Insulin-like Growth factor-I Binding Sites Research Articles

Characterization of insulin-like growth factor I and epidermal growth factor receptors in meningioma

Receptors for insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) were localized and characterized in eight samples of human meningioma (four fibrous, two meningothelial, and two angioblastic types), using quantitative autoradiographic techniques. Effects of both growth factors on deoxyribonucleic acid (DNA) synthesis in the cultured meningioma cells were examined. High numbers of specific binding sites for both IGF-I and EGF were homogeneously present in tissue sections derived from fibrous and meningothelial types of meningiomas, whereas binding sites for these growth factors were not detectable in adjacent leptomeninges. While relatively large numbers of IGF-I binding sites were located in the wall of the intratumoral vasculature, the number of binding sites in the stromal component was lower in angioblastic-type meningiomas, including a low number of EGF binding sites detected only in the stromal portion. Scatchard analysis revealed the presence of a single class of high-affinity binding sites for both IGF-I and EGF in the meningiomas examined (dissociation constant (Kd) = 0.6 to 2.9 nM, and the maximum number of binding sites (Bmax) = 16 to 80 fmol/mg for IGF-I; and Kd = 0.6 to 4.0 nM, Bmax = 3 to 39 fmol/mg for EGF). Both growth factors increased the synthesis of DNA, in a dose-dependent manner, as measured by 3H-thymidine incorporation. The combination of IGF-I and EGF synergistically stimulated the synthesis of DNA, and the effects seen with 10% fetal bovine serum could be reproduced at a concentration of 10(-10) M. These observations can be interpreted to mean that both IGF-I and EGF may be involved in the growth modulation of meningiomas, possibly through paracrine or autocrine mechanisms.

Receptor autoradiographic analysis of insulin-like growth factor-I (IGF-I) binding sites in rat forebrain and pituitary gland.

1. Specific 125I-labeled insulin-like growth factor-I [( 125I] IGF-I) binding sites in the rat forebrain and pituitary gland were investigated using quantitative receptor autoradiography. 2. High densities of [125I]IGF-I binding sites were present in the olfactory nerve layer, olfactory glomerular layer, choroid plexus, CA3 and CA4 of the hippocampus, basolateral amygdaloid nucleus, and endopiriform nucleus. Moderate to high binding densities were found in the cerebral cortex (II, VI), bed nucleus stria terminalis, accumbens nucleus, lateral septum, median preoptic nucleus, supraoptic nucleus, paraventricular hypothalamic nucleus, and ventroposterior thalamic nucleus. In the circumventricular organs, subfornical organ, vascular organ of the lamina terminalis, and median eminence, the binding sites were numerous. High densities of [125I]IGF-I binding sites were also observed in the anterior pituitary gland. 3. In kinetic experiments, [125I]IGF-I binding sites in the olfactory glomerular layer, choroid plexus, median eminence, and anterior pituitary gland were found to be single and of a high affinity. 4. Noteworthy was the difference in the potency of insulin in inhibiting the binding among the areas examined, a finding which suggests heterogeneity of IGF-I receptors. 5. The possibility that IGF-I plays the role of a neurotransmitter and/or neuromodulator in the central nervous system warrants further investigation.

Insulin-Like Growth Factor-I: Autocrine Secretion by Human Thyroid Follicular Cells in Primary Culture*

Insulin-like growth factor-I (IGF-I) stimulates the growth of thyroid cells of different animal species. However, conflicting results have been reported as regards the function and mechanism of the action of IGF-I at the thyroid level. This study was designed to determine whether normal human thyroid cells have IGF-I receptors and whether IGF-I could act on such cells through an autocrine mechanism. Human thyroid follicular cells in primary culture, not contaminated by fibroblasts, were used. They had specific and saturable binding sites for IGF-I, as revealed by radiolabeled binding method, and displayed an average receptor number of 2000/cell. Under the same experimental conditions, insulin receptors were not detectable. Human thyroid follicular cells secreted IGF-I into the culture medium, as assessed by a specific chemiluminescent immunoassay. The IGF-I secretory process was detectable for at least 12 days of culture, but a high degree of variability has been found among individual samples. Acid-gel chromatography demonstrated that IGF-I and a higher mol wt IGF-I, most likely IGF-I bound to its binding protein, were secreted by human thyroid cells. TSH stimulated the secretion of the two molecules in normal human thyroid cells. The TSH effect on IGF-I secretion was concentration dependent between 0.1 nmol/L and 0.1 mumol/L. GH stimulated IGF-I synthesis by thyroid cells in a concentration range from 20-200 micrograms/mL. Since binding studies demonstrated the presence of IGF-I receptors on human thyroid cells, IGF-I probably regulates human thyroid function through an autocrine mechanism.

Insulin-like growth factor I binding in hepatocytes from human liver, human hepatoma, and normal, regenerating, and fetal rat liver.

Insulin-like growth factor-I (IGF-I) in human hepatoma cells (HEP-G2) has, in addition to its effect on cell growth, short-term metabolic effects acting through its own receptor. We have demonstrated that normal human hepatocytes, compared with HEP-G2 cells, have virtually no IGF-I binding sites. Because the rate of growth is the major difference between the hepatoma and the normal liver, we asked if normal liver might express IGF-I binding sites under physiologic growth conditions. Indeed, whereas adult rat hepatocytes have low IGF-I binding sites similar to those in human liver, hepatocytes from regenerating liver after 3 d subtotal hepatectomy have an approximately sixfold increase (P less than 0.005) and those from fetal rat liver a approximately 12-fold increase (P less than 0.005), to levels comparable to those in the HEP-G2 cells. The specificity of 125I IGF-I binding to its receptor was demonstrated by competition studies with monoclonal antibodies directed toward the IGF-I and the insulin receptors, with unlabeled IGF-I and insulin and by affinity labeling experiments. Thus, if IGF-I has any short-term metabolic functions in the adult human liver, it is not through interaction with its own receptor. Autocrine regulation by IGF-I of liver growth appears possible since IGF-I binding sites are expressed under pathological and physiological conditions of growth. The mechanism that couples these two phenomena remains to be elucidated.

