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Mol Endocrinol. May 2008; 22(5): 1213–1225.
Published online Feb 7, 2008. doi:  10.1210/me.2007-0536
PMCID: PMC2366183

Insulin-Like Growth Factor (IGF) Binding Protein-4 Is Both a Positive and Negative Regulator of IGF Activity in Vivo

Abstract

IGFs are required for normal prenatal and postnatal growth. Although actions of IGFs can be modulated by a family of IGF-binding proteins (IGFBPs) in vitro, these studies have identified a complicated pattern of stimulatory and inhibitory IGFBP effects, so that understanding relevant aspects of IGFBP action in vivo has been limited. Here we have produced a null mutation of one specific IGFBP, IGFBP-4, which is coexpressed with IGF-II early in development. Surprisingly, mutation of IGFBP-4, believed from in vitro studies to be exclusively inhibitory, leads to a prenatal growth deficit that is apparent from the time that the IGF-II growth deficit first arises, which strongly suggests that IGFBP-4 is required for optimal IGF-II-promoted growth during fetal development. Mice encoding a mutant IGFBP-4 protease (pregnancy-associated plasma protein-A), which facilitates IGF-II release from an inactive IGF-II/IGFBP-4 complex in vitro, are even smaller than IGFBP-4 mutant mice. However, the more modest IGFBP-4 growth deficit is completely restored in double IGFBP-4/pregnancy-associated plasma protein-A-deficient mice. Taken together these results indicate not only that IGFBP-4 functions as a local reservoir to optimize IGF-II actions needed for normal embryogenesis, but also establish that IGFBP-4 proteolysis is required to activate most, if not all, IGF-II mediated growth-promoting activity.

THE IGFs (IGF-I and IGF-II) regulate cellular proliferation, survival, and differentiation (1,2). IGF bioactivity is modulated by a family of IGF-binding proteins (IGFBPs) (3) that can modulate IGF activity in vitro by high-affinity binding to IGFs, which often prevents IGFs from binding to their cell membrane receptors. In addition, some IGFBPs bind to components of the extracellular matrix (IGFBP-2, -3, and -5) or the cell membrane (IGFBP-1, -2, -3, and -5), thus providing a potential mechanism to concentrate IGF activity within discrete regions (4). IGFs bound to IGFBPs can be released after proteolytic cleavage of IGFBPs by specific proteases (5,6). Proteases that cleave IGFBP-2 (7), IGFBP-3 (8), IGFBP-4 (9), and IGFBP-5 (10) have been reported, although the absolute specificity for IGFBPs has not been established.

IGFBP-4 is the smallest IGFBP and is unique in having two extra Cys residues in the variable L-domain encoded by exon 2 (11). IGFBP-4 also contains an N-linked glycosylation site and commonly exists in biological fluids as a doublet: a 24-kDa nonglycosylated form and a 28-kDa glycosylated form (12). Other characteristics of IGFBP-4 are that it inhibits IGF action in vitro and in vivo (13,14,15), that it lacks the ability to associate with cell surface, and that it is generally coexpressed with IGF-II during development (12). IGFBP-4 can bind both IGF-I and IGF-II although it is not known whether either or both of these interactions have relevance in vivo. Because mutations of IGF-I and IGF-II lead to growth deficits with quite different times of onset and progression (16), comparison of any IGFBP-4 knockout (KO) growth phenotype with the IGF growth deficits could begin to indicate potential specificity in IGFBP-4 interactions with the IGFs.

Recent studies indicate that proteolysis is a major regulatory mechanism used to alter the binding affinity of IGFBP-4 for IGFs. Pregnancy-associated plasma protein-A (PAPP-A) has been identified as an IGF-dependent IGFBP-4 protease (17,18) and belongs to the metzincin superfamily of metalloproteinases (19,20). The PAPP-A is secreted as a dimer of 400 kDa (9,21), but during pregnancy the majority of serum PAPP-A (>99%) is found as a 2:2 disulfide bound complex with the proform of eosinophil major basic protein (pro-MBP), which functions to inhibit PAPP-A activity (21). Except for a recently identified homolog, PAPP-A2, which shares 45% sequence homology with PAPP-A and cleaves IGFBP-5 independently of IGF binding (21), PAPP-A does not show global similarity to any known metalloproteinase because of an unique Val residue directly following the zinc-binding motif and the unusually long distance between the zinc-binding motif and the Met-turn (17). PAPP-A proteolytic activity has been detected in the conditioned media from human osteoblasts (22,23), vascular smooth muscle cells (24), granulosa cells (25), and trophoblast and decidualized endometrial stromal cells (26), as well as in ovarian follicular fluid (27) and human pregnancy serum (28). PAPP-A cleaves intact 24-kDa IGFBP-4 to fragments with reduced (N-terminal fragment) or no affinity (C-terminal fragment) for IGFs (29). This reaction is strongly enhanced by adding IGFs into the medium (28).

