High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.     

Effects of calcium-antagonists and calmodulin inhibitors on DNA synthesis in cultured rat vascular smooth muscle cells

To investigate the role of intracellular Ca2+ in the mechanism of cellular proliferation of vascular smooth muscle cells (VSMC), the effects of Ca2+-antagonists and calmodulin (CaM) inhibitors on DNA synthesis stimulated by serum-derived growth factors were studied in cultured VSMCs derived from rat aorta. DNA synthesis assessed by incorporation of [3H]thymidine into the cells was significantly stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF) or fetal bovine serum (FBS), of which the effects were dose-dependently inhibited by a variety of Ca2+-antagonists, such as verapamil, diltiazem and nicardipine. Trifluoperazine and W-7, both specific CaM inhibitors, similarly inhibited DNA synthesis stimulated by EGF, PDGF or FBS in a dose-dependent manner, whereas W-5, a less specific CaM inhibitor, was minimally effective. These data suggest that the Ca2+-CaM system plays an important role in the mechanism of growth factor-induced DNA synthesis in VSMCs.     

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta

Specific binding site for atrial natriuretic factor (ANF), a potent natriuretic and vasorelaxant polypeptide recently isolated from mammalian atria, was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. Binding studies of 125I-labeled-synthetic alpha-human natriuretic peptide (alpha-hANP) revealed the presence of a non-interacting, single class of high affinity binding sites for alpha-hANP on VSMC in culture: the apparent dissociation constant (Kd) was approximately 1-2 X 10(-9)M and the number of maximal binding sites was approximately 200,000-300,000 sites/cell. A variety of vasoactive substances and other polypeptide hormones did not affect the binding of 125I-labeled-alpha-hANP to its binding sites. alpha-hANP significantly increased the concentrations of intracellular cyclic GMP in VSMC in a dose-dependent manner (3.2 X 10(-9)-1.6 X 10(-7)M). These data indicate that the specific receptor for ANF is present in VSMC and suggest that intracellular cyclic GMP may be involved in its vasorelaxant effect.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells

DNA sequences encoding the human epidermal growth factor (EGF) receptor and various EGF-receptor deletion mutants were transfected into chinese hamster ovary (CHO) cells devoid of endogenous EGF receptors. A functional human EGF-receptor is expressed on the surface of heterologous CHO cells with the following properties: it exhibits typical high affinity (10%; Kd = 3 X 10(-10) M) and low affinity (90%; Kd = 3 X 10(-9) M) binding sites for 125I-EGF; it is expressed as a polypeptide of 170,000 molecular weight with intrinsic protein tyrosine kinase activity. EGF stimulates the kinase activity leading to self-phosphorylation and to phosphorylation of exogenous substrate; 125I-EGF is rapidly internalized into the CHO cells by receptor mediated endocytosis and; EGF stimulates DNA synthesis in the cells expressing the human EGF-receptor. Deletion of 63 amino acids from the C-terminal end of EGF-receptor, which removes two autophosphorylation sites, abolishes the high affinity state of the receptor. Nevertheless, this receptor mutant is able to undergo endocytosis and to respond mitogenically to EGF to a similar extent as the "wild type" receptor. Further deletions from the cytoplasmic domain give rise to low affinity endocytosis-defective receptor mutants. Finally, deletion of the transmembrane domain of the human receptor yields an EGF-receptor ligand binding domain which is secreted from the cells.     

Decreased susceptibility of cultured smooth muscle cells from SHR rats to growth inhibition by heparin

Smooth muscle cell proliferation is regulated through the coordinated action of growth inhibitors and growth factors/mitogens; a specific heparin-epidermal growth factor (EGF) complementation has been proposed (Reilly et al., 1987, J. Cell. Physiol., 131:149-157). In culture, vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate more rapidly than VSMC from control Wistar Kyoto rats (WKY). We observed that, compared with WKY-derived VSMC, cells from SHR were markedly less susceptible to growth inhibition both by heparin and its homopolysaccharide analogue pentosan polysulphate (PPS). SHR-derived VSMC exhibited a reduced capacity for binding of [3H]heparin to specific extracellular surface receptors, whereas affinities for heparin were comparable between both VSMC isolates. The early (0-2 hr at 37 degrees C) kinetics of internalization did not differ between SHR- and WKY-derived VSMC, but both internalized equivalent proportions (approximately 10%) of initially surface-bound heparin. VSMC from SHR exhibited a greater capacity, without a changed affinity, for [I125]EGF binding than VSMC from WKY. Pre-exposure of VSMC to heparin or PPS decreased, in a time-dependent manner, the EGF binding capacity for both SHR and WKY (by 40-50% after 72 hr). However, in absolute terms, the EGF-binding capacity of VSMC from SHR exposed to heparinoids was similar to that of nonexposed VSMC from WKY.     

