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Departments of Physiology (J.V.S., Y.G., S.M., A.L., L.J.M.) and Internal Medicine (L.J.M.), University of Manitoba, Winnipeg, Canada R3E 0W3; Center of Reproductive Medicine, Shenzhen Hospital of Peking University (Y.G.), Shenzhen 518036, Peoples Republic of China; and Division of Endocrinology, Department of Pediatrics, Mattel Children’s Hospital, University of California (P.C.), Los Angeles, California 90095-1752
Address all correspondence and requests for reprints to: Dr. L. J. Murphy, Department of Physiology, University of Manitoba, Winnipeg, Canada R3E 0W3. E-mail: ljmurph{at}cc.umanitoba.ca.
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Introduction |
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The mechanisms that mediate these IGF-independent effects of IGFBP-3 are unclear. It has been assumed that these IGF-independent effects of IGFBP-3 are mediated via specific cell surface binding sites that have been reported by some groups (13, 14), although this remains to be proven. In addition, IGFBP-3 can be transported to the nucleus (15, 16), where it may interact with transcription factors (17). The interaction of IGFBP-3 with nuclear transcription factors has also been implicated in the IGF-independent actions of IGFBP-3 (17, 18).
We have previously demonstrated that overexpression of IGFBP-3 in transgenic (Tg) mice is associated with a growth-retarded phenotype and impaired glucose homeostasis (19, 20). These phenotypic manifestations of IGFBP-3 overexpression were attributed to modulation of free IGF-I in the circulation and tissues. However, the in vitro reports of IGF-independent effects of IGFBP-3 provide justification for other possible interpretations of our previously published data (19, 20). The IGF-independent actions of IGFBP-3 may mediate, at least in part, the growth retardation associated with overexpression of IGFBP-3. To this end, we have generated Tg mice that overexpress a non-IGF-binding, human mutant IGFBP-3 cDNA that retains proapoptotic effects in vitro (21, 22), with the aim of investigating the IGF-independent effects of IGFBP-3 in vivo.
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Materials and Methods |
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Tg mice were generated by pronucleus injection of the linearized transgene fragment, devoid of plasmid sequences, into fertilized CD-1 zygotes. The microinjected embryos were transferred into CD-1 foster mice using standard techniques. The founders were bred with CD-1 mice. For comparisons between Tg and wild-type (Wt) mice, homozygous male Tg mice were breed with female mice, and Tg and Wt mice from the same litter were compared. Thus, the Wt, non-Tg control mice were of an identical genetic background as the Tg mice and were derived from the same litters. All experiments were performed in accordance with protocols approved by the animal care committee of the Faculty of Medicine, University of Manitoba.
Expression of IGFBP-3 and mutant IGFBP-3 in COS-1 cells
PGKBP-3 and PGKmBP-3 plasmids were transiently expressed in COS-1 cells. The cells were grown in 24-well plates and transfected with 1 µg plasmid DNA using 2.5 µl Lipofectamine 2000 reagent (Invitrogen Life Technologies, Inc., Burlington, Canada) in Opti-MEM1 medium. After a 24-h incubation, the conditioned medium (CM) was collected and concentrated in Centricon centrifugal filter units (Millipore Corp., Bedford, MA). The concentrated CM was mixed with Laemmli sample buffer and heated for 5 min at 95 C, then separated on a 10% SDS-PAGE gel. Separated proteins were transferred to nitrocellulose membranes (MSI, Westborough, MA). For Western blotting, membranes were blocked with 3% skim milk in TBST [5 mM Tris-HCl (pH 7.4), 136 mM NaCl, and 0.05% Tween 20], then incubated with 1:200 diluted goat antihuman IGFBP-3 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 C. After washing with TBST buffer, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-linked donkey antigoat immunoglobulin G (Santa Cruz Biotechnology, Inc.) at a dilution of 1:10,000. Detection of immune complexes was achieved using an ECL Western blotting kit (Amersham Biosciences, Baie d’Urfe, Canada). For ligand blotting, the membranes were incubated with [125I]IGF-I (106 cpm/ml; NEN Life Science Products, Inc., Boston, MA) overnight at 4 C. The washed filters were subsequently exposed to x-ray film overnight.
Southern blot analysis
The presence of the transgene was detected by Southern blot analysis of tail DNA. Filters were hybridized with a fragment of the transgene under stringent conditions. After washing, filters were exposed to Kodak XAR film (Eastman Kodak Co., Rochester, NY) at –70 C for 24–72 h.
