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• W2049453433 abstract "The present studies aimed to identify the 70-kDa nuclear protein that binds to an insulin-responsive element in the rat angiotensinogen gene promoter and to define its action on angiotensinogen gene expression. Nuclear proteins were isolated from rat kidney proximal tubular cells and subjected to two-dimensional electrophoresis. The 70-kDa nuclear protein was detected by Southwestern blotting and subsequently identified by mass spectrometry, which revealed that it was identical to 65-kDa heterogeneous nuclear ribonucleoprotein K (hnRNP K). hnRNP K bound to the insulin-responsive element of the rat angiotensinogen gene was revealed by a gel mobility shift assay and chromatin immunoprecipitation assay. hnRNP K inhibited angiotensinogen mRNA expression and promoter activity. In contrast, hnRNP K down-expression by small interference RNA enhanced angiotensinogen mRNA expression. Moreover, hnRNP K interacted with hnRNP F in pulldown and co-immunoprecipitation assays. Co-transfection of hnRNP K and hnRNP F further suppressed angiotensinogen mRNA expression. Finally, in vitro and in vivo studies demonstrated that high glucose increases and insulin inhibits hnRNP K expression in rat kidney proximal tubular cells. In conclusion, our experiments revealed that hnRNP K is a nuclear protein that binds to the insulin-responsive element of the rat angiotensinogen gene promoter and modulates angiotensinogen gene transcription in the kidney. The present studies aimed to identify the 70-kDa nuclear protein that binds to an insulin-responsive element in the rat angiotensinogen gene promoter and to define its action on angiotensinogen gene expression. Nuclear proteins were isolated from rat kidney proximal tubular cells and subjected to two-dimensional electrophoresis. The 70-kDa nuclear protein was detected by Southwestern blotting and subsequently identified by mass spectrometry, which revealed that it was identical to 65-kDa heterogeneous nuclear ribonucleoprotein K (hnRNP K). hnRNP K bound to the insulin-responsive element of the rat angiotensinogen gene was revealed by a gel mobility shift assay and chromatin immunoprecipitation assay. hnRNP K inhibited angiotensinogen mRNA expression and promoter activity. In contrast, hnRNP K down-expression by small interference RNA enhanced angiotensinogen mRNA expression. Moreover, hnRNP K interacted with hnRNP F in pulldown and co-immunoprecipitation assays. Co-transfection of hnRNP K and hnRNP F further suppressed angiotensinogen mRNA expression. Finally, in vitro and in vivo studies demonstrated that high glucose increases and insulin inhibits hnRNP K expression in rat kidney proximal tubular cells. In conclusion, our experiments revealed that hnRNP K is a nuclear protein that binds to the insulin-responsive element of the rat angiotensinogen gene promoter and modulates angiotensinogen gene transcription in the kidney. Diabetic nephropathy is a leading cause of end stage renal disease, accounting for 30–50% of all new end stage renal disease cases in North America (1Groggel G.C. Arch. Family Med. 1996; 5: 513-520Crossref PubMed Scopus (7) Google Scholar, 2Bernege R.T.V. Brancati F.L. Welten P.K. Klag M.J. Ann. Intern. Med. 1996; 121: 912-918Google Scholar, 3NIDDK, National Institutes of Health United States Renal Data System: USRDS Annual Data Report. National Institutes of Health, Bethesda, MD2005Google Scholar). Both clinical and animal studies indicate that intensive insulin therapy and prolonged treatment with angiotensin-converting enzyme inhibitors or angiotensin II-AT1 receptor blockers delay the progression of nephropathy in diabetes, but neither strategy cures nephropathy (4Bakris G.L. Ann. Intern. Med. 1993; 118: 643-644Crossref PubMed Scopus (49) Google Scholar, 5The Diabetes Control and Complications Trial Research Group N. Engl. J. Med. 1993; 329: 977-986Crossref PubMed Scopus (22784) Google Scholar, 6The Diabetes Control and Complications Trial Research Group Kidney Int. 1995; 47: 1703-1720Abstract Full Text PDF PubMed Scopus (746) Google Scholar, 7Nakamura T. Takahasi T. Fukui M. Ebihara I. Osada S. Tomino Y. Koide H. J. Am. Soc. Nephrol. 