Insulin-like Growth factor-I Binding Sites Research Articles

Enhanced insulin‐like growth factor‐I (IGF‐I) cell association at reduced pH is dependent on IGF binding protein‐3 (IGFBP‐3) interaction

The cellular microenvironment impacts how signals are transduced by cells and plays a key role in tissue homeostasis. Although pH is generally well regulated, there are a number of situations where acidosis occurs and our work addresses how low pH impacts cell association of insulin-like growth factor-I (IGF-I) in the presence of IGF binding protein-3 (IGFBP-3). We have previously shown that IGF-I cell binding was enhanced in the presence of IGFBP-3 at low pH and now show that this binding is IGFBP-mediated as it is inhibited by Y60L-IGF-I, a mutant with reduced affinity for the IGF receptor (IGF-IR), and unaffected by insulin, which binds but not IGFBPs. Using surface plasmon resonance (SPR), we show that direct binding between IGF-I and IGFBP-3 is pH sensitive. Despite this, the key step in the process appears to be IGFBP-3 cell surface association as Long-R(3)-IGF-I, a mutant with reduced affinity for IGFBPs, shows a similar increase in cell association at pH 5.8 in the presence of IGFBP-3 but does not exhibit pH-dependent binding by SPR. Further, analysis indicates a large increase in low-affinity binding sites for IGF-I in the presence of IGFBP-3 and an elimination of IGF-I enhanced binding when a non-cell associating mutant of IGFBP-3 is added in place of IGFBP-3. That the IGFBP-3-mediated binding localizes IGF-I away from IGF-IR is suggested by triton-solubility testing and indicates additional complexities to IGF-I regulation by IGFBP-3. Identifying the pH-dependent binding partner(s) for IGFBP-3 is a necessary next step in deciphering this process.

Modulation of brain insulin-like growth factor I (IGF-I) binding sites and hypothalamic GHRH and somatostatin levels by exogenous growth hormone and IGF-I in juvenile rats.

The effect of exogenous growth hormone (GH) and insulin-like growth factor I (IGF-I) on brain IGF-I binding sites (IGF-IR), and on the levels of growth hormone-releasing hormone (GHRH) and somatostatin was studied in hypophysectomized and intact juvenile male rats. Animals were injected subcutaneously twice daily (n = 5 each) with recombinant GH (rGH) (2.5 U/kg per day) or rIGF-I (500 microg/kg per day). In the hypophysectomized rats, serum GH and IGF-I levels were markedly suppressed and IGF-I levels were partially restored by GH treatment. There was a significant increase in IGF-IR binding capacity in the IGF-I-treated hypophysectomized rats compared to the saline-treated hypophysectomized animals (150.61 +/- 45.66 vs 41.32 +/- 12.42 fmol/mg, p < 0.05) but no significant difference in IGF-IR mRNA levels. GHRH levels in the saline-treated hypophysectomized group were significantly lower than in the saline-treated intact rats (31.2 +/- 11.2 vs 140.6 +/- 48.1 pg/mg tissue, respectively, p < 0.01); no effect was induced by GH or IGF-I (37.5 +/- 26.8 and 53.8 +/- 22.5 pg/mg tissue, respectively). However, in the intact rats, GH and IGF-I injection led to a decrease in GHRH content, which was significant in the GH-treated compared to the saline-treated animals (33.1 +/- 16.2 vs 140.6 +/- 48.1 pg/mg tissue, p < 0.01). No difference was found in somatostatin levels between intact and hypophysectomized rats (631.2 +/- 81.2 and 625.0 +/- 62.5 pg/mg tissue, respectively). However, in the hypophysectomized animals, GH and IGF-I treatment induced a significant increase in somatostatin levels (1300 +/- 193.7 pg/mg tissue, p < 0.01, and 912.5 +/- 81.2 pg/mg tissue, p < 0.05, respectively). Our findings suggest that the bioavailability of exogenous IGF-I is greater than that of GH-stimulated endogenous IGF-I. Because IGF-I is a potent neurotrophic agent, this effect may have important implications for states of neurodegenerative diseases.

