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Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins MARLON R. SCHNEIDER, HARALD LAHM, MINYAO WU, ANDREAS HOEFLICH, AND ECKHARD WOLF1 Institute of Molecular Animal Breeding, Gene Center, D-81377 Munich, Germany ABSTRACT The insulin-like growth factor-binding proteins (IGFBPs) comprise a family of six related peptides that interact with high affinity with IGFs. IGFBPs compete with IGF receptors for IGF binding, and as a consequence of this competition they can affect cell growth. In addition, IGF-independent regulatory mechanisms of IGFBPs have been described. Despite their common property to interact with IGFs every IGFBP is expressed in a tightly regulated time- and tissue-specific manner suggesting that each protein may have its own distinct functions. Several transgenic mouse models overexpressing IGFBP-1, -2, -3, or -4 were developed in the past few years. Brain abnormalities were a common feature of IGFBP-1 transgenic models. Individual strains showed alterations in glucose homeostasis, reproductive performance, and a reduction of somatic growth as the most prominent phenotypes. The latter was also the main effect observed in IGFBP-2 transgenic mice. The overexpression of IGFBP-3 under the control of an ubiquitous promoter resulted in selective organomegaly, whereas mammary gland-targeted expression of this protein caused an altered involution after pregnancy in this organ. Tissue-specific overexpression of IGFBP-4 resulted in hypoplasia and reduced weight of smooth muscle-rich tissues such as bladder, aorta, and stomach. This review summarizes the current knowledge about the actions of IGFBPs in vivo based on the presently established transgenic mice.—Schneider, M. R., Lahm, H., Wu, M., Hoeflich, A., Wolf, E. Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins. FASEB J. 14, 629 – 640 (2000)

Key Words: IGFBP 䡠 insulin-like growth factors 䡠 transgenic mice 䡠 overexpression 䡠 knockout

Growth factors are multifunctional cell signaling molecules, typically multicellular in origin, that may act in an autocrine, paracrine, and endocrine fashion (1). These features, which hamper their analysis using classical experimental approaches of endocrinology, make the use of transgenic models specifically attractive for unraveling the determinants of 0892-6638/00/0014-0629/$02.25 © FASEB

growth factor expression and their target cell response during embryonic and fetal development as well as in the adult animal (2). The insulin-like growth factors (IGFs), IGF-I and IGF-II, are a pair of small single-chain peptides that share significant structural homology with insulin (3). They are involved in regulation of cell growth, differentiation, and metabolism, eliciting diverse effects in a plethora of biological processes (4). The biological effects of IGFs are achieved through binding to high-affinity receptors on the cell surface. The type I IGF receptor (IGF-I-R) is, like the insulin receptor, a heterotetrameric protein complex containing a tyrosine kinase domain that mediates signal transduction (5). Its inactivation leads invariably to severe neonatal growth deficiencies as a consequence of hypoplasia in several tissues. Nevertheless, these animals are born alive but die within minutes due to respiratory failure (6). The structurally distinct IGF-II-R is a monomeric protein lacking tyrosine kinase activity and is also known as the cationindependent mannose-6-phosphate receptor. There is no known signal transduction mechanism that is initiated by this receptor, and all proliferative effects of the IGFs are thought to be mediated through the IGF-I-R (7). However, the IGF-II-R plays an important role for the degradation of IGF-II. Inactivation of IGF-II-R expression in mice by gene targeting results in fetal overgrowth, skeletal abnormalities, and perinatal death due to overexposition of fetuses to IGF-II (8 –10). Unlike insulin, the IGFs are bound in serum and other biological fluids to a family of six structurally and evolutionary related binding proteins, termed IGF-binding proteins (IGFBP-1 to IGFBP-6) (Table 1). In the circulation, ⬃75– 80% of the IGFs are present in a complex of 150 kDa, composed of one molecule of IGF-I or IGF-II, a 85 kDa acid-labile subunit (ALS), and IGFBP-3. A smaller proportion (20 –25%) of the IGFs is associated with other IG1 Correspondence: Institute of Molecular Animal Breeding, Gene Center, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany. E-mail: [email protected]

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TABLE 1. Expression of IGFBPs in rodent tissues Detection of transcripts Embryonic tissuesa E12