1995; 5: 1492-1497Crossref PubMed Google Scholar, 8Ravid M. Lang R. Rachmani R. Lishner M. Arch. Intern. Med. 1996; 156: 286-289Crossref PubMed Scopus (0) Google Scholar, 9The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Groups N. Engl. J. Med. 2000; 342: 381-389Crossref PubMed Scopus (1406) Google Scholar, 10Andersen S. Tarnow L. Ressing P. Hansen B.V. Parving H.H. Kidney Int. 2000; 57: 601-606Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 11Laverman G.D. Remuzzi G. Ruggenenti P. J. Am. Soc. Nephrol. 2004; 15: 60-70Google Scholar, 12Wolf G. Ritz G. Kidney Int. 2005; 67: 798-812Abstract Full Text Full Text PDF Scopus (257) Google Scholar). Whereas such results support the concept that hyperglycemia and the renin-angiotensin system (RAS) 2The abbreviations used are: RAS, renin-angiotensin system; AGT, angiotensinogen; rAGT, rat angiotensinogen; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation; GMSA, gel mobility shift assay; hnRNP F, heterogeneous nuclear ribonucleoprotein F; hnRNP K, heterogeneous nuclear ribonucleoprotein K; IRE, insulin-responsive element; IRE-BP, IRE-binding protein; IRPTC, immortalized renal proximal tubular cell; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; RPT, renal proximal tubule; RPTC, renal proximal tubular cell; RT, reverse transcription; siRNA, small interfering RNA; STZ, streptozotocin; TBP, TATA-binding protein; GST, glutathione S-transferase; RIPA, radioimmune precipitation. activation are involved in the development and progression of diabetic nephropathy, the molecular mechanism(s) linking hyperglycemia to RAS activation remain largely undefined. Angiotensinogen (AGT), a glycoprotein consisting of 452 amino acid residues with an apparent molecular mass of 62–65 kDa, is the sole substrate in the RAS cascade (13Ohkubo H. Kageyama R. Ujihara M. Hirose T. Inayama S. Nakanishi S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2196-2200Crossref PubMed Scopus (210) Google Scholar, 14Kageyama R. Ohkubo H. Nakanishi S. Biochemistry. 1984; 23: 3603-3609Crossref PubMed Scopus (125) Google Scholar). AGT is principally produced by the liver and cleaved by renin from the kidney to form angiotensin I, which is then further processed by angiotensin-converting enzyme to form angiotensin II. The existence of an intrarenal RAS is now generally accepted (15Dzau V.J. Ingelfinger J.R. J. Hypertens. 1989; 7: 53-58Google Scholar, 16Johnston C.I. Fabris B. Jandeleit K. Kidney Int. 1993; 44: 559-563Google Scholar). Renal proximal tubules (RPTs) contain all components of the RAS, including messenger RNAs and proteins, such as AGT, renin, angiotensin-converting enzymes, and angiotensin II receptors (17Ingelfinger J.R. Zuo W.M. Fon E.A. Ellison K.E. Dzau V.J. J. Clin. Invest. 1990; 85: 417-423Crossref PubMed Scopus (305) Google Scholar, 18Wolf G. Neilson E.G. Kidney Int. 1992; 43: 100-107Google Scholar, 19Anderson S. Jung F.F. Ingelfinger J.R. Am. J. Physiol. 1993; 265: F477-F486Crossref PubMed Google Scholar, 20Chen M. Harris M.P. Rose D. Smart A. He X.R. Kretyler M. Briggs J.P. Schnermann J. J. Clin. Invest. 1994; 94: 237-243Crossref PubMed Scopus (59) Google Scholar, 21Tang S.S. Jung F. Diamant D. Brown D. Bachinsky D. Hellman P. Ingelfinger J. Am. J. Physiol. 1995; 268: F435-F446PubMed Google Scholar, 22Loghman-Adham M. Rohrwasser A. Helin C. Zhang S. Terreros D. Inoue I. Lalouel J.M. Kidney Int. 1997; 52: 229-239Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 23Li N. Zimpelman J. Chang K. Wilkins J.A. Burns K.D. Am. J. Physiol. 2005; 288: F353-F362Crossref PubMed Scopus (142) Google Scholar, 24Wang L. Lei C. Zhang S.L. Roberts K.D. Tang S.S. Ingelfinger J.R. Chan J.S.D. Kidney Int. 1998; 53: 287-295Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Most recently, we reported that RAS blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat AGT (rAGT) gene in the kidney (25Sachetelli S. Liu Q. Zhang S.