the role of the insulin-like growth factor binding proteins and the igfbp proteases in modulating...

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THE ROLE OF THE INSULIN-LIKE GROWTH FACTOR BINDING

PROTEINS AND THE IGFBP PROTEASES IN MODULATING

IGF ACTION

Paulo F. Collett-Solberg, MD, and Pinchas Cohen, MD

The insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and the IGFBP proteases (BP-Pr) are involved in the regulation of somatic growth and cellular proliferation in vivo and in vitro. The growth effects of growth hormone are mediated primarily through the hepatic production of IGF-I. IGFs are potent mitogenic agents, and their actions are determined by the availability of free IGFs to interact with the IGF receptors. The levels of free IGFs in a system are modulated by the rate of IGF production, clearance, and degree of binding to the IGFBPs. The IGFBPs are a family of proteins that bind to IGFs with high affinity and specificity. IGFBPs not only regulate IGFs bioavailability but also seem to have their own receptors that mediate IGF-independent actions. IGFBPs are produced by a variety of different tissues, each of which has specific levels of several IGFBPs. In addition, several enzymes capable of proteolyzing IGFBPs have been identified. The cleavage of IGFBPs by BP-Pr has a key role in modulating free IGF and IGFBP levels and actions (Fig. 1). The authors thus propose two main mechanisms for the action of IGFBPs and their proteases to influence IGF action: (1) IGFBP binding to IGFs decrease the concentration of free IGFs available to interact with the IGF receptors and (2) cleavage of the

This work was supported by grants lROlDK 47591, lROlFD 1181, and lROlCA 58110 from the National Institutes of Health.

From the Pediatric Endocrine Training Program, Division of Endocrinology (PC), Depart- ment of Pediatrics (PFC), University of Pennsylvania, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA

VOLUME 25 - NUMBER 3 SEPTEMBER 1996 591

592 COLLET'i-SOLBERG & COHEN

Figure 1. The components of the IGF axis.

IGFBPs into fragments with lower affinity to IGFs allows for increased IGF receptor activation.

IGFs AND IGFBPs IN SERUM

In serum, most of the IGF-I and IGF-I1 is found in the ternary complex, formed by IGFs, IGFBP-3, and the glycoprotein known as the acid labile subunit (ALS)." Only small amounts of IGFs are carried by other IGFBPs, and less than 1% circulate in the free form (Fig. ,).I1 The ternary complex does not cross the capillary barrier, and ALS is found only in the intravascular space." The formation of the ternary complex protects, and consequently prolongs, the half-life of both IGFBP-3 and IGFs. The half-life of unbound IGFBP-3 is between 30 and 90 minutes, the half-life of free IGF-I is less then 10 minutes, and the half-life of the 150 kd complex is approximately 12 hours.ih The binding of IGFs to IGFBP- 3 and ALS maintain IGFs in the intravascular space for steady delivery of IGF-I in contrast with the pulsatile levels of GH."

The liver is the main source of circulating IGFs even though physiologically important production occurs in other tissues with various autocrine and para- crine functions.w The liver is also the main source of circulating IGFBP-3 and ALS,2K although different components of the liver produce different components of the ternary complex. IGFBP-3 is produced by the hepatic endothelia and by Kupffer cells, and ALS and IGF-I are produced by hepatocytes." 2'1 The hepatic production of all three components of the 150-kd complex is regulated by growth hormone,'? and the serum levels of all three components of the 150-kd

THE ROLE OF IGFBPs AND IGFBP PROTEASES IN MODULATING ICF ACTION 593

IGF-I bound to ternary complex

IGF-I bound to other IGFBPs

Free IGF-I

Figure 2. Distribution of IGFs in serum. Ninety-five percent of the IGFs are found as part of the ternary complex of IGF-I or IGF-II bound to IGFBP-3 and the acid labile subunit (ALS). Less than 5% are bound to other IGFBPs, and a very small component circulates as the free form.

complex are reduced in growth hormone deficiency, resistance, or both and elevated in growth hormone excess.8, 29. 57, 72 ')', "I7

IGFBP-3 is the most abundant IGFBP in postnatal serum, and its levels do not change acutely.hh IGFBP-1 and IGFBP-2 levels are variable during the day, depending on the metabolic state.'"', Iu7 The regulators of IGFBP-4 through -6 in serum have not been well studied; however, their levels have been shown to be age dependent.12. '', Hfl F' igure 3 shows the relative levels of IGFBPs in serum in the fed and fasting states. IGFBP-3 is the predominating IGFBP, with levels (2000-5000 ng/mL) that are an order of magnitude higher then the levels of the

Figure 3. Concentration of IGFBPs in serum of adolescents in the fed and fasting state IGFBP-3 has the highest concentration in serum IGFBP-1 levels rise drastically in the fasting state, preventing the potential hypoglycemic effects of IGFs The levels of IGFBPP also rise during fasting, although not as much as IGFBP-1 l 1 j2 27 66 79 97 lo' '07

594 COLLETT-SOLBERG & COHEN

other IGFBPs. During fasting, IGFBP-1 has the lowest levels (5-20 ng/mL), tenfold lower than IGFBP-2, -4, -5, and -6.

Evolutionary and Physiologic Aspects of Insulin-Like Growth Factor Binding Proteins

Six IGFBPs have been identified to date, and at least one more has been proposed. All six IGFBPs have at least 50% homology among themselves and 80% homology among different species.72, y9 Most of the homology is conserved in the N- and C- terminal regions, whereas the middle region has little similarity among the different 1GFBPs.l’ The IGFBPs have a highly conserved set of at least 16 cysteines that shape their structure.hh

The evolutionary conservation of the IGFBPs supports their importance in the regulatory processes. As shown in Figure 4, IGFBP genes are in close proximity to the homeobox gene clusters (Hox-A through Hox-D) and seem to have evolved together. It is speculated that both Hox and IGFBP genes originated from single genes which underwent coordinated duplications and translocations several times. The Hox genes are DNA binding proteins and, like some of the IGFBPs, are transcriptionally regulated by retinoic acid. Chromosomes 2 and 7 encode two binding proteins each, and all of these four binding proteins have 18 cysteines.’, 6o IGFBP-1 and IGFBP-3 genes are in chromosome 7, next to the Hox-A gene. IGFBP-2 has an Arg-Gly-Asp (RGD) sequence and is related to carbohydrate metabolism, whereas IGFBP-3 is involved primarily in growth.% Similarly, on the long arm of chromosome 2, next to Hox-D, IGFBP-2 also has an RGD sequence and correlates with the metabolic state, whereas IGFBP-5

IGFBP Hox - IGFBP-I,ZS,S Hox-A,D IGFBP-4,6 HOX-B,C

I Duplication

IGFBP-1,2 IGFBP-3,5 Hox-A,D

IGFBP-1 IGFBP-3 ’Hox-A IGFBP-; IGFBP-S Hox-D 1GFBP-4 Hox-B IGFBP-6 Hox-C -1Ic1cIc--

Chromosome-7 Chromosome-2 Chromosome-17 Chromosome-12

Figure 4. Co-evolution of the IGFBP and Hox genes.

THE ROLE OF IGFBPS AND IGFBP PROTEASES IN MODULATING IGF ACTION 595

functions are primarily growth related, as can be expected because of its high homology with IGFBP-3. The IGFBP-6 gene, with 16 cysteines and next to Hox- C, is found on chromosome 12,'. 58, 71, 78 and lGFBP-4, with 20 cysteines, is localized at the long arm of chromosome 17, next to Hox-B.1,58,60,71,78

The IGFBPs have several potential functions: (1) prolongation of IGFs half- life in the circulation; (2) prevention of IGF-induced hypoglycemia; (3) regulation of the passage of IGFs from the intravascular to the extravascular space; (4) limitation of the bioavailability of free IGFs to interact with the IGF receptors; (5) enhancement of IGF actions by the formation of a pool of slow-release IGFs; and (6) direct cellular actions mediated through their own receptors, acting independently of IGFs.

