The Matrix Metalloproteinase-9 Regulates the Insulin-like Growth Factor-triggered Autocrine Response in DU-145 Carcinoma Cells
1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês
10.1074/jbc.274.11.6935
ISSN1083-351X
AutoresSantos Mañes, Mercedes Llorente, Rosa Ana Lacalle, Concepción Gómez‐Moutón, Leonor Kremer, E. Mira, Carlos Martínez‐A,
Tópico(s)Sexual Differentiation and Disorders
ResumoThe androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34cdc2 levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1–3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9. The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34cdc2 levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1–3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9. In normal cells, proliferation is a coordinated process involving intercellular communication through soluble regulatory molecules known as polypeptide growth factors (1Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (973) Google Scholar). In contrast, neoplastic cells are characterized by a relative autonomy of growth, a consequence of the constitutive expression of growth factors and receptors involved in autocrine loops (2Sporn M.B. Roberts A.B. Nature. 1985; 313: 745-747Crossref PubMed Scopus (1190) Google Scholar). Examples of constitutive autocrine growth factor loops have been reported for different cancer cells and growth factors, such as transforming growth factor α, insulin-like growth factors (IGF-I and IGF-II), 1The abbreviations used are: IGF, insulin-like growth factor; IGFBP, IGF-binding protein; IGF-1R, IGF type-1 receptor; 3T3-IGF-1R, 3T3 fibroblasts overexpressing the human IGF-1R AS antisense; CM, conditioned medium; DMEM, Dulbecco's modified Eagle's medium; mAb, monoclonal antibody; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PO, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; SFM, serum-free medium; TIMP, tissue inhibitor of metalloproteinase; TdR, thymidine; BSA, bovine serum albumin; RT-PCR, reverse transcriptase-polymerase chain reaction; FACS, fluorescence-activated cell sorter; h, human and platelet-derived growth factors, among others (3Macaulay V.M. Br. J. Cancer. 1992; 65: 311-320Crossref PubMed Scopus (506) Google Scholar). IGF-I and IGF-II are potent mitogens for several non-transformed and cancer cell types, and viral and nonviral oncogenes appear capable of interfering with the IGF autocrine loop (4Drummond I.A. Madden S.L. Rohwer-Nutter P. Bell G.I. Sukhatme V.P. Rauscher III, F.J. Science. 1992; 257: 674-678Crossref PubMed Scopus (512) Google Scholar). Indeed, c-MYB increases IGF-I secretion and IGF type-1 receptor (IGF-1R) production. It has also been suggested that the IGF-1R is critical in the establishment and maintenance of the transformed phenotype. Mouse embryo cells with a targeted disruption of the IGF-1R gene (5Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2622) Google Scholar, 6Baker J. Liu J.P. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 73-82Abstract Full Text PDF PubMed Scopus (2100) Google Scholar) cannot be transformed by SV40 large T antigen alone or in conjunction with Ha-ras (7Sell C. Dumenil G. 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In serum and in extracellular fluids, both IGF-I and -II are bound with high affinity to soluble IGF-binding proteins (IGFBP), seven of which have been identified to date (12Oh Y. Endocrine. 1997; 7: 111-113Crossref PubMed Google Scholar). Many tumor cell types secrete one or more of these proteins (13Cohick W.S. Clemmons D.R. Annu. Rev. Physiol. 1993; 55: 131-153Crossref PubMed Scopus (580) Google Scholar). The relevance of the IGFBP lies in their potential to modify the metabolic and mitogenic effects of IGF. In fact, IGFBP may either inhibit and/or enhance IGF activity (14Adashi E.Y. Resnick C.E. Ricciarelli E. Hurwitz A. Kokia E. Tedeschi C. Botero L. Hernández E.R. Rosenfeld R.G. Carlsson-Skwirut C. Francis G.L. J. Clin. Invest. 