Comparison of insulin-like growth factor 1 and insulin effects on prolactin-induced lactogenesis in the rabbit mammary gland in vitro

In organ culture of pregnant rabbit mammary gland, a low casein synthesis occurs with prolactin (PRL) alone. Insulin markedly potentiates the effect of PRL. Only pharmacological concentrations of insulin (5 micrograms/ml) exert the maximal enhancement, suggesting a possible interaction with the insulin-like growth factor 1 (IGF1) receptor. The presence of IGF1 and insulin binding sites was analyzed and the biological effects of both peptides were compared. Binding of iodinated human IGF1 or porcine insulin to mammary microsomes prepared from mid-pregnant rabbits revealed distinct high affinity binding sites for both peptides (Kd approximately 2 nM). In rabbit mammary explants, we confirmed that only non-physiological concentrations of insulin (greater than or equal to 100 ng/ml) exerted a significant stimulation of the PRL effect. Surprisingly, IGF1 was not found to be more potent than insulin on a molar basis, which did not provide evidence for the exclusive involvement of the IGF1 receptor. Near-physiological concentrations of IGF1 (approximately 100 ng/ml), however, exerted a significant enhancement which suggested a possible action for IGF1 on PRL-induced lactogenesis in vivo.     

Specificity and mechanism of biphasic action of glucocorticoids on alpha-lactalbumin production by rat mammary gland explants

RU26988 [11 beta, 17 beta-dihydroxy-17 alpha-(1-propynyl)androsta-1,4,6-trien-3-one], a specific type II (glucocorticoid) ligand with negligible affinity for type I (mineralocorticoid) receptors, increased alpha-lactalbumin production in mammary gland explants from midpregnant rats when cultured in vitro in the presence of insulin (5 micrograms/ml) and rat PRL (1 microgram/ml). The dose-response curve was biphasic, over a 3-300 nM dose range, with maximum at 3-10 nM, followed by a progressive decline toward or to control levels at 300 nM. A similar dose-response curve was obtained with another type II-specific ligand, RU28362 [11 beta, 17 beta-dihydroxy-6-methyl-17 alpha-(1-propynyl)androsta-1,4,6-trien-3-one], and with dexamethasone. This biphasic response had been previously reported for mouse mammary gland explants using cortisol, which binds to both type I and type II receptors. Our experiments, therefore, show that occupancy of type II receptors alone can be responsible for the stimulation at low steroid concentrations, followed by the decrease from peak levels observed at higher steroid concentrations. On the basis of these data, we propose a model to account for these findings, based on ligand binding to a single class of receptors and the putative existence of both turn-on and turn-off glucocorticoid regulatory elements in the nucleus.     

Adrenergic effects on exocrine secretion of rat submandibular epidermal growth factor

The present study was undertaken to investigate the effect of alpha- and beta-adrenergic agonists on secretion of epidermal growth factor (EGF) from the rat submandibular glands and to test the possibility of intestinal absorption of EGF. Alpha-adrenergic agonists increased the concentration of salivary EGF by approximately a hundred times, while the serum concentration of EGF was unchanged. The contents of EGF in the submandibular glands decreased upon administration of the alpha-adrenergic agonist noradrenaline, and this was confirmed on immunohistochemical investigation of the glands. Beta-adrenergic agonists had no effect on secretion of EGF from the submandibular glands. Intestinal absorption of EGF could not be confirmed, as stimulation by noradrenaline with free passage of saliva to the gastrointestinal tract and intrajejunal infusion of EGF had no influence on the concentration of EGF in serum. This study shows that alpha-adrenergic agonists stimulate exocrine secretion of submandibular EGF and that EGF in physiological amounts are not absorbed in the gastrointestinal tract.     

