Expression and release of insulin-like growth factor binding proteins in isolated epiphyseal growth plate chondrocytes from the ovine fetus

Insulin-like growth factor-II (IGF-II) is an autocrine modulator of epiphyseal chondrogenesis in the fetus. The cellular availability of IGFs are influenced by the IGF-binding proteins (IGFBPs). In this study, we investigated the control of expression and release of IGFBPs from isolated epiphyseal growth plate chondrocytes from the ovine fetus by hormones and growth factors implicated in the chondrogenic process. Chondrocytes were isolated from the proliferative zone of the fetal ovine proximal tibial growth plate and maintained in monolayer culture at early passage number. Culture media conditioned by chondrocytes under basal conditions released IGFBPs of 24, 34, and 29 kDa, and a less abundant species of 39-43 kDa that were identified immunologically as IGFBP-4, IGFBP-2, IGFBP-5, and IGFBP-3, respectively. Messenger RNAs encoding each species were identified by Northern blot analysis within chondrocytes, as was mRNA encoding IGFBP-6. Exposure to IGF-I or IGF-II (13 or 26 nM) caused an increase in expression and release of IGFBP-3. The release of IGFBP-2 and IGFBP-5 were also potentiated without changes to steady state mRNA, and for IGFBP-5 this was due in part to a release from the cell membrane in the presence of IGF-II. Insulin (16.7 or 167 nM) selectively increased mRNA and the release of IGFBP-3, while cortisol (1 or 5 microM) inhibited both mRNA and release of IGFBP-2 and IGFBP-5. Transforming growth factor-beta1 (TGF-beta1) (0.1 or 0.2 nM) increased the expression and release of IGFBP-3, and caused an increase in mRNAs encoding IGFBP-2 and IGFBP-5. Neither growth hormone (GH), fibroblast growth factor-2, nor thyroxine (T(4)) had any effect on IGFBP expression or release. The results suggest that IGFBP expression and release within the developing growth plate can be modulated by IGF-II and other trophic factors, thus controlling IGF availability and action.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Purification and characterization of native human insulin-like growth factor binding protein-6

Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.     

Inhibition of Caco-2 cell proliferation by all-trans retinoic acid: role of insulin-like growth factor binding protein-6.

The present study examined the effects of all-trans retinoic acid (tRA) on proliferation and expression of the IGF system in Caco-2 human colon adenocarcinoma cells. tRA inhibited Caco-2 cell proliferation in a dose-dependent manner, with a 40 +/- 2% decrease in cell number observed 48 h after the addition of 1 microM tRA. Ligand blot analysis of IGFBPs in conditioned media revealed that Caco-2 cells produced three IGFBPs of M(r): 34,000 (IGFBP-2), 24,000 (IGFBP-4), and 32,000 (IGFBP-6). The concentrations of IGFBP-2 and IGFBP-4 decreased by 48 +/- 6 and 70 +/- 13%, respectively, whereas that of IGFBP-6 increased by 698 +/- 20% with 1 microM tRA. tRA decreased mRNA levels of IGFBP-2 and IGFBP-4 by 20 +/- 3 and 50 +/- 8%, respectively, whereas tRA increased IGFBP-6 mRNA by 660 +/- 20%. tRA did not alter levels of IGF-II mRNA or peptide. To examine if endogenous IGFBP-6 inhibits cell proliferation, Caco-2 cells were transfected with an IGFBP-6 cDNA expression construct or pcDNA3 vector only and stable clones were selected. Clones overexpressing IGFBP-6 grew more slowly than vector controls and achieved final densities 30-55% lower than those of vector controls. Accumulation of IGFBP-6 mRNA and concentrations of IGFBP-6 peptide in conditioned media were increased by 200-250 and 220-250%, respectively, in the IGFBP-6 clones compared with controls. Increased expression of IGFBP-6, which has a high binding affinity for IGF-II, following tRA treatment suggests that the decreased proliferation caused by tRA may result, at least in part, from IGFBP-6-mediated disruption of the IGF-II autocrine loop in these colon cancer cells.     

Human insulin-like growth factor binding protein-6 is O-glycosylated.

