Insulin-like growth factor-I (IGF-I) and IGF binding protein-5 in Schwann cell differentiation

Schwann cells (SCs) are the myelin producing cells of the peripheral nervous system. During development, SCs cease proliferation and differentiate into either a myelin-forming or non-myelin forming mature phenotype. We are interested in the role of insulin-like growth factor-I (IGF-I) in SC development. We have shown previously SCs proliferate in response to IGF-I in vitro. In the current study, we investigated the role of IGF-I in SC differentiation. SC differentiation was determined by morphological criteria and expression of myelin proteins. Addition of 1 mM 8-bromo cyclic AMP (cAMP) or growth on Matrigel matrix decreased proliferation and induced differentiation of SCs. IGF-I enhanced both cAMP and Matrigel matrix-induced SC differentiation, as assessed by both morphological criteria and myelin gene expression. Cultured SCs also express IGF binding protein-5 (IGFBP-5), which can modulate the actions of IGF-I. We examined the expression of IGFBP-5 during SC differentiation. Both cAMP and Matrigel matrix treatment enhanced IGFBP-5 protein expression and cAMP increased IGFBP-5 gene expression five fold. These findings suggest IGF-I potentiates SC differentiation. The concomitant up-regulation of IGFBP-5 may play a role in targeting IGF-I to SCs and thus increase local IGF-I bioavailability. J. Cell. Physiol. 171:161–167, 1997. © 1997 Wiley-Liss, Inc.     

Insulin-like growth factor (IGF) binding protein-5 blocks skeletal muscle differentiation by inhibiting IGF actions

Signaling through the IGF-I receptor by locally produced IGF-I or -II is critical for normal skeletal muscle development and repair after injury. In most tissues, IGF action is modulated by IGF binding proteins (IGFBPs). IGFBP-5 is produced by muscle cells, and previous studies have suggested that when overexpressed it may either facilitate or inhibit IGF actions, and thus potentially enhance or diminish IGF-mediated myoblast differentiation or survival. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5, we studied its effects in cultured muscle cells. Purified wild-type (WT) mouse IGFBP-5 or a variant with diminished extracellular matrix binding (C domain mutant) each prevented differentiation at final concentrations as low as 3.5 nm, whereas analogs with reduced IGF binding (N domain mutant) were ineffective even at 100 nm. None of the IGFBP-5 variants altered cell number. An IGF-I analog (R(3)IGF-I) with diminished affinity for IGFBPs promoted full muscle differentiation in the presence of IGFBP-5(WT), showing that IGFBP-5 interferes with IGF-dependent signaling pathways in myoblasts. When IGFBP-5(WT) or variants were overexpressed by adenovirus-mediated gene transfer, concentrations in muscle culture medium reached 500 nm, and differentiation was inhibited, even by IGFBP-5(N). As 200 nm of purified IGFBP-5(N) prevented activation of the IGF-I receptor by 10 nm IGF-II as effectively as 2 nm of IGFBP-5(WT), our results not only demonstrate that IGFBP-5 variants with reduced IGF binding affinity impair muscle differentiation by blocking IGF actions, but underscore the need for caution when labeling effects of IGFBPs as IGF independent because even low-affinity analogs may potently inhibit IGF-I or -II if present at high enough concentrations in biological fluids.     

Insulin-like growth factor (IGF)-I, IGF-I receptor, and IGF binding protein-3 messenger ribonucleic acids and protein in corpora lutea from prostaglandin F(2alpha)-treated gilts

Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F(2alpha) (PGF(2alpha)) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF(2alpha) (or saline) on Days 5-9 of the cycle to induce premature luteolysis. CL were collected on Days 6-9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF(2alpha)-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8-9, while IGFBP-2 was higher on Days 6-7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF(2alpha)-treated animals vs. control animals (P < 0.05). In conclusion, PGF(2alpha)-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6-7), it is possible that they may mediate the luteolytic actions of PGF(2alpha) by sequestering IGF-I and preventing its interaction with the IGF-IR.     

Insulin-like growth factor binding protein (IGFBP-3), an inhibitor of serum growth factors other than IGF-I and -II.

Our results show that an insulin-like growth factor binding protein, IGFBP-3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF-I as well as by human serum. Rat IGFBP-3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP-3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti-IGF-I IgG was able to completely inhibit stimulation induced by added IGF-I, it did not decrease stimulation induced by 1% human serum. Anti-IGF-II IgG inhibited the stimulation induced by added IGF-II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP-3 than by IgG anti IGF-II; 3) after separation of IGF-I and IGF-II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF-I and -II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP-3.     

Insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3, phosphoisoforms of IGFBP-1 and postnatal growth in very-low-birth-weight infants

Small preterm infants experience a unique postnatal period characterized by slow growth, inadequate nutrition and growth inhibiting treatments. Many have already been growth-restricted in utero. Studying this period is important when developing growth optimizing strategies for these infants and, in a broader context, as a model of extreme conditions that restrict growth. By following short-term growth of 48 very-low-birth-weight (VLBW; birth weight <1,500 g) infants for 9 postnatal weeks, we found that circulating insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 levels are low and reflect rigorously measured (knemometry and weight) concurrent growth velocity. Moreover, weight growth velocity is correlated with the ratio of lesser to highly phosphorylated IGFBP-1 but not with absolute IGFBP-1 concentrations. Thus, IGF-I, IGFBP-3 and the phosphorylation status of IGFBP-1 in circulation are likely to be involved in growth regulation during the postnatal period in VLBW infants.     

Insulin-like growth factor binding protein (IGFBP) inhibits IGF action on human osteosarcoma cells.

The influence of a human insulin-like growth factor binding protein, hIGFBP-1, on the action of IGFs on human osteosarcoma cells was examined. hIGFBP-1 was found to block binding of IGFs to their receptors on MG-63 cells and subsequent IGF stimulation of DNA synthesis. Concurrent incubation of hIGFBP-1 with either 125I-IGF-I or 125I-IGF-II prevented the binding of both 125I-IGFs to cells in a dose-dependent manner. hIGFBP-1 inhibition of IGF binding occurred similarly under both 4 degrees and 37 degrees C conditions. Additionally, hIGFBP-1 facilitated the dissociation of IGFs bound to cells. The inhibitory effect of hIGFBP-1 on IGF-1 mediated 3H-thymidine incorporation into DNA was dose dependent. hIGFBP-1 did not inhibit binding to or stimulation of growth in MG-63 cells by des3-IGF-1, an IGF-I analog with a 100-fold less affinity for hIGFBP-I. This confirmed that hIGFBP-1 competed for IGF receptor binding sites on MG-63. Since hIGFBP-1 did not bind to cells, inhibition of IGF action was indirect, presumably through the formation of extracellular soluble bioinactive IGF-BP complexes.     

Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors

The primary structure of an insulin-like growth factor (IGF) binding protein produced by human HEP G2 hepatoma cells has been deduced from the cDNA sequence. The 234 amino acid protein has a predicted molecular mass of 25,274 and contains a single, distinctive cysteine-rich region. The N-terminal sequence of this protein is quite similar to the limited sequence data available for a rat IGF binding protein produced by BRL-3A cells and suggests a common ancestral origin. In contrast, the HEP G2 IGF binding protein sequence bears no similarity to the N-terminal 15 amino acids of a 53 kilodalton binding protein purified from human plasma. Comparison of full-length protein sequences for the IGF-I and IGF-II receptors with that of the HEP G2 IGF binding protein also fails to demonstrate any significant similarities among these three proteins, and suggests that each contains a unique binding domain for the IGF peptides.     

Preferential enhancement of myoblast differentiation by insulin-like growth factors (IGF I and IGF II) in primary cultures of chicken embryonic cells

Cells prepared from the body walls of chicken embryos were plated in the absence of serum. Insulin-like growth factors (IGFs) barely stimulated cell replication, but preferentially enhanced the differentiation of muscle cells. Myoblast fusion was favoured in the presence of IGF (or insulin). Concomitantly, acetylcholinesterase activity increased. IGF I and IGF II were equipotent and active in low physiological concentrations, in contrast to insulin, which was known for a long time to exert such effects at pharmacological concentrations.     