BIOCHEMISTRY, MOLECULAR BIOLOGY, AND REGULATION OF IGFBPS

Insulin-Like Growth Factor Binding Protein 1

IGFBP-1 is a 25-kd protein with an RGD sequence in its s t r u c t ~ r e . ~ ~ ~ ~ ~ RGD is a recognition sequence for membrane integrin receptors, which suggests the possibility of an IGF-independent action via these r e c e p t o r ~ . ~ ~ IGFBP-1 is found in several phosphorylated states that determine its affinity to IGFS.~*

IGFBP-1 is produced in the liver, decidua, and kidneys and is the most abundant IGFBP in amniotic fluid. Serum IGFBP-1 levels are regulated mainly by metabolic factors. After meals, IGFBP-1 levels fall to less then 10 ng/mL, whereas during fasting, IGFBP-1 levels rise to more than 100 ng/dL.h6 In children with ketotic hypoglycemia who underwent diagnostic fasting studies, the IGFBP-1 levels were as high as 700 ng/dL at the time of the hypoglycemia (Paulo F. Collett-Solberg, MD, personal communication, 1996). Insulin and corti- costeroids are the main regulators of serum IGFBP-1 levels, through the transcriptional control of the hepatic production of IGFBP-1.2R Several studies have shown that insulin inhibits the synthesis of IGFBP-1, resulting in elevated levels during low insulin states, such as intrauterine growth retardation, fasting, or poorly controlled type I diabetes.'", 45, The inverse also has been shown, with decreased levels in conditions of hyperinsulinemia, such as the postprandial period, obesity, large-for-gestational-age babies, insulinomas, and congenital hyperinsulinism with hypoglycemia (Fig. 3) . In patients with type I1 diabetes, IGFBP-1 levels are either elevated or suppressed, depending on the insulin levels and the degree of insulin resistance. Other hormones also influence IGFBP-1 levels. Glucocorticoids and glucagon stimulate IGFBP-1 production in synergism with low levels of In chronic renal failure caused by increased renal production, IGFBP-1 levels increase.66 In patients with diabetes and renal failure, high IGFBP-1 levels are thought to have a pathophysiologic role, presumably decreasing the levels of free IGFs to interact with the IGF-I receptor, and may be responsible for the decreased linear growth in these conditions.

74, R9,


IGFBP-2 is a 31-kd pr~te in . '~ Like IGFBP-1, IGFBP-2 has an RGD sequence although it has not been demonstrated to bind integrin-type receptors; however, IGFBP-2 has been shown to be cell surface-associated via other unknown mecha- n i s m ~ . ~ ~ IGFBP-2 is neither phosphorylated nor glycosylated.

596 COLLETl-SOLBERG & COHEN

The levels of IGFBP-2 are age dependent, with high levels in infancy and older age and low levels in young IGFBP-2 is the major IGFBP in cerebrospinal fluid because of its production by multiple neural tissues,11 and its levels are elevated in the spinal fluid of patients with some forms of tumors of the central nervous system.RZ The concentration of IGFBP-2 in seminal plasma is greater than that of any IGFBP in any biologic fluid, approximately 10,000 ng/ mL.y7 Growth hormone deficiency causes an increase in the levels of IGFBP- 2 in even though growth hormone seems to have no direct effects on ICFBP-2 gene expression in cultured cells. The regulation of IGFBP-2 levels is also dependent on the metabolic state and the insulin level, albeit to a lesser degree than IGFBP-Lh8 IGFBP-2 levels increase with prolonged fasting and are more sensitive to protein restriction than to caloric restriction, being elevated in malnutrition and anorexia nervosa.1"6 In patients with low levels of insulin, such as in untreated insulin-dependent diabetes mellitus, levels of IGFBP-2 are elevated and return toward normal with chronic insulin therapy.I0' In vitro, IGFBP-2 is produced in multiple cell systems.x2 6x The in vitro regulators in most cell systems remain largely unknown; however, in neuroblastoma cells, retinoic acid downregulated IGFBP-2 expression.I3

Insulin-like Growth Factor Binding Protein 3

The molecular weight of IGFBP-3 in its nonglycosylated form is 29 kd.Io2 IGFBP-3 has three glycosylation sites and is present in the circulation in the glycosylated state, with a molecular weight between 40 and 44 kd.6h

Serum levels of IGFs and IGFBP-3, are age dependent, being low at birth, increasing during childhood to reach a peak during puberty, and decreasing thereafter.27, 6h Nutritional status has an important role in controlling serum IGF- I, with low IGF-I levels observed in patients with chronic disease or malnutri- t i ~ n . ~ ~ , IU1, I"' The levels of IGFBP-3, however, are less affected by these condi- t ion~." '~ Estrogens, parathormone (PTH), and glucocorticoids are other regulators of IGFs and IGFBPs in the circulation.4", hh

The mechanism by which growth hormone stimulates IGFBP-3 production is still under investigation. The two proposed mechanisms are (1) a direct effect of growth hormone on Kupffer cells and (2) an indirect effect mediated by IGF- I. In a large population of growth hormone receptor-deficient patients from Equador, the administration of IGF-I stimulated growth but did not change IGFBP-3 levels by radioimmunoassay (RIA)?, lo9 In a similar population from Israel, IGF-I caused increased levels of IGFBP-3 by Western ligand blotting; however, IGFBP-3 levels by RIA were not performed." One of the explanations for this finding is that IGF-I did not stimulate the production of IGFBP-3 but instead protected IGFBP-3 from proteolysis. In some animal models, IGF-I induces the serum levels of IGFBP-3 detected by Western ligand blotting.I13 In vitro studies have shown production of IGFBP-3 in liver cells after IGF-I but not after growth hormone stimulation.Im In bone cells, both unchanged levels of IGFBP-3 in response to growth hormoneh-5 and an increase in IGFBP-3 expression after growth hormone (but not IGF-I) stimulation have been rep~rted. '~ IGFBP- 3 is produced in a variety of systems,4' and its expression in vitro is regulated by PTH, retinoic acid, transforming growth factor beta (TGF-P), and several other hormone^.^", h5, 85,

THE ROLE OF IGFBPs AND IGFBP PROTEASES IN MODULATlNG IGF ACTION 597


IGFBP-4 is found with its predicted molecular weight of 24 kd or in the glycosylated form with a molecular weight of 28 kd.66 IGFBP-4 has been identified in all biologic fluids.28 Different cell types produce IGFBP-4 locally, including fibroblasts, neuroblastoma cells, prostate cells, and bone cells." Although there is demonstrable binding of IGFBP-4 to cell membranes (the function of which is unknown), IGFBP-4 is found mainly as the extracellular soluble form.5h The regulatory mechanisms affecting IGFBP-4 expression are still poorly under- stood, but at least in bone, IGFBP-4 is regulated by vitamin D and PTH,H'. '' whereas in neuroblastoma cells, retinoic acid inhibits IGFBP-4."

insulin-Like Growth Factor Binding Protein 5

IGFBP-5 has a molecular weight of 29 kd, and it can be found in several glycosylation forms with a molecular weight between 29 and 32 kd."h As is true for IGFBP-3, IGFBP-5 levels decrease with age, starting after puberty. The levels in older women are approximately 30% of those for teenager^.^', "(I Fetal tissues have high levels of IGFBP-5 during rapid whereas levels in adult tissues vary. Cerebrospinal fluid (CSF) and connective tissues have substantial concentrations of IGFBP-5, and IGFBP-5 is the main IGFBP expressed in the kidneyshh Unlike the other IGFBPs, IGFBP-5 strongly binds to bone cells because of its high affinity for hydroxyapatite.6R Like IGFBP-3, IGFBP-5 binds to endothelial cell monolayers and is found in large concentrations in the extracellular matrix. The binding to the endothelial cells is competitively inhibited by heparin and heparan sulfate, and alterations in the C-terminal region of IGFBP-5 inhibit the binding to the cells but not to the extracellular matrix.I7 IGFBP-5 binds to the extracellular matrix on an ionic basis. When bound to the extracellular matrix, the affinity of IGFBP for IGF-I is sevenfold to twelvefold reduced when compared with the intact IGFBP-5 in solution.z4, 6'

The mechanisms by which IGFBP-5 is regulated are still being unraveled. Bone cells produce large amounts of IGFBP-5, and levels decrease with the progression of maturation because of protease activity, as is discussed later. Treatment of osteoblastic cells with fibroblast growth factor, TGF-P, and platelet- derived growth factor BB causes a decrease in the IGFBP-5 expression." In the same cell system, treatment with IGF-I and retinoic acid causes the inverse effect, with increased levels of IGFBP-5 mRNA?7 The levels of IGFBP-5 in fibroblasts and osteoblast-like cells decreased when glucocorticoids were added to the cells.", 87 In smooth muscle cells, IGFBP-5 mRNA expression is stimulated by IGF-I by a mechanism that is time and dose dependent and cell type specific.?"


