Smad4-dependent Regulation of Urokinase Plasminogen Activator Secretion and RNA Stability Associated with Invasiveness by Autocrine and Paracrine Transforming Growth Factor-β
2006; Elsevier BV; Volume: 281; Issue: 45 Linguagem: Inglês
10.1074/jbc.m607010200
ISSN1083-351X
AutoresSheng-Ru Shiou, Pran K. Datta, Punita Dhawan, Brian K. Law, Jonathan M. Yingling, Dan A. Dixon, R. Daniel Beauchamp,
Tópico(s)Renal and related cancers
ResumoMetastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-β (TGF-β) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-β supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-β at higher concentrations than autocrine TGF-β further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-β is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-β-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-β maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-β further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells. Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-β (TGF-β) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-β supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-β at higher concentrations than autocrine TGF-β further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-β is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-β-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-β maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-β further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells. Malignant tumors are characterized by their ability to metastasize to distant organs. The initial steps of metastasis involve invasive growth of tumors across the basement membrane and migration through the extracellular matrix (ECM). 2The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TGF-β, transforming growth factor-β;TβRI, -II, transmembrane types I and II; ERK, extracellular signal-regulated kinase; DNIIR, dominant-negative type II receptor; uPA, urokinase plasminogen activator; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, analysis of variance. 2The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TGF-β, transforming growth factor-β;TβRI, -II, transmembrane types I and II; ERK, extracellular signal-regulated kinase; DNIIR, dominant-negative type II receptor; uPA, urokinase plasminogen activator; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, analysis of variance. Because the enzymatic degradation of both the basement membrane and ECM barriers requires a number of ECM-degrading proteases (1Mignatti P. Robbins E. Rifkin D.B. Cell. 1986; 47: 487-498Abstract Full Text PDF PubMed Scopus (635) Google Scholar, 2Ellenrieder V. Hendler S.F. Ruhland C. Boeck W. Adler G. Gress T.M. Int. J. Cancer. 2001; 93: 204-211Crossref PubMed Scopus (134) Google Scholar) and is a critical early event in metastasis, invasiveness may be modulated by the expression of ECM-degrading proteases in tumor cells in response to autonomous and microenvironmental signals. Among the increasing number of ECM-degrading proteases implicated in metastasis, considerable attention has been focused on the family of matrix metalloproteinases (MMPs) and the plasminogen activator system. One of the regulators of these ECM-degrading proteases is transforming growth factor-β (TGF-β). TGF-β is a multifunctional cytokine that regulates cell proliferation, differentiation, plasticity, and migration in a context-dependent manner (reviewed in Refs. 3Zavadil J. Bottinger E.P. Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1357) Google Scholar and 4Huang S.S. Huang J.S. J. Cell. Biochem. 2005; 96: 447-462Crossref PubMed Scopus (189) Google Scholar). TGF-β transduces signaling through a transmembrane type II receptor (TβRII), a constitutively active serine/threonine kinase receptor (5Lin H.Y. Wang X.F. Ng-Eaton E. Weinberg R.A. Lodish H.F. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (963) Google Scholar). Upon ligand binding, the TβRII recruits and transphosphorylates intracellular TGF-β type I receptor (TβRI), thereby stimulating TβRI serine/threonine kinase activity (6Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2088) Google Scholar). The TβRI then activates the downstream effectors, Smad2 and Smad3, by phosphorylation. The activated Smad proteins form complexes with the common Smad mediator, Smad4, and then translocate to the nucleus, where the Smad complexes regulate transcription of TGF-β target genes in conjunction with various transcriptional or co-transcriptional regulators. In addition to the Smad pathway, other signaling pathways, including the extracellular signal-regulated kinases (ERK1/2) (7Frey R. Mulder K. Cancer Res. 1997; 57: 628-633PubMed Google Scholar, 8Mulder K.M. Cytokine Growth Factor Rev. 2000; 11: 23-35Crossref PubMed Scopus (383) Google Scholar), the mitogen-activated protein kinase (p38) (9Bakin A.V. Rinehart C. Tomlinson A.K. Arteaga C.L. J. Cell Sci. 2002; 115: 3193-3206Crossref PubMed Google Scholar, 10Bhowmick N.A. Zent R. Ghiassi M. McDonnell M. Moses H.L. J. Biol. Chem. 2001; 276: 46707-46713Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), the Src (11Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and the phosphatidylinositol 3-kinase (PI3K) (12Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar) pathways can be activated by TGF-β in a context-dependent manner. The precise molecular mechanisms of regulation of these pathways for TGF-β signaling and the physiological and pathological roles of TGF-β in normal tissues and cancer have not been completely defined. The importance of autocrine TGF-β in tumor progression and metastatic behavior has been documented previously. For instance, disruption of autocrine TGF-β signaling by a dominant-negative type II receptor (DNIIR) inhibited the invasive and metastatic potential of mammary and colon carcinoma cells (13Oft M. Heider K.-H. Beug H. Curr. Biol. 1998; 8: 1243-1252Abstract Full Text Full Text PDF PubMed Google Scholar). This was attributed to prevention of autocrine TGF-β-induced epithelial-to-mesenchymal transition, a process believed to promote tumor cell migration and invasion (12Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). In a different study, overexpression of a soluble TGF-β type III receptor antagonized autocrine TGF-β activity and resulted in inhibition of tumor cell proliferation and induction of apoptosis (14Lei X. Bandyopadhyay A. Le T. Sun L. Oncogene. 2002; 21: 7514-7523Crossref PubMed Scopus (68) Google Scholar). The urokinase plasminogen activator (uPA) is a serine protease capable of initiating cascades of activation of ECM-degrading enzymes (15Carmeliet P. Moons L. Lijnen R. Baes M. Lemaitre V. Tipping P. Drew A. Eeckhout Y. Shapiro S. Lupu F. Collen D. Nat. Genet. 1997; 17: 439-444Crossref PubMed Scopus (567) Google Scholar) and eliciting intracellular signaling through receptor binding. Clinically, elevated uPA expression in tumors is associated with tumor aggressiveness and poor outcome in patients (16Duffy M.J. O'Grady P. Devaney D. O'Siorain L. Fennelly J.J. Lijnen H.J. Cancer. 1988; 62: 531-533Crossref PubMed Scopus (309) Google Scholar, 17Duffy M.J. Maguire T.M. McDermott E.W. O'Higgins N. J. Surg. Oncol. 1999; 71: 130-135Crossref PubMed Scopus (184) Google Scholar) and numerous studies have linked uPA to invasive and metastatic phenotype of tumors in vitro and in animal models (18Andreasen P.A. Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (829) Google Scholar, 19Reuning U. Magdolen V. Wilhelm O. Fischer K. Lutz V. Graeff H. Schmitt M. Int. J. Oncol. 1998; 13: 893-906PubMed Google Scholar, 20Farina A.R. Coppa A. Tiberio A. Tacconelli A. Turco A. Colletta G. Gulino A. Mackay A.R. Int. J. Cancer. 