG GGC CCT GAG G-3') and N + 1228 to N + 1245 (5'-TCG TAC CCA CCT ATA CTA ATC-3') of rat hnRNP F cDNA (35) were used in PCR. In addition, the Not 1 enzyme restriction site was added on the 5' and 3' ends of sense and antisense primers, respectively. hnRNP F cDNA then was subcloned in sense and antisense orientation at the polyclonal site (Not 1) of the bacterial expression vector pGex 4T-3 or mammalian expression vector pcDNA 3.1 by conventional method.

Purification of Recombinant hnRNP F from Bacteria
Escherichia coli BL-21 cells (Amersham-Pharmacia Biotech) were transformed by pGex 4T-3 that contained rat hnRNP F cDNA. Expression of the fusion protein (GST fused with hnRNP F [GST-hnRNP F]) in BL-21 cells was induced by the addition of 0.5 mM isopropylthiogalactoside into the culture medium with incubation for 4 h. The bacteria then were harvested, and GST-hnRNP F fusion proteins were purified from the bacterial extracts by GST affinity column chromatography according to the manufacturer‘s protocol (Amersham-Pharmacia Biotech). The purified GST-hnRNP F fusion proteins were used in gel mobility shift assays (GMSA).

GMSA
GMSA were performed according to method described elsewhere (31,41), using the labeled monomeric ANG-IRE motif DNA as probe. Briefly, the rANG-IRE DNA fragment was 5' end labeled with [-³²P]ATP by T₄ polynucleotide kinase. Purified GST-hnRNP F fusion proteins (1 µg) or GST (5 µg) or IRPTC nuclear proteins (5 µg) in the presence of 0.3 units of poly(dI/dC) in 20 mM HEPES (pH 7.6), 1 mM EDTA, 50 mM KCl, 2 mM spermidine, 1 mM DTT, 0.5 mM PMSF, and 10% glycerol (vol/vol) were incubated for 30 min at room temperature. Then, the 5'-labeled probe (approximately 0.1 pmol) was added and incubated further for 30 min at room temperature. After being chilled on ice, the mixture was run on 5% (wt/vol) nondenaturing PAGE and exposed for autoradiography.

In competition assays, 100- to 300-fold molar excess of unlabeled DNA fragments was added to the reaction mixture and incubated for 30 min at room temperature before incubation with the labeled probe. In supershift assays, 0.5 to 2 µl of polyclonal antibodies against hnRNP F was added in the reaction mixture and incubated for 30 min on ice before incubation with the labeled probe.

Mammalian Expression of Recombinant hnRNP F
Mammalian expression vector pcDNA 3.1 that contained rat hnRNP F cDNA in sense (pcDNA 3.1/hnRNP F [+]) or antisense (pcDNA 3.1/hnRNP F [–]) orientation was transfected into IRPTC with Fugene 6 reagent according to the instruction manual provided by the supplier (Roche Diagnostics). We optimized the DNA concentration for gene transfection at 2 µg per 0.5 to 1 x 10⁶ cells. Forty-eight hours after transfection, the cells were harvested and assayed for hnRNP F protein and ANG mRNA by Western blotting and RT-PCR, respectively.

Western Blotting for hnRNP F
Briefly, the cellular proteins and nuclear extracts were dissolved in 700 µl of lysis buffer (62.5 mM Tris-HCl [pH 6.8] that contained 2% [wt/vol] SDS, 10% glycerol, 50 mM DTT, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.1% [wt/vol] BPB), sonicated for 15 s, heated at 95°C for 5 min, and centrifuged at 12,000 x g for 5 min. Thirty-five microliters of the supernatants was subjected to 10% PAGE-SDS gel and then transferred onto a polyvinylidene difluoride membrane (Hybond-P; Amersham-Pharmacia Biotech). The membrane was initially blotted for anti-hnRNP F antibody (1:30,000 dilution) and then reblotted for anti–-actin antibody (1:10,000 dilutions) using chemiluminescent developing reagent (Roche Diagnostics). The relative densities of the hnRNP F and -actin bands were quantified by computerized laser densitometry (ImageQuant software, version 5.1; Molecular Dynamics, Amersham-Pharmacia Biotech).