IGFBP-6 is an 0-glycosylated protein7 with a predicted molecular weight of 34 kd.28 IGFBP-6 is found predominantly in CSF and serum.hh It is the only IGFBP that preferentially binds to IGF-I1 by more than two orders of magnitude better than to IGF-I.7, 12,22 In osteoblastic cells, the addition of excessive concentration of IGFBP-6 inhibited IGF-11-stimulated DNA and glycogen synthesis but had minimal effects on inhibiting IGF-I actions.I2 IGFBP-6 is expressed in ovarian cells, prostatic cells, fibroblasts, and other cells.7 The expression of IGFBP-6 is regulated by IGF-I1 and other hormones. Studies in breast cancer cell lines


showed that, in the cells studied, only estrogen receptor-negative cells produced IGFBP-6 and that its expression was not changed by IGF-I but was enhanced by retinoic In osteoblast cells, retinoic acid increases IGFBP-6 expression by more then 1000%.114

A Candidate for Insulin-Like Growth Factor Binding Protein 7

Mac-25 is a gene that was originally found in normal leptomeningeal cells and found to be absent in meningioma tumors. Later, mac-25 was also found in high levels in senescent human breast epithelial cells but in low levels in breast cancer cell lines. The mac-25 gene is localized on chromosome 4q12. Mac-25 has 40% to 45% homology with the six known IGFBPs and conserves 12 of the 16 common cysteines and thus has been speculated to be a member the IGFBP family. Like IGFBP-3 and IGFBP-5, its expression is stimulated by TGF-P, IGFs, and retinoic acid.lo4 It has yet to be demonstrated that mac-25 actually binds IGFs.

IGFBP FUNCTIONS

Figure 5 proposes how IGFBPs can inhibit or enhance IGF actions. It also shows that IGFBPs can have IGF-independent activity mediated through their own receptors. In most systems tested to date, IGFBPs decrease IGF function by binding to IGF and consequently reducing the levels of free IGFs to interact with IGF receptors.

IGFBPs Inhibiting IGF Actions

Insulin-Like Growth factor Binding Protein I The effects of IGFBP-1 are still under investigation; however, both inhibitory

and stimulatory activity have been reported. IGFBP-1 has serine residues that can be phosphorylated. Phosphorylation of IGFBP-1 substantially increases its affinity for IGF-I and is probably involved in the regulation of IGFBP-1 function. Phosphorylated forms, in general, inhibit IGF-I action, whereas the dephosphor- ylated forms seem to have stimulatory In vivo, IGFBP-1 helps to protect the organism from the hypoglycemic effects of IGF-I. Without this mechanism, IGF-I could cause hypoglycemia during fasting. ICFBP-1 levels rise in the fasting state (see Fig. 3), suppressing the hypoglycemic effect by binding to IGFs and decreasing the levels of free IGF-I. To corroborate this theory, Lewitt and B a ~ t e r ~ ~ , 76 administrated IGFBP-1 to rats and registered a transient elevation in the blood glucose. Another proposed inhibitory function for IGFBP-1 may occur during fetal development in certain conditions. In fetuses with poor placental supply, low availability of nutritional factors cause insulin levels to be low, and, consequently, levels of IGFBP-1 increase. The increase in IGFBP-1 levels sequesters free IGF-I from the circulation so the use of nutrients for fetal growth decrease in detriment to survival. Similarly, large-for-gestational-age fetuses who have low serum IGFBP-1 because of high levels of insulin have a decreased inhibition of IGFs and thus are large.

Consistent with the in vivo effects of IGFBP-1 inhibiting the hypoglycemic effects of IGF-I, several studies in vitro have shown that IGFBP-1, when added

THE ROLE OF IGFBPs AND IGFBP PROTEASES IN MODULATING IGF ACTION 599

Figure 5. IGFBP functions. A, Prolongation of IGF half-life in the circulation. €3, Prevention of IGF-induced hypoglycemia. C, Facilitation of the passage of IGFs from the intravascular to the extravascular space. D, Limitation of bioavailability of free IGFs to interact with the IGF receptors. €, Enhancement of IGF actions by the formation of a pool of slow-release IGFs and presentation of IGFs to the IGF receptor. F, Direct cellular effect mediated through their own receptors.

to cells in culture, inhibits IGF-I-stimulated growth. The inhibition of IGF-I action by IGFBP-1 does not occur when IGF-I analogues with low affinity to the IGFBPs are added to the system, demonstrating an IGF-dependent mechanism of inhibition.h0

Insulin-Like Growth factor Binding Protein 2

IGFBP-2 has mainly inhibitory effects on IGF-mediated functions. Studies performed with multiple cell lines demonstrated that IGFBP-2 inhibits cell growth and multiplication.60 In IEC-6 cells, the inhibition of IGFBP-2 mRNA expression caused stimulation of growth," and the reverse also occurred because the inhibition of growth by TGF-P is associated with the induction of IGFBP-2 mRNAJ3 In prostatic stromal cells from patients with benign prostatic hyperplasia, the IGFBP-2 levels are reduced, probably causing increased levels of free IGFs, thereby facilitating tissue Nevertheless, targeted disruption of the IGFBP-2 gene in mice did not result in an altered phenotype.2x


Different cell systems have shown different effects of IGFBP-3 on IGF-I function. Multiple in vitro studies showed that IGFBP-3 in solution decreases the stimulatory effects of the IGFs, in most of the cases by preventing IGFs from


binding to their receptor because the affinity of IGF to IGFBP-3 is higher than to the IGF-I receptor.41 The authors showed that the growth of cells transfected with IGFBP-3 gene is slower then the wild type.31 The addition of IGFBP-3 to a variety of cells, in competition with IGFs, results in attenuation of IGF-mediated mitosis and metabolic actions.28, 93 The inhibitory effects of several hormones, such as TGF-P and retinoic acid, may be mediated by stimulation of IGFBP-3.Rs


All studies using IGFBP-4 in different cell lines reported so far showed an inhibitory effect of IGF.24, 81, lo" In solution, IGFBP-4 binds to IGF-I competitively, decreasing its binding to the type I IGF receptor. It does not affect IGF-induced cell proliferation when IGF-I analogs with low affinity for IGFBP-4 but with normal affinity for the IGF-I receptor are used.H' The inhibition of IGF activity by IGFBP-4 was confirmed to be a consequence of the diminution of free IGF-I from the extracellular environment by the addition of recombinant IGFBP-424, "'"'' and by the transfection of IGFBP-4 into cell lines.'"" The regulation of the IGF inhibitory IGFBP-4 function is protease dependent, as is discussed later.


IGFBP-S inhibits the growth of a variety of cells. In granulosa cells, the levels of IGFBP-5 increase during the degenerative phase.6* Because IGF-I promotes maturation and proliferation of the follicles, it was speculated that IGFBPS inhibits IGF-I action.68 It also has been shown that follicle-stimulating hormone (FSH) promotes granulosa cell proliferation by reducing intact IGFBP-5 levels (in a process that involves induction of a specific binding protein protease).6R Ling and colleagues77 showed that IGFBP-5 inhibited granulosa cell steroidogen- esis stimulated by IGF-I. In the kidneys, IGFBP-S also may inhibit growth because it is expressed inversely with renal tissue growth status.@ IGFBP-S inhibits glycogen and DNA synthesis in osteosarcoma cells, a process that is IGF-I dependent.hy


In vitro studies have shown that, when added to tissue culture media, IGFBP-6 inhibits IGF-I1 binding and suppresses IGF-11-dependent myoblast differentiation and proliferation but does not affect IGF-I-dependent function^.^, In osteoblastic cells, the addition of excessive concentration of IGFBP-6 also inhibited IGF-I1 stimulated DNA and glycogen synthesis but had only relatively lower effect on inhibiting the IGF-I effects."

Candidate for Insulin-Like Growth Factor Binding Protein 7 (mac-25)

Mac-25 is found in normal cells but is absent in corresponding tumor cells, demonstrating that it may inhibit growth.lo4 Cytokines such as TGF-P and retinoic acid may inhibit growth via the stimulation of IGFBP-3, IGFBP-5, and mac-25. Although mac-25 seems to be associated with inhibition of cell growth, no evidence yet proves that these effects are mediated through IGF-binding and inhibition.

