Reduction In Epidermal Growth Factor Binding Research Articles

Ultraviolet A Decreases Epidermal Growth Factor (EGF) Processing in Cultured Human Fibroblasts and Keratinocytes: Inhibition of EGF-Induced Diacylglycerol Formation

The binding, uptake, and degradation of epidermal growth factor (EGF) has been studied in MRC5 human fibroblasts and NCTC 2544 human keratinocytes following ultraviolet A (UVA) irradiation at doses up to 18.9 J/cm2, which are not lethal to cells under our experimental conditions. A dose-dependent reduction in EGF binding was observed, with an approximately 75% decrease at the maximal studied UVA dose. At lower doses (6 to 12 J/cm2), EGF binding was more affected by ultraviolet A in fibroblasts than in keratinocytes. In both cell types, this effect of UVA appeared to be related to a reduction of the affinity of the EGF receptor for EGF. Kinetic studies by pulse-chase experiments indicated that EGF is more rapidly internalized by keratinocytes than by fibroblasts, and that UVA exposure resulted in a slower decay of EGF intracellular content. A 24-h pretreatment of cells with 5 x 10(-5) M vitamin E strongly reduced the appearance of light-induced lipid peroxidation products, measured via assay of thiobarbituric acid reactive substances formation, but only partially prevented the UVA-induced alterations of EGF processing by cells. Finally, UVA exposure almost completely abolished the EGF-induced increase in diacylglycerol production from 14C-arachidonic acid-labeled lipids in both cell types. These results demonstrate that UVA radiation induces important changes in EGF processing and could participate in the light-induced degenerative processes of the skin.

Bradykinin-stimulated transient modulation of epidermal growth factor receptors in A-431 human epidermoid carcinoma cells.

Of nine biological factors (ATP, bradykinin, vasopressin, substance P, angiotensin II, norepinephrine, epinephrine, 12-tetradecanoylphorbol 13-acetate (TPA), and A23187 calcium ionophore) examined, bradykinin, as well as ATP, TPA, and A23187, significantly increased the phosphorylation of epidermal growth factor (EGF) receptors and reduced the binding of EGF to their high-affinity site. The reduction in EGF binding by bradykinin, ATP, and TPA was similarly reversed by concomitant incubation with staurosporine, a protein kinase C inhibitor, implying that the phosphorylation of EGF receptors was catalyzed probably by a protein kinase C of the same or similar type in each case. This possibility was confirmed by the fact that the major phosphorylation site of EGF receptors by the stimulation with either bradykinin, ATP, or TPA was the same (Thr-654). Different from the stimulations with ATP and TPA, the effect of bradykinin of decreasing the high-affinity EGF binding was transient (a minimum binding at 2.5 min); the reduced EGF binding was, however, sustained for up to 30 min in the presence of calyculin A, a phosphoprotein phosphatase inhibitor. Moreover, the homogenate prepared from bradykinin-stimulated A-431 cells had stronger dephosphorylation activity for phosphorylated EGF receptors than that from control cells. These results suggest that bradykinin stimulates both the protein kinase C system and a phosphoprotein phosphatase(s) activity in A-431 cells. Such biphasic effects of bradykinin to phosphorylate and dephosphorylate EGF receptors via protein kinase C and a phosphoprotein phosphatase, respectively, imply a homeostatic control of receptor function in regulating phosphorylation level by the same bioactive factor.

Modulation of the epidermal growth factor receptor by basic fibroblast growth factor

Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C. Treatment of cells with bFGF stimulated the phosphorylation of the EGF receptor, which persisted for several hours. The inhibition of EGF receptor autophosphorylation by bFGF was reduced in the presence of cycloheximide. However, cycloheximide had no effect on the reduction of EGF binding by bFGF. In contrast to these results with Swiss 3T3 fibroblasts, treatment of PC12 cells with bFGF lead to a reduction in EGF binding but no inhibition of EGF receptor autophosphorylation. Thus inhibition of EGF receptor autophosphorylation and inhibition of EGF binding can be uncoupled.

The effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin on epidermal growth factor binding and protein kinase activity in the RTH-149 rainbow trout hepatoma cell line

The time- and dose-dependent effects of TCDD on epidermal growth factor receptor (EGF-r) and protein kinase activities were studied in the RTH-149 rainbow trout hepatoma cell line. A single exposure to 150 pM TCDD in isooctane caused a reduction in epidermal growth factor (EGF) binding to RTH-149 plasma membranes. Reduction was first observed at 6 h and continued throughout the experimental period (96 h). EGF binding was also reduced in a dose-dependent manner with an EC 50 of 27.5 pM. The reduction in binding of EGF was accompanied by an increase in EGF receptor phosphorylation. The decrease in binding of EGF to cell membranes and the increase in EGF receptor phosphorylation were also correlated with greater activities of membrane bound protein kinase C and protein tyrosine kinase. The TCDD-induced reduction in EGF binding was also statistically correlated with an increase in cell number and [ 3H]thymidine incorporation, both of which were statistically correlated with an increase in EGF-r phosphorylation and protein kinase activity. The results suggest that TCDD-induced alteration of EGF-mediated effects has some toxicological significance in the regulation of normal growth and differentiation of RTH-149 cells.