RT-PCR for Quantification of ANG mRNA in IRPTC
Total RNA, isolated with TRIzol reagent (InVitrogen) according to the supplier’s protocol and quantified by its absorbance at 260 nm, was deployed in RT-PCR to quantify the amount of ANG mRNA expressed in IRPTC as described previously (25,26). The sense and antisense rat ANG primers were 5'-CCT CGC TCT CTG GAC TTA TC-3' and 5'-CAG ACA CTG AGG TGC TGT TG-3', corresponding to N + 729 to N + 748 of exon 2 and N + 111 to N + 130 of exon 3 of rANG gene (9), respectively. The sense and antisense rat -actin primers were 5'-ATG CCA TCC TGC GTC TGG ACC TGG C-3' and 5'-AGC ATT TGC GGT GCA CGA TGG AGG G-3', corresponding to the nucleotide sequences of +155 to +179 of exon 3 and nucleotide sequences of +115 to +139 of exon 5 of the rat -actin gene (44), respectively.

For identifying rANG and -actin cDNA fragments, 10 µl of the PCR product were electrophoresed on 1.2% agarose gels and transferred onto a Hybond XL nylon membrane (Amersham-Pharmacia Biotech). Digoxigenin-labeled oligonucleotide 5'-GAG GGG GTC AGC ACG GAC AGC ACC-3', corresponding to nucleotide +775 to +798 of rANG cDNA (9) (i.e., nucleotide +828 to +851 of exon 2 of rANG gene [44]), prepared with a digoxigenin oligonucleotide 3' end labeling kit (Roche Diagnostics), was used to hybridize the PCR products on the membrane. After stringent washing, the membrane was detected with a digoxigenin luminescence kit (Roche Diagnostics) and exposed to Kodak BMR film (Eastman Kodak Co., Rochester, NY). After rANG mRNA analysis, the same membrane was stripped and rehybridized with a -actin oligonucleotide probe (sequence: 5'-TCC TGT GGC ATC CAT GAA ACT ACA TTC-3', corresponding to nucleotides +9 to +35 of exon 4 of the rat -actin gene [44]). ANG mRNA levels were normalized by corresponding -actin mRNA levels.

CAT Assay
The method of construction of the rANG-CAT fusion gene pOCAT/rANG N-1498/+18 and mutant pOCAT/rANG N-1498/+18 with mutated IRE has been described previously (31,32). Control plasmid or ANG-CAT fusion gene were transfected into IRPTC using lipofectamine (InVitrogen). We optimized the DNA concentration for gene transfection at 2 to 3 µg per 0.5 to 1 x 10⁶ cells. Thus, in the present studies, a total of 2 µg of supercoiled DNA (i.e., 2 µg of pRSV/CAT, 1 µg of pOCAT/rANG N-1498/+18, or mutant pOCAT/rANG N-1498/+18 plus 1 µg of pcDNA 3.1 or pcDNA 3.1/hnRNP F) was used routinely in cell transfection. Twenty-four hours after the transfection, the media were replaced with fresh 5 mM D-glucose DMEM and incubated for another 24 h. The cells then were harvested and assayed for CAT activity (30).

For normalizing the efficiency of transfection, 0.5 µg of pTK/hGH (a vector with thymidine kinase enhancer/promoter fused to 5' human growth hormone gene) was co-transfected with pRSV/CAT or pOCAT/rANG N-1498/+18 as described previously (30). The plasmid pRSV/CAT served as a positive control to monitor the efficiency of transfection of rANG-CAT fusion gene. The level of transfection efficiency for pRSV/CAT in IRPTC ranged from 60 to 90%, i.e., the percentage of conversion of ¹⁴C chloramphenicol to mono- and diacetyl chloramphenicol. The transfection efficiency of pOCAT/rANG N-1498/+18 in IRPTC ranged from 25 to 35% compared with pRSV-CAT. The inter- and intra-assay coefficients of variation of transfection for pOCAT/rANG N-1498/+18 in IRPTC are 25 and 12% (n = 10), respectively. The method for CAT assay has been described previously (30).