The physiological relevance of PAPP-A has been shown by the production of PAPP-A-null mice, which are viable but 60% the size of wild-type littermates at birth. Thus, PAPP-A is clearly involved in optimal fetal growth and development (30) although no prior studies have examined the PAPP-A phenotype at prenatal ages. Because PAPP-A is the only identified protease known to cleave IGFBP-4, it became important not only to explore the possible effect of IGFBP-4 mutation on growth but also to explore the relationship between IGFBP-4 and PAPP-A using gene-targeted mouse models. In this study we first generated IGFBP-4-null mice and demonstrated a required growth-regulatory role for this protein during fetal development. We further investigated the relationship between IGFBP-4 and PAPP-A in growth by cross-breeding IGFBP-4- and PAPP-A-null mice to produce several genotypes of mutant mice that expressed different levels of IGFBP-4 and PAPP-A. Growth curves and levels of IGFBP expression in PAPP-A-null mice, IGFBP-4-null mice, and IGFBP-4/PAPP-A double-null mice were then determined. Together, these data demonstrated that IGFBP-4 can both promote and inhibit growth, presumably by altering IGF activity, and that these IGFBP-4 effects are critically regulated by PAPP-A during fetal and postnatal growth and development.

RESULTS

Production of IGFBP-4-Deficient Mice and Lack of Compensatory Changes in the Expression of Other IGFBPs in Mutant Mice

IGFBP-4-null mice were generated by standard procedures (see Materials and Methods). Deletion of most of exon 1 of the IGFBP-4 gene, including the translation start site (Fig. 11,, A–C), resulted in the complete loss of IGFBP-4 protein and mRNA, which was confirmed by using both Western ligand blotting and in situ hybridization (Fig. 22,, A–C). IGFBP-4 mutant mice were viable and fertile, which allowed us to assess the effect of the mutation on IGFBP-4 expression at both pre- and postnatal stages. IGFBP-4 wild-type, heterozygous, and homozygous embryos were coembedded, and the expression patterns of the IGFBP genes at embryonic d 13.5 (e13.5) and e16.5 were explored using in situ hybridization. As shown in Fig. 22,, A and B, IGFBP-4 mRNA expression was not detectable in IGFBP-4(−/−) embryos at e13.5 and e16.5, demonstrating that we had successfully disrupted the IGFBP-4 gene. Interestingly, the intensity of the IGFBP-4 signal in heterozygous embryos was decreased approximately 50% compared with that of wild-type embryos (Fig. 2B2B),), indicating that both IGFBP-4 alleles contribute to the expression levels of IGFBP-4. We also determined that expression of the other IGFBP genes and the IGF-II gene, as shown in Fig. 22,, A and B, was not altered in IGFBP-4(−/−) embryos at e13.5 and e16.5.

Figure 1
Production of IGFBP-4-Null Mice
Figure 2
There Is No Compensation in the Remaining IGFBPs and IGF-II in IGFBP-4-Null Embryo

To test whether loss of IGFBP-4 was accompanied by compensatory changes in circulatory IGFBPs, we analyzed the IGFBP profiles in serum from adult IGFBP-4 wild-type, heterozygous and homozygous mouse serum by Western ligand blotting (Fig. 2C2C).). IGFBP-4 was not detectable in serum obtained from IGFBP-4-deficient mice, as expected, and no significant changes in any of the other serum IGFBPs were observed. In addition, the intensity of the IGFBP-4 band was decreased by approximately 50% in serum from IGFBP-4 heterozygous mice, which was consistent with the lowered mRNA expression levels observed in IGFBP-4 heterozygotes, and also indicated that significant changes in the levels of other serum IGFBPs would have been detected if present.

