The Association of Early Dietary Supplementation with Vitamin E with the Incidence of Ulcerative Dermatitis in Mice on a C57BL/6 Background: Diet and Ulcerative Dermatitis in Mice.

The purpose of this study was to ascertain if prophylactic ingestion of a diet rich in vitamin E would prevent or impede the development of ulcerative dermatitis in mice on a C57BL/6 background. Mice were fed either a standard mouse diet, vitamin E (99 IU/kg), or a mouse diet fortified with vitamin E (3000 IU/kg) after weaning. Cases of ulcerative dermatitis were recorded by individuals unmasked to the diet assignment. The incidence of ulcerative dermatitis in a retrospective cohort of mice on standard diet was compared with the group on the diet fortified with vitamin E. Age was associated with ulcerative dermatitis in standard diet and vitamin E fortified diet groups, r = 0.43, p-value < 0.0001 and r = 0.18, p-value < 0.02, respectively. The average age of incidence for ulcerative dermatitis in the mice fed the standard diet was 89 weeks and for the mice fed the vitamin E diet it was 41 weeks. The unadjusted odds ratio comparing the incidence of ulcerative dermatitis between the two diet groups was 4.6 with a 95% confidence interval of (2.44, 8.58), χ(2) p-value < 0.0001. Therefore, there was an association between the diets and ulcerative dermatitis, with the mice on the vitamin E fortified diet having almost five times the odds of having ulcerative dermatitis compared with mice on the standard diet. Incidence of ulcerative dermatitis was not influenced by sex or genotype. Our study results show that a diet fortified in vitamin E initiated at weaning does not prevent or impede the development of ulcerative dermatitis in mice on a C57BL/6 background and may accelerate development when administered to young mice.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

Insulin-like growth factor I and II (IGF-I and IGF-II) associate with specific IGF binding proteins (IGFBPs) present in plasma and extracellular fluids that can modulate the anabolic effects of these peptides. IGF-I has been shown to increase IGFBP concentrations in vivo and in vitro, but the mechanism and significance of this action are unknown. We examined these issues using normal and simian virus 40-transformed adult human fibroblasts (SV40-HF) in culture. Treatment with IGF-I markedly stimulated the appearance of IGFBP-3 (42/38 kD doublet), a 36 kD IGFBP, and 28-32 kD IGFBPs in the medium of these cells, as assessed by Western ligand blotting; IGF-I decreased levels of 24 kD IGFBP in normal HF cultures. The IGF-I-induced change in IGFBP levels was not a type I IGF receptor-mediated effect on IGFBP synthesis because (a) high concentrations of insulin did not mimic IGF-I's effect; (b) IGF-II and IGF-I analogues having reduced affinity for the IGF-I receptor were equipotent with IGF-I in increasing medium IGFBPs; (c) [QAYL]IGF-I, and IGF-I analogue having normal receptor affinity and decreased affinity for IGFBPs, had no effect; and (d) alpha IR-3, a monoclonal antibody specific for the type I IGF receptor, did not block IGF-I-stimulated increases in IGFBPs. In physiological studies, preincubation with 1 nM IGF-I had no effect on type I IGF receptor binding in normal HF and SV40-HF. In contrast, preincubation of cells with an equivalent concentration of [QAYL]IGF-I downregulated the receptors 40-50%. Changes in cell surface receptor number were reflected in cell responsiveness to IGF-I-stimulated [3H]thymidine incorporation and [3H]aminoisobutyric acid uptake. In conclusion, IGF-I regulates the availability of specific IGFBPs in cultured human fibroblasts by a novel receptor-independent mechanism. Rapid changes in levels of soluble IGFBPs as a direct response to extracellular IGF-I, in turn, modulate IGF-I peptide and receptor interaction, and may constitute an important level of control in IGF cellular physiology.

Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies.

Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4 degrees C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation.