Statistical Analyses
Three to five separate independent experiments were performed per protocol, and each treatment group was run in duplicate. The data were analyzed by one-way ANOVA and the Bonferroni test. P 0.05 was regarded as statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of Rat IRE-BP in IRPTC
Figure 1A shows the staining of nuclear proteins after 2-D electrophoresis. Southwestern blotting of IRE-BP after 2-D electrophoresis is displayed in Figure 1B. It is apparent that two positive spots with an apparent molecular weight of 46 to 48 kD were identified, and these spots were cut out and subjected to MALDI-MS. The MS results are displayed in Figure 2. The two spots identified a common protein designated as RIKEN (Accession no. 4833420I20 [Mus musculus]). Database analysis revealed that RIKEN is identical to the rat hnRNP F cDNA sequence reported by Yoshida et al. (34).

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Rat angiotensinogen (ANG) insulin-responsive element binding proteins (IRE-BP) detected by two-dimensional (2-D) electrophoresis and Southwestern and Western blots. (A) Rat immortalized renal proximal tubular cells (IRPTC) nuclear extracts were subjected to 2-D electrophoresis and then stained with Coomassie Brilliant Blue R-250. M, Amersham Pharmacia Biotech’s rainbow molecular mass markers; N.E., rat IRPTC nuclear extracts without isoelectrofocusing. (B) Southwestern blot analysis of ANG IRE-BP from IRPTC nuclear proteins after 2-D electrophoresis. After 2-D electrophoresis, the nuclear proteins were transferred onto a Hybond C-extra membrane, hybridized with radioactively labeled ANG-IRE (N-878/N-864), washed, and subjected to autoradiography. The arrowheads indicate the proteins that were determined to be heterogeneous nuclear ribonucleoprotein F (hnRNP F) by later mass spectrometry (MS). The broken box denotes the proteins that were subjected to MS, but their identities could not be determined. (C) Western blot analysis of the 2-D electrophoresis membrane from B using anti–hnRNP F antibody.

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. MS analysis of proteins detected by Southwestern blot. Spot 1 was isolated from 2-D gel and then subjected to tryptic digestion and matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis. (A) A typical MALDI-MS peptide fingerprint of spot 1. (B) Peptide sequence homology with hnRNP F identified by MALDI-MS and database search (bold letters). The sequence coverage of hnRNP F reached 32%. Similar results were obtained from spot 2 (not shown).

GMSA
Bacterially expressed recombinant hnRNP F was used to study the interaction of putative rANG-IRE (N-878 to N-864) with hnRNP F. Figure 3 illustrates the analysis of GST-hnRNP F fusion proteins by 10% PAGE-SDS gel. Three major bands with apparent molecular weights of 26, 44, and 69 to 72 kD were induced by isopropylthiogalactosidase (Figure 3A). The 44- and 69- to 72-kD species interacted with rabbit antiserum against hnRNP F but not the 26-kD species (Figure 3B). The 26-kD molecular species was the induced GST protein, according to information provided by the supplier (Amersham-Pharmacia Biotech). The 69- to 72-kD molecular species had the molecular weight of hnRNP F (46 kD) fused with GST (26 kD), whereas the 44-kD molecular species was likely partially degraded GST-hnRNP F fusion proteins. Partially purified GST-hnRNP F (Figure 3, lane 7) was used in subsequent GMSA.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. SDS- PAGE and Western blotting analysis of glutathione S-transferase (GST)-rat hnRNP F (GST-hnRNP F) fusion proteins from bacterial culture. (A) Bacterial extracts were subjected to 10% SDS-PAGE and then stained with Coomassie Brilliant Blue R-250. (B) Western blot analysis of GST-hnRNP F fusion proteins from bacterial culture with rabbit anti–hnRNP F antiserum. Lane 1, Amersham-Pharmacia Biotech’s rainbow markers; lanes 2 and 3, 30 µg, crude bacterial extract (Escherichia coli transformed with control plasmid pGex 4T-3 without and with 0.5 M isopropylthiogalactoside [IPTG] induction, respectively); lanes 4 and 5, 30 µg, crude bacterial extract (E. coli transformed with the plasmid pGex 4T-3 that contained hnRNP F cDNA without and with 0.5 M IPTG induction, respectively); lane 6, 10 µg, purified GST protein after the GST affinity column chromatography; and lane 7, 10 µg, purified GST-hnRNP F fusion protein after the GST affinity column chromatography. Similar results were obtained in two additional experiments.

