Regulation of osteoprotegerin production by androgens and anti-androgens in human osteoblastic lineage cells

BACKGROUND: Estrogens and androgens have anti-resorptive effects on bone, although recent evidence indicates that, even in men, estrogen is the dominant sex steroid regulating bone resorption. The receptor activator of NF-kappaB ligand is essential for osteoclastic bone resorption, and its effects are blocked by the decoy receptor, osteoprotegerin (OPG). While estrogen has been shown to induce osteoblastic OPG production, the effects of androgens on OPG production have not been defined. METHODS: In this study, we assessed the regulation of OPG by androgens in hFOB/AR-6, an immortalized fetal osteoblastic cell line stably transfected with the human androgen receptor (AR), and MSC cells, primary human pluripotent marrow stromal cells capable of differentiating towards mature osteoblasts. RESULTS AND CONCLUSIONS: 5Alpha-dihydrotestosterone (DHT) dose-dependently decreased OPG mRNA levels and protein concentrations in hFOB/AR-6 cells by up to 50 and 60% respectively (P<0.001). Inhibition of OPG mRNA levels and protein production by 5alpha-DHT was completely abrogated by the AR antagonist, hydroxyflutamide (OHF), indicating that these effects are directly mediated by the AR. Of note, OHF alone increased OPG mRNA levels and protein secretion by 2- to 3-fold. Moreover, 5alpha-DHT and testosterone also decreased OPG protein secretion by 40-46% in the untransformed MSC cells, while OHF stimulated it. In conclusion, we demonstrate that androgens specifically inhibit OPG mRNA levels and protein secretion by osteoblastic cells.     

Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft

The insulin-like growth factor (IGF) binding proteins (IGFBPs) are important modulators of IGF action in many tissues including human prostate. IGFBPs and the androgen receptor (AR) are expressed in CWR22, an androgen-dependent epithelial cell human CaP xenograft that retains biological characteristics of human CaPs, including regression following androgen withdrawal and recurrent growth of AR-containing cells in the absence of testicular androgens beginning several months after castration. Northern blot and in situ hybridization analyses demonstrated that IGFBP-5 is androgen-regulated in CWR22. IGFBP-5 messenger RNA (mRNA) decreased by 90% following castration of tumor-bearing mice compared with noncastrate androgen-stimulated mice. Testosterone treatment of CWR22 tumor-bearing mice 6 or 12 days after castration increased IGFBP-5 mRNA 10- to 12-fold. Levels of other IGFBP mRNAs did not change following androgen withdrawal and replacement. IGFBP-5 protein in tumor extracts bound 125I-labeled IGF-I in ligand blot assays and the amounts of IGFBP-5 measured by immunoblotting paralleled the levels of IGFBP-5 mRNA. Androgen-induced expression of IGFBP-5 was at a maximum level within 24 h after testosterone replacement, whereas the major increase in cell proliferation as measured by Ki-67 immunostaining occurred between 24-48 h. This time course suggested IGFBP-5 may be a mediator of androgen-induced growth of CWR22. In tumors that recurred several months following castration, IGFBP-5 mRNA and protein increased to levels that approached those in androgen-stimulated CWR22 tumors from noncastrate mice. IGFBP-5 immunohistochemical staining of prostate tissue specimens from patients was stronger in androgen-dependent and androgen-independent CaP than in areas of intraepithelial neoplasia (PIN) or benign prostatic hyperplasia (BPH). IGFBP-5 mRNA in these specimens was localized predominantly to stromal cells and IGFBP-5 protein to epithelial cell membranes.     

Insulin-like growth factor I (IGF-I) and long R(3)IGF-I differently affect development and messenger ribonucleic acid abundance for IGF-binding proteins and type I IGF receptors in in vitro produced bovine embryos