E18

Adult tissuesb

mRNA size

Liver Generally expressed except in snout Not detected

Liver Generally expressed

1.6 kb 1.4–1.8 kbc

30 kDa 32 kDa

11 12–14

2.4–2.6 kbc

16–30 kDad

14–16

IGFBP-4

Generally expressed except in nasale placode

2.0–2.6 kb

16–28 kDad

14, 17–19

IGFBP-5

Tongue, sclerotomes, nasal placode

Widely expressed, kidney, muscle, ovaries, liver

5.0–6.0 kb

21–31 kDad

14, 20, 21

IGFBP-6

Not detected

Widely expressed except in heart, meninges, and choroid plexus Widely expressed except in liver, heart, and choroid plexus Vertebrae, lung, liver

Liver Widely expressed, liver, kidney, muscle Widely expressed, kidney, testes Widely expressed, kidney, liver, spleen testes, ovaries

Widely expressed, lung, heart, muscle, testes, ovaries, kidney

1.3 kb

32 kDa

22, 23

IGFBP-1 IGFBP-2 IGFBP-3

a For further details, see ref 24. in italics; for further details, see ref 25.

Vertebrae, liver

b

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References

Main sources of the respective IGFBP are indicated in boldface, nonexpressing tissues are indicated c d Results were obtained with rat tissues. Proteolytic fragments have been described.

FBPs forming a 50 kDa complex, and less than 1% is found in free form of ⬃7.5 kDa (26). The IGFBPs are expressed in a tissue-specific manner and have different affinities for the IGFs. Besides their role in preventing insulin-like side effects of the relatively large concentrations of IGFs in the circulation, IGFBPs are able to modulate different IGF actions positively or negatively; recent evidence suggests they can also exert IGF-independent effects (26 –28). Actually, these proteins are part of a superfamily that comprises the six high-affinity IGFBPs and at least four other low-affinity IGF binders, termed insulin-like growth factor binding proteinrelated proteins (IGFBP-rP) (29). Finally, at least three mechanisms have been shown to alter the affinity of the IGFBPs to IGFs, making the network of regulatory components in the IGF system even more complex: IGFBP proteolysis, phosphorylation, and adherence to the cell surface or to the extracellular matrix (30). The accumulation of experimental evidence of the intrinsic importance of IGFBPs in modulating IGF actions caused them to be an object of great interest, now occupying a front position in IGF and growth factor research. However, the precise roles of individual IGFBPs are still unknown, due mainly to the great complexity of their actions and their regulation, but also to the fact that the overwhelming majority of information about the IGFBPs is derived from in vitro studies. Nonphysiological concentrations of the IGFs and their binding proteins as well as the lack of other components such as the IGFBP-rPs may lead to artificial culture conditions, which can 630

Protein size

explain confusing and sometimes contradictory results obtained from these studies (31). In many situations of physiological or pathological growth, multiple components of the IGF system may be dysregulated (4, 32–35). It is therefore difficult to predict which components are responsible for altered growth and which ones are only coregulated without an intrinsic effect on growth and differentiation. Transgenic and knockout strategies in model organisms provide a unique opportunity of performing gain of function, partial loss of function, and loss of function studies for a particular gene. Consequently, effects of altered expression of a member of the IGF system on the other components of the system and the phenotypic consequences can be precisely studied. More recently, this can be performed in an inducible and/or tissue-specific or stage-specific manner. For technical details, the reader is referred to recent reviews and textbooks (36 – 40). Many transgenic and knockout mouse models for the ligands (41, 42), receptors (43), and binding proteins of the IGF system were developed in the last decade. The knockout approach, when applied to ligands and receptors, resulted in exuberant phenotypes. In contrast, when applied to the binding proteins, the results were rather disappointing: in IGFBP-2 knockout mice, a reduction in spleen weight of adult males was the only morphological alteration (44); IGFBP-4 mutants appear to be slightly smaller than normal counterparts, and the genetic ablation of IGFBP-6 has not resulted in any apparent phenotypical manifestation (45). It is rea-

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sonable to speculate that functional compensation by other members of the IGFBP family (including the recently described IGFBP-rPs) may prevent the appearance of dramatic phenotypes. The alternative approach, namely, the overexpression of IGFBPs in transgenic mice, has been far more successful in producing both expected and unexpected phenotypes. This review presents an overview of transgenic models that have been developed to date for the study of IGFBP functions in vivo and discusses the data obtained from these experiments. IGFBP-1 IGFBP-1 was originally isolated from human amniotic fluid and temporarily termed amniotic fluidbinding protein (46). With the publication of its complete cDNA in 1988 (47), it became the first characterized member of the IGFBP family. In the developing mouse, IGFBP-1 expression can be detected in day 13.5 whole embryo preparations. In the adult mouse, its expression appears to be restricted to the liver, being high during the first days of postnatal life and decaying thereafter (Table 1). Although it may stimulate IGF actions under some conditions, the vast majority of in vitro studies indicates that IGFBP-1 mainly inhibits IGF-dependent cellular growth and differentiation (27). The biological actions of IGFBP-1 are associated with basically three aspects of physiology: carbohydrate metabolism, female reproductive functions, and fetal growth. Further information about IGFBP-1 actions, gene structure and expression can be found in a