Receptors for epidermal growth factor in the rat uterus

Uterine membranes contain high affinity [dissociation constant (Kd) = 0.36 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I] iodo-EGF is specific since it is abolished by excess unlabeled EGF but not by excess unlabeled insulin, fibroblast growth factor, or multiplication-stimulating activity. Incubation of [125I] iodo-EGF with uterine membranes, followed by chemical cross-linking with disuccinimidyl suberate and detergent extraction reveals a major species of specifically bound EGF (mol wt = 170,000) and a minor species (mol wt = 150,000) visualized by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the extracts. In detergent-solubilized preparations EGF also stimulates the phosphorylation of major (mol wt = 170,000) and minor (mol wt = 150,000) species of identical molecular weight. The increased phosphorylation produced by incubation of membrane extracts with EGF occurs largely at tyrosine residues, as indicated by phosphoamino acid analysis. These results indicate that the rat uterus contains high affinity EGF binding sites with the properties expected of EGF receptors.     

A sensitive enzyme immunoassay system of rat epidermal growth factor in biological fluids and tissue extracts

Rat epidermal growth factor was purified from rat submandibular glands to obtain specific antiserum for the establishment of an immunoassay system. Purified rat epidermal growth factor showed a single peak on reverse phase HPLC and a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis at mol wt 5100 in the presence of 2-mercaptoethanol and at mol wt 41,000 in the absence of 2-mercaptoethanol. Antibody against rat epidermal growth factor showed cross-reactivities with mouse and human epidermal growth factors on soft agar-double immunodiffusion test. The established sandwich enzyme immunoassay for rat epidermal growth factor had a high sensitivity (500 fg/tube), which made it possible to measure minute amounts of endogenous rat epidermal growth factor without pretreatment. Physiological concentrations of rat epidermal growth factor in rat biological fluids and tissues were determined. Species differences in physiological distributions of epidermal growth factor are discussed.     

Distribution of epidermal growth factor receptors in the rat ovary

The distribution of epidermal growth factor (EGF) was studied in the ovary using light microscope radioautography which was performed at different time intervals (2-60 min) after intravenous (i.v.) injection of [125I]EGF into adult rat at random stages of the estrous cycle and also after topical localization of iodinated EGF on slide-mounted frozen ovarian sections. After i.v. injection of label, the labeling was mostly observed in the theca interna cells of secondary, preovulatory and atretic follicles and luteal cells of corpus luteum. Primordial and primary follicles did not show any significant labeling. The time-course study performed on luteal cells showed that, 2 min after injection, most silver grains were found at the periphery of the cells. At the 10 min time interval, silver grains were found both at the periphery and over the cytoplasm of these cells. The number of grains was very reduced over the cytoplasm at the 60 min time interval. In the in vitro study, a positive radioautographic reaction was seen in the same cellular elements as found in vivo, with the additional labeling of the granulosa cells of growing and preovulatory follicles. Control experiments indicated that the radioautographic labeling was due to specific interaction of [125I]EGF with its receptor. These results clearly indicate that EGF binding sites are present in luteal, thecal and granulosa cells, and provide support for the inhibitory and stimulatory actions of EGF on different parameters of ovarian cells.     

Epidermal growth factor and insulin-like growth factor I upregulate the expression of the epidermal growth factor system in rat liver