Receptors for insulin-like growth factor-I and tumor necrosis factor-α are hormonally regulated in bovine granulosa and thecal cells

Mastitis induces release of tumor necrosis factor-α (TNFα) and has been linked with reduced reproductive performance. To further elucidate the role and mechanism of action of TNFα on ovarian cells, the effect of TNFα on insulin-like growth factor-I (IGF-I)-induced steroidogenesis and IGF-I binding sites in granulosa and thecal cells as well as the hormonal regulation of TNFα receptors were evaluated. Granulosa and thecal cells were obtained from small (1–5 mm) and large (≥8 mm) bovine ovarian follicles, respectively, and cultured for 3–4 days. During the last 2 days of culture, cells were treated with various hormones and steroid production and specific binding of 125 I -IGF-I and 125 I -TNFα was determined. Two-day treatment with 30 ng/ml of TNFα decreased ( P<0.05) IGF-I-induced estradiol production by granulosa cells and IGF-I-induced androstenedione production by thecal cells. Two-day treatment with 10 and 30 ng/ml of TNFα decreased ( P<0.05) specific binding of 125 I -IGF-I to thecal cells, but had no effect on specific binding of 125 I -IGF-I to granulosa cells, or on specific binding of 125 I -IGF-II to thecal cells. TNFα did not compete for 125 I -IGF-I binding to granulosa or thecal cells whereas unlabeled IGF-I suppressed 125 I -IGF-I binding. Insulin inhibited ( P<0.10) whereas FSH had no effect on the number of specific 125 I -TNFα binding sites in granulosa cells. In contrast, LH increased ( P<0.10) whereas insulin had no effect on specific 125 I -TNFα binding sites in thecal cells. These results suggest that IGF-I and TNFα receptors in granulosa and thecal cells are regulated by hormones differentially.

Insulin-like growth factor I in the anterior pituitary of the clawed frog Xenopus laevis: immunocytochemical and autoradiographic indication for a paracrine action and corelease with prolactin.

Insulin-like growth factor I (IGF-I) and its receptor are present in human and rat anterior pituitary. However, few data exist on the potential presence of IGF-I or its receptor in the non-mammalian pituitary and the cellular sites of IGF-I production have not been identified in any species. Thus, we investigated the anterior pituitary of the clawed frog Xenopus laevis which is widely used to study growth and differentiation. The study was performed with antisera against mammalian insulin-like growth factor I (IGF-I), prolactin (PRL) and growth hormone (GH) using immunohistochemical and immunocytochemical techniques. IGF-I binding was determined by in-vitro receptor autoradiography. The PRL-and GH-immunoreactive cells exhibited distinct distribution patterns. Neither at the light nor the electron microscopical level any colocalization of PRL-and GH-immunoreactivities was apparent. The PRL-immunoreactive cells exhibited round granules of medium electron density (mean diameter: 312 nm) and the GH-immunoreactive cells spherical granules of medium electron density (mean diameter: 165 nm). By the use of serial semithin sections IGF-I-immunoreactivity was exclusively located in PRL-immunoreactive cells. At the ultrastructural level, IGF-I-immunoreactivity was confined to the secretory granules in coexistence with PRL-immunoreactivity using the double labelling immunogold technique. Specific IGF-I binding sites were localized throughout the pituitary. The results provide evidence for a concomitant release of PRL and IGF-I and suggest autocrine/paracrine actions of IGF-I in the anterior pituitary.

Growth hormone, insulin-like growth factor I and cognitive function in adults.

This review focuses on the possible contribution of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis to cognitive function. Binding sites for GH and IGF-I are found in various areas of the brain. Their distribution suggests that GH and IGF-I contribute to the function of the hippocampus, a brain structure important for the maintenance of cognitive functions such as learning and memory. Evidence for cognitive deficits in GH-deficient individuals has been found in various studies, some of which have shown that these deficits can be reversed by GH substitution therapy. In addition to examining conditions of GH deficiency, this article reviews studies evaluating the correlation between the cognitive deficits associated with ageing and age-related decreases in GH or IGF-I secretion. Based on the available data, one might hypothesize that relative GH or IGF-I deficiency could contribute to the deterioration of cognitive functions observed in the elderly.

DECREASED INSULIN-LIKE GROWTH FACTOR-I RECEPTOR SITES ON CIRCULATING MONONUCLEAR CELLS FROM CHILDREN WITH ACUTE LEUKEMIA

Insulin-like growth factor-I (IGF-I) is a known mitogen for various cell types, including those of the hematopoietic cell system. To study the role of IGF-I in the neoplastic process of leukemia in children, the authors have determined the number of IGF-I binding sites on circulating mononuclear cells of children with acute leukemia as compared to normal children, using binding assays. The IGF-I binding sites per cell on peripheral mononuclear cells of children with leukemia decreased compared to those of the control group (411 +/- 73 and 1334 +/- 227, respectively, p < .001), while their affinity increased (Kd = 0.14 +/- 0.04 and 0.43 +/- 0.16, respectively, p = .05). Furthermore, in the patients, the number of the IGF-I binding sites was significantly lower in the subgroup of the peripheral mononuclear cells, which included lymphocytes and monocytes, as compared to their number in the peripheral blast cells (254 +/- 43.6 and 536 +/- 98.6, respectively, p = .02). A significant reduction was found in serum GHBP levels in the patients as compared to the controls (28.21 +/- 1.93 and 35.83 +/- 2.90, respectively, p = .02), while serum IGF-I and growth hormone levels were similar in patient and control groups. These results suggest a possible involvement of IGF-I in childhood acute leukemia, but further studies are needed to establish whether IGF-I plays a role in this disease.