IGFBP-3, IGF-I, and GH assays
Human IGFBP-3 was measured using an immunoradiometric assay from Diagnostic Systems Laboratories (Webster, TX). Total plasma IGF-I was measured using a rat IGF-I RIA assay kit (Diagnostic Systems Laboratories). To detect the presence of human IGFBP-3 in the ternary complex, 1 ml pooled sera from Tg mice was chromatographed on a Sephacryl S-200HR 16/60 column at room temperature. The column was eluted with Tris-buffered saline, pH 7.5, and 1-ml fractions were collected. Murine IGFBP-3 was measured using an in-house murine IGFBP-3-specific assay with less than 0.1% cross-reaction with human IGFBP-3. Cross-reaction of mutant IGFBP-3 in this assay was assumed to be of the same order as that of human IGFBP-3. The murine IGFBP-3 assays uses a recombinant full-length mouse IGFBP-3 protein as a standard and a specific antibody pair generated against it. GH was measured using a rat GH enzyme immunoassay kit purchased from Spi-Bio (Massy, France). The cross-reactivity with mouse GH is 91%. All samples were analyzed in a single assay, where the intraassay coefficient of variation was 9%.
RNA extraction and ribonuclease (RNase) protection assays (RPAs)
Total RNA was extracted from a variety of tissues using TRIzol reagent (Invitrogen Life Technologies, Inc.). The concentration of RNA was determined spectrophotometrically, and the integrity of the RNA in all samples was documented by visualization of the 18S and 28S ribosomal bands after electrophoresis through a 0.8% formaldehyde/agarose gel. Maxiscript SP6/T7 and RPAIII kits (Ambion, Austin, TX) were used for the RPA. Using the PGKBP-3 plasmid as a template, a 267-bp fragment containing the sequence corresponding to the 3' end of the human IGFBP-3 cDNA and the bovine GH polyadenylation signal of the transgene was subcloned into pCRII vector by PCR with the primers 5'-AGA AAA AGC AGT GTC GCC CTT-3' and 5'-TAG GAA AGG ACA GTG GGA GTG-3' (19). The plasmid was linearized by digestion with BamHI and used as templates for riboprobe synthesis. Total RNA (10–20 µg) from mouse tissues was hybridized with approximately 3 x 105 cpm 32P-labeled cRNA by incubation overnight at 45 C. After hybridization, single-stranded RNA was digested with RNase A/T at 37 C for 30 min. The undigested RNA duplexes were separated on 5% polyacrylamide/8 M urea gels. Dried gels were exposed to Kodak XAR film at –70 C for 12–24 h. A mouse cyclophilin riboprobe was used as the internal standard, and century RNA markers from Ambion were used to determine the size of the protected fragment. The protected sizes for the transgene-derived RNA and cyclophilin fragments were 267 and 103 bp, respectively.
Western and ligand blotting of sera
Sera (2 µl) from Tg and Wt mice were analyzed on 10% SDS-PAGE and transferred to a nitrocellulose membrane. For immunodetection, membranes were incubated with biotinylated goat antihuman IGFBP-3 antibody (Diagnostic Systems Laboratories) at 4 C overnight. After washing, the membrane was incubated with streptavidin-horseradish peroxidase conjugate (Invitrogen Life Technologies, Inc.). Detection of immune complexes was achieved using an ECL Western blotting kit (Amersham Biosciences). For ligand blotting, the membrane was incubated with [125I]IGF-I (500,000 cpm; NEN Life Science Products, Inc.) at 4 C overnight. The membrane was subsequently washed four times with Tris-buffered saline, pH 7.6, and 0.1% Tween 20 and exposed to Kodak XAR film at –70 C for 24–72 h.
Apoptosis and proliferating cell nuclear antigen (PCNA) immunohistochemistry
Pregnant mice, generated by crossing 5012 Tg mice with Wt mice, were killed by exsanguination on embryonic d 18. The fetuses were removed and fixed in 4% neutral buffered formalin overnight. Embryos were genotyped as described above, and six Wt and six Tg embryos were paraffin-embedded for immunohistochemical investigation. Deparaffinized 6-µm-thick whole fetal sections were subsequently stained for PCNA using a commercially available kit (PCNA Staining Kit, Zymed Laboratories, San Francisco, CA) or for apoptosis by the terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling assay (ApopTag Peroxidase In Situ Apoptosis Detection Kit, Chemicon Laboratories, Inc., Temecula, CA). Negative controls were obtained by omitting the primary antibodies. Positive controls were provided by the respective manufacturers. Images of the sectioned tissue were acquired through a x40 objective of a phase contrast light microscope (Eclipse E800, Nikon, Melville, NY) and digitized by means of a Sony color video camera (Sony, Tokyo, Japan). To determine the degree of proliferation and apoptosis, the numbers of cells positive for PCNA and apoptotic cells were counted on four randomly selected images of the liver, kidney, periventricular region of the brain, spleen, and lungs and expressed as a percentage of the total number of cells.