Specific, high affinity insulin-like growth factor (IGF) binding proteins are secreted by human fibroblasts in culture. By multiple criteria, the species of IGF binding proteins produced by human fibroblasts are distinct from the HepG2/amniotic fluid IGF binding protein, but share many characteristics with the growth hormone-dependent IGF binding protein forms predominant in normal adult human plasma. Treatment of cultured human fibroblasts with growth hormone produced an increase in IGF binding protein activity in the medium, while addition of glucocorticoids markedly diminished IGF binding activity. Insulin, epidermal growth factor, platelet-derived growth factor, and progesterone had no effect on IGF binding activity in fibroblast media. In comparison, HepG2 IGF binding activity was enhanced by progesterone, decreased by insulin, and unaffected by growth hormone or glucocorticoid treatment. Five molecular forms of IGF binding proteins were identified by Western ligand blots in human fibroblast conditioned medium, with Mr = 41,500, 37,000, 32,000, 28,000, and 23,000. In human fibroblast conditioned medium, the Mr = 41,500 and 37,000 IGF binding protein species were abundant, as in normal human plasma, with a major Mr = 23,000 form which was a minor component in plasma.

Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults.

Hepatitis C-associated osteosclerosis (HCAO) is a rare disorder characterized by a marked increase in bone mass during adult life. Despite the rarity of HCAO, understanding the mediator(s) of the skeletal disease is of great interest. The IGFs-I and -II have potent anabolic effects on bone, and alterations in the IGFs and/or IGF-binding proteins (IGFBPs) could be responsible for the increase in bone formation in this disorder. Thus, we assayed sera from seven cases of HCAO for IGF-I, IGF-II, IGF-IIE (an IGF-II precursor), and IGFBPs. The distribution of the serum IGFs and IGFBPs between their ternary ( approximately 150 kD) and binary (approximately 50 kD) complexes was also determined to assess IGF bioavailability. HCAO patients had normal serum levels of IGF-I and -II, but had markedly elevated levels of IGF-IIE. Of the IGFBPs, an increase in IGFBP-2 was unique to these patients and was not found in control hepatitis C or hepatitis B patients. IGF-I and -II in sera from patients with HCAO were carried, as in the case of sera from control subjects, bound to IGFBP-3 in the approximately 150-kD complex, which is retained in the circulation. However, IGF-IIE was predominantly in the approximately 50-kD complex in association with IGFBP-2; this complex can cross the capillary barrier and access target tissues. In vitro, we found that IGF-II enhanced by over threefold IGFBP-2 binding to extracellular matrix produced by human osteoblasts and that in an extracellular matrix-rich environment, the IGF-II/IGFBP-2 complex was as effective as IGF-II alone in stimulating human osteoblast proliferation. Thus, IGFBP-2 may facilitate the targeting of IGFs, and in particular IGF-IIE, to skeletal tissue in HCAO patients, with a subsequent stimulation by IGFs of osteoblast function. Our findings in HCAO suggest a possible means to increase bone mass in patients with osteoporosis.

The insulin-like growth factor axis: A review of atherosclerosis and restenosis.

Insulin-like growth factors I and II (IGF-I and -II) and their regulatory proteins are secreted by cells of the cardiovascular system. They are growth promoters for arterial cells and mediators of cardiovascular disease. IGFs are bound to IGF binding proteins (IGFBPs), which modulate IGF ligand-receptor interaction and consequently to IGF action. IGFBPs are in turn posttranslationally modulated by specific proteases. This dynamic balance (IGFs, IGFBPs, and IGFBP proteases) constitutes the IGF axis and ultimately determines the extent of IGF-dependent cellular effects. Dysregulated actions of this axis influence coronary atherosclerosis through effects on vascular smooth muscle cell growth, migration, and extracellular matrix synthesis in the atherosclerotic plaque. IGF-I promotes macrophage chemotaxis, excess LDL cholesterol uptake, and release of proinflammatory cytokines. Endothelial cells also receive the effects of IGFs stimulating their migration and organization forming capillary networks. Neointimal hyperplasia of restenosis after coronary artery injury is also modulated by the IGF axis. IGFs stimulate vascular smooth muscle cell proliferation and migration to form the neointima and upregulate tropoelastin synthesis after disruption of the elastic layer. Understanding IGF axis regulation establishes a scientific basis for strategies directed to limit or reverse plaque growth and vulnerability in atherosclerosis and in the neointimal hyperplasia of restenosis.

Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer.

