Adult transgenic mice overexpressing human insulin-like growth factor-binding protein-1 in the liver present reproductive abnormalities in both sexes. In the present work, we have investigated the mechanisms responsible for limiting breeding capacity in these transgenic male mice. Homozygous adult transgenic male mice (3-6 months old) exhibited irregular copulatory behavior and a reduction of the number of pregnancies per female as well as of litter size per pregnancy. Genital tract weight, more specifically epididymal and seminal vesicle weights, were reduced by 45% in homozygous transgenic vs. nontransgenic mice. Homozygous transgenic mice exhibited a 30% reduction of the length of seminiferous tubules (P = 0.007), a 30% decrease in daily sperm production per testis (P = 0.019), and a 50% decrease in the number of spermatozoa in testis (P = 0.037), associated with morphological abnormalities of the sperm heads leading to an approximately 50% reduction of fertilized two-cell eggs (P = 0.002) and of implanted embryos on d 5.5 after mating (P = 0.004). The round spermatids also appeared altered in their morphology. In addition, Leydig cells in homozygous transgenic mice exhibited an altered appearance, with a 1.8-fold increase in lipid droplets in their cytoplasm (P < 0.001). Moreover, the concentration of 3 beta-hydroxysteroid dehydrogenase was 66% lower in testis from transgenics compared with those from normal mice (P = 0.01), leading to a tendency toward lower plasma testosterone levels (P = 0.1). Interestingly, LH concentrations were increased by 40% in transgenic pituitary extracts (P = 0.02), and basal LH secretion by pituitary explants in vitro was increased by 60% in homozygous transgenic vs. normal mice (P = 0.04), suggesting an alteration of LH pulsatile secretion in vivo. In conclusion, these data suggest that the breeding impairment of human insulin-like growth factor-binding protein-1 transgenic males is due at least in part to an alteration of the process of spermatogenesis, leading to a diminution of sperm production and of its quality. Minor impairment of steroidogenesis may also contribute to the reduced reproductive capacity of these animals. Our observations are consistent with the idea that normal spermatogenesis and perhaps also steroidogenesis are dependent on the actions of sufficient concentrations of unbound IGF-I.

Insulin-like growth factor binding protein-3 (IGFBP-3) is the most abundant IGFBP in serum and other biological fluids. Apart from its capacity for specific and high-affinity binding to IGFs, it also has so-called "IGF-independent" activities that modulate cell proliferation and survival/apoptosis. However, the molecular elements of the IGFBP-3 signalling pathway remain obscure. In this study, we investigated the possible implication of phosphatidylinositol 3-kinase (PI 3-kinase) activity in MCF-7 breast carcinoma cells. In cells incubated with IGFBP-3, both total and insulin receptor substrate-1 (IRS-1)-associated PI 3-kinase activities were rapidly stimulated, with maximal effects after 3 and 10min of incubation, respectively. IGFBP-3-induced PI 3-kinase activity was unaffected by the state of IRS-1 tyrosine phosphorylation. Since IGFBP-3 failed to stimulate PI 3-kinase activity in MDA-MB 231 breast carcinoma cells, its effects in MCF-7 cells could be considered as cell-type-specific. Pertussis toxin abolished IGFBP-3-stimulation of PI 3-kinase activity, suggesting that this IGFBP-3 signalling pathway depends upon a pertussis toxin-sensitive G protein. Our results provide further evidence that IGFBP-3 directly triggers a specific intracellular signal in MCF-7 cells.

In an earlier study, we reported that an N-terminal proteolytic fragment (1–95IGFBP-3) corresponding to the first 95 residues of human insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation in a variety of fibroblasts. With a view to investigating its cytostatic capacity in carcinoma cells, we transiently transfected MCF-7 breast adenocarcinoma cells with an expression vector containing 1–95IGFBP-3 cDNA. The transfected cells secreted a hyper-glycosylated form of 1–95IGFBP-3. Twenty-four hours after transfection, cell morphology and viability were similar in control and 1–95IGFBP-3-secreting cells. However, after 48 h, 1–95IGFBP-3-secreting cells were apoptotic, with marked cytoplasmic vacuolation and increased free histones in the cytoplasm. Culture media conditioned by 1–95IGFBP-3-secreting cells also induced morphological changes and apoptosis in wild-type MCF-7 cells, indicating that 1–95IGFBP-3 was responsible for the effects observed. These results provide further evidence that the N-terminal proteolytic fragment of IGFBP-3 has a functional role.

