Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential
Masayuki AzumaTetsuya TamataniKazuhiro FukuiTokuyuki YukiHideo YoshidaTakashi BandoMohammad Obaidul HoqueTakashi KamogashiraKōichi OginoNaoki NishinoToshimitsu SuzukiMitsunobu Sato
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Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.
Platelet-derived growth factor
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Autocrine growth due to dysregulated growth factor production may have a role in the development of neoplasia. Whether autocrine growth is stimulated by growth factor secretion in an autocrine loop or by intracellular binding of the growth factor to a receptor has been unclear. The carboxyl-terminus coding sequence for murine interleukin-3 (IL-3) was extended with an oligonucleotide encoding a four-amino acid endoplasmic reticulum retention signal. IL-3-dependent hematopoietic cells became growth factor-independent when the modified IL-3 gene was introduced by retroviral gene transfer, despite lack of secretion of the modified IL-3. Hence autocrine growth can occur as a result of the intracellular action of a growth factor and this mechanism may be important in neoplastic and normal cells.
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The reduced growth factor requirements of murine fibroblasts transformed by simian virus 40 (SV 40) have been attributed to insulin-like growth factor (IGF)-I induction by T antigen and consequent activation of IGF-I receptor signaling. The present study shows that the autonomous growth of SV 40-transformed human fibroblasts also requires type-I IGF-I receptor activation but that this is not due to de novo induction of IGF-I gene expression since untransformed human fibroblasts, which fail to proliferate in the absence of serum, also showed IGF-I gene expression under serum-free conditions. DNA synthesis assays confirmed that untransformed cells were responsive to exogenous IGF and indicated that transformed cells were already maximally stimulated. In untransformed fibroblasts, IGF binding was principally to abundant membrane-associated IGFBP-5, whereas in transformed fibroblasts this protein was minimally expressed, and IGF binding was to IGF receptors. Loss of detectable membrane-associated IGFBP-5 in transformed cells was associated with diminished IGFBP-5 gene expression and with loss of IGF-II gene expression. Exogenous IGFBP-5 associated with the membranes of transformed cells and inhibited the autocrine growth of these cells. These findings suggest that loss of IGFBP-5 in SV 40-transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor and increases their sensitivity to autocrine stimulation. The reduced growth factor requirements of murine fibroblasts transformed by simian virus 40 (SV 40) have been attributed to insulin-like growth factor (IGF)-I induction by T antigen and consequent activation of IGF-I receptor signaling. The present study shows that the autonomous growth of SV 40-transformed human fibroblasts also requires type-I IGF-I receptor activation but that this is not due to de novo induction of IGF-I gene expression since untransformed human fibroblasts, which fail to proliferate in the absence of serum, also showed IGF-I gene expression under serum-free conditions. DNA synthesis assays confirmed that untransformed cells were responsive to exogenous IGF and indicated that transformed cells were already maximally stimulated. In untransformed fibroblasts, IGF binding was principally to abundant membrane-associated IGFBP-5, whereas in transformed fibroblasts this protein was minimally expressed, and IGF binding was to IGF receptors. Loss of detectable membrane-associated IGFBP-5 in transformed cells was associated with diminished IGFBP-5 gene expression and with loss of IGF-II gene expression. Exogenous IGFBP-5 associated with the membranes of transformed cells and inhibited the autocrine growth of these cells. These findings suggest that loss of IGFBP-5 in SV 40-transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor and increases their sensitivity to autocrine stimulation.
