Expression of pleiotrophin an embryonic growth and differentiation factor in rheumatoid arthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 48, No. 3, March 2003, pp 660–667 DOI 10.1002/art.10839 © 2003, American College of Rheumatology Expression of Pleiotrophin, an Embryonic Growth and Differentiation Factor, in Rheumatoid Arthritis Thomas Pufe, Michaela Bartscher, Wolf Petersen, Bernhard Tillmann, and Rolf Mentlein Objective. Pleiotrophin (PTN), a 15.3-kd heparinbinding peptide, is expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. Since developmentally regulated factors often reappear during disease, we sought to determine whether there was PTN expression in the synovial membranes of patients with rheumatoid arthritis (RA). Methods. PTN messenger RNA expression was assayed by quantitative reverse transcriptase– polymerase chain reaction. The protein was localized by immunohistochemistry and quantified by enzymelinked immunosorbent assay (ELISA). Effects of PTN on cell proliferation in vitro were determined by DNA measurements. Results. PTN expression in normal adult synovial membranes and cartilage was barely detectable. However, PTN was strongly up-regulated in synovial tissues from patients with RA. In contrast, samples from patients with pyogenic arthritis had moderate PTN levels, and those from patients with osteoarthritis had only a slight increase in PTN, as measured by ELISA. In RA patients, PTN was localized primarily in synoviocytes but was also found in endothelial cells of blood vessels. In cultured mouse fibroblasts used as a model, PTN expression was up-regulated by tumor necrosis factor ␣ and was more weakly up-regulated by epidermal growth factor. Recombinant PTN stimulated the proliferation of cultured human synoviocytes and the monocyte cell line THP-1, but not human dermal fibroblasts, in which PTN increased the synthesis of vascular endothelial growth factor. Conclusion. In addition to certain types of cancer, the embryonic growth and differentiation factor PTN is expressed in adults with inflammatory diseases, in particular, RA. Proinflammatory cytokines enhance the expression of PTN. Thus, we propose that PTN is a further paracrine angiogenesis and growth factor for synovial cells in RA. Pleiotrophin (PTN), which is also known as heparin-binding growth-associated molecule, heparinbinding growth factor 8, heparin-binding neurotrophic factor, and osteoblast-specific factor 1, is an 136–amino acid (15.3-kd) secreted growth/differentiation cytokine that is developmentally regulated (for review, see refs. 1–3). Mature PTN is a nonglycosylated lysine-rich peptide that migrates anomalously as an 18-kd band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and is therefore called p18 (4). The mature form of PTN is derived from a 168-residue precursor with a 32–amino acid signal sequence. The molecule is called “pleiotrophin” because of its function as a differentiation factor or growth factor for various cell types. PTN promotes angiogenesis, stimulates neurite outgrowth from cultured neurons, induces a bipolar form of glial cell precursors, and induces cell migration (5–9). Together with midkine, PTN forms a family of heparin-binding proteins that are normally expressed during embryogenesis but not (or only at low levels) in healthy adult tissues (10). However, PTN is reexpressed in some human tumors, e.g., meningiomas (11), gliomas (12), some breast cancers (13), and pancreatic cancers (14). There is also evidence that PTN is involved in tumorigenesis by enhancing the angiogenesis or proliferation of the tumor cells themselves (13–16). The hyperplastic synovial pannus in rheumatoid arthritis (RA) resembles a solid tumor in certain ways, Supported by grants from the Muskel- und Skelettsystem of the Medical Faculty of the University Kiel and the Verein zur Förderung und Bekämpfung Rheumatischer Erkrankungen, Bad Bramstedt, Germany. Thomas Pufe, PhD, Michaela Bartscher, Wolf Petersen, MD, Bernhard Tillmann, MD, Rolf Mentlein, PhD: University of Kiel, Kiel, Germany Dr. Pufe and Ms Bartscher contributed equally to this work. Address correspondence and reprint requests to Professor Rolf Mentlein, PhD, Department of Anatomy, University of Kiel, Olshausenstrasse 40, Kiel D-24098, Germany. E-mail: rment@ anat.uni-kiel.de. Submitted for publication June 7, 2002; accepted in revised form December 2, 2002. 