1998; 75: 721-730Crossref PubMed Scopus (115) Google Scholar, 21Frandsen T.L. Holst-Hansen C. Nielsen B.S. Christensen I.J. Nyengaard J.R. Carmeliet P. Brunner N. Cancer Res. 2001; 61: 532-537PubMed Google Scholar). The metastatic MDA-MB-231 breast cancer cells secrete active TGF-β (22Martinez-Carpio P.A. Mur C. Rosel P. Navarro M.A. Cell. Signal. 1999; 11: 753-757Crossref PubMed Scopus (20) Google Scholar, 23Martinez-Carpio P.A. Mur C. Fernandez-Montoli M.E. Ramon J.M. Rosel P. Navarro M.A. Cancer Lett. 1999; 147: 25-29Crossref PubMed Scopus (13) Google Scholar) and are TGF-β-responsive (24Chen C.-R. Kang Y. Massague J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 992-999Crossref PubMed Scopus (274) Google Scholar). These cells also express both the matrix metalloproteinases-9 (MMP-9) and uPA (20Farina A.R. Coppa A. Tiberio A. Tacconelli A. Turco A. Colletta G. Gulino A. Mackay A.R. Int. J. Cancer. 1998; 75: 721-730Crossref PubMed Scopus (115) Google Scholar, 25Matrisian L.M. Curr. Biol. 1999; 9: R776-R778Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 26Matrisian L.M. Wright J. Newell K. Witty J.P. Princess Takamatsu Symp. 1994; 24: 152-161PubMed Google Scholar). We hypothesized that autocrine TGF-β may function as a tumor promoter by regulating MMP-9 or uPA activity in MDA-MB-231 cells. The present study provides evidence that autocrine TGF-β regulates both cell invasiveness and uPA secretion. Inhibition of uPA activity is sufficient to suppress tumor cell invasion to the same extent as inhibition of autocrine TGF-β signaling, suggesting that autocrine TGF-β stimulation of invasiveness occurs via its regulation of uPA release. The Smad pathway appears to be required for the regulation of uPA release as silencing of Smad4 protein expression suppressed uPA secretion. Interestingly, although autocrine TGF-β regulates uPA production through protein secretion, exogenous TGF-β further increases uPA expression through RNA stabilization also through a Smad4-dependent fashion. Finally, this work demonstrates that a pharmacological kinase inhibitor of TGF-β receptors inhibits both uPA secretion and tumor cell invasiveness, thereby providing evidence for the potential efficacy of targeting TGF-β signaling for therapeutic intervention in cancer and suggests that uPA expression or secretion may be an important mediator of such effects. Cell Cultures and Reagents—MDA-MB-231 cells from the American Type Culture Collection (Manassas, VA) were maintained in the Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 37 °C incubator with 5% CO2. TGF-β was purchased from R & D Systems (Minneapolis, MN). The recombinant active human PAI-1 (cat. no. 1092) and human urokinase (cat. no. 124) were from American Diagnostica, Inc. (Greenwich, CT). The pharmacological inhibitor of the TGF-β type I receptor (LY364947) (27Peng S.B. Yan L. Xia X. Watkins S.A. Brooks H.B. Beight D. Herron D.K. Jones M.L. Lampe J.W. McMillen W.T. Mort N. Sawyer J.S. Yingling J.M. Biochemistry. 2005; 44: 2293-2304Crossref PubMed Scopus (86) Google Scholar, 28Li H.Y. Wang Y. Heap C.R. King C.H. Mundla S.R. Voss M. Clawson D.K. Yan L. Campbell R.M. Anderson B.D. Wagner J.R. Britt K. Lu K.X. McMillen W.T. Yingling J.M. J. Med. Chem. 2006; 49: 2138-2142Crossref PubMed Scopus (43) Google Scholar) was provided by Eli Lilly (Indianapolis, IN). Immunoblot Analysis—To harvest protein lysates, cells were washed with cold phosphate-buffered saline (PBS) and lysed in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, and 1 mm sodium fluoride) for 20 min on ice. Lysates were sonicated and then clarified by centrifugation at 15,000 × g for 15 min at 4 °C. Protein contents of lysates were determined by the Bradford Assay (Bio-Rad). Proteins in the lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% milk PBS-T (0.1% Tween 20 (v/v) in PBS) for 1 h at room temperature and then probed with primary antibodies in 5% milk PBS-T overnight at 4 °C. After several washes with PBS-T, membranes were incubated in PBS-T containing horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and washed again with PBS-T. Immunoreactive bands were visualized by chemiluminescence reaction using ECL reagents (Amersham Biosciences) followed by exposure of the membranes to XAR5 films (Kodak, Rochester, NY). To detect secreted uPA, conditioned media were collected, centrifuged at 15,000 × g for 5 min to remove cell debris, and then subjected to immunoblotting under non-reducing conditions. 