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Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell. Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell. Vascular smooth muscle cell (SMC)1 migration and proliferation are necessary events that contribute to the formation of neointimal hyperplasia in atherosclerosis, restenosis, and venous bypass graft disease (1Luscher T.F. Turina M. Braunwald E. Coronary Artery Graft Disease: Mechanisms and Prevention. Springer, Heidelberg, Germany1994: 42-52Crossref Google Scholar, 2Ross R. N. Engl. J. Med. 1986; 314: 488-500Crossref PubMed Scopus (4043) Google Scholar). SMC mitogenesis is triggered by a large number of growth factors, cytokines, and extracellular matrix proteins that participate in the pathogenic process (3Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10041) Google Scholar). The induction of various mitogenic factor expressions in mechanically injured arteries (4Majesky M.W. Lindner V. Twardzik D.R. Schwartz S.M. Reidy M.A. J. Clin. Invest. 1991; 8: 904-910Crossref Scopus (475) Google Scholar, 5Inoue S. Orimo A. Hosoi T. Matsuse T. Hashimoto M. Yamada R. Ouchi Y. Orimo H. Muramatsu M. Arterioscler. Thromb. 1993; 13: 1859-1864Crossref PubMed Scopus (23) Google Scholar, 6Li Z. Moore S. Alavi M.Z. Exp. Mol. Pathol. 1995; 63: 77-86Crossref PubMed Scopus (13) Google Scholar, 7Igura T. Kawata S. Miyagawa J. Inui Y. Tamura S. Fukuda K. Isozaki K. Yamamori K. Taniguchi N. Higashiyama S. Matsuzawa Y. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1524-1531Crossref PubMed Scopus (59) Google Scholar) suggests that hemostatic and thrombotic events occurring in the early phase of vascular response to injury might be involved. After arterial injury, tissue factor is expressed in SMCs (8Marmur J.D. Rossikhina M. Guha A. Fyfe B. Friedrich V. Mendlowitz M. Nemerson Y. Taubman M.B. J. Clin. Invest. 1993; 91: 2253-2259Crossref PubMed Scopus (224) Google Scholar) and forms a high affinity complex with factor VII/VIIa, thereby initiating an extrinsic blood coagulation pathway leading to the formation of factor Xa and thrombin (9Carmeliet P. Collen D. Int. J. Biochem. Cell Biol. 1998; 30: 661-667Crossref PubMed Scopus (59) Google Scholar). In addition to its procoagulant effects, thrombin itself can act as a mitogen for fibroblasts (10Kahan C. Seuwen K. Meloche S. Pouyssegur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar), lymphocytes (11Naldini A. Carney D.H. Bocci V. Klimpel K.D. Asuncion M. Soares L.E. Klimpel G.R. Cell. Immunol. 1993; 147: 367-377Crossref PubMed Scopus (84) Google Scholar), mesenchymal cells (12Chohisy D.R. Erdmann J.M. Wilner G.D. J. Biol. Chem. 1990; 265: 7729-7732Abstract Full Text PDF PubMed Google Scholar) and SMCs (13Bar-Shavit R. Benezra M. Eldor A. Hy-Am E. Fenton Jr., J.W. Wilner G.D. Vlodavsky I. Cell Regul. 1990; 1: 453-463Crossref PubMed Scopus (133) Google Scholar, 14Herbert J. Lamarche I. Dol F. FEBS Lett. 1992; 301: 155-158Crossref PubMed Scopus (67) Google Scholar, 15McNamara C.A. Sarembock I.J. Gimple L.W. Fenton Jr., J.W. Coughlin S.R. Owens G.K. J. Clin. Invest. 1993; 91: 94-98Crossref PubMed Scopus (466) Google Scholar). Thrombin also regulates cellular responses during inflammation (16Bar-Shavit R. Benezra M. Sabbah V. Bode W. Vlodavsky I. Am. J. Respir. Cell Mol. Biol. 1992; 6: 123-130Crossref PubMed Scopus (80) Google Scholar) and inhibits the migration and invasion of breast cancer cells (17Kamath L. Meydani A. Foss F. Kuliopulos A. Cancer Res. 2001; 61: 5933-5940PubMed Google Scholar). After stimulation by thrombin, SMCs produce and secrete autocrine growth factors, including basic fibroblast growth factor (18Weiss R.H. Maduri M. J. Biol. Chem. 1993; 268: 5724-5727Abstract Full Text PDF PubMed Google Scholar), PDGF (19Stouffer G.A. Sarembock I.J. McNamara C.A. Gimple L.W. Owens G.K. Am. J. Physiol. 