Statistical analysis
Data are expressed as the mean ± SEM. A t test was used for single comparisons between Tg and Wt mice. For determining statistical differences between multiple groups, an ANOVA, followed by Dunnett’s t test, was used.
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Results |
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. Approximately equal amounts of human IGFBP-3 and mutant IGFBP-3 were apparent in immunoblots of CM from cells transfected with either of the transgenes (Fig. 1, upper panel, lanes 5 and 6). Ligand blotting with [125I]IGF-I failed to detect IGFBP-3 in CM from pPGKmBP-3-transfected cells (Fig. 1, lower panel). A smaller molecular weight binding protein, possibly IGFBP-2, was detected in CM from untransfected COS-1 cells; however, no IGFBP-3 was apparent in CM from untransfected cells.
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A transgene-specific RPA was used to examine transgene expression in various tissues (Fig. 2). The level of transgene expression varied between strains, between tissues, and even between genders within the same tissue and strain. Generally transgene-derived mRNA abundance was higher in male than in female mice. The highest levels of transgene expression were detected in reproductive tissues and fat.
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). IGF-I levels were not significantly different in Wt and Tg mice of the 5005 strain, but were significantly elevated in male Tg mice of the 5012 strain, and the same trend was apparent in female mice of the 5012 strain. Mouse GH levels were similar in all three groups of mice. Human IGFBP-3 was measured using an assay that predominantly measures intact binding protein. This assay showed some cross-reaction with endogenous murine IGFBP-3. Using the assay for intact IGFBP-3, serum from Wt mice gave a reading on the order of 90 ng/ml, representing cross-reaction with endogenous murine IGFBP-3, whereas serum from the PGKmBP-3 Tg mice had levels of approximately 150–450 ng/ml. The levels of murine IGFBP-3 in the circulation were similar in Wt and Tg mice of the 5005 strain, but were significantly elevated in both male and female mice of the 5012 strain (Table 1). Although mutant IGFBP-3 levels were higher in mice of the 5012 Tg strain, cross-reaction did not appear to completely account for the increased levels of IGFBP-3 detected in the murine IGFBP-3 assay, because PGKBP-3 and CMVBP-3 Tg mice previously generated in the laboratory had markedly higher levels of human IGFBP-3 present in the circulation, but had similar levels of murine IGFBP-3 as Wt mice (Fig. 3). In the Tg mice, there was a significant positive correlation between intact mutant IGFBP-3 levels and murine IGFBP-3 levels (r2 = 0.27; n = 80; P < 0.001; Fig. 4). There was also a positive correlation between mutant IGFBP-3 levels and IGF-I levels (r2 = 0.20; n = 80; P < 0.001).
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2-macroglobulin. Additional immunoreactivity was apparent in fractions that eluted after albumin, possibly representing free intact or partially degraded mutant IGFBP-3 (Fig. 5). Negligible amounts of mutant IGFBP-3 were present in the 150-kDa fractions, which constituted the major peak of IGFBP-3 cross-reacting immunoreactivity in sera from Wt mice.
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). Because mutant IGFBP-3 does not bind IGF-I under these conditions (Fig. 1
), the IGFBP-3 detected by ligand binding represents endogenous murine IGFBP-3. Immunoblotting with antihuman IGFBP-3-specific antibody detected human IGFBP-3 in sera from Tg mice, but not Wt mice (Fig. 6B).
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). Postnatal growth, as assessed by weight gain, was identical in Tg and non-Tg littermates. Linear growth was assessed by measuring the nose to tip of tail length and the tail length in anesthetized animals at 16 wk of age (Table 2). There was a tendency for male and female Tg mice of both strains to be shorter than comparable Wt mice; however, the differences between Tg and Wt mice did not achieve statistical significance.
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).
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). There was no significant difference in apoptosis between Wt and Tg mice observed in any of the organs examined. Cell proliferation assessed by PCNA-positive cells was also examined in these embryos. The percentage of PCNA-positive cell was similar in all organs examined, with the exception of the periventricular region of the brain, where a significant reduction in PCNA-positive cells was observed in Tg mice.