2- to 8-h conditioned media were concentrated using Microcon YM-10 centrifugal filter devices from Millipore (Billerica, MA). The volumes of conditioned media loaded on gels were normalized to the protein concentrations of cell lysates. The fibronectin antibody was purchased from BD Transduction Laboratories, Inc. The Smad2 and phospho-Smad2 antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The uPA antibody (cat. no. 394) was obtained from American Diagnostica, Inc. The polyadenosine diphosphate ribose polymerase and Rho GDI (guanine nucleotide dissociation inhibitor) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The actin and FLAG antibodies were purchased from Sigma-Aldrich, Inc. Transient Transfection and Luciferase Reporter Assay—Cells at 50–70% confluence on 12-well plates were co-transfected with 0.5 μg of a firefly luciferase promoter-reporter construct and 0.01 μg of the Renilla reniformis luciferase reference reporter construct, phRL-TK (Promega, Madison, WI) using Lipofectamine Plus reagents (Invitrogen). Four hours after transfection, cells were cultured back in regular media. Forty-eight hours after transfection, firefly and R. reniformis luciferase activities were measured using the Dual Luciferase Reporter Assay System kit (Promega) in an Optocomp II Luminometer (MGM Instruments, Inc., Hamden, CT). Normalized firefly luciferase activity was plotted as mean ± S.D. from three independent experiments. The phuPA-Luc reporter containing the nucleotide sequence –2345 to +30 of the human uPA promoter was kindly provided by Dr. Shuji Kojima (29Suzuki Y. Shimada J. Shudo K. Matsumura M. Crippa M.P. Kojima S. Blood. 1999; 93: 4264-4276Crossref PubMed Google Scholar). The p3TP-Lux reporter was a generous gift from Dr. Joan Massague (30Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1365) Google Scholar). Matrigel Invasion Assay—A modified Boyden chamber assay was performed using Transwells (12-μm pore size, 12 mm in diameter) from Costar (Cambridge, MA) and Matrigel (BD Biosciences). Each Transwell insert was first coated with 100 μl of 2.5 mg/ml Matrigel diluted in serum-free media for 1 h at 37 °C, and then 10 μl of Matrigel was added in the center of the Transwell 2 h before use. Cells were trypsinized, washed with serum-free media twice, re-suspended in 0.2% bovine serum albumin serum-free medium, seeded in Transwell inserts (150,000 cells/insert), and grown in the presence of 10% fetal bovine serum media in the lower chamber. After 16 h of incubation, Matrigel and cells remaining inside the inserts were removed with Q-tips, and the cells that had traversed to the reverse side of the inserts were rinsed with PBS, fixed in 4% formaldehyde for 30 min at room temperature, and stained with 1% crystal violet for 1 h to overnight at room temperature. Cells were counted under a light microscope (at 200× power), and invasive cell numbers were the averages of those from five areas on each insert. Each invasion assay was performed in triplicate and repeated three times. Adenovirus-mediated Overexpression of a Dominant-negative TGF-β Type II Receptor (DNIIR)—To amplify adenoviruses, 293T cells cultured in 5% serum/Dulbecco's modified Eagle's medium at 80% confluence on P100 plates, were infected with adenoviruses in 1 ml of fresh 5% serum medium with rocking. After 3 h, 9 ml of 5% serum medium was added in each plate