1993; 265: C806-C811Crossref PubMed Google Scholar), heparin-binding epidermal growth factor (EGF) (20Nakano T. Raines E.W. Abraham J.A. Wenzel F.G. 4th Higashiyama S. Klagsbrun M. Ross R. J. Biol. Chem. 1993; 268: 22941-22947Abstract Full Text PDF PubMed Google Scholar), and transforming growth factor-β (21Bachhuber B.G. Sarembock I.J. Gimple L.W. Owens G.K. J. Vasc. Res. 1997; 34: 41-48Crossref PubMed Scopus (37) Google Scholar). Factor Xa acts as the major mediator of thrombus formation at the sites of vascular injury by activating prothrombin to thrombin and as a potent mitogen for endothelial cells (22Bono F. Herault J.P. Avril C. Schaeffer P. Lormeau J.C. Herbert J.M. J. Cell. Physiol. 1997; 172: 36-43Crossref PubMed Scopus (35) Google Scholar), fibroblasts (23Blanc-Brude O.P. Chambers R.C. Leoni P. Dik W.A. Laurent G.J. Am. J. Physiol. Cell Physiol. 2001; 281: C681-C689Crossref PubMed Google Scholar), and aortic SMCs (24Gasic G.P. Arenas C.P. Gasic T.B. Gasic G.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2317-2320Crossref PubMed Scopus (185) Google Scholar, 25Ko F.N. Yang Y.C. Huang S.C. Ou J.T. J. Clin. Invest. 1996; 98: 1493-1501Crossref PubMed Scopus (75) Google Scholar, 26Herbert J. Bono F. Herault J. Avril C. Dol F. Mares A. Schaeffer P. J. Clin. Invest. 1998; 101: 993-1000Crossref PubMed Scopus (70) Google Scholar). Factor Xa also induces the production of cytokines and adhesion molecules such as interleukin-6, interleukin-8, monocyte chemotactic protein-1, E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1 in human umbilical vein endothelial cells, leading to pro-inflammatory responses (27Senden N.H. Jeunhomme T.M. Heemskerk J.W. Wagenvoord R. van't Veer C. Hemker H.C. Buurman W.A. J. Immunol. 1998; 161: 4318-4324PubMed Google Scholar, 28Papapetropoulos A. Piccardoni P. Cirino G. Bucci M. Sorrentino R. Cicala C. Johnson K. Zachariou V. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4738-4742Crossref PubMed Scopus (61) Google Scholar). Although some aspects of the effect of factor Xa on aortic SMCs through PDGF have been investigated (25Ko F.N. Yang Y.C. Huang S.C. Ou J.T. J. Clin. Invest. 1996; 98: 1493-1501Crossref PubMed Scopus (75) Google Scholar, 26Herbert J. Bono F. Herault J. Avril C. Dol F. Mares A. Schaeffer P. J. Clin. Invest. 1998; 101: 993-1000Crossref PubMed Scopus (70) Google Scholar), the PDGF isoform that mediates factor Xa-induced SMC mitogenesis has not been identified yet. Moreover, no information is available about any particular autocrine production that is associated with factor Xa-induced SMC mitogenesis. In this work we have demonstrated that factor Xa-induced mitogenesis of rat aortic smooth muscle cells (RASMCs) is independent of PDGF. More importantly, we have identified epiregulin as the major element responsible for factor Xa-induced RASMC mitogenesis. It was also revealed that autocrine production of epiregulin is essential in the mitogenesis. Epiregulin is the newest member of the family of EGF-like ligands, and it was first identified from the cultured medium of mouse fibroblast-derived tumor cell line NIH3T3/clone T7 (29Toyoda H. Komurasaki T. Uchida D. Takayama Y. Isobe T. Okuyama T. Hanada K. J. Biol. Chem. 1995; 270: 7495-7500Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). It was subsequently shown that epiregulin acts as a mitogen for various cell types, including fibroblasts, aortic SMCs, hepatocytes (29Toyoda H. Komurasaki T. Uchida D. Takayama Y. Isobe T. Okuyama T. Hanada K. J. Biol. Chem. 1995; 270: 7495-7500Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), keratinocytes (30Shirakata Y. Komurasaki T. Toyoda H. Hanakawa Y. Yamasaki K. Tokumaru S. Sayama K. Hashimoto K. J. Biol. Chem. 2000; 275: 5748-5753Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), and pancreatic cancer cells (31Zhu Z. Kleeff J. Friess H. Wang L. Zimmermann A. Yarden Y. Buchler M.W. Korc M. Biochem. Biophys. Res. Commun. 2000; 273: 1019-1024Crossref PubMed Scopus (80) Google Scholar). Epiregulin was also purified from the cultured medium of angiotensin II-stimulated RASMCs and was identified to be a major mitogenic constituent (32Taylor D.S. Cheng X. Pawlowski J.E. Wallace A.R. Ferrer P. Molloy C.