In humans, intrauterine growth retardation (hIUGR) is correlated with an overexpression of insulin-like growth factor binding protein 1 (IGFBP-1). The affected children also present a delay in bone mineralization. In this study, transgenic 12-day-old mutant mice overexpressing human IGFBP-1 hepatospecifically showed a severe growth retardation. Alcian blue and alizarin red S staining of the skeleton revealed mineralization defects at the posterior level of the skull (delayed suture closure) and in appendicular and axial skeleton. Furthermore, microradiographic analysis showed a reduced bone density in the same areas. Thus, overexpressing of hIGFBP-1 demonstrates early postnatal life growth retardation and a delay in mineralization in transgenic mutant mice. These data show the involvement of the IGF/IGFBP system and more particularly IGFBP-1 in the biomineralization process.

The proliferative action of insulin-like growth factors (IGF-I and -II) is mediated via the type I IGF receptor (IGF-IR) and is modulated by their association with high affinity binding proteins, IGFBP-1 to -6. We recently found that, in addition to its ability to bind IGFs, IGFBP-3 also inhibits IGF-IR activation independently of IGF binding and without interacting directly with IGF-IR. Here, we show that IGFBP-3 is capable of blocking the signal triggered by IGFs. Breast carcinoma-derived cells (MCF-7) were stimulated by des(1-3)IGF-I or [Gln(3),Ala(4),Tyr(15),Leu(16)]IGF-I, two IGF analogues with intact affinity for IGF-IR, but with weak or virtually no affinity for IGFBPs, then incubated with IGFBP-3. The activated IGF-IR was desensitized through reversal of its autophosphorylation, following which both phosphatidylinositol 3-kinase and p42(MAPK) activities were depressed. Direct measurement of phosphotyrosine phosphatase activity and reconstitution experiments using tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1) indicated that IGFBP-3 activated a phosphotyrosine phosphatase (PTPase). This action appeared to be peculiar to IGFBP-3 among the IGFBPs, since neither IGFBP-1 nor IGFBP-5 (structurally the closest to IGFBP-3), had any such effect. Several cell lines derived from normal or tumor cells responsive to IGF-I were used to show that IGFBP-3-stimulated PTPase is cell type-specific. Although the precise nature of the phosphatase remains to be determined, the results of this study demonstrate that IGFBP-3 stimulates a phosphotyrosine phosphatase activity that down-regulates the IGF-I signaling pathway, suggesting a major role for IGFBP-3 in regulating cell proliferation.

The mechanisms responsible for reproductive abnormalities in transgenic female mice overexpressing human IGF binding protein-1 (IGFBP-1) in the liver have been investigated. At 2 months of age, none of these transgenic mice exhibited ovarian cyclicity. Genital tract and ovary tissue weights were reduced in transgenic mice, this weight reduction being disproportionate with the reduction of body weight. Examination of ovarian follicular population revealed a marked decrease in the number of corpora lutea and gonadotropin-dependent follicles, suggesting an alteration of terminal follicular growth and ovulation. Stimulation of ovaries by exogenous gonadotropins revealed that ovaries from transgenic mice ovulated less oocytes than nontransgenic mice. This lower responsiveness of ovaries from transgenic mice to gonadotropins was not associated with a decrease in FSH-, LH- or IGF-I receptor expression. Transgenic and nontransgenic mice have similar circulating LH and FSH concentrations at dioestrus, after castration, 46 h after equine CG administration, or 15 min after GnRH injection. However, LH concentrations were 8-fold higher in pituitaries from transgenic vs. nontransgenic mice. Moreover, the size of LH-immunoreactive cells was reduced and their number was increased, suggesting a subtle alteration of LH secretion.Overall, these data indicate that reduced fertility in transgenic female mice overexpressing human IGFBP-1 are mainly due to an alteration of terminal follicular growth leading to a decrease in natural and induced ovulation rate, likely due to an impairment of IGF-I action on follicular cells. Increased circulating IGFBP-1 concentrations may additionally lead to altered GnRH and LH pulsatility and thereby exacerbate the ovulation defect.