comprehensive review (48) and its recent update (49). Three different transgenic models overexpressing this protein have been developed (Table 2). D’Ercole et al. (50) reported the generation of three strains of transgenic mice harboring a fusion gene in which the human IGFBP-1 cDNA was placed under the control of the mouse metallothionein-I promoter (MT). In addition to a wide tissue expression (51) all three lines ectopically express IGFBP-1 in the brain. The generation of four strains of transgenic mice expressing the rat IGFBP-1 gene under the control of the mouse phosphoglycerate kinase promoter (PGK) was reported by Rajkumar et al. (52). High levels of transgene expression were detected in the offspring, especially in brain, uterus, lung, and kidney. Gay et al. (11) reported the development of two strains of transgenic mice carrying the human IGFBP-1 cDNA under the control of the human ␣1-antitrypsin (␣1 AT) promoter, which directs the expression of the transgene to the liver. Phenotypical consequences of IGFBP-1 transgene expression Brain development and growth Reduced brain weight associated with specific morphological alterations was a common feature of all three IGFBP-1 transgenic models (Table 3). The MT-hIGFBP-1 mice exhibit brain growth retardation beginning in the second week of postnatal life, which persists when brain weight is calculated as a function of body weight. Further analyses confirmed multiple

TABLE 2. Characteristics of IGFBP-transgenic mouse lines Expression characteristics Transgene

Mouse strain

Tissue specificity

MT-hIGFBP-1

C3B ⫻ C57BL

Brain, heart, kidney, liver, lung, testes

PGK-rIGFBP-1

B6CBAF1 ⫻ CD-1

␣1 AT-hIGFBP-1

B6D2F2

Brain, uterus, lung, kidney, spleen Liver

CMV-mIGFBP-2

B6D2F2 ⫻ C57BL/6

MT-hIGFBP-3

B6CBAF1 ⫻ CD-1

WAP-hIGFBP-3 SMP-rIGFBP-4

B6SJLF2 ⫻ C57BL/6 FVB/N

IGFBP-TRANSGENIC MOUSE MODELS

Pancreas, stomach, skeletal muscle, heart, colon, spleen, brain Kidney, small and large intestine Mammary gland Bladder, aorta, stomach, uterus

Serum IGFBP levels

References

line A: 56 ng/ml (10 mM ZnSO4) 262 ng/ml (20 mM ZnSO4) line B: 127 ng/ml (uninduced) 1024 ng/ml (5 mM ZnSO4) 2470 ng/ml (25 mM ZnSO4) 11.5–42.2 ng/ml

50, 51

1.21–2.99 ng/ml (hemizygous) 2.41–13.69 ng/ml (homozygous) females: 843 ng/ml males: 1199 ng/ml

11

65–180 ng/ml (25 mM ZnSO4, homozygous) N.D. N.D.

55

52, 53

54

56 57

631

TABLE 3. Major phenotypical characteristics of IGFBP transgenic mouse lines Transgenic line

Alterations

References

MT-hIGFBP-1

Abnormal brain development Increased tolerance to ethanol Impaired brain development Reduced birth weight and postnatal growth retardation Altered glucose homeostasis and pancreas structure Impaired fecundity Reduced brain weight with several alterations Reduced body weight gain Glucose tolerance affected Impaired fecundity Proteinuria and glomerulus lesions Reduced body weight Selective organomegaly (spleen, liver, heart) Altered involution of the mammary gland, reduced size of alveoli Hypoplasia of smooth muscle cell-rich tissues (bladder, aorta, stomach)