View larger version (46K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Gel mobility shift assay (GMSA) of the radioactively labeled rANG-IRE DNA fragment with GST-hnRNP F fusion protein(s). (A) The labeled DNA probe (0.1 pmol) was incubated with GST (5 µg) or GST-hnRNP F fusion protein(s) (0.1 to 2. 0 µg each) in the presence of 0.3 units of poly dI-dC. (B) Competition with 100- and 300-fold molar excess of unlabeled hGAPDH-IRE (N-473/-446), rGlucagon-IRE (N-266/-242), consensus SP1 sequence, and rANG-IRE motif is shown in lanes 4 to 5, lanes 6 to 7, lanes 8 to 9, and lanes 10 to 11, respectively. Similar observations were made in two other experiments.

View larger version (61K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Autoradiography in GMSA of the radioactively labeled rANG-IRE DNA fragment with GST-hnRNP F fusion protein(s). The labeled DNA probe (0.1 pmol) was incubated with GST (5 µg, lane 2) or GST-hnRNP F fusion protein(s) (1 µg each; lanes 3 to 13) in the presence of 0.3 units of poly dI-dC. Competition with 100- and 300-fold excess of unlabeled ANG-IRE motif and mutants of rANG N-882/-854 (M1, M2, M3 and M4) is shown in lanes 4 to 5, lanes 6 to 7, lanes 8 to 9, lanes 10 to 11, and lanes 12 to 13, respectively. Similar observations were made from two other experiments.

View larger version (44K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Supershift GMSA of the radioactively labeled rANG-IRE DNA fragment with GST-hnRNP F fusion protein(s). The labeled DNA probe (0.1 pmol) was incubated with GST (5 µg) or GST-hnRNP F fusion protein(s) (1 µg each, lanes 3 to 9) in the presence of 0.3 units of poly dI-dC. Rabbit anti-hnRNP F antiserum (0.5 to 2 µl, lanes 4 to 6) or rabbit IgG (0.5 to 2 µg, lanes 7 and 9) was added to the reaction mixture and incubated for 30 min on ice before incubation with the labeled probe. Similar observations were made from two other experiments.

View larger version (47K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Supershift GMSA of the radioactively labeled rANG-IRE with IRPTC nuclear protein(s). The labeled DNA probe (0.1 pmol) was incubated with BSA (5 µg) or IRPTC nuclear protein(s) (5 µg each, lanes 2 to 7) in the presence of 0.3 units of poly dI-dC. Excessive rat ANG-IRE (100-fold) was added for competition with the labeled probe (lane 3). Rabbit IgG (10 and 5 µg, lanes 4 and 5, respectively) or rabbit anti-hnRNP F antiserum (1.0 and 0.5 µl, lanes 6 and 7, respectively) was added the reaction mixture and incubated for 30 min on ice before incubation with the labeled probe. Similar observations were made from two other experiments. SS, supershift band.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Expression of hnRNP F and ANG mRNA in IRPTC that were transiently transfected with pcDNA 3.1, pcDNA 3.1/hnRNP F (+), or pcDNA 3.1/hnRNP F (–). (A) Western blot analysis of hnRNP F and -actin expression in IRPTC. Twenty-four hours after gene transfection, the cells were incubated for 24 h in 5 mM DMEM that contained 5% FBS. Then, cells were collected, extracted, and assayed for hnRNP F and -actin by Western blot. The relative densities of the hnRNP F band were compared with the -actin band. The hnRNP F level in pcDNA 3.1-transfected cells represents the control level (100%). (B) Reverse transcriptase–PCR (RT-PCR) analysis of endogenous rat ANG and -actin mRNA expression in IRPTC. Twenty-four hours after gene transfection, the cells were cultured for 24 h in 5 mM DMEM that contained 5% FBS. Then, cells were harvested and assayed for rat ANG mRNA by RT-PCR as described in the Materials and Methods section. The relative densities of the PCR band of ANG mRNA were compared with the -actin band. The ANG mRNA level in pcDNA 3.1-transfected cells represents the control level (100%). Results were expressed as the means ± SD of three determinations (*P 0.05, **P 0.01). Similar results were obtained in three other experiments.