Uric acid stimulates vascular smooth muscle cell proliferation by increasing platelet-derived growth factor A-chain expression

Recent data suggest that uric acid is generated locally in the vessel wall by the action of xanthine oxidase. This enzyme, activated during ischemia/reperfusion by proteolytic conversion of xanthine dehydrogenase, catalyzes the oxidation of xanthine, thereby generating free radicals and uric acid. Because of the potential role of ischemia/reperfusion in vascular disease, we studied the effects of uric acid on rat aortic vascular smooth muscle cell (VSMC) growth. Uric acid stimulated VSMC DNA synthesis, as measured by [3H]thymidine incorporation, in a concentration-dependent manner with half-maximal activity at 150 microM. Maximal induction of DNA synthesis by uric acid (250 microM) was approximately 70% of 10% calf serum and equal to 10 ng/ml platelet-derived growth factor (PDGF) AB or 20 ng/ml fibroblast growth factor. Neither uric acid precursors (xanthine and hypoxanthine) nor antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) were mitogenic for VSMC. Uric acid was mitogenic for VSMC but not for fibroblasts or renal epithelial cells. The time course for uric acid stimulation of VSMC growth was slower than serum, suggesting induction of an autocrine growth mechanism. Exposure of quiescent VSMC to uric acid stimulated accumulation of PDGF A-chain mRNA (greater than 5-fold at 8 h) and secretion of PDGF-like material in conditioned medium (greater than 10-fold at 24 h). Uric acid-induced [3H]thymidine incorporation was markedly inhibited by incubation with anti-PDGF A-chain polyclonal antibodies. Thus uric acid stimulates VSMC growth via an autocrine mechanism involving PDGF A-chain. These findings suggest that generation of uric acid during ischemia/reperfusion contributes to atherogenesis and intimal proliferation following arterial injury.     

Transforming growth factor (TGF beta) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding

Human bone marrow fibroblasts were cultivated and characterized by immunofluorescent staining and electron microscopy. Their interactions with PDGF and TGF beta were studied. While a positive intracellular antifibronectin staining was observed, the cultured cells were not labeled with specific antibodies toward factor VIII von Willebrand factor (F VIII/vWF), desmin, and macrophage antigen. Moreover, electron microscopy excluded the presence of endothelial cells by the absence of Weibel-Palade bodies. The binding of pure human PDGF to the cultured bone marrow fibroblasts was investigated. Addition of an excess of unlabeled PDGF decreased the binding to 75 and 80%, which means that the nonspecific binding represented 20-25% of total binding, whereas epidermal growth factor (EGF) had no effect. Two classes of sites were detected by Scatchard analysis with respectively 21,000 and 37,000 sites per cell, with a KD of 0.3 x 10(-10) M and KD of 0.5 x 10(-9) M. The stimulation of DNA synthesis by PDGF was quantified by [3H]thymidine incorporation. When PDGF was added alone at a concentration of 15 ng/ml, it induced a maximal DNA synthesis of 400%, which increased up to 900%, in the presence of platelet-poor plasma (PPP). On the other hand, PDGF-induced fibroblast proliferation was inhibited in a dose-dependent manner by TGF beta. This inhibition was related to a significantly decreased binding of 125I-labeled PDGF observed in the presence of TGF beta. Our results suggested that PDGF and TGF beta could modulate the growth of bone marrow fibroblasts.     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

Inhibition of epidermal growth factor binding to Swiss 3T3 cells by human platelet release-products

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.     

Differential inhibitory effects of TGF-beta on EGF-, PDGF-, and HBGF-1-stimulated MG63 human osteosarcoma cell growth: possible involvement of growth factor interactions at the receptor and postreceptor levels

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or heparin-binding growth factor-1 (HBGF-1). The mitogenic effect of EGF and PDGF is completely blocked by TFG-beta at 1 ng per ml and the effect of HBGF-1 is attenuated by 75-80%. Treatment of MG63 cells with TGF-beta reduces HBGF-1 receptor binding affinity from 1.24 x 10(-11) M to 3.51 x 10(-11) M with no change on the receptor number (1.1 x 10(3) per cell). The receptor-binding affinity of EGF and PDGF is not altered by TGF-beta treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF-beta pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF-1-stimulated cells with and without TGF-beta pretreatment. These data suggest that TGF-beta inhibits EGF and PDGF mitogenicity by blocking EGF- and PDGF-stimulated tyrosine kinase activity and attenuates HBGF-1 mitogenicity by decreasing its receptor affinity.     

Distinct effects of transforming growth factor-beta on EGF receptors and EGF-induced DNA synthesis in primary cultured rat hepatocytes

Previous studies showed that transforming growth factor-beta (TGF-beta, 1 ng/ml) strongly inhibited DNA synthesis induced by epidermal growth factor (EGF) in primary cultures of adult rat hepatocytes. This paper reports that TGF-beta (4 ng/ml) caused marked increase of EGF-binding to cultured rat hepatocytes. The binding increased biphasically with time to a maximum after treatment with TGF-beta for 12 h. Scatchard analysis showed that adult rat hepatocytes had a single class of non-cooperative binding sites with a Kd of 1.5 nM, that there were 1.4 X 10(5) binding sites/cell, and that TGF-beta increased the number of binding sites without changing the Kd value. The increase in EGF binding sites by TGF-beta was dose-dependent and the dose that elicited the maximum increase was about 10 times that which inhibited DNA synthesis stimulated by EGF. These findings suggest that the effect of TGF-beta in modulating the EGF receptor is not directly related to that in inhibiting DNA synthesis in adult rat hepatocytes.     

Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor

Pretreatment of $\text{Balb}\text{c}\text{-3T3}$ cells with platelet-derived growth factor (PDGF) has been shown to decrease binding sites for ¹²⁵I-labelled epidermal growth factor (EGF) (1,2,3). Agents which elevate cellular cyclic AMP concentrations enhance this ability, and the change in EGF binding is inversely proportional to the elevation of cyclic AMP. In quiescent density arrested cells, the sensitivity of cells to down regulation of EGF receptors by PDGF is proportional to the cyclic AMP content of the cultures in three different cell lines. Agents which elevate cyclic AMP and which potentiate PDGF mediated heterologous down regulation of EGF receptors are able, like cholera toxin (3), to stimulate cells to synthesize DNA in defined medium in the absence of EGF. Down regulation of EGF receptors by PDGF in combination with agents elevating cyclic AMP effectively mimics the action of EGF.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

Heterologous regulation of EGF receptors in fibroblastic cells

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.     

Specific epidermal growth factor receptors on porcine and human thyroid membranes

Specific, high affinity, saturable receptors for epidermal growth factor (EGF) have been demonstrated both on porcine and on human thyroid membranes. The binding affinities of porcine (Ka 3.0 X 10(-9) M) and human thyroid EGF receptors (Ka 1.75 X 10(-9) M) are very similar. TSH does not inhibit the binding of 125I-EGF to either membrane. These results suggest the possibility that EGF may be involved in the regulation of human as well as porcine thyroid follicular cell growth and function.     

Thymic epithelium in vitro. IV. Regulation of growth and mediator production by epidermal growth factor

The regulation of thymic epithelial cell function has been examined using pure cultures of morphologically distinct thymic epithelial cells and the ubiquitous hormone epidermal growth factor (EGF). Small thymic epithelial cells, TECS, had receptors for EGF with high affinity, Kd = 1.2 X 10(-9) M, and exhibited increased DNA synthesis and increased RNA synthesis upon stimulation with EGF. In addition, incubation of TECS monolayers with EGF resulted in enhanced production of prostaglandin E2. In contrast, large thymic epithelial cells, TECL, did not express receptors for EGF and demonstrated no biological response to the hormone. These results suggest the possibility that intrathymic regulation of lymphoid cells may occur via the action of "nonimmunologic" mediators on thymic epithelial cells. They further suggest the more general possibility that immunologic and nonimmunologic hormonal systems may be linked via intersecting cellular pathways.     

Binding sites for epidermal growth factor in nuclear fraction from rat liver

Binding sites for mouse epidermal growth factor (EGF) were observed in purified nuclear fraction from rat liver. The binding data yielded a curvilinear Scatchard plot which was decomposed into two binding components with different binding affinities. The binding component with higher affinity (Kd approximately 10(-10) M) represented approximately 15% of the total binding, while the high affinity binding sites were less than 5% of the total in the microsomes. The ligand-dependent autophosphorylation, one of the characteristic features of EGF receptor in the plasma membrane, was not observed at the site of the receptor (170 KDa) in the nuclear fraction. The binding characteristics for EGF fluctuated during the course of liver regeneration after partial hepatectomy; the binding capacity in the nuclear fraction increased in contrast to the decrease in the microsomes. However, the binding sites in the nuclear fraction obtained in the early period after partial hepatectomy consisted only of low affinity ones.     

Regulation of ovarian progestin production by epidermal growth factor in cultured rat granulosa cells

The modulation of ovarian steroidogenesis by epidermal growth factor (EGF) was investigated in cultured rat granulosa cells. Granulosa cells, obtained from ovaries of immature, hypophysectomized, estrogen-treated rats, were incubated for 2 days with EGF, follicle-stimulating hormone (FSH), or EGF plus FSH. Treatment with EGF did not affect estrogen production, but stimulated progestin (i.e. progesterone and 20 alpha-hydroxy-pregn-4-en-3-one) production in a dose-dependent manner. Stimulation of progestin production by EGF appears to be the result of an increase in pregnenolone biosynthesis as well as increases in the activities of 20 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase/isomerase. Treatment with FSH increased both estrogen and progestin production by cultured granulosa cells. When cells were treated concomitantly with EGF, FSH-stimulated estrogen production was inhibited, while progestin production was further enhanced. The EGF enhancement of FSH-stimulated progestin production appears to be the result of synergistic increases in pregnenolone biosynthesis and 20 alpha-hydroxysteroid dehydrogenase activity, resulting in substantial increases in 20 alpha-hydroxypregn-4-en-3-one but not progesterone production. The effects of EGF were shown to be time-dependent. The concept of a direct action of EGF on rat granulosa cells is reinforced by the demonstration of high affinity (Kd approximately 3 X 10(-10) M), low capacity (approximately 5,000 sites/cell) EGF binding sites in these cells. Thus, EGF interacts with specific granulosa cell receptors to stimulate progestin but to inhibit estrogen biosynthesis.