The insulin-like growth factor (IGF) system is a complex network, including ligands (IGF-I and -II), binding proteins (IGFBP-1 to -6), and receptors, of which the type I IGF receptor (IGF-I-R) is important for transmission of most biological effects of IGFs. As IGFs are secreted in large amounts by the female reproductive tract, it has been hypothesized that maternal IGFs may affect embryonic growth and differentiation in a fine-tuned manner, involving modulation of IGF effects by embryonic IGFBP and IGF-I-R expression. To address this point, we cultured in vitro produced bovine embryos in a chemically defined culture system in the presence (100 ng/ml) of recombinant human IGF-I, long R(3)IGF-I (LR(3)), or without IGF supplementation (control). The affinity of LR(3) to IGFBPs measured by competition assays and Western ligand blots is at least 3 orders of magnitude lower than that of IGF-I. LR(3) was most efficient in stimulating early embryonic cleavage, whereas further development was most potently supported by IGF-I. Total cell numbers of blastocysts were highest in the presence of LR(3) (105 +/- 4), followed by IGF-I (96 +/- 5), and the control group (91 +/- 3; P < 0.05). Differential cell staining of blastocysts revealed that these differences were mainly represented by trophectoderm cell numbers. Analysis of messenger RNA (mRNA) expression for IGFBPs and IGF-I-R was performed by RT-real-time PCR, using expression of the nonregulated housekeeping gene glyceraldehyde-3-phosphate dehydrogenase for normalization. Embryonic IGFBP-2 mRNA levels in the LR(3) treatment group were 1.7-fold (P < 0.001) and 2.8-fold (P < 0.001) higher than those in the IGF-I and control groups, respectively. IGFBP-5 mRNA levels were about 2-fold (P < 0.001) elevated in both IGF treatment groups, with slightly (P < 0.05) higher levels in IGF-I- than in LR(3)-treated embryos. Similarly, IGFBP-3 mRNA abundance was increased (P < 0.05) in embryos from the IGF-I vs. the LR(3) culture system. IGF-I-R mRNA levels were reduced by IGF-I (80% of control; P < 0.01), but increased by LR(3) (1.3-fold vs. control; P < 0.001). These data show that the affinity for IGFBPs of IGF peptides is relevant for their effects on preimplantation embryos and affects different parameters, i.e. development, cell numbers, and mRNA expression for components of the IGF system, in different directions.     

Testosterone and insulin-like growth factor (IGF) I interact in controlling IGF-binding protein production in androgen-responsive foreskin fibroblasts

The growth of the male external genitalia is primarily regulated by androgens. However, human genital fibroblast growth is also stimulated by insulin-like growth factor (IGF) I. In this study, we report that IGF-binding protein (IGFBP) production in human foreskin fibroblasts is regulated by androgens and IGF-I. Human foreskin fibroblasts secrete IGFBP-3, IGFBP-4, and IGFBP-5. IGF-I increased the abundance of both intact IGFBP-3 and -5 in the culture medium. Testosterone increased IGFBP-3, and the combination of IGF-I and testosterone had an additive effect. Following its secretion, IGFBP-5 was degraded, but the effect of IGF-I on IGFBP-5 peptide abundance in conditioned media did not seem to be due to inhibition of proteolysis. Testosterone had no effect on IGFBP-5 degradation. Intact IGFBP-4 was decreased by IGF-I, and the combination resulted in a similar reduction. The mechanism seemed to be decreased synthesis, since IGFBP-4 messenger RNA was also decreased. The increase in IGFBP-5 synthesis was associated with an increase in the abundance of intact IGFBP-5 in the extracellular matrix. The combination of testosterone and IGF-I resulted in a synergistic stimulation of total protein synthesis by the fibroblast cultures, suggesting that a maximum anabolic response requires both hormones. These observations suggest that combined exposure to androgen and IGF-I altered the abundance of some forms of IGFBPs and that the IGFBPs that are regulated may play a role in modulating the effects of IGF-I on the anabolic response.     

1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2, -3 and -4 in human bone marrow stromal cells

BACKGROUND: 1,25-Dihydroxyvitamin D3 (calcitriol) inhibits proliferation and stimulates differentiation of multiple cell types, including osteoblasts. Human (h) bone marrow stromal cells (MSCs) are a homogenous non-hematopoietic population of cells present in the bone marrow and exhibit a less differentiated osteoblastic phenotype. The IGF system, including IGFs-I, and -II and IGF binding proteins (IGFBPs), plays an important role in osteoblast cell proliferation and differentiation. OBJECTIVE: To examine the pattern of expression of the IGF system in hMSCss and its regulation by calcitriol. METHODS AND RESULTS: hMSCs express mRNA of both IGFs-I, and -II and IGFBPs-1 to -6 as shown by RT-PCR and northern blot analysis. As assessed by western ligand blotting (WLB) and western immmunoblot analysis, hMSCs secrete 38-42 kDa IGFBP-3, 24-28 kDa IGFBP-4 and a 33 kDa IGFBP-2. Calcitriol (dose range 10-10 mol/l) exerted no consistent dose-dependent effects on either IGF-I or IGF-II mRNA levels. In contrast, calcitriol treatment increased steady-state mRNA levels of IGFBPs-2, -3 and -4, but had no effect on IGFBP-5 or -6. Similarly, calcitriol increased the secretion of IGFBPs-2, -3 and -4 as determined by WLB. We found no detectable basal IGFBP-3 or IGFBP-4 protease activities in the absence or presence of calcitriol treatment. CONCLUSIONS: Our results demonstrate that hMSCs expressed a distinct pattern of IGFs and IGFBPs that may be related to their stage of differentiation. The observed increase in production of IGFBPs-2, -3 and -4 by hMSCs upon treatment with calcitriol may be an important mechanism mediating the effects of calcitriol on MSC proliferation and differentiation.     