660 PLEIOTROPHIN IN RHEUMATOID ARTHRITIS especially its intrinsic cellular proliferation and invasive properties and the association of angiogenesis with the development of this highly vascularized tissue (17–19). Some oncogenes and angiogenesis factors constitutively produced by malignant cells have been detected in cells of the synovium and synovial fluid in RA (20–22). We therefore investigated whether PTN, a growth and angiogenesis factor of tumors, is produced in RA and whether it also affects the proliferation of synoviocytes. MATERIALS AND METHODS Peptides and antibodies. Goat anti-PTN (affinitypurified IgG fraction; catalog no. AF-252-PB) and biotinylated anti-PTN (catalog no. BAF252) were purchased from R&D Systems (Minneapolis, MN). Secondary antibodies were obtained from Sigma (Taufkirchen, Germany). Recombinant human PTN, tumor necrosis factor ␣ (TNF␣), and epidermal growth factor (EGF) were obtained from PeproTech (Rock Hill, NJ). Tissues and cell cultures. Synovial tissues and synoviocytes were obtained from patients with RA, pyogenic arthritis (PA), and osteoarthritis (OA) at the time of therapeutic synovectomy, which was performed at the Department of Orthopaedic Surgery, Christian-Albrechts University (Kiel, Germany). Samples from RA patients were obtained during the early phase of disease (1–5 years from disease onset) from joints with radiographic Larsen scores of 0–2. These patients had been treated with sulfasalazine and a low dose of cortisone (5 mg/day). Healthy control samples were obtained during routine autopsies performed at the Department of Anatomy, University of Kiel (Kiel, Germany). Synovial fluids were obtained from RA, PA, and OA patients who were undergoing arthroscopy at the Ambulantes Operationszentrum am Eichkoppelweg (Kiel, Germany). Human THP-1 cells were obtained from the National Institutes of Health (Bethesda, MD) and normal human dermal fibroblasts from PromoCell (Heidelberg, Germany). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). Synoviocytes were isolated without enzymatic digestion from synovial membranes obtained from cadavers. Synovium was cut into small pieces, transferred to petri dishes, and kept in DMEM–50% FCS for 24 hours. Then, the pieces were removed, and the cells that migrated out of the tissue and adhered to the bottoms of the petri dishes were cultured for 48 hours in DMEM–10% FCS. Cells were subcultured in trypsin–EDTA and used for proliferation assays after a further 48 hours of culture. Reverse transcriptase–polymerase chain reaction (RTPCR). RNA was isolated by a standard guanidinium thiocyanate–phenol–chloroform extraction method, and digested with DNase. For standard RT-PCR, complementary DNA was generated and amplified for PTN with the primers 5⬘-CCT-TCT-TGG-CAT-TCA-TTT-TCA-TAC-3⬘ (sense) and 5⬘-GAG-GTT-TGG-GGT-TGG-TCA-GT-3⬘ (antisense) at an annealing temperature of 60°C for 40 cycles, according to previous protocols (23). The RT-PCR product was identified by agarose gel electrophoresis and by direct sequencing with 661 the DyeDeoxy Terminator Cycle Sequencing method using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) as described elsewhere (23). A separate RT-PCR for GAPDH with an intron-spanning primer pair served as control for the intactness of RNA and the absence of contaminating DNA (23). For real-time RT-PCR, 100 ng of total RNA was reverse transcribed and amplified in the presence of SYBR Green with a commercial 1-step system (QuantiTect SYBR Green RT-PCR; Qiagen, Hilden, Germany) using the above primers. Amplification was monitored with an iCycler (BioRad, Munich, Germany) according to standard procedures. PCR was performed using Hot StarTaq DNA polymerase, which is activated by an initial heating step, whereas Omniscript Reverse Transcriptase is deactivated. The temperature profile included an initial denaturation for 15 minutes at 95°C, followed by 37 cycles at 95°C for 15 seconds, annealing at 60°C for 30 seconds, elongation at 72°C (elongation time depended on the size of the fragment; the number of basepairs divided by 25 yielded the time in seconds), and fluorescence monitoring at 72°C. For analysis of the PCR data, iCycler Data Analysis software (Bio-Rad) was used. The specificity of the amplification reaction was determined