Insulin-like growth factor (IGF) parameters and tools for efficacy: the IGF-I generation test in children

Serum levels of growth hormone (GH)-dependent peptides could provide important and valuable measures of GH sensitivity and, potentially, responsiveness. In normal individuals, serum insulin-like growth factor I (IGF-I) concentrations are dependent on the dose of GH given, with IGF-I responsiveness not decreasing with age. Individuals heterozygous for the E180 GH receptor (GHR) splice mutation have normal IGF-I generation, but those homozygous for the E180 splice mutation have very low basal and stimulated IGF-I concentrations. Similar results are observed for the serum IGF-binding protein 3 (IGFBP-3) response to GH, with a correlation between changes in serum concentrations of IGF-I and changes in IGFBP-3 in normal, heterozygotic, GH-insensitive and GH-deficient participants. In individuals with the E180 splice mutation, IGF-I and IGFBP-3 tests show sensitivity and specificity for detecting GH insensitivity (GHI). In children with idiopathic short stature, it appears that some individuals have selective resistance to GH, with their ability to generate IGF-I more impaired than their ability to generate other GH-dependent peptides. This heterogeneous group may require individualization of GH dosage. IGF generation tests remain the best short-term, in vivo test for classic GHI, although diagnostic tests will undoubtedly require further modification to identify milder pathophysiologic abnormalities.     

Insulin-like growth factor (IGF)-I and IGF-II binding to an IGF binding protein. An investigation using chemical modification of tyrosine residues as a structural probe for the sites of interaction.

We have investigated insulin-like growth factor (IGF)-I and IGF-II binding to bovine insulin-like growth factor binding protein-2 (bIGFBP-2) using chemical modification to locate sites on the IGF involved in the binding interaction. bIGFBP-2 was incubated with either recombinant human (hIGF-I) or purified ovine (oIGF-II) to form a mixture of bound and free IGF. Sites of interaction between the binding protein and IGF were then probed by iodination of the available tyrosine residues. Subsequently, the mixture of free IGF and IGF.bIGFBP-2 complex was resolved by neutral chromatography, and the IGF component of the complex with bIGFBP-2 was recovered by reverse-phase high performance liquid chromatography at pH 2.1. The tyrosine labeling patterns of the two populations of IGF, one iodinated while free and the other iodinated while associated with binding protein, were determined following endoproteinase Glu-C peptide mapping. Binding of hIGF-I or oIGF-II to bIGFBP-2 resulted in reduced iodination of the tyrosines in both hIGF-I and oIGF-II that are near the carboxyl-terminal, Tyr-60 and Tyr-59, respectively. The reduction in labeling of these tyrosine residues was 2-fold and 6-fold for hIGF-I and oIGF-II, respectively. On the other hand, labeling of the other 2 tyrosines in hIGF-I and oIGF-II was not different between the free and complexed growth factors. From these results we conclude that Tyr-60 and Tyr-59 in the carboxyl-terminal regions of hIGF-I and oIGF-II, respectively, are either directly involved in the binding reaction or lie in a region of the IGF molecule encompassed by the association with bIGFBP-2. Conversely, the labeling pattern of the other tyrosines, Tyr-24 and Tyr-31 in hIGF-I and Tyr-2 and Tyr-27 in oIGF-II, implies that they are not involved in binding to bIGFBP-2. To examine the role of IGF tyrosine residues in the association with bIGFBP-2, we prepared nonradioactive 127I-labeled oIGF-II. In bIGFBP-2 competition binding assays, 127I-labeled oIGF-II was 2.5-fold and 5-fold less potent than native oIGF-II when competing for binding of 125I-labeled IGF-I or IGF-II tracers, respectively. We interpret these results as indicating that at least 1 tyrosine in oIGF-II is involved in binding to bIGFBP-2.     

Chromatin protein kinases and phosphoproteins during myoblast growth and differentiation

A photolabile derivative of α-bungarotoxin which binds specifically to $\text{Torpedo}\text{californica}$ acetylcholine receptor has been used to investigate the topography of the membrane associated protein. It is shown that the toxin can be crosslinked to a polypeptide of 40,000 daltons, to which it is known to bind, and in addition to another polypeptide of 65,000 daltons which is a major constituent of the membrane. The results substantiate the notion that this nicotinic acetylcholine receptor is composed of different polypeptides and that some of these interact with each other or are in close proximity on the exterior surface of the post-synaptic membrane.     