THE ROLE OF IGFBPs AND ICFBP PROTEASES IN MODULATING IGF ACTION 601

IGFBP Stimulating IGF Actions


Although IGFBP-1 inhibits mitosis by removing free IGF-I from the extracellular space, in some in vitro systems IGFBP-1 stimulates growth. In the presence of a low concentration of platelet-poor plasma and IGF-I, IGFBP-1 stimulated DNA synthesis in porcine aortic smooth muscle cells, chick embryo fibroblasts, and mouse embryo fibroblasts.*" 49 Koistinen and colleague^^^ concluded that IGFBP-I caused slow and steady release of IGF-I when concentrations that can inhibit IGF-I binding to its receptor sometimes enhance IGFs stimulated thymi- dine incorporation. The inhibition did not occur when IGFBP-1 was added without IGF-I, suggesting an effect caused by the slow release of IGF-I and not the result of a direct effect of IGFBP-1.26, 70 Other investigators showed that IGFBP-1 phosphorylation alters its effects. Phosphorylated IGFBP-1 has high affinity for IGF-I and probably inhibits IGF-I action, whereas nonphosphorylated forms, such as those in pregnancy serum, have low affinity and stirnulatory activity.z4, h3 IGFBP-1 may be involved in IGF-I transport through the capillary barrier, a process that, in certain tissues, seems to involve an insulin-dependent me~hanism.~, 7h

insulin-Like Growth Factor Binding Protein 3

Studies by Baxter and by Conover showed that, although in the same cell system the addition of IGFBP-3 to culture media caused inhibition of cell growth, preincubation with IGFBP-3 followed by removal of this IGFBP-3 caused potentiation of the effects of IGF-I.", 46 The conclusion was that the presence of large amounts of IGFBP-3 caused reduction in the free IGF levels, whereas small amounts of IGFBP-3 protected IGFs and intensified its effects. Conover and co- workers4' showed that IGFBP-3 inhibits IGF-I action when added to the conditioned media, probably because of removal of free IGF-I. When cells were preincubated with IGFBP-3 and then washed, low molecular weight forms of IGFBP-3, probably representing proteolysed fragments, were bound to the cell membrane.41 The proposed mechanism for the difference between the effects of preincubated and soluble IGFBP-3 is that the IGFBP-3 fragments bound to the cell membrane have an order-of-magnitude lower affinity to IGF-I than the intact, soluble IGFBP-3, which has a higher affinity to the IGFs than the IGF receptor. In fact, the affinity of IGF-I to the membrane-bound IGFBP-3 fragment is lower than that of the type I IGF receptor. Thus, IGFBP-3 might function as a reservoir of IGF-I, presenting and slowly releasing IGF-I to interact with its receptor all the while, protecting the receptor from downregulation. In support of this latter concept, Conover and Powell37 showed that the IGF-I receptor downregulation induced by IGF-I can be prevented by IGFBP-3, which may protect the receptor by regulating the availability of IGF-I to bind to its receptor.

In vivo studies have shown that the topical use of IGFBP-3 in association with IGF-I causes better wound healing than the use of IGF-I alone.54 In another study, the administration of IGFBP-3 and IGF-I to growth-deficient rats caused better growth than the administration of IGF-I alone.2' In the same study, however, IGFBP-3 protected the rats from the hypoglycemic effects of IGF-I. Thus, it seems that IGFBP-3 targets IGF-I toward growth provocation and away from the insulin-sensitive, glucose-consuming tissues.



Several in vitro studies showed that IGFBP-5 stimulated IGF-I actions when compared with IGF-I 24 The IGF-enhancing actions of IGFBP-5 are partic- ularly evident in bone cells. Studies suggest a need for IGFBP-5 to be bound to the cell membrane or to extracellular matrix to cause this potentiator effect.*, 24

When bound to the extracellular matrix, IGFBPS has lower affinity for IGF-I than the free form but has a prolonged half-life.z4,61 The binding of IGF-I to the matrix-bound IGFBP-5 facilitates subsequent binding of IGF-I to its receptor^.^^,^^

IGFBPs with IGF Independent Function

Several studies have shown a direct effect of the IGFBPs on cell growth that is independent of the IGFs. Only IGFBP-1 and IGFBP-3 have been shown to have independent functions, but such actions are postulated for IGFBP-2, -4, and -5 also,"', Rh all of which seem to be cell-surface associated in some systems.

A study by Jones and colleagues62 showed a direct effect of IGFBP-1 stimulating cell migration in a monolayer-wounding assay. The direct effect of IGFBP- 1 and some of the other stimulatory activity of IGFBP-1 are dependent on the interaction of the RGD sequence with the integrin receptor in the cell membrane.

In cultured ovarian granulosa cells, DNA synthesis is stimulated by FSH. In those cells, IGFBP-3 inhibited DNA synthesis independently of the presence of IGFs. To further exclude the possibility of an effect mediated through IGFs, IGFBP-2, which should also bind IGFs, was added, but similar inhibition was not obtained.14 The synthesis of DNA by embryonic fibroblasts is inhibited by the addition of IGFBP-3 even in the presence of high concentrations of insulin, which should provoke effects mediated through the type I IGF receptor. The authors concluded that IGFBP-3 had inhibitory action, independent of the IGFS."~ The authors also have shown that IGFBP-3 had independent inhibitory effects on cell growth, using a cell transfection system.31 Recently, Valentinis and colleagues11o showed inhibition of growth by IGFBP-3 in cells derived from an IGF-I receptor "knock-out" mouse and demonstrated that the IGF-IGF receptor interaction is not involved in this IGFBP-3 effect. Oh et a1 showed that IGFBP-3 caused a direct inhibition of cell growth and that this inhibition was blocked by the addition to the culture media of IGF-I1 (but not analogues of IGF-I1 with normal IGF-R binding capacity but with no affinity to IGFBP-3). Oh and c o - ~ o r k e r s ~ ~ , 85, R6 showed that IGFBP-3 binds to a cell membrane receptor and that it is the interaction with the receptor that causes the direct inhibition of cell growth.

IGFBP-4 also may have IGF-independent actions compatible with its membrane binding. When the IGFBP-4 gene was transfected into colon cancer cell line HT-29, growth inhibition that was not reversed by IGFs or serum was observed.Io0 IGFBP-5 has been shown to stimulate bone cell proliferation in vitro both in the absence of IGFs and in the presence of IGF analogs that do not bind to IGFBP-5, demonstrating an IGF independent function, probably involving cell-surface binding sites.2, *5

IGFBP PROTEASES

The BP-Prs were first described in pregnancy serum as a proteolytic activity against IGFBP-3.I5 Since then, BP-Prs have been described in many other clinical


situations and in various body fluids and have been shown to cleave IGFBP-2 through IGFBP-6 with varying specificity. The molecular nature of some of these proteases is being unraveled, and at least three classes of BP-Prs have been identified, including kallikreins, cathepsins, and matrix metalloproteinases (MMP).

The proteolysis of the IGFBPs is probably an essential piece of the intricate and complex regulation of IGF action. In general, fragmented binding proteins have decreased affinity for IGFs, causing release of free IGF and subsequent mitogenesis (Fig. 6). In serum, the proteolysis of IGFBP-3 releases IGF-I and may allow IGF-I to be transported to the extravascular space. In the extravascular space, IGFs are bound to various IGFBPs, and specific proteases presumably promote the release of IGFs at the tissue level. As mentioned earlier, the IGFBP- 3 bound to the cell membrane which causes stimulatory effects over IGF action, is a fragment caused by local proteolysis, with lower affinity for IGFs.4I

Kallikrein-like IGFBP Proteases

The first biochemically identified BP-Pr, prostate-specific antigen (PSA), is a protease found in seminal plasma and is characterized as a kallikrein-like serine p r ~ t e a s e . ~ ~ PSA causes proteolysis of IGFBP-3 and IGFBP-5 but not the proteolysis of IGFBP-1, -2, -4, or -6.30 PSA cleaves IGFBP-3 into fragments that had an order-of-magnitude lower affinity to IGF-I.91 IGFBP-3 inhibits the stimulatory effects of IGF on the growth of prostate cells in vitro. The addition of PSA to the cell culture media had no effect on its own but blocked the IGFBP-

Figure 6. BP-Prs-modifying IGFBP function. A, In the intravascular space, the cleavage of IGFBPs releases IGFs, which cross the capillary barrier and reach certain tissues. B, At the tissue level, the cleavage of IGFBPs allows the IGFs to interact with their receptors.


3 inhibitory activity and retained the IGF stimulatory effecP (Fig. 7). The prostate cancer cell line PC-3 produces IGF-11, IGFBP-3, and an IGFBP-3 protease (identified as plasmin). Incubation with anti-IGF or anti-IGF receptor antibodies reduced the growth of these cells. The use of plasmin-specific protease inhibitors, which cause decreased proteolysis of IGFBP-3, also inhibited growth because of decreased availability of free IGFs to interact with the IGF-I receptor.'

Other kallikrein-like IGFBP proteases include the y-nerve growth factor (y- NGF or 75 NGF), which shares a common domain with, and has a high DNA sequence homology to, PSA."' 2.5s NGF and other kallikreins have little or no proteolytic activity against IGFBPs, suggesting that the y-subunit of NGF is the proteolytically active component.y1 Unlike PSA, y-NGF also displays potent proteolytic activity against IGFBP-4 and IGFBP-6."' Thus, y-NGF may be involved in the growth of cells by more than one mechanism. In addition to binding to its own receptors, NGF is capable of cleaving IGFBPs and enhancing IGF action." The synergistic interaction between NGF and the IGF axis may have important implications on cell growth, development, and repair in brain and other tissues.