Insulin-like growth factor binding protein-3, IGFBP-3, specifically binds to IGFs with high affinity, but it is also capable of modulating the IGF-I signalling pathway or inducing apoptosis independently of its binding to IGFs. The molecular mechanisms underlying the action of IGFBP-3 have not been elucidated. In this study, we have demonstrated that binding of IGFBP-3 to a cell surface receptor in MCF-7 breast carcinoma cells induces a rapid and transient increase in intracellular free calcium. This increase was mediated via a pertussis toxin-sensitive pathway, indicating that the IGFBP-3 receptor may be specifically coupled to a Gi protein. The effect of IGFBP-3 on calcium concentrations was dose-dependent and also occurred when IGFBP-3 was complexed with either IGF-I or heparin, suggesting that the receptor binding site is probably located in the least conserved central domain of IGFBP-3. Neither IGFBP-1, nor IGFBP-5 (structurally the closest to IGFBP-3) altered intracellular calcium concentrations. These results provide evidence that a specific intracellular signal is triggered by IGFBP-3 binding to a cell surface receptor.

BACKGROUND:The insulin-like growth factors (IGFs) are involved in the growth and differentiation of neuroblastoma cells. In all biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBP-1 to -6) which modulate their bioavailability. We previously showed that IGFBP-6 expression is linked to the arrest of growth in neuroblastoma cells, whereas IGFBP-2 is associated with proliferation.PROCEDURE:To study the role of IGFBP-6 in cell growth, we stably IGR-N-91 neuroblastoma cells with a plasmid containing sequences coding for IGFBP-6 under the control of the cytomegalovirus (CMV) promoter.RESULTS:The incidence and size of tumors generated by injecting IGFBP-6-expressing cells into nude mice were reduced by factors of 2 and 5, respectively, as compared with those generated by injection by control cells. Northern blot analyses if xenografts revealed weaker expression of IGF-II, type 2 IGF receptor and IGFBP-2 mRNAs in IGFBP-6-expressing cthan in control xenografts. IGFBP-6 may therefore reduce the expression of IGF-II (which induces tumour development) at a transcriptional level. Conversely, containing IGFBP-2 cDNA under the control of CMV promoter grew three to four times as fast as normal control xenografts. Northern blot analyses revealed weaker expression of intact IGFBP-3 and IGFBP-1 in IGFBP-2-expressing than in control xenografts.CONCLUSIONS:IGFBP-1 and intact IGFBP-3 expression both enhance IGF bioavailability which promotes tumour growth. Although the mechanisms of action of IGFBP-2 and IGFBP-6 remain to be elucidated, an inverse relationship appears to exist between the two binding proteins, IGFBP-2 being involved in proliferation and IGFBP-6 in its arrest.

