Hepatocyte Growth Factor Receptor Signaling Mediates the Anti-Fibrotic Action of 9-cis-Retinoic Acid in Glomerular Mesangial Cells
2005; Elsevier BV; Volume: 167; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)61185-6
ISSN1525-2191
AutoresXiao‐Yan Wen, Yingjian Li, Kebin Hu, Chunsun Dai, Youhua Liu,
Tópico(s)Pregnancy and preeclampsia studies
ResumoRetinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the regulation of cell proliferation, survival, and differentiation. RA action is primarily mediated through its receptors, ligand-dependent transcription factors of the steroid/thyroid/vitamin D nuclear receptor superfamily. Recent studies indicate that administration of RA mitigates progressive kidney disease, underscoring its renoprotective potential. In this study, we investigated the effects of 9-cis-RA on glomerular mesangial cell activation induced by transforming growth factor (TGF)-β1 using an in vitro cell culture system. In human mesangial cells 9-cis-RA suppressed TGF-β1-induced α-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression, but it did not significantly affect cell proliferation and survival. Interestingly, 9-cis-RA induced hepatocyte growth factor (HGF) mRNA expression and protein secretion, stimulated HGF promoter activity, and activated c-met receptor phosphorylation. Similar to HGF, 9-cis-RA induced expression of the Smad transcriptional co-repressor TGIF in mesangial cells. Overexpression of exogenous TGIF by transfection or 9-cis-RA treatment suppressed trans-activation of the TGF-β-responsive promoter. Moreover, conditional ablation of the c-met receptor completely abolished the anti-fibrotic effect of 9-cis-RA and abrogated TGIF induction. Collectively, these results indicate that 9-cis-RA possesses anti-fibrotic ability by antagonizing TGF-β1 in mesangial cells and that 9-cis-RA activity is likely mediated through a mechanism dependent on HGF/c-met receptor signaling. Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the regulation of cell proliferation, survival, and differentiation. RA action is primarily mediated through its receptors, ligand-dependent transcription factors of the steroid/thyroid/vitamin D nuclear receptor superfamily. Recent studies indicate that administration of RA mitigates progressive kidney disease, underscoring its renoprotective potential. In this study, we investigated the effects of 9-cis-RA on glomerular mesangial cell activation induced by transforming growth factor (TGF)-β1 using an in vitro cell culture system. In human mesangial cells 9-cis-RA suppressed TGF-β1-induced α-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression, but it did not significantly affect cell proliferation and survival. Interestingly, 9-cis-RA induced hepatocyte growth factor (HGF) mRNA expression and protein secretion, stimulated HGF promoter activity, and activated c-met receptor phosphorylation. Similar to HGF, 9-cis-RA induced expression of the Smad transcriptional co-repressor TGIF in mesangial cells. Overexpression of exogenous TGIF by transfection or 9-cis-RA treatment suppressed trans-activation of the TGF-β-responsive promoter. Moreover, conditional ablation of the c-met receptor completely abolished the anti-fibrotic effect of 9-cis-RA and abrogated TGIF induction. Collectively, these results indicate that 9-cis-RA possesses anti-fibrotic ability by antagonizing TGF-β1 in mesangial cells and that 9-cis-RA activity is likely mediated through a mechanism dependent on HGF/c-met receptor signaling. Retinoic acid (RA) is an active metabolite of vitamin A (retinol) that plays an imperative role in regulating a wide range of biological processes such as cell proliferation and survival, embryonic development, immune modulation, and tissue homeostasis.1Chambon P A decade of molecular biology of retinoic acid receptors.FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2623) Google Scholar, 2Wagner J Potential role of retinoids in the therapy of renal disease.Nephrol Dial Transplant. 