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Discussion |
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We have previously reported that generalized overexpression of IGFBP-3 in Tg mice is associated with impaired intrauterine and postnatal growth (19). Although these effects are most likely explicable on the basis of reduced free IGF-I and -II and impaired IGF action, the possibility that some or all of the growth retardation observed in these IGFBP-3-overexpressing mice could be due to IGF-independent effects of IGFBP-3 was not directly addressed in previous reports from this laboratory. In this study we report that overexpression of Gly56/Gly80/Gly81-IGFBP-3, a mutant IGFBP-3 with negligible affinity for IGF-I and -II (21, 22), has no observable effect on either pre- or postnatal growth in the mouse.
The Gly56/Gly80/Gly81-mutant IGFBP-3 was previously reported to have a very low affinity for IGF-I (21), and this was confirmed in our experiments by both ligand blotting of CM from COS-1 cells transfected with transgene and the observation that circulating IGF-I levels were not markedly elevated in the Tg mice. Although other mutants, such as Ala56/Ala57/Ala75/Ala77/Ala80/Ala81-mutant IGFBP-3 (24), may have an even lower affinity for IGF-I while still retaining the IGF-independent, proapoptotic, antiproliferative effects of IGFBP-3, we chose to investigate the in vivo effects of a minimally modified IGFBP-3 mutant that retained IGF-independent effects (22). More extensively modified IGFBP-3 molecules may have additional unknown effects and potentially confound the interpretation of data generated in vivo.
The growth effect of overexpression of mutant IGFBP-3 was examined in Tg and Wt litter mates derived from crossing hemizygous Tg male mice to CD1 Wt female mice. This paradigm was chosen to eliminate both the potential effect of variable litter size on birth weight and postnatal growth and the possible confounding effects of maternal transgene expression on intrauterine growth. In addition, this comparison is probably the most sensitive method of detecting subtle effects, because non-Tg littermates serve as controls for the hemizygous Tg mice. Under the conditions used in this study, any apparent differences in growth would be solely due to transgene expression. In the two independent Tg strains we observed no significant effect on either birth weight or postnatal growth. Furthermore, our impression was that homozygous PGKmBP-3 mice were similar in size to hemizygous Tg mice.
The failure to demonstrate any effect on birth weight or growth argues strongly against the IGF-independent effects of IGFBP-3 playing a major physiological role in growth in vivo. Most of the in vitro demonstrations of the IGF-independent proapoptotic effects of IGFBP-3 have used cancer cell lines; however, these effects have also been reported with mouse embryo fibroblast (8, 9), porcine embryonic mitogenic cells (25), and human umbilical vein endothelial cells (26). Thus, the IGF-independent effects of IGFBP-3 do not appear to be restricted to cancer cell lines and should have been demonstrable in our Tg mice.
The possibility that other mechanisms could have masked or compensated for the potential negative IGF-independent effects of overexpression of mutant IGFBP-3 in Tg mice needs to be considered. Despite the fact that mutant IGFBP-3 did not bind IGF-I to any significant extent, there was a significant positive correlation between the level of mutant IGFBP-3 and IGF-I levels and between the level of mutant IGFBP-3 and mouse IGFBP-3 levels. This positive correlation does not appear to be completely explained by cross-reactivity between the murine and human IGFBP-3 assays, because PGKBP-3 and CMVBP-3 Tg mice, which have very high levels of human IGFBP-3 in the circulation (19), had murine IGFBP-3 levels similar to those in Wt mice. However, there is the remote possibility that mutant human IGFBP-3 cross-reacted markedly more in the murine IGFBP-3 assay than native human IGFBP-3. Furthermore, cross-reactivity between the murine and human IGFBP-3 assays could not account for the correlation between IGF-I and mutant IGFBP-3 levels, which does not bind IGF-I. Thus, the positive correlation between murine IGFBP-3 and mutant IGFBP-3 levels may indicate increased GH secretion or action in Tg mice. It appears that IGFBP-3 may participate in the regulation of GH secretion or action in an IGF-independent manner. These effects, which are in the opposite direction to those of IGF-I, may be mediated by mutant IGFBP-3 occupying binding sites in extracellular matrix in pituitary tissue and limiting uptake of circulating IGF-I. GH was measured in random single plasma samples from individual mice. There were no significant differences between any of the groups of mice. However, GH is secreted in a pulsatile fashion, and random levels do not necessarily reflect 24-h secretory patterns.