Growth Deficit of IGFBP-4-Null Mice

The successful disruption of the IGFBP-4 locus, combined with the observation that its loss was not accompanied by obvious compensatory alterations in the expression of other IGFBPs, enabled us to determine the consequence of IGFBP-4 deficiency during mouse development. Mating of IGFBP-4 heterozygous mice generated all genotypes in the normal Mendelian ratios (26%+/+, 48%+/−, 26%−/−) indicating the absence of embryonic lethality. Weight measurements indicated that IGFBP-4-deficient mice are born with a body mass 10–15% less than wild-type littermates. After birth, IGFBP-4-deficient mice remained 10–15% smaller throughout at least 14 wk of age (Fig. 3A3A).). Specifically, there was no additional decline in growth at the time (beginning ~20 d after birth) when IGF-I-null mice exhibit the second of two pronounced growth deficits (31). This result indicates that absence of IGFBP-4 does not disrupt at one major phase of IGF-I-stimulated growth. Interestingly the IGFBP-4 heterozygous exhibit similar body weight as wild-type littermates at all ages, despite the 50% reduction in its serum level, indicating that reduced levels are able to support normal growth.

Figure 3
Deficits in Embryonic and Postnatal Growth of IGFBP-4-Null Mice

To determine the time during development when the growth deficit in IGFBP-4-deficient mice begins, we isolated and weighed embryos obtained from IGFBP-4 heterozygous matings at e12.5, e14.5, and e16.5. As shown in Fig. 3B3B,, IGFBP-4-deficient mice were significantly (12%) smaller than their wild-type littermates at e16.5 and 11% smaller at e14.5. Even at e12.5, IGFBP-4-deficient mice already weighed significantly (7%) less than wild-type littermates, indicating that the growth deficit of IGFBP-4-deficient mice is already apparent by the time that IGF-II mutation leads to growth deficiency (12,16).

Growth Deficit of PAPP-A-Null Mice

The growth deficit of IGFBP-4-null mice indicated that IGFBP-4 is required to attain normal body size. We thus initially expected that preventing ongoing cleavage and turnover of IGFBP-4 by mutation of the relevant protease might not only reverse the IGFBP-4 growth deficit, but perhaps even increase mouse size. In contrast, however, mutation of the PAPP-A gene, which abolished IGFBP-4 proteolytic activity in cultured fibroblasts in vitro, surprisingly led to an even greater growth deficiency than that seen in the IGFBP-4-null mice (30). To determine whether this additional growth deficit might be elicited by altered proteolysis of additional substrates, such as additional IGFBPs, we examined offspring from matings of mice heterozygous for both IGFBP-4 and PAPP-A genes.

Mating of PAPP-A/IGFBP-4 heterozygous mice generated all possible genotypes in the normal Mendelian ratios (Table 11),), indicating the absence of embryonic lethality of any genotype. Consistent with previously reported studies, all mice that had total or partial PAPP-A deficiency were born with a decreased body size compared with wild-type mice and remained smaller throughout the first 7 postnatal weeks. For example, PAPP-A(−/−)BP4(+/+) mice were 34% smaller at postnatal d 30 and PAPP-A(+/−)BP4(+/+) also exhibited a slight growth deficit 7% (Fig. 4A4A),), which confirmed that PAPP-A is essential to growth in a wild-type IGFBP-4 background. To determine whether the absence of PAPP-A led to an alteration in IGFBP-4 levels in vivo, Western ligand blotting was performed and demonstrated that significantly more IGFBP-4 was accumulated in adult PAPP-A homozygote and heterozygous mutant serum compared with wild-type serum (Fig. 4B4B).

Figure 4
PAPP-A Is Required for Normal Prenatal and Postnatal Growth
Table 1
Genotypes from IGFBP-4(+/−)PAPP-A(+/−) Matings

To begin to determine when the PAPP-A is required in prenatal growth, we measured the body weights of embryos from heterozygous matings at e18.5. All mice homozygous for the PAPP-A mutation were already significantly smaller than the wild-type mice by this age (Fig. 44,, C and D), demonstrating the critical role of PAPP-A in fetal development.

PAPP-A Regulates Growth via Proteolysis of IGFBP-4

Although IGFBP-4 protein was absent from the serum of IGFBP-4(−/−) mice, and high levels of IGFBP-4 accumulated in serum of PAPP-A(−/−) mice, both IGFBP-4(−/−) mice and PAPP-A(−/−) mice are smaller than wild-type mice, which left unclear the specific roles of both IGFBP-4 and PAPP-A in growth.