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1633-1638Crossref PubMed Scopus (69) Google Scholar). Materials—Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen, and [3H]thymidine was from PerkinElmer Life Sciences. Bovine factor Xa, bovine thrombin, and pGEX-2T vector were purchased from Novagen, Inc., and Pefabloc®TH (Pefa-3204) was from Pentapharm Ltd. (Switzerland). Recombinant tick anticoagulant protein (rTAP) was kindly provided by Dr. Y. Jang (Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Korea). NuPAGE™ 4–12% Bis-Tris gel was purchased from Invitrogen, and DC protein assay reagent from Bio-Rad. An antibody to phospho-p44/42 mitogen-activated protein kinase (ERK-1/2) and an antibody that recognizes both phosphorylated and nonphosphorylated forms of p44/42 mitogen-activated protein kinase were obtained from Cell Signaling Technology. ECL reagent, benzamidine-Sepharose, and heparin-Sepharose were purchased from Amersham Biosciences. Anti-PDGF-A (E-10) and -B (H-55) antibodies came from Santa Cruz Biotechnology Inc. Tyrphostin AG 1296 was purchased from Sigma-Aldrich, whereas neutralizing anti-PDGF-AB antibodies PDGF-AA and PDGF-BB were purchased from Upstate Biotechnology. Quantikine human PDGF-AB and human PDGF-BB immunoassay kits and anti-mouse epiregulin antibody came from R&D Systems. Ultraspect-II RNA system was purchased from Biotecx Laboratories, Inc., whereas avian myeloblastosis virus reverse transcription system and pGEM-T Easy vector were obtained from Promega. PCR primers and phosphorothiolated oligodeoxynucleotides (ODNs) were synthesized from Genotech, Inc. (Korea). All other reagents were of the highest commercial purity. Cell Culture—RASMCs were isolated by a modification of the method of Chamley-Campbell et al. (33Chamley-Campbell J.H. Campbell G.R. Ross R. Physiol. Rev. 1979; 59: 1-61Crossref PubMed Scopus (1270) Google Scholar). The thoracic aortas from 6–8-week-old Sprague-Dawley rats were removed and transferred onto ice in serum-free DMEM containing 1% penicillin/streptomycin. The aorta was incubated in 5 ml of the enzyme mixture containing DMEM with 1 mg/ml collagenase type 1 and 0.5 mg/ml elastase for 30 min at 37 °C. Then the aorta was transferred into DMEM with 2% FBS, and the adventitia were stripped off under a binocular microscope. The aorta was then transferred into a plastic tube containing 5 ml of the enzyme dissociation mixture and incubated for 1 h at 37 °C. The suspension was centrifuged at 3000 rpm for 10 min, and the pellet was resuspended in DMEM with 10% FBS. RASMCs were cultured in DMEM supplemented with 10% FBS, 100 international units/ml penicillin, 100 μg/ml streptomycin in 5% CO2. RASMCs were used with passages 4–9. [3H]Thymidine Incorporation—To evaluate DNA synthesis in cells, the incorporation of [3H]thymidine into DNA was determined. RASMCs (2.5 × 104) were seeded into a 24-well plate and incubated for 24 h in DMEM containing 10% FBS. After the cells were further incubated in DMEM containing 0.2% FBS for 48 h, they were treated with inhibitors or antibodies before stimulation. After an additional 24-h incubation, the cells were then pulsed with 2 μCi of [3H]thymidine for 16 h at 37 °C and washed twice with phosphate-buffered saline. The cells were fixed in methanol at 4 °C for 5 min and washed twice with 5% trichloroacetic acid. The acid-insoluble material was dissolved in 0.3 n NaOH at room temperature followed by radioactivity measurement with liquid scintillation counter. Cell Proliferation Assay—RASMCs (1 × 104) were plated onto 24-well culture plates and incubated at 37 °C for 24 h. After the cells were rendered quiescent in DMEM containing 0.2% FBS for 48 h, they were treated for 30 min with inhibitors or antibodies. The cells were stimulated with factor Xa for 48 h. The medium was removed, and the cell number/well was counted using a hemocytometer. Immunoblotting—For the ERK-1/2 activation experiment, RASMCs were allowed to grow to confluence in 6-well plates and made quiescent in DMEM containing 0.2% FBS for 48 h. After stimulation with