Insulin-like growth factor-I and its receptor in the frontal cortex, hippocampus, and cerebellum of normal human and alzheimer disease brains.

Assimilated evidence indicates that the neurotoxic potential of amyloid beta (Abeta) peptide and an alteration in the level of growth factor(s) may possibly be involved in the loss of neurons observed in the brain of patients suffering from Alzheimer disease (AD), the prevalent cause of dementia in the elderly. In the present study, using receptor binding assays and immunocytochemistry, we evaluated the pharmacological profile of insulin-like growth factor-I (IGF-I) receptors and the distribution of IGF-I immunoreactivity in the frontal cortex, hippocampus, and cerebellum of AD and age-matched control brains. In control brains, [(125)I]IGF-I binding was inhibited more potently by IGF-I than by Des(1-3)IGF-I, IGF-II or insulin. The IC(50) values for IGF-I in the frontal cortex, hippocampus, and cerebellum of the normal brain did not differ significantly from the corresponding regions of the AD brain. Additionally, neither K(D) nor B(max) values were found to differ in the hippocampus of AD brains from the controls. At the regional levels, [(125)I]IGF-I binding sites in the AD brain also remained unaltered compared to the controls. As for the peptide itself, IGF-I immunoreactivity, in normal control brains, was evident primarily in a subpopulation of astrocytes in the frontal cortex and hippocampus, and in certain Purkinje cells of the cerebellum. In AD brains, a subset of Abeta-containing neuritic plaques, apart from astrocytes, exhibit IGF-I immunoreactivity. These results, taken together, suggest a role for IGF-I in compensatory plasticity and/or survival of the susceptible neurons in AD brains.

Impact of neonatal kainate treatment on hippocampal insulin-like growth factor receptors

The insulin-like growth factors-I and -II have neurotrophic properties and act through specific membrane receptors. High levels of binding sites for these growth factors are distributed discretely throughout the brain, being concentrated in the hippocampal formation. Functionally, the insulin-like growth factors, in addition to their growth-promoting actions, are considered to play important roles in normal cell functions, as well as in response to pharmacological or surgical manipulations. In adult rats, we have previously shown that systemic injection of kainate produces an overall decrease, in a time-dependent manner, in insulin-like growth factor-I and -II receptor binding sites in the hippocampus [Kar S. et al. (1997) Neuroscience 80, 1041–1055]. Given the evidence that insulin-like growth factors play a critical role during the early stages of brain development, the present study is a logical extension of this earlier report and established the effect of neonatal kainate injection on the developmental profile of insulin-like growth factor receptors. We have evaluated the time-course alteration of these receptors following systemic injection of kainate to newborn rats. After injection of a sublethal dose of kainate (5 mg/kg, i.p.) to postnatal one-day-old pups, [ 125I]insulin-like growth factor-I, [ 125I]insulin-like growth factor-II and [ 125I]insulin binding sites were studied at different postnatal days (7, 14, 21, 28 and 35) using receptor autoradiography. In the developing hippocampus, insulin-like growth factor-I and insulin binding sites are concentrated primarily in the dentate gyrus and the CA2/CA3 subfields, whereas insulin-like growth factor-II binding is discretely localized to the pyramidal layer and the granular layer of the dentate gyrus. Following kainate injection, we observed a slight increase in insulin-like growth factor-I binding sites in given hippocampal subfields starting at postnatal day 14, being significant at day 21. At later days, a progressive decrease was noted. This transient increase may represent an attempt for neuronal plasticity by up-regulating receptor levels. In contrast, insulin-like growth factor-II and insulin receptor binding sites are found to be decreased in various regions of the hippocampus in kainate-treated pups. Taken together, these results provide further evidence for the existence and differential alterations of insulin-like growth factor-I, insulin-like growth factor-II and insulin receptors in the developing rat hippocampus following kainate-induced lesion, suggesting possible involvement of these growth factors in brain plasticity.