To the editor:Lassarre and Binoux (1) recently described an assay for intact IGFbinding protein-3 (IGFBP-3) in which serum is acidified to destroy the acid-labile subunit (ALS) and dissociate IGF-IGFBP complexes, IGFs are removed by ultrafiltration, and active IGFBP-3 is quantitated by radiolabeled IGF-I binding.This assay is described as opening new perspectives in investigating IGFBP-3 proteolysis and IGF bioavailability.The authors state that proteolyzed IGFBP-3 loses affinity for IGFs, which are then "redistributed towards the 40-kDa binary IGFBP-IGF complexes" (1), and point out that in human pregnancy, "virtually all circulating IGFBP-3 is degraded."There is some disagreement as to the changes in total serum IGFs through pregnancy, with increases in both IGF-I and IGF-II (2), an increase in IGF-I but not IGF-II (3), and a 15% decrease in IGF-I (4) reported.The proportion of total IGF-I detectable in "free IGF-I" assays approximately doubles in pregnancy from the value of about 1% reported for nonpregnant women (4, 5), although the biological significance of these measurements is unclear.Abundant published evidence refutes the concept that there is a substantial redistribution of IGFs in pregnancy serum away from IGFBP-3-ALS complexes; direct measurement indicates that the proportion of total IGFs in these complexes is unchanged or even slightly increased in pregnancy (6, 7).The size distribution of IGFBP-3 is also unchanged throughout the course of pregnancy, with 85-90% remaining in high molecular weight fractions (8), whereas the total immunoreactive IGFBP-3 concentration is reported to increase ( 8).This provides important information about the distribution of IGFs, since human IGFBP-3 has an unmeasurably low affinity for ALS unless IGF-I or IGF-II is bound to the IGFBP-3 (9).Thus, the unaltered proportion of pregnancy serum IGFBP-3 in high molecular weight complexes indicates that IGFs are also in these complexes.It can be demonstrated that, if there is a substantial loss of affinity in the IGF-IGFBP interaction, there would be a decreased binding of IGFs, and consequently a loss of ALS binding (10).In contrast, IGFBP-3 isolated from nonpregnancy or pregnancy serum by immunoaffinity chromatography, and not depleted of its endogenous IGFs, shows normal affinity for ALS (11).Although IGFBP-3 in pregnancy serum, or isolated from pregnancy serum in the presence of endogenous IGFs, seems to bind and transport IGFs normally, depletion of endogenous IGFs under acidic conditions leads to a loss of affinity as determined by radioligand binding (12)-indeed, this is the basis of the new assay of Lassarre and Binoux (1).While this may in part be explained by the selective effect of altering tyrosine residues 24 and 60 of IGF-I during the iodination process (13), it has been clearly demonstrated that IGF-I affinity is decreased by up to 8-fold, and IGF-II affinity by 2.5-fold, in IGFdepleted pregnancy serum compared with nonpregnancy serum (12,13).Similarly, IGFBP-3 in pregnancy serum is undetectable by ligand blotting after SDS-PAGE, as first demonstrated over a decade ago (14,15).Because an 8-fold loss of affinity from the intact IGFBP-3 affinity of 20 liters/nmol would be insufficient to abolish IGF binding on a ligand blot, it must be assumed that further damage to the proteolyzed protein during electrophoresis, or to the IGF-I during iodination ( 16), contributes to this observation.What is the biological consequence of this limited proteolysis of IGFBP-3, detectable by radioligand binding in vitro after depletion of bound IGFs, but apparently without effect on IGF transport in the circulation?It is certainly possible that this could alter the bioavailability of IGFs to the tissues, and this may eventually turn out to be true, but there has been no definitive experimental proof of this to date, in which pregnancy-proteolyzed IGFBP-3, not subjected to destructive isolation and processing, has been shown to allow IGFs to pass from circulating complexes to their target tissues more readily than unproteolyzed IGFBP-3.Indeed, the definitive experiment is difficult to envisage.Even if this accelerated release of IGFs does occur following proteolysis, it is unclear whether the released IGFs would have increased availability to tissue receptors (local IGFBPs might rapidly re-bind them), or would simply be lost through degradative clearance, for example through the kidneys.Therefore, it appears that there is insufficient evidence at present to state, as Lassarre and Binoux (1) do, that "limited proteolysis of IGFBP-3 is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream."

