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Insulin-Like Growth Factor-Binding Protein-5 Inhibits Growth and Induces Differentiation of Mouse Osteosarcoma Cells

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Insulin-Like Growth Factor-Binding Protein-5 Inhibits Growth and Induces Differentiation of Mouse Osteosarcoma Cells Biochemical and Biophysical Research Communications 288, 435–442 (2001) doi:10.1006/bbrc.2001.5785, available online at http://www.idealibrary.com on Insulin-like Growth Factor-Binding Protein-5 Inhibits Growth and Induces Differentiation of Mouse Osteosarcoma Cells Marlon R. Schneider,*,1 Rui Zhou,* Andreas Hoeflich,* Ottheinz Krebs,* Jo¨rg Schmidt,† Subburaman Mohan,‡ Eckhard Wolf,* and Harald Lahm* *Institute of Molecular Animal Breeding, Gene Center of the Ludwig-Maximilian University, Munich, Germany; †Department of Comparative Medicine, National Research Center for Environment and Health, Neuherberg, Germany; and ‡Musculoskeletal Disease Center, Jerry L. Pettis Veterans Administration, Loma Linda, California 92357 Received September 11, 2001 The insulin-like growth factors (IGF-I and IGF-II) The precise role of insulin-like growth factor-binding are recognized stimulators of cellular growth and dif- protein-5 (IGFBP-5) in regulating the growth of tumor ferentiation in several tissues (1). These effects are cells, especially of bone-derived malignant cells, is not regulated systemically and locally by a group of six well understood. We have investigated the biological ac- structurally related proteins designated IGF-binding tivity of IGFBP-5 by transfecting OS/50-K8 mouse osteo- proteins (IGFBP-1 to -6) which have affinities for IGFs sarcoma cells with an expression vector containing the comparable to that of the type I IGF receptor. Although osteocalcin promoter and the complete mouse IGFBP-5 structurally related, each IGFBP has an individual cDNA (OC-IGFBP-5). Overexpression of IGFBP-5 mRNA expression pattern and exerts different functions in- and secretion of increased amounts of bioactive protein cluding stimulation or inhibition of IGF-bioactivity as in conditioned media were demonstrated in different well as IGF-independent actions (2, 3). The relative clones. For the analysis of cell proliferation, three clones concentrations of IGFBPs in a tissue and posttransla- exhibiting high levels of IGFBP-5 expression were se- tional mechanisms like phosphorylation, glycosylation lected and compared to a mock clone and to nontrans- or cleavage by specific proteases are the major deter- fected parental cells. IGFBP-5-secreting clones displayed minants of IGFBP actions (4, 5). reduced proliferation under both anchorage-dependent IGF-II is the most abundant peptide growth factor and -independent conditions (P < 0.05). The increase in stored in bone (6) and the IGFs regulate the growth of proliferation observed in IGFBP-5-secreting clones after both normal and transformed bone cells under physio- addition of exogenous IGF was significantly lower than logical and pathological conditions (7–9). In bone the that observed in mock-transfected or parental cells. A degree of IGF-mediated cellular proliferation appears similar result was obtained with long[R3]IGF-I which to be the result of the interaction with mainly two has a low affinity for all IGFBPs, suggesting that the binding proteins, IGFBP-4 and IGFBP-5. IGFBP-4 in- inhibitory effect of IGFBP-5 is only partially IGF-depen- hibits the IGF-stimulated bone cell proliferation in dent. OC-IGFBP-5-transfected clones expressed signifi- vitro (10, 11) and bone formation in vivo (12). In con- cantly higher amounts of osteocalcin mRNA (P 0.05) < trast, several in vitro studies reported that IGFBP-5 and secreted more osteocalcin protein than a mock clone enhances IGF-induced stimulation of proliferation of or parental OS-50/K8 cells. Thus, part of the growth- osteoblasts (11, 13–17), an effect that was confirmed by inhibiting effect of IGFBP-5 may be due to an induction in vivo studies (18, 19). IGFBP-5 has the unique prop- of differentiation in these cells. © 2001 Academic Press Key Words: differentiation; IGF; IGFBP-5; OS/50-K8; erty of binding to bone matrix, due to its strong affinity osteocalcin; osteosarcoma; overexpression; transfection. for hydroxyapatite thereby mediating storage of IGFs in bone. When necessary, these growth factors can be released to participate in physiological bone formation or to accelerate repair processes after injury (13). 