without removing infection medium. Three days after infection, cells were trypsinized, collected in 1 ml of 5% fetal bovine serum medium, and subjected to three freeze/thaw cycles at –20 °C/37 °C. Adenovirus-containing supernatant was obtained from the cell suspension by centrifugation at 15,000 × g for 20 min at 4 °C and stored at –80 °C. The relative values of multiplicity of infection (m.o.i.) of adenoviral suspension were determined by examining the cytopathic effect in 293T (adenovirus at m.o.i. values of 10–20 causes a total CPE in confluent 293T cells 3 days post infection). To perform adenoviral infection, 70–80% confluent cells were washed with PBS once, incubated with adenovirus-containing medium for 3 h, and then grown in regular serum media. Lysates and conditioned media were harvested 48–72 h post infection. Cell Proliferation Assay—Cells were seeded in 96-well plates (50,000/well), and the relative viable cell numbers were determined by MTT assay using the CellTiter 96 Non-radioactive Cell Proliferation Assay kit (Promega), following the manufacturer's protocol. MTT hydrolysis was determined by measuring the absorbance at 570 nm using a plate reader. Preparation of Plasma Membrane Fractions—Cells were collected into buffer containing 0.15 m NaCl, 20 mm HEPES, 2 mm CaCl2, 100 μg/ml leupeptin, 2.5 mg/ml pepstatin A, and 1 mm phenylmethylsulfonyl fluoride (pH 8.0) by scraping and then lysed by freeze/thaw (liquid N2/42 °C) cycles. Nuclei were isolated from the suspension of lysed cells by centrifugation at 500 × g for 20 min at 4 °C, washed three times, and re-suspended in radioimmune precipitation assay buffer. The nucleus-free supernatant was spun at 100,000 × g for 1 h at 4 °C. The resulting supernatant was compose of cytoplasmic fractions, and the pellets were subsequently washed three times with 3 ml of the cell resuspension buffer and dissolved in radioimmune precipitation assay buffer as membrane fractions. MMP Zymography—Serum-free conditioned medium was mixed with 2× sample buffer (0.5 m Tris-HCl, pH 6.8, 5% SDS, 20% glycerol, and 1% bromphenol blue) and subject to SDS-PAGE using 10% SDS-gelatin (1 mg/ml final concentration) gels under a non-reducing condition. After electrophoresis, gels were soaked in washing buffer (50 mm Tris-HCl, pH 7.5, 0.1 m NaCl, and 2.5% Triton X-100) for 1 h at room temperature to remove SDS and then in reaction buffer (50 mm Tris-HCl, pH 7.5, and 5 mm CaCl2, pH 8.0) overnight at 37 °C. Subsequently, gels were stained in staining buffer (0.15% Coomassie Blue R250 in 10% acetic acid and 30% methanol) and de-stained in the same staining solution without Coomassie Blue R250. Clear bands of pro- and active MMP-9 (92 and 84 kDa, respectively) were observed against the blue background of stained gels. Northern Blot Analysis—Total RNA isolated with the TRIzol reagent was resolved on formaldehyde-agarose gels, transferred, and immobilized onto Hybond-N nylon membranes (Amersham Biosciences). Blots were blocked in the ULTRAHyb buffer (Ambion, Austin, TX) for 3 h at 65°C and then probed with 32P-labeled antisense Riboprobes (5 × 105 cpm/ml) in the ULTRAHyb buffer overnight at 68 °C. After several washes with low stringency (2× SSC/0.1% SDS) and high stringency (0.1× SSC/0.1% SDS) buffers at 68 °C, images were acquired by autoradiography using a PhosphorImager. To prepare Riboprobes, cDNA plasmids were linearized, purified, and then subject to in vitro transcription using the MAXIscript kit (Ambion) in the presence of 50 μCi of [α-32P]UTP (800 Ci/mmol) for 1 h at 37 °C. Unincorporated nucleotides were removed using Nuc-Away columns (Ambion). Nuclear Run-on Assay—Cell were collected, washed twice with PBS, and then re-suspended in lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, and 1 mm dithiothreitol). Nonidet P-40 was then added to a final concentration of 0.2–0.5%, depending on cell