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Original Studies |
Endocrine Genetics Laboratory, the Montreal Children’s Hospital Research Institute, and Department of Pediatrics, McGill University, Montreal, Quebec, Canada H3H 1P3
Address all correspondence and requests for reprints to: Constantin Polychronakos, M.D., F.R.C.P. (C), Endocrinology and Metabolism, Montreal Children’s Hospital, 2300 Tupper Street, Montreal, Quebec, Canada H3H 1P3. E-mail: mc97{at}musica.mcgill.ca
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Abstract |
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By quantitative immunoblot, the soluble IGF-II/MPR in serum and AF was found to undergo developmental regulation that parallels that of the rodent, although it is much less pronounced quantitatively. The highest levels are seen in midgestation, decreasing at term in both serum and AF. In serum, they further decline to one-third of the preterm levels by adulthood.
As part of characterizing AF IGF-II binding, we also show that the prominent high-molecular mass IGF-II-binding protein in preterm AF is GPC3, a protein of the glypican family, recently cloned because its mutations predispose to Wilms’ tumor. For the first time, we show that IGF-II binding to this protein is saturable, and therefore specific. These findings should promote understanding of the role of IGF-II and its binding proteins in human development.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the mouse, the genes encoding both IGF-II (Igf2) and IGF-II/MPR (Igf2r) are parentally imprinted in the opposite direction: Igf2 is expressed exclusively from the paternal copy in most tissues (12), whereas Igf2r expressed only from the maternal one (13). In humans, imprinting of the IGF2 gene is conserved (14), but imprinting of the IGF2R gene is polymorphic: IGF2R is expressed from both copies in most individuals (15, 16), although quantitatively, the maternal copy is expressed at considerably higher levels in about one-third of normal human fetuses (15).
It seems that differences between species also exist in the patterns of gene expression. In the rat and mouse, a marked developmental regulation is seen in the expression of IGF-II/MPR. Abundance of the molecule rises in late gestation to values 20–40 times higher than in the adult animal, with a rapid fall to adult levels in the first 2 weeks of postnatal life (17, 18).
The human IGF-II/MPR does not seem to follow a similar developmental regulation. Funk et al. (19) have shown no substantial differences in levels of immunoreactive tissue receptor between gestational ages ranging from 18 weeks to term and postnatal ages up to 18 months. This study, however, used samples from stillborn fetuses and infants dying of various pathological conditions, and may be subject to confounding from the pathological processes themselves, terminal events before death, and postmortem autolysis. The study reported here aimed at reexamining this question in samples from healthy individuals, using the soluble form of IGF-II/MPR.
Most of the IGFII/MPR molecule is extracytoplasmic. This portion is enzymatically cleaved at the carboxy-terminal domain in tissues, resulting in a soluble molecule that retains the ability to bind IGF-II and M6P (20, 21, 22). Developmentally regulated changes in the levels of membrane-associated receptor are reflected in the abundance of the soluble form in rat serum (20).
Soluble IGF-II/MPR has been found in human serum (21), but its developmental regulation has not been examined and neither has the presence of soluble IGF-II/MPR in amniotic fluid (AF). In this study, we demonstrate the presence of a 220-kDa soluble IGF-II/MPR form in AF identical to that previously described in serum, which is subject to developmental regulation parallel to that seen in the rat, albeit of much lesser magnitude. In addition, in preterm AF, we detected a soluble 250-kDa form of GPC3, an IGF-II-binding glypican (23), recently identified because its mutations may predispose to Wilms’ tumor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Second-trimester AF was obtained at 16 weeks of gestation for diagnostic amniocentesis, which subsequently did not reveal any abnormality in the fetus. Term AF (38–42 weeks of gestation) was obtained at the time of repeat cesarean sections in otherwise normal pregnancies, and term fetal blood was collected from the placental side of the umbilical cord. Preterm fetal blood was collected at the time of induced abortions, performed for reasons other than fetal pathology. For all samples studied, consent was obtained from the mothers.