Endothelial differentiation gene (edg)-encoded G protein-coupled receptors (Edg Rs)-1, -3, and -5 bind sphingosine 1-phosphate (S1P), and Edg-2 and -4 bind lysophosphatidic acid (LPA). Edg Rs transduce signals from LPA and S1P that stimulate ras- and rho-dependent cellular proliferation, enhance cellular survival, and suppress apoptosis. That high levels of LPA in plasma and ascitic fluid of patients with ovarian cancer correlate with widespread invasion suggested the importance of investigating expression and functions of Edg Rs in ovarian cancer cells (OCCs) as compared with nonmalignant ovarian surface epithelial cells (OSEs). Analyses of Edg Rs by semiquantitative reverse transcription-PCR, a radioactively quantified variant of PCR, and Western blots developed with monoclonal antibodies showed prominent expression of Edg-4 R in primary cultures and established lines of OCCs but none in OSEs. In contrast, levels of Edg-2, -3, and -5 were higher in OSEs than OCCs. LPA stimulated proliferation and signaled a serum response element-luciferase reporter of immediate-early gene activation in OCCs but not OSEs, whereas S1P evoked similar responses in both OSEs and OCCs. Pharmacological inhibitors of Edg R signaling suppressed OCC responses to LPA. A combination of monoclonal anti-Edg-4 R antibody and phorbol myristate acetate, which were inactive separately, evoked proliferative and serum response element-luciferase responses of OCCs but not OSEs. Thus the Edg-4 R may represent a distinctive marker of OCC that transduces growth-promoting signals from the high local concentrations of LPA characteristic of aggressive ovarian cancer.

Physiological concentrations of insulin induce cellular desensitization to the mitogenic effects of insulin-like growth factor I.

Insulin and insulin-like growth factor I (IGF-I) are structurally related peptides capable of stimulating a variety of metabolic and mitogenic processes. In this study, we investigated the interaction between these peptides and their receptor-mediated pathways in an untransformed cell line. Cultured bovine fibroblasts specifically bound IGF-I and insulin, and each peptide could stimulate DNA synthesis and cell replication through its own receptor. Preincubation of bovine fibroblasts with concentrations of insulin that did not bind to the IGF-I receptor resulted in complete but reversible cellular desensitization to IGF-I-stimulated mitogenesis. Preincubation with as little as 0.1 nM insulin was sufficient to inhibit subsequent IGF-I action. Various insulin analogs produced desensitization in direct relation to the affinity of the insulin for the insulin receptor. Desensitization required > 4 h of cell exposure to insulin and was blocked in the presence of cycloheximide. Neither serum-stimulated mitogenesis nor IGF-I-stimulated glucose uptake were affected by insulin pretreatment. 125I-labeled IGF-I affinity cross-linking experiments indicated that preincubation with insulin did not affect labeling of the 130,000-M(r) alpha-subunit of the IGF-I receptor, but was associated with the loss of IGF-I- and insulin-inhibitive bands at M(r) = 100,000, 85,000, 58,000, and 34,000. These studies suggest that insulin, via interaction with insulin receptors on bovine fibroblasts, regulates IGF-I action at a step distal to IGF-I receptor binding and are consistent with desensitization occurring at an intracellular step in the mitogenic pathway shared by insulin and IGF-I.

Regulation of ornithine decarboxylase in skeletal muscle: evidence for the involvement of an insulin-dependent serum factor.

Previous studies in vivo have shown that the activity of ornithine decarboxylase (ODC), the rate-controlling enzyme in polyamine biosynthesis, is markedly decreased in muscle of diabetic rats and is restored to normal by insulin therapy. Also, muscle ODC is diminished by starvation and increased by refeeding. To investigate the basis for these findings, the regulation of ODC was studied in vitro using rat soleus and extensor digitorum longus muscles. Incubation of muscles from fed rats in Krebs-Henseleit solution resulted in a 75% decrease in ODC activity within 1 h. Addition of insulin and amino acids had no effect; however, 50% rat serum increased ODC activity four- to seven-fold after the initial decrease. Rat serum also increased ODC in muscles from starved rats. The effect of serum was blocked by both cycloheximide and antinomycin D. Serum from diabetic rats was only 50% as effective as serum from normal rats in increasing ODC activity. Addition of physiologic levels of insulin to diabetic serum had no effect; however, treatment of diabetic rats with insulin in vivo restored serum activity to normal. These findings suggest that insulin modulates the synthesis of ODC via production of a second circulating factor, the activity of which is diminished in serum of diabetic rats. They also suggest that the stimulation of polyamine biosynthesis by this factor may be an integral component of the growth-promoting effect of insulin on muscle in vivo.