1992; 90: 1593-1599Crossref PubMed Scopus (52) Google Scholar, 15Jones J.I. Gockeman A. Busby W.H. Camacho-Hubner C. Clemmons D.R. J. Cell Biol. 1993; 121: 679-697Crossref PubMed Scopus (488) Google Scholar, 16Cohen P. Lamson G. Okajima T. Rosenfeld R.G. 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In this study, we characterize the role of IGF, IGF-1R, and IGFBP in tumor cell proliferation using DU-145 cells, a human androgen-independent prostate adenocarcinoma cell line (24Mickey D.D. Stone K.R. Wunderli H. Mickey G.G. Vollmer R.T. Paulson D.F. Cancer Res. 1977; 37: 4049-4058PubMed Google Scholar). Earlier studies indicated that this cell line expresses several components of the IGF axis (25Iwamura M. Sluss P.M. Casamento J.B. Cockett A.T. Prostate. 1993; 22: 243-252Crossref PubMed Scopus (203) Google Scholar, 26Figueroa J.A. Lee A.V. Jackson J.G. Yee D. J. Clin. Endocrinol. & Metab. 1995; 80: 3476-3482Crossref PubMed Scopus (75) Google Scholar). Our results suggest the existence of an IGF autocrine loop in DU-145 cells and its specific modulation by IGFBP-3 proteolysis. This proteolytic activity may be ascribed to the matrix metalloproteinase (MMP)-9, which is also produced by this cell line in an autocrine manner. Furthermore, we find MMP-9 both in soluble form and in a membrane-associated form on the DU-145 cell surface. The expression of an MMP-9 antisense cDNA leads to an 80% inhibition in DU-145 proliferation, which is reversed by the addition of exogenous IGF-I. This growth inhibition is associated with the abrogation of IGFBP-3 proteolysis as well as with a decrease in IGF-1R-promoted cell signals. MMP-9 therefore controls DU-145 cell proliferation by interacting, at least partially, with the IGF-I autocrine loop in this cell line. BALB/c 3T3 fibroblasts overexpressing the human IGF-1R (3T3-IGF-1R, a gift of Drs. A. Ullrich and R. Lammers) and DU-145 cells (ATCC HTB-81, American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal calf serum. Murine interleukin-3-dependent Ba/F3 cells (27Palacios R. Steinmetz M. Cell. 1985; 41: 727-734Abstract Full Text PDF PubMed Scopus (628) Google Scholar) were cultured in RPMI, 10% fetal calf serum, 1 mm sodium pyruvate, 2 mml-glutamine, and 10% conditioned medium from the interleukin-3-producing cell line WEHI-3B. The monoclonal antibodies (mAb) KM5A1 and BB9E10 were raised in our laboratory by immunization with the IGF-I-(55–70) synthetic peptide or recombinant human IGF-I, respectively (28Mañes S. Kremer L. Albar J.P. Mark C. Llopis R. Martı́nez-A C. Endocrinology. 1997; 138: 905-915Crossref PubMed Scopus (22) Google Scholar, 29Mañes S. Kremer L. Vangbo B. López A. Gómez-Moutón C. Peiró E. Albar J. Mendel-Hartvig I. Llopis R. Martı́nez-A C. J. Endocrinol. 1997; 154: 293-302Crossref PubMed Scopus (6) Google Scholar). KM5A1 is IGF-I-specific, with less than 0.1% cross-reactivity with IGF-II, and recognizes IGF-I when bound to IGFBP or the IGF-1R. BB9E10 has approximately 3% cross-reactivity with IGF-II and binds to an IGF-I epitope hidden by IGFBP and the IGF-1R. We also used an anti-IGF-II mAb raised in our laboratory, with an apparent affinity constant of 1011m−1 and less than 0.1% cross-reactivity with IGF-I, which is an IGF-II antagonist. 2S. Mañes, M. Llorente, R. A. Lacalle, C. Gómez-Moutón, L. Kremer, E. Mira, and C. Martı́nez-A., unpublished data. Total RNA, derived from either starved or control cultured cells, was isolated by ultracentrifugation through a cesium chloride cushion (30Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (18696) Google Scholar). This material (5 μg) was then reverse-transcribed using a first strand cDNA synthesis kit (Pharmacia AB, Stockholm, Sweden). One-tenth of the transcription product was amplified for either 40 PCR cycles with hIGF-I-specific primers or for 30 cycles with primers specific for hIGF-II or the IGF-1R. The primers for hIGF-I were 5′-GGTGGATGCTCTTCAGTTCGTGTGT-3′ and 5′-GCAATACATCTCCAGCCTCCTTAGA-3′. The primers for hIGF-II were 5′-CTTACCGCCCCAGTGAGACCCTGTG-3′ and 5′-CTCTCGGACTTGGCGGGGGAGCAC-3′. For amplification of the IGF-1R, the primers were used as described previously (31Telford N.A. Hogan A. Franz C.R. Schultz G.A. Mol. Reprod. Dev. 1990; 27: 81-92Crossref PubMed Scopus (71) Google Scholar). Finally, 10 μl of the PCR products were resolved on 2% agarose gels. DU-145-conditioned medium (DU145-CM) was recovered from subconfluent cells cultured for 5 days in serum-free DMEM (SFM) and concentrated 10-fold using Centricon filters with a 3,000 molecular weight cut-off (Amicon, Danvers, MA). Ligand blots were performed basically as described (32Hossenlopp P. Seurin D. Segovia-Quinson B. Hardouin S. Binoux M. Anal. Biochem. 