To explore further the relationship between IGFBP-4- and PAPP-A-stimulated growth, the postnatal growth curves from PAPP-A(−/−)/BP4(−/−) double-mutant mice were determined (Fig. 5A5A),), which clearly indicated that double-mutant mice were larger than PAPP-A mice alone. Statistical comparisons over a range of ages indicated that PAPP-A(−/−)/BP4(−/−) double-KO mice were significantly larger than PAPP-A(−/−) mice at d 20, 30, 40, and 50 (Fig. 5B5B).). These results indicated that most of the growth deficit of PAPP-A(−/−) single-mutant mice is reversed with the concurrent loss of IGFBP-4 and demonstrated that IGFBP-4 is required for most, if not all, of the growth deficit elicited by PAPP-A mutation. Moreover, PAPP-A(−/−)/BP4(−/−) mice had body weights similar to PAPP-A(+/+)BP4(−/−) mice at d 0, 10, 20, 30, 40, and 50 (Fig. 5C5C).). The identical growth curves shown by PAPP-A(−/−)/BP4(−/−) and PAPP-A(+/+)/BP4(−/−) mice on the mixed genetic background, which were both 20% smaller than wild-type mice at all ages, demonstrated that the effect of PAPP-A in growth depends on the IGFBP-4 pathway. We then examined whether the reversal of the PAPP-A growth deficit by deletion of IGFBP-4 occurred prenatally. We found that PAPP-A(−/−)/BP4(−/−) mice had body weights similar to IGFBP4(−/−) mice at e18.5 (Fig. 5D5D)) as at postnatal ages. Taken together, these data indicated that when IGFBP-4 was deleted in the mouse, the detrimental effects of PAPP-A on growth were abolished. Therefore PAPP-A regulates growth via its interaction with IGFBP-4, presumably by regulating its proteolysis.

Figure 5
PAPP-A Regulation of Normal Growth Requires IGFBP-4

IGFBP-4 Needs Optimal Concentration to Regulate Normal Growth

The multiple genotypes from the double-heterozygous mice mating were expected to exhibit different levels of IGFBP-4 and PAPP-A. Therefore, those genotypes provided additional information regarding the relationship between IGFBP-4 and PAPP-A during growth. Most interestingly, the PAPP-A-null mice that expressed different levels of IGFBP-4 showed different growth curves. Thus, PAPP-A(−/−)/BP4(+/−) mice were intermediate in size between IGFBP-4 single-mutant mice and double-mutant mice at all ages examined (Fig. 6A6A),), which reached significance from the double homozygous KO at older ages (Fig. 6B6B)) and approached significance compared with the PAPP-A KO. Thus, the growth deficit of the PAPP-A mutation was only partially reversed in the heterozygous IGFBP-4 +/− mice; this indicated that the growth deficit resulting from PAPP-A absence is proportional to the amount of IGFBP-4 present. A similar pattern was seen in the embryo at e18.5. Mice carrying PAPP-A KO were significantly smaller than wild-type mice. PAPP-A(−/−)/BP4(+/+) mice were significantly smaller than PAPP-A(−/−) BP4(−/−) mice, and the IGFBP-4 heterozygous mice were midway between wild-type and mutant IGFBP-4 mice on a PAPP-A null background (Fig. 6C6C).

Figure 6
IGFBP-4 Needs an Optimal Concentration to Regulate Normal Growth

Western ligand blots continued to show the significant accumulation of IGFBP-4 in serum of PAPP-A mutant mice from this cohort of offspring and the complete loss of IGFBP-4 in IGFBP-4 mutant mice, compared with wild-type mice. Moreover, levels of IGFBP-4 in serum from PAPP-A(−/−) BP4(+/−) mice were between the levels of IGFBP-4 present in wild-type and PAPP-A(−/−) mice (Fig. 6D6D)) although this difference did not reach significance with either group in the cohort of mice analyzed.