Cathepsins as IGFBP Proteases

Cathepsins are lysosomal proteinases found in aqueous extracts of a variety of normal and malignant tissues.36 These proteases are active at acid pH ranging from pH 5.5 to pH 4 and are considered important in many physiologic and pathologic processes, including neoplastic infiltration.'h Cathepsins have been shown to cleave IGFBP-1 through IGFBP-5 in proteolytic studies using

W IGFBPQ

IGF-II

U I I IGF-II+IGFBP-3

IGF-II+IGFBP-3+PSA

*

T *

Figure 7. Regulation of cell growth by IGF axis components. The effects of IGF-II (2 nM), recombinant IGFBP-3 (4 nM), and PSA (2 kM) on prostate epithelial cell growth. Results are expressed as mean ? SEM relative to control (cells grown in basal media); * = P < 0.0001 relative to control; 5 = P < 0.001 relative to IGF stimulation but not significantly different from control.

THE ROLE O F IGFBPs AND IGFBP PROTEASES IN MODULATING IGF ACTION 605

purified cathepsin D and conditioned media from prostate and breast cancer cell 43 Furthermore, immunoadsorption of cathepsin D from the media attenuated the acid-activated IGFBP hydrolysis.", The authors have shown that cathepsin D is an acid IGFBP protease present in both normal and malignant prostatic epithelial cells (but not stromal cells) in primary ~ul ture ."~ The authors also demonstrated the presence and activity of cathepsin D in seminal ~ l a s m a . " ~ The physiologically relevant role of cathepsins as IGFBP proteases is much in debate. The abnormally elevated release of H+ ions from membrane-bound proteins in cancer cells may provide an acidic environment for extracellular cathepsin action and thereby may permit an accelerated rate of cell growth secondary to the release of free IGFs because of the cleavage of IGFBPs.

Matrix Metalloproteinases as IGFBP Proteases

The MMPs are peptide hydrolases that require a metal ion for their catalytic activity. This group of enzymes is inactivated by metal chelating agents and by naturally occurring The BP-Pr activity in pregnancy serum is caused by members of the MMP family.s1 The MMP family includes a number of collagenases, such as interstitial collagenase (MMP-l), gelatinase A (MMP-2), stromolysin 1 (MMP-3), and gelatinase B (MMP-9). Although MMPs seems to have an important pathologic role in inflammation and in the invasion and metastasis of cancer cells, little is known about the role of these proteases as comitogens. The first suggestion of MMPs as IGFBP proteases was the demonstration of a Zn(2 + )-dependent IGFBP protease(s) produced by dermal fibroblasts in vitro and present in mouse pregnancy serum.sn, 51 These proteases were inhibitable by the MMP-specific tissue inhibitor of metalloproteinases-1 (TIMP- 1). Removal of MMP-1, MMP-2, and MMP-3 from conditioned medium by sequential immunoaffinity and gelatin-Sepharose chromatography resulted in the complete loss of IGFBP-3-degrading proteinase activity. Those studies sug- gested that MMPs may have a role in regulating cellular growth and proliferation via degradation of IGFBP-3, thus enhancing IGF bioavailability. The authors recently identified BP-Pr activity in airway smooth muscle cell conditioned media and characterized it to be MMP-1 by immunologic techniques.32

IGFBP-5 proteases are present in osteoblast-conditioned media following differentiation and maturation. Studies have shown that in MC3T3-El osteoblasts, the IGFBP-5 mRNA is present and increases during the maturation process, even though the concentration in the conditioned media gradually decreases, The concentrations of MMP-1, MMP-2 and another unidentified protease increase during the same period of time,"I5 suggesting a role for MMPs in IGFBP-5 proteolysis and cell maturation.

Effects of Proteases on the IGF-IGFBP Axis

Asthma-associated airway hyperplasia has been shown to be primarily a result of smooth muscle cell pr~l i ferat ion.~~ This condition is a common feature of chronic asthma and is a major cause of mortality and morbidity because of the associated bronchial narrowing. Suspecting that inflammatory agents are responsible for this hyperplasia via mechanisms that may involve the IGF axis, the authors studied the effects of leukotriene D, (LTD?) on bronchial smooth muscle (BSM) cells. LTD, mediated the induction of a BP-Pr that was identified as MMP-1. The activity of this BP-Pr resulted in loss of intact IGFBP-2 and


IGFBP-3 in conditioned media without changes in the mRNA expression, and the generation of low affinity fragments. The authors also observed that IGFBP- 2 is highly inhibitory to IGFs actions on BSM growth.35 Furthermore, the authors demonstrated a synergism between IGF and LTD, in BSM cell proliferation and concluded that this effect is mediated through the induction of MMP-1 by LTD4, leading to IGFBP-2 proteolysis and increased free IGF-I1 in the BSM environment9' (Fig. 8).

150,

0

lz L .L = I 8 100

m B

B o

LL

LTD, concentration

Figure 8. Effects of LTD, on IGF-stimulated cell growth and IGFBP-2 levels. A, Synergistic effect of IGF-I and LTD, on airway smooth muscle proliferation. Cell counts were determined on day 4 postinoculation for cells refed at 24 hours postinoculation with either serum-free medium (SFM) alone or SFM containing IGF-I, LTD,, or both. Values statistically significant by ANOVA are P < 0.05 (*) relative to control and P < 0.02 (9) relative to control and P < 0.05 relative to IGF-I alone. B, Dose response of LTD, on airway smooth muscle IGFBP-2 levels in conditioned media. Mean IGFBP-2 levels by densitometric analysis of autoradiographs of Western ligand blots expressed as percentage of control. = P < 0.01; ** P < 0.001. Airway smooth muscle cells were treated with LTD, for 48 hours at the molar concentrations indicated.

Mustration continued on opposite page


68- MMP-1

C 1 2 3 4 5 6 7 0 9

- 0 Q,

* * * *

Figure 8 (Continued). Demonstration of MMP-1 in ASM-CM. 50 (LL of ASM-CM (48 hours) from control and treated (LTD,) ASM cells electrophoresed on 10% SDS-PAGE and blotted on nitrocellulose membrane. MMP-1 was visualized using monoclonal anti-MMP-1 and ECL detection system. Lane 1 shows the positive control (conditioned media from dexametha- sone-treated fibrosarcoma cells). Lanes 2, 3, and 9 show ASM-CM from cells incubated with serum-free conditioned media for 48 hours. Lanes 4 to 8 show ASM-CM from cells incubated with serum-free conditioned media and LTD, in various concentrations. D, Inhibi- tor profile of the IGFBP-2 protease activity in LTD, treated airway smooth muscle (ASM) cells conditioned media (CM). Means of IGFBP-2 protease activity derived using densitometric analysis of autoradiographs expressed as percentage of control. Means and SDs of quadruplicate experiments are plotted. Control or LTD,-treated ( M) ASM-CM was incubated with or without inhibitors and V-IGFBP-2 for 6 hours at 37°C with and without inhibitors. Cells were conditioned for 48 hours. The EDTA (5mM), TIMP-1 (2(~g/mL) and BB-94 (5 kg/mL) effect on BP-2 proteolysis, when calculated as percent proteolysis, demonstrated significant (* = P < 0.05, ** = P < 0.01) inhibitory effect compared with LTD,-treated CM without inhibitors. Anti-MMP-1 antibody eliminated almost 95% of the protease activity in LTO, CM (*** = P < 0.001).


In prostate epithelial cells, the authors showed that PSA blocks the inhibitory effects of IGFBP-3, and they speculated that this process may be involved in the pathogenesis of prostate hyperplasia and proliferation. Cleavage of IGFBP- 3 by PSA caused a modest decrease in the affinity of IGFBP-3 fragments for IGF-11, but the affinity of IGF-I for the PSA-derived IGFBP-3 cleavage products fell by an order of magnitude. This represents an important mechanism by which PSA can enhance IGF action because decreased affinity of a PSA-gener- ated IGFBP-3 fragment for IGFs results in an increased availability of free IGF to interact with the IGF receptor. To test the latter theory at the prostate epithelial cell level, clonal assays for prostate epithelial cell growth in the presence and absence of IGF, IGFBP-3, and exogenous PSA were performed. It should be noted that prostate epithelial cells in this culture system do not produce substantial amounts of any of these three proteins; however, in vivo, prostate cells produce PSA and secrete IGFBP-3 and IGF-11. When compared with cells grown under serum-free conditions without insulin or IGFs for 5 days, the growth rate of cells grown in the presence of submaximally stimulating concentrations of IGFs doubled. The addition of IGFBP-3 at concentrations of 200 ng/mL had no effect when added alone (see Fig. 7); however, IGFBP-3 completely blocked the stimulatory effects of simultaneously added IGFs.” The most noteworthy phenomenon observed in this experiment was the reversal of the inhibitory effects of IGFBP-3 on IGF-stimulated prostate epithelial cell growth when cells were grown in the presence of PSA. It seems, therefore, that PSA functions as a prostate epithelial cell growth enhancer in the presence of IGFs and IGFBP-3 (as found in serum) by a mechanism directly related to its IGFBP-3 protease characteristics.