Lack of growth hormone effect on insulin-associated suppression of insulinlike growth factor binding protein 1 in humans.

Insulinlike growth factor binding protein 1 (IGFBP-1) has been shown to modulate the metabolic and mitogenic actions of the growth hormone (GH)-dependent peptide insulinlike growth factor I. Previous studies showed that levels of IGFBP-1 are regulated by insulin. The relative role of GH in the regulation of IGFBP-1 levels is less well defined and was examined in our study with a contiguous two-part protocol. Overnight (part A) and pre- and post-morning meal (part B) blood samples were obtained from eight healthy adults during a constant infusion of saline (SAL) or 4 micrograms.kg-1.min-1 GH. Five of eight subjects were restudied with glucose (GLUC) infused during part B (SAL + GLUC) to match glucose and insulin to levels observed during GH infusion. During SAL infusion, IGFBP-1 levels measured by specific radioimmunoassay showed a marked immediate decline after the evening meal in part A, with a subsequent nocturnal rise of 2.4- to 17.3-fold. GH infusion resulted in a similar meal-induced fall in IGFBP-1 levels but led to a delayed nocturnal rise in IGFBP-1, which was associated with elevated postprandial insulin concentrations. During part B, changes in plasma IGFBP-1 levels showed a similar pattern, with a delayed postprandial increase observed during both GH and SAL + GLUC infusions. The half-life of IGFBP-1 disappearance was calculated at approximately 2 h for all three infusion groups. Comparison of venous and arterialized blood samples showed no consistent pattern of difference, arguing against peripheral tissue clearance or compartmentalization as the mechanism for the rapid rise and fall in IGFBP-1 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor binding protein-4 protease produced by smooth muscle cells increases in the coronary artery after angioplasty.

Insulin-like growth factor (IGF)-I stimulates vascular smooth muscle cell (VSMC) migration and proliferation, which are fundamental to neointimal hyperplasia in postangioplasty restenosis. IGF-I action is modulated by several high-affinity IGF binding proteins (IGFBPs). IGFBP-4 is the predominant IGFBP produced by VSMCs and is a potent inhibitor of IGF-I action. However, specific IGFBP-4 proteases can cleave IGFBP-4 and liberate active IGF-I. In this study, we document IGFBP-4 protease produced by human and porcine coronary artery VSMCs in culture as pregnancy-associated plasma protein-A (PAPP-A). This was shown by a distinctive IGFBP-4 cleavage pattern, specific inhibition of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition of PAPP-A by monoclonal antibodies. Furthermore, we found a 2-fold increase in IGFBP-4 protease activity in injured porcine VSMC cultures in vitro (P<0.05). We also evaluated IGFBP-4 protease/PAPP-A expression in vivo after coronary artery balloon injury. Twenty-five immature female pigs underwent coronary overstretch balloon injury, and vessels were examined at defined time points after the procedure. Abundant PAPP-A expression was observed in the cytoplasm of medial and neointimal cells 7, 14, and 28 days after angioplasty (P<0.01 vs control). The highest PAPP-A labeling indices were located in the neointima (36.1+/-2.1%) and the media (31.7+/-1.2%) 28 days after injury. Western blot analysis confirmed increased PAPP-A in injured vessels. PAPP-A, a regulator of IGF-I bioavailability through proteolysis of IGFBP-4, is thus expressed by VSMCs in vitro and in restenotic lesions in vivo. These results suggest a possible role for PAPP-A in neointimal hyperplasia.

Evaluation of continuous infusion suramin in metastatic breast cancer: impact on plasma levels of insulin-like growth factors (IGFs) and IGF-binding proteins.