-L. Liu F. Hsieh T.-J. Brezniceanu M.-L. Guo D.-F. Filep J.G. Ingelfinger J.R. Sigmund C.D. Hamet P. Chan J.S.D. Kidney Int. 2006; 69: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). These observations indicate that the local formation of angiotensin II may play an important role in the development of nephropathy in diabetes. Our laboratory has established that high glucose (i.e. 25 mm) stimulates rAGT gene expression in IRPTCs (26Zhang S.L. Filep J.G. Hohman T.C. Tang S.S. Ingelfinger J.R. Chan J.S.D. Kidney Int. 1999; 55: 454-464Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 27Zhang S.L. Tang S.S. Chen X. Filep J.G. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2000; 141: 4637-4646Crossref PubMed Scopus (51) Google Scholar, 28Hsieh T.J. Zhang S.L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2002; 143: 2975-2985Crossref PubMed Scopus (147) Google Scholar, 29Hsieh T.J. Fustier P. Zhang S.L. Filep J.G. Tang S.S. Ingelfinger J.R. Fantus I.G. Hamet P. Chan J.S.D. Endocrinology. 2003; 144: 4338-4349Crossref PubMed Scopus (86) Google Scholar). RAS blockers and stable transfer of antisense rAGT cDNA into IRPTCs inhibit transforming growth factor-β1 gene expression and cellular hypertrophy in high glucose (30Zhang S.L. Chen X. Filep J.G. Tang S.S. Ingelfinger J.R. Carrière S. Chan J.S.D. Exp. Nephrol. 2001; 9: 109-117Crossref PubMed Scopus (35) Google Scholar, 31Zhang S.L. To C. Chen X. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2002; 13: 302-312Crossref PubMed Google Scholar). These investigations strongly indicate that rAGT and transforming growth factor-β1 gene expression are essential for the high glucose effect on IRPTC hypertrophy and kidney injury. We have also established that insulin inhibits the stimulatory effect of high glucose levels on rAGT gene expression and the induction of hypertrophy in IRPTCs (32Zhang S.L. Chen X. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 1999; 140: 5285-5292Crossref PubMed Google Scholar, 33Zhang S.L. Chen X. Wei C.C. Filep J., G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2002; 143: 4627-4635Crossref PubMed Scopus (22) Google Scholar, 34Chen X. Zhang S.L. Pang L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2001; 142: 2577-2585Crossref PubMed Scopus (30) Google Scholar, 35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar, 36Seuningen I.V. Ostrowski J. Bustelo X.R. Sleath P.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 37Takimoto M. Tomonaga T. Matunis M. Avigan M. Krutzsch H. Dreyfuss G. Levens D. J. Biol. Chem. 1993; 268: 18249-18258Abstract Full Text PDF PubMed Google Scholar, 38Nasrin N. Ercolani L. Denaro M. Kong X.F. Kang I. Alexander M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5273-5277Crossref PubMed Scopus (137) Google Scholar, 39Philippe J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7224-7227Crossref PubMed Scopus (100) Google Scholar, 40Dynan W.S. Tjian R. Cell. 1983; 35: 79-87Abstract Full Text PDF PubMed Scopus (911) Google Scholar, 41Ingelfinger J.R. Jung F. Diamant D. Haveran L. Lee E. Brem A. Tang S.S. Am. J. Physiol. 1999; 276: F218-F227PubMed Google Scholar, 42Hennighausen L. Lubon H. Methods Enzymol. 1987; 152: 721-735Crossref PubMed Scopus (165) Google Scholar, 43Kwast-Welfeld J. Belle I. Walker P.R. Whitfield J.F. Sikorska M. J. Biol. Chem. 1993; 268: 19581-19585Abstract Full Text PDF PubMed Google Scholar). Moreover, a putative insulin-responsive element (IRE) containing nucleotides –878 to –864 (5′-CCT TCC CGC CCT TCA-3′) upstream of the transcription start site of the rAGT gene promoter has been identified, and it binds to two major nuclear proteins with apparent molecular mass of ∼48 and 70 kDa from IRPTCs (34Chen X. Zhang S.L. Pang L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2001; 142: 2577-2585Crossref PubMed Scopus (30) Google Scholar). We recently reported that the 48-kDa nuclear protein is identical to the 46-kDa heterogeneous