Action of retinoic acid on the diacylglycerol-induced ornithine decarboxylase activity, reduction in EGF binding and protein kinase C activation in rat tracheal epithelial 2C5 cells

We have shown previously that in rat tracheal epithelial 2C5 cells the induction of ornithine decarboxylase (ODC) activity and the reduction in the binding of epidermal growth factor (EGF) by diacylglycerol is related to the activation of protein kinase C. In this paper we analyse the action of retinoic acid (RA) on these two parameters in order to determine whether RA acts on the level of protein kinase C. RA inhibits the induction of ODC activity by diacylglycerol (sn-1,2-dioctanoylglycerol) in a dose- and time-dependent manner. A biologially inactive analog of RA has no effect on this induction. RA does not affect the activation of protein kinase C by diacylglycerol in an in vitro assay. In contrast to the effect on ODC induction, RA does not counteract the reduction in EGF binding induced by diacylglycerol. These results are consistent with the concept that RA does not act at the level of protein kinase C and inhibits ODC induction during a stage following protein kinase C activation.

Platelet-derived growth factor modulates epidermal growth factor receptors by a mechanism distinct from that of phorbol esters.

Platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) decrease high affinity binding of 125I-labeled epidermal growth factor (EGF) and potentiate mitogenesis in BALB/c 3T3 cells, and both have been shown to induce the phosphorylation of the EGF receptor at threonine residues. These similarities suggest that the actions of PDGF on EGF binding may be mediated by protein kinase C, the cellular effector of PMA. We show that in density-arrested BALB/c 3T3 cells PDGF and PMA induce a rapid, transient, cycloheximide-independent loss of EGF binding activity. As has been previously shown for PDGF, the ability of PMA to reduce EGF binding was enhanced by cholera toxin, a potent activator of adenylate cyclase. In contrast to PMA, however, PDGF induced a further reduction in EGF binding that was strictly dependent upon continued protein synthesis. Furthermore, PDGF effectively reduced EGF binding in cells refractory to PMA. Cells desensitized to PMA, presumably due to the loss of protein kinase C activity, also remained mitogenically responsive to PDGF. These data suggest that the mechanism by which PDGF modulates EGF binding differs from that of PMA and thus, at least in part, is independent of protein kinase C.

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) modulates epidermal growth factor (EGF) binding to basal cells from a human keratinocyte cell line

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) modulates the proliferation, differentiation, or both, of epidermal keratinocytes in vivo and in culture. The growth of epidermal cells in culture is regulated by several biochemical mediators including epidermal growth factor (EGF). In this report the actions of TCDD on EGF binding in a basal cell population from a human keratinocyte cell line were examined. TCDD decreased the specific binding of 125I-EGF to basal cells by 40% within 96 hr. this reduction in EGF binding could not be attributed to changes in the state of differentiation as assessed by cell size and morphology, and cornified envelope competence, a marker of terminal differentiation. Modulation of EGF binding by TCDD was concentration-dependent (EC50 = 1 n m) and stereospecific, suggesting involvement of the Ah receptor. Scatchard analysis of EGF binding to the basal cells indicated a single class of high-affinity sites in both control ( K d = 0.14 n m) and treated ( K d = 0.11 n m) cultures and confirmed a decrease in the number of these sites in response to TCDD. The reduction in EGF binding correlated with a decrease in EGF-stimulated DNA synthesis and cell proliferation. Comparison of differentiation-competent squamous cell carcinoma (SCC) lines treated with TCDD supported an association between modulation of EGF binding and enhanced differentiation. The data indicate that basal cells are a target for TCDD. We propose that the modulation of EGF binding in basal keratinocytes by TCDD is one of the critical regulatory events resulting in enhanced differentiation.

Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding.