2001; 16: 441-444Crossref PubMed Scopus (19) Google Scholar, 3Xu Q Lucio Cazana J Kitamura M Ruan X Fine LG Norman JT Retinoids in nephrology: promise and pitfalls.Kidney Int. 2004; 66: 2119-2131Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar The natural RA exists in different forms and represents a group of active metabolic derivatives of vitamin A, which include all-trans RA, 9-cis-RA, 13-cis-RA, and others. The action of RA is mediated through binding to its receptors, the ligand-dependent transcription factors that belong to the steroid/thyroid/vitamin D nuclear receptor superfamily. Two subfamilies of RA receptors with different ligand specificities are found, namely retinoic acid receptor (RAR) and retinoid X receptor (RXR), each of them has three isotypes (-α, -β, and -γ), respectively. Although RARs are stimulated by the all-trans RA and 9-cis-RA, the RXRs can be activated exclusively by 9-cis-RA. On ligand binding, the retinoid receptors undergo nuclear translocation, and subsequently bind to specific cis-acting RA- and RX-responsive elements in the promoter regions of their target genes, thereby directly modulating gene transcription. Retinoid receptors may also heterodimerize with the other members of the nuclear receptor superfamily, including the vitamin D receptor and peroxisome proliferator-activated receptors.2Wagner J Potential role of retinoids in the therapy of renal disease.Nephrol Dial Transplant. 2001; 16: 441-444Crossref PubMed Scopus (19) Google Scholar This will result in significant cross-talks among the different hormonal systems. In addition, retinoid receptors may modulate gene expression by interacting with other general transcription factors, such as activator protein-1 (AP-1) and nuclear factor (NF)-κB.4Zhou XF Shen XQ Shemshedini L Ligand-activated retinoic acid receptor inhibits AP-1 transactivation by disrupting c-Jun/c-Fos dimerization.Mol Endocrinol. 1999; 13: 276-285Crossref PubMed Scopus (0) Google Scholar, 5Na SY Kang BY Chung SW Han SJ Ma X Trinchieri G Im SY Lee JW Kim TS Retinoids inhibit interleukin-12 production in macrophages through physical associations of retinoid X receptor and NFkappaB.J Biol Chem. 1999; 274: 7674-7680Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar In such way, retinoids may integrate the signaling cascade of multiple regulatory pathways and control the distinctive cellular responses in a cell- and context-specific manner. Kidney cells express all major isotypes of retinoid receptors and respond to their stimulation.6Manzano VM Munoz JCS Jimenez JR Puyol MR Puyol DR Kitamura M Cazana FJL Human renal mesangial cells are a target for the anti-inflammatory action of 9-cis retinoic acid.Br J Pharmacol. 2000; 131: 1673-1683Crossref PubMed Scopus (35) Google Scholar, 7Liebler S Uberschar B Kubert H Brems S Schnitger A Tsukada M Zouboulis CC Ritz E Wagner J The renal retinoid system: time-dependent activation in experimental glomerulonephritis.Am J Physiol. 2004; 286: F458-F465Crossref PubMed Scopus (22) Google Scholar In the embryonic stage, retinoid is a major modulator of renal tubulogenesis and plays an essential role in determining the total number of nephrons per kidney.8Vilar J Gilbert T Moreau E Merlet-Benichou C Metanephros organogenesis is highly stimulated by vitamin A derivatives in organ culture.Kidney Int. 1996; 49: 1478-1487Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 9Lelievre-Pegorier M Vilar J Ferrier ML Moreau E Freund N Gilbert T Merlet-Benichou C Mild vitamin A deficiency leads to inborn nephron deficit in the rat.Kidney Int. 1998; 54: 1455-1462Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 10Merlet-Benichou C Vilar J Lelievre-Pegorier M Gilbert T Role of retinoids in renal development: pathophysiological implication.Curr Opin Nephrol Hypertens. 1999; 8: 39-43Crossref PubMed Scopus (51) Google Scholar Double knockout of the RARs and RXRs result in several malformations, including kidney agenesis, hypoplasia, or aplasia of the ureteral bud.11Mendelsohn C Lohnes D Decimo D Lufkin T LeMeur M Chambon P Mark M Function of the retinoic acid receptors (RARs) during development (II). 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139: 823-831Crossref PubMed Scopus (55) Google Scholar, 19Kiss E Adams J Grone HJ Wagner J Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google Scholar, 20Kinoshita K Yoo BS Nozaki Y Sugiyama M Ikoma S Ohno M Funauchi M Kanamaru A Retinoic acid reduces autoimmune renal injury and increases survival in NZB/W F1 mice.J Immunol. 