50, 58–60 61

PGK-rIGFBP-1

␣1 AT-hIGFBP1

CMV-mIGFBP-2 MT-hIGFBP-3 WAP-hIGFBP-3 SMP-rIGFBP-4

52, 62 52 52, 63–67 68 11 11 11 11 11

Somatic growth

54 55 56 57

alterations in brain development and morphology (58 – 60). In the PGK-rIGFBP-1 model, the brain was the only organ showing significant weight reduction (52). The neuroanatomical deficits and other parameters such as cell proliferation and apoptosis in the brain of PGK-rIGFBP-1 mice were further evaluated by Ni et al. (62). Hydrocephalus was observed in 50% of ␣1 AThIGFBP-1 homozygotes, which also had lower brain weights than controls. Examination of brain sections showed dilation of the ventricles, reduced width of the cerebral cortex, and disorganization of its architecture, together with atrophy of the corpus callosum and hippocampus (11). The IGFs are potent agents that stimulate proliferation and/or survival of neural cells in vitro and are essential factors for normal development of the nervous system in vivo (4). Indeed, the mammalian nervous system is a site of intense and well-documented IGF activity (69). In the mouse, the brain is normally not a site of IGFBP-1 expression (25), and despite its identification in the neuromuscular synapse (70), no significant functions are attributed to IGFBP-1 in the central nervous system. Several lines of evidence indicate conclusively that brain growth retardation observed in these transgenic models is a direct and specific consequence of high levels of IGFBP-1 in the brain and related to its capacity to bind and to inhibit the stimulatory actions of IGF. 1) 632

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The same effect was observed in all three transgenic models, each carrying a different fusion gene. 2) The effect was associated with a high level of hIGFBP-1 expression in the brain of MT-hIGFBP-1 and PGKrIGFBP-1 models, and with high and constant serum IGFBP-1 levels in the ␣1 AT-hIGFBP-1 model. 3) Transgene expression in the MT-hIGFBP-1 model occurred in regions (cerebral and piriform cortex, hippocampus, thalamus, and hypothalamus) known to be sites of IGF-I expression during the first month of postnatal life (71), exactly at the same time the alteration was observed. 4) Mice overexpressing IGF-I in the brain under the control of the MT promoter display a phenotype inverse to that of MT-hIGFBP-1 transgenic mice in many aspects (72).

In PGK-rIGFBP-1 transgenic mice, the birth weight was significantly reduced to ⬃83–92% of the weight of wild-type animals (52). The growth retardation was also present postnatally, particularly after weaning. Except for brain (which was disproportionately reduced; see above) and spleen (which was heavier), the absolute weights of individual organs were reduced in transgenic mice, but similar to those of control mice when considered as a function of body weight. ␣1 AT-hIGFBP-1 transgenic animals show growth retardation within the first weeks of postnatal life. Body weight of adult mice was negatively correlated with IGFBP-1 plasma concentrations, further supporting a relationship between phenotype and transgene expression (11). It remains to be elucidated whether overexpression of IGFBP-1 has different effects in specific organs as observed in the PGKrIGFBP-1 model. The IGFs are undoubtedly involved in the regulation of fetal growth. Knockout mice lacking IGF-I, IGF-II or IGF-I-R are born with marked growth retardation (73). In humans, there is a direct correlation between cord blood IGF-I concentration and fetal size and birth weight (74). Likewise, IGFBP-1 is involved in intrauterine growth regulation in humans (49) and rodents (75, 76). Insulin, whose inactivation results in growth retardation and neonatal lethality in mice (77), positively regulates the expression of IGFBP-1 (49) as does hypoxia (78). Taken together, these data indicate that IGFBP-1 may be an important regulator of fetal growth, probably by limiting the availability of IGFs to their target tissues under conditions of hypoxia or restricted substrate availability. In the ␣1 AT-hIGFBP-1 transgenic mice, abnormally high and constant levels of IGFBP-1 in the bloodstream are likely to be responsible for impaired somatic growth. Although serum levels of IGFBP-1 in

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PGK-rIGFBP-1 transgenic mice were not markedly higher than in control animals, the elevated tissue levels can be responsible for inhibiting IGF actions, thereby causing overall weight reduction. The fact that growth retardation was present at birth in PGK-rIGFBP-1 animals may be a consequence of the ubiquitous expression provided by the PGK promoter and the consequent stronger IGFBP-1 inhibitory effect. Glucose homeostasis Rajkumar et al. (63) reported that in addition to the fasting hyperglycemia observed in homozygous male PGK-rIGFBP-1 transgenic mice (52), these animals also demonstrated fasting hyperinsulinemia and glucose intolerance. The sensitivity to insulin, however, was not altered. The relative weight of the pancreas was increased. They also observed that pancreatic islets were significantly larger and more numerous than in wild-type mice, although pancreatic insulin content was reduced. Alterations of pancreas structure and function, as well as of glucose metabolism, in the PGK-rIGFBP-1 model were investigated in more detail in further studies (64 – 67). In ␣1 AThIGFBP-1 homozygous transgenic mice, glucose tolerance was also affected (11). In addition to the general role of IGFBPs in limiting the potential hypoglycemic effects of IGFs, a specific role in the regulation of carbohydrate metabolism has been proposed for IGFBP-1 (48). When administered intravenously together with IGF-I in rats, IGFBP-1 blocked its hypoglycemic effects and increased blood glucose levels when administered alone (79). In addition, the promoter region of the IGFBP-1 gene is similar to that of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme in the regulation of gluconeogenesis. In contrast to glucagon and glucocorticoids, which stimulate transcription, insulin is a potent inhibitor of both basal and stimulated IGFBP-1 expression (80). Although the hyperglycemia observed in transgenic mice is easily explained by IGFBP-1 inhibiting the hypoglycemic effect of free IGFs, the reason for the abnormalities observed in the pancreas of PGKrIGFBP-1 animals is not that obvious. However, as suggested by the authors, the impaired pancreatic function may be a consequence of persistent hyperglycemia, which would also explain the insulin depletion (63). Reproductive performance The observation that litter size was reduced during the generation of PGK-rIGFBP-1 homozygous strains led Huang et al. (68) to examine this aspect in detail in one strain that strongly expressed IGFBP-1. The IGFBP-TRANSGENIC MOUSE MODELS