1986; 154: 138-143Crossref PubMed Scopus (1225) Google Scholar). Briefly, 40 μl of concentrated DU145-CM were fractionated in 12.5% SDS-PAGE under nonreducing conditions and transferred to nitrocellulose (Schleicher & Schuell). IGFBP species were detected with biotin-labeled IGF-II, followed by peroxidase (PO)-labeled streptavidin (Sigma), and the ECL chemiluminescence detection system (Amersham, Aylesbury, UK). Immunoblots were performed as above, but IGFBP species were detected with specific anti-IGFBP-1 mAb or anti-IGFBP-3 (raised in our laboratory), anti-hIGFBP-2, hIGFBP-4, hIGFBP-5, or hIGFBP-6 (Austral Biologicals; San Ramon, CA) polyclonal antibodies, followed by PO-labeled goat anti-mouse or anti-rabbit IgG and ECL. Either DU145-CM or conditioned medium from the phorbol 12-myristate 13-acetate-stimulated human fibrosarcoma HT-1080 cell line was recovered from subconfluent cultures after 2 days in serum-free DMEM supplemented with 0.5% BSA (RIA Grade; Sigma; SFM/BSA) and concentrated using Centricon filters. Zymography was performed in SDS-PAGE gels containing gelatin (1 mg/ml) as previously reported (33Quesada A.R. Barbacid M.M. Mira E. Fernández-Resa P. Márquez G. Aracil M. Clin. Exp. Metastasis. 1997; 15: 26-31Crossref PubMed Scopus (38) Google Scholar). The same samples were electrophoresed in SDS-PAGE under reducing conditions, blotted to nitrocellulose, and probed with anti-MMP-9 Ab3 antibody (Calbiochem), followed by PO-labeled goat anti-mouse antibody and ECL. DU-145 cells were detached using 0.05% trypsin, 0.02% EDTA (Life Technologies, Inc.) and plated at several cell densities in 96-well plates (for proliferation experiments) or in 24-well plates (for cell cycle analysis). Twenty-four hours later, the cells were washed extensively with PBS and cultured for 24 h SFM/BSA. Thereafter, medium was renewed with SFM/BSA with or without recombinant human IGF-I (rhIGF-1, Pharmacia & Upjohn), recombinant human des-(1–3)-IGF-I (kindly provided by Dr. Pär Gellerfors, Pharmacia & Upjohn, Stockholm, Sweden), IGFBP-3 (Calbiochem), blocking anti-hIGF-I and -II mAb, anti-IGF-1R mAb αIR-3 (Oncogene Science, Uniondale, NY), aprotinin (Sigma), tissue inhibitor of metalloproteinases (TIMP)-2, or Batimastat (BB-94, kindly provided by Dr. F. Colotta, Pharmacia & Upjohn, Milan, Italy). Cell lysates from IGFBP-3- or anti-hIGF-I mAb-treated DU-145 cells were prepared and analyzed for IGF-1R autophosphorylation as described below. For proliferation experiments, DU-145 cells were pulsed for 8 h with 0.5 μCi/well of [3H]thymidine ([3H]TdR, Amersham Pharmacia Biotech) at various times during the course of the experiment, and nuclei were harvested using a cell harvester (LKB-Wallac, Sweden). [3H]TdR incorporation was determined on a liquid scintillation counter. Ba/F3 cell proliferation assays were performed as described (28Mañes S. Kremer L. Albar J.P. Mark C. Llopis R. Martı́nez-A C. Endocrinology. 