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream. Its measurement by Western immunoblotting provides only semiquantitative estimation. We have developed a ligand immunofunctional assay (LIFA) for quantifying human (h) intact IGFBP-3 in biological fluids. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration, and a monoclonal antibody specific to the first 160 amino acids of IGFBP-3 is used to capture hIGFBP-3 in a solid-phase assay. The complex is then incubated with (125)I-IGF-I, which binds to intact IGFBP-3 but not to its proteolytic fragments. Binding specificity was demonstrated in competition experiments with unlabeled IGF. Nonspecific binding was 1.4%. The fragments comprising residues 1-160 and 1-95 of recombinant hIGFBP-3 [corresponding to the major proteolytic fragments of approximately 30 kDa and (glycosylated) 20 or (nonglycosylated) 16 kDa detected in serum by Western immunoblotting, respectively] fail to bind (125)I-IGF-I when complexed with the monoclonal antibody. Similarly, no binding of (125)I-IGF-I was obtained in the LIFA when applied to plasmas from pregnant women during the final 3 months of pregnancy, where the characteristic 42- to 39-kDa doublet of intact IGFBP-3 is undetectable. The standard curve was established using a pool of plasmas (EDTA) from healthy adults, for which standardization with glycosylated recombinant hIGFBP-3 yielded an intact IGFBP-3 content of 2 microg/mL. The dynamic range of the LIFA was 0.50-3.75 microL equivalent of the plasma pool in a total volume of 300 microL per assay tube, with a sensitivity threshold of approximately 1 ng intact IGFBP-3. Unknown plasma samples were studied at three concentrations. Intra- and interassay variations were 3.6% and 4%, respectively. In 31 healthy adults, the mean plasma concentration of intact IGFBP-3 was 2.24 +/- 0.08 (SEM) mg/L, and that of total IGFBP-3 measured by immunoradiometric assay was 3.27 +/- 0.14 mg/L. The calculated mean proportion of proteolysed IGFBP-3 was 29.4 +/- 1.9%. In these subjects, a close correlation was found between intact and total IGFBP-3 (r = 0.71, P = 0.0001). The LIFA for IGFBP-3, therefore, provides accurate and sensitive measurement of intact IGFBP-3, the form with the functional capacity to sequester IGF-I in the bloodstream by association with the acid-labile subunit in 140-kDa complexes. In combination with total IGFBP-3 and IGF-I assays, the LIFA opens new perspectives in investigating the regulation of IGFBP-3 proteolysis and IGF-I bioavailability in man.

Insulin-like growth factors (IGFs) produced in the brain are known to participate in brain development via activation of the type 1 IGF receptor. IGF binding proteins (IGFBPs) modulate the cellular action of IGFs and some are expressed in the fetal brain. Under normal conditions IGFBP-1 is not one of these, but IGFBP-1 expression obtained via transgenesis using ubiquitous promoters affects brain development. In earlier work, we established a model of transgenic mouse in which liver-specific IGFBP-1 expression begins during fetal life. The repercussions of this IGFBP-1 over-expression include reproductive defects, ante- and perinatal mortality and post-natal growth retardation, the extent of which is related to the degree of transgene expression. Unexpectedly, during the first 2 months of postnatal life, there were some cases of head enlargement revealing hydrocephalus among homozygotes, frequently associated with motor disorders. Brain sections showed dilatation of the lateral ventricles in 10 out of 15 homozygotes examined. Histologically, dilatation was evident in four out of nine heterozygotes. Brain weight in transgenics was relatively less reduced than the weights of other organs. Hence, brain weight/body weight ratios were normal in heterozygotes and on average higher than normal in homozygotes. The width of the cerebral cortex was reduced in homozygotes, with disorganized neuronal layers. The corpus callosum was underdeveloped, particularly in homozygotes. The area of the hippocampus was reduced in homozygotes and one-third of the heterozygotes, with a short and thick dentate gyrus in the former. Similar anomalies have been reported in mice with disruption of the igf-I gene and in a model of transgenic mice over-expressing IGFBP-1 in all tissues, including the brain. Hydrocephalus was not mentioned in these reports, raising the possibility that insertional mutagenesis may have been involved in our mice. Nevertheless, our observations indicate that hepatic over-expression of IGFBP-1 may have endocrine effects on brain development.

Background. The growth hormone (GH)/insulin-like growth factor (IGF) system is thought to participate in the glomerulosclerosis process. Because IGF-binding proteins (IGFBPs) modulate IGF actions and hence GH secretion, this study assessed whether mice transgenic for human IGFBP-1 have altered susceptibility to glomerulosclerosis.Methods. A line of transgenic mice that express human IGFBP-1 mRNA in the liver under the control of the alpha 1-antitrypsin promoter has been obtained, and morphological changes in the kidney tissue were assessed. Glomerulosclerosis was identified using light microscopy, light microscopic morphometry, and electron microscopy. Extracellular matrix components were analyzed by immunohistochemistry.Results. There was a marked increase in mesangial extracellular matrix area in homozygous transgenic mice at three months of age as compared with heterozygous transgenic mice and nontransgenic littermates. These changes were not associated with alterations in glomerular volume or cellularity. The expansion of extracellular matrix area was related to a marked increase in laminin and type IV collagen and to the appearance of type I collagen.Conclusions. These observations indicate that the enhanced expression of IGFBP-1 may result in the development of glomerulosclerosis without glomerular hypertrophy. The changes are potentially related to a decrease in IGF-I availability and/or to an IGF-I-independent role of IGFBP-1.