Insulin-like growth factor binding protein (IGFBP)-3-bound IGF-I and IGFBP-3-bound IGF-II in growth hormone deficiency

In blood, circulating IGFs are bound to six high-affinity IGFBPs, which modulate IGF delivery to target cells. Serum IGFs and IGFBP-3, the main carrier of IGFs, are upregulated by GH. The functional role of serum IGFBP-3-bound IGFs is not well understood, but they constitute the main reservoir of IGFs in the circulation. We have used an equation derived from the law of mass action to estimate serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II, as well as serum free IGF-I and free IGF-II, in 129 control children and adolescents (48 girls and 81 boys) and in 13 patients with GHD. Levels of serum total IGF-I, total IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 were determined experimentally, while those of IGFBP-4, IGFBP-5 and IGFPB-6, as well as the 12 affinity constants of association of the two IGFs with the six IGFBPs, were taken from published values. A correction for in vivo proteolysis of serum IGFBP-3 was also considered. In controls, serum total IGF-I, total IGF-II, IGFBP-3, IGFBP-3-bound IGF-I, IGFBP-3-bound IGF-II and free IGF-I increased linearly with age, from less than 1 to 15 years, in the two sexes. The concentrations of serum free IGF-I and free IGF-II were approximately two orders of magnitude below published values, as well as below the affinity constant of association of IGF-I with the type-1 IGF receptor. Therefore, it is unlikely that these levels can interact with the receptor. In the 13 patients with GHD, mean (+/- SD) SDS of serum IGFBP-3-bound IGF-I was -2.89 +/- 0.97. It was significantly lower than serum total IGF-I, free IGF-I or IGFBP-3 SDSs (-2.35 +/- 0.83, -1.12 +/- 0.78 and -2.55 +/- 1.07, respectively, p = 0.0001). The mean SDS of serum total IGF-II, IGFBP-3-bound IGF-II and free IGF-II were -1.25 +/- 0.68, -2.03 +/- 0.87 and 0.59 +/- 1.10, respectively, in GHD. In control subjects, 89.8 +/- 4.47% of serum total IGF-I and 77.3 +/- 9.4% of serum total IGF-II were bound to serum IGFBP-3. In patients with GHD, the mean serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II were 8.63 +/- 8. 53 and 19.1 +/- 14.7% below the respective means of control subjects (p < 0.02). In conclusion, in GHD there was a relative change in the distribution of serum IGFs among IGFBPs, due to the combined effects of the decrease in both total IGF-I and IGFBP-3. As a result, serum IGFBP-3-bound IGF-I and IGFBP-3 bound IGF-II, the main reservoirs of serum IGFs, were severely affected. This suggests that the decrease in serum IGFPB-3-bound IGF-I and IGFBP-3-bound IGF-II might have a negative effect for growth promotion and other biological effects of IGF-I and IGF-II. Finally, the estimation of serum IGFBP-3-bound IGF-I, or the percentage of total IGF-I and IGF-II bound to IGFBP-3, might be useful markers in the diagnosis of GHD.     

Insulin-like growth factor binding protein-3 mediates IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction

IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. In addition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell lines were developed that constitutively express IGFBP-3. MAC-T cells transfected with IGFBP-3 (+BP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in +BP3 cells compared to Mock cells. However, DNA synthesis was increased by IGF-I (1-50 ng/ml) relative to untreated controls to a greater extent in +BP3 cells compared to Mock cells. IGF-I (20 ng/ml) increased DNA synthesis 11- and threefold in +BP3 and Mock cells, respectively. Additionally, +BP3 cells were more sensitive to the lower concentrations of IGF-I (1-5 ng/ml). In contrast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance responsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the differences observed between +BP3 and Mock cells were not attributable to sequestration of endogenous IGF-I by IGFBP-3. There were no differences between +BP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis was also increased in +BP3 cells, compared to controls, in response to 5 microg/ml insulin and 2.5 ng/ml Long R(3)IGF-I, indicating that the potentiated response did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does require an IGF-I-IGFBP-3 interaction.     

Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation

The identification of relevant protein kinase–protein substrate partners remains a serious challenge on a genome-wide scale. The design and synthesis of a photo-activatable nucleotide reagent to crosslink protein kinases with their substrates is described in which an azido group is appended to the γ-phosphoryl and purine moieties of ATP. In the absence of UV, compounds of this class were shown to act as competitive inhibitors versus ATP and non-competitive inhibitors versus peptide substrate for the protein tyrosine kinase Csk, suggesting that they can form a ternary complex with kinase and protein substrate. In vitro experiments with protein kinases indicate the bifunctional reagent can induce covalent protein–protein crosslinking that is dependent on UV irradiation. That significant kinase–substrate crosslinking occurs is suggested by the fact that this crosslinking is competitively inhibited by ATP. The crosslinked adducts can be readily cleaved by phosphodiesterase which supports the model for crosslinking and provides a simple method to deconvolute the linked protein partners.     

Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs)

The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.     

Insulin-like growth factor binding protein secretion by muscle cells: effect of cellular differentiation and proliferation

Several cell types have been shown to secrete insulin-like growth factor binding proteins (IGF-BP) in vitro. Since IGF-BP influences cell responsiveness to IGF, three muscle cell types were investigated to determine if they produced IGF-BP and to identify factors that regulate IGF-BP secretion. Porcine smooth muscle cells (pSMC), rat L6 skeletal muscle cells, and mouse BC3H-1 myocytes were used. IGF-BP activity in serum-free conditioned media was quantitated with a polyethylene glycol precipitation method. All three cell types secreted IGF-BP activity into the medium. Insulin was a potent stimulant of IGF-BP secretion for each cell type. Specifically, 1 microgram/ml insulin increased the IGF-BP concentration in conditioned media from 10.5 +/- 1.3 to 15.0 +/- 1.5 ng/ml in confluent L6 myotubes, from 42.5 +/- 11.1 to 90.5 +/- 9.8 ng/ml in confluent BC3H-1 cells, and from 2.1 +/- 0.1 to 3.8 +/- 0.1 ng/ml in confluent pSMC. L6 myotubes required more insulin (8 micrograms/ml) to achieve a half-maximal stimulation of IGF-BP secretion than confluent pSMC, differentiation deficient L6.DD cells or BC3H-1 cells, where half-maximal stimulation occurred between 125 and 300 ng/ml. L6 myoblasts were 40-fold more sensitive to insulin stimulation of IGF-BP secretion than L6 myotubes. IGF-I, although it interferes with the assay and thereby lowers the amount of detectable IGF-BP, stimulated the secretion of IGF-BP from all three cell types. Dexamethasone, (10(-7) M) decreased IGF-BP secretion into the media by approximately 50% for all three cell types. Affinity cross-linking and ligand blotting of 125I-IGF-I to conditioned media from each cell type showed (IGF-BP)-(IGF-I) complexes with molecular weights ranging 32-40 kDa (24-32 kDa for IGF-BP and 7.5 kDa for IGF-I). Insulin stimulated cell proliferation for both L6 myoblasts and BC3H-1 myocytes. This cell proliferative response was associated with an increase in IGF-BP secretion/cell in response to insulin. In contrast dexamethasone decreased L6 myoblast proliferation and decreased IGF-BP secretion/cell. We conclude that IGF-BP is secreted by each muscle cell type and that the state of cellular differentiation or quiescence influences its basal and insulin-stimulated secretion. Insulin and IGF-I are stimulators of IGF-BP secretion, whereas dexamethasone inhibits IGF-BP secretion. Because these hormones control muscle cell growth and differentiation, the IGF-BP may play an important regulatory role in these processes.     

Insulin-like growth factor (IGF)-independent effects of IGF binding protein-4 on human granulosa cell steroidogenesis

The ovarian insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system operates to permit maximal stimulation of steroidogenesis in the dominant follicle. In atretic follicles, the predominant IGFBPs are IGFBP-2 and IGFBP-4, which appear to be selectively cleaved in healthy follicles. We have recently demonstrated potent inhibition by IGFBP-4 of both theca and granulosa cell steroid production. The degree to which the inhibition occurred suggested that it was greater than might be expected by sequestration of IGF alone. Our study was designed to test this idea. Granulosa cells were harvested from follicles dissected intact from patients undergoing total abdominal hysterectomy and bilateral salpingoophorectomy. Granulosa cells were incubated with or without gonadotropins and IGFBP-4 in the presence or absence of either the IGF type I receptor blocker alphaIR3 or excess IGFBP-3 to remove the effects of endogenous IGF action. Steroid accumulation in the medium was assessed. IGFBP-4 continued to exert potent inhibitory effects when the action of endogenous IGF was removed from the system, demonstrating that its actions are independent of IGF binding. There was no effect on cell metabolism, and the effects on steroidogenesis were reversible after IGFBP-4 removal from the culture medium. No similar effects were seen with IGFBP-2. These reasults are the first evidence of IGF-independent IGFBP-4 actions and the first evidence of IGF-independent actions of any IGFBPs in the ovary.