Suramin represents a new class of antitumor drugs that targets growth factor networks. In this Phase II trial, suramin was administered by continuous infusion to 10 patients with advanced breast cancer. The target level of 280 microgram/ml suramin was achieved in a median of 10 days; toxicities in this patient group were low. We monitored the insulin-like growth factor (IGF) network in these patients because of the previously defined growth-promoting role of the IGFs in breast cancer. Plasma levels of total IGF-I and total IGF-II showed variable responses to suramin with median decreases of 24 and 23%, respectively, for the 10 patients; for total IGF-I levels, this did not reach statistical significance. On the other hand, free IGF-I plasma levels were consistently and dramatically increased (over 250%) after suramin infusion. IGF-binding proteins (IGFBPs), modulators of IGF bioavailability, were also measured. Levels of IGFBP-3, the major carrier of IGFs in the circulation, were decreased 21% after suramin treatment when measured by immunoradiometric assay. However, the majority of the plasma IGFBP-3 remaining after suramin was not the intact high-affinity IGF-binding form but rather a 30-kDa fragment with markedly reduced affinity for IGF-I. IGFBP-3 protease activity was evident in the plasma of 3 of 10 patients after suramin. Measurements of plasma IGFBP-1, IGFBP-2, and IGFBP-4 revealed no significant changes in response to suramin. The dramatic increase in active free IGF-I seen after suramin raises concern and underscores the importance of measuring relevant biomarkers in clinical trials.

Regulation of insulin-like growth factor binding protein 4 by a specific insulin-like growth factor binding protein 4 proteinase in normal human osteoblast-like cells: implications in bone cell physiology.

Insulin-like growth factor binding protein 4 (IGFBP-4) is secreted by normal human osteoblast-like cells (hOB) and is a potent inhibitor of insulin-like growth factor (IGF) action in vitro. In previous studies, IGF treatment of hOB in culture led to markedly reduced medium levels of IGFBP-4 as detected by western ligand blotting. In the present study, incubation of hOB-conditioned medium (hOB-CM) with IGF under cell-free conditions resulted in a similar loss of IGFBP-4. Both IGF-I and IGF-II were capable of inducing a decrease in IGFBP-4; however, IGF-II was more effective. When the six characterized IGFBP were added to hOB-CM, only IGFBP-4 disappeared in response to IGF-II addition. This IGF-regulated loss of IGFBP-4 was inhibited by metalloproteinase inhibitors and appeared to be due to a proteinase that cleaved IGFBP-4 in 18 and 14 kD fragments identified by western immunoblotting. Conditioned media from eight of eight different donor hOB lines tested exhibited IGFBP-4 proteinase activity. To assess the biologic consequences of IGF-II-induced IGFBP-4 proteolysis, we treated hOB with IGF-II for 5 h, which decreased medium IGF-BP-4 by 70%, and then measured IGF-I and insulin stimulation of [3H]thymidine incorporation. IGF-II itself was not mitogenic and had no effect on insulin-stimulated [3H]thymidine incorporation. However, pretreatment of cultured hOB with IGF-II enhanced IGF-I-stimulated [3H]thymidine incorporation threefold.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal human osteoblast-like cells consistently express genes for insulin-like growth factors I and II but transformed human osteoblast cell lines do not.

Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3E. coli) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3CHO) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3E. coli or hIGFBP-3CHO produced a dose-dependent inhibition of IGF-I but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGAFBP-3E. coli or hIGFBP-3CHO potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3E. coli for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3H]AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3E. coli with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 Mr hIGFBP-3E. coli seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3E. coli forms of 12,000-27,000 Mr. Cell-associated IGFBP-3E. coli (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3E. coli in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.

Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: studies of underlying mechanism.