agonists for 5 min in the presence or absence of antagonists, the cells were rinsed in ice-cold phosphate-buffered saline and treated with lysis buffer (1% Triton X-100, 0.1% β-mercaptoethanol, 1 mm EDTA, 50 mm Tris-HCl (pH 7.0), 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride) on ice. Cell lysates were collected into microcentrifuge tubes, sonicated, and centrifuged at 13,000 rpm for 20 min. Protein concentration was measured in the supernatants using DC protein assay reagent according to the manufacturer's instructions and equalized for all samples. For PDGF and epiregulin immunoblotting, RASMCs were allowed to grow to confluence and made quiescent for 48 h. After stimulation with factor Xa, the cultured supernatants (5 ml) were concentrated using a Vivaspin concentrator with a 3-kDa cut-off molecular size (Vivascience). Reduced samples were resolved on NuPAGE™ 4–12% Bis-Tris gel and electrotransferred to nitrocellulose membranes. The membranes were incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibody. ECL detection method was employed for color development. Enzyme-linked Immunosorbent Assay—RASMCs were allowed to grow to confluence in 24-well plates and made quiescent for 48 h. After washing and replacing with fresh medium, the cells were incubated with 100 nm factor Xa. After incubation, the cultured medium was collected and centrifuged at 13,000 rpm for 20 min to remove debris. The concentrations of PDGF-AB and PDGF-BB in the medium were determined using Quantikine® immunoassay kits. Purification of Epiregulin from the Cultured Medium of Factor Xa-stimulated RASMCs—Epiregulin was purified from the RASMC-cultured medium that was treated with 50 nm factor Xa in serum-free DMEM for 6 h. The cultured medium (2 liters) was concentrated and subjected to diafiltration against 20 mm Tris-HCl (pH 7.5) (buffer A) with Amicon high output stirred cells using an ultrafiltration disc with a 3-kDa cut-off molecular size (Millipore). The medium was applied to a benzamidine-Sepharose column (1.5 × 5 cm) equilibrated with buffer A, and then proteins were eluted with the same buffer. Mitogenic activity in each fraction was assayed by determining [3H]thymidine incorporation into DNA in RASMCs. Active fractions were pooled and loaded onto a heparin-Sepharose column (1.5 × 4 cm) equilibrated with buffer A containing 0.15 m NaCl. The adsorbed proteins were eluted with a linear gradient of 0.15 ∼ 2 m NaCl in buffer A. Active fractions were pooled, concentrated, and separated in a Superose 12 HR 10/30 gel filtration column (Amersham Biosciences) equilibrated with buffer A containing 0.15 m NaCl at a flow rate of 0.4 ml/min. Fractions containing mitogenic activity were concentrated and applied to SYMMETRY C8 HPLC column (Waters, 3.9 × 150 mm) at a flow rate of 0.5 ml/min. Bound proteins were eluted from the column with a linear gradient of 5–50% acetonitrile containing 0.1% trifluoroacetic acid. SDS-PAGE was subsequently performed on NuPAGE™ 4–12% Bis-Tris gel for each active fraction. The gel was visualized by silver staining. The highly purified protein was used for N-terminal amino acid sequencing after alkylation with iodoacetamide. Reverse Transcription-PCR Analysis—The expression level of epiregulin mRNA was analyzed using the reverse transcription-PCR method. For the RNA preparation, RASMCs were incubated for 48 h in DMEM containing 0.2% FBS and then treated with factor Xa for the times indicated. Total RNA was prepared by Ultraspect-II RNA system, and single-stranded cDNA was then synthesized from the isolated total RNA using avian myeloblastosis virus reverse transcriptase. A 20-μl reverse transcription reaction mixture containing 1 μg of total RNA, 1× reverse transcription buffer (10 mm Tris-HCl (pH 9.0), 50 mm KCl, 0.1% Triton X-100), 1 mm deoxynucleoside triphosphates, 0.5 unit of RNase inhibitor, 0.5 μg of oligo(dT)15, and 15 units of avian myeloblastosis virus reverse transcriptase was incubated at 42 °C for 15 min. After denaturation at 99 °C for 5 min, the cDNA was subjected to PCRs. PCRs