It is of particular interest that, PAPP-A(−/−)/BP4(+/+) mice exhibits similar size as IGF-II-null mice (32) (~60% of normal body weight), indicating the accumulation of IGFBP-4 in a noncleavable form may inhibit most, if not all, of the IGF-II growth effects. Conversely, total deletion of IGFBP-4 may also result in a partial loss of IGF-II function, as reflected in the similar size of the IGFBP-4(−/−) mice and PAPP-A(−/−) BP4(−/−) mice. Taken together with the facts that IGFBP-4 is coexpressed with IGF-II in many tissues (30,33,34) and mice carrying IGFBP-4 or IGF-II mutation are both viable and fertile (32), an optimal level of IGFBP-4 appears necessary to regulate the bioavailability of IGF-II, as further discussed below.

DISCUSSION

This study demonstrates that a complex and carefully integrated network of IGF system components regulates growth. We have shown, for the first time, that a specific IGFBP, IGFBP-4, is required for prenatal growth, and that its loss cannot be compensated by other IGFBPs expressed in the embryo. Moreover, the kinetics of growth disruption suggests that IGFBP-4 is required for IGF-II-, rather than IGF-I-mediated prenatal and postnatal growth. Finally, we have shown that the more severe growth effect that accompanies deletion of the IGFBP-4 protease, PAPP-A, is completely dependent on the presence of IGFBP-4. We thus conclude that the major role of PAPP-A is to adjust the bioavailability of IGF-II by proteolysis of IGFBP-4, which itself is needed for normal growth.

IGFBP-4 Is Required for Normal Prenatal Growth

IGFBP-4 was first identified and isolated based on its ability to inhibit IGF action (35). Previous in vitro evidence suggested that IGFBP-4 functions as an inhibitor of IGF-induced cell proliferation and differentiation in all cell types studied including bone cells (36), muscle cells (37), rat neuroblastona cells (38), and human prostate cancer cells (39). However, previous in vivo studies of IGFBP-4 have been more controversial. For example, locally overexpressed IGFBP-4 in muscle cells functions to antagonize IGF actions (40) whereas systemic administration of IGFBP-4 was reported to have a growth-stimulatory effect in bone formation (41). The IGFBP-4 KO model is the first to explore the consequences of IGFBP-4 ablation in vivo. The observation that IGFBP-4-deficient mice are smaller than their wild-type and heterozygous littermates beginning at e12.5 indicates that IGFBP-4 is required for normal prenatal growth. This is in contrast to the expectation from in vitro studies that would predict an increased size due to the absence of an IGF inhibitor. Importantly, this result also shows that remaining IGFBPs expressed in the embryo (42,43) cannot compensate for the absence of IGFBP-4, probably because of their differential expression patterns.

Both IGF-I and IGF-II are essential for normal embryonic growth (16). During development, IGFBP-4 transcripts are coexpressed with IGF-II in many regions (30,33,34), indicating a potential interaction between IGF-II and IGFBP-4 in the growth process. The growth deficit of IGFBP-4-null mice, although less severe than that resulting from disruption of IGF-II, nonetheless shows striking similarities to that of the IGF-II mutants. Growth deficits in both strains begin at e12.5 and reach their maximal extents soon thereafter. Moreover, both IGFBP-4- and IGF-II-null mice stay proportionally smaller through adulthood (44), while remaining fully viable and fertile. Preliminary studies also show that double IGFBP-4/IGF-II mutant mice are no smaller than the IGF-II-null mice alone. Taken together, these results strongly suggest that the growth deficit of the IGFBP-4-deficient mice is due to a partial loss of IGF-II stimulation. We thus hypothesize that IGFBP-4 acts to localize and stabilize IGF-II after secretion from fetal cells and that its absence leads to increased IGF-II degradation or diffusion leading to a growth deficit. Although more direct measurements of local IGF-II levels are needed to demonstrate this conclusively, the presumptive stabilization and sequestering of IGF-II by IGFBP-4 may also protect the embryo from the detrimental effects of excess IGF-II stimulation that have been demonstrated by previous studies in H19- and IGF-II receptor-mutant mice (45,46). As discussed below, however, mice lacking the ability to degrade IGFBP-4 and release IGF-II exhibit an even greater growth deficit that likely reflects the inhibitory aspect of the IGF-II/IGFBP-4 interaction.

