Binding of 125I-epidermal growth factor in human uterus

Summary Quantitative light-microscopic autoradiography was used on five human uteri at two different phases of the menstrual cycle to ascertain the cell types with binding sites for epidermal growth factor (EGF). The results revealed that stromal cells, glandular epithelium of endometrium, elongated and circular muscle cells of myometrium, smooth muscle and endothelial cells of arterioles in the basal endometrium and myometrium contained numerous silver grains following incubation with ¹²⁵I-EGF. Coincubation with 100-fold excess unlabeled EGF resulted in a complete disappearance of silver grains from all cell types. Quantitative grain analysis indicated that stromal cells contained the highest number of EGF-binding sites (P<0.05) with no significant differences among the others (P>0.05). There was no cyclic variation of EGF-binding to any of the uterine cell types. The present data demonstrate that all the cell types of human uterus, including arterioles, contain EGF-binding sites. This suggests that all the cells in human uterus subserving different functions are targets of EGF action.     

Ligand-induced desensitization of 125I-epidermal growth factor internalization

The internalization of 125I-epidermal growth factor (EGF) by A431 cells was investigated. Control cells were able to internalize over 80% of receptor-bound 125I-EGF. By contrast, cells treated with EGF before incubation with 125I-EGF internalized only 50% of the surface-bound radioligand. The ligand-induced decrease in 125I-EGF internalization showed a dose response to EGF with half-maximal effect occurring at 3 nM. The alteration in the extent of 125I-EGF internalization did not require extended treatment with high concentrations of the hormone. When the internalization of picomolar versus nanomolar concentrations of EGF were compared, the lower concentrations of 125I-EGF were more completely internalized than the higher concentrations of radioligand. These data are consistent with the hypothesis that occupation of the EGF receptor by hormone rapidly leads to the activation of cellular processes which effectively desensitize the system to further ligand-induced internalization. The decrease in the extent of ligand internalization occurred in cells in which the protein kinase C (Ca2+/phospholipid-dependent enzyme) activity had been down-regulated by prolonged treatment with 12-O-tetradecanoyl-phorbol-13-acetate implying that the desensitization process is independent of protein kinase C. However, the effects of EGF on the extent of hormone internalization could be mimicked by the addition of A23187 and could be prevented by pretreatment of the cells with calmodulin antagonists suggesting the possibility that Ca2+-calmodulin is involved in the regulation of EGF receptor internalization in A431 cells.     

Chloroquine allows the secretion of internalized 125I-epidermal growth factor from fibroblasts

Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse-chase paradigm in which cell surface receptors were labelled with 125I-EGF at 4 degrees C, it was found that when 100 microM chloroquine was present in the 37 degrees C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor-mediated endocytosis and subcellular distribution of 125I-EGF-receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I-EGF released from the cells could rebind to surface receptors and be re-internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I-EGF whose rebinding was inhibited. Thus, the 125I-EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I-EGF and mechanisms of its preferential rebinding are discussed.     

Intracellular processing of epidermal growth factor. I. Acidification of 125I-epidermal growth factor in intracellular organelles

We previously reported that 125I-labeled epidermal growth factor is processed intracellularly to acidic macromolecules in Rat-1 fibroblasts. The present study defines the precursor-product relationship and localization of the processing steps to subcellular organelles by the use of a single isoelectric species of 125I-epidermal growth factor and Percoll gradient fractionation. The native pI 4.55 125I-epidermal growth factor was rapidly processed to a pI 4.2 species on or near the cell surface and in organelles corresponding to clathrin-coated vesicles, Golgi, and endoplasmic reticulum. This species was then processed to a pI 4.35 species in similar organelles. The pI 4.2 and 4.35 species were converted to a pI 4.0 species in dense, lysosome-like organelles. This species was ultimately degraded and exocytosed from the cell as low molecular weight products.     

Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing

When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium ¹²⁵iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized ¹²⁵I-labelled molecules revealed processing of all the ¹²⁵I-EGF species to macromolecules with more acidic isoelectric points. The ¹²⁵I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.     

Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts

Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 125I-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 125I-EGF both bound preferentially to coated pits at 4 degrees C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 125I-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but 125I-EGF bound to coated pits and was internalized just as in normal fibroblasts.     

Energetics of heparin binding to human acidic fibroblast growth factor

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.     