expression of the transgenic IGFBP-1 in the ovary was detected by Northern analysis. The authors observed that compared to wild-type mice, naturally mated transgenic mice had a significantly reduced number of corpora lutea and significantly fewer blastocysts could be harvested by flushing the uterine horns. Basal DNA synthesis and total DNA content of wild-type and transgenic blastocysts were similar, but the latter did not respond to exogenous IGF-I with DNA synthesis. The reproductive performance of ␣1 AT-hIGFBP-1 transgenic mice was impaired in several aspects, especially in homozygous females. The changes included a reduced percentage of matings resulting in pregnancy, interrupted or prolonged pregnancies with fetal or neonatal death (accordingly, homozygotes represented two-thirds of the dead pups), and a reduced litter size. Although present in the human ovary and apparently involved in its physiology and pathology (49), IGFBP-1 normally is not expressed at detectable levels in the mouse ovary. This circumstance associated with the characteristics of impaired fertility indicates that the main element affecting the reproductive performance of PGK-rIGFBP-1 transgenic mice is reduced ovulation as a consequence of the ectopical expression of IGFBP-1 in the ovaries. The origin of the impaired fertility of ␣1 AThIGFBP-1 transgenic mice remains unclear. Several studies indicate that IGFBP-1 is not only involved in cyclic alterations of ovary and uterus, but also in blastocyst implantation, acting as a maternal ‘restraint’ on trophoblast invasion (49). This could be an alternative or an additional mechanism leading to impaired fertility in these animals. However, it appears to be a consequence of high IGFBP-1 serum levels, since no transgene expression was detected in ovary or uterus, and several other systems were affected without showing any expression of the transgene. Other effects Besides the main effects discussed above, several other alterations were observed in IGFBP-1 transgenic mice (Table 3). For instance, ␣1 AT-hIGFBP-1 animals show proteinuria and glomerulus lesions (11). This is a very interesting finding since some components of the IGF system are known to be aberrantly expressed in pathophysiological processes associated with diabetic renal disease (83). The relative increase of spleen weight observed in PGKrIGFBP-1 animals (52) may be a consequence of IGF-independent actions of IGFBP-1. However, this does not apply to the altered tolerance and sensitivity to ethanol in MT-hIGFBP-1 mice (61). 633

IGFBP-2 IGFBP-2 is the second most abundant IGFBP in the circulation and binds IGF-II with higher affinity than IGF-I (for a review, see ref 26). Serum IGFBP-2 levels are increased in transgenic mouse models overexpressing IGF-II, suggesting a regulation of IGFBP-2 expression by its primary ligand (82, 83). Increased levels of serum IGFBP-2 are found after fasting and in a number of pathological conditions, including non-islet cell tumor hypoglycemia, diabetes mellitus, chronic renal failure, liver cirrhosis, and certain types of leukemia (reviewed in ref 84). Knockout mice lacking IGFBP-2 expression did not show overt phenotypic alterations. The only morphological difference appeared to be the size of the adult male spleen, which was reduced by ⬃30%. However, the concomitant up-regulation of other IGFBPs and the functional redundancy within this family of related proteins might have hidden intrinsic effects of IGFBP-2 (44, 85, 86). There is indirect evidence for a role of IGFBP-2 as an inhibitor of IGF actions. First, reduced growth of mice selected for low body weight was associated with increased hepatic IGFBP-2 mRNA expression and IGFBP-2 serum levels (87). Second, transgenic rabbits expressing high levels of recombinant human IGF-I in their mammary glands did not show any phenotypic alterations. Ligand blot analysis of milk from these transgenic rabbits revealed a marked increase in the activity of IGFBP-2 that might have buffered effects of excess IGF-I (88). Direct evidence for an IGF-inhibitory role of IGFBP-2 comes from cell culture studies. IGFBP-2 inhibited IGF-II-dependent autocrine growth stimulation of human colon carcinoma cells but did not affect proliferation of IGFresistant cells (89). To characterize effects of excess IGFBP-2 in vivo, we generated transgenic mice expressing a mouse IGFBP-2 cDNA under the control of the CMV promoter (54). Transgene expression was detectable in nearly every organ tested with exception of the liver. Transgenic mice were characterized by threefold increased IGFBP-2 serum levels if compared to nontransgenic littermates (Table 2). Despite abundant transgene expression in most of the organs, organ weights were only moderately reduced (⬇3%) in IGFBP-2 transgenic mice. The weight reductions were most prominent in pancreas, spleen, and liver. It is important to point out that it is precisely these organs that showed the greatest increase in weight in IGF transgenic mice (90). A similar inverse phenotype was seen for the carcass weight: IGFBP-2 overexpression decreased carcass weight by 12–13% (54) whereas IGF-I overexpression resulted in a 20% increase (90). IGFBP-2 transgene expression was detected at birth and active throughout the postnatal period. Despite early transgene expression around birth, body weight gain was reduced only at an age later than 3 wk postnatally (Fig. 1). We have 634