1 To whom correspondence and reprint requests should be ad- In bones IGFBP-5 is exposed to specific proteases dressed at Institute of Molecular Animal Breeding, Gene Center, (20). Although the exact function of this mechanism is Ludwig-Maximilian University, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany. Fax: ϩ49-89-2180-6849. E-mail: schnder@lmb. unknown it certainly alters the binding affinity of uni-muenchen.de. IGFBP-5 for IGFs and consequently influences IGF 435 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Vol. 288, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 中国科技论文在线___________________________________________________________________________http://www.paper.edu.cn Technologies, Paisley, Scotland). Individual clones were picked and subcultured in the presence of geneticin for 2 months. Genomic DNA from selected clones was isolated using the Wizard Genomic DNA purification system (Promega, Mannheim, Germany). Preparation of conditioned media. Cells were seeded in 10-cm plastic petri dishes and allowed to grow to 80–90% confluence. Me- FIG. 1. Schematic representation of OC-IGFBP-5. The construct dium was removed and cells were washed two times with PBS. After was cloned as described under Materials and Methods. The BamHI- 4 days of culture in 9 ml serum-free DMEM conditioned media were linearized construct used for the transfection includes the osteocal- collected, centrifuged at 600g for 10 min to remove cellular debris cin promoter (367 bp), a chimeric intron (132 bp), the mouse IGFBP-5 and stored in aliquots at Ϫ20°C until use. Cells were trypsinized and cDNA (888 bp), the SV40 late polyadenylation signal (221 bp), and their numbers were determined for normalization of samples. the sequence for neomycin phosphotransferase with a synthetic poly- PCR analysis. To detect the integration of the construct in indi- adenylation signal (1369 bp). vidual clones a PCR analysis using primers which amplify a se- quence of the IGFBP-5 cDNA (sense, 5Ј-ATG GTG ATC AGC GTG GTC CTC-3Ј; antisense, 5Ј-TCC TGT CTC ATC TCA GGT GCA-3Ј)or actions. A further possibility is the generation of spe- a sequence of the neomycin resistance gene (sense, 5Ј-CCT GAA TGA cific fragments with IGF-independent activity (2). The ACT GCA GGA CGA-3Ј; antisense, 5Ј-TGC GAA TCG GGA GCG first proof of an IGF-independent action of IGFBP-5 GCG ATA-3Ј) was performed. The integrity of the genomic DNA was ␤ Ј was the stimulation of osteoblast mitogenesis (14) and confirmed by amplifying a sequence of the -actin gene (sense, 5 - GGC ATC GTG ATG GAC TCC-3Ј; antisense, 5Ј-GTC GGA AGG experiments with osteoblasts from IGF-I knockout TGG ACA GGG-3Ј). All PCR analyses were carried out in 20-␮l mice confirmed that this IGFBP functions as an inde- reactions containing 16 mM (NH4)2SO4, 50 mM Tris–HCl, pH 8.8, pendent bone growth factor (19). The identification of a 0.01% Tween 20, dNTPs (50 ␮M each) and 0.5 U Taq polymerase putative IGFBP-5 receptor (21, 22) and the intra- (PAN Systems, Aidenbach, Germany). Sense and antisense primers nuclear localization of IGFBP-5 (23, 24) support this were added at a final concentration of 0.1 ␮M for all investigations. concept. The amplification steps were performed as follows: 94°C for 4 min, followed by 36 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for Recently, several lines of evidence indicated a role 2 min. After a final extension period of 10 min at 72°C the amplified for IGFBP-5 in the regulation of growth of different products were separated in 2% agarose gels and visualized by malignant cell types (25–31). In addition, IGF-inde- ethidium bromide staining under UV light. pendent effects of IGFBP-5 on apoptotic signaling RT-PCR analysis. RNA was extracted using the TriPure reagent pathways were described (32). The precise role of (Roche Diagnostics, Mannheim, Germany). DNase-digested total IGFBP-5 in growth of cancer cells, especially of bone- RNA (2.5 ␮g) was used as a template for cDNA synthesis. Reverse derived malignant cells, is not well understood. Here transcription was performed for 60 min at 37°C in RT buffer (50 mM we describe the biological activity of IGFBP-5 on OS/ Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 mM DTT, dNTPs (1 mM each), random hexamer primers (0.6 ␮g) and 20 U M-MLV 50-K8 mouse osteosarcoma cells which possess strong reverse transcriptase (Gibco, Karlsruhe, Germany). The reaction osteogenic properties. We present evidence that clones was terminated by incubation for 10 min at 95°C. The PCR condi- which strongly overexpress IGFBP-5 display impaired tions and the visualization of the products were as described above. growth properties. Furthermore, these clones exert The integrity of cDNA was confirmed by a ␤-actin-specific RT-PCR an increased expression of osteocalcin, a marker of analysis. osteoblast differentiation, suggesting an influence of Real-time RT-PCR. RNA samples were digested with DNase I IGFBP-5 on the differentiation status of these cells. and reverse transcription was performed with the avian myeloblas- tosis virus (AMV) reverse transcriptase (Roche Diagnostics) at 42°C for 60 min. The cDNA was diluted 1:35 in aqua bidest, and 5 ␮l was MATERIALS AND METHODS assessed in duplicate in a 20-␮l PCR using the LightCycler system (Roche Diagnostics). Serially diluted plasmid preparations (1 ϫ 107 Cells and culture conditions. The OS/50-K8 cell line was origi- to 1 molecules) containing the appropriate cDNA were run in parallel nally established from a spontaneously arising osteosarcoma in and used as standards. After an initial step of 10 min at 95°C, BALB/c mice (33).
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