Insulin-like growth factor binding protein-6 is abundant in cerebrospinal fluid and has a marked preferential binding affinity for IGF-II over IGF-I. The present study demonstrates that IGFBP-6 is O-glycosylated but not N-glycosylated. Carbohydrate analysis revealed the presence of approximately 20-30 carbohydrate residues/molecule. Galactosamine, galactose and sialic acid were most abundant, with glucosamine and fucose present in lower concentrations. Mannose was not detected. Enzymatic deglycosylation did not alter the high affinity of IGF binding protein-6 for IGF-II (Ka 4.4 +/- 2.2 x 10(11) M-1) or its preference for IGF-II over IGF-I. Glycosylation of IGFBP-6 may affect its secretion, in vivo stability or localization, but does not affect its ligand binding properties.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

C-terminal domain of insulin-like growth factor (IGF) binding protein 6: conformational exchange and its correlation with IGF-II binding

Insulin-like growth factor binding proteins (IGFBPs) function as carriers and regulators of the insulin-like growth factors (IGF-I and -II). Within the family of six binding proteins, IGFBP-6 is unique in having a 20-100-fold higher affinity for IGF-II over IGF-I and appears to act primarily as an inhibitor of IGF-II actions. We have recently determined the solution structure of the C-terminal domain of IGFBP-6 (C-BP-6), which shows the presence of substantial flexible regions, including three loop regions. In this paper, we report results from (15)N relaxation measurements carried out in both the laboratory and rotating frames. Analysis of conventional (15)N relaxation data (R(1), R(2), and steady-state (15)N-[(1)H] nuclear Overhauser effect) indicated that there was a considerable number of residues involved in conformational/chemical exchange. Measurements of off-resonance (15)N R(1)(rho) in the rotating frame and (15)N relaxation dispersion using an in- and antiphase coherence-averaged Carr-Purcell-Meiboom-Gill sequence were thus carried out to gain further insight into the solution dynamics of C-BP-6. Although the off-resonance (15)N relaxation data showed no clear evidence for residues undergoing microsecond motion, the (15)N relaxation dispersion data allowed us to identify 15 residues that clearly exhibit submilli- to millisecond motion. A good correlation was observed between residues exhibiting motion at submilli- to millisecond time scales and those affected by IGF-II binding, as identified through the perturbation of nuclear magnetic resonance (NMR) spectra of C-BP-6 following IGF-II addition. A complete NMR relaxation study of C-BP-6 dynamics in complex with IGF-II was hampered by peak broadening and disappearance of C-BP-6 in the presence of IGF-II. Nonetheless, current results strongly suggest possible conformation switching or population shifting between pre-existing conformations in C-BP-6 upon binding to IGF-II.     

The N-terminal subdomain of insulin-like growth factor (IGF) binding protein 6. Structure and interaction with IGFs

Insulin-like growth factor binding proteins (IGFBPs) modulate the activity and distribution of insulin-like growth factors (IGFs). IGFBP-6 differs from other IGFBPs in being a relatively specific inhibitor of IGF-II actions. Another distinctive feature of IGFBP-6 is its unique N-terminal disulfide linkages; the N-domains of IGFBPs 1-5 contain six disulfides and share a conserved GCGCC motif, but IGFBP-6 lacks the two adjacent cysteines in this motif, so its first three N-terminal disulfide linkages differ from those of the other IGFBPs. The contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties and their structure-function relationships have been characterized in part, but the structure and function of the distinctive N-terminal subdomain of IGFBP-6 are unknown. Here we report the solution structure of a polypeptide corresponding to residues 1-45 of the N-terminal subdomain of IGFBP-6 (NN-BP-6). The extended structure of the N-terminal subdomain of IGFBP-6 is very different from that of the short two-stranded beta-sheet of the N-terminal subdomain of IGFBP-4 and, by implication, the other IGFBPs. NN-BP-6 contains a potential cation-binding motif; lanthanide ion binding was observed, but no significant interaction was found with physiologically relevant metal ions like calcium or magnesium. However, this subdomain of IGFBP-6 has a higher affinity for IGF-II than IGF-I, suggesting that it may contribute to the marked IGF-II binding preference of IGFBP-6. The extended structure and flexibility of this subdomain of IGFBP-6 could play a role in enhancing the rate of ligand association and thereby be significant in IGF recognition.     

Role of the insulin-like growth factor system in adrenocortical growth control and carcinogenesis.

Clinically silent adrenocortical adenomas are the most frequent abnormalities in the adrenal gland. In contrast, adrenocortical carcinoma is a rare tumor with an extremely poor prognosis. The factors responsible for the frequent occurrence of benign adrenocortical tumors on one hand and the rare malignant transformation on the other are not known. Several genetic alterations such as loss of imprinting or loss of heterozygosity of the 11p15 gene locus causing a strong IGF-II overexpression have been demonstrated in the majority of adrenocortical carcinomas. In addition to IGF-II overexpression, increased levels of the IGF-I-receptor and IGFBP-2 have been found in advanced human adrenocortical carcinomas, suggesting an important role for the IGF-system in adrenocortical carcinogenesis. IGFs are potent mitogens regulating growth and apoptosis through interaction with the IGF-I-receptor, and overexpression of the human IGF-I-receptor promotes ligand-dependent neoplastic transformation in a variety of different cell systems. It is evident, therefore, that high levels of IGF-II in combination with overexpression of the IGF-I-receptor can provide a significant growth advantage for adrenocortical carcinoma cells and thus contribute to the highly malignant phenotype of this rare type of cancer. Additionally, it has been shown that overexpression of IGFBP-2 can promote malignant transformation of Y1 mouse adrenocortical tumor cells through unknown IGF-independent mechanisms. As one possible mechanism, we have recently found altered expression of catalase in IGFBP-2-overexpressing tumor cells, thus implicating IGFBP-2 in influencing intracellular peroxide levels. However, since transgenic mice with IGF-II or IGFBP-2 overexpression in the adrenal gland do not show an increased frequency of adrenal tumors, IGF-II or IGFBP-2 may act as progression factors but not as initiation factors in adrenocortical tumorigenesis.     

Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3.

In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma. IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions.     

The expression pattern of an insulin-like growth factor (IGF)-binding protein gene is distinct from IGF-II in the midgestational rat embryo.

Insulin-like growth factor-II (IGF-II), the predominant form of IGF in fetal and neonatal serum and tissues, is found in vivo complexed with IGF-binding proteins. One of these binding proteins, IGFBP-2, is present at high levels in fetal rat plasma and binds both IGF-I and IGF-II with high affinity. We here have used in situ hybridization to compare the distribution of IGFBP-2 mRNA with that of IGF-II mRNA in embryonic day 13.5-15 rat embryos. The spatial patterns of IGF-II and IGFBP-2 expression in the fetal trunk were distinct and, in general, nonoverlapping. Most mesoderm derivatives that express IGF-II at high levels contained little, if any, IGFBP-2 mRNA. Instead, IGFBP-2 mRNA was expressed at high levels in many cell types derived from ectoderm and endoderm. The expression of IGFBP-2 mRNA in the central nervous system (CNS) during this developmental period was examined in particular detail. The three most prominent sites of IGFBP-2 expression in the CNS were comprised of cells with nonneuronal phenotypes: 1) the epithelium of the choroid plexus, a tissue that produces cerebrospinal fluid; 2) the floor plate, an area that can guide axonal outgrowth from commissural neurons of the spinal cord in vitro; and 3) the infundibulum, the progenitor of the posterior pituitary that is believed to influence differentiation of the adjacent intermediate pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)     

O-glycosylation of insulin-like growth factor (IGF) binding protein-6 maintains high IGF-II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis.

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans, IGFBP-6 had approximately 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-affinity, soluble form and so contributing to its inhibition of IGF-II actions.     

Insulin-like growth factors and insulin-like growth factor binding proteins in adult patients with severe liver disease before and after orthotopic liver transplantation

INTRODUCTION: The liver is the main source of serum insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) and the concentration of these proteins might reflect liver function. METHODS: In a retrospective longitudinal study we examined serum levels of total and free IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 in 21 adult patients with end-stage liver disease before and after orthotopic liver transplantation (LTX) by sensitive and specific RIAs. In each patient, the mean value of at least three measurements before and after LTX was calculated. RESULTS: Before LTX, serum levels of total and free IGF-I, IGF-II, IGFBP-3 were low and showed a rapid and significant increase in almost all patients after successful LTX (total IGF-I: 30 +/- 7 vs. 256 +/- 30 ng/ml, p < 0.001; free IGF-I: 1.3 +/- 0.3 vs. 3.5 +/- 0.6 ng/ml, p < 0.01; IGF-II: 177 +/- 28 vs. 618 +/- 30 ng/ml, p < 0.001; IGFBP-3: 1,230 +/- 136 vs. 3,665 +/- 264 ng/ml, p < 0.001). In contrast, IGFBP-1 was found to be high immediately before LTX, and declined to normal levels after LTX (210 +/- 40 vs. 90 +/- 15 ng/ml, p < 0.01), while IGFBP-2 did not show any significant changes (1,154 +/- 296 vs. 1,303 +/- 192 ng/ ml). Positive correlations were found between IGF-I, IGF-II or IGFBP-3, and serum pseudocholinesterase (R = 0.50, 0.72 and 0.61 respectively, p < 0.001). Negative correlations were found between IGF-I, IGF-II or IGFBP-3, and prothrombin time (R = 0.50, 0.59 and 0.51 respectively, p < 0.001). CONCLUSION: Patients with severe liver disease show decreased levels of total and free IGF-I, IGF-II and IGFBP-3, and increased levels of IGFBP-1. These abnormalities are promptly normalized after successful LTX. Thus, serum levels of IGF-I, IGF-II and IGFBP-3 might be useful parameters for the assessment of liver function.     

Insulin-like growth factor binding protein-6: the "forgotten" binding protein?