In contrast to the similarities between the IGFBP-4 and IGF-II growth deficits, the growth pattern of IGF-I deficient mice is quite different. The growth deficit of the IGF-I-null mice increases gradually, rather than abruptly, from e13.5 until birth. IGF-I-null mice are born approximately 60% of normal size, and the few that survive display a second pronounced growth deficit that begins at 3 wk of age and decreases body weight to 30% of the normal adult (47). In addition, both sexes of IGF-I-null mice are infertile (44). None of these characteristic IGF-I phenotypes were observed in IGFBP-4-deficient mice, indicating that there is little if any contribution of IGF-I deficiency to the IGFBP-4 growth deficit, although the requirement for IGFBP-4/IGF-I interaction in other functions cannot be eliminated.

Proteolysis of IGFBP-4 Is Required for Normal Growth

PAPP-A is the only physiological protease known thus far to cleave IGFBP-4. The resulting fragments have decreased affinity for IGFs and, in vitro, release IGFs from an inactive, local reservoir to an active form that can activate IGF receptors. Prior studies indicated an important role for PAPP-A activity in growth. For example, the absence of PAPP-A expression in the placenta of patients with Cornelia de Lange syndrome, a condition involving incomplete fetal development and subsequent deformities, provided initial evidence for a role of PAPP-A in human development (48). Recently PAPP-A-null mice were found to exhibit a severe growth deficit similar to that of IGF-II mutants (30), which demonstrated that PAPP-A is also required for normal mouse growth. Despite the demonstrated activity of PAPP-A against IGFBP-4 in vitro, the growth effects in vivo could potentially be explained by altered cleavage of additional substrates other than IGFBP-4.

Production of IGFBP-4/PAPP-A double mutant mice allowed us to further explore the mechanism of PAPP-A-dependent growth and its relationship to IGFBP-4. Because the growth difference between PAPP-A and IGFBP-4 mice is totally abolished in PAPP-A(−/−)BP4(−/−) mice, this result indicates that the growth effect of the PAPP-A mutation is mediated solely by the absence of IGFBP-4 cleavage. Moreover, PAPP-A(−/−)BP4(+/−) mice, which showed half the level of increased IGFBP-4 accumulation in serum compared with PAPP-A(−/−)BP4(+/+) mice, also displayed only half the growth deficit compared with PAPP-A(−/−)BP4(+/+) mice. This result indicates not only that PAPP-A regulates growth via proteolysis of IGFBP-4, but also that the inhibitory effect of IGFBP-4 is dose dependent.

Previous in vitro studies have suggested that IGF-II is a cofactor for PAPP-A proteolytic activity because the IGFBP-4-IGF-II complex has increased susceptibility for PAPP-A (49,50). In addition, secretion of high levels of IGF-II by fetal mouse fibroblasts promotes PAPP-A-mediated proteolysis of exogenous IGFBP-4 in these cultures (30). Whole-embryo in situ hybridization indicates that the PAPP-A transcripts, like those of IGFBP-4, are extensively colocalized with IGF-II transcripts (30). These observations, together with the similarity between the IGF-II and PAPP-A growth deficits, lead us to hypothesize that PAPP-A is necessary to elicit IGF-II-promoted growth by proteolysis of the IGF-II/IGFBP-4 complex in vivo. Thus complex and tightly regulated interactions between IGF-II, IGFBP-4, and the IGFBP-4-specific protease PAPP-A occur both during intracellular biosynthesis and after secretion. The precise nature of these regulatory pathways is also indicated by the fact that disruption of IGF-II imprinting during embryonic development, which increases IGF-II level 2-fold, rescues the growth deficit of both PAPP-A-null mice (51) and IGFBP-4 mutant mice (Schuller, A. G. P., and J. E. Pintar, unpublished).

Biochemical studies have demonstrated that, under normal circumstances, IGFBPs bind IGFs with high affinity and stabilize the IGFs, whereas proteases cleave IGFBPs into low binding-affinity fragments to allow release of IGFs to their receptors followed by rapid degradation. As a consequence, IGFs levels are kept in balance in the circulation, with an interruption of this process expected to lead to an alteration in IGF effects. In our study, both accumulation of IGFBP-4 in PAPP-A(−/−)-null mice and deletion of IGFBP-4 in IGFBP-4(−/−)-null mice resulted in growth retardation. Thus IGFBP-4 can have both stimulatory and inhibitory effects on growth. We thus hypothesize that an optimal level of IGFBP-4 stabilizes and maintains a local reservoir of IGFs after secretion. The modest growth deficit of IGFBP-4 KO mice indicates that this local stabilization is relatively minor but nonetheless required to optimize IGF action. In contrast, a more significant effect is seen when the normal turnover of IGFBP-4 is abolished. The absence of PAPP-A leads to accumulation of IGFBP-4, which sequesters IGF-II, prevents its release, and abolishes most, if not all, of IGF-II-stimulated growth. Nonetheless, further work will be needed to exclude the possibility that effects on other IGFBPs may also contribute to the growth deficits because, for example, PAPP-A can cleave IGFBP-5 into fragments (52) as well.