1997; 138: 905-915Crossref PubMed Scopus (22) Google Scholar). For cell cycle analysis, DU-145-treated cells were detached, washed with PBS, and stained with propidium iodide using the DNA-Prep Stain kit (Coulter Corp., Miami, FL). Cell cycle analysis was carried out in a flow cytometer equipped with a pulse processing facility to enable discrimination of cell doublets (Epics XL, Coulter). Cell number was determined in some experiments by manual counting on a hemocytometer. DU-145 cells were washed and cultured in SFM/BSA alone or supplemented with different amounts of human insulin (Life Technologies, Inc.). Cells were detached after 72 h, washed twice in ice-cold PBS, and resuspended at 2 × 106 cells/ml in PBS plus 0.5% BSA, 0.01% NaN3. Biotinylated anti-IGF-I, anti-MMP-9 Ab3, or anti-MMP-2 (Calbiochem) mAb was added, followed by phycoerythrin-labeled avidin or fluorescein isothiocyanate-labeled goat anti-mouse IgG (Southern Biotechnologies, Birmingham, AL), respectively. An irrelevant isotype-matched mouse antibody was used as control. Cell-associated fluorescence was visualized by flow cytometry. To eliminate cell surface-bound IGF, detached cells received an acid wash (34Leamon C.P. Low P.S. Biochem. J. 1993; 291: 855-860Crossref PubMed Scopus (131) Google Scholar) prior to staining as above. Subconfluent DU-145 cells were cultured in SFM/BSA for 3 days and pulsed with IGFBP-3 for 24 h, with the anti-IGF-1R mAb αIR-3 for the times indicated, or with IGF-I (10 nm) for 5 min at 37 °C. After washing with ice-cold PBS, cells were lysed at 4 °C for 30 min using 20 mm Tris-HCl, pH 7.5, 130 mm NaCl, 1 mm MgCl, 1% Nonidet P-40, 10% glycerol, a proteinase inhibitor mixture, 1 mm sodium orthovanadate, and 10 mm NaF. Lysates were centrifuged for 25 min at 4 °C, and their protein concentration was determined using the micro-BCA kit (Pierce). Cell lysates (50 μg) were immunoprecipitated with αIR-3 or anti-IRS-1 mAb (Upstate Biotechnology Inc., Lake Placid, NY) for 3 h at 4 °C, followed by goat anti-mouse IgG1-agarose (Sigma), and then fractionated in 7.5% SDS-PAGE under reducing conditions. Tyrosine-phosphorylated proteins were developed with the PY-20 mAb (Santa Cruz Biochemicals, Santa Cruz, CA), and IRS-1 specifically with anti-IRS-1 mAb, followed by peroxidase-labeled goat anti-rabbit Ig antibody (ICN, Costa Mesa, CA) and ECL. A similar protocol was followed for control 3T3-IGF-1R cells. The level of cell division cycle 2 (Cdc-2) was estimated in DU-145 cell extracts treated with IGFBP-1, IGFBP-3, anti-IGF-I mAb, or BSA as described above. Samples containing equivalent amounts of protein were fractionated in 12.5% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and incubated for 2 h with anti-Cdc-2 (Transduction Laboratories, Lexington, KY) or anti-p53 antibodies (PharMingen, San Diego, CA). After washing, the filter was incubated with a PO-labeled goat anti-mouse antibody and ECL. The entire MMP-9 cDNA was cloned in the XbaI site of pEFBOS in the antisense direction, as determined by restriction analysis (pEFBOS-MMP-9AS). DU-145 cells were transfected with the pEFBOS-MMP-9AS or the pEFBOS empty vector using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Transfection efficiency was determined by cotransfecting an equal amount of green fluorescent protein-pEFBOS plasmid and subsequent FACS analysis determination of the percentage of cells expressing green fluorescent protein at 48 h. Expression was maximal between 48 and 96 h. After 24 h, DU-145-transfected cells were detached and plated in 96- or 24-well plates, allowed to adhere, washed three times with PBS, and starved overnight in SFM/BSA. Cells were then washed extensively with PBS and cultured for 24 h in SFM/BSA alone or supplemented with different amounts of IGF-I or 20 nm IGFBP-3 and then processed for proliferation experiments as described above. DU-145-transfected cells in 24-well plates were maintained in SFM without BSA for an additional 48 h, after which conditioned medium was collected and analyzed for IGFBP-3 proteolysis by Western blot or for MMP-9 activity by zymography. To analyze the effects of MMP-9 antisense expression on IGF-induced cell signaling, DU-145-transfected cells, either with the pEFBOS-MMP-9AS or the empty vector, were plated on 35-mm2dishes and treated as above. After 24 h starvation, dishes were incubated for 5 min in SFM/BSA with or without IGF-I (1 μg/ml). Cell lysates were obtained and immunoprecipitated with αIR-3 as described above and proteins resolved in 7.5% SDS-PAGE gels, and after blotting, nitrocellulose membranes were incubated sequentially with the PY-20 mAb and anti-IGF-1R β-subunit rabbit polyclonal antiserum (Santa Cruz Biotechnology) and ECL. DU-145 cells, untransfected or transfected with pEFBOS-MMP-9AS or empty pEFBOS plasmid, were starved for 48 h, washed five times with PBS, and then incubated with the fluorogenic peptide 2,4-dinitrophenol-Pro-β-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-N-methyl-aminobenzoyl) (Bachem, Bubendorf, Switzerland) at a final concentration of 5 μm, as described (33Quesada A.R. Barbacid M.M. Mira E. Fernández-Resa P. Márquez G. Aracil M. Clin. Exp. Metastasis. 