by performing a melting curve analysis. Relative quantification of the signals was performed by normalizing the signals of the different genes with a ␤-actin signal, with the primers 5⬘-TGC-CAT-CCT-AAA-AGCCAC-3⬘ (sense) and 5⬘-TCA-ACT-GGT-CTC-AAG-TCAGTG-3⬘ (antisense). Western blots and immunohistochemistry. Western blotting was performed as previously described (12,23). Briefly, samples were reduced and boiled in SDS sample buffer with 2-mercaptoethanol, separated on 15% SDS-PAGE gels, and transferred by semidry method onto nitrocellulose membranes. Bands were detected by chemiluminescence. For immunohistochemistry, tissues were fixed with 4% formaldehyde in phosphate buffered saline (PBS; 30 minutes at room temperature), dehydrated, and embedded in paraffin. Sections (8 m) were deparaffinized in xylol (3 times for 10 minutes each), organic solvent was removed with decreasing concentrations of ethanol (100%, 90%, 80%, 70%, 60%, and 50%; 2 minutes each), then sections were immersed in doubledistilled water for 10 seconds. After washing 3 times with Tris buffered saline (TBS; pH 7.4), sections were demasked with trypsin (0.1% in TBS with 0.1% CaCl2), washed 3 times with TBS, and incubated overnight at 4°C with anti-PTN (diluted 1:500 in PBS). Sections were then washed 3 times with TBS, incubated for 30 minutes at room temperature with peroxidase-labeled anti-goat IgG (1:250 in PBS), washed another 3 times with TBS, stained for 7 minutes at room temperature with 3-amino-9-ethylcarbazole/H2O2 (Universal Peroxidase Detection kit; Coulter Immunotech, Hamburg, Germany), and then counterstained with Mayer’s hemalum. Enzyme-linked immunosorbent assay (ELISA). For ELISA, tissues were homogenized in 0.14M NaCl, 20 mM HEPES (pH 7.4), centrifuged at 15,000g for 15 minutes at 4°C, and the supernatants were analyzed. The ELISA was performed with Nunc-Immuno Maxisorp plates (Nunc, Roskilde, Denmark) that had been coated overnight at room temperature with anti-PTN (50 ng/well), washed 3 times with 0.05% Tween 20 in PBS (washing buffer), blocked for 1 hour at room 662 PUFE ET AL temperature with 1% BSA in PBS, and washed with washing buffer. Wells were incubated for 2 hours at room temperature with samples, standards, or blanks (300 l, diluted with PBS), washed 3 times with washing buffer, and incubated for another 2 hours at room temperature or overnight at 4°C with biotinylated anti-PTN (50 ng/well). After washing 3 times with washing buffer, wells were incubated for 20 minutes at room temperature with a 1:20,000 dilution of a 1.25-mg/ml solution Figure 2. Pleiotrophin (PTN) protein concentrations in synovial membranes from patients with rheumatoid arthritis (RA), pyogenic arthritis (PA), and osteoarthritis (OA), as well as in normal cadavers. PTN protein concentrations are highly elevated in synovial membranes from RA patients, moderately elevated in those from PA patients, but only slightly elevated in those from OA patients. Tissues were homogenized, and PTN concentrations were determined by enzyme-linked immunosorbent assay (related to wet tissue weight). Values are the mean ⫾ SD of triplicate determinations of samples from individual subjects. The following between-group comparisons were statistically significant: RA versus normal (P ⬍ 0.01), PA versus normal (P ⬍ 0.05), and RA versus OA (P ⬍ 0.05). Figure 1. Detection of pleiotrophin (PTN) mRNA in synovial tissues by A, reverse transcriptase–polymerase chain reaction (RT-PCR) in inflamed synovial tissue and B, real-time RT-PCR, showing higher expression in rheumatoid arthritis (RA) synovium. RNA was extracted from the synovial membranes of normal cadavers and patients with osteoarthritis (OA), pyogenic arthritis (PA), or RA, and then reverse transcribed. A, For RT-PCR, cDNA was amplified with primers specific for PTN or GAPDH (control for intactness of RNA and absence of contaminating DNA). Products were separated by agarose gel electrophoresis and stained with ethidium bromide. C ⫽ control (without RNA). The PTN PCR products correspond to the predicted size of 402 bp, as estimated from the 100-bp markers (M); their identity was further verified by sequencing. B, Real-time RT-PCR was performed in the presence of SYBR Green as described in Materials and Methods. Relative quantification of the PTN signals was performed by normalizing