Purification
The soluble IGF-II/MPR was purified from crude AF by chromatography on a phosphomannan affinity column provided to us by Dr. S. Kornfeld’s laboratory, Washington University, St. Louis, MO. Preterm AF with added protease inhibitors (1 mmol/L phenylmethylsulfonylfluoride, 1–2 µg/mL aprotinin, and 1–2 µg/mL leupeptins) was centrifuged at 2,000 x g for 5–10 min to remove cells and cell debris. After extensive washing of the column by washing buffer [50 mmol/L Imidozole (pH 7), 150 mmol/L NaCI, 0.05% Triton X-100, 5 mmol/L Na-ß-glycerophosphate, and 0.02% sodium azide], 10–15 mL treated AF was applied to 2 mL of the gel at a speed of 2–3 drops/min at 4 C. After washing with 15 column vol of the buffer, the immobilized IGF-II/MPR was eluted with 0.01 mol/L M6P (Sigma, St. Louis, MO) in washing buffer, collecting fractions of 1 mL. The pooled eluate was either used for IGF-II binding assay or further desalted and concentrated by spin filter (10,000 NMWL, Millipore, Nepean, Ontario) at 2,000 x g at 4 C. The concentrated receptor was lyophilized and redissolved in 60–80 µL loading buffer for Western blot.
IGF-II binding
Human recombinant IGF-II (a gift from Eli Lilly, Indianapolis, IN) was radiolabeled to an SA of 100–150 nCi/ng, using the chloramine-T method followed by purification by gravity flow chromatography (Econo-Pac columns, Bio Rad, Missisauga, Ontario).
Samples of 40 µL purified IGF-II/MPR were incubated with 20,000 cpm radiolabeled IGF-II and 0–100 ng/mL unlabeled peptide in a total vol of 0.12 mL for 60 min, at room temperature, in a 25-mmol/L Tris-HCl buffer, (pH 7.4), containing 10 mmol/L Mg++.
Bound IGF-II was separated from free by precipitation of the
receptor-ligand complex with 3 mL of 12% polyethylene glycol, in the
presence of 0.2 mg bovine serum globulin as a carrier. The mixture was
centrifuged at 2,000 x g for 30 min, the supernatant
was decanted, and the pellet was counted in a 
counter.
Radioactivity found in the presence of 100 ng/mL of unlabeled IGF-II
was considered nonspecific and was subtracted from all values.
The saturation analysis data were expressed as Scatchard plot, by plotting the bound to free ratio as a function of bound.
Western ligand blot
This technique visualizes soluble IGF binding proteins or the IGF-II/MPR, by Western blotting using radiolabeled IGF-II as a probe. The protocol used by Hardouin et al. (24) was employed. Briefly, 6 µL crude AF or 5 µL of the purified IGF-II-R (equal to 1.5 mL unpurified AF) or 3 µL serum was boiled in 2% SDS (nonreduced) and subjected to SDS-PAGE, using a 7.5% polyacrylamide gel in a Bio Rad miniapparatus. The gel was electroblotted to a nitrocellulose membrane using the Bio Rad apparatus. After washing and blocking with 5% skim milk, the membrane was incubated overnight at 4 C with 1.35 x 106 cpm radiolabeled IGF-II in 10 mL Tris-Mg buffer, pH 7.4. Nonspecific bands were identified by absence of suppression when coincubated with 100–150 ng/mL unlabeled IGF-II.
After extensive washing in PBS buffer, the membrane was applied on photographic film for autoradiographic visualization of the bands.
Western immunoblot
This technique was used to distinguish IGF-II/MPR from other proteins with IGF-II-binding site and similar molecular mass, and to quantitate the amount of the receptor during different developmental stages. After transfer, the membranes were briefly washed in 50 mM tris-buffered saline, pH 7.5 (TBS), and blocked with 5% skim milk in TBS at room temperature for 1 h. Then, the membranes were immunoblotted by 1–2 µg/mL of either anti-IGF-II/MPR antibody (antibovine polyclonal antibody, highly cross-reacting with the human receptor, kindly provided by Dr. S. Kornfeld’s Laboratory, Royal College of Physicians, Canada) or anti-GPC3 antibody (kindly provided by Dr. D. Schlessinger, Washington University) at 4 C in 5% skim milk. The time of incubation with the antibody was 2–3 days for AF and 1–2 days for serum. After two washes with TBST (TBS buffer containing 0.1% tween 20) and two additional washes in 5% milk solution, the membranes were incubated with donkey antirabbit Ig peroxidase-conjugated second antibody (Jackson ImmunoResearch, West Grove, PA), diluted 1:10,000 in 5% milk, at room temperature for 1 h. After incubation with the second antibody, the membranes were washed four times in TBST for 15 min each and processed for chemiluminescence with the Boehringer Mannheim (Indianapolis, IN) chemiluminescent kit, according to the manufacturer’s instructions. The membranes were exposed to x-ray films for visualization of chemiluminescence of the bands.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When eluted fractions from the phosphomannan affinity column were
analyzed for IGF-II binding, a sharp peak was seen after the
application of free M6P (Fig. 1
). This
directly demonstrates the presence of functional binding sites for both
M6P and IGF-II in the soluble form of the receptor in AF.