were performed for 25 cycles at 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 1 min with each 3′ and 5′ primers based on the sequence of the rat epiregulin gene (5′-GTGTTGATTACAAAGTGTAGCTCTG-3′ and 5′-AGAAAGAAGTGTTCACACCGCAGACC-3′). The glyceraldehyde-3-phosphate dehydrogenase was used as the internal standard (primer sequences, 5′-GACAACTTTGGCATCGTGGA-3′ and 5′-ACAACCTGGTCCTCAGTGTA-3′). The amplified PCR product was subcloned into pGEM-T Easy Vector, and its nucleotide sequence was analyzed using the ALF automatic sequencing system (Amersham Biosciences). Expression Construct for Rat Epiregulin—The rat epiregulin cDNA was modified by PCR for pGEX-2T vector. The N-terminal primer (5′-GGATCCGTGTTGATTACAAAGTGTAGCTCTG-3′) contains a BamHI site. The C-terminal primer (5′-GAATTCTCACAGAAAGAAGTGTTCACACCGCAGACC-3′) contains a stop codon and an EcoRI restriction site. The thermal cycling reaction was performed with 30 cycles of steps (denaturation at 94 °C for 30 s, annealing at 61 °C for 30 s, and extension at 72 °C for 1 min). The modified PCR product was subcloned into pGEM-T Easy Vector, and its nucleotide sequence were analyzed. For the expression construct for rat epiregulin, the pGEM-T-rat epiregulin vector was digested with BamHI and EcoRI, and cloned into the expression pGEX-2T vector, which forms a fusion of glutathione S-transferase to the mature form of rat epiregulin. Expression and Purification of Recombinant Rat Epiregulin—The expression vector was transformed into Escherichia coli strain BL21. The transformants were grown in LB medium containing ampicillin to an absorbance of 0.5 at A600 nm, induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside, and incubated for an additional 4 h at 37 °C. The expressed inclusion bodies of glutathione S-transferase-rat epiregulin were refolded. Briefly, the inclusion bodies were solubilized in 8 m urea solution (8 m urea, 50 mm Tris-HCl, pH8.5, 50 mm dithiothreitol). The unfolded fusion protein was refolded with rapid dilution against a refolding buffer (20 mm Tris-HCl, pH8.5, 10 mm cysteine, 1 mm cystine) at 4 °C for 48 h. The refolded fusion protein was bound to glutathione-Sepharose and then incubated with thrombin for 12 h at room temperature while rocking. The eluted recombinant epiregulin was concentrated and applied to a SYMMETRY C8 column (Waters, 3.9 × 150 mm) at a flow rate of 0.5 ml/min. Bound proteins were eluted from the column with a linear gradient of 5–50% acetonitrile containing 0.1% trifluoroacetic acid. Antisense Oligodeoxynucleotide to Epiregulin—Sense (S-) (5′ to 3′) and antisense (AS-) (3′ to 5′) ODNs with phosphorothiolate linkages were designed to symmetrically cover the translation initiation sites of rat epiregulin. For rat epiregulin, the AS-ODN sequence was 5′-AAGTCTCCATCCTTCTC-3′, and S-ODN was 5′-GAGAAGGATGGAGACTT-3′. The specificity of the ODN for rat epiregulin was tested by Western blot analysis. Statistical Analysis—Data represented the mean ± S.E. of n experiments. Statistical analysis was performed using an unpaired t test. A p value less than 0.05 was considered to be statistically significant. Mitogenesis of Factor Xa-stimulated RASMCs—Factor Xa-induced mitogenesis of RASMCs was determined by measuring [3H]thymidine incorporation and the cell number in RASMCs stimulated by the protease. As expected, factor Xa markedly increased [3H]thymidine incorporation into the cell and increased the cell number compared with unstimulated RASMCs. A similar mitogenic effect was also observed with thrombin-stimulated RASMCs (Fig. 1). To determine whether the mitogenic effect of factor Xa on RASMCs is dependent on its proteolytic activity, a competitive factor Xa inhibitor, rTAP, was employed. As shown in Fig. 1, rTAP significantly reduced the mitogenesis of RASMCs that was induced by factor Xa, indicating that the induction of RASMC mitogenesis is closely associated with the proteolytic enzyme activity of factor Xa. rTAP on its own did not influence the [3H]thymidine incorporation and proliferation of unstimulated