BACKGROUND/AIM: Both epidermal growth factor and insulin-like growth factor I play a role in connection with the liver. In the present study, the possible interaction of these two growth factor systems was studied by investigating the effect of epidermal growth factor or insulin-like growth factor I treatment on the expression of the epidermal growth factor receptor, and its activating ligands, transforming growth factor-alpha and epidermal growth factor. METHODS: Fifty-five male rats received no treatment, human recombinant epidermal growth factor or human recombinant insulin-like growth factor I for either 3 or 7 days. The amount of epidermal growth factor receptor, transforming growth factor-alpha, and epidermal growth factor mRNA was quantitated by a calibrated user-friendly RT-PCR assay (CURT-PCR), and the expression of transforming growth factor-alpha and epidermal growth factor peptides was quantitated by ELISA. RESULTS: Control liver (n=16) contained a mean (+/-SD) value of 12.7+/-7.4x10(-18) mol epidermal growth factor receptor mRNA, 3.8+/-2.0x10(-18) mol transforming growth factor-alpha mRNA and 0.8+/-0.4x10(-18) mol epidermal growth factor mRNA per microg total RNA and 9.8+/-1.6 fmol/mg protein epidermal growth factor and 144+/-22 fmol/mg protein transforming growth factor-alpha. Both epidermal growth factor and insulin-like growth factor I treatment increased the expression of mRNA for transforming growth factor-alpha and epidermal growth factor receptor, as well as the expression of transforming growth factor-alpha peptide. The level of epidermal growth factor receptor and transforming growth factor-alpha mRNA expression was found to correlate both in control and growth factor-treated animals, whereas the expression of epidermal growth factor receptor and epidermal growth factor showed no correlation. Marked differences were seen upon activation of the two growth factor systems, as epidermal growth factor, but not insulin-like growth factor I treatment, increased the plasma concentration of urea and decreased the concentration of insulin-like growth factor I and the liver enzymes, alanine aminotransferase and alkaline phosphatase. CONCLUSION: Our results show that epidermal growth factor and insulin-like growth factor I, which belong to two different growth factor systems, both induce a correlated upregulation of transforming growth factor-alpha and epidermal growth factor receptor mRNA in rat liver. Although marked differences were observed after treatment with either epidermal growth factor or insulin-like growth factor I on the liver as reflected in the plasma concentrations of e.g. liver enzymes, a common motif in their action involves an upregulation of the expression of the epidermal growth factor system.     

Comparative effects of epidermal growth factor, an insulin-glucagon combination, and a hepatocyte growth factor preparation on epidermal growth factor receptors.

We investigated the changes in cell surface epidermal growth factor (EGF) receptors in the liver after partial hepatectomy, and in primary adult rat hepatocyte cultures following stimulation with either EGF, or a preparation of hepatocyte growth factor, or an insulin-glucagon combination. We confirmed a reduction in EGF receptors on hepatocytes after partial hepatectomy and a rapid down-regulation of EGF receptors on normal hepatocytes in vitro following exposure to EGF. Insulin and glucagon and hepatocyte growth factor, whilst initiating hepatocyte DNA synthesis, had only slight effects on their EGF binding capacity and EGF-receptor affinity. These results indicate that changes in cell membranes early in proliferation have only non-specific effects on EGF receptors, and, therefore, support the role of ligand binding to the EGF receptor as an important component of hepatocyte proliferation in vivo.     

Antibodies to the epidermal growth factor receptor block the biological activities of sarcoma growth factor.

The role of the epidermal growth factor (EGF) receptor system in mediating the biological activities of sarcoma growth factor (SGF) has been assessed by using specific anti-EGF receptor antibodies. There are two classes of anti-EGF receptor antibodies, those that block binding of 125I-labeled EGF (125I-EGF) and those that do not block binding but do interact with a portion of the EGF receptor on the surface of intact cells. Antisera of both types have been assayed for their capacity to affect the biological activities of SGF. The antisera that block 125I-EGF binding to its receptor block the induction of DNA synthesis in human fibroblasts by either EGF or SGF but not by other polypeptide mitogens. Titration of the anti-EGF receptor antiserum indicates the presence of one population of antibody that blocks the site of both EGF and SGF action. Antisera to the EGF receptor that block 125I-EGF binding also inhibited the SGF-dependent anchorage-independent growth of normal cells in soft agar. The antisera to the EGF receptor that does not block 125I-EGF binding or EGF activity did not inhibit any of the biological activities of SGF. The results suggest that occupation of the EGF receptor is required for both the mitogenic and colony-forming activity of SGF.     