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When purified receptor was diluted back to the original
concentration in AF and compared with the crude sample, total binding
was much lower (data not shown), probably because of the presence of a
large amount of IGF-BP1 and other IGF-II binding proteins (see below),
which is also precipitated by 12% polyethylene glycol, albeit
incompletely. The equilibrium affinity constant of the pure material
was estimated at 1.57 ± 0.29 x 109
mol/L-1 (mean ± SE, n = 4). This is
in the same range as that for the membrane-associated receptor (Fig. 2).
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The soluble form of IGF-II/MPR in human AF was characterized by
Western immunoblot with an antibody specific for IGF-II/MPR. A single
220-kDa band was observed in both purified receptor and crude AF (Fig. 3B). Immunoblot in serum also showed the
same 220-kDa band as that seen in AF (Fig. 3C).
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). The m6p-affinity
purified receptor preparation showed a single 220-kDa IGF-II-binding
band (R1 in Fig. 3A) with complete removal of all other IGF-II binding
proteins, including IGF-BP1. However, careful examination of the
high-molecular mass band showed that the size of the IGF-II-binding
band in purified receptor was only 220 kDa, distinctly smaller than
that in crude AF (Fig. 3A). The larger (
250 kDa) band seen in
crude AF is unlikely to be an isoform of IGF-II/MPR because it
cannot bind to phosphomannan column and does not react with the
anti-IGF-II/MPR antibody. It is the prominent high-molecular IGF-II
binding protein in preterm AF; and, in ligand blots, it overshadows the
220-kDa band corresponding to immunoreactive receptor, which can be
seen only in the purified and concentrated preparations (R1 and R2 in
Fig. 3A). A soluble form of GPC3 is present in human AF
The characteristics of the 250-kDa protein discussed above are
compatible with GPC3, a protein of the glypican family, recently shown
to bind IGF-II (23). To better characterize the identity of the two
IGF-II-binding bands, we examined preterm AF by Western immunoblot,
using anti-IGF-II/MPR and anti-GPC3 antibodies. As predicted, when the
parallel membranes were matched, the single band of 220 kDa was
visualized by antibody specific for the receptor, and the band of
approximately 250-kDa was visualized by the specific anti-GPC3 antibody
(Fig. 4A). In addition, the 250-kDa band
labeled by 125I-IGF-II in the ligand blot matched the band
labeled by anti-GPC3 antibody (Fig. 4B, lanes 3–6). These results
confirm the presence of a soluble form of GPC3 molecule in preterm AF.
It has been shown that GPC3 binds IGF-II, but competition studies were
not done to demonstrate specificity of binding (23). In our experiment,
we showed that 125I-IGF-II binding to the GPC3 band was
displaced by approximately 10-8 mol/L unlabeled IGF-II,
indicating that it is a saturable, high-affinity interaction (Fig. 4B, lanes 1–4).
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We first compared the concentration of the soluble receptor in AF
in midgestation vs. term (Fig. 5A). The level of the receptor was one
fourth at term, compared with the level at 16 weeks of gestation, by
quantitative analysis (Fig. 6A). Although
some variation of the level was observed in individuals, a
statistically significant decrease between 16 and 40 weeks was obvious.