2003; 170: 5793-5798Crossref PubMed Scopus (78) Google Scholar In anti-Thy1.1 glomerulonephritis, treatment with all-trans RA or 13-cis-RA effectively ameliorates renal damage and mesangial cell proliferation, attenuates capillary occlusion and glomerular inflammation, and reduces albuminuria.13Schaier M Liebler S Schade K Shimizu F Kawachi H Grone HJ Chandraratna R Ritz E Wagner J Retinoic acid receptor alpha and retinoid X receptor specific agonists reduce renal injury in established chronic glomerulonephritis of the rat.J Mol Med. 2004; 82: 116-125Crossref PubMed Scopus (47) Google Scholar, 21Wagner J Dechow C Morath C Lehrke I Amann K Waldherr R Floege J Ritz E Retinoic acid reduces glomerular injury in a rat model of glomerular damage.J Am Soc Nephrol. 2000; 11: 1479-1487Crossref PubMed Google Scholar, 22Dechow C Morath C Peters J Lehrke I Waldherr R Haxsen V Ritz E Wagner J Effects of all-trans retinoic acid on renin-angiotensin system in rats with experimental nephritis.Am J Physiol. 2001; 281: F909-F919Crossref PubMed Google Scholar, 23Schaier M Lehrke I Schade K Morath C Shimizu F Kawachi H Grone HJ Ritz E Wagner J Isotretinoin alleviates renal damage in rat chronic glomerulonephritis.Kidney Int. 2001; 60: 2222-2234Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 24Lehrke I Schaier M Schade K Morath C Waldherr R Ritz E Wagner J Retinoid receptor-specific agonists alleviate experimental glomerulonephritis.Am J Physiol. 2002; 282: F741-F751Crossref PubMed Scopus (60) Google Scholar The beneficial effects of retinoids are also clearly evident in many other models of renal diseases, such as anti-glomerular basement membrane glomerulonephritis,17Oseto S Moriyama T Kawada N Nagatoya K Takeji M Ando A Yamamoto T Imai E Hori M Therapeutic effect of all-trans retinoic acid on rats with anti-GBM antibody glomerulonephritis.Kidney Int. 2003; 64: 1241-1252Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar lupus nephritis,14Perez de Lema G Lucio-Cazana FJ Molina A Luckow B Schmid H de Wit C Moreno-Manzano V Banas B Mampaso F Schlondorff D Retinoic acid treatment protects MRL/lpr lupus mice from the development of glomerular disease.Kidney Int. 2004; 66: 1018-1028Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 20Kinoshita K Yoo BS Nozaki Y Sugiyama M Ikoma S Ohno M Funauchi M Kanamaru A Retinoic acid reduces autoimmune renal injury and increases survival in NZB/W F1 mice.J Immunol. 2003; 170: 5793-5798Crossref PubMed Scopus (78) Google Scholar obstructive nephropathy,16Schaier M Jocks T Grone HJ Ritz E Wagner J Retinoid agonist isotretinoin ameliorates obstructive renal injury.J Urol. 2003; 170: 1398-1402Crossref PubMed Scopus (26) Google Scholar puromycin aminonucleoside-induced nephrosis,15Suzuki A Ito T Imai E Yamato M Iwatani H Kawachi H Hori M Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats.J Am Soc Nephrol. 2003; 14: 981-991Crossref PubMed Scopus (80) Google Scholar, 18Moreno-Manzano V Mampaso F Sepulveda-Munoz JC Alique M Chen S Ziyadeh FN Iglesias-de la Cruz MC Rodriguez J Nieto E Orellana JM Reyes P Arribas I Xu Q Kitamura M Lucio Cazana FJ Retinoids as a potential treatment for experimental puromycin-induced nephrosis.Br J Pharmacol. 2003; 139: 823-831Crossref PubMed Scopus (55) Google Scholar and acute renal allograft nephropathy.19Kiss E Adams J Grone HJ Wagner J Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google Scholar Despite increasing evidence demonstrating that RA is a potent renoprotective agent that may have therapeutic potential for human kidney diseases, relatively little is known regarding the mechanisms of its action. In light of the interactions between retinoid receptors and the AP-1 or NF-κB, previous studies are primarily emphasized on its anti-proliferative and anti-inflammatory capacity.2Wagner J Potential role of retinoids in the therapy of renal disease.Nephrol Dial Transplant. 2001; 16: 441-444Crossref PubMed Scopus (19) Google Scholar, 25Wagner J Nuclear (receptor) power: retinoids in rat mesangioproliferative disease.Nephrol Dial Transplant. 2002; 17: 81-83Crossref PubMed Scopus (5) Google Scholar It remains ambiguous whether RA can directly block the fibrogenic processes of the kidney cells. Likewise, it is also to be determined whether RA counteracts the action of transforming growth factor (TGF)-β1, the principal profibrotic cytokine that has been implicated in the pathogenesis of virtually all kinds of fibrotic disorders.26Border WA Noble NA TGF-beta in kidney fibrosis: a target for gene therapy.Kidney Int. 1997; 51: 1388-1396Abstract Full Text PDF PubMed Scopus (382) Google Scholar, 27Bottinger EP Bitzer M TGF-β1 signaling in renal disease.J Am Soc Nephrol. 