胃癌患者血清表皮生长因子的变化

目的探讨人血清表皮生长因子（ｈＥＧＦ）水平与胃癌的关系．方法胃癌患者３３例，男３０例，女３例，年龄３５岁～８１岁，平均６４９岁；慢性胃炎患者３８例，男２５例，女１３例，年龄１８岁～７５岁，平均４３２岁；正常人２０例，男１０例，女１０例．应用ＲＩＡ法检测血清中的ｈＥＧＦ．结果胃癌组血清ｈＥＧＦ（μｇ／Ｌ）为１３５±０６７，正常对照组为０９０±０３３，统计学差异非常显著（ｔ＝２８２５，Ｐ＜００１），慢性胃炎组血清ｈＥＧＦ为０８６±０３３，与正常对照组比较差异无显著性（ｔ＝０４１，Ｐ＞００５）．结论胃癌患者血清ｈＥＧＦ水平升高可能与胃癌发生密切相关．     

Progesterone regulates epidermal growth factor receptor on mucous secreting cells in the rabbit endocervix

During postnatal differentiation, epidermal growth factor (EGF) receptor is expressed by all major cell types of the cervix. Computer-assisted image analysis confirmed the highest concentration of EGF receptor is in the epithelial cells. Flow cytometric analysis of subpopulations of epithelial cells from estrous rabbits showed the mucous secreting cells had the highest concentration of EGF receptor, i.e. 1-1.5 x 10(5) receptors per cell. Because the mucous secreting cells are targets for steroid hormones it seemed likely that steroids regulate EGF receptor expression. To investigate this possibility, hormone-dependent changes in EGF receptor expression were quantified by flow cytometry. Ovariectomy and the treatment of ovariectomized animals with estradiol altered forward angle light scatter and side scatter signals which correlated with cell size and secretory granule content, respectively. However, the number of epithelial cells and the number of EGF receptors per cell were unaffected. Progesterone treatment of ovariectomized animals dramatically reduced the number of EGF receptors on the mucous secreting cells, accounting for a 43% reduction in the total EGF receptor content of the epithelium. The treatment of neonates with diethylstilbestrol did not change the number of EGF receptors in endocervical epithelial cells when examined in adulthood. However, the number of mucous secreting cells was decreased, thereby reducing the EGF receptor content of the epithelium 19-36% compared to estrous and estradiol-treated animals. These results provide the first evidence that progesterone regulates EGF receptor on mucous secreting cells in the endocervix and that diethylstilbestrol treatment alters the EGF receptor content of the epithelium by altering its cellular composition.     

Prolactin-dependent accumulation of alpha-lactalbumin in mammary gland explants from the pregnant tammar wallaby (Macropus eugenii)

The minimal hormonal requirements for the in-vitro accumulation of alpha-lactalbumin have been investigated in a marsupial, the tammar (Macropus eugenii). Mammary gland explants from 24-day pregnant tammars cultured in medium containing bovine insulin, cortisol and ovine prolactin showed a progressive increase in accumulation of alpha-lactalbumin during 4 days of incubation. No increment was observed if prolactin was omitted from the medium. However, a similar rate of increase was observed after 3 days of culture in medium containing prolactin alone. This induction of alpha-lactalbumin was maximal at a prolactin concentration of approximately 0.02 mg/l, which corresponds to physiological levels during pregnancy and early lactation. The absence of an effect of bovine insulin on tammar explants is not due to a general unresponsiveness to this hormone since insulin-stimulated DNA synthesis and amino acid uptake was evident after 3 days of culture. The inclusion of tri-iodothyronine and raised concentrations of cortisol in culture media have been shown to modulate alpha-lactalbumin synthesis in eutherian mammals but were without effect in the tammar. In addition, increased levels of progesterone did not inhibit the induction of alpha-lactalbumin, confirming an earlier in-vivo study suggesting that progesterone withdrawal may not be the lactogenic trigger in this species. Thus the pregnant tammar is the only species yet described in which alpha-lactalbumin is induced maximally in vitro in response to a single hormone.     