Insulin-like growth factor binding protein-6 (IGFBP-6) differs from IGFBPs 1-5 in that it binds IGF-II with marked preferential affinity over IGF-I. Human and rat IGFBP-6 lack 2 and 4 N-terminal cysteines and therefore the Gly-Cys-Gly-Cys-Cys motif present in IGFBPs 1-5. IGFBP-6 is O-glycolsylated, and five serine/threonine glycosylation sites in the non-conserved mid-region of human IGFBP-6 have been identified. O-Glycosylation inhibits proteolysis of IGFBP-6 by chymotrypsin and trypsin, but has no effect on high affinity IGF binding. IGFBP-6 is a relatively specific inhibitor of IGF-II actions; it has not been shown to potentiate IGF actions. IGFBP-6 is only cell-associated to a very limited extent, if at all. IGFBP-6 is often expressed in non-proliferative, quiescent states in vitro and differentiating agents increase its expression. IGFBP-6 expression is associated with inhibition of growth of tumour cells in vitro and in vivo. Although many questions remain regarding the biological role of IGFBP-6, its major function appears to be the regulation of IGF-II actions. This could be especially significant since IGF-II has been implicated as an autocrine tumour growth factor.     

Cervical scrapes levels of insulin-like growth factor-II and insulin-like growth factor binding protein 3 in women with squamous intraepithelial lesions and cervical cancer

A growing number of studies have demonstrated an association between serum levels of insulin-like growth factors (IGFs) and IGF binding protein-3 (IGFBP-3) and increased risk for various cancers. The aim of this study was to evaluate the relationship between levels of IGF-II or IGFBP-3 in cervical scrapes with cervical cancer and precancerous lesions: low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL). 4 groups of cases were examined: LSIL (n=20), HSIL (n=28), cervical cancer (n=45), and controls (n=51). Control subjects were women with normal, HPV DNA-negative Papanicolau (Pap) test. IGF-II and IGFBP-3 levels in cervical scrapes were measured by ELISA. Results show that median protein levels of IGF-II were significantly lower in cervical cancer cases vs. controls (446.5 ng/mg vs. 1,168.6 ng/mg, p<0.001). Significantly higher values of IGFBP-3 were found in HSIL vs. controls (median: 549.5 ng/mg vs. 216 ng/mg; p=0.018), and were not affected by HR HPV infection, meanwhile no significant differences were observed in IGFBP-3 levels between LSIL or cervical cancer as compared to controls. These data suggests that the progression to cervical cancer is associated with alterations in the IGF system and not affected by HR HPV infection. More studies are needed to understand the possible role of IGFBP-3 in cervical carcinogenesis.     

Characterization of insulin-like growth factor binding proteins (IGFBPs) secreted by bovine adrenocortical cells in primary culture: regulation by insulin-like growth factors (IGFs) and adrenocorticotropin (ACTH).

In previous studies, we have shown that insulin-like growth factor II (IGF-II) stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I [1]. The steroidogenic effect of both IGFs is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we therefore characterized the IGFBPs secreted by bovine adrenocortical cells in primary culture, and investigated the effect of corticotropin (ACTH) and recombinant human IGF-I and IGF-II on the regulation of IGFBP synthesis. By Western ligand blotting, four different molecular forms of IGF-binding proteins were identified in conditioned medium of bovine adrenocortical cells with apparent molecular weights of 39-44 kDa, 34 kDA, 29-31 kDa, and 24 kDa. In accordance to their electrophoretic mobility, glycosylation status and binding affinity, these bands were identified by immunoprecipitation and immunoblotting as IGFBP-3, IGFBP-2, IGFBP-1, and deglycosilated IGFBP-4, respectively. Quantification of the specific bands by gamma counting revealed that, in unstimulated cells, IGFBP-3 accounts for approximately half of the detected IGFBP activity, followed by IGFBP-1, IGFBP-2 and IGFBP-4. ACTH treatment predominantly increased the abundance of IGFBP-1 and to a lesser extent IGFBP-3 in a time and dose-dependent fashion. In contrast, IGF-I or IGF-II (6.5 nM) preferentially induced the accumulation of IGFBP-3 (1.9-fold) and to a lesser extent of IGFBP-4, but did not show any effect on IGFBP-1. When ACTH and IGFs were combined, an additive stimulatory effect on the accumulation of IGFBP-3 and IGFBP-4 was observed. In contrast to their different steroidogenic potency, no significant difference in the stimulatory effect of IGF-I and IGF-II on IGFBP secretion was found. In conclusion, bovine adrenocortical cells synthesize IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4, and their secretion is regulated differentially by ACTH and IGFs. These results, together with earlier findings, suggest that IGF-binding proteins play a modulatory role in the regulation of differentiated adrenocortical functions. Therefore, bovine adult adrenocortical cells provide a useful tissue culture model in which the complex interactions between two IGF-ligands, at least four IGF binding proteins and two IGF-receptors can be evaluated.