Visualization of epidermal growth factor receptors in human epidermis

The localization of epidermal growth factor (EGF) receptors in normal human epidermis was examined with two independent experimental methods. The distribution of EGF receptor sites was studied using light microscopic autoradiography with [125I]EGF and direct immunocytochemical techniques with EGF receptor antibodies and protein A-colloidal gold complexes. Direct visualization by autoradiography indicated that the concentration of EGF receptors was greatest in the lower epidermal layers. Ultrastructural morphometric analysis of protein A-gold complexes showed that EGF receptors were primarily associated with the plasma membranes although intranuclear and cytoplasmic localization was also evident. This postembedment immunolocalization method also confirmed the relative differences in the number of EGF receptors found in individual epidermal layers (basalis greater than spinosum greater than granulosum greater than corneum layers). This inverse relationship between numbers of EGF receptors and the degree of epidermal differentiation and/or keratinization may suggest a physiologic role for EGF in these processes in human epidermis.     

Epidermal growth factor receptors in different skin tumors

Specific binding of 125I-labeled epidermal growth factor (EGF) was measured in 62 skin tumors of different severity. Within a group of 28 benign tumors, 11 of 15 condylomata acuminata were receptor positive, whereas the investigated mesenchymal tumors and normal skin as a control were receptor negative. 6 of 18 basal cell epitheliomas bound EGF specifically. In the group of precancerous and malignant skin tumors, 7 of 8 squamous cell carcinomas had the highest number of EGF binding sites and a high affinity state, whereas 5 malignant melanomas were receptor negative. The clinical relevance of these findings is not yet clear due to the short follow-up of the patients.     

Epidermal growth factor receptor in human epidermal cells: direct demonstration in cultured cells

The interaction of [125I] labeled murine epidermal growth factor (EGF)( with cultured human keratinocytes has been studied. Epidermal cells from neonatal foreskins were propagated to confluence in 24-well culture trays and incubated with [125I] EGF for binding assays. Association reached equilibrium within 2-4 hr at 4 degrees and slightly earlier at 37 degrees. EGF bound at 37 degrees dissociates very slowly from cells, since it can be shown to enter cells and is degraded to trichloracetic-acid-soluble material. Cells exposed to chloroquine, an inhibitor of lysosomal enzymes, fail to degrade internalized [125I] EGF. Scatchard plots of the binding data yield a dissociation constant of 1 X 10(-9) m and show that epidermal cells bind approximately 3-4 X 10(4) molecules of EGF. Cells exposed to EGF alter their ability to bind EGF by decreasing the number of binding sites in a time- and concentration-dependent manner. Differentiation of epidermal cells in culture poses a problem in assessment of binding of EGF and possibly of other biologically active ligands, since cells lose the ability to bind EGF as they differentiate. These findings indicate that isolated epidermal cells possess a functional receptor for EGF which binds and responds to EGF in a manner similar to that described for other cells.     

Epidermal growth factor and transferrin receptor expression in human embryonic and fetal epidermal cells

Summary Epidermal growth factor receptors (EGFR) and transferrin receptors (TFR) are known to be involved in cell proliferation and to be expressed in normal human epidermis. To date little is known about EGFR and TRF expression in human skin during embryonic and fetal development. In the present work, we studied skin specimens from 30 aborted embryos and fetuses ranging from 7 to 31 weeks estimated gestational age. Monoclonal antibodies to EGFR and TFR were applied on frozen skin sections using an amplification biotin-streptavidin-fluorescein technique. TFR was faintly expressed on epidermal basal cells throughout embryonic and fetal development, as it is in adult epidermis. Up to week 12, EGFR was uniformly expressed on cells of the basal, intermediate and periderm cell layers. From the midfetal period onwards, the suprabasal cell layers showed a decreased staining compared with the basal layer. During the third trimester the cornified cell layer was completely negative. The hair germ and heir peg cells were positive. Later, the outer root sheath and hair bulb remained labelled, with less staining of the hair cone. The sebaceous and eccrine sweat glands were also labelled. These results suggest that in embryonic and fetal epidermis, TFR expression is not correlated with cellular proliferation, whereas EGFR appear to be associated with proliferating and undifferentiated cells.Presented at the 18th Annual Meeting of the European Society for Dermatological Research, München, 19–22 June 1988     

Epidermal growth factor/transforming growth factor alpha receptors and psoriasis

The abnormal growth and differentiation in psoriasis is reflected in the abnormal regulation of Epidermal Growth Factor/Transforming Growth Factor Alpha (EGF/TGF alpha) receptor metabolism. In psoriasis and other hyperproliferative skin conditions these receptors are persistently expressed throughout the interfollicular epidermis as long as the growth stimulatory signal persists. One of the first biochemical signs of effective therapy of psoriasis is the return of the EGF/TGF alpha receptor pattern toward the primarily basilar distribution seen in normal human adult skin. Whether the abnormal expression of TGF alpha in the involved skin induces the persistent expression of EGF receptors is not known nor is the signal that causes the increased production of TGF alpha. Studies to determine what factors regulate EGF receptor expression and TGF alpha induction may yield important new insights into the pathogenesis and therapy of psoriasis.     

Immunolocalization of epidermal