Further studies showed that this developmental regulation also was
reflected in serum (Fig. 5B, C). In the serum, a constant decrease was
seen from midgestation to adulthood (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown, for the first time, that there is developmental down-regulation of the IGF-II/MPR in human serum and AF, albeit not as dramatic as in the rat. In the rat, the soluble receptor in serum accurately parallels the developmental regulation of the abundance of the membrane-associated form in virtually all tissues (20), and a correlation between the two in cultured cells has been found in vitro (25, 26). If proteolytic cleavage is a disposal and degradation mechanism proceeding at a steady, unmodulated rate, then the soluble form can be used as an index of the overall abundance of the membrane-associated receptor. If such is the case, then this developmental regulation of the soluble receptor in the human circulation reflects the pattern of developmental regulation of the receptor in tissues. Using autopsy samples, Funk et al. (19) found evidence suggesting subtle developmental regulation in kidney and probably in other tissues. However, because of the variability of the results (likely the consequence of autolysis and/or underlying disease), this trend was not statistically significant in their hands. Our use of the soluble receptor, obtained from healthy subjects, allowed us to observe an overall trend at a statistically significant level.
The developmental regulation of IGF-II/MPR suggests a specific functional importance in fetal life and may be related to parental imprinting of IGF2R, which is also subject to developmental regulation (15, 27) and whose conservation between rodent and human is similarly weak (15, 27, 28). The biological purpose of silencing the gene copy derived from the parent of a specific sex is not known; but Haig and Graham (29) have speculated that, in the specific case of the imprinted Igf2-Igf2r pair, it is related to nutrient exchange between mother and fetus. IGF-II promotes fetal growth, whereas IGF-II/MPR inhibits it; large size is one of the features of the Igf2r knockout (11). The very high level of IGF-II expression in the rodent fetus has the evolutionary advantage of a larger newborn, but it may be deleterious to the mother by depleting too much of her resources. If so, allelic variants that promote higher fetal IGF-II levels would most efficiently prevail in evolution by developing mechanisms of silencing themselves when transmitted by the mother. By a converse reasoning, genes such as Igf2r, which favor the mother in this competition for nutrients, would gain evolutionary advantage by silencing themselves when paternally transmitted. Special considerations in humans (anatomy of birth canal, single-fetus gestations) obviates much of the evolutionary advantage of enhanced fetal growth. This may be the evolutionary basis of much smaller differences between fetal and adult IGF2 expression in humans (30, 31, 32) than in rodents (33) and also of the similarly blunted developmental IGF2R regulation reported in this paper, as compared with that seen in the rat (17, 20). The complete silencing of the paternal Igf2r copy in the mouse embryo (13) [although in the human, only about one third of fetuses repress it partially (15, 27)] may reflect the same evolutionary process. In both species, the repressed paternal copy seems to be reactivated at birth (11, 27).
Another important finding reported here is the detection, in preterm AF, of a soluble form of GPC3 that specifically binds IGF-II and is 20- to 30-kDa larger than the soluble IGF-II receptor. GPC3 is a proteoglycan of the glypican family. It was positionally cloned as the gene whose mutations cause the Simpson-Golabi-Behmel syndrome, a state of fetal overgrowth similar to the Beckwith-Wiedeman Syndrome (23). Besides increased fetal growth, a common feature of the two syndromes is a high incidence of Wilms’ tumor, an embryonic kidney neoplasm in which IGF-II overexpression seems to play an etiologic role (reviewed in Ref.27). The demonstration, by Pilia et al. (23), that GPC3 binds IGF-II in a ligand blot, is consistent with a central role for IGF-II in both fetal growth and Wilms’ tumor. However, the specificity of the GPC3-IGF-II interaction was not demonstrated by competition with unlabeled hormone, an important consideration for such a sticky peptide as IGF-II. The present study provides the first experimental evidence that IGF-II binding by GPC3 is specific.
Our demonstration of the presence of two soluble IGF-II-binding proteins in human body fluid would provide insights into understanding the role of IGF-II and the proteins that modulate its action in fetal growth and embryonic malignancy.
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Acknowledgments |
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Footnotes |
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2 Performed this work as a research fellow of the McGill
University-Montreal Children’s Hospital Research Institute.
Received April 17, 1997.
Revised October 1, 1997.
Accepted October 23, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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