In this study we investigated the mechanism(s) by which insulin-like growth factor-binding protein-3 (IGFBP-3) potentiates IGF-I action in cultured bovine fibroblasts. Preincubation of cells with glycosylated or nonglycosylated recombinant human IGFBP-3 enhanced responsiveness to IGF-I in a time-dependent manner. A preincubation period of at least 24 h with IGFBP-3 was required to see a significant effect. Pretreatment with IGFBP-3 for 72 h resulted in a 2- to 4-fold augmentation of IGF-I-stimulated [3H]aminoisobutyric acid uptake; IGFBP-3 had no effect on basal [3H]aminoisobutyric acid uptake. During the preincubation period, exogenous IGFBP-3 associated with the fibroblast surface and exhibited time-dependent processing to lower mol wt forms that retained the ability to bind radiolabeled IGF-I. Initial surface adherence (preincubation time of 24 h or less) was readily reversible. However, IGFBP-3, once processed, appeared to be closely associated with the cell. After 72 h of exposure to bovine fibroblasts, cell-associated IGFBP-3 had a 10-fold lower affinity for IGF-I compared to intact IGFBP-3 in solution. In addition, incubation of bovine fibroblasts with IGFBP-3 had modifying effects on type I IGF receptor-mediated signalling because 1) the bioeffectiveness of [Gln3,Ala4,Tyr15,Leu16]IGF-I and insulin, IGF-I receptor activators with little or no affinity for IGFBP-3, was potentiated by preincubation with IGFBP-3; and 2) fibroblast responsiveness to IGF-I analogs with different affinities for the type I IGF receptor was enhanced in direct relation to the ability of the peptide to bind to the receptor. There was no evidence for an increase in receptor number or affinity as a result of IGFBP-3 treatment. These data suggest that IGFBP-3 potentiation of IGF-I action in bovine fibroblasts may involve changes in IGFBP-3 and type I IGF receptor responsiveness. Thus, cell-associated IGFBP-3 may provide a mechanism for optimal presentation of IGF-I to its receptor as well as a means to heighten receptor reactivity to IGF-I and related peptides.

Regulation of insulin-like growth factor (IGF)-binding protein synthesis by insulin and IGF-I in cultured bovine fibroblasts.

Specific insulin-like growth factor-binding proteins (IGFBPs) are synthesized and secreted by bovine fibroblasts in vitro. By Western ligand blotting, three molecular forms of IGFBP were identified in conditioned medium from control cultures with mol wt (Mr) of 34,000, 28,000, and 24,000. Concentrations of these three IGFBP forms increased with time in serum-free conditioned medium without benefit of hormonal supplementation. Insulin and IGF-I were potent stimuli for IGFBP production by bovine fibroblasts, whereas bovine GH, epidermal growth factor, or steroid treatment had little or no effect. Insulin and IGF-I enhanced the production of 24,000, 28,000, and 34,000 Mr IGFBPs in a dose-dependent fashion. Moreover, addition of low nanomolar concentrations of insulin or IGF-I to bovine fibroblast cultures specifically induced the secretion of a 42,000/38,000 Mr species of IGFBP, which corresponded in size to the IGF-binding subunit of the principal 150,000 Mr IGFBP complex in serum. After stimulation with insulin or IGF-I, bovine fibroblasts (3 X 10(5) cells) secreted approximately 30 ng/24 h 42,000/38,000 Mr IGFBP. Subunits of 42,000/38,000 Mr in bovine fibroblast-conditioned medium did not form macromolecular complexes in either the absence or presence of bovine GH.

Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts.

Insulin-like growth factor-I (IGF-I) initiates its diverse biological effects by binding to type I IGF receptors on cells. In addition, IGF-I associates with distinct proteins that can modulate its actions. One of these IGF-binding proteins, IGFBP-3, is the major circulating form in adults and is produced by many cells in culture. We investigated the effect of purified bovine IGFBP-3 on IGF-I binding and IGF-I stimulation of amino acid uptake and DNA synthesis in cultured bovine fibroblasts, a cell culture system highly suitable for these types of studies. Incubation of cells with IGF-I resulted in time- and dose-dependent decreases in [125I]IGF-I binding and IGF-I stimulated [3H]aminoisobutyric acid uptake and [3H]thymidine incorporation. Preincubation with 4 nM IGF-I resulted in a 50-60% decrease in IGF-I receptor binding, accompanied by marked decreases in IGF-I-stimulated [3H]aminoisobutyric acid uptake (50-60%) and [3H]thymidine incorporation (80-90%). Preincubation with the IGF-I analog [QAYL]IGF-I (4 nM) or with 100 nM insulin, growth factors that bind and activate type I IGF receptor signalling but have little or no affinity for IGFBP-3, had effects comparable to IGF-I, decreasing both IGF-I binding and action 50-95%. The addition of IGFBP-3 during the preincubation period with IGF-I blocked the decrease in receptor availability and prevented the cells from becoming desensitized. IGFBP-3 did not prevent the [QAYL]IGF-I- or insulin-induced receptor loss and cellular resistance to IGF-I. These data indicate that IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I. Thus, cell-derived IGFBP-3 may function in a buffering capacity to restrict IGF-I and target cell interaction, thereby modulating the biological response to changes in local IGF-I levels.