Insulin-like growth factor-I binds in the inner plexiform layer and circumferential germinal zone in the retina of the goldfish.

Results of the previous study suggest that insulin-related peptides regulate proliferation of retinal progenitors in the adult goldfish. Because of their known roles in retinal neurogenesis, we have chosen to focus future studies on insulin-like growth factor I (IGF-I) and the IGF-I receptor. In the study described here, we characterized the spatial distribution and specificity of IGF-I binding sites in the retina of the adult goldfish by performing receptor-binding autoradiography with [125I]-IGF-I alone and with unlabeled IGF-I-related molecules (IGF-I, IGF-II, insulin, and des-[1-3]-IGF-I) as competitive inhibitors of [125I]-IGF-I binding. The results of these experiments show that IGF-I binds in two locations in the retina of the adult goldfish, within the inner plexiform layer of the differentiated retina and the circumferential germinal zone. The competition experiments suggest that [125I]-IGF-I binds at sites specific for IGF-I, and that both IGF-I receptors and IGF-I binding proteins are present in the retina.

Physiological changes in extracellular sodium directly control human proximal tubule growth and transport.

In order to examine the nature and potential mechanisms of action of extracellular sodium on human proximal tubule growth and transport, quiescent primary cultures of human proximal tubule cells (PTC) were incubated for 24 h in serum-free, growth-factor-free culture media containing low (130 mmol/l), control (140 mmol/l) or high (150 mmol/l) Na+. Compared to control conditions, cells exposed to a high Na+ concentration demonstrated stimulated thymidine incorporation (121.8 +/- 7.6%, P < 0.05) and increased cellular protein content (139.7 +/- 9.9%, P < 0.05); the latter arising from suppressed protein degradation ([3H]valine release 72.3 +/- 2.5%, P < 0.01) and unchanged protein synthesis ([3H]valine incorporation 98.5 +/- 2.6%, P > 0.1). Substitution of choline chloride for NaCl did not replicate these effects. Conversely, cells incubated in low-Na+ media showed reduced thymidine incorporation (77.2 +/- 4.4%, P < 0. 05), reduced protein synthesis (60.6 +/- 4.3%, P < 0.01), reduced protein degradation (79.5 +/- 1.8%, P < 0.01) and an unaltered protein content (102.4 +/- 8.8%). A role for apical Na+/H+ exchange (NHE) activity in mediating Na+-dependent alterations in PTC growth was suggested by the findings of increased apical, ethylisopropylamiloride- (EIPA)-sensitive 22Na+ uptake in the presence of a high Na+ concentration (159 +/- 19% of control, P < 0. 05) and concentration-dependent inhibition of cellular growth by EIPA at levels corresponding to those producing inhibition of apical NHE. Conditioned media from low Na+, control or high Na+ PTC contained comparable amounts of platelet-derived growth factor-AB (1. 19 +/- 0.23, 1.14 +/- 0.22 and 1.28 +/- 0.20 ng/mg protein, P > 0.1) and transforming growth factor-beta1 (1.76 +/- 0.32, 1.73 +/- 0.33 and 1.45 +/- 0.28 ng/mg protein, P > 0.1), and did not exhibit autocrine growth factor activity on separate PTC following adjustment of Na+ concentrations to 140 mmol/l by dialysis. Similarly, low-Na+, control or high-Na+ media did not modify the mitogenic responsiveness of PTC to insulin-like growth factor-I (IGF-I) or alter the affinity or number of PTC IGF-I binding sites. The results confirm that physiological increases in extracellular Na+ concentration directly stimulate human proximal tubule growth and Na+ transport. Such stimulation does not appear to be mediated by altered PTC secretion of, or responsiveness to, cytokines known to affect tubule growth and transport.

Diabetes-induced suppression of IGF-1 and its receptor mRNA levels in rat superior cervical ganglia

Insulin-like growth factor-I (IGF-I) is implicated in the development, survival and maintenance of function of sympathetic and sensory neurons. These neurons are affected at an early stage during the course of diabetes. Reverse transcriptase polymerase chain reaction (RT-PCR) based assay revealed that rat superior cervical ganglia (SCG) express mRNA transcripts for IGF-I and its receptor. Moreover, specific membrane protein binding sites for IGF-I within the SCG have also been demonstrated using competition–inhibition and affinity cross-linking techniques. An induction of diabetes with streptozotocin (STZ, 55 mg/kg, i.v.) produced a marked decrease in the SCG levels of mRNA transcripts for IGF-I and its receptor. Concentrations of circulating IGF-I and its receptor protein within the SCG were also reduced in this disease state. Insulin treatment partially prevented diabetes-related alterations in circulating IGF-I and the SCG-IGF-I system. Overall, the data described in this study may be of value in understanding the pathogenetic mechanism(s) responsible for the development of diabetic sympathetic neuropathy.