The insulin-like growth factor (IGF) system is a major regulator of somatic growth in vertebrates. Both ligands (IGF-I and IGF-II) signal via the same IGF receptor (IGF-IR). Classical IGF-IR invalidation is lethal at birth, so that conditional models are needed to study the postnatal role of this receptor. To establish a genetically inducible invalidation of IGF-IR, we targeted the IGF-IR gene using a construct that introduced a neomycin resistance cassette into intron 2, leaving the rest of the gene intact. This neomycin resistance cassette interfered with the processing of the primary transcript, resulting in there being 12% fewer IGF-binding sites at the cell surface in heterozygous mice and 41% fewer in homozygous mice. Hetero- and homozygous offspring grew more slowly than their wild-type littermates. This difference was noticeable from 4 weeks after birth and was significant from 5 weeks after birth in males. In females, the effect on postnatal growth of insertion of the neo cassette was not significant. In males, IGF-I levels increased moderately (+26%) but significantly, indicating effective feedback regulation of the IGF system. IGF-binding protein-4 (IGFBP-4) levels, estimated by Western ligand blotting, were low in homozygotes (-38%), whereas IGFBP-1, -2, and -3 levels were unaffected. In females, IGF-I and IGFBP-1, -2, -3, and -4 levels did not differ significantly among heterozygous, homozygous, and wild-type animals. We investigated the molecular mechanism involved and characterized two RNA-splicing events that could account for the decrease in IGF-IR. The phenotype of these mice developed exclusively postnatally, and body proportions were maintained. IGF-IRneo mice constitute a new model for human postnatal growth deficiency.

The regulation of insulin-like growth factor binding protein (IGFBP)-4 proteolytic degradation by insulin-like growth factors (IGFs) has been largely studied in vitro, but not in vivo. The aim of this study was to investigate the involvement of IGFs, IGFBP-2, IGFBP-3, and IGFBP-3 proteolytic fragments in the regulation of IGFBP-4 proteolytic activity in ovine ovarian follicles. Follicular fluid from preovulatory follicles contains proteolytic activity degrading exogenous IGFBP-4. The addition of an excess of IGF-I enhanced IGFBP-4 proteolytic degradation, whereas addition of IGFBP-2 or -3 or monoclonal antibodies against IGF-I and -II dose dependently inhibited IGFBP-4 proteolytic degradation. IGF-I and IGF-II, but not LongR3-IGF-I, reversed this inhibition in a dose-dependent manner. C-terminal, but not N-terminal, proteolytic fragments derived from IGFBP-3 (aa 161-264), as well as heparin-binding domain-containing peptides derived from the C-terminal domain of IGFBP-3 and -5 also induced the inhibition of IGFBP-4 proteolytic degradation. Other heparin-binding domain-containing peptides derived from the connective tissue growth factor (CTGF) and from proteins not related to IGFBP, heparan/heparin interacting protein (HIP) and vitronectin, but not from p36 subunit of annexin II tetramer, inhibited IGFBP-4 degradation. Furthermore, IGFBP-3, mutated on its heparin-binding domain, was not able to inhibit IGFBP-4 proteolytic degradation. So, in ovine preovulatory follicles, IGFBP-4 proteolytic degradation both 1) depends on IGFs, and 2) is inhibited by IGFBP-3 via its C-terminal heparin-binding domain as well as by heparin-binding domain containing peptides. These data suggest that in early atretic follicles, the increase in IGFBP-2 participates in the decrease in IGFBP-4 degradation. In late atretic follicles, the increase in the levels of C-terminal IGFBP-3 proteolytic fragments, generated by IGFBP-3 degradation, as well as the increase in IGFBP-5 expression would strengthen the inhibition of IGFBP-4 degradation. This inhibition might be partly mediated by direct interaction of IGFBP-4 proteinase(s) and heparin-binding domain within the C-terminal region from IGFBP-3 and -5.