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demonstrated high endogenous IGFBP-2 expression during the early postnatal period in nontransgenic controls, suggesting absence of a clear phenotype due to minor differences of IGFBP-2 serum levels. Adult IGFBP-2 transgenic mice displayed ⬃10 –13% reduced body weight (Fig. 1), which again represents an inverse phenotype to IGF-I transgenic mice. In pancreas, transgene expression was highest and restricted to the islets but absent in the exocrine pancreas (Fig. 2). The immunohistochemical staining pattern within the islets suggested ␤ cell-specific transgene expression. This finding is particularly interesting with respect to potential actions of IGFBP-2 in glucose homeostasis. Indeed, involvement of IGFBP-2 with glucose homeostasis cannot be excluded, since glucose levels were reduced in IGFBP-2 transgenic mice with borderline significance (P⫽0.06) if compared to nontransgenic mice (54). This supports the concept that there are distinct functions for different IGFBPs, since IGFBP-1 transgenic mice were hyperglycemic whereas IGFBP-2 transgenic mice certainly were not. However, the potential involvement of IGFBP-2 with glucose homeostasis deserves further investigation. Taking into account diverse phenotypes in many parameters, it is tempting to speculate that IGFBP-2 acts mainly by an inhibition of IGF-I bioactivity in IGFBP-2 transgenic mice.

IGFBP-3 In adult tissues, IGFBP-3 is most abundantly found in kidney and to a lesser extent in liver, lung, heart, spleen, and muscle. The molecular weight of the mature protein may vary considerably due to differences in N-glycosylation (Table 1). The concentra-

Figure 1. Body weight gain of IGFBP-2 transgenic mice (tg; hemizygous mice from the F1 and F2 generations) and nontransgenic littermates (co). Mice were weighed twice a week and body weight data were calculated by pooling the weight data for the various ages (weeks). In addition least square means were calculated. Marked by an asterisk, significant differences (P⬍0.05) were present at an age later than 3 wk postnatally in both sexes (n⫽9).

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alveoli and ducts and confirmed its absence in myoepithelial and stromal cells of mammary tissue. Despite high transgene expression, no abnormalities were obvious in mammary development during pregnancy. The only structural difference was an overall reduced size of alveoli in transgenic animals. However, the involution of the mammary gland after pregnancy was altered. The remodeling of mammary tissue was retarded in IGFBP-3 transgenic animals possibly as a consequence of decreased apoptosis. IGFBP-3 might exert this effect directly in an IGFindependent manner. Alternatively, IGFBP-3 might direct IGF-I, which is known to have antiapoptotic properties (94). Figure 2. Immunohistochemical analysis of transgene expression in the pancreas. Photomicrographs of pancreatic tissue of an F2 CMV-IGFBP-2 transgenic mouse. Formaldehydefixed pancreata were embedded in paraffin wax and serial paraffin sections were cut at a thickness of 3 ␮m. A) The histological sections were then stained with hematoxylin and eosin. B) IGFBP-2 was detected by an indirect immunoperoxidase technique using peptide-induced antiserum specific for murine IGFBP-2 as described previously (54). The immunostained section was counterstained with hematoxylin. Within the pancreata of transgenic mice, IGFBP-2 immunostaining was exclusively observed in the islets with the majority of endocrine islet cells, demonstrating intensive cytoplasmic staining. No immunostaining was seen in pancreatic tissue of control animals (not shown).