nuclear ribonucleoprotein F (hnRNP F) (35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar). Furthermore, transient transfer of sense and antisense hnRNP F cDNA, respectively, inhibits and enhances rAGT gene expression in IRPTCs (35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar). Thepresentstudiesaimedtoidentify the 70-kDa nuclear protein and to investigate its action on rAGT gene expression. We identified the 70-kDa nuclear protein as 65-kDa heterogeneous nuclear ribonucleoprotein K (hnRNP K) by two-dimensional electrophoresis and mass spectrometry (MS). Recombinant hnRNP K bound to rAGT-IRE, as shown by a gel mobility shift assay (GMSA) and chromatin immunoprecipitation assays. Overexpression of hnRNP K attenuated rAGT mRNA expression and rAGT gene promoter activity in IRPTCs. In contrast, down-expression of hnRNP K by small interference RNA enhanced rAGT gene expression. Moreover, hnRNP K was pulled down and co-immunoprecipitated with hnRNP F. Co-transfection of hnRNP K and hnRNP F further suppressed rAGT mRNA expression. Finally, in vitro and in vivo studies revealed that high glucose or hyperglycemia increased and insulin inhibited hnRNP K expression in rat kidney proximal tubular cells. These experiments demonstrated that 65-kDa hnRNP K is a nuclear protein that binds to the rat AGT gene promoter and modulates AGT gene expression in the kidneys. d(+)-glucose, d-mannitol, and insulin were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Canada). Insulin implant (Linplant) and [γ-32P]ATP (3,000 Ci/mol) were obtained from Linshin Ltd. (Scarborough, Canada) and Amersham Biosciences, respectively. Plasmid containing full-length hnRNP K cDNA (pcDNA 3/FLAG-hnRNP K) and rabbit polyclonal anti-serum (number 54) recognizing hnRNP K (QNSVKQYADVEGF, corresponding to amino acids 452–464 of human hnRNP K) were generated (in the laboratory of K. Bomsztyk) as described previously (36Seuningen I.V. Ostrowski J. Bustelo X.R. Sleath P.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Mouse monoclonal antibody against human hnRNP K/L (clone 3C2), a gift from Dr. Gideon Dreyfuss (Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA), has been reported elsewhere (37Takimoto M. Tomonaga T. Matunis M. Avigan M. Krutzsch H. Dreyfuss G. Levens D. J. Biol. Chem. 1993; 268: 18249-18258Abstract Full Text PDF PubMed Google Scholar). Rabbit polyclonal anti-TATA box-binding protein (TBP) (sc-273) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The bacterial expression vector pGex 4T-3 and mammalian expression vectors pcDNA 3.1 and pRC/RSV were purchased from Amersham Biosciences and Invitrogen, respectively. Restriction-modified enzymes were acquired from either Invitrogen, Amersham Biosciences, or La Roche Biochemicals (Laval, Canada). Oligonucleotides for rAGT-IRE –882 to –855 (5′-CCT CCC TTC CCG CCC TTC ACT TTC TAG T-3′) (34Chen X. Zhang S.L. Pang L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2001; 142: 2577-2585Crossref PubMed Scopus (30) Google Scholar), mutants of rAGT –882 to –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′), concatemeric wide type rAGT-IRE motif (3×–878 to –864, 5′-CCT TCC CGC CCT TCA CCT TCC CGC CCT TCA CCT TCC CGC CCT TCA-3′), concatemeric mutant rAGT-IRE motif (3× –878 to –864, 5′ CCT TCT TAT TCT TCA CCT TCT TAT TCT TCA CCT TCT TAT TCT TCA-3′), IRE of the human glyceraldehyde phosphate dehydrogenase gene (–473 to –477, 5′-CCA ACT TTC CCG CCT CTC AGC CTT TGA A-3′) (38Nasrin N. Ercolani L. Denaro M. Kong X.F. Kang I. Alexander M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5273-5277Crossref PubMed Scopus (137) Google Scholar), IRE of the rat glucagon gene (–267 to –242, 5′-AGT TTT CAC GCC TGA CTG AGA TTG A-3′) (39Philippe J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7224-7227Crossref PubMed Scopus (100) Google Scholar), and the consensus Sp1-binding site (5′-TCG CCC CGC CCC CGA TCG AAT-3′) (40Dynan W.S. Tjian R. Cell. 1983; 35: 79-87Abstract Full Text PDF PubMed Scopus (911) Google Scholar) were synthesized