2002; 13: 2600-2610Crossref PubMed Scopus (673) Google Scholar, 28Schnaper HW Hayashida T Hubchak SC Poncelet AC TGF-beta signal transduction and mesangial cell fibrogenesis.Am J Physiol. 2003; 284: F243-F252Crossref PubMed Scopus (36) Google Scholar In this study, we have demonstrated that 9-cis-RA effectively suppresses TGF-β1-mediated glomerular mesangial cell activation. Further examination reveals that 9-cis-RA also induces anti-fibrotic hepatocyte growth factor (HGF) expression. Interestingly, conditional ablation of HGF receptor, c-met, completely abolishes the action of 9-cis-RA in mesangial cells. These studies establish that HGF/c-met signaling is essential for mediating the anti-fibrotic activity of 9-cis-RA. The mouse monoclonal anti-α-smooth muscle actin (α-SMA, clone 1A4) was obtained from Sigma (St. Louis, MO). Mouse monoclonal anti-fibronectin (clone 10) was purchased from BD Pharmingen (San Jose, CA). The anti-TGIF (sc-17800), anti-plasminogen activator inhibitor-1 (PAI-1) (sc-5297), and anti-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Phospho-Met (Tyr1234/1235) antibody that detects Met only when phosphorylated at tyrosine 1234/1235 and monoclonal anti-Met antibody (25H2) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Antibody against Cre recombinase was purchased from EMD Biosciences, Inc. (San Diego, CA). Monoclonal antibody against human HGF (clone H-14) was prepared by our laboratory and described elsewhere.29Yang J Chen S Huang L Michalopoulos GK Liu Y Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar This antibody specifically recognizes human HGF protein. Affinity-purified secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). The TGF-β-responsive reporter vector p3TP-Lux, Smad2, Smad3, and TGIF expression vectors were kindly provided by Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York, NY).30Lo RS Wotton D Massague J Epidermal growth factor signaling via Ras controls the Smad transcriptional co-repressor TGIF.EMBO J. 2001; 20: 128-136Crossref PubMed Scopus (152) Google Scholar Adenoviral vectors containing Cre recombinase (Ad.Cre) or green fluorescent protein (Ad.GFP) were provided by Dr. A. Gambotto at the Vector Core Facility of the University of Pittsburgh. Recombinant human TGF-β1 was purchased from R&D Systems Inc. (Minneapolis, MN). Recombinant human HGF was provided by Genentech Inc. (South San Francisco, CA). 9-cis-Retinoic acid (9cRA) was obtained from Sigma. Cell culture media, fetal bovine serum, and supplements were obtained from Invitrogen (Carlsbad, CA). All other chemicals were of analytic grade and were obtained from Sigma or Fisher (Pittsburgh, PA) unless otherwise indicated. Human glomerular mesangial cells (HMCs) and media were purchased from ScienCell Research Laboratories (San Diego, CA). These cells are characterized by the manufacturer using morphological appearances and immunofluorescent method with various antibodies, including anti-Thy-1 and fibronectin. Rat mesangial cells were provided by Dr. C. Wu at the University of Pittsburgh and described previously.31Guo L Sanders PW Woods A Wu C The distribution and regulation of integrin-linked kinase in normal and diabetic kidneys.Am J Pathol. 2001; 159: 1735-1742Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar HMCs were cultured in HMC medium and rat glomerular mesangial cells were seeded in Dulbecco's modified Eagle's medium-F12 medium supplemented with 5% fetal bovine serum. Twenty-four hours later, the cells were changed to serum-free medium and then incubated for 16 hours. Cells were then treated with various agents at the concentrations specified. The supernatants or whole cell lysates were collected at different time points for various analyses. Cell proliferation was assessed by using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]-based cell growth determination kit (Sigma). Briefly, HMCs were seeded in 96-well plates at the same concentration (2 × 103/well). After incubation in complete medium containing 5% fetal bovine serum overnight, cells were changed to serum-free medium for 24 hours. HMCs were then treated with 9-cis-RA in the absence or presence of TGF-β1 for 48 hours. MTT (5 mg/ml) was added to the culture at 10 μl per well, followed by incubation at 37°C for 2 hours. Medium was aspirated, and cells were lysed with dimethyl sulfoxide. Absorbance of each well