types. After a 5-min incubation on ice, nuclei were pelleted at 500 × g for 5 min, washed once with nuclear freezing buffer (50 mm Tris-HCl, pH 8.3, 40% glycerol, 5 mm MgCl2, 1 mm dithiothreitol), and re-suspended in fresh nuclear freezing buffer. In vitro run-on transcription was performed using 2 × 107 nuclei in 150 μl of reaction buffer (5 mm Tris-HCl, pH 8.0, 2.5 mm MgCl2, 150 mm KCl, 1 mm of ATP, CTP, or GTP, 150 μCi of [α-32P]UTP (800 Ci/mmol), 80 units of RNasin, and 2.5 mm dithiothreitol) for 30 min at 30 °C. Transcription was terminated by adding 350 μl of deoxyribonuclease I solution (20 mm Tris-HCl, pH 7.4, 10 mm CaCl2, and 300 units of RNase-free DNase I) and a 30-min incubation at 37 °C. Next, proteins were digested by adding 50 μl of proteinase K solution (1% SDS, 5 mm EDTA, 1 mm Tris-HCl, pH 7.4, and 300 μg/ml proteinase K) and a 30-min incubation at 50 °C. The 32P-labeled RNAs were phenol/chloroform purified and precipitated in 10% trichloroacetic acid plus 20 μg of yeast tRNA. After centrifugation at 15,000 × g for 1 h, the RNAs were re-suspended in RNase-free H2O, denatured for 10 min at 65 °C, and then chilled on ice. The radiolabeled RNAs were hybridized to cDNAs pre-immobilized on membranes in hybridization buffer (50% formamide, 5× SSC, 5 mm EDTA, 5× Denhardt's solution, 0.1% SDS, and 100 μg/ml denatured salmon sperm DNA) for 48 h at 42 °C. Next, the membranes were washed several times in 2× SSC (1× SSC = 0.15 m NaCl and 12.5 mm sodium citrate, pH 7) and then in 2× SSC plus 10 μg/ml RNase A for another 30 min or not depending on intensity of background. Signals were acquired and quantified by a PhosphorImager. To immobilize cDNAs to nitrocellulose membranes, 1 μg of linearized plasmid was denatured in 0.2 m NaOH for 30 min at room temperature and then neutralized with 10 volumes of 6× SSC. The DNAs were applied onto nitrocellulose membranes using a slot blot apparatus and immobilized by UV cross-linking. RNA Interference—To perform Smad4 silencing, 50% confluent cells were transfected with 50–200 pm of a pool of four Smad4 or scrambled siRNAs (Dharmacon, Lafayette, CO) using Oligofectamine (Invitrogen) according to manufacturer's guideline. Conditioned media (48–72 h post transfection) and protein lysates (72 h post transfection) were harvested and subjected to immunoblotting for Smad4 and uPA. To determine uPA mRNA stability under a Smad4-silencing condition, cells were first transfected with 100 nm Smad4 siRNA for 6 h and grew in serum media overnight, and then uPA mRNA stability was determined following treatment of TGF-β overnight. Statistical Analysis—p values for multiple comparison tests were derived by an analysis of variance (ANOVA) with a Bonferroni correction. Autocrine TGF-β Signaling Contributes to MDA-MB-231 Cell Invasion—The metastatic MDA-MB-231 breast cancer cells secrete active TGF-β (22Martinez-Carpio P.A. Mur C. Rosel P. Navarro M.A. Cell. Signal. 1999; 11: 753-757Crossref PubMed Scopus (20) Google Scholar, 23Martinez-Carpio P.A. Mur C. Fernandez-Montoli M.E. Ramon J.M. Rosel P. Navarro M.A. Cancer Lett. 1999; 147: 25-29Crossref PubMed Scopus (13) Google Scholar) and are TGF-β-responsive (24Chen C.-R. Kang Y. Massague J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 992-999Crossref PubMed Scopus (274) Google Scholar). The importance of autocrine TGF-β signaling in regulation of MDA-MB-231 cell invasiveness was assessed by Matrigel invasion assays following abrogation of autocrine TGF-β signaling using the LY364947 compound (27Peng S.B. Yan L. Xia X. Watkins S.A. Brooks H.B. Beight D. Herron D.K. Jones M.L. Lampe J.W. McMillen W.T. Mort N. Sawyer J.S. Yingling J.M. Biochemistry. 