Differential expression of heparin-binding epidermal growth factor-like growth factor in the rat ovary

We investigated the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors in the rat ovary to define the role of HB-EGF in the ovarian function. The expression pattern of HB-EGF mRNA and protein were studied by semi-quantitative RT-PCR and immuno-histochemistry using an antibody that was specifically stained for the precursor form of HB-EGF in naturally cycling rats and immature pseudo-pregnant rat models. The immuno-histochemical study showed that in naturally cycling rats, HB-EGF was expressed in most granulosa cells of early follicles and all the developing follicles but not in preovulatory follicles. This was supported by the semi-quantitative RT-PCR results in that the lowest level of HB-EGF mRNA during the estrous cycle was found in the evening of proestrous when the HB-EGF negative preovulatory follicles were most prominent. The results suggest that HB-EGF might be a mitogen for granulosa cells and down regulation of its expression may be necessary for the final maturation of follicles. In corpora lutea, luteal cells of older generation stained stronger than those of younger generation. Moreover, luteal cells of late luteal phase stained stronger than those of the mid and early luteal phases in the immature pseudo-pregnant rat models, indicating that the precursor form may be associated with death of luteal cells. Finally, of the two cognate receptors for HB-EGF, erbB1 was expressed in the rat ovary, but erbB4 was specifically not expressed in this organ. The spatial and temporal pattern of HB-EGF expression suggest that HB-EGF may an important local regulator of ovarian function and structure.     

Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor.

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A-431 cells and selected according to their ability to inhibit the binding of radiolabeled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerful tool to study the structure, processing, and mode of action of EGF receptors.     

Synergistic effects of bombesin and epidermal growth factor on cancers.

Bombesin and gastrin-releasing peptide act as autocrine mitogens in various cancers. Bombesin antagonist RC-3095 inhibited growth in some cancers and slowed the progression of premalignant lesions, possibly by down-regulating epidermal growth factor (EGF) receptors. Since the EGF receptor mitogen response involves tyrosine kinase stimulation, we tested the hypotheses that bombesin stimulates, and RC-3095 inhibits, phosphorylation; EGF and bombesin promote the phosphorylation of the same substrates; and EGF and bombesin act synergistically on phosphorylation. Therefore, in vitro assays for phosphorylation were performed in the presence or absence of EGF, bombesin, RC-3095, and combinations in samples derived from tumor, tissue surrounding tumor, cell lines, and normal and transforming tissue derived from the 9,10-dimethyl-1,2-benzanthracene-induced squamous cell lesions of the hamster cheek pouch. Bombesin increased, and RC-3095 decreased, phosphorylation in these samples. In the human hepatoma sample and surrounding tissue, these ligands altered the phosphorylation of the same substrates affected by EGF. EGF and bombesin stimulated phosphorylation synergistically in the hamster samples and the hepatoma. Bombesin-induced phosphorylation was greater in tissue surrounding the hepatoma, whereas RC-3095 was more effective in inhibiting phosphorylation in the hepatoma itself. This cancer, therefore, could be endogenously stimulated by gastrin-releasing peptide. These observations support the hypothesis that bombesin stimulates growth of tissues and tumors by amplifying the phosphorylation response to EGF. The growth inhibitory response to RC-3095, or other bombesin analogues, of individual tumors may be prognosed by in vitro phosphorylation assays using the samples from the patient's tumor.     

Effect of epidermal growth factor on the development of rat gastric mucosa

Epidermal growth factor (EGF) has been shown to stimulate the growth of adult rat gastric mucosa and to increase DNA synthesis of mouse small and large intestinal mucosa. This study examines whether EGF affects the functional and structural development of the rat gastric mucosa. Rats were injected with 20 micrograms/kg EGF three times/day for 5 days beginning on the 10th day after birth. A control group of animals received saline injections of identical volume. All rats were killed on day 15. EGF significantly increased the weight of the whole stomach and the DNA, RNA, and protein content of the oxyntic gland mucosa, but had no effect on the RNA/DNA ratio, or antral and serum gastrin levels. Two groups of similarly treated rats, were anesthetized with ether, pylorus-ligated, and injected with either saline or pentagastrin (250 micrograms/kg) after they had recovered from anesthesia. EGF-treated rats had significantly higher rates of basal acid secretion and pentagastrin-stimulated acid secretion than the saline-treated controls. EGF, however, did not alter basal or pentagastrin-stimulated pepsin secretion nor did it change mucosal pepsinogen content. These results indicate that EGF stimulates oxyntic mucosal growth in unweaned rats but that it does not lead to precocious maturation or functional development.     