Identification and characterization of the insulin-like growth factor I receptor in mature rabbit osteoclasts.

In this study, the insulin-like growth factor I (IGF-I) receptor was identified in rabbit osteoclasts at mRNA and protein levels by in situ hybridization and autoradiography, respectively. Using highly purified mature osteoclasts, the IGF-I receptor was characterized on the molecular level according to its size and its affinity and number per osteoclast by isolation of the receptor-ligand complex and by binding studies, respectively, and on the cellular level according to the response of mature osteoclasts to IGF-I stimulation. In situ hybridization and autoradiography experiments showed that osteoclasts express IGF-I receptor mRNA and IGF-I binding sites. Chemical cross-linking of 125I-IGF-I bound to the purified mature osteoclasts and subsequent sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the specific binding of 125I-IGF-I in complexes with molecular masses of 130 and 230-RD consistent with binding to the IGF-I receptor. In competition experiments, 125I-IGF-I binding to mature osteoclasts was dose-dependently reduced by unlabeled IGF-I in the picomolar range, whereas 20 nM insulin did not reduce the binding of 125I-IGF-I binding. The calculated receptor number was 6000 per osteoclast, and the Kd was 0.10 nM. Searching for a role of the IGF-I receptor in mature osteoclasts, we found no significant influence of IGF-I on the levels of the proform of matrix metaloproteinase 9 and tartrate-resistant acid phosphatase. However, the induction of nuclear fragmentation in serum-depleted cultures of purified mature osteoclasts was dose-dependently inhibited by IGF-I in the picomolar range, but not by 1 nM insulin. These data show that functionally active IGF-I receptor is present in mature osteoclasts.

Functional active receptors for insulin-like growth factor-I (IGF-I) and IGF-II on insulin-, glucagon-, and somatostatin-producing cells

Insulin-like growth factors I and II (IGF-I and IGF-II) are expressed at high levels in the endocrine pancreas during development and tissue regeneration. However, their effects at the endocrine pancreas are poorly understood. We searched for receptors of IGF-I and IGF-II and possible biological effects on clonal insulin-secreting (HIT), glucagon-secreting (INR1G9), and somatostatin-secreting (RIN 1027 B2) cell lines. Our data showed that HIT cells and RIN 1027 B2 cells express specific type I and type II IGF receptors. INR1G9 cells possess type II IGF receptors and IGF-I binding sites with the same affinity for both IGF-I and IGF-II. In HIT cells, insulin secretion was not influenced by either peptide. Proinsulin gene transcription was stimulated by IGF-II but not by IGF-I. IGF-I potently inhibited proglucagon gene transcription and glucagon secretion in INR1G9 cells, whereas IGF-II only inhibited glucagon release. In RIN 1027 B2 cells, IGF-I but not IGF-II increased somatostatin output, whereas both stimulated somatostatin gene expression. These data demonstrate the presence of classic type I and type II IGF receptors on insulin-, glucagon-, and somatostatin-secreting cells. Both peptides may be important regulators of endocrine pancreatic function in terms of islet hormone release and gene expression. Therefore, both peptides may be involved in the regulation of intraislet cellular homeostasis.

Interactions among basic fibroblast growth factor, epidermal growth factor, insulin, and insulin-like growth factor-I (IGF-I) on cell numbers and steroidogenesis of bovine thecal cells: role of IGF-I receptors.

The objectives of the present study were to determine the interaction among basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, and insulin-like growth factor-I (IGF-I) on cell numbers, steroidogenesis, and IGF-I binding sites in cultured thecal cells. Cells from large (> or = 8 mm) follicles were collected from cattle and cultured for 4 days. Treatment with 1-30 ng/ml of bFGF for 2 days increased (p < 0.05) thecal cell numbers but inhibited (p < 0.05) androstenedione and progesterone production in the presence of insulin alone or insulin plus LH. Treatment with 1-30 ng/ml of bFGF for 2 days also inhibited (p < 0.05) the number of specific 125I-IGF-I binding sites in thecal cells, with maximal inhibition being detected at 1 ng/ml; coculture with 100 ng/ml LH did not influence this effect. In the absence of bFGF and the presence of LH, 30 ng/ml of IGF-I increased thecal cell numbers and production of androstenedione and progesterone. However, cotreatment with 10 ng/ml bFGF inhibited thecal cell response to IGF-I. Cotreatment consisting of 3-100 ng/ml EGF with insulin increased (p < 0.05) thecal cell numbers but decreased (p < 0.05) androstenedione and progesterone production and the number of specific 125I-IGF-I binding sites in thecal cells, with maximal inhibition observed at 3 ng/ml of EGF. Also, cotreatment consisting of 3 and 10 ng/ml of EGF with IGF-I plus LH inhibited (p < 0.05) cell numbers and production of androstenedione and progesterone by thecal cells. These results suggest that bFGF, EGF, insulin, and IGF-I may interact to play a significant role in basal and LH-modulated thecal cell steroidogenesis and mitogenesis during follicular development in cattle.