tion of IGFBP-3 in serum exceeds that of other IGFBPs by severalfold. Consistent with its function as a carrier of IGFs in the circulation, the affinity of IGFBP-3 for the IGFs is higher than those of most other serum IGFBPs. The interaction with IGFs may result in inhibitory and stimulatory responses (91). In addition, IGFBP-3 may exert growth regulatory effects that occur independently of IGFs (92, 93). Two different IGFBP-3 transgenic mouse models have been established. In one model, hIGFBP-3 is expressed under the control of the MT promoter (Table 2; 55). Transgene expression was consistently found in kidney and to a lower extent in small and large intestine. Surprisingly, both transgenic lines showed selective organomegaly that developed at places different from the major expression sites. Weights of spleen, liver, and heart were significantly increased in transgenic animals, the effect being most prominent in spleen (two- to fourfold). Serum of transgenic animals contained immunoreactive IGFBP-3 in lower molecular mass fractions (30 –75 kDa). This nonternary IGFBP-3 might account for the phenotype in these organs. In the second model, the hIGFBP-3 cDNA was placed under the control of the whey acidic protein promoter (Table 2; 56), which directs expression of the transgene to mammary tissue during late pregnancy and throughout lactation. In situ hybridization showed transgene expression in epithelial cells of IGFBP-TRANSGENIC MOUSE MODELS

IGFBP-4 IGFBP-4 was originally cloned from a human osteosarcoma cell line (95). It exists as a 24 kDa nonglycosylated and a 28 kDa N-glycosylated protein (Table 1). During embryonic development, IGFBP-4 is expressed in the zone of polarizing activity of the developing limb bud. It is also detected in the vicinity of precartilage elements, and it is speculated that IGFBP-4 expression in interdigital tissue might counteract the antiapoptotic effect of IGF-I (96). In adult murine tissues, IGFBP-4 is found most abundantly in liver, kidney, and spleen and to a lesser extent in lung, heart, brain, and muscle (Table 1). In the adult skeleton, IGFBP-4 is a major player of bone formation and resorption (97). In vitro IGFBP-4 has consistently been shown to inhibit IGF-I- and IGF-IImediated actions (29, 98 –101). Only very recently the first transgenic mouse strain was published in which rIGFBP-4 expression is driven by two different promoter constructs (Table 2; 57). The first construct consisting of 724 bp of the mouse smooth muscle ␣-actin promoter (SMP), followed by the rIGFBP-4 cDNA and SV40 small T intron, enhanced IGFBP-4 expression only moderately and was not specifically targeted to smooth muscle cell (SMC) tissues. In an alternative approach, an extended fragment of the 5⬘-flanking region and the 2.5 kb intron 1 of the SM ␣-actin gene were placed upstream of the rIGFBP-4 cDNA. This new construct (SMP8-IGFBP-4) contains further motifs that are in favor of high SMC-specific expression (102). Transgenic animals showed targeted expression of rIGFBP-4 in SMC-rich tissues, being highest in bladder and aorta whereas transgene expression was lower in stomach and in uterus. Western blotting proved the more abundant presence of glycosylated and unglycosylated forms of IGFBP-4 in SMC-rich tissues. However, this locally increased biosynthesis was not accompanied by a rise in serum IGFBP-4 levels. Whereas total body weight was indistinguish635

able from that of control animals, wet weights of bladder, aorta, and stomach were significantly decreased as a consequence of hypoplasia in SMC tissues. Transgenic mice that overexpress IGF-I under the control of the same promoter (SMP8-IGF-I) exhibit a hypertrophy in SMC tissue (103). However, this hypertrophic reaction was only modestly decreased in IGF-I/IGFBP-4 double transgenic animals. In addition, the decrease of SMC mass in SMP8-IGFBP-4 transgenic occurred without interference with IGF-I abundance or gene expression (57). One might speculate that the inhibitory action of IGFBP-4 might occur through IGF-independent mechanisms, though a satisfactory explanation for this surprising finding is still pending. Alternatively, the activation of an IGF-I dependent SMC-derived IGFBP-4 protease (104) might account for these results. In marked contrast to in vitro experiments and the overexpression of IGFBP-4 in SMC are preliminary results of IGFBP-4 knockout mice: homozygous mutants are clearly smaller than wild-type littermates, supporting the idea that IGFBP-4 may be required for optimal action of IGF-II in vivo rather than act as an inhibitor (45).