them to a ␤-actin signal. Values are the mean and SD of 3 experiments. of streptavidin–peroxidase (catalog no. 43-4323; Zymed, Burlingame, CA), washed 3 times with washing buffer, and then incubated for 30 minutes at room temperature with 100 l of tetramethylbenzidine–H2O2 (TMB Substrate kit, catalog no. 34021; Pierce, Rockford, IL). The reaction was stopped by the addition of 50 l of 0.5M H2SO4, and the yellow dye was measured at an absorbance of 450 nm. The assay was linear for 0.05–1.5 ng of PTN (detection limit ⬃0.02 ng). Stimulation of cells and assays for proliferation and vascular endothelial growth factor (VEGF). Cells (106) were seeded into fresh dishes and cultured for 24 hours in DMEM plus 10% FCS. The medium was replaced with DMEM plus 0.5% FCS, and the cells were exposed to the stimulators for 24 hours. Conditioned medium was removed, and aliquots were assayed for PTN or VEGF content. The cells were then washed with PBS, lysed, and the DNA content was measured fluorometrically with the CyQuant reagent (Molecular Probes, Eugene, OR) and related to a measurement of microscopically counted number of trypsinized cells. VEGF content was PLEIOTROPHIN IN RHEUMATOID ARTHRITIS 663 Figure 3. Strong immunostaining of pleiotrophin (PTN) in synovial membranes of rheumatoid arthritis (RA) patients, with much weaker staining in osteoarthritis (OA) and normal synovium. In normal synovium, only a few single cells, mostly endothelial cells, are PTN positive (red). The synovial surface and the lining cells are immunonegative. In OA synovium, the lining cells are PTN immunoreactive. Staining in RA synovium is strongly immunopositive, with immunoreactivity in lining cells as well as endothelial cells. RA synovium is immunonegative after absorption of the antibody with recombinant PTN (antibody control [Co]). Blue areas indicate nuclear counterstaining with Mayer’s hemalum. Bars ⫽ 10 m. determined with a sandwich ELISA as described previously (24). Proliferation assays were performed in DMEM plus 0.5% FCS. 3H-thymidine incorporation (72 hours of stimulation; 6 hours of incorporation in the presence of stimulators) was measured in adherent subconfluent cells, as described elsewhere (12). Nonadherent THP-1 cells were subjected to 48 hours of stimulation, and DNA was quantified with the CyQuant method (24). Statistical analysis. Statistical significance was evaluated by Dunnett’s multiple comparisons test or by t-test. RESULTS Detection of pleiotrophin in RA synovial tissue by RT-PCR and quantification by real-time RT-PCR and ELISA. Using sequence-specific primers, we detected PTN messenger RNA (mRNA) in synovial tissues from RA patients (Figure 1A), as well as from patients with PA and OA. Quantitative measurements by realtime RT-PCR showed considerably higher PTN mRNA expression in RA synovium than in normal synovium, whereas in OA synovium, only a moderate increase was detected (Figure 1B). Quantification of PTN protein by ELISA yielded similar results (Figure 2). Synovial tissues from controls (cadavers without signs of RA or joint diseases) showed a low, but measurable, PTN content. In samples from RA patients, the PTN content was elevated 3–5-fold over that in the normal controls. In samples from PA patients, a lower (up to 2.5-fold) increase in PTN concentration was detected, whereas the PTN content was increased only in some of the samples from patients with OA. Immunostaining of PTN in the synovium of RA patients. Consistent with the ELISA measurements, synovial tissues from RA patients showed intense immunostaining for PTN (Figure 3). Immunostaining of OA synovium was much weaker. In normal adult synovium (obtained from cadavers), only single cells, mostly endothelial cells, were PTN immunopositive. Reactivity in 664 Figure 4. Staining of 18-kd and 36-kd bands in Western blots of rheumatoid arthritis (RA) synovial proteins by pleiotrophin (PTN) antibody. Synovial tissue from RA patients was boiled and reduced in sodium dodecyl sulfate sample buffer. Equal amounts of protein from 2 different samples (lanes 1 and 2) were separated on 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, blotted onto nitrocellulose paper, stained with anti-PTN followed by a peroxidaselabeled secondary antibody, and detected by chemiluminescence. A control with PTN-inactivated antibody