Stimulatory effects of epidermal growth factor on steroidogenesis in Leydig cells

We studied the effects of epidermal growth factor (EGF) on steroidogenesis in freshly prepared Percoll-purified Leydig cells from prepubertal and adult rats and mice, and in interstitial cells from immature rats cultured in the presence or absence of LH. It is demonstrated that EGF directly stimulates the output of C19-steroids (testosterone and androstenedione) as well as C21-steroids (progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-hydroxypregn-4-en-3-one) in all systems studied. When combined with LH, EGF has little effect on freshly isolated cells but still stimulates steroidogenesis in cells cultured in the presence of LH. The strong inhibitory effects of EGF on androgen production that have been reported previously are only observed when cells that have lost part of their steroidogenic potential by prolonged culture in the absence of LH are acutely challenged with LH (or cholera toxin or dbcAMP) and EGF. Under the latter conditions EGF blocks the conversion of C21-steroids into C19-steroids. The stimulatory effects of EGF on androgen production are evident within the first hours of incubation and occur at ED50 values of 0.3 up to 2.5 ng/ml. They are not accompanied by any measurable change in the production of cAMP. The effects of EGF are compared to those of LHRH and those of SCF, a putative paracrine factor produced by Sertoli cells. Although there are many similarities between the effects of these three polypeptides on steroidogenesis in Leydig cells, our data indicate that they must act by different mechanisms. Moreover, SCF is the only one of these agonists that markedly stimulates androgen production in the presence of LH.     

Metabolism and effects of epidermal growth factor and related growth factors in mammals

The initial observations of Stanley Cohen in the 1960s established that EGF induced in vivo effects such as precocious eyelid opening and tooth eruption. Subsequently the actions of EGF have been extensively explored in cell culture systems. The receptor for EGF was characterized as a prototype model for other growth factors and the now extensive in vitro data indicate multiple functions for EGF. Moreover, EGF and EGF receptors have been characterized in many tissues, and EGF has been identified in most body fluids of several mammalian species. Interestingly, neither EGF antibody administration to newborn animals nor passive immunization of pregnant rodents against EGF has caused major deleterious effects (except the delay in epidermal maturation events), as might be expected from the in vitro studies. This is in contrast to the effects of nerve growth factor antiserum in developing rodents. Also, to date, no pathological EGF deficiency disorder has been characterized. However, the EGF family of growth factors appears to be important in mammalian development and function, although the precise roles and significance are not yet clear. Members of the family may have a role in embryogenesis and fetal growth since receptors have been identified in fetal tissues. Available evidence suggests that TGF alpha subserves the growth factor family roles in fetal development. In the developing postnatal animal pro-EGF mRNA, immunoreactive EGF, immunoreactive TGF alpha, and EGF receptors are present in many tissues. EGF also is produced and secreted by the maternal mammary gland, and mammary derived EGF appears to be important in gut development in the neonatal rodent. There is now extensive data to indicate important hormone-EGF interactions. In the postnatal period, thyroid and steroid hormones including retinoic acid have been shown to modulate EGF and/or EGF receptors in several tissues. GH increases EGF binding in liver and increases urine EGF concentrations. Moreover, EGF stimulates secretion of several hypothalamic and pituitary hormones, increases placental production of hCG and human chorionic somatomammotropin, increases adrenal cortisol production, and inhibits testicular, ovarian, and thyroid hormone secretions. As summarized in this review EGF has been implicated in a number of developmental events including palate and skin differentiation, growth of hair follicles, eye opening and tooth eruption, lung maturation, gut and liver growth, and differentiation of neurons. These EGF actions probably are mediated via autocrine, paracrine, and endocrine routes. A role for salivary and urine EGF in the maintenance of adult stomal, gut, and urinary epithelial surface integrity seems likely, although not yet proven.(ABSTRACT TRUNCATED AT 400 WORDS)