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Effect of hnRNP F on rANG gene promoter activity in IRPTC. Forty-eight hours after transfection, cells were harvested and assayed for chloramphenicol acetyltransferase (CAT) activity. The relative activity in cells that were transfected with 1 µg of pOCAT/rANG N-1498/+18 or mutant pOCAT/rANG N-1498/+18 was given a relative value of 100% (control). Each point represents the mean ± SD of three independent experiments (*P 0.05; **P 0.01).

View larger version (40K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Effect of high glucose and insulin on hnRNP F expression in IRPTC. (A) Southwestern blot analysis. Cells were incubated in 5 mM plus 20 mM D-mannitol, 25 mM D-glucose, or 25 mM D-glucose plus insulin (10–7 M) and 5% FBS for 24 h. Then, cells were harvested and nuclear proteins (100 µg) were subjected to Southwestern blotting as described in the Materials and Methods section. (B) Western blot analysis. After Southwestern blot analysis, the same membrane was blotted with rabbit polyclonal antibodies against hnRNP F and -actin control and ECL-chemiluminescent developing reagent. The relative densities of hnRNP F bands were compared with the -actin control. The level of hnRNP F expressed in IRPTC in 5 mM glucose medium was considered to be the control (100%). Each bar represents the mean ± SD of three independent experiments (*P 0.05, **P 0.01).

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 11. Distribution of the hnRNP F protein in various rat tissues as analyzed by Western blot analysis. Nuclear proteins (100 µg) were subjected to Western blotting as described in the Materials and Methods section with rabbit polyclonal antibodies against hnRNP F and -actin and ECL-chemiluminescent developing reagent. The relative densities of hnRNP F bands were compared with the -actin control. The level of hnRNP F expressed in IRPTC was considered to be the control (100%). Each bar represents the mean ± SD of three independent experiments (**P 0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A broad spot with an apparent molecular weight of 68 to 70 kD (denoted by broken rectangle in Figure 1) was detected by Southwestern blotting. Subsequent MALDI-MS analysis of this spot, however, could not yield a definite peptide with a significantly high Masscot score. These data suggest that the positive broad spot detected by Southwestern blotting is a cluster of several different proteins.

To demonstrate that hnRNP F interacts with rANG-IRE, we cloned hnRNP F from IRPTC by conventional RT-PCR and expressed it in a bacterial system for use in GMSA. Our GMSA revealed that labeled rANG-IRE DNA interacted with GST-hnRNP F fusion protein(s) but not with GST proteins. The addition of unlabeled rANG-IRE DNA effectively displaced labeled ANG-IRE at or greater than a 100-fold molar excess of unlabeled DNA, whereas unlabeled hGAPDH-IRE, rat glucagon-IRE, SP1 consensus sequence, and mutants M1 and M2 of rANG-IRE were not effective in displacing labeled rANG-IRE. In contrast, M3 and M4 were effective in competing with labeled ANG-IRE. These studies demonstrate that nucleotides N-878 to N-864 represent the rANG-IRE motif, which is essential for binding to hnRNP F fusion proteins.

Most convincing, the addition of specific rabbit polyclonal antibodies against hnRNP F yielded a supershift of labeled rANG-IRE binding with hnRNP F fusion proteins in a dose-dependent manner. Moreover, the addition of rabbit polyclonal antibodies against hnRNP F yielded a supershift of labeled rANG-IRE binding with IRPTC nuclear proteins but not with rabbit IgG. Taken together, these data demonstrate unequivocally that hnRNP F binds to rANG-IRE.