Localization of binding sites for insulin-like growth factor-I (IGF-I) in the rat brain by quantitative autoradiography

In vitro quantitative autoradiography was used to localize IGF-I binding sites in rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.01 nM 125I[Thr 59]IGF-I, alone or mixed with 10 nM unlabeled [Thr 59]IGF-I or insulin, for 22 h at 4 °C and apposed to LKB Ultrofilm. Measurement of labeled [Thr 59]IGF-I binding by computer digital image analysis of the autoradiographic images indicated that high affinity IGF-I binding sites are widely distributed at discrete anatomical regions of the brain microarchitecture. The highest concentration of specific binding sites was in the choroid plexus of the lateral and third ventricles. Unlabeled porcine insulin was less potent than unlabeled IGF-I in competing for binding sites on brain slices. Regions of the olfactory, visual, and auditory, as well visceral and somatic sensory systems were labeled, in particular the glomerular layer of the olfactory bulb, the anterior olfactory nucleus, accessory olfactory bulb, primary olfactory cortex, lateral-dorsal geniculate, superior colliculus, medial geniculate, and the spinal trigeminal nucleus. High concentrations of IGF-I-specific binding sites were present throughout the thalamus and the hippocampus, (dentate gyrus, Ca1, Ca2, Ca3). The hypothalamus had moderate binding in the paraventricular, supraoptic, and suprachiasmatic nucleus. Highest binding in the hypothalamus was in the median eminence. The arcuate nucleus showed very low specific binding, approaching the levels found in optic chiasm and white matter regions. Layers II and VI of the cerebral cortex also had moderate IGF-I binding. The results suggest that the development and functions of brain sensory and neuroendocrine pathways may be regulated by IGF-I.

Presence of somatomedin receptors on primary human breast and colon carcinomas

Competitive binding techniques were used to study the interaction of insulin-like growth factor I (IGF-I) with a plasma membrane-enriched subcellular fraction purified from primary breast and colon carcinoma specimens obtained at surgery. The presence of specific binding sites for IGF-I was detected in all tumour specimens studied. Scatchard analysis and competition studies with insulin and insulin-like growth factor-II (IGF-II) revealed the presence of specific IGF-I receptors, showing a K d -value of approximately 2 nM. These results are consistent with the hypothesis that somatomedins play a role in determining the proliferative behaviour of human breast and colon tumors, and suggest that recent laboratory studies showing dependence of neoplastic cells on somatomedins for optimum proliferation may have clinical relevance.

Decrease in IGF-I binding sites on human promyelocytic leukemia cell line (HL-60) with differentiation.

Specific insulin-like growth factor I (IGF-I) receptors on human promyelocytic leukemia cell line (HL-60) were identified and characterized. [125I]IGF-I specifically bound to the cells, and [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. [125I]IGF-I binding to the cells were displaced by multiplication stimulating activity (MSA) and porcine insulin, with potencies that were 10 and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present on the HL-60 cells. After the cells were differentiated to the macrophage-like cells by 12-o-tetra-decanoyl-phorbol-13-acetate (TPA) and 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), [125I]IGF-I binding to the cells decreased significantly. By Scatchard analysis, it was found to be due to a decrease in the number of IGF-I receptors. Thus, the differentiation of HL-60 cells to the macrophage-like cells was accompanied by a decrease in IGF-I receptors.

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells).

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.

The human placenta contains two distinct binding and immunoreactive species of insulin-like growth factor-I receptors.

Two species of insulin-like growth factor-I (IGF-I) receptors in human placenta have been delineated on the basis of their immunoreactivity with an autoantiserum (B-2) to the insulin receptor. When all the IGF-I binding sites in solubilized human placenta were assayed by polyethylene glycol precipitation, a curvilinear Scatchard plot was obtained which could be resolved into two single classes of binding sites: one immunoprecipitable by B-2 IgG and the other, nonimmunoprecipitable. The B-2 reactive sites bound IGF-I with lower affinity (Kd = 7.1 X 10(-10) M) than the B-2 nonreactive sites (Kd = 2.1 X 10(-10) M) and cross-reacted more readily with insulin, the IGF-I/insulin-binding potencies being congruent to 120 and congruent to 1100, respectively. Both receptor subtypes bound IGF-I with congruent to 30-fold higher affinity than multiplication-stimulating activity, and, after affinity cross-linking with 125I-IGF-I, migrated as specific reduced bands of Mr = 138,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit sizes of the B-2 reactive IGF-I receptor were similar to those of the insulin receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors immunoprecipitated by autoantiserum B-2 or autoantiserum B-10 (which recognizes only insulin receptors) revealed, in both cases, specific reduced bands of Mr = 130,000 and 90,000; the same bands were also seen after sequential precipitation with B-10 and B-2 antisera to enrich the proportion of IGF-I receptors recovered. The presence of two distinct binding and immunoreactive species of IGF-I receptors in human placenta raises the possibility that cell- or tissue-specific isotypes of the IGF-I receptor could mediate the different biological actions of IGF-I.