was recorded in a microplate reader at 540 nm. Data are presented as means ± SEM. Three wells per treatment were used. The cytotoxicity of various agents to mesangial cells was assessed by measuring lactate dehydrogenase release using a cytotoxicity detection kit (Roche Diagnostics GmbH, Penzberg, Germany). HMCs were treated with various agents for 48 hours as indicated. The supernatants of the cultures were harvested and transferred to an optically clear 96-well plate. After incubation with 100 μl of the reaction mixture for 30 minutes at room temperature, absorbance of each well was read at 490 nm. Data are presented as means ± SEM. Three wells per treatment were used. Apoptotic cell death was detected by using TUNEL staining with an apoptosis detection system (Promega, Madison, WI), as described previously.32Dai C Yang J Liu Y Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in mice.J Am Soc Nephrol. 2002; 13: 411-422Crossref PubMed Google Scholar Briefly, HMCs after various treatments for 48 hours were fixed and permeabilized with methanol at −20°C for 15 minutes. After washing with phosphate-buffered saline (PBS), cells with incubated with 100 μl of equilibration buffer at room temperature for 10 minutes, followed by incubation with terminal deoxynucleotidyl transferase at 37°C for 60 minutes. The reaction was terminated by immersing the slides in 2× standard saline citrate for 15 minutes at room temperature. The slides were mounted in Vectashield mounting medium with propidium iodide (Vector Laboratories Inc., Burlingame, CA) and observed on a Nikon Eclipse E600 epifluorescence microscope equipped with a digital camera (Nikon Inc., Melville, NY). Apoptotic cells were counted in high-power (×400) fields and expressed as apoptotic cells per field. Whole cell lysates were prepared as described previously.33Yang J Liu Y Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar Cells were lysed with sodium dodecyl sulfate sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 50 mmol/L dithiothreitol, and 0.1% bromophenol blue). Samples were heated at 100°C for ∼5 to 10 minutes before loading and separated onto 10% sodium dodecyl sulfate-polyacrylamide gels. After the proteins were electrotransferred to Hybond-P polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ), nonspecific binding to the membrane was blocked for 1 hour at room temperature with 5% Carnation nonfat milk (Nestlé, Wilkes-Barre, PA) in TBST buffer (20 mmol/L Tri-HCl, 150 mmol/L NaCl, and 0.1% Tween 20). The membranes were then incubated for 16 hours at 4°C with various primary antibodies in blocking buffer containing 5% milk at the dilutions specified by the manufacturers. After extensive washing in TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature in 5% nonfat milk dissolved in TBST. Membranes were then washed with TBST buffer and the signals were visualized using the SuperSignal West Pico chemiluminescent substrate kit (Pierce Biotechnology, Rockford, IL). Indirect immunofluorescence staining was performed using an established procedure.34Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar Briefly, cells cultured on coverslips were washed twice with cold PBS and fixed with cold methanol/acetone (1:1) for 10 minutes at −20°C. After three extensive washings with PBS containing 0.5% bovine serum albumin, the cells were blocked with 20% normal donkey serum in PBS buffer for 30 minutes at room temperature and then incubated with the specific primary antibody described above. Slides were then incubated with fluorescein-conjugated secondary antibody for 1 hour before being extensively washed with PBS. As a negative control, the primary antibody was replaced with nonimmune IgG, and no staining occurred. For some samples, cells were double stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Stained cells were mounted and viewed with a Nikon Eclipse E600 epifluorescence microscope. Real-time quantitative RT-PCR was performed to determine the steady-state levels of HGF c-met mRNA. Briefly, total RNA was extracted from HMCs using TRIzol RNA isolation system according to the manufacturer's instructions (Invitrogen). RNA samples were quantified by determination of ultraviolet absorbance at 260 nm. The first strand cDNA synthesis was performed by using a reverse transcription system kit according to the instructions of the manufacturer (Promega, Madison, WI). Real-time