Regulation of insulin-like growth factor binding protein-3 messenger ribonucleic acid expression by insulin-like growth factor I.

Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator of the anabolic and mitogenic actions of the insulin-like growth factor (IGF) peptides. Previous studies have shown that the IGFs themselves can elevate levels of IGFBP-3 in vivo and in vitro. However, the regulatory mechanisms responsible for IGF-induced increases in IGFBP-3 are unclear. In this study we examined the expression of messenger RNA (mRNA) encoding IGFBP-3 in cultured bovine and human fibroblasts, two cell lines that secrete IGFBP-3 under IGF-I control. Northern analysis of bovine fibroblast RNA hybridized with a specific bovine IGFBP-3 complementary DNA probe indicated a single 2.8-kilobase (kb) transcript readily detectable within 2 h in IGF-I- or insulin-treated, but not in untreated, cells. IGFBP-3 mRNA abundance was maximal around 6 h, and remained elevated after 24 h of treatment. Secreted IGFBP-3 protein appeared more slowly. By Western ligand blotting, IGFBP-3 was not detected in medium from bovine fibroblasts incubated with IGF-I for 2, 4, or 6h, but was apparent after 24 h IGF-I treatment. Induction of IGFBP-3 mRNA was blocked when RNA synthesis was inhibited by actinomycin D. Furthermore, IGFBP-3 mRNA and protein was induced by different IGF-I analogs in direct relation to the ability of the peptides to bind to the type I IGF receptor, indicating a receptor-mediated process. GH had no effect on IGFBP-3 mRNA or protein levels in these cells. In contrast to its effect in bovine fibroblasts, IGF-I had no significant effect on steady state levels of IGFBP-3 mRNA in cultured human fibroblasts. A human IGFBP-3 complementary DNA probe hybridized to a single 2.8-kilobase mRNA species abundant in normal and SV40-transformed human fibroblasts under all culture conditions, and IGFBP-3 protein was secreted by these cells in the absence of exogenous stimuli. In human fibroblast cultures, IGF-I rapidly increased levels of IGFBP-3 in the medium without influencing transcript levels. Steady state levels of induced or constitutively expressed IGFBP-3 mRNA did not change significantly after 6h in the presence of actinomycin D, even though general RNA synthesis was inhibited more than 98%. These data demonstrate that expression of mRNA encoding IGFBP-3 is differentially controlled by IGF-I in bovine and human fibroblasts. Whereas cultured human fibroblasts may be suitable to study posttranscriptional regulation of IGFBP-3 availability, cultured bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3 gene expression and regulation by IGF-I.

Glucocorticoid regulation of insulin-like growth factor-binding protein expression in normal human osteoblast-like cells.

Glucocorticoid (GC) modulates insulin-like growth factor (IGF) action in bone through mechanisms that are complex and not well understood. Because the family of IGF-binding proteins (IGFBP-1 through -6) is important in the regulation of IGF availability and bioactivity, we examined the effect of GC on IGFBP expression in normal human osteoblast-like (hOB) cells. As assessed by Western ligand blot, hOB cells release IGFBP-3, IGFBP-4, and a 31-kilodalton IGFBP, which appeared to be IGFBP-5. Northern analysis revealed that hOB cells express abundant IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6 mRNA, with barely detectable IGFBP-1 mRNA. GC treatment resulted in time- and dose-dependent decreases in IGFBP-3, IGFBP-4, and 31-kilodalton IGFBP levels in culture medium, with corresponding decreases in IGFBP-3, IGFBP-4, and IGFBP-5 mRNA levels. In addition, GC treatment increased steady state levels of IGFBP-1 mRNA and did not alter IGFBP-6 mRNA levels. Although hOB cells secrete an acid-activated IGFBP-3 protease and an IGF-dependent IGFBP-4 protease, GC had little effect on these protease activities and did not alter degradation of the secreted IGFBPs. Our results indicate that GC has dramatic effects on IGFBP gene expression and suggest that differential regulation of IGFBPs by GC may modulate hOB cell responsiveness to IGFs.