Pregnancy serum has elevated levels of proteases, more specifically, MMPs.~’ Protease activity regulating the IGF-IGFBP axis is present from early pregnancy,s1 with MMPs and serine proteinases secretion being documented during trophoblast in~asion.~’ During pregnancy, serum levels of MMPs pro- gressively increase, causing reduction in the levels of intact IGFBP-3. IGFBP-2, - 4, and -5 also undergo proteolysis during pregnancy.23, 52, h7 The increased activity of BP-Prs during pregnancy probably causes increased levels of free IGF-I and consequently stimulation of placental and fetal growth. When chick embryo fibroblasts were exposed to IGF-I and IGFBP-3, the IGFBP-3 inhibitory effect on IGF-I action was more pronounced in normal serum than in pregnancy serum. The authors concluded that the fragmented IGFBP-3 released IGF-I faster.’”, 71

IGFBP-4 proteases have been identified in neuroblastoma cells, smooth muscle cells, and fibroblast^.^^ The fragments of IGFBP-4 have no IGF binding capacity; consequently, the protease activity increases tissue levels of free IGFs. IGFBP-4 proteases are activated by the IGFs, predominantly by IGF-II.4Z, 59

IGFBP-5 activity is also modulated by proteases. Similar to IGFBP-3 and unlike IGFBP-4, IGFBP-5 is protected from protease activity when bound to IGFs. IGFBP-5 is found in high concentrations in the extracellular matrix. When bound to the matrix, IGFBP-5 is protected from BP-Pr activity.2q

SUMMARY

Over the past few years, there has been an explosion of data in the scientific literature regarding the various components of the IGF axis. IGFBPs and related molecules are now believed to be critical elements in numerous cellular processes and key factors in several disease states related to abnormal tissue and somatic growth. Recently, the BP-Prs were included in this complex system, and


their importance is being unraveled. The upcoming years will undoubtedly bring even more information on the molecular biology of these key cellular regulators. These discoveries are likely to lead to better understanding of growth and cellular regulation and to development of novel therapeutic approaches to a variety of diseases.

References

1. Allander SV, Bajalica S, Larsson C, et al: Structure and chromosomal localization of human insulin-like growth factor-binding protein genes. Growth Regul 3:3-5, 1993

2. Andress DL, Birnbaum RS: Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action. J Biol Chem 26722467-22472, 1992

3. Angelloz-Nicoud P, Binoux M: Autocrine regulation of cell proliferation by insulin- like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3). Endocrinology 136:5485-5492, 1995

4. Arany E, Afford S, Strain AJ, et al: Differential cellular synthesis of insulin-like growth factor binding protein 1 and IGFBP-3 within human liver. J Clin Endocrinol Metab 79:1871-1876, 1994

5. Bach LA, Hsieh S, Brown AL, et al: Recombinant human insulin-like growth factor (1GF)-binding protein-6 inhibits IGF-I1 induced differentiation of L6A1 myoblasts. Endocrinol 1352168-2176, 1994

6. Bach LA, Salemi R, Leeding K S Roles of insulin-like growth factor (IGF) receptors and IGF-binding proteins in IGF-I1 induced proliferation and differentiation of L6A1 rat myoblasts. Endocrinology 136:5061-5069, 1995

7. Bach LA, Thotakura NR, Rechler MM: Human insulin-like growth factor binding protein-6 is 0-glycosylated: Growth Regul 3:59-62, 1993

8. Ballard J, Baxter R, Binoux M, et al: Report on the nomenclature of the IGF binding proteins. Endocrinology 130:1736-1737, 1992

9. Bar RS, Boes M, Clemmons DR, et al: Insulin differentially alters transcapillary movement of intravascular IGFBP-1, IGFBP-2 and endothelial cell IGF binding proteins in rat heart. Endocrinology 127497499, 1990

10. Batch JA, Baxter RC, Werther G: Abnormal regulation of IGFBPs in adolescents with IDDM. J Clin Endocrinol Metab 73:964-968, 1991

11. Baxter RC: Insulin-like growth factor binding proteins in the human circulation, a review. Horm Res 42:140-144, 1994

12. Baxter RC, Saunders H: Radioimmunoassay of insulin-like growth factor-binding protein-6 in human serum and other body fluids. J Endocrinol 134:133-139, 1992

13. Bemardini S, Cianfarani S, Spagnoli A, et al: Expression and down-regulation by retinoic acid of IGF binding protein-2 and -4 in medium from human neuroblastoma cells. J Neuroendocrinol 6:409413, 1994

14. Bicsak TA, Shimonaka M, Malkowski, et al: Insulin-like growth factor binding protein (IGFBP) inhibition of granulosa cell function, effect on cyclic adenosine 3’,5’-mono- phosphate, deoxyribonucleic acid synthesis, and comparison with the effect of an IGF-I antibody. Endocrinology 126:2184-2189, 1990

15. Binoux M, Lalou C, Lassarre C, et al: Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3): A physiological mechanism in the regulation of IGF bioavailability. In LeRoith D, Raizada MK (eds): Current directions in insulin-like growth factor research. New York, Plenum Press, 1994, pp 293-300

16. Blat C, Villaudy J, Binoux M: In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells. J Clin Invest 93:2286-2290, 1994

17. Booth BA, Boes M, Andress DL, et al: IGFBP-3 and IGFBP-5 association with endothelial cells: Role of C-terminal heparin binding domain: Growth Regul 5:l-17, 1995

18. Canalis E, Gabbitas 8: Skeletal growth factors regulate the synthesis of insulin-like growth factor binding protein-5 in bone cell cultures: J Biol Chem 270:10771-10776, 1995


19. Chin E, Bondy C: Insulin-like growth factor system gene expression in human kidney. J Clin Endocrinol Metab 75:962-968, 1992

20. Chin E, Zhou J, Dai J, et al: Cellular localization of gene expression for components of the IGF ternary complex. Endocrinology 1342498-2504, 1994

21. Clark RG, Mortensen D, Reifsynder D, et al: Recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3): Effects on the glycemic and growth promoting activities of rhIGF-I in the rat. Growth Regul 3:50-52, 1993

22. Claussen M, Buergisser D, Schuller AG, et al: Regulation of insulin like growth factor (1GF)-binding protein-6 and mannose 6-phosphate/IGF-II receptor expression in IGF- I1 overexpressing NIH 3T3 cells. Mol Endocrinol 9:902-912, 1995

23. Claussen M, Zapf J, Braulke T: Proteolysis of Insulin-like growth factor binding protein-5 by pregnancy serum and amniotic fluid. Endocrinology 134:1964-1966,1994

24. Clemmons DR Role of post-translational modifications in modifying the biologic activity of insulin like growth factor binding proteins. In LeRoith D, Raizada MK (eds): Current directions in insulin-like growth factor research. New York, Plenum Press, 1994, pp 245-253

25. Clemmons DR, Gardner Ll: A plasma factor is required for IGFBP-1 to potentiate DNA synthesis. J Cell Physiol 145:129-135, 1990

26. Clemmons DR, Cascieri MA, Camacho-Hubner C, et al: Discrete alterations of the IGF-I molecule that alter its affinity for IGF binding proteins result in changes in bioactivity. J Biol Chem 265:12210-12216, 1990

27. Cohen P, Rosenfeld RG: Physiological and clinical relevance of the insulin-like growth factor binding proteins. Curr Opin Pediatr 6:462467, 1994

28. Cohen P, Rosenfeld RG: The IGF axis. In Rosenbloom AL (ed): Human Growth Hormone, Basic and Scientific Aspects. Boca Raton, FL, CRC Press, 1995, pp 43-58

29. Cohen P, Fielder PJ, Hasegawa Y , et al: Clinical aspects of insulin-like growth factor binding proteins. Acta Endocrinol 124:74-85, 1991

30. Cohen P, Graves HC, Peehl DM, et al: Prostate-specific antigen (PSA) is an insulin- like growth factor binding protein-3 protease found in seminal plasma. J Clin Endocri- no1 Metab 75:1046-1053, 1992