Inhibition of growth of human osteosarcomas by antagonists of growth hormone-releasing hormone.

Insulin-like growth factor I (IGF-I) may be involved in the proliferation of human osteosarcomas. Most of the IGF-I found in blood is produced in the liver, where transcription of the IGF-I gene is regulated by growth hormone (GH). Recently, we synthesized various potent antagonists of GH-releasing hormone (GH-RH), including [Ibu0, D-Arg2, Phe(4-Cl)6, Abu15, Nle27]hGH-RH(1-28)Agm, which is also called MZ-4-71. We investigated the effects of this antagonist on the growth of the human osteosarcoma cell lines SK-ES-1 and MNNG/HOS, transplanted into nude mice or cultured in vitro. Nude male mice bearing SK-ES-1 and MNNG/HOS tumors were treated for 4 and 3 weeks, respectively, with MZ-4-71 administered from osmotic minipumps at a dose of 40 micrograms per animal per day. Tumor volume, tumor weight, and levels of receptors for IGF-I were determined. IGF-I levels in serum, tumor, and liver tissue were measured by radioimmunoassay. In other experiments, tumor-bearing nude mice were treated subcutaneously for 3 weeks with the GH-RH agonist hGH-RH(1-29)NH2 or with MZ-4-71 for 13 days at doses of 50 micrograms per animal per day. Effects of MZ-4-71, hGH-RH(1-29)NH2, and human GH (hGH) on cell proliferation and on the production of IGF-I and cyclic adenosine monophosphate were also evaluated in SK-ES-1 and MNNG/HOS cells in vitro. The growth of SK-ES-1 and MNNG/HOS tumors in nude mice was significantly inhibited by MZ-4-71, as measured by a reduction in tumor volume and weight (all P values < .05). MZ-4-71 treatment of either SK-ES-1 or MNNG/HOS tumor-bearing animals decreased tumor tissue IGF-I levels. The growth of MNNG/HOS xenografts was stimulated by hGH-RH(1-29)NH2 (P < .01). IGF-I levels in serum of tumor-bearing nude mice treated subcutaneously for 13 days with MZ-4-71 were decreased (both P values < .01). High-affinity binding sites for IGF-I were demonstrated on cell membranes of SK-ES-1 and MNNG/HOS tumors. In cell cultures of both osteosarcomas, IGF-I production was stimulated by 25 ng/mL hGH but was not changed by 10 ng/mL hGH-RH(1-29)NH2 or 5 microM MZ-4-71. Incorporation of [3H]thymidine into DNA in SK-ES-1 (but not MNNG/HOS) cells was increased by 25 ng/mL IGF-I (P < .01). The proliferation rate of the two cell lines was not affected by 5-50 ng/mL hGH-RH(1-29)NH2 or 1-80 ng/mL hGH but was suppressed by 10(-6)-10(-5) M MZ-4-71. Our findings demonstrate that the GH-RH antagonist MZ-4-71 can significantly inhibit the growth of SK-ES-1 and MNNG/HOS osteosarcomas in mice.

Increased specific binding of insulin‐like growth factor‐I in healing cutaneous wounds

