Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat lung cells is due to enhanced receptor synthesis

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung (FRL) cells is increased approximately 2 to 3-fold within 18 h of retinoic acid addition. Analysis of 125I-EGF binding assays at 0 C reveals approximately 25,000 receptors per cell, while analysis of growth factor binding to retinoic acid-treated cells demonstrates an increase in receptor levels to approximately 70,000 receptors per cell with no detectable changes in receptor affinities. We show by immunoprecipitation of 35S-methionine labeled EGF receptors that retinoic acid addition produces an increase in the accumulation of EGF receptor protein. Using brief pulses of 35S-methionine, an increase in EGF receptor synthesis can be identified within 3 h after retinoic acid addition. These results are the first to demonstrate that a retinoic acid-induced increase in 125I-EGF binding capacity is due to increased EGF receptor protein synthesis. Also, we find that a transient decrease in the rate of EGF receptor turnover occurs when retinoic acid is initially added to FRL cells. On the basis of our data, we conclude that the retinoic acid-induced accumulation of EGF receptors in FRL cells is primarily due to increased receptor synthesis. The effect of retinoic acid on EGF receptor turnover may be a secondary factor, influencing the rate at which receptors accumulate.     

Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA.     

EGF-induced PGE2 release is synergistically enhanced in retinoic acid treated fetal rat lung cells

Retinoic acid has been shown to induce a 2.5-fold increase in 125I-EGF binding capacity through increased EGF receptor synthesis in a fetal rat lung (FRL) cell line (1). In FRL cells, incubation with either EGF or retinoic acid induces a modest increase in PGE2 secretion (80% or 40%, respectively). However, in the presence of both EGF and retinoic acid, FRL cells exhibit a 6.4-fold increase in PGE2 secretion. Retinoic acid and EGF dose-response curves demonstrate that the effect on PGE2 secretion correlates with the retinoic acid induced increase in EGF receptors. These data suggest a relationship between increased EGF receptor expression and increased EGF responsiveness. Furthermore, these data indicate a potential mechanism by which EGF and retinoic acid may interact in lung physiology.     

Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions.

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.     

Endocytosis and intracellular transport of epidermal growth factor after destruction of the actin cytoskeleton by cytochalasin B

Endocytosis of the epidermal growth factor (EGF) was investigated in three cell lines--A431, 3T6 and Swiss 3T3--after their incubation with cytochalasin B (CB). CB was introduced into culture medium (10 mkg/ml) 1.5-2 hours before addition of 125I-EGF (20-40 ng/ml). The label uptake rate was measured after a 35-40 minutes incubation of cells with 125I-EGF. It appeared that disorganization of microfilamentous network caused by CB exerted no influence on the binding of EGF to the surface membrane receptors and its internalization. Nevertheless, the experiments performed on A431 cells using a fluorescent label--rhodamine--bound to EGF (EGF-R) indicate that CB, though not influencing the initial steps of endocytosis, inhibits the next step--the intracellular transport of EGF-receptor complexes from the trans-Golgi region to lysosomes. As was shown elsewhere (Barkan, Nikol'sky, 1986), CB inhibits the mitogenic effect of EGF on resting Swiss 3T3 cells. So, the process of EGF-receptor uptake and delivery to the trans-Golgi region is evidently not enough to stimulate the cell proliferation; next steps of transport and degradation of ligand-receptor complexes are presumably needed.     

Binding, internalization, and degradation of epidermal growth factor by balb 3T3 and BP3T3 cells: relationship to cell density and the stimulation of cell proliferation.

Epidermal growth factor (EGF) stimulates the growth of both benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) and untransformed Balb 3T3 cells. We describe here the binding, internalization, and degradation of [125I]-EGF by BP3T3 cells and 3T3 cells. Binding of [125I]-EGF reaches a maximum after 45 to 90 minutes incubation at 37 degrees C. In both BP3T3 and 3T3 cells the extent of EGF binding required to stimulate DNA synthesis is density dependent; sparse cultures require a 15-30% occupancy to elicit a maximal response whereas dense cultures require a 70-85% occupancy. At physiological concentrations the total binding of [125I]-EGF to 3T3 cells is higher than to BP3T3 cells, and this difference increases at higher cell densities. The rate of degradation of [125I]-EGF is directly proportional to the total [125I]-EGF binding in each cell type. This supports the hypothesis that one cause of the diminished serum requirement of BP3T3 cells is a reduced rate of utilization of serum growth factors.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Characterization of the action of retinoids on mouse fibroblast cell lines

Retinoic acid affects 3T6 and 3T3 cells by inhibiting growth, causing a morphological change and increasing cell-to-substratum adhesiveness. Retinoic acid does not exert such effects on virus-transformed 3T3SV cells. Retinoic acid treatment of 3T6 cells causes a concentration-dependent increase in generation time and a reduction in saturation density. Analysis of cell surface proteins shows that a high molecular weight band of 230 000 D, corresponding to the position of the LETS glycoprotein, is more intensely labeled by iodination of cells treated with retinoic acid compared to control cells. Retinoic acid substantially stimulates the incorporation of ³⁵SO₄ into cell-associated glycosaminoglycans and causes a less dramatic increase in glycosaminoglycans excreted into the medium. The relationship between the increase in these cell surface components and the enhanced adhesiveness is discussed. A retinoic acid binding protein is detectable in the cytosol of 3T6 and 3T3 cells but not in 3T3SV cells, suggesting that the action of retinoids on these cells is mediated via this protein.     

