Involvement of multiple subcellular compartments in intracellular processing of epidermal growth factor

The intracellular translocation and processing of epidermal growth factor (EOF) in 3T3 cells has been studied utilizing Percoll density gradients. EGF is internalized and rapidly becomes associated with two types of intracellular compartments. The extent to which EGF is delivered to these two compartments is apparently regulated depending upon the cell's physiological condition. In growth medium, an increased proportion of EGF is taken up into a Golgi-like element. Uptake through this pathway correlates with a decrease in degradation of the ligand. In the absence of scrum and amino acids, an increased proportion of EGF is taken up into a component which has a density of 1.05. Uptake through this pathway correlates with increased degradation of the ligand. The ligand taken up through both pathways is transferred to dense vesicles which comigrate with lysosomes. In the presence of growth medium, however, dense vesicles containing EGF can be shown to be lysosomal enzyme-deficient upon further fractionation. In addition, in the presence of serum, a portion of the internalized EGF is apparently released from the cells, intact, and then re-bound. The processes described may be important in the production of a mitogenic response and the ability of cells to self-regulate their responsiveness to the growth factor.     

Mitogenic action of tumor necrosis factor in human fibroblasts: interaction with epidermal growth factor and platelet-derived growth factor

We have previously shown that tumor necrosis factor (TNF) can increase the number of epidermal growth factor (EGF) receptors on human FS-4 fibroblasts and that this increase may be related to the mitogenic action of TNF in these cells. Here we show that TNF stimulated the growth of FS-4 fibroblasts in a chemically defined, serum-free medium in the absence of EGF. Anti-EGF receptor antibody, which blocked the mitogenic effects of EGF in FS-4 cells, did not inhibit the mitogenic action of TNF in serum-free or serum-containing medium, indicating that EGF or an EGF-like molecule was not responsible for the mitogenic effects of TNF. However, the simultaneous addition of TNF and EGF to cells grown in serum-free medium resulted in a synergistic stimulation of DNA synthesis and cell growth. The actions of TNF and EGF were also examined in growth-arrested FS-4 cells and were compared with the action of platelet-derived growth factor (PDGF). In the absence of other growth factors, TNF was a relatively weak mitogen in growth-arrested cells, compared with EGF or PDGF. Nevertheless, TNF synergized with EGF or high doses of PDGF in stimulating DNA synthesis. Furthermore, antibodies specific for TNF or the EGF receptor were used to selectively inhibit the actions of these two factors, after specific incubation periods, in growth-arrested cells treated concurrently with EGF and TNF. To produce an optimal stimulation of DNA synthesis, EGF had to be present for a longer period of time than TNF. We conclude that in their synergistic action on growth-arrested FS-4 cells, EGF was responsible for driving the majority of the cells into S phase, while TNF appeared to make the cells more responsive to the mitogenic action of EGF. The findings indicate that TNF can cooperate with, and enhance the actions of, EGF in promoting DNA synthesis and cell division.     

Effect of recombinant IGF binding protein-1 on primary cultures of human keratinocytes and fibroblasts: selective enhancement of IGF-1 but not IGF-2-induced cell proliferation.

The present report describes the mitogenic effect of recombinant IGF-2 on cultured human keratinocytes and fibroblasts compared to that of IGF-1. Furthermore, the modulating effect of a recently expressed recombinant form of placental-derived IGF-binding protein 1 (IGFBP-1) on IGF-induced proliferation was examined. A dose-dependent increase, up to 100%, in cell proliferation was seen in cultured human keratinocytes with IGF-2 and -1 and the proliferative response was comparable to the effect of epidermal growth factor (EGF). In human fibroblasts, IGF-1 stimulated DNA synthesis up to 300% for IGF-1 and up to 200% for IGF-2. The mitogenic effect of IGF-1 was enhanced by IGFBP-1 in both cell types. In contrast, the IGF-2-induced mitogenic effect was unperturbed. These findings indicate that the interaction between IGFs and their binding proteins may induce different responses depending upon the ligand and the target cell.     

Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: The endocytic activation hypothesis

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity ¹²⁵I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of ³H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6–8 h period as an obligatory step in production of “second messenger” in the action of this hormone.     

A nonmitogenic analogue of epidermal growth factor induces early responses mediated by epidermal growth factor

Cyanogen bromide-cleaved epidermal growth factor (CNBr-EGF) binds to EGF receptors with reduced affinity compared to the native hormone but fails to induce DNA synthesis. However, at similar receptor occupancy, CNBr-EGF is as potent as EGF in activating early cell responses to the hormone. The phosphorylation of membrane proteins, the stimulation of Na+-K+-ATPase as reflected by the ouabain-sensitive uptake of 86Rb of fibroblasts, changes in the organization of microfilaments and in cell-morphology, and the activation of the enzyme ornithine-decarboxylase are all induced by CNBr-EGF as well as EGF Our results are consistent with the notion that EGF-induced phosphorylation could act as a "second messenger" for the action of various EGF-induced responses such as activation of Na+-K+-ATPase, changes in the cytoskeleton and cell morphology, and the activation of the enzyme ornithine decarboxylase. However, the stimulation of phosphorylation of membrane proteins and other early responses are either not required or necessary but insufficient for the induction of DNA synthesis. Suboptimal concentrations of EGF together with CNBr-EGF stimulate DNA synthesis in human fibroblasts. Other growth factors such as insulin, fibroblast growth factor, and prostaglandin F2 alpha, which potentiate the mitogenic response of EGF, do not effect the response to CNBr-EGF. This suggests that the restoration of the mitogenic properties of CNBr-EGF by suboptimal doses of EGF occurs at the level of EGF receptors or during their processing.     

Hyperthermia can enhance the initiation of DNA synthesis stimulated by growth factors in Swiss mouse 3T3 cells

Confluent quiescent Swiss mouse 3T3 cells can be stimulated to initiate DNA synthesis and to divide by epidermal growth factor (EGF) and prostaglandin F2 alpha (PGF2 alpha), two mitogens of unrelated structure. Heat treatment at 46 degrees C for up to 20 min of confluent quiescent cells, which has no mitogenic effect, can enhance the stimulatory effect of suboptimal concentrations of EGF or PGF2 alpha on the initiation of DNA synthesis. Furthermore, insulin, which is not mitogenic in these cells, enhances the effect of these mitogens, but this effect is not further enhanced by heat treatment. Likewise the combination of EGF and PGF2 alpha is synergistic on DNA synthesis, and this effect is also not enhanced by the heat treatment. Incubation at 46 degrees C for longer than 20 min was inhibitory in all cases. These results suggest that heat treatment induces events which affect the regulation of the initiation of DNA synthesis in a manner depending on the duration of the heat treatment and the stimulation of the cells.     

A comparison of the responses of cultured myoblasts and chondrocytes to fibroblast and epidermal growth factors.

The effects of fibroblast and epidermal growth factors on proliferation and differentiation of cultured myoblasts and chondrocytes have been compared. FGF stimulated myoblast proliferation, as determined by monitoring levels of DNA synthesis during seven days growth in vitro and by the morphology of the cultures after myotube formation. EGF has relatively little effect on myoblast proliferation. With chondrocytes, both FGF and EGF are mitogenic and FGF's, but not EGF's effect is potentiated by dexamethasone. One implication of these results is that in the course of differentiation cell types which develop from the same embryonic origin as fibroblasts are controlled by different sets of mitogenic factors. Myoblasts become primarily dependent on mitogenic agents such as FGF while chondrocytes can respond to both FGF and EGF.     

Reinitiation of DNA synthesis in quiescent mouse keratinocytes; Regulation by polypeptide hormones,cholera toxin,dexamethasone, and retinoic acid

Summary Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration <0.1 mM, supplemented only with insulin and transferrin. In this quiescent state, greater than 95% of the cell population is in the G_0/1 phase of the cell cycle. Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity. Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA. Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone. In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone. A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells. Finally, transforming growth factor type beta (TGFβ) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC₅₀∶10pM). This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells. In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGFβ. Supported by PHS Award CA-41556 from the National Cancer Institute, Bethesda, MD.     

Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways

The synthesis of specific protein has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr approximately 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid-G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell-cycle-dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr approximately 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase-delta. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S-phase. These data support the view that the cAMP-mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1-phase.     

Intracellular degradation of asialoglycoproteins in hepatocytes starts in a subgroup of lysosomes

Isolated rat hepatocytes take up and degrade [125I]tyramine-cellobiose-labelled asialofetuin [( 125I]TC-AF). The labelled degradation products are trapped at the site of degradation. The intracellular transport of [125I]TC-AF was studied by means of cell fractionation in Nycodenz gradients. The labelled ligand was kept in a small, slowly sedimenting vesicle during the first minutes after uptake in the cells, and was then transferred to a larger endosome. Labelled degradation products first appeared in an organelle with the same density distribution as the larger endosome and then in a denser organelle. These observations suggest that two types of lysosome, 'light' lysosomes and 'dense', are sequentially involved in the degradation of the asialoglycoprotein. The bulk of the lysosomal enzymes is associated with the dense lysosome.     

Inhibition by epidermal growth factor (EGF) of epidermal DNA synthesis in cultured chick embryonic skin pretreated with retinol and/or hydrocortisone: specific increment in EGF binding activity in both retinol- and hydrocortisone-pretreated epidermis without correlation to EGF-mediated inhibition of cell growth.

When tarsometatarsal skin of 13-day-old chick embryos that had been cultured in medium containing 5% delipidized FCS with or without retinol (20 microM) and/or hydrocortisone (20 nM) for 1 day was cultured in a chemically defined medium without either the hormone or retinol for 1 day, epidermal DNA synthesis of hydrocortisone- and/or retinol-pretreated skin was inhibited when compared to that of control skin. The addition of epidermal growth factor (EGF, 10 ng/ml) to retinol- or hydrocortisone-pretreated skin further inhibited the epidermal DNA synthesis. Epidermal DNA synthesis in retinol- and hydrocortisone-pretreated skin was more strongly inhibited than in retinol- or hydrocortisone-pretreated skin, but was not further inhibited by EGF. In epidermis which was induced to differentiation toward keratinization by hydrocortisone or mucous metaplasia by retinol, EGF inhibited DNA synthesis. The extent of [125I]-EGF binding to the epidermis of retinol- and hydrocortisone-pretreated skin was 160-180% that in control skin, with no change in affinity. Hence there is no correlation between EGF-binding and the mitogenic activity of EGF.     

The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments

Cytolytic lymphocytes contain specialized lytic granules whose secretion during cell-mediated cytolysis results in target cell death. Using serial section EM of RNK-16, a natural killer cell line, we show that there are structurally distinct types of granules. Each type is composed of varying proportions of a dense core domain and a multivesicular cortical domain. The dense core domains contain secretory proteins thought to play a role in cytolysis, including cytolysin and chondroitin sulfate proteoglycan. In contrast, the multivesicular domains contain lysosomal proteins, including acid phosphatase, alpha-glucosidase, cathepsin D, and LGP-120. In addition to their protein content, the lytic granules have other properties in common with lysosomes. The multivesicular regions of the granules have an acidic pH, comparable to that of endosomes and lysosomes. The granules take up exogenous cationized ferritin with lysosome-like kinetics, and this uptake is blocked by weak bases and low temperature. The multivesicular domains of the granules are rich in the 270-kD mannose-6-phosphate receptor, a marker which is absent from mature lysosomes but present in earlier endocytic compartments. Thus, the natural killer granules represent an unusual dual-function organelle, where a regulated secretory compartment, the dense core, is contained within a pre-lysosomal compartment, the multivesicular domain.     