There are at least 20 to 30 independent hnRNP species in mammalian cells (45,46). Because antibodies against hnRNP affect splicing (47–49) and hnRNP are associated with poly (A)-containing RNA, i.e., premRNA and mRNA, and found to be constituents of the splicesome (47), they are believed to be important in splicing events. HnRNP F is a member of a subfamily of hnRNP proteins that includes at least F, H, and H' proteins (33,50). These proteins are highly related; hnRNP H and H' are 96% identical and 78 and 75% identical to hnRNP F, respectively (33,50). HnRNP F has the unique nucleotide binding property of binding to (G/C)-stretch of double-stranded DNA and RNA sequences via its GY-rich motif and RNA-binding domains, respectively (34). Studies on hnRNP F and H/H' have revealed that these proteins participate at various steps in the processing of cellular mRNA, e.g., in alternating splicing of the c-src gene (51), the -tropomyosin gene (52), and the thyroid hormone receptor gene (53). Furthermore, Veraldi et al. (54) demonstrated that differential changes in expression levels of hnRNP F and H/H' are involved in controlling the synthesis of membrane-bound versus secreted antibodies during the development of memory B cells into plasma cells. Most interesting, studies by Yoshida et al. (55) and Gamberi et al. (56) demonstrated that hnRNP F could bind to TATA-binding protein, associates with RNA polymerase II, and interacts directly with nuclear cap-binding protein complex, suggesting that hnRNP F could modulate gene expression at the transcriptional and posttranscriptional levels. Thus, these data strongly indicate that the members of this subfamily are important for the control of gene expression at both the transcriptional and the posttranscriptional levels in various cells.

Surprising, overexpression and downexpression of hnRNP F inhibits and augments ANG mRNA expression and ANG gene promoter activity in IRPTC, respectively. To the best of our knowledge, the present report is the first to demonstrate that hnRNP F can modulate ANG gene expression in kidney proximal tubular cells in vitro. At present, the molecular mechanism(s) of hnRNP F action on ANG gene expression is not known. One possibility is that hnRNP F behaves like a negative transacting protein and inhibits the binding of other positive transacting factor(s) (e.g., the p70 kD IRE-BP) to TATA-binding protein and RNA polymerase II, subsequently attenuating ANG gene expression. The second possibility is that hnRNP F overexpression exhausts the availability of nuclear scap-binding protein for capping premRNA, which would subsequently attenuate the formation of mature ANG mRNA in the cytoplasm. The third possibility is that hnRNP F could bind to unidentified splicing silencer(s) in ANG premRNA and subsequently alter the normal splicing of ANG premRNA in the nucleus. Finally, it is possible that hnRNP F is a negative regulatory protein, but its negative effect is normally neutralized by interaction (heterodimerization) with the unidentified 70-kD IRE-BP (a hypothetical positive regulatory transacting factor that binds to rANG-IRE) in cells. HnRNP F overexpression then will exceed the neutralization ability of the unidentified 70-kD IRE-BP and subsequently attenuate ANG mRNA expression. More studies are definitely needed along these lines to elucidate the mechanism(s) of action of hnRNP F on ANG gene expression in IRPTC.

Finally, our studies reveal that high glucose stimulated and insulin inhibited the expression of hnRNP F in IRPTC. To the best of our knowledge, this is the first report that hnRNP F could be modulated by high glucose and insulin in kidney proximal tubular cells in vitro. Our tissue distribution analysis revealed that the hnRNP F protein is detectable in the nuclear extracts of rat liver, kidney, testis, brain, and lung but not in the spleen. Rat liver, kidney, testis (epididymis), brain, and lung are known to express ANG mRNA (57,58). These observations raise the possibility that the expression of hnRNP F might have a role in regulating ANG gene expression in these tissues.

In summary, we have identified a nuclear protein that binds to rANG-IRE by a combination of Southwestern blotting and proteomics. This IRE-BP is identified as hnRNP F. hnRNP F modulates ANG gene expression in IRPTC. Finally, it seems that high glucose and insulin regulate hnRNP F expression in IRPTC, and hnRNP F is detected in various rat tissues that express ANG mRNA. The present studies raise the possibility that the expression of hnRNP and the unidentified 70-kD IRE-BP may play an important role in regulating local intrarenal RAS activation. Dysregulation of the expression of hnRNP F and unidentified 70-kD IRE-BP may contribute to renal injury in diabetes via altering local intrarenal RAS activation.

	Acknowledgments

 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR, MOP-13420 and MOP-62920 to J.S.D.C and MT-14726 to D.F.G), Canadian Diabetes Association (grant 1061), Kidney Foundation of Canada, and the National Institutes of Health (HL-48455 to J.R.I.). S.-L.Z. is the recipient of a CIHR Fellowship.

The data were presented as free communication in part at the 37th meeting of the American Society of Nephrology in St. Louis, MO, October 27 to November 1, 2004.

We thank Ovid M. Da Silva, Editor, Bureau d’aide à la recherche, Research Centre, CHUM, for editing this manuscript.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

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