cells (data not shown). To rule out the possibility that the mitogenic function of factor Xa may be caused by the local production of thrombin, the factor Xa-induced mitogenesis was examined in the presence of Pefabloc®TH, a specific thrombin inhibitor. Experimental results clearly indicate that Pefabloc®TH did not affect factor Xa-induced mitogenesis, whereas the thrombin-stimulated mitogenic effect was markedly suppressed by the inhibitor (Fig. 1). Pefabloc®TH did not influence the [3H]thymidine incorporation and proliferation of unstimulated RASMCs (data not shown). PDGF Independence of Factor Xa-induced [3H]Thymidine Incorporation and ERK-1/2 Activation—To determine whether the factor Xa-induced mitogenesis is associated with the secretion of PDGF, RASMCs were stimulated with factor Xa in the presence of anti-PDGF-AB polyclonal antibody, which neutralizes all three isoforms of PDGF, i.e. PDGF-AA, -AB, and -BB. As demonstrated in Fig. 2, the antibody failed to affect the increase in [3H]thymidine incorporation and ERK-1/2 phosphorylation in factor Xa-stimulated cells. To further investigate the requirement of autocrine PDGF for this particular mitogenic pathway, RASMCs were stimulated by factor Xa in the presence of Tyrphostin AG 1296, a selective tyrosine kinase inhibitor of PDGF receptors (34Kovalenko M. Gazit A. Bohmer A. Rorsman C. Ronnstrand L. Heldin C.H. Waltenberger J. Bohmer F.D. Levitzki A. Cancer Res. 1994; 54: 6106-6114PubMed Google Scholar). Experimental results indicate that neither the factor Xa-induced [3H]thymidine incorporation nor ERK-1/2 activation was influenced by the reagent (Fig. 2). In control experiments, it was demonstrated that the neutralizing antibody or tyrphostin AG 1296 completely suppressed the mitogenic effects induced by PDGF-BB (Fig. 2). The influence of the antibody or the inhibitor itself in the unstimulated cells was negligible on such mitogenic effects (data not shown). Analysis of Autocrine PDGF in the Cultured Medium—To assess whether factor Xa stimulates the secretion of PDGF from RASMCs, Western blot analysis was carried out. For this investigation, the cultured supernatant from untreated or factor Xa-treated RASMCs was concentrated and analyzed by Western blot using anti-PDGF-A or -B antibody. The obtained results indicate that the immunoreactive band recognized by the PDGF-A or -B antibody was not detected in the supernatants (data not shown). To further confirm the presence of PDGF-AB and -BB in the cultured medium, the concentrations of these growth factors were measured using Quantikine® immunoassay kits. The stimulation of RASMCs with factor Xa for up to 48 h did not result in any detectable secretion of PDGF-AB and -BB in the cultured medium (data not shown). The detection limits of all enzyme-linked immunosorbent assay systems used in the experiments were below 15 pg/ml. Autocrine-dependent DNA Synthesis and Cell Proliferation—To determine whether the factor Xa-induced mitogenesis is autocrine-dependent, the cultured supernatant was harvested from the factor Xa-stimulated RASMCs at various time intervals up to 6 h and then added to the serum-starved RASMCs after rTAP treatment to prevent the direct effect of factor Xa. The cells were then assayed for [3H]thymidine incorporation and cell proliferation. Experimental results demonstrate that the factor Xa-treated conditioned medium was successful in significantly increasing DNA synthesis and cell number in RASMCs (Fig. 3), whereas the medium of unstimulated cells failed to exert such mitogenic effects (data not shown). Anti-PDGF-AB antibody or tyrphostin AG 1296 did not affect the [3H]thymidine incorporation induced by the cultured medium containing rTAP, indicating that there is no requirement for autocrine PDGF in the particular mitogenesis induced by factor Xa (Fig. 3A). The mitogenic activity appeared 1 h after factor Xa stimulation and reached its plateau in 4 h (Fig. 3). Purification and Identification of the Autocrine Factor—Autocrine factor, which is responsible for the factor