Interestingly, PAPP-A can reversibly bind to the cell surface of several cell types, which does not affect its proteolytic activity (53). Thus, PAPP-A adhesion to the cell surface could precisely localize IGF bioavailability near IGF receptors in specific cells, decreasing the probability that the IGF, after PAPP-A activity, would be either degraded or bound by other IGFBPs before receptor binding.

To summarize, we have shown that highly integrated interactions between a growth-promoting ligand (IGF-II), one of its binding proteins (IGFBP-4), and an IGFBP-specific protease (PAPP-A) are essential to normal fetal growth. IGFBP-4 is necessary for growth promoted by IGF-II, which is summarized in Fig. 77 and consistent with all the growth curve data. 1) IGFBP-4 binds and stabilizes the IGF after secretion. 2) PAPP-A can reversibly bind to the cell surface to target IGF-II/IGFBP-4 complex to the vicinity of the IGF receptor. 3) PAPP-A releases most, if not all, IGF-II from the IGFBP-4/IGF-II complex via proteolysis of IGFBP-4 either near the specific cell membrane or pericellularly and thus regulates the IGF bioavailability. In PAPP-A-null mice, IGFBP-4 cannot be cleaved and accumulates to a higher level, which inhibits the growth-promoting effect of IGF-II and results in small mice. In IGFBP-4-null mice, loss of IGFBP-4 results in the loss of its pericellular reservoir function and results in smaller mice as well. Overall, IGFBP-4 serves as both a positive and negative regulator of IGF activity, and its function is critically regulated by PAPP-A in vivo.

Figure 7
Proposed Model of IGFBP-4 Functions in Growth

MATERIALS AND METHODS

Experimental Animals

All animal experimentations described below were conducted in accord with accepted standards of humane animal care

Generation of IGFBP-4-Deficient Mice

Gene targeting experiments were performed essentially as described elsewhere (54). Four partially overlapping IGFBP-4 genomic clones were isolated from a 129/ReJ genomic library after screening with a digoxigenin-labeled rat IGFBP-4 cDNA probe containing exon 1 and part of exon 2, obtained from Dr. Shimasaki, and characterized by restriction mapping. One IGFBP-4 genomic clone, containing exons 1, 2, 3, and part of exon 4, was characterized in more detail and served as a template to generate the IGFBP-4 targeting construct. A 4-kb NotI/NruI fragment, containing a region 5′ of the translation start site of the IGFBP-4 gene, was subcloned into the NotI/ XbaI blunted sites of a targeting vector already containing a neomycin resistance cassette and the thymidine kinase gene. From this 4-kb fragment, the most 5′ 1.5 kb was deleted by digesting with NotI and BstXI followed by blunt ending and self-ligation, to enable screening for a homologous recombination event with a 5′-probe located outside of the targeting construct. Then, a 5-kb HindIII/BstXI blunted fragment containing part of intron 1 was cloned into the HindIII/SalI blunted sites between the neomycin cassette and the thymidine kinase gene. The resulting targeting construct was purified on CsCl, linearized with XhoI and introduced into CCE embryonic stem (ES) cells by electroporation. G418- and gancyclovir-resistant ES clones were picked, expanded, and split in duplicate wells for freezing and genomic DNA extraction. Genomic DNA from double-resistant clones was digested with BamHI, separated on 0.7% agarose gels, transferred to nylon membranes and screened for the occurrence of a homologous recombination event using a radiolabeled 0.8-kb HinFI/BstXI fragment. Targeted ES cells, evident from the presence of both a 9-kb wild-type and 5.5-kb mutant BamHI fragment, were injected into C57Bl/6J blastocysts and transferred into pseudopregnant females to generate germline-transmitting chimeras. Male chimeras were bred with C57Bl/6J females to obtain mice carrying the IGFBP-4 mutation. IGFBP-4 heterozygous offspring were bred to obtain IGFBP-4-deficient mice and wild-type control littermates.