Transmembrane delivery of polypeptide growth factors bypassing the intrinsic cell surface receptors: synthesis and biological activity of a conjugate of epidermal growth factor with alpha 2-macroglobulin

Epidermal growth factor (EGF) was derivatized at the amino terminus with N-succinimidyl 3-(2-pyridyldithio)propionate and then cross-linked to the cysteinyl residues of alpha 2-macroglobulin (alpha 2M) via disulfide bonds. The EGF-alpha 2M conjugate delivered EGF into dense lysosomal fractions through binding to alpha 2M receptors in a variant of mouse Swiss/3T3 fibroblasts, NR-6, which are deficient in EGF receptors. The conjugate stimulated DNA synthesis in Swiss/3T3 cells, but it did not stimulate DNA synthesis in NR-6 cells. This differential stimulation was due to the conjugate's binding to EGF receptors since bacitracin, which completely inhibits [125I]alpha 2M binding to its receptors, inhibited conjugate binding by approximately 80%. Thus, EGF bound to and internalized through alpha 2M receptors does not function as a mediator for DNA stimulation. The mechanisms of action of the conjugate are discussed in relation to the role of receptor-mediated endocytic pathways.     

Secreted proteins induced by epidermal growth factor and transforming growth factor beta in EL2 rat fibroblasts. Role in the mitogenic response

Most growth active hormones and peptides are mitogenic only in the presence of other growth factors [e.g., Platelet Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) in "competence-progression" fibroblast model]. We have previously described that EGF alone is able to induce the signals which appear necessary for the mitogenic stimulation of EL2 rat embryo fibroblast line. Recently, we have demonstrated that Transforming Growth Factor beta (TGF beta) slightly stimulates the mitogenic response in EL2 cells. Here, we show that in EGF-treated EL2 cells the induction of at least four inducible-secreted proteins (ISPs, range from 29,000 to 68,000 Mr) is accompanied by a marked increase in DNA synthesis. In contrast, TGF beta or different concentrations of EGF induce a slow increase of the ISPs proportional to slow induction in DNA synthesis. Our results suggest that the mitogenic response in EL2 cell line may be connected with the qualitative and quantitative induction of these secreted proteins.     

Further evidence for a cell surface proteinase essential to the growth of cultured fibroblasts

Specific antibodies and protein proteinase inhibitors will inhibit cell-surface proteinase activity on human fibroblasts and cause a concomitant inhibition of DNA synthesis and of cell multiplication. An insolubilized proteinase inhibitor also inhibits cell multiplication. The same reagents partially inhibit the multiplication of mouse L cells, both in monolayer and suspension culture, and inhibit the mitogenic effect of epidermal growth factor (EGF) on both types of cell.     

Effect of cell-to-cell contact on in vitro deoxyribonucleic acid synthesis and apoptosis responses of bovine granulosa cells to insulin-like growth factor-I and epidermal growth factor

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)-I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA + sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [(3)H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of cell-cell contact was a major factor reducing cell proliferation and inducing apoptosis among both subsets of GC.     

Synthesis and migration of glycoproteins in cells of the rat thymus, as shown by radioautography after 3H-fucose injection

Young male rats received a single intravenous injection of 3H-fucose and were killed after various time-intervals. Light- and electron-microscopic radioautographic studies of the thymus in animals killed shortly after injection showed that all of the different cell types present incorporated 3H-fucose label. The heaviest uptake occurred in macrophages and in hypertrophic epithelial cells located near the cortico-medullary border. Somewhat lighter incorporation was observed in medullary and cortical stellate epithelial cells and in cells designated as special cells, while the lightest reaction appeared over lymphocytes. In all cells the label was localized initially to the Golgi apparatus, where, presumably, it was incorporated into glycoproteins. With time, some of the labeled putative glycoproteins in all cell types migrated to the plasma membrane. In macrophages, much of the label migrated to lysosomal bodies, while in the special cells the label migrated to dense bodies which may also be of lysosomal nature. In stellate and hypertrophic epithelial cells much of the label migrated to characteristic vacuoles. The possible relationship between the observed glycoprotein synthesis in these cells and hormone production is discussed.     

Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

Control of proliferation of bovine vascular endothelial cells.

The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.