Insulin-like growth factor-I is a polypeptide hormone structurally related to insulin. It is a potent mitogen that promotes growth and differentiation in many tissues. A role for insulin-like growth factor-I in wound healing is suggested by its rapid rise in levels and increased insulin-like growth factor-I messenger RNA expression in tissue after wounding. We designed our study to characterize possible changes in insulin-like growth factor-I receptor binding during wound healing. Surgical wounds created on the abdominal skin of anesthetized New Zealand White rabbits were either left open or closed primarily. Size- and weight-matched specimens were harvested at wounding time (day 0), and at 1, 4, 7, 38, and 50 days after wounding. Preliminary experiments showed that the greatest difference in specific binding occurred between day 0 and day 7. (125)I-insulin-like growth factor-I binding studies were performed on frozen tissue specimens and autoradiography was performed and analyzed by computerized densitometry. Scatchard analysis of the binding data showed a single class of insulin-like growth factor-I binding sites whose affinity that is, binding constant (K(d) = 0.6 x 10(-9)) did not change significantly over time; in contrast there was a threefold increase in the number of receptors per milligram tissue in day 7 wound tissue versus normal skin harvested at day 0 (17.3 +/- 2.6 x 10(10) versus 4.7 +/- 2.5 x 10(10), respectively, p < 0.05). Binding inhibition experiments showed that (125)I-insulin-like growth factor-I binding was most specific to insulin-like growth factor-I with insulin-like growth factor-I > insulin-like growth factor-II > insulin. This increase in binding was due to upregulation of insulin-like growth factor-I receptors rather than increased levels of insulin-like growth factor-I binding protein as less than 20% of the threefold increase in binding at day 7 could be attributed to insulin-like growth factor-I binding protein in membrane-free extracts. The presence of specific, high-affinity insulin-like growth factor-I receptors in the skin and their upregulation at day 7 after wounding suggest that insulin-like growth factor-I plays an important role during wound healing.

Insulin- and insulin-like growth-factor-I receptor tyrosine-kinase activities in human renal carcinoma.

We studied expression and functional characteristics of the insulin- and insulin-like-growth-factor-I (IGF-I) receptors in human renal carcinoma. Ligand-binding properties and tyrosine-kinase activity of both receptors, as well as the expression of the 2 isoforms of the human insulin receptor (HIR-A and -B) were analyzed in renal carcinoma and normal adjacent kidney tissue of 8 adult patients. Partially purified insulin- and IGF-I receptors from normal and renal cell carcinoma tissue possessed identical affinities for their ligands. Renal cell carcinoma, however, contained 3- to 4-fold more specific insulin-binding sites and 2-fold more IGF-I binding sites than adjacent normal kidney tissue. In addition, we determined the relative content of insulin/IGF-I receptor hybrids in both tissues. Renal cell carcinoma and adjacent normal tissue revealed similar amounts of insulin/IGF-I receptor hybrids, i.e., 44 +/- 8.2% of tracer IGF-I binding in normal tissue and 46 +/- 12.0% in renal cell carcinoma. When equal amounts of insulin- and IGF-I receptor protein were studied, we found significantly increased receptor autophosphorylation and elevated substrate phosphorylation in carcinoma tissue. To assess whether the differences in insulin-receptor tyrosine-kinase activity were caused by an altered pattern of insulin receptor isoform expression, we determined mRNA levels for HIR-A and -B. The 2 insulin receptor isoforms were, however, expressed in highly variable ratios in both normal and tumor tissue. Our experiments show that renal carcinoma expresses an elevated amount of insulin- and IGF-I receptor protein with increased specific autophosphorylation and tyrosine-kinase activity each. The increase of insulin-receptor tyrosine-kinase activity in renal carcinoma cannot be explained by an altered expression pattern of insulin receptor isoforms.

Activation of phosphorylation of plasma membrane insulin-like growth factor-I receptors in the kidney of Syrian hamsters by diethylstilbestrol.

In the present work we have investigated activation of phosphorylation of plasma membrane insulin-like growth factor-I receptors (IGF-IR) by diethylstilbestrol (DES). Insulin-like growth factor-I (IGF-I) stimulated the activity of membrane protein tyrosine kinase(s) (PTK) in both normal and DES-treated hamster kidneys. The level of IGF-I-stimulated PTK(s) almost doubled after 15 days of DES treatment. Autophosphorylation experiments revealed that phosphorylation of a 95 kDa band (presumably the beta subunit of IGF-IR) was 2-fold higher in the membranes of kidney from DES-treated animals compared with controls. To understand the mechanism of activation of IGF-I-dependent PTK by DES, we investigated the relationship between the binding capacity of IGF-I to membrane proteins and the level of IGF-IR. The binding of [125I]IGF-I to membranes from the DES-treated group was 30% higher than that of age-matched normal kidney (P < 0.001). Scatchard analysis of the binding data for both normal and DES-treated hamster kidney revealed a single class binding site for IGF-I with a dissociation constant (Kd) of 4.1 and 4.6 nM and a maximum binding capacity (Bmax) of 1786 and 2086 fmol/200 micrograms protein respectively. Therefore, the difference observed in [125I]IGF-I binding between DES-treated and normal kidney membranes may be partially due to an increase in the number of IGF-I binding sites, with no change in the affinity of the receptors for IGF-I. An enhanced level of IGF-IRs in membranes from DES-treated animals was visualized by autoradiography following affinity labeling of membrane proteins subjected to SDS-PAGE. Under reducing conditions a molecular band of 132 kDa was evident. The 132 kDa band represents the alpha-subunit of IGF-IRs. Northern blot analyses revealed that DES treatment increased the level of IGF-IR mRNA 2-fold compared with that of controls. These findings suggest that an enhanced level of IGF-IR coupled with qualitative changes may be responsible for the activation of IGF-I-dependent PTK on DES exposure. Whether the stimulation of IGF-IR phosphorylation by exposure to a carcinogenic dose of DES may be a factor in the induction of renal cancer in Syrian hamsters is not clear.