by Invitrogen, as reported previously (35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar). The plasmid containing the concatemeric wide type and mutant rAGT-IRE motif DNAs were constructed by inserting the double-stranded concatemeric wide type or mutant rAGT-IRE motif oligonucleotide with the NotI enzyme restriction site added on both termini into the polyclonal site of pcDNA 3.1 by conventional methodology. The double-stranded concatemeric wide type or mutant rAGT-IRE motif DNA fragment was then excised from the plasmid and treated with alkaline phosphatase and used for labeling as probe. Cellular Nuclear Extract Preparation—IRPTCs from passages 12–18 were utilized. The characteristics of IRPTCs, which express the mRNA and protein of rAGT, renin, angiotensin-converting enzyme, and angiotensin II receptors, have been described previously (41Ingelfinger J.R. Jung F. Diamant D. Haveran L. Lee E. Brem A. Tang S.S. Am. J. Physiol. 1999; 276: F218-F227PubMed Google Scholar). IRPTC nuclear extracts were prepared from 20 plates (150 × 20 mm), each containing confluent IRPTCs previously incubated in Dulbecco's modified Eagle's medium with 5 mm glucose plus 20 mm d-mannitol, 25 mm glucose, or 25 mm glucose plus insulin (10–7 m) for 24 h according to the method of Hennighausen and Lubon (42Hennighausen L. Lubon H. Methods Enzymol. 1987; 152: 721-735Crossref PubMed Scopus (165) Google Scholar) with slight modifications (34Chen X. Zhang S.L. Pang L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2001; 142: 2577-2585Crossref PubMed Scopus (30) Google Scholar, 35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar). Two-dimensional Electrophoresis—Two-dimensional electrophoresis was carried out with the IPGphor isoelectric focusing unit (Amersham Biosciences) as previously described (35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar). For two-dimensional separation, the IPG strips were placed above 10% polyacrylamide gel containing SDS and electrophoresed (SDS-PAGE) (35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar). Amersham Biosciences rainbow markers served as molecular weight markers. IRPTC nuclear extracts (100 μg) were run on the same 10% SDS-PAGE as the controls. Each sample was divided into two strips for two-dimensional electrophoresis. One gel was stained with Coomassie Brilliant Blue R-250 (Amresco Inc., Solon, OH) to visualize proteins. The other was electrotransferred to a Hybond C-extra membrane (Amersham Biosciences) for Southwestern blotting. Southwestern Blotting—Southwestern blotting was performed according to the procedure of Kwast-Welfeld et al. (43Kwast-Welfeld J. Belle I. Walker P.R. Whitfield J.F. Sikorska M. J. Biol. Chem. 1993; 268: 19581-19585Abstract Full Text PDF PubMed Google Scholar) with slight modifications (34Chen X. Zhang S.L. Pang L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2001; 142: 2577-2585Crossref PubMed Scopus (30) Google Scholar, 35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar). Briefly, IRPTC nuclear proteins (200 μg) were resolved on a 4–20% SDS-PAGE gradient or on 10% SDS-PAGE (44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207180) Google Scholar) and then electrotransferred to a Hybond C-extra mem-brane. The membrane was in-cubated with 10% (w/v) nonfat milk proteins and then washed at least twice with binding buffer containing 0.25% nonfat milk proteins. Subsequently, it was hybridized overnight with 32P-labeled rAGT-IRE DNA (∼1.0–2.0 pmol; 106 cpm/ml) in binding buffer containing 0.25% non-fat 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 Spectrometry (MALDI-MS)—Spots on the gel corresponding to positive signals of the Southwestern blot membrane were picked up for MALDI-MS. All MALDI-MS analyses were performed at the Quebec Genome Centre (McGill University, Montreal, Canada). Briefly, protein samples were first 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 finger-prints were searched by the Mascot program developed by Matrix Science Ltd. (freely accessible on the World Wide Web at www.matrixscience.com). Expression of Recombinant hnRNP K—Murine hnRNP K cDNA (36Seuningen I.V. Ostrowski J. Bustelo X.R. Sleath P.