1997; 15: 26-31Crossref PubMed Scopus (38) Google Scholar). The fluorescence increase after incubation at 37 °C was evaluated at 360 and 460 nm excitation and emission wavelengths, respectively. When inhibitors were tested, they were preincubated for 30 min with DU-145 before addition of the fluorogenic substrate. DU-145 cell monolayers were rinsed twice with minimal essential medium without amino acids plus 20 mm HEPES, pH 7.3, and cell-surface proteins were cross-linked by adding 0.5 mm disuccinimidyl suberate (Pierce). After 30 min at 4 °C, the cross-linking reagent was removed, and the reaction was terminated by rinsing and subsequent cell incubation with 37.5 mm Tris-HCl, pH 7.4, plus 150 mm NaCl for 10 min. The cells were then lysed with 100 mm n-octyl glucopyranoside (Calbiochem) in 37.5 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm benzamidine hydrochloride, and a protease inhibitor mixture as above. Cell lysates were resolved in 7% SDS-PAGE under reducing conditions, blotted, and probed with anti-MMP-9 mAb (Calbiochem) and, after stripping according to manufacturer's specifications, with antibodies specific for different integrin chains. To analyze DU-145 growth factor requirements, we tested the effect of several serum concentrations and cell densities in proliferation experiments. As reported previously, DU-145 cells grow in serum-free medium (36Pietrzkowski Z. Wernicke D. Porcu P. Jameson B.A. Baserga R. Cancer Res. 1992; 52: 6447-6451PubMed Google Scholar), determined both by [3H]TdR incorporation and the increase in the number of viable cells (data not shown). A sensitive, specific RT-PCR assay shows that DU-145 cells express mRNA coding for IGF-I and IGF-II and the IGF-1R (Fig. 1A). IGFBP secreted by DU-145 cells were analyzed by Western ligand and immunoblot (Fig. 1B). Labeled IGF-II binds to three proteins in the DU145-CM as follows: a protein of approximately 40 kDa, corresponding to the 39–42-kDa doublet characteristic of IGFBP-3, and two proteins of 34 and 25 kDa, identified in immunoblot as IGFBP-2 and IGFBP-4, respectively. A weak band of approximately 30 kDa is also occasionally visible, although no reactivity was observed with anti-IGFBP-1, -IGFBP-5, or -IGFBP-6 antibodies. The anti-IGFBP-3 antibody confirmed that the 39–42-kDa doublet is IGFBP-3, but it also developed a 31-kDa protein and, weakly, another of 19 kDa, which are not detected in ligand blot. This suggests that IGFBP-3 is proteolyzed in DU145-CM, as it is in other biological fluids (37Hossenlopp P. Segovia B. Lassarre C. Roghani M. Bredon M. Binoux M. J. Clin. Endocrinol. & Metab. 1990; 71: 797-805Crossref PubMed Scopus (384) Google Scholar). This proteolytic processing was not observed for either IGFBP-2 or -4. To demonstrate that the IGF secreted by DU-145 cells binds to cell-surface receptors, we used the IGF-I-specific mAb KM5A1, which recognizes the growth factor complexed either to the IGF-1R or to IGFBP-1 or -3 (28Mañes S. Kremer L. Albar J.P. Mark C. Llopis R. Martı́nez-A C. Endocrinology. 1997; 138: 905-915Crossref PubMed Scopus (22) Google Scholar). KM5A1 binds specifically to DU-145 cells that have been starved for 72 h (Fig. 2A); KM5A1 mAb binding to DU-145 cells is lost when the antibody is preincubated with IGF-I before being added to the cells (Fig. 2A). Other anti-IGF-I mAb (BB9E10) recognizing an epitope occult in the IGF-I·IGF-1R complex (28Mañes S. Kremer L. Albar J.P. Mark C. Llopis R. Martı́nez-A C. Endocrinology. 