Insulin‐like growth factors (IGF‐I and ‐II) play an active role in cell proliferation. In biological fluids, they are non‐covalently bound to high‐affinity binding proteins (IGFBPs), at least 6 species of which have been identified to date, but with poorly defined functions. One of these IGFBPs, IGFBP‐2, is secreted by most cell lines and appears to be involved in cell proliferation. A human epidermoid carcinoma cell line, KB 3.1, which produces IGFBP‐1 and ‐3 and small amounts of IGFBP‐4, but no IGFBP‐2, was stably transfected with an expression vector comprising IGFBP‐2 complementary DNA (cDNA), whose expression was placed under the control of the constitutive and ubiquitous cytomegalovirus promoter. After an s.c. injection of these IGFBP‐2‐expressing KB 3.1 cells into nude mice, tumours developed more quickly than in controls, they were 3 to 4 times larger and grew about 3 times as fast. Concomitant with IGFBP‐2 expression in these tumours, were a decrease in IGFBP‐1 expression and an increase in IGFBP‐3 proteolysis, both of which increase the bioavailability of the IGF‐II produced by the cells. The increased IGFBP‐3 proteolysis most probably resulted from amplified expression of tissue‐type plasminogen activator (t‐PA) and depression of its inhibitor (PAI‐1) observed in IGFBP‐2‐expressing xenografts. Our findings suggest that IGFBP‐2 plays a role in this model of experimental tumorigenesis via a mechanism that remains unclear, but appears to involve increased protease activity and IGF‐II bioavailability. Int. J. Cancer 77:874‐879, 1998.© 1998 Wiley‐Liss, Inc.

In a previous study, the biphasic effect of increasing dosages of recombinant human insulin-like growth factor binding protein-3 (rhlGFBP-3) on proliferation in the prostate carcinoma PC-3 cell line (stimulation followed by depression) was shown to reflect changes in the bioavailability of IGF-II secreted by the cells, IGF-II being the major factor responsible for their autocrine growth. These changes depend on the extent of IGFBP-3 proteolysis induced by serine proteases, in particular, plasmin. In order to examine the mechanism of action of IGFBP-3, we investigated the effects of its two major fragments isolated by HPLC following limited proteolysis by plasmin in vitro. The predominant fragment with an apparent molecular mass of 22–25 kDa in SDS-PAGE (under non-reducing conditions) had previously been shown to retain weak affinity for IGFs, whereas the other fragment of 16 kDa lost all such affinity. From their recently determined amino acid sequences, these fragments correspond to the first 160 and 95 residues, respectively, of IGFBP-3. 0.5–5 nM intact rhlGFBP-31–264, when pre-incubated with 5 nM rhlGF-II, dose-dependently inhibited (up to 100%) its mitogenic effect, via sequestration owing to its strong affinity for IGF-II. The same concentrations of the larger fragment (IGFBP-31–160) elicited only weak inhibition (up to 30%), coherent with its weak affinity. The smaller fragment (IGFBP-31–95) provoked total inhibition despite its lack of affinity for IGFs and therefore by an IGF-independent mechanism. PC-3 cells in serum-free medium were weakly stimulated by 5 nM intact IGFBP-3. This had previously been shown to be related to its proteolysis and the ratio of proteolysed to intact IGFBP-3. At the same concentration, IGFBP-31–160 stimulated this proliferation by a factor of 5–7, whereas IGFBP-31–95 totally suppressed it. 5 nM IGFBP-31–95 inhibited the mitogenic action of 1% fetal calf serum by 80%, but by only 25% in the presence of an antibody blocking the type 1 IGF receptor. Its inhibition is therefore exerted principally, but not exclusively, via the IGF signalling pathway. Our data indicate that the IGFBP-3 fragments composed of residues 1–160 and 1–95 are biologically active on PC-3 cells and that their opposite actions may account for the events observed when IGFBP-3 is proteolysed in the cell environment. These proteolytic fragments may therefore play a role in the development of prostate adenocarcinomas in vivo.