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), with the NotI enzyme restriction site added on the 5′ and 3′ ends of sense and antisense primers, respectively, was subcloned at the polyclonal site (NotI) of the bacterial expression vector pGex 4T-3 by conventional methodology. E. coli BL-21 cells (Amersham Biosciences) were transformed by pGex 4T-3 containing hnRNP K cDNA. Expression of the fusion protein (GST fused with hnRNP K (GST-hnRNP K)) in BL-21 cells was induced by the addition of 1 mm isopropylthiogalactoside in the culture medium with incubation for 4 h at 37°C. The bacteria were then harvested, and GST-hnRNP K fusion proteins were purified from the bacterial extracts by GST affinity column chromatography according to the manufacturer's protocol (Amersham Biosciences). The purified GST-hnRNP K fusion proteins were tested in GMSAs. GMSAs—These assays were performed according to the methodology described elsewhere (34Chen X. Zhang S.L. Pang L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2001; 142: 2577-2585Crossref PubMed Scopus (30) Google Scholar, 35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar), employing labeled rAGT-IRE DNA as probe. Briefly, the rAGT-IRE DNA fragment was 5′-end-labeled with [γ-32P]ATP by T4 polynucleotide kinase. Purified GST-hnRNP K fusion proteins (1.5 μg) or GST (5 μg) in the presence of 1 μg of poly(dI/dC) in 20 mm Hepes (pH 7.6), 1 mm EDTA, 50 mm KCl, 2 mm spermidine, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 10% glycerol (v/v) were incubated for 30 min on ice. Subsequently, the 5′-labeled probe (∼0.1 pmol) was added and further incubated for 45 min on ice. The mixture was run on 6% (w/v) nondenaturing PAGE and exposed for autoradiography. In competition assays, a 100-fold molar excess of unlabeled DNA fragments was added to the reaction mixture and incubated for 30 min on ice before incubation with the labeled probe. Chromatin Immunoprecipitation (ChIP)—ChIP analysis was performed according to the methodology of Kuo and Allis (45Kuo M.H. Allis C.D. Methods. 1999; 19: 425-433Crossref PubMed Scopus (486) Google Scholar) with slight modifications (46Ostrowski J. Kawata Y. Schullery D.S. Denisenko O.N. Bomsztyk K. Nucleic Acids Res. 2003; 31: 3954-3962Crossref PubMed Scopus (45) Google Scholar). Briefly, 0.4 ml of 37% formaldehyde was added to 10 ml of overlaying medium of IRPTC culture for 15 min at 4 °C. After cross-linking, the cells were harvested, washed twice with 1 ml of phosphate-buffered saline in Eppendorf tubes, and then lysed with 0.5 ml of immunoprecipitation buffer (150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 50 mm Tris-HCl, pH 7.5, 0.5 mm dithiothreitol) containing the following inhibitors: 10 μg/ml leupeptin, 0.5 mm phenylmethylsulfonyl fluoride, 30 mm p-nitrophenol phosphate, 10 mm NaF, 0.1 mm Na3VO4, and 10 mm β-glycerophosphate. After one wash with immunoprecipitation buffer, the pellet was suspended in 1 ml of immunoprecipitation buffer and sheared in a Branson sonicator with 10-s cycles, 1 pulsed and 1 continuous, for 10 min at an output 3 and 80% duty cycle. Pulldowns were done using anti-K protein antibody with or without blocking peptide (100 μm) and protein A beads (Amersham Biosciences). The beads were washed five times with 1 ml of immunoprecipitation buffer without inhibitors. DNA was eluted twice from the beads with 250 μl of elution buffer (1% SDS, 0.1 m NaHCO3) for 15 min with periodic vortexing (at room temperature). Cross-linking was reversed by adding 1 μl of 10 mg/ml RNase and 5 m NaCl to a final concentration of 0.3 m and incubating the tubes at 65 °C for 4 h. After adding 2.5 volumes of 100% ethanol, the pellet was precipitated overnight at –20 °C. DNA was pelleted and resuspended in 100 μl of Tris-EDTA buffer, pH 8.0. Then 11 μl of 10× protein K buffer (0.1 m Tris, pH 7.8, 50 mm EDTA, 5% SDS) and 1 μl of 20 μg/μl proteinase K were added and incubated at 45 °C for 2 h. DNA was extracted with phenol/chloroform and precipitated with ethanol, and the final DNA pellet was dissolved in 20 μl of Tris-EDTA buffer. PCR amplifications were done in 50 μl of 1× PCR buffer containing DNA, 0.5 μm primers (forward primer (5′-CCT TGA TGC CTC CAA CAA CT-3′) and backward primer (5′-GGT GGG AGC TGA GAA GAC AG-3′), corresponding to nucleotides –1043 to –1026 and –718 to –698 of the rAGT gene promoter (47Chan J.S.D. Chan A.H.H. Jiang M. Nie Z.R. Lachance S. Carrière S. Pediatr. Nephrol. 