31. Cohen P, Lamson G, Okajima T, et al: Transfection of the human insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular growth. Mol Endocrinol 7380-386, 1993

32. Cohen P, Noveral JP, Bhala A, et al: Leukotriene D, facilitates airway smooth muscle cell proliferation via modulation of the IGF axis. Am J Physiol 269:L151-L157, 1995

33. Cohen P, Peehl DM, Graves HCB, et al: Biological effects of prostate specific antigen (PSA) as an IGF binding protein-3 protease: J Endocrinol 142:407415, 1994

34. Cohen P, Peehl DM, Hintz RL, et al: Insulin-like growth factor axis abnormalities in cultures of prostate stromal cells from patients with benign prostatic hypertrophy. J Clin Endocrinol Metab 79:1410-1415, 1994

35. Cohen P, Peehl DM, Stamey TA, et al: Elevated levels of insulin-like growth factor- binding protein-2 in the serum of prostate cancer patients. J Clin Endocrinol Metab

36. Conover CA, De Leon DD: Acid-activated insulin-like growth factor-binding protein- 3 proteolysis in normal and transformed cells. Role of cathepsin D. J Biol Chem 2691:7076-7080, 1994

37. Conover CA, Powell DR Insulin-like growth factor (1GF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts. Endocrinology 129:710-716, 1991

38. Conover CA, Divertie GD, Lee PD: Cortisol increases plasma insulin-like growth factor binding protein-1 in humans. Acta Endocrinol 128:140-143, 1993

39. Conover CA, Bale LK, Clarkson JT, et a1 Regulation of insulin-like growth factor binding protein-5 messenger ribonucleic acid expression and protein availability in rat osteoblast-like cells. Endocrinology 1322525-2530, 1993

40. Conover CA, Clarkson JT, Bale LK: Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF-binding protein expression in fibroblasts. Endocrinol-

76:1031-1035, 1993

ogy 1361403-1410, 1995


41. Conover CA, Clarkson JT, Durham SK, et al: Cellular actions of insulin-like growth factor binding protein-3. In LeRoith D, Raizada MK (eds): Current Directions in Insulin-Like Growth Factor Research. New York, Plenum Press, 1994, pp 255-265

42. Conover CA, Kiefer MC, Zapf J: Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies. J Clin Invest 91:1129-1137, 1993

43. Conover CA, Perry J, Tindall D, et al: Endogenous cathepsin D-mediated hydrolysis of insulin-like growth factor-binding proteins in cultured human prostatic carcinoma cells. J Clin Endocrinol Metab 80:987-993, 1995

44. Corkins MR, Vanderhoof JA, Slentz DH, et al: Growth stimulation by transfection of intestinal epithelial cells with an antisense insulin-like growth factor binding protein- 2 construct. Biochem Biophys Res Commun 211:707-713, 1995

45. Counts DR, Gwirtsman H, Carlsson LMS, et al: The effect of anorexia nervosa and refeeding on growth hormone binding protein, the insulin-like growth factors (IGFs) and the IGF-binding proteins: J Clin Endocrinol Metab 75:762-767, 1992

46. DeMellow JSM, Baxter RC: Growth hormone-dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGFOl stimulated DNA synthesis in human skin fibroblasts. Biochem Biophys Res Commun 156:199-204, 1988

47. Dong Y, Canalis E: Insulin-like growth factor (IGF) I and retinoic acid induce the syntGesis of IGF-binding protein-5 in rat osteoblastic cells. Endocrinology 136:2000- 2006, 1995

48. Duan C, Hawes SB, Prevette T, et al: Insulin-like growth factor-I (IGF-I) regulates IGF-binding protein-5 synthesis through transcriptional activation of the gene in aortic smooth muscle cells. J Biol Chem 271:42804288, 1996

49. Elgin RG, Busby WH, Clemmons DR: An insulin-like growth factor binding protein enhances the biologic response to IGF-I. Proc Natl Acad Sci USA 84:3254-3258, 1987

50. Fowlkes JL, Enghild JJ, Suzuki K, et al: Matrix metalloproteinases degrade insulin- like growth factor binding protein-3 in dermal fibroblasts cultures. J Biol Chem

51. Fowlkes JL, Suzuki K, Nagase H, et al: Proteolysis of insulin-like growth factor binding protein-3 during rat pregnancy: A role for matrix metalloproteinases. Endo- crinology 135:2810-2813, 1994

52. Giudice LC, Farrel EM, Lamson G, et al: Insulin-like growth factor binding proteins in maternal serum throughout gestation and in the puerperium: Effects of a pregnancy- associated serum protease activity: J Clin Endocrinol Metab 71:806-816, 1990

53. Guo YS, Townsend CM Jr, Jin GF, et al: Differential regulation by TGF-b 1 and insulin of insulin-like growth factor binding protein-2 in IEC-6 cells. Am J Physiol

54. Hamon GA, Hunt TK, Spencer EM: In vivo effects of systemic insulin-like growth-I alone and complexed with insulin-like growth factor binding protein-3 on corticoste- roid suppressed wounds. Growth Regul 3:55-56, 1993

55. Hardouin S, Gurmelem M, Noguiez, et al: Molecular forms of serum insulin-like growth factor (1GF)-binding proteins in man: Relationship with growth hormone and IGFs and physiologic significance. J Clin Endocrinol Metab 69:1291-1301, 1989

56. Hasegawa T, Cohen P, Rosenfeld RG: Characterization of the insulin-like growth factor axis in TM 3 Leydig cells. Growth Regul 5:151-159, 1995

57. Holly JMP, Martin JL: IGFBPs: A review of methodological aspects of their purifica- tion, analysis and regulation. Growth Regul 4(Suppl):20-30, 1994

58. Hong YS, Kim SY, Bhattacharya A, et al: Structure and function of the HOX A1 human homeobox gene cDNA. Gene 159:209-214,1995

59. Irwin JC, Dsupin BA, Giudice LC: Regulation of insulin-like growth factor binding protein-4 in human endometrial stromal cell cultures: Evidence for ligand-induced proteolysis. J Clin Endocrinol Metab 80619426, 1995

60. Jones JI, Clemmons DR Insulin-like growth factors and their binding proteins: Biolog- ical actions. Endocr Rev 163-34, 1995

61. Jones JI, Gockerman A, Busby WH Jr, et al: Extracellular matrix contains insulin-like growth factor binding protein-5: Potentiation of the effects of IGF-I. J Cell Biol

26925742-25746, 1994

268:E1199-1204, 1995

121 :679-687, 1993

612

62

63

64

65

66

67

68

69

70

71

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

COLLEP-SOLBERG & COHEN

Jones JI, Gockerman A, Busby WH, et al: Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the a5pl integrin by means of its Arg-Gly- Asp sequence. Proc Natl Acad Sci USA 90:10553-10557, 1993 Jyung RW, Mustoe TA, Busby WH, et al: Increased wound breaking strength induced by insulin-like growth factoi-I in combination with IGF binding ;rote&-1. Surgery 115: 133-239, 1994 Kanety H, Karasik A, Klinger B, et al: Long-term treatment of Laron type dwarfs with insulin-like growth factor I increases serum IGFBP-3 in the absence of growth hormone activity. Acta Endocrinol 128:144-149, 1993 Kanzaki S, Baxter RC, Knutsen R, et al: Evidence that human bone cells in culture secrete insulin-like growth factor (1GF)-I1 and IGF binding protein-3 but not acid- labile subunit both under basal and regulated conditions. J Bone Miner Res 10:854- 858, 1995 Katz LEL, Rosenfeld RG, Cohen I? Clinical significance of insulin-like growth factor binding proteins (IGFBPs). The Endocrinologist 5:36-43, 1995 Katz LEL, Stamey D, Peehl DM, et al: Neutral and acid IGFBP Proteases in serum from pregnant and nonpregnant adults and patients with prostate cancer. Program and Abstracts of the 77th Meeting of the Endocrine Society, Washington, DC, June