Insulin-like growth factor II binding to cultured human chondrosarcoma cells

Cultured cells originally derived from a human chondrosarcoma (A1684) were used to investigate somatomedin binding in terms of kinetics and specificity. In this study, the rat somatomedin, multiplication-stimulation activity (MSA) was utilized. While the human chondrosarcoma cells did not exhibit a mitogenic response to MSA, the rate of transport of glucose and amino acids was significantly increased. In competitive binding experiments a specific insulin-insensitive MSA receptor was identified which showed half maximal displacement of tracer at a concentration of 250 ng/ml of MSA using whole cells. This receptor had an affinity constant of 4.8 X 10(7) M-1. Kinetic analysis of MSA binding to membrane preparations and to Triton X-100 solubilized membranes revealed an increase in the binding affinity to 1.28 X 10(8) M-1 and 2.8 X 10(8) M-1, respectively. Of particular significance is the observation that these cells have especially high levels of MSA receptors. Determination of binding capacity revealed that these cells contain approximately 1.9 X 10(6) MSA receptors per cell and therefore are an excellent model system for the characterization and purification of somatomedin receptors. Affinity labeling of the MSA receptor using the chemical crosslinking reagent, disuccinimidyl suberate, confirmed that this receptor was of the type II class of somatomedin receptors and exhibited a molecular weight of 218,000 under nonreducing conditions.     

Biphasic effects of type beta transforming growth factor on epidermal growth factor receptors in NRK fibroblasts. Functional consequences for epidermal growth factor-stimulated mitosis

Exposure of confluent NRK cells to transforming growth factor-beta (TGF-beta) results in distinct alterations in subpopulations of plasma membrane epidermal growth factor (EGF) receptors. The low affinity sites increase in number, whereas the high affinity sites undergo a transient decrease in affinity followed by a prolonged increase in number. Cycloheximide inhibits both of these effects. Functional assays measuring EGF-stimulated thymidine incorporation in the presence of TGF-beta show that the resulting long-term stimulation of EGF receptor binding is associated with an increased sensitivity to EGF. Similarly, the initial, transient decrease in EGF binding is associated with a temporary inhibition of EGF-stimulated thymidine incorporation. The results describe a bifunctional effect of TGF-beta at the biochemical level consistent with the action of this peptide on NRK cell growth.     

Cytosine arabinoside increases the binding of125I-labelled epidermal growth factor and125I-transferrin and enhances the in vitro targeting of human tumour cells with anti-(growth factor receptor) mAb

We report that cytosine arabinoside (Ara-C), a cytosine analogue that at low doses causes phenotypical changes on human leukemia cells in vitro and in vivo, induces growth inhibition of oropharyngeal cancer KB and lung adenocarcinoma A549 cell lines. An increase in the number of epidermal growth factor and transferrin receptors (EGFR, TrfR) is induced by Ara-C on these cells. Maximal EGFR up-regulation occurs 96 h after the beginning of Ara-C exposure while maximal TrfR up-regulation is detected 24 h later. These effects occur without changes in the affinity of EGFR and TrfR for their ligands. Two classes of EGF-binding sites with aK _d of 0.055 nM and 2.3 nM respectively, and one class of transferrin-binding sites with aK _d of about 4 nM are detected on both untreated and Ara-C-treated KB cells. [³H]Thymidine uptake is clearly stimulated on KB cells by nanomolar concentrations of EGF and transferrin, whereas in Ara-C-treated cells [³H]thymidine uptake is not increased by EGF and transferrin under conditions where maximal EGFR and TrfR up-regulation occurs. The enhanced EGF and transferrin binding is paralleled by a twofold increase of in vitro targeting of Ara-C-treated KB and A549 cells with anti-EGFR 108.1 mAb and anti-TrfR OKT9 mAb. We propose that Ara-C could provide a new approach for the improvement of the therapeutic index of anti-EGFR and anti-TrfR immunoconjugates.This work has been supported by the Italian Association for Cancer Research (A.I.R.C.) and by the National Council of Research (C.N.R.) of Italy, contract 92.02274.PF39     

Specific binding sites for 125I-nerve growth factor in peripheral tissues and brain

Specific binding of ¹²⁵I-nerve growth factor (NGF), defined as that part of the total binding of the iodinated derivative displaced by 15–30 μg/ml native NGF, is found at significant levels in many peripheral tissues of chick embryos and rats. Destruction of the sympathetic innervation of tissues by treatment of newborn rats with guanethidine does not materially alter the ¹²⁵I-NGF specific binding capacity of tissues, indicating that these binding sites for NGF are part of the tissues themselves and not a property of the sympathetic nerve terminals which innervate them. Specific binding of ¹²⁵I-NGF which is also resistant to guanethidine treatment exists in chick embryonic and rat brain. The time course of the development of this specific binding in chick embryonic heart and brain suggests a developmental role for these peripheral and central nervous system NGF binding sites.     