1997; 138: 905-915Crossref PubMed Scopus (22) Google Scholar) do not bind to DU-145 cells. As an additional specificity control, we performed an acid wash of cells to remove any receptor-bound ligand; this wash completely abolishes KM5A1 reactivity with the cells (Fig. 2B), which can be restored by incubation of cells with IGF-I (Fig. 2C). To determine whether KM5A1 recognizes IGF-I bound either to IGF-1R or to membrane-associated IGFBP, DU-145 cells were cultured for 72 h in serum-free medium supplemented with BSA and then pulsed for 24 h with different amounts of human insulin (Fig. 2D). Since insulin binds to the IGF-1R and to insulin receptor with different affinities and does not bind to IGFBP, the effect of insulin dose on KM5A1 mAb cell binding may indicate the receptor to which IGF-I ligates. KM5A1 mAb reactivity is completely abolished when the cells are cultured in 1 μm insulin and significantly reduced (but not lost) at 0.1 μm, indicating that autocrine-secreted IGF-I binds mainly to the IGF-1R. We were not able to analyze IGF-II binding in DU-145, although it probably is similar to that of IGF-I. Since secreted IGF (or at least IGF-I) binds to IGF-1R, we examined IGF-1R tyrosine kinase activity in serum-starved DU-145 cells. After incubation of 3T3-IGF-1R cells with IGF-I, αIR-3 immunoprecipitates a 98-kDa phosphoprotein band that represents the autophosphorylated IGF-1R β-subunit, not observed in the absence of IGF-I (Fig. 3A). IGF-I-treated or -starved DU-145 cells also show this phosphoprotein band. Upon autophosphorylation, the IGF-1R β-subunit associates with the insulin receptor substrate-1 (IRS-1), a docking protein with a key role in IGF-1R signal transduction (38Lee C.-H. Li W. Nishimura R. Zhou M. Batzer A.G. Myers M.G. White M.F. Schlessinger J. Skolnik E.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11713-11717Crossref PubMed Scopus (206) Google Scholar). The αIR-3-immunoprecipitated cell lysates were assayed with anti-IRS-1 antibody (Fig. 3B), showing that the IGF-1R is associated with IRS-1 in both IGF-I-treated and -starved DU-145 cells. Similar cell lysates, immunoprecipitated with an anti-IRS-1 antibody, show a 98-kDa phosphoprotein band corresponding to the IGF-1R β-subunit (data not shown). The relevance of the autocrine IGF/IGF-1R loop in DU-145 cell growth was analyzed by blocking the activation of IGF-1R induced upon IGF/IGF-1R interaction. To block the IGF autocrine loop, we first used anti-IGF-I and anti-IGF-II mAb, which inhibit the binding of both ligands to the IGF-1R (29Mañes S. Kremer L. Vangbo B. López A. Gómez-Moutón C. Peiró E. Albar J. Mendel-Hartvig I. Llopis R. Martı́nez-A C. J. Endocrinol. 1997; 154: 293-302Crossref PubMed Scopus (6) Google Scholar). Incubation of cells with these mAb at concentrations as high as 100 μg/ml for 3 days does not inhibit DU-145 serum-free growth, as measured by [3H]TdR incorporation (Fig. 4A). The failure of anti-IGF-I mAb to inhibit DU-145 cell proliferation is not a consequence of lack of activity, since this mAb inhibits IGF-I-induced Ba/F3 cell proliferation at the same level as IGFBP-3 in Ba/F3 cells (Fig. 4C). The most probable explanation for the difference in the results with the IGF mAb in DU-145 and Ba/F3 cell lines is the presence of IGFBP in the DU145-CM, which may compete with antibodies for IGF binding. The antagonistic anti-IGF-1R mAb αIR-3 inhibits cell proliferation by 40% at 10 nm (Fig. 4A). This contradicts earlier data showing that αIR-3 blocks IGF-II-dependent growth but not DU-145 serum-free proliferation (26Figueroa J.A. Lee A.V. Jackson J.G. Yee D. J. Clin. Endocrinol. & Metab. 1995; 80: 3476-3482Crossref PubMed Scopus (75) Google Scholar). We also analyzed the effect of exogenous hIGFBP-3 on DU-145 cell proliferation. IGFBP-3 at 20 nm causes a 40% inhibition of DNA synthesis (Fig. 4A), and proliferation is partially restored when IGFBP-3 is preincubated with a molar excess of IGF-I (Fig. 4B). The des-(1–3)-IGF-I variant, which binds to the IGF-1R as does IG
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