Kelley KM, Oh Y, Gargosky SE, et al: Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics. Intern J Bioch Cell Biol 28:619-637, 1996 Kiefer MC, Schmid C, Waldvogel M, et a 1 Recombinant human insulin-like growth factor binding proteins 4,5, and 6: Biological and physiochemical characterization. Growth Regul 3:56-59, 1993 Koistinen R, Itkinen P, Selenius P, et al: Insulin-like growth factor binding protein-1 inhibits binding of IGF-I on fetal skin fibroblasts but stimulates their DNA synthesis. Biochem Biophys Res Commun 173:408415, 1990 Kou K, Jenkins NA, Gilbert DJ, et al: Organization, expression, and chromosomal location of the mouse insulin-like growth factor binding protein 5 gene. Genomics 20412418, 1994 Lamson G, Giudice LC, Rosenfeld RG: Insulin-like growth factor binding proteins: Structural and molecular relationships. Growth Factors 5:19-28, 1991 Lassarre C, Binoux M: Insulin-like growth factor binding protein-3 is functionally altered in pregnancy plasma. Endocrinology 134:1254-1262, 1994 Lee PDK, Conover CA, Powel DR: Minireview: Regulation and function of insulin- like growth factor binding protein-1. Proc Soc Exp Biol Med 204:4-29, 1993 Lewitt MS, Denyer GS, Cooney GJ, et al: Insulin-like growth factor binding protein- 1 modulates blood glucose levels. Endocrinology 129:2254-2256, 1991 Lewitt MS, Saunders H, Cooney GJ, et al: Effect of human insulin-like growth factor binding protein-1 on the half life and action of administered insulin-like growth factor in rats. J Endocrinol 136:253-260, 1993 Ling NC, Liu XJ, Malkowski M, et al: Structural and functional studies of insulin-like growth factor binding proteins in the ovary. Growth Regul 3:70-74, 1993 Matsui T, Hirai M, Hirano M, et al: The HOX complex neighbored by the EVX gene, as well as two other homeobox-containing genes, the GBX-class and the EN-class, are located on the same chromosomes 2 and 7 in humans. FEBS Lett 336:107-110, 1993 Mohan S, Baylink DJ: Age-related changes in IGFBP-4 and IGFBP-5 levels in human serum and bone: Implications for bone loss with aging. Prog Growth Factor Res, in press Mohan S, Libanati C, Dony C, et al: Development, validation, and application of a radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids. J Clin Endocrinol Metab 80:2638-2645, 1995 Mohan S, Nakao Y, Honda Y, et al: Studies on the mechanisms by which insulin-like

1995, pp 1-204

growth factor (IGF) binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells. J Biol Chem 270:20424-20431, 1995

82. Muller HL, Oh Y, Lehrnbecher T, et al: Insulin-like growth factor binding protein-2 concentrations in cerebrospinal fluid and serum of children with malignant solid tumors or acute leukemia. J Clin Endocrinol Metab 79:428434, 1994


83. Nunn S, Peehl DM, Cohen P: Cathepsin D is an acid activated IGFBP protease produced in normal and malignant epithelial cells. Cancer Res, in press

84. Oh Y, Muller LM, Lamson G, et al: Insulin-like growth factor (1GF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition. J Biol Chem 268:14964-14971, 1993

85. Oh Y, Muller HL, Ng L, et al: Transforming growth factor-beta-induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-binding protein3 action. J Biol Chem 270:13589-13592, 1995

86. Oh Y, Muller HL, Pham H, et al: Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells. J Biol Chem 268:26045- 26048, 1993

87. Okazaki R, Riggs BL, Conover CA: Glucocorticoid regulation of insulin-like growth factor-binding protein expression in normal human osteoblast-like cells: Endocrinol-

88. Pintar JE, Schuller A, Wood TL, et al: Targeted disruption of IGFBPs. Abstract presented at the third symposium on insulin-like growth factors. Prog Growth Factor Res, in press

89. Price WA, Stiles AD, Moats-Staats BM, et al: Gene expression of insulin-like growth factors (IGFs), the type I IGF receptor, and the IGF-binding proteins in dexametha- sone-induced fetal growth retardation. Endocrinology 130:1424-1432, 1990

90. Rajah R, Bhala A, Nunsse: 75 nerve growth factor is an insulin-like growth factor- binding protein protease. Endocrinology 137:2676-2682, 1996

91. Rajah R, Katz LL, NUM S, et al: Insulin-like growth factor binding protein (IGFBP) proteases: Functional regulators of cell growth. Prog Growth Factor Res, in press

92. Rajah R, NUM S, Herrick D, et al: LTD, induces matrix metalloproteinase-l which functions as an IGFBP protease in airway smooth muscle cells. Am J Physiol, in press

93. Rosenfeld RG, Pham H, Cohen P, et al: Insulin-like growth factor binding proteins and their regulation: Acta Paediatr Suppl 339:154-158, 1994

94. Ruoslathi E, Pierschlaber MD: New perspectives in cell adhesions: RGD and integrins. Science 238:491-497, 1987

95. Scharla AH, Strong DD, Mohan S, et al: 1,25-Dihydroxivitamin D3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts. Endocrinology 129:3139-3146, 1991

96. Schmid C, Schlapfer I, Peter M, et al: Growth hormone and parathyroid hormone stimulate IGFBP-3 in rat osteoblasts. Am J Physiol 267E226-E233, 1994

97. Schwander J, Mary JL: The RIA for IGFBP-2 in man: A meagre catch? Growth Regul

98. Sheikh MS, Shao ZM, Hussain A, et al: Regulation of insulin-like growth factor- binding protein-1, 2, 3, 4, 5, 6: Synthesis, secretion, and gene expression in estrogen receptor-negative human breast carcinoma cells: J Cell Physiol 155:556-567, 1993

99. Shimasaki S, Ling N Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4,-5, and -6). Prog Growth Factor Res 3:243-266, 1991

100. Singh P, Dai B, Dhruva B, et al: Episomal expression of sense and antisense insulin- like growth factor (1GF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I. Cancer Res 54:6563-6570, 1994

101. Smith WJ, Underwood LE, Clemmons DR Effects of caloric or protein restriction on insulin-like growth factor-I (IGF-I) and IGF-binding proteins in children and adults. J Clin Endocrinol Metab 80:443449, 1995

102. Sommer A, Spratt SK, Tatsuno GP, et al: Properties of glycosylated and nonglycosylated human recombinant IGF binding protein-3 (IGFBP-3). Growth Regul 3:4649, 1992

103. Strasser-Vogel B, Blum WF, Past R, et al: Insulin-like growth factor (1GF)-I and -11 and IGF-binding proteins-1, -2, and -3 in children and adolescents with diabetes mellitus: Correlation with metabolic control and height attainment. J Clin Endocrinol Metab 80:1207-1213, 1995

104. Swisshelm K, Ryan K, Tsuchiya K, et a1 Enhanced expression of an insulin growth

ogy 134:126-132, 1994

3:105-109, 1993


factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid. Proc Natl Acad Sci USA 92:44724476, 1995

105. Thrailkill KM, Quarles LD, Nagase H, et al: Characterization of insulin-like growth factor-binding protein 5-degrading proteases produced throughout murine osteoblast differentiation: Endocrinology 136:3527-3533, 1995

106. Uchijima Y, Takenaka A, Takahashi S, et a1 Production of insulin-like growth factors and their binding proteins in primary cultures of rat liver parenchymal and nonparenchymal cells. Bioscience, Biotechnology, and Biochemistry 59:1503-1515, 1995

107. Underwood LE, Thissen JP, Lemozy, et al: Hormonal and nutritional regulation of IGF-I and its binding proteins. Horm Res 42:145-151, 1994

108. Unterman TG, Oehler DT, Murphy LJ, et al: Multihormonal regulation of insulin-like growth factor binding protein-1 in rat H411E hepatoma cells: The dominant role of insulin. Endocrinology 128:2693-2701, 1991

109. Vaccarello MA, Diamond FB, Guevara-Aguirre J, et al: Hormonal and metabolic effects and pharmacokinetics of recombinant insulin-like growth factor-I in growth hormone receptor deficiency/Laron syndrome. J Clin Endocrinol Metab 77273-280, 1993

110. Valentinis B, Bhala A, DeAngelis T, et al: The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I gene. Mol Endocrinol 9:361-367, 1995

111. Villafuerte BC, Koop BL, Pao C, et al: Coculture of primary rat hepatocytes and nonparenchymal cells permits expression of insulin-like growth factor binding protein-3 in vitro. Endocrinology 134:2044-2050, 1994

112. Villaudy J, Delbe J, Blat C, et al: An IGF binding protein is an inhibitor of FGF stimulation. J Cell Physiol 149:492496, 1991

113. Zapf J, Hauri C, Waldvogel M, et al: Recombinant human IGF-I induces its own specific carrier proteins in hypophysectomized and diabetic rats. Proc Natl Acad Sci

114. Zhou Y, Mohan S, Linkhart TA, et al: Retinoic acid regulates insulin-like growth factor-binding protein expression in human osteoblast cells. Endocrinology 137975- 983, 1996

USA 86:3813-3817, 1989

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Division of Endocrinology and Diabetes Abramson Research Center, Room 410 D The Children’s Hospital of Philadelphia 34th Street and Civic Center Boulevard

Philadelphia, PA 19104

the role of the insulin-like growth factor binding proteins and the igfbp proteases in modulating...

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