High affinity, specific binding of [125I] beta nerve growth factor to glass beads

The binding of [¹²⁵I] β nerve growth factor to glass beads was studied. It was found that [¹²⁵I] β nerve growth factor exhibited high affinity, specific binding to glass beads. This binding cannot be explained as radioactivity being occluded in the spaces between the glass beads. The binding appears to be nonsaturable under the conditions used. Binding is complete in less than ten minutes with a half-time of two minutes. The binding appears similar to that seen for receptor binding on responsive cells.     

Characterization of the rebinding of 125I-epidermal growth factor released from BALB/c-3T3 cells following accumulation in the presence of chloroquine

Lysosomotropic amines, such as chloroquine and methylamine, increase the intracellular accumulation of 125I-EGF by inhibiting lysosomal degradation. It has been shown previously that BALB/c-3T3 cells, prelabeled at 4 degrees C with 125I-EGF for 3 h and subsequently chased at 37 degrees C in the presence of chloroquine, internalized the surface bound 125I-EGF which was subsequently released into the extracellular medium in a high molecular weight form which co-migrated with native 125I-EGF. The secreted 125I-EGF rebound to the cells from which it was released more efficiently than does peptide in the extracellular media. We now show that when the BALB/c-3T3 cells were prelabeled at 37 degrees C for 2 h in the presence of chloroquine, the internalized 125I-EGF released into the medium was in a high molecular weight form which co-migrated with native 125I-EGF and did not rebind anymore efficiently than did peptide in the extracellular media. This lack of rebinding was not due to an alteration in the 125I-EGF molecule since it was still capable of rebinding to naive A431 cells, nor was it due to the exhaustion of EGF receptors on the BALB/c-3T3 cells. The inhibition of rebinding was observed only when the cells were treated with EGF in the presence of chloroquine, and was not due to a general down-regulation of membrane receptors. The differences between the rebinding of 125I-EGF at 4 degrees C and 37 degrees C suggest that EGF may be processed via different pathways in the cell.     

Biphasic effects of dexamethasone on growth hormone release in vitro

The effect of dexamethasone (Dex) on growth hormone (GH) release was examined in vitro in monolayer culture of normal rat pituitary cells and human somatotropinoma cells from patients with acromegaly. In either cell strain, Dex, at a concentration of 50 nM initially inhibited, but later (48 less than or equal to h) potentiated, the release of GH into the medium, with or without growth hormone releasing hormone (GHRH). The intracellular GH was significantly increased by 4-hour incubation with Dex in rat cell cultures. These results indicate a biphasic effect of glucocorticoids on GH release, irrespective of the origin of somatotrophs, and that the initial inhibitory effect is probably caused by inhibition of the release.     

Overexpression of midkine contributes to anti-apoptotic effects in human meningiomas

Meningiomas are the second most common intracranial tumours. Most meningiomas grow slowly; however, atypical and anaplastic meningiomas show an aggressive biological behaviour. Overexpression of growth factors is considered to be a cause of carcinogenesis. Midkine and pleiotrophin are heparin-binding growth factors that promote growth, survival, migration and differentiation of various target cells. Both molecules are highly expressed during human embryogenesis but are rarely seen in the adult. We show that in relation to normal dura and arachnoid tissues, midkine was overexpressed in meningiomas on the mRNA and protein level, whereas pleiotrophin was not. Thereby, not only the intact but also the truncated form of midkine could be observed. The expression of midkine receptors was variable in different samples. Midkine stimulation of cultured meningioma cells induced phosphorylation of Akt, whereas no increase in phosphorylation of p42/44 MAPK or p38 MAPK could be detected. Midkine did not influence the proliferation of meningioma cells in vitro, but it did protect meningioma cells from camptothecin-mediated apoptotic cell death through reduction in the amounts of active caspase-3. These findings provide evidence for the overexpression of midkine in meningiomas which contributes to protection from cell death in these second most common intracranial tumours.     

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.     

Ability of growth hormone fragments to compete with 125I-iodinated human growth hormone for specific binding to isolated adipocytes of hypophysectomized rats

Several noncovalent complexes of large fragments of human GH, which are less active than native human GH in stimulating glucose metabolism in adipose tissue of hypophysectomized rats, were tested for their ability to compete with ¹²⁵I-iodinated human GH for specific binding to isolated adipocytes of hypophysectomized rats. The complexes tested were A (residues 1–134 + residues 141–191; S-carbamidomethylated), B (residues 1–134 + residues 135–191; S-carbamidomethylated) and C (residues 1–134 + residues 135–191; S-carboxymethylated). When compared to native human GH, the complexes were less active in competing with ¹²⁵I-iodinated human GH for specific binding to adipocytes, and their order of potency in the binding assay (A > B > C) was similar to that of their respective activities in stimulating glucose metabolism in isolated adipose tissue of hypophysectomized rats.