1990; 4: 429-435Crossref PubMed Scopus (28) Google Scholar), or forward primer (5′-TCC TGT GGC ATC CAT GAA ACT AC-3′) and backward primer (5′-AGC ATT TGC GGT GCA CGA TGG AG-3′), corresponding to nucleotides +808 to +830 and +1120 to +1098 of the rat β-actin (48Nudel U. Zakut R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acids Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1018) Google Scholar), respectively), a 40 μm concentration of each deoxynucleotide triphosphate, 1.5 mm MgCl2, and 1 unit of Taq DNA polymerase (Invitrogen). The PCR products were resolved on 2% agarose gel and transferred onto a Hybond XL nylon membrane (Amersham Biosciences). Digoxigenin-labeled oligonucleotide 5′-CCT CCC TTC CCG CCC TTC ACT TTC TAG T-3′ and 5′-CCA ACT CTC TTG GCT TAA GGA-3′, corresponding to nucleotides –882 to –855 of the rAGT gene promoter (47Chan J.S.D. Chan A.H.H. Jiang M. Nie Z.R. Lachance S. Carrière S. Pediatr. Nephrol. 1990; 4: 429-435Crossref PubMed Scopus (28) Google Scholar) and intron 4 nucleotides +56 to +77 of the β-actin gene (48Nudel U. Zakut R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acids Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1018) Google Scholar), respectively, prepared with a digoxigenin oligonucleotide 3′-end labeling kit (La Roche Biochemicals), served to hybridize the PCR products on the membrane. After stringent washing, the membrane was detected with a digoxigenin luminescence kit (La Roche Biochemicals) and exposed to Eastman Kodak Co. BMR film. Mammalian Expression of Recombinant hnRNP K—Murine hnRNP K cDNA with FLAG tag at the N terminus in mammalian expression vector pRC/RSV (36Seuningen I.V. Ostrowski J. Bustelo X.R. Sleath P.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) was transfected into IRPTCs with Lipofectamine according to the instruction manual provided by the supplier (Invitrogen). We optimized the DNA concentration for gene transfection at 2–3 μg per 0.5–1 × 106 cells. 48 h after transfection, total RNAs and nuclear proteins were isolated from IRPTCs and assayed for rAGT mRNA by RT-PCR (34Chen X. Zhang S.L. Pang L. Filep J.G. Tang S.S. Ingelfinger J.R. Chan J.S.D. Endocrinology. 2001; 142: 2577-2585Crossref PubMed Scopus (30) Google Scholar, 35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar) or for hnRNP K protein by Western blotting, respectively. Small Interfering RNA (siRNA) of hnRNP K—IRPTCs were transfected with 40 nm scrambled Silencer® Negative Control number 1 siRNA (Ambion Inc., Austin, TX) or 40 nm siRNA for hnRNP K (sense, 5′-CCA GAU GUA AUG UUU UAG Utt-3′; antisense, 5′-ACU AAA ACA UUA CAU CUG Gtg-3′; hnRNP K siRNA ID 195920 (Ambion)) or 40 nm siRNA for hnRNP F (sense, 5′-GCA UGG GAC ACC GGU AUA Utt-3′; antisense, 5′-AUA UAC CGG UGU CCC AUG Ctt-3′; hnRNP F siRNA ID 192101 (Ambion)). Transfections were accomplished by using siPORT Amine (Ambion) according to the manufacturer's instructions. Total cellular RNA and protein were harvested at 48 h post-transfection and then analyzed for rAGT and β-actin mRNA and hnRNP K protein expression by RT-PCR (35Wei C.C. Guo D.F. Zhang S.L. Ingelfinger J.R. Chan J.S.D. J. Am. Soc. Nephrol. 2005; 16: 616-628Crossref PubMed Scopus (22) Google Scholar) and Western blotting, respectively. Western Blotting for hnRNP K—Briefly, the cell pellets were lysed in 100 μl of RIPA buffer and centrifuged, and 30 μg o" @default.
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