Generation of IGFBP-4/PAPP-A-Deficient Mice

IGFBP-4-null mice on a mixed 129/C57 background were cross-bred with PAPP-A-null mice. The heterozygous mutants were intercrossed to generate IGFBP-4/PAPP-A-null mice. The homozygous IGFBP-3–4 double-null mice were identified by Southern analysis of tail tip DNA. For testing IGFBP-4, we expected to see both a wild-type 11-kb fragment and a mutant 8-kb fragment of BamHI-digested DNA in IGFBP-4 heterozygous mice. For testing PAPP-A, we expected to see both a wild-type 16-kb fragment and a mutant 2.8-kb fragment of BamHI-digested DNA in PAPP-A heterozygous mice.

In Situ Hybridization

In situ hybridizations were performed essentially as described elsewhere (55). Briefly, fresh frozen cryostat sections were mounted onto TESPA coated microscope slides and stored at −80 C until use. Sections were fixed in 4% paraformaldehyde in PBS, dehydrated in ethanol series, acetylated in 0.25% acetic anhydride/50 mm triethanolamine, washed in 0.2× saline sodium citrate (SSC), dehydrated in ethanol series, and prehybridized at room temperature. Then, sections were hybridized overnight at 50 C with 2 × 104 cpm/μl 35S-labeled antisense RNA probes, washed in 50% formamide/10 mm dithiothreitol/1× SSC, RNase treated in 100 μg/ml Rnase A, washed in 0.5× SSC, dehydrated, and subjected to autoradiography. 35S-labeled antisense RNA probes were transcribed using SP6, T7, or T3 RNA polymerases in the presence of [35S]UTP from linearized plasmids pRBP1–501 [rat BP1: nucleotide (nt) 486–892] (56), pG3-2-11 (rat BP2: nt 502-1087) (57), pRBP3-AR (rat BP3: nt 163–861) (58), pRBP4-SH (rat BP4: nt 435–878) (59), pGEM3Z/mBP5, 2–3 (mouse BP5: nt 512–988) (60), pRBP6-PP (rat: BP6 nt 229–475) (61), and pRIGF-II-BP (rat IGF-II, 551 nt BamHI/PstI fragment) (62).

Western Ligand Blotting

Western ligand blots were prepared essentially as described by Hossenlopp et al. (63). Serum samples (3 μl) were applied to a 9% sodium dodecyl sulfate-polyacrylamide gel and run under nonreducing conditions. Separated proteins were transferred onto a nitrocellulose filter by electroblotting, washed in 3% Nonidet P-40/0.9% NaCl/100 mm Tris, pH 7.5 (TBS), blocked in 1% BSA/TBS, washed in 1% Tween 20/TBS, and incubated with 4 × 105 cpm [125I]IGF-I in 1% BSA/1% Tween 20/TBS overnight at 4 C. Then, filters were washed in 0.1% Tween 20/TBS and in TBS followed by autoradiography.

Growth Curves

Offspring from three separate rounds of IGFBP-4 heterozygote matings (at F2, F3, and F4) were weighed daily for 3 wk, every other day until 6 wk of age and once (F3 and F4) or twice (F2) a week thereafter. Offspring from four separated rounds of IGFBP-4(+/−)PAPP-A(+/−) heterozygote matings were weighted daily for 4 wk, twice per week for another month. Weights were entered in Prism and graphed as the average of all mice (males and females separate). Statistical analysis was performed using Student's t test at 0, 1, 2, 4, 8, and 12 wk of age.

Data Analysis

Data were reported as means ± sem. Where appropriate, statistical analysis was performed using a two-tailed, unpaired Student's t test. Values of P < 0.05 were considered statistically significant.

Footnotes

This work was supported by National Institutes of Health Grant NS-21970 (to J.P.) and the Mayo Foundation (to C.C.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 7, 2008

Abbreviations: e13.5, Embryonic d 13.5; ES, embryonic stem; IGFBP, IGF-binding protein; KO, knockout; nt, nucleotide; PAPP-A, pregnancy-associated plasma protein-A; SSC, saline sodium citrate.

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