Although the levels of mutant IGFBP-3 mRNA in tissues were high, the levels of mutant IGFBP-3 protein in the circulation were less than those previously observed in PGKBP-3 Tg mice, where the same promoter was used to drive overexpression of intact human IGFBP-3 (19). The concentration of mutant IGFBP-3 protein in the circulation approached approximately 30% the level of endogenous murine IGFBP-3. However, because the mutant IGFBP-3 transgene product is unable to bind IGF-I and form stable ternary complexes, the lower levels of mutant IGFBP-3 may reflect increased clearance of mutant IGFBP-3 from the circulation. After correction for the apparent cross-reaction of murine IGFBP-3 in the human IGFBP-3 assay in Wt mice, the increase in total IGFBP-3 in mutant IGFBP-3 Tg mice was on the order of 10–20%.
Mutant IGFBP-3 was present in the circulation as a high molecular mass complex. Under normal circumstances, the majority of IGFBP-3 in the circulation is present as an approximately 150-kDa ternary complex; however, both IGFBP-3 and IGFBP-5 are capable of forming high molecular mass multimers (27). IGFBP-3 can interact with a variety of known and unidentified serum proteins (28, 29, 30, 31). Because the mutant IGFBP-3 cannot bind IGF-I, it is unable to form stable ternary complex and therefore may be more liable to bind to other serum proteins with lower binding affinities. These higher molecular mass proteins may have limited the distribution to tissues and/or limited the action of mutant IGFBP-3 in certain tissues.
The only significant phenotypic difference between Tg and Wt mice was the modest reduction in brain size observed in the 5012 Tg strain. Interestingly, this observation was supported by a reduction in PCNA-staining cells in the periventricular region of the cerebral hemisphere in embryonic d 18 embryos expressing the mutant IGFBP-3. We chose to specifically examine this region of brain because it is known to be rich in dividing cells that populate other brain areas. Although circulating levels of mutant IGFBP-3 were higher in the 5012 Tg strain, the abundance of transgene-derived mRNA appears to be similar in brain tissue from 5004 and 5012 Tg strains. Buckway and colleagues (22) were unable to demonstrate any significant binding of IGF-II to the Gly56/Gly80/Gly81-mutant IGFBP-3 using dot-blot analysis, Western ligand blotting, solution binding assays, and BIAcore analysis. However, it is possible that the mutant IGFBP-3 has a very low affinity for mouse IGF-II. The modest effect of mutant IGFBP-3 overexpression on brain size could be due to sequestration of mouse IGF-II by the mutant IGFBP-3 that may be important in this tissue, which is particularly sensitive to IGF deficiency (19). Alternatively, the mutant IGFBP-3 may occupy binding sites in the extracellular matrix normally occupied by IGFBP-3/IGF-I binary complex. This latter mechanism could effectively limit the IGF-I or IGF-II available for the proliferating cells of the developing brain.
In summary, the data presented here demonstrate that the IGF-independent effects of IGFBP-3 demonstrable in vitro are unlikely to play a major role in normal growth in vivo and are unlikely to contribute to the growth-retarded phenotype observed when IGFBP-3 is overexpressed in Tg mice. However, it may not be possible to demonstrate IGF-independent effects of mutant IGFBP-3 with the Tg paradigm used here because IGF binding appears to stabilize IGFBP-3, reduces clearance from the circulation, and may reduce proteolysis in tissues. In our Tg model, rapid degradation of mutant IGFBP-3 may have attenuated the IGF-independent effects. Additional studies with non-IGF-binding IGFBP-3 mutants that are able to bind acid-labile subunit and are resistant to rapid clearance and degradation are necessary to fully explore the role of the IGF-independent effects of IGFBP-3 in vivo.
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Footnotes |
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First Published Online November 18, 2004
Abbreviations: CM, Conditioned medium; IGFBP, IGF-binding protein; PCNA, proliferating cell nuclear antigen; PGK, phosphoglycerate kinase I; PGKBP, phosphoglycerate kinase I-binding protein; RNase, ribonuclease; RPA, ribonuclease protection assay; Tg, transgenic; Wt, wild-type.
Received July 14, 2004.
Accepted for publication November 9, 2004.
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regulate transcriptional signaling and apoptosis. J Biol Chem 275:33607–33613This article has been cited by other articles:
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, Murine IGFBP-3; 
, human IGFBP-3. Note the differences in scale for murine and human IGFBP-3. The data represent the mean ± SEM for five to 20 mice/group.