Growth hormone directly and indirectly stimulates articular chondrocyte cell growth

Although growth hormone (GH) is known to regulate cartilage growth and differentiation during development, it is still unclear whether the cell growth of articular chondrocytes is stimulated directly by GH or mediated by GH-induced insulin-like growth factor-I (IGF-I). In the present study, we focused on whether GH directly or indirectly stimulates articular chondrocyte proliferation. Monolayer articular chondrocytes from 5-week-old male Sprague-Dawley rats were cultured in Ham's F-12/Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. Stimulation of DNA synthesis by GH was dose-dependent between 0.1 and 1 microg/ml, and the maximum active concentration of GH was 500 ng/ml, which induced a 3.5-fold increase over control values. Anti-IGF-I antiserum neutralized about 80% of GH-induced DNA synthesis. GH stimulated the secretion of IGF-I into the conditioned medium in a dose-responsive manner. To determine whether GH stimulated DNA synthesis directly, we investigated the time-course changes in mRNA expression of IGF-I and the proto-oncogene c-myc. Induction of IGF-I mRNA occurred at 4 h, and reached a maximum level at 12 h, whereas the expression of c-myc mRNA was induced within 4 h, and continued to increase until 72 h after GH treatment. Furthermore, administration of cycloheximide, an inhibitor of protein synthesis, resulted in the superinduction of both IGF-I and c-myc mRNAs. These results suggest that early induction of c-myc is due to a direct stimulatory effect of GH, and that long-term induction of c-myc was attributable to an indirect effect of GH in which GH-induced secondary proliferative factors may act in an autocrine/paracrine manner. The superinduction of c-myc gene by cycloheximide also indicates that fresh protein synthesis of an intermediate protein was not required for GH-induced c-myc expression. Western ligand blot analysis of IGF-binding proteins revealed that cultured rat articular chondrocytes produced a predominant 41 kDa and a faint 32 kDa form, and that GH significantly stimulated the secretion of the 41 kDa form without affecting expression of the 32 kDa form. Furthermore, a specific IGF-I binding study suggested that the increase in DNA synthesis induced by GH was not associated with changes in affinity or in the number of IGF-I binding sites. These results support the conclusion that the stimulatory effect of GH was mainly mediated by GH-induced IGF-I production in monolayer rat articular chondrocytes. However, it is likely that GH may also have a direct stimulatory effect by inducing c-myc proto-oncogene expression.

The insulin-like growth factor I system in the rat cerebellum: developmental regulation and role in neuronal survival and differentiation.

The developmental regulation of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins (IGFBPs) was studied in the rat cerebellum. All the components of the IGF-I system were detectable in the cerebellum at least by embryonic day 19. Levels of IGF-I receptor and its mRNA were highest at perinatal ages and steadily decrease thereafter, although a partial recovery in IGF-I receptor mRNA was found in adults. Levels of IGF-I and its mRNA also peaked at early ages, although immunoreactive IGF-I showed a second peak during adulthood. Finally, levels of IGFBPs were also highest at early postnatal ages and abruptly decreased thereafter to reach lower adult levels. Since highest levels of the different components of the IGF-I system were found at periods of active cellular growth and differentiation we also examined possible trophic effects of IGF-I on developing cerebellar cells in vitro. We found a dose-dependent effect of IGF-I on neuron survival together with a specific increase of the two main neurotransmitters used by cerebellar neurons, GABA and glutamate. Analysis of cerebellar cultures by combined in vitro autoradiography and immunocytochemistry with cell-specific markers indicated that both Purkinje cells (calbindin-positive) and other neurons (neurofilament-positive) contain IGF-I binding sites. These results extend previous observations on a developmental regulation of the IGF-I system in the cerebellum and reinforce the notion of a physiologically relevant trophic role of IGF-I in cerebellar development.