remained negative (lane C). The positions of molecular mass markers (from the top, chicken ovalbumin, bovine erythrocyte carbonic anhydrase, soybean trypsin inhibitor, bovine milk ␣-lactalbumin, and bovine lung aprotinin) are shown on the left. The estimated apparent molecular masses for PTN and its putative dimer are shown on the right. OA and RA synovium was confined to the lining cells, immunostaining both type A (macrophage-like) and type B (fibroblast-like) cells. These cells were negative in normal synovium. Endothelial cells in RA and OA synovium, as well as normal synovium, were also immunopositive for PTN (Figure 3). There was no immunoreactivity in control samples subjected to preadsorption of the antibody with recombinant PTN (Figure 3), those in which the primary antibody had been omitted (results not shown), and those incubated with anti-goat IgG control antibody (results not shown). The specificity of the antibody was further evaluated by Western blotting experiments with RA synovium (Figure 4). The antibody stained an 18-kd protein (an apparent molecular mass corresponding to the value previously reported for PTN ) as well as a 36-kd protein (probably the dimer [for comparison, see ref. 3]). Staining of both bands could be suppressed by preadsorption of the antibody with recombinant PTN. No other strong bands of staining were identified. Induction of PTN in fibroblasts by TNF␣. Several cytokines are involved in the inflammatory process of RA, in particular, TNF␣. Using human dermal fibroblasts and the human monocyte cell line THP-1 as a model for fibroblast-like and macrophage-like synovial PUFE ET AL cells, respectively, we determined whether TNF␣ could induce the expression of PTN. Indeed, stimulation of dermal fibroblasts with this cytokine yielded an ⬃9-fold induction of PTN secretion into the culture medium (Figure 5). Treatment with EGF had a considerably smaller induction effect. Thus, expression of PTN in fibroblasts (and probably cells derived from them) can be induced by proinflammatory cytokines and growth factors. Stimulation of proliferation and gene expression in synoviocytes by PTN. To identify target cells for PTN, we investigated the effects of PTN on the proliferation of synoviocytes as well as on the gene expression of human dermal fibroblasts and a monocyte cell line that served as a model for fibroblast-type and monocyte/ macrophage-type synovial cells. PTN increased the proliferation of synoviocytes ⬃2.5-fold at concentrations of 10–100 ng/ml (Figure 6). Proliferation of the human monocyte cell line THP-1 was also stimulated by PTN, but proliferation of human dermal fibroblasts was not increased. As an example of the effects of PTN on gene induction, we investigated its possible effects on the expression of the angiogenesis factor VEGF. Synthesis of VEGF, as measured by its release into the culture medium after 24 hours of stimulation, was increased by PTN in dermal fibroblasts, but not in THP-1 cells Figure 5. Induction of pleiotrophin (PTN) secretion in human dermal fibroblasts by tumor necrosis factor ␣ (TNF␣) and epidermal growth factor (EGF), and negligible PTN production in the human monocyte cell line THP-1, which cannot be stimulated. Cells were stimulated without (control [C]) and with 10 ng/ml of the cytokines for 24 hours in medium containing 0.5% fetal calf serum, and PTN was measured in the supernatants by enzyme-linked immunosorbent assay. Values are the mean and SD of 4 samples. P ⬍ 0.01 for cytokines versus control in the fibroblast studies. PLEIOTROPHIN IN RHEUMATOID ARTHRITIS 665 Figure 6. Induction of the proliferation of synoviocytes and monocytes, but not fibroblasts, by pleiotrophin (PTN). Human synoviocytes, the monocyte cell line THP-1, and human dermal fibroblasts were stimulated without (control [C]) and with different concentrations of PTN for 24 hours in medium containing 0.5% fetal calf serum, and cell numbers were determined by 3H-thymidine incorporation (synoviocytes, fibroblasts) or DNA quantification (THP-1). Values are the mean and SD of 6 individual cultures each. P ⬍ 0.01 for PTN versus control in the synoviocytes and in the THP-1 cells at 100 ng/ml of PTN. (Figure 7). The results of these experiments show not only that PTN is produced in synoviocytes, but also that synoviocytes are the targets of PTN. Figure 