Xa-stimulated mitogenesis of RASMCs, was purified to homogeneity from two liters of the conditioned medium in four steps. After diafiltration against buffer A, the cultured medium was fractionated by affinity chromatography on a column of benzamidine-Sepharose. Mitogenic activity was recovered in the washing step of the column with buffer A, whereas factor Xa was retained in the column. When the pooled active fractions were applied to a heparin-Sepharose column, the mitogenic activity was recovered by washing the column with buffer A containing 0.15 m NaCl. The active fractions were then concentrated and fractionated in a gel filtration column (Fig. 4A). Finally, the mitogenic activity was further purified to homogeneity in a C8 reversed phase HPLC column (Fig. 4B). The protein eluted in a gradient of 28 ∼ 29% acetonitrile migrated as a single band with an apparent molecular mass of 5 kDa under the reducing condition of SDS-PAGE (Fig. 4C). When the N-terminal amino acid sequence of the purified 5-kDa protein was determined through Edman degradation, it was revealed to be VLITKC-SSDM, which is identical to that of epiregulin. Expression of Epiregulin mRNA in Factor Xa-stimulated RASMCs—Reverse transcription-PCR was used to analyze the mRNA level of epiregulin in factor Xa-stimulated RASMCs. PCR primers were designed on the basis of the complete sequence of the rat epiregulin gene. After stimulation of the quiescent RASMCs with factor Xa, it was interesting to observe a rapid and transient induction of epiregulin mRNA. Expression of the mRNA reached its maximal level at 1 h and returned to the basal level in 6 h (Fig. 5). Cloning, Expression, and Purification of Recombinant Rat Epiregulin—The rat epiregulin cDNA was cloned and expressed in E. coli as a form of inclusion body. The expressed fusion protein containing glutathione S-transferase was refolded, adsorbed to the glutathione-Sepharose column, and then enzymatically cleaved at a specific site to remove the fusion partner. Recombinant epiregulin was further purified to homogeneity using C8 reversed phase HPLC column. The finally purified recombinant epiregulin migrated as a single band, which corresponded to native epiregulin, on SDS-PAGE (Fig. 4C). The molecular identity of the recombinant epiregulin with the native protein was confirmed by mass spectrometric analysis and by N-terminal amino acid sequencing. Then we examined the recombinant epiregulin for its functional ability to stimulate RASMC mitogenesis under serum-free conditions. As expected, the recombinant epiregulin was able to successfully stimulate the RASMC mitogenesis in a dose-dependent manner (Fig. 6). Epiregulin Dependence of Factor Xa-induced RASMC Mitogenesis—To investigate the role of epiregulin in factor Xa-stimulated mitogenesis, we examined the effect of anti-epiregulin antibo
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Abstract Human prostate cancer (PC) cell lines possess epidermal growth factor (EGF) receptors and secrete EGF‐related polypeptides. We used an EGF receptor‐blocking antibody (anti‐EGF.R) to demonstrate a functional autocrine loop, as well as the interaction between this and the effects of linoleic acid (LA), an omega‐6 fatty acid, on PC cell growth. The anti‐EGF.R competed effectively with [ 125 I]EGF for receptors on DU145 PC cells, and on a high‐passage DU145 variant (DU145M); when added to the culture medium, it suppressed both DU145 and DU145M cell growth in a dose‐dependent manner. LA, a precursor for eicosanoid synthesis, had little effect on DU145 cell growth rate but stimulated DU145M growth in a concentration‐related manner over a range of 0.25–2.0 μg/ml. Anti‐EGF.R (10 −9 M) caused suppression of LA‐stimulated growth of DU145M cells in serum‐free medium, which was prevented by the addition of 2 nM EGF. We conclude that an EGF.R‐mediated autocrine loop is involved in PC cell growth regulation and that at least one site of action may be the synthesis of eicosanoids from their LA precursor.
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