IGFBP-5 IGFBP-5 is the most abundant IGFBP in bone extracts and it appears to be essential for the storage of the IGFs, especially IGF-II, in this tissue due to its strong affinity to hydroxyapatite (105). Furthermore, it is involved in the regulation of several physiological processes in ovary and kidney (27). Recently, IGFBP-5 was also identified as one component of a ternary complex with IGF-I or -II and ALS in the human circulation (106). This finding requires a re-evaluation of the role of this IGFBP in regulating both systemic and local IGF actions. Despite these unique characteristics, no transgenic models that overexpress IGFBP-5 have been reported so far. We have generated two independent transgenic lines that have stably integrated the murine IGFBP-5 cDNA under the control of the CMV promoter (Fig. 3). Preliminary examinations revealed limited expression of the transgene in some organs (M. R. Schneider et al., unpublished results). If these animals show consistent overexpression of the transgene in certain tissues, they should provide powerful models to elucidate regulatory and developmental properties of IGFBP-5 in vivo.

IGFBP-6 IGFBP-6 differs from IGFBPs 1 to 5 in that it binds IGF-II with marked preferential affinity over IGF-I. 636

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Figure 3. Analysis of CMV-mIGFBP-5 transgenic mice. A) Schematic representation of the transgene. CMV: CMV promoter, int: rat insulin II intron A sequences, hGH: terminating sequences of the human growth hormone gene, ori: SV40 origin of replication. The microinjection fragment was excised with SpeI and XhoI and injected into fertilized oocytes. B) Southern blot analysis of genomic DNA obtained from tail tips. The purified DNA was digested with PstI and transgene integration was detected using a fluorescein-labeled mIGFBP-5 cDNA as a probe. 2,3 ⫽ transgenic; wt - wild-type

IGFBP-6 expression has been associated with inhibition of tumor cell growth in vitro and in vivo, most likely by inhibition of IGF-II, which has been implicated as an autocrine tumor growth factor (107, 108). During mouse development, strong IGFBP-6 immunoreactivity was found in ossifying bones of the cranial base, in cell clusters of the pancreas anlage, in the trigeminal ganglion, in myoblasts, and in motoneurons of the spinal cord. In tissues of adult mice, strong IGFBP-6 immunostaining was present in epidermal and peridermal layers of the skin, in meningeal layers, in long-striated skeletal muscle, and in the Langerhans islets of the pancreas (Table 1; 109). IGFBP-6 expression is markedly up-regulated in pancreatic tumors of SV40-transgenic mice re-expressing IGF-II (T. Braulke, personal communication). However, mice lacking both IGF-II and IGF-II-R expression also show strong IGFBP-6 immunoreactivity in all pancreatic islet cells, secretory granules of acinar cells, and interlobar connective tissue of exocrine pancreas (110). IGFBP-6 knockout mice do not appear to differ significantly from control animals (45), and to date no IGFBP-6 transgenic mouse model exists. Thus, the role of IGFBP-6 in vivo remains unclear and must be the subject of future studies.

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CONCLUSIONS AND PERSPECTIVES FOR FUTURE RESEARCH To date, six different IGFBPs have been characterized in mammals. They have coevolved with the IGFs and are essential elements in the regulation of their actions. Despite intense research and the accumulation of a lot of data from in vitro experiments, the precise role of each IGFBP and their degree of mutual functional compensation in vivo remain largely undefined. To gain further insight into the biology of these molecules and to unravel some of their activities in certain tissues knockout mice and transgenic mouse models overexpressing different IGFBP fusion genes have been established within the past few years. Albeit a rather wide expression of IGFBPs (except IGFBP-1) in adult murine tissues, each of the transgenic strains exhibited a specific phenotype that might be directly or indirectly related to the overexpression of a specific binding protein. These phenotypes might be the consequence of a tightly regulated interplay of many factors in vivo. A slight imbalance could either produce pathological phenotypes in one tissue or alter the activity of a given IGFBP in another. Transgenic mouse models have clearly confirmed a role of IGFBPs as regulators of IGF-activity in vivo. However, they also revealed the complexity of interactions in which a single molecule is involved in an intact organism. IGFBPs clearly mediate biological responses that occur independently of IGFs. In addition, molecules outside the IGF-system such as proteases can modulate availability and activity of IGFBPs. The current research focuses on unraveling specific functions of individual IGFBPs, which may yield essentially new information about their biological properties. This knowledge could open a wide field for therapeutic applications in human medicine for diseases like diabetes mellitus and cancer.

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M.R.S. is a recipient of a grant from the German Academic Exchange Service (DAAD) (grant no. A/97/14831). 19.

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