7. Induction of vascular endothelial growth factor (VEGF) synthesis in fibroblasts by pleiotrophin (PTN). Human dermal fibroblasts or human THP-1 monocytes used as a model for fibroblast-type or macrophage-type cells were stimulated without (control [C]) and with 10 ng/ml of PTN for 24 hours in medium containing 0.5% fetal calf serum, and VEGF was measured in the supernatants by enzymelinked immunosorbent assay. Values are the mean and SD of 4 samples. P ⬍ 0.05 for PTN versus control in the fibroblasts. TNF␣ ⫽ tumor necrosis factor ␣; EGF ⫽ endothelial growth factor. DISCUSSION Chronic inflammation is characterized by the production of various cytokines and angiogenic factors, e.g., TNF␣, interleukins 1 and 6, VEGF, and basic fibroblast growth factor (20,21,25). We have shown that PTN, a developmentally expressed growth and differentiation factor, is reexpressed and up-regulated in RA synovial membranes, whereas it is not expressed or is only weakly expressed in tissues from normal controls or from patients with other joint diseases (PA and OA). Induction experiments with fibroblasts showed that proinflammatory cytokines such as TNF␣ up-regulate the expression of PTN and that growth factors such as EGF or platelet-derived growth factor (see ref. 26) have smaller effects. The stimulatory effects of PTN on the proliferation of synoviocytes demonstrated that this peptide acts as a paracrine growth factor. Experiments with a monocyte cell line as a model suggested that PTN targets the macrophage-type cells in the synovial membrane in RA, whereas recombinant PTN was not mitogenic for dermal fibroblasts in our experiments. Moreover, gene expression (e.g., expression of mRNA for VEGF) was upregulated by PTN. Since PTN has repeatedly been reported to induce the proliferation of endothelial cells (5,6,9,26), we think that besides other factors such as VEGF (21), PTN could be involved in the extensive 666 PUFE ET AL vascularization of the synovium that is observed in RA. Thus, PTN is a further paracrine mitogenic and angiogenic factor that contributes to the inflammatory process in RA. Receptors for PTN and their signal transduction mechanisms have not yet been fully characterized. So far, interaction of PTN with the receptor-type protein tyrosine phosphatase /␤, anaplastic lymphoma kinase, and syndecan 3 have been shown (2,28,29). By disruption of the normal balance between tyrosine kinase and phosphatase activities, phosphorylation of intracellular proteins has been found to be increased, particularly the phosphorylation of ␤-catenin (28), which is involved in transcription and transformation of the cytoskeletal architecture and the phosphorylation of downstream effector molecules, such as insulin receptor substrate 1, Shc, phospholipase C␥, and phosphatidylinositol 3⬘kinase (29). However, the mechanisms and biologic effects differ significantly between the various target cell types. The expression and effects of PTN have been primarily studied during embryogenesis. So far, clinical interest in PTN has focused on its expression in certain types of cancer, especially breast cancer, pancreatic cancer, and melanoma (11–16). Chronic inflammatory diseases such as RA should be added to the list of pathologic conditions in which there is PTN expression. Whether PTN functions as a repair factor or an amplifier of inflammation may depend on the tissue. ACKNOWLEDGMENTS We thank Martina Burmester, Dagmar Freier, Miriam Lemmer, Frank Lichte, Karin Stengel, and Regine Worm for their expert technical assistance and Clemens Franke for drawing the illustrations. REFERENCES 1. Kurtz A, Schulte AM, Wellstein A. Pleiotrophin and midkine in normal development and tumor biology. Crit Rev Oncol 1995;6: 151–77. 2. Zhang N, Deuel TF. Pleiotrophin and midkine, a family of mitogenic and angiogenic heparin-binding growth and differentiation factors. Curr Opin Hematol 1999;6:44–50. 3. Muramatsu T. Midkine and pleiotrophin: two related proteins involved in development, survival, inflammation and tumorigenesis. J Biochem (Tokyo) 2002;132:359–71. 4. Corbley MJ. Transformation by Ras suppresses expression of the neurotrophic growth factor pleiotrophin. J Biol Chem 1997;272: 24696–702. 5. Li YS, Milner PG, Chauhan AK, Watson MA, Hoffman RM, Kodner CM, et al. 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