PCR amplification was performed on ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). The PCR reaction mixture at a 25-μl volume contained 12.5 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems), 5 μl of diluted RT product (1:20), and 0.5 μmol/L sense and anti-sense primer sets. The primer sequences were as follows: HGF, 5′-AAGGTGACTTTGAATGAGTC (sense), and 5′-GGCACATCCACGACCAGGAACAATG (anti-sense); β-actin, 5′-AGGCATCCTCACCCTGAAGTA (sense), and 5′-CACACGCAGCTCATTGTAGA (anti-sense). Each sample was added in duplicate. PCR reaction was run by using standard conditions. After sequential incubations at 50°C for 2 minutes and 95°C for 10 minutes, respectively, the amplification protocol consisted of 50 cycles of denaturing at 95°C for 15 seconds, annealing and extension at 60°C for 60 seconds. The standard curve was made from a series of dilutions of template cDNA. Expression levels of HGF mRNA were calculated after normalizing with β-actin. The reporter construct 0.9HGF-Luc, which contains 0.9 kb of the 5′-flanking region of mouse HGF gene and the coding sequence for firefly luciferase, was described previously.35Liu Y Michalopoulos GK Zarnegar R Structural and functional characterization of the mouse hepatocyte growth factor gene promoter.J Biol Chem. 1994; 269: 4152-4160Abstract Full Text PDF PubMed Google Scholar At 24 hours before transfection, rat mesangial cells were seeded onto six-well plates at 4 × 105 cells per well. The cells were then transfected with 0.9HGF-Luc (0.9 μg). A fixed amount (0.1 μg) of internal control reporter Renilla reniformis luciferase driven under thymidine kinase (TK) promoter (pRL-TK; Promega) was also co-transfected for normalizing the transfection efficiency. After transfection with Lipofectamine 2000 reagent (Invitrogen), the cells were incubated for an additional 48 hours in the absence or presence of 10−6 mol/L 9cRA. For investigating the effect of 9cRA on the transcription of TGF-β1-responsive genes, reporter plasmid p3TP-Lux (1.0 μg) together with or without Smad2 (0.5 μg)/Smad3 (0.5 μg), and/or TGIF (1.0 μg) expression vectors were co-transfected into mesangial cells. A fixed amount (0.1 μg) of control reporter pRL-TK was also co-transfected. The transfected cells were pretreated with 9cRA (10−6 mol/L) for 16 hours, followed by incubation with or without TGF-β1 (1 ng/ml) for another 36 hours. After being washed in PBS, the cells were pelleted, resuspended in 250 μl of lysis buffer, and then disrupted by three freeze-thaw cycles. The protein suspension was clarified by centrifugation at 15,000 × g for 5 minutes at 4°C, and the supernatant was collected and analyzed. Luciferase assay was performed using the dual luciferase assay system kit essentially according to the manufacturer's protocols (Promega). Relative luciferase activity of each construct (arbitrary unit) was reported as fold induction after normalizing for transfection efficiency. The c-met-floxed mice in which the exon 16 of the c-met gene was flanked by loxP sites were kindly provided by Dr. S. Thorgeirsson of the National Cancer Institute, National Institutes of Health (Bethesda, MD).36Huh CG Factor VM Sanchez A Uchida K Conner EA Thorgeirsson SS Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair.Proc Natl Acad Sci USA. 2004; 101: 4477-4482Crossref PubMed Scopus (639) Google Scholar Mouse primary cultured mesangial cells from outgrowths of isolated whole glomeruli were prepared essentially as described previously.37Liu Y Tolbert EM Sun AM Dworkin LD Primary structure of rat HGF receptor and induced expression in glomerular mesangial cells.Am J Physiol. 1996; 271: F679-F688PubMed Google Scholar Briefly, glomeruli were isolated from the c-met-floxed mice under sterile conditions by differential sieving of cortical tissue. After the final wash, the isolated glomeruli were resuspended in RPMI 1640 medium supplemented with 20% fetal bovine serum and 5 μg/ml of insulin, and plated on culture dishes for outgrowth of the mesangial cells. When the cells reached confluency, they were subcultured at 1:4 dilution. The cells showed the typical smooth muscle cell-like morphology and positive staining for Thy1.1 and α-SMA, while negative staining for cytokeratin. Passages 4 through 10 of subcultured mesangial cells were infected with adenoviral vectors (Ad.GFP or Ad.Cre) in s
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