Study of the in vivo functions of the insulin-like growth factor binding proteins (IGFBPs) is complicated by their variety (six molecular species) and the differences in their expression related to tissue of origin and stage of development. To investigate the physiological role of IGFBP-1 in the bloodstream, we induced hepatic overexpression of IGFBP-1 in transgenic mice, placing human IGFBP-1 (hIGFBP-1) cDNA under the control of the alpha1-antitrypsin promoter so as to obtain liver-specific expression. Five transgenic founder mice were raised, only two of which (lines 124 and 149) produced transgenic offspring. Northern blotting revealed transgene expression exclusively in the liver during fetal life and unchanged through to adulthood, whereas expression of the endogenous gene was undetectable beyond 10-15 days postnatally. hIGFBP-1 was detected by western immunoblotting in the plasma of transgenic mice and IRMAs yielded mean concentrations of 2.41 +/- 0.33 ng/ml and 13.69 +/- 1.42 ng/ml in homozygous animals of lines 124 and 149, respectively. In the latter, IGFBP-1 levels were distinctly higher than in heterozygotes (2.99 +/- 0.39 ng/ml), P < 0.0001. These levels remained stable in each given animal and did not change with age. Plasma concentrations of IGF-I measured in line 149 exhibited the well-known profile of an increase from birth up to puberty. Values for heterozygotes were similar to those for wild-type mice, with adult levels (544 +/- 98 ng/ml) slightly below those of controls (630 +/- 56 ng/ml), P < 0.05. In homozygotes they were distinctly lower, with adult levels of 370 +/- 75 ng/ml, P = 0.001. In heterozygous and homozygous adults, there was a negative correlation between IGF-I and IGFBP-1 concentrations (r = 0.8, P < 0.0001), suggesting a link between transgene expression and IGF-I levels. Study of body weight gain in line 149 revealed growth retardation within the first weeks after birth, which was marked in homozygous males and females (P < 0.001) but also present in heterozygous males (P = 0.002), indicating some relationship with transgene expression. In addition, body weight in adult mice was negatively correlated to plasma concentrations of IGFBP-1 (r = 0.7, P < 0.0001). Reproductive function also appeared to be severely affected, especially in homozygous females: mating that failed to result in pregnancy in half of the homozygous females crossed with nontransgenic males, suggestive of impaired fertilization or implantation; interrupted or prolonged pregnancies with fetal and neonatal death. Litter size was reduced in transgenic females (by about half in homozygotes) and in nontransgenic females mated with homozygous males, resulting from pre- or neonatal mortality. Moreover, deaths occurred within the first 5 days of life, with an incidence of approximately 50% in the litters of homozygous females, 12-18% among heterozygotes mated with nontransgenic or heterozygous males, respectively, and 30% among those mated with homozygous males. These results, suggesting that fetal transgene expression largely accounted for ante- and perinatal mortality, were confirmed by the predominance of homozygotes among those that could be analyzed genetically. Similarly impaired reproductive function was seen in line 124, but to a lesser degree. Although the mechanisms responsible for these disorders remain to be determined, our results indicate that permanent and uncontrolled hepatic expression of IGFBP-1, even at low levels, affects fertility in females and both ante- and postnatal development.

Is the title of this Commentary a question which is still relevant? The dependence of insulin-like growth factor-I (IGF-I) on growth hormone (GH), which formed the basis of the classical somatomedin hypothesis (1), is now almost old hat. After insulin-like growth factor- binding protein-3 (IGFBP-3) was identified in serum as the carrier protein for most circulating IGFs, its variations were shown also to be regulated by GH secretion (2, 3). Serum ALS (acid-labile subunit), which binds IGFBP-3 associated with IGF-I or -II to form 140 kDa complexes (4), is age- and GH-dependent in the same way as are IGF-I and IGFBP-3. However, over recent years, the use of recombinant IGF-I in both experimental investigation and treatment of dwarfism resulting from genetic disorders of the GH receptor (Laron syndrome) has raised controversy as to the respective physiological roles of GH and IGF-I. It was recently proclaimed that 'long-term IGF-I therapy in patients with Laron syndrome consistently failed to