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of 125I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two types of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

The role of EGF receptor transmodulation in embryonal carcinoma-derived growth factor-induced mitogenesis.

Exposure of quiescent 10T1/2 fibroblast cells to embryonal carcinoma-derived growth factor (ECDGF) results in a rapid temperature and ECDGF concentration-dependent inhibition of [125I]EGF binding to the epidermal growth factor (EGF) receptor (transmodulation). ECDGF predominantly inhibits the association of [125I]EGF with a high affinity subclass of EGF receptors, and induces increased phosphorylation of the EGF receptor on serine and threonine residues. No mitogenic effect of EGF can be detected in the presence of ECDGF concentrations which induce maximal EGF receptor transmodulation. ECDGF-induced EGF receptor transmodulation is sensitive to phorbol ester-induced desensitization whereas ECDGF-induced DNA synthesis is unaffected by prolonged pre-treatment with biologically active phorbol ester. These findings suggest that EGF receptor transmodulation is not essential for ECDGF mitogenicity but may inhibit EGF-induced DNA synthesis.     

Retinoic acid modulates attachment of mouse fibroblasts to laminin substrates

The effect of retinoic acid treatment on cell attachment to plastic substrates precoated with fibronectin, gelatin, laminin, and type IV collagen was investigated. Both retinoic acid-treated and control cells attached efficiently to fibronectin or gelatin substrates without any significant difference. In contrast, retinoic acid-treated cells attached to laminin or type IV collagen substrates, while control cells showed little or no attachment. The minimal effective concentration of retinoic acid for pretreatment to yield a significant increase in the attachment assay was higher than 10(-8) M. The attachment of retinoic acid-treated cells to laminin substrates reached a maximum at 60 min, while that to type IV collagen substrates had a time lag and did not reach a maximum by 60 min. The effect of retinoic acid treatment reached a maximum at 2 days and was partly reversible. These results suggest that retinoic acid may increase NIH/3T3 cell adhesion through an effect on laminin receptors. Other mouse fibroblast lines, 3T3-Swiss, 3T6-Swiss, Balb/3T3, and Balb/3T12-3 (spontaneously transformed Balb/3T3), responded to retinoic acid treatment in a manner similar to that of NIH/3T3 cells. However, the virus-transformed Balb/3T3 lines, SV-T2 and M-MSV, showed significant attachment to laminin substrates without retinoic acid treatment, and retinoic acid did not affect or slightly decreased the cell attachment to laminin substrates.     

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of ¹²⁵I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two type of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

Effects of retinoic acid on protein synthesis in cultured melanoma cells

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G₁ phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis anlaysis of L-[³⁵S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.     

Platelet-derived growth factor. II. Specific binding to cultured cells

We have prepared radioiodinated purified platelet-derived growth factor (125I-PDGF) which retains full mitogenic activity. The binding of 125I-PDGF to Swiss 3T3 cells is saturable and highly competed by whole blood serum, purified unlabeled PDGF, and by material from each stage in the purification of PDGF from platelet-rich plasma. Other purified mitogens and substances tested do not compete. 125I-PDGF binding to fibroblasts, 3T3 cells, and arterial smooth muscle cells shows an apparent dissociation constant of 10(-11) M, comparable to the range in which PDGF is mitogenic. A clone of Swiss 3T3 cells obtained from a population selected repeatedly against mitogenic response to PDGF shows a greatly reduced mitogenic response to PDGF and binds only 5% as much 125I-PDGF/cell. The binding capacity of the different cell types tested ranges from 2,500 binding sites/cell on the poorly responding variant to 390,000 binding sites/cell on one strain of Swiss 3T3 cells. Cell types that do not respond to PDGF do not show specific high affinity binding of 125I-PDGF. At 4 degrees C, 125I-PDGF binding to monolayer cultures is relatively slow. Equilibrium binding of low concentrations of 125I-PDGF is not achieved during 7 h unless the binding medium is constantly mixed. 125I-PDGF binding at 4 degrees C shows a broad pH optimum between 6.3 and 8.0. Binding does not seem to require Ca2+ or Mg2+ but is reduced more than 6-fold if both monovalent and divalent salts are omitted. The initial rate of 125I-PDGF binding is greater at 37 degrees C than at 4 degrees C but cell-associated 125I begins to decline soon after reaching a peak value at 30-60 min. Coincident with this decline, trichloroacetic acid-soluble 125I appears in the medium and the binding capacity of the cells declines. These phenomena suggest that PDGF and its receptor may be internalized and degraded.     

Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to approximately 1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of (125)I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the up-regulation of the endocytosis of EGFR.     

The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.