2005; 44: 2293-2304Crossref PubMed Scopus (86) Google Scholar, 28Li H.Y. Wang Y. Heap C.R. King C.H. Mundla S.R. Voss M. Clawson D.K. Yan L. Campbell R.M. Anderson B.D. Wagner J.R. Britt K. Lu K.X. McMillen W.T. Yingling J.M. J. Med. Chem. 2006; 49: 2138-2142Crossref PubMed Scopus (43) Google Scholar), a kinase inhibitor of TGF-β type I receptor (TβRI) by functioning as a potent ATP competitive inhibitor. The inhibitory effect of LY364947 on TGF-β signaling was first validated by examining Smad2 phosphorylation. Basal levels of Smad2 phosphorylation were undetectable by immunoblotting. However, LY364947 abolished Smad2 phosphorylation induced by exogenous TGF-β without altering total Smad2 protein levels (Fig. 1A). Expression of fibronectin is induced by TGF-β through a Smad-independent pathway (31Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar). LY364947 decreased both basal and exogenous TGF-β-induced fibronectin expression (Fig. 1B). In addition, we evaluated the effect of LY364947 on TGF-β-induced promoter activation by reporter assays using p3TP-Lux, a luciferase reporter construct highly responsive to TGF-β (30Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1365) Google Scholar) and observed that LY364947 significantly inhibited both basal and exogenous TGF-β-induced promoter activation (Fig. 1C). To investigate whether autocrine TGF-β has a role in regulation of MDA-MB-231 invasiveness, Matrigel invasion assays were performed with or without LY364947 treatment. LY364947 inhibited cell invasion in a dose-dependent manner (Fig. 1D). These data suggest that autocrine TGF-β plays a role in basal invasive growth of MDA-MB-231 cells. A Dominant-negative TGF-β Type II Receptor Suppresses Autocrine TGF-β Signaling and Cell Invasion—To substantiate the results obtained using the TGF-β receptor kinase inhibitor, we assessed invasiveness of MDA-MB231 cells following suppression of autocrine TGF-β signaling with a dominant-negative TGF-β type II receptor (TβRII). TβRII is the receptor responsible for ligand binding and for activation of TGF-β type I receptor through its kinase activity. TβRII devoid of the kinase domain (DNIIR) acts as a dominant-negative mutant by competing with wild-type receptors for TGF-β ligands (32Chen R.H. Ebner R. Derynck R. Science. 1993; 260: 1335-1338Crossref PubMed Scopus (356) Google Scholar). Expression of FLAG-tagged dominant-negative DNIIR was achieved using an adenoviral vector and was confirmed by immunoblotting for FLAG (Fig. 2A). DNIIR expression was increased with increasing amounts of adenovirus, whereas no DNIIR was detected in parental or the β-galactosidase adenovirus-infected cells. Expression of DNIIR inhibited TGF-β-stimulated Smad2 activation (Fig. 2B) and decreased both basal and exogenous TGF-β-induced fibronectin expression (Fig. 2C). DNIIR also suppressed basal and exogenous TGF-β-stimulated p3TP-Lux promoter activation (Fig. 2D), demonstrating the inhibitory effects of DNIIR on TGF-β signaling. As expected, DNIIR expression significantly decreased MDA-MB-231 cell invasion (Fig. 2E). This effect is not a result of inhibition of cell proliferation as determined by MTT assay (data not shown). Thus, consistent with the results using the pharmacological inhibitor, inhibition of autocrine TGF-β signaling by the dominant-negative TGF-β type II receptor (DNIIR) down-regulated MDA-MB-231 invasiveness. Disruption of Autocrine TGF-β Signaling Suppresses uPA Secretion—The MDA-MB-231 cells secrete uPA (20Farina A.R. Coppa A. Tiberio A. Tacconelli A. Turco A. Colletta G. Gulino A. Mackay A.R. Int. J. Cancer. 1998; 75: 721-730Crossref PubMed Scopus (115) Google Scholar). To address whether autocrine TGF-β can modulate uPA expression, uPA levels were determined after blockade of autocrine TGF-β signaling using LY364947. Immunoblotting for uPA was conducted under non-reducing conditions, which detect both uPA and complexes of uPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1) (33Wun T. Reich E. J. Biol. Chem. 1987; 262: 3646-3653Abstract Full Text
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