Tumor-promoting phorbol esters induce ornithine decarboxylase (ODCase) activity and reduce epidermal growth factor (EGF) binding in rat tracheal epithelial 2C5 cells. Phorbol esters activate protein kinase C by interacting at the same site as sn-1,2-diacylglycerols, the presumed physiological regulators. The effects of added sn-1,2-diacylglycerols and those generated by phospholipase C treatment of 2C5 cells on ODCase induction and EGF binding were investigated to establish a role for protein kinase C in these cellular responses. Treatment of 2C5 cells with phospholipase C induced ODCase activity and reduced EGF binding, whereas phospholipases A2 and D were inactive. When sn-1,2-diacylglycerols containing fatty acids 3-10 carbons in length were added to 2C5 cells, those diacylglycerols containing fatty acids 5-10 carbons in length caused ODCase induction and reduction in EGF binding. sn-1,2-Dioctanoylglycerol was one of the most active compounds tested. It induced ODCase in a dose- (50-500 microM) and time-dependent manner. The reduction of binding of 125I-labeled EGF by sn-1,2-dioctanoylglycerol was also time and dose dependent and appeared to result from a change in EGF affinity and not the number of receptor sites. This series of sn-1,2-diacylglycerols showed similar structure-function relationships in their ability to induce ODCase activity, to decrease EGF binding, to stimulate protein kinase C, and to inhibit [3H]phorbol dibutyrate binding to the phorbol ester receptor. These data demonstrate biological activities for a number of diacylglycerols and indicate that protein kinase C activation is implicated in ODCase induction and decreased EGF binding.

Epidermal growth factor-receptor interaction in rat pheochromocytoma (PC12) and human epidermoid A431 cells: Biochemical and ultrastructural studies

Pheochromocytoma cells (clone PC12) have specific plasmamembrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). These growth factors have however, opposite biological effects in PC12 cells; EGF acts mitogenically, while NGF induces differentiation and causes arrest of cell proliferation. EGF-receptor interaction in PC12 cells was characterized biochemically by ¹²⁵I-EGF binding studies. EGF binding was concentration-dependent and saturable. Scatchard graph analysis indicated that two different classes of EGF binding sites may be present in PC12 cells. Addition of NGF to PC12 cells caused a reduction in EGF binding, the result of a decrease of the number of binding sites and in their affinity for EGF. The reduction of EGF binding was dependent upon the duration and concentration of NGF exposure.

Production of growth factors by type 5 adenovirus transformed rat embryo cells.

Transformation of Sprague-Dawley rat embryo (RE) cells and a cloned Fischer rat embryo cell line (CREF) with wild-type (Ad5) or a temperature-sensitive DNA-minus mutant (H5ts125) of type 5 adenovirus results in a reduction in binding of epidermal growth factor (EGF) to cell surface receptors. A reduction in EGF binding is also seen in a Syrian hamster embryo cell line transformed by a hexon mutant of Ad5. In contrast, a human embryonic kidney cell line (293) transformed by sheared Ad5 DNA or transfected clones of KB cells expressing the E1 transforming region of Ad5 do not show a decrease in receptor binding. When cocultivated, the adenovirus transformed rat cells were able to induce the growth in agar of normal CREF cells. Medium from Ad5 transformed RE cells stimulated the growth in agar of CREF cells and also inhibited [125I]-EGF binding in CREF cells. When fractionated by gel filtration, two peaks of [125I]-EGF inhibiting activities were obtained with apparent molecular weights of 35,000 and 16,000. These results provide the first evidence that cells transformed by an adenovirus can produce a growth factor(s) that inhibits EGF-receptor binding and induces anchorage-independent growth of normal cells.

Modulation of the epidermal growth factor receptor by platelet-derived growth factor and choleragen: effects on mitogenesis.

The addition of fresh medium supplemented with partially purified platelet-derived growth factor (PDGF) to quiescent density-arrested cultures of BALB/c-3T3 cells decreases the subsequent binding of radiolabeled epidermal growth factor (EGF). The decrease in EGF binding can be observed 1 hr after the addition of PDGF. This effect is maximal in 2-3 hr, and binding remains diminished for at least 6 hr. These effects can be accounted for by a decrease in the number of EGF receptors with no change in receptor affinity. The action of PDGF is concentration dependent, but even at very high concentrations of PDGF the reduction in EGF binding is never more than 50%. Similar decreases in EGF binding are produced by other treatments that render BALB/c-3T3 cells competent, such as the addition of fibroblast growth factor or medium previously exposed to the macrophage-like cell line P388D(1). Cholera toxin (choleragen), which alone had no effect on EGF binding, dramatically potentiated the ability of PDGF to down regulate EGF receptors. Two to three hours after the addition of PDGF and choleragen, EGF binding was reduced by 80-90% compared with control values. The ability of PDGF and choleragen together to decrease EGF binding was substantially inhibited by cycloheximide. Autoradiography of [(3)H]thymidine-labeled cells shows that choleragen potentiates the action of PDGF; lower concentrations of PDGF are required to make cells competent after choleragen treatment. Furthermore, cells treated with PDGF and choleragen no longer require EGF for traverse of G(1) phase and initiation of DNA synthesis in defined medium. The reduction in receptor number produced by choleragen and PDGF, which may be due to internalization of the EGF receptor, may mimic the action of EGF and thereby remove the EGF requirement for DNA synthesis.