Regulation of insulin-like growth factor-I and of insulin-like growth factor binding protein-1, -3 and -4 in cocultures of rat hepatocytes and Kupffer cells by interleukin-6.

BACKGROUND/AIMS: Catabolism is associated with decreased serum concentrations of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 associated with elevated IGFBP-3 protease activity and increased concentrations of IGFBP-1 and -4. The effects of the acute phase mediators interleukin (IL)-6, IL-1beta and tumor necrosis factor alpha (TNFalpha) on the biosynthesis of IGF-I and IGFBPs were studied in primary rat liver cells. METHODS: mRNA levels of IGF-I and of IGFBPs were analyzed by Northern blotting, secretion of IGFBPs by [(125)I]IGF-I ligand blotting. Proteolytic activity was measured using iodinated recombinant IGFBP-3 as the substrate. RESULTS: In hepatocytes, Kupffer cells (KC) and cocultures of hepatocytes with KC, IL-6 reduced IGF-I biosynthesis dose-dependently. IL-6 stimulated mRNA expression and protein secretion of IGFBP-1 and -4 in hepatocytes and that of IGFBP-3 in KC, respectively. In cocultures, biosynthesis of IGFBP-1, -3 and -4 was increased dose-dependently by IL-6, while the effects of IL-1beta or TNFalpha were less prominent. At neutral pH, proteolytic activity against IGFBP-3 was not detected in media of cocultures treated with IL-6. CONCLUSIONS: The alterations of IGF-I, IGFBP-1 and -4 observed in catabolism correlate with the effects of IL-6 on the biosynthesis of these components in primary rat liver cells, while a neutral IGFBP-3 protease was not detectable.     

Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes

Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.     

Identification and characterization of insulin-like growth factor (IGF)-binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotropin

In previous studies we have shown that IGF-II stimulates basal as well as ACTH-induced cortisol secretion from adult human adrenocortical cells more potently than IGF-I, and that both IGFs predominantly stimulate androgen biosynthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we identified and characterized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I and -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in both adrenocortical tissue and monolayer cell cultures of adrenocortical cells. Using Western ligand and immunoblotting and two-dimensional Western ligand blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adrenocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost half the binding activity in conditioned medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15%), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induced only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In contrast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 by ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult human adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cells. Furthermore, the expression and secretion of IGFBP-1 is upregulated by ACTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier findings, these results suggest that IGFBPs play an important modulatory role in the regulation of the differentiated adrenocortical function.     

Serum levels of insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 to -6 and their relationship to bone metabolism in osteoporosis patients

BACKGROUND: Insulin-like growth factor (IGF) system components are important regulators of bone formation. Alterations of individual IGF system components have been described in osteoporosis (OP) patients; however, no study has addressed changes in free IGF-I and in all six IGF binding proteins (IGFBPs). METHODS: A cross-sectional study was performed in 45 OP patients and 100 healthy matched controls. Serum levels of free and total insulin-like growth factor I (IGF-I), IGFBP-1 through -6, intact parathyroid hormone (PTH), 25-OH-vitamin D(3) (25OHD(3)), 1,25-(OH)(2)-vitamin D(3) (1,25-(OH)(2)D(3)), osteocalcin (OSC), bone alkaline phosphatase (B-ALP), and carboxyterminal propeptide of type-I procollagen (PICP) were measured with specific assays. Bone mineral density (BMD) of the lumbar spine was determined by dual-energy X-ray absorptiometry (DEXA). RESULTS: Compared with age- and sex-matched control subjects, OP patients showed a 73% decrease in free IGF-I, a 29% decrease in total IGF-I, a 10% decrease in IGFBP-3, and a 52% decrease in IGFBP-5 levels; they had higher levels of IGFBP-1 (4.1-fold), IGFBP-2 (1.8-fold), IGFBP-4 (1.3-fold), and IGFBP-6 (2.1-fold). Alterations in IGF system components were most evident in 13 OP patients with vertebral fractures in the past 4 years compared to patients without fractures. In OP patients with fractures, the ratio between IGFBP-4 and IGFBP-5 was increased whereas levels of OSC were decreased. CONCLUSIONS: Our data provide strong indirect evidence for a functional connection between circulating IGF system components and bone metabolism and the susceptibility to fractures in OP patients.     

Regulation of insulin-like growth factor binding protein synthesis and secretion in a bovine epithelial cell line.

The Madin-Darby bovine kidney cell line was used to examine regulation of insulin-like growth factor binding protein (IGFBP) synthesis by epithelial cells. Ligand and immunoblot analysis of conditioned media indicated that IGFBP-2 was the predominant IGFBP secreted by untreated cells. Treatment with forskolin decreased secretion of IGFBP-2 by 75 +/- 3% and induced the appearance of IGFBP-3 and 24,000 Mr IGFBP. Although insulin alone did not induce the appearance of either band, in the presence of forskolin it increased the IGFBP-3 and 24,000 Mr bands 4.2 +/- 1.1 and 7.3 +/- 0.9-fold, respectively, above the values for forskolin treatment alone. Exposure to forskolin resulted in a 3-fold decrease in the abundance of IGFBP-2 messenger RNA (mRNA), and a 30-fold increase in IGFBP-3 mRNA. An additional 2- to 3-fold increase in IGFBP-3 mRNA was observed when cells were treated with insulin plus forskolin. Treatment with insulin plus forskolin increased cell number 2-fold, compared to small increases (26%) observed with forskolin treatment alone. Since treatment with IGF-I or -II did not result in similar responses to those of insulin, IGF analogs with differing affinities for IGFBP and IGF type I receptor were tested. B-chain IGF-I (decreased affinity for IGFBP) increased cell number and enhanced forskolin's effects on IGFBP-3 secretion and mRNA abundance to the same extent as insulin, whereas [Leu24,1-62]IGF-I (decreased affinity for the type I IGF receptor) did not. Therefore, activation of the type I IGF receptor was required to elicit increases in cell number and IGFBP synthesis and secretion, and the actions of IGF-I and II were likely blocked by binding to the large amounts of IGFBP-2 that were secreted. These results are in direct contrast to studies with human fibroblasts in which IGF-I and [Leu24,1-62]IGF-I stimulate IGFBP-3 secretion, whereas B-chain IGF-I has only a minimal effect. The ability to differentially regulate secretion of different forms of IGFBPs by epithelial cells and the finding that regulation is distinct from that of fibroblasts may have important implications for understanding mechanisms by which IGFs and IGFBPs interact to regulate epithelial cell growth.     

Characterization of insulin-like growth factor binding proteins (IGFBP) and regulation of IGFBP-4 in bone marrow stromal cells

Summary. Bone marrow stromal cells synthesize and secrete insulin-like growth factor (IGF)-I and IGF-binding proteins (IGFBP). IGFBPs may modulate the action of IGF-I or IGF-II on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP-1 to -6 mRNA expression. [¹²⁵I]IGF-I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kD species, consistent with IGFBP4 and IGFBP5, respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP-2 to -6 mRNAs, with IGFBP4, IGFBP5 and IGFBP6 mRNAs predominating. Since agents that modulate cAMP levels may influence haemopoiesis via the release of stromal-derived cytokines, we determined the effect of forskolin, a cAMP agonist, on IGFBP4 expression in TC-1 cells. Forskolin (10 ⁵ M) up-regulated IGFBP4 mRNA and protein secretion in a time-dependent manner. These findings suggest that IGFBP-4, -5 and -6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment.     

Spatial and temporal regulation of GH-IGF-related gene expression in growth plate cartilage

Previous studies of the GH-IGF system gene expression in growth plate using immunohistochemistry and in situ hybridization have yielded conflicting results. We therefore studied the spatial and temporal patterns of mRNA expression of the GH-IGF system in the rat proximal tibial growth plate quantitatively. Growth plates were microdissected into individual zones. RNA was extracted, reverse transcribed and analyzed by real-time PCR. In 1-week-old animals, IGF-I mRNA expression was minimal in growth plate compared with perichondrium, metaphyseal bone, muscle, and liver (70-, 130-, 215-, and 400-fold less). In contrast, IGF-II mRNA was expressed at higher levels than in bone and liver (65- and 2-fold). IGF-II expression was higher in the proliferative and resting zones compared with the hypertrophic zone (P < 0.001). GH receptor and type 1 and 2 IGF receptors were expressed throughout the growth plate. Expression of IGF-binding proteins (IGFBPs)-1 through -6 mRNA was low throughout the growth plate compared with perichondrium and bone. With increasing age (3-, 6-, 9-, and 12-week castrated rats), IGF-I mRNA levels increased in the proliferative zone (PZ) but remained at least tenfold lower than levels in perichondrium and bone. IGF-II mRNA decreased dramatically in PZ (780-fold; P < 0.001) whereas, type 2 IGF receptor and IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4 increased significantly with age in growth plate and/or surrounding perichondrium and bone. These data suggest that IGF-I protein in the growth plate is not produced primarily by the chondrocytes themselves. Instead, it derives from surrounding perichondrium and bone. In addition, the decrease in growth velocity that occurs with age may be caused, in part, by decreasing expression of IGF-II and increasing expression of type 2 IGF receptor and multiple IGFBPs.     

Tissue-specific regulation of IGF-I and IGF-binding proteins in response to TNFalpha.

The circulating concentration of insulin-like growth factor-I (IGF-I) is regulated by both its rate of synthesis and its ability to form stable complexes with IGF-binding proteins (IGFBPs). An equilibrium between IGF-I and IGFBPs is thought to help maintain muscle protein balance. In contrast, catabolic conditions disrupt the IGF system and result in the loss of skeletal muscle protein. We have examined the mechanisms by which tumour necrosis factor alpha (TNFalpha), a catabolic cytokine, alters the IGF system. Conscious rats were infused intravenously with recombinant human TNFalpha or vehicle for 24 h. TNFalpha decreased the concentration of both total and free IGF-I in the plasma (30-40%). This change was associated with a reduction in IGF-I mRNA expression in liver (39%), gastrocnemius (73%), soleus (46%) and heart (63%), but a 2.5-fold increase in the whole kidney. In contrast, TNFalpha did not alter IGF-II mRNA expression in skeletal muscle. TNFalpha also increased IGFBP-1 in the blood (4-fold) and this response was associated with an increase in IGFBP-1 mRNA expression in both liver (3-fold) and kidney (9-fold). In contrast, IGFBP-3 levels in the blood were reduced 38% in response to the infusion of TNFalpha. This change was accompanied by a 60-80% reduction of IGFBP-3 mRNA in liver and kidney but no significant change in muscle. Hepatic mRNA levels of the acid-labile subunit were also reduced by TNFalpha (46%). Finally, tissue expression of mac25 (also referred to IGFBP-related protein-1) mRNA was increased in gastrocnemius (50%) but remained unchanged in liver and kidney. These results more fully characterize the changes in various elements of the IGF system and, thereby, provide potential mechanisms for the alterations in the circulating IGF system as well as for changes in tissue metabolism observed during catabolic insults associated with increased TNFalpha expression.     

Contributions of the N- and C-terminal domains of IGF binding protein-6 to IGF binding

Insulin-like growth factors IGF-I and IGF -II are important mediators of growth. A family of six high affinity IGF binding proteins (IGFBPs) modulate IGF action. IGFBPs have three domains, of which the N- and C-domains are involved in high affinity IGF binding. IGFBP-6 is unique in its 20-100-fold IGF-II binding specificity over IGF-I. The aim of this study was to determine the contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties. We confirmed that differential dissociation kinetics are responsible for the IGF-II binding preference of IGFBP-6. The N-domain has rapid association kinetics, similar to full-length IGFBP-6, but both IGF-I and -II dissociate rapidly from this domain, thereby reducing its binding affinity for IGF-II approximately 50-fold. However, the N-domain binds IGF-I and -II with similar affinities and it has a similar IGF-I binding affinity to full-length IGFBP-6. This suggests that the C-domain confers the IGF-II binding preference of IGFBP-6; indeed, IGF-I bound inconsistently with very low affinity to the C-domain. Coincubation studies showed that isolated N- and C-domains of IGFBP-6 do not strongly cooperate to enhance IGF binding. The results of the binding studies are supported by the effects of the IGFBP-6 domains on IGF-induced colon cancer cell proliferation; the N-domain inhibited IGF-II induced proliferation with approximately 20-fold lower potency than IGFBP-6 and it was equipotent in inhibiting IGF-I- and IGF-II-induced proliferation. Coincubation of C-domain had no additional effect on N-domain-induced inhibition of proliferation. In conclusion, both the N- and C-domains of IGFBP-6 are involved in IGF binding, the C-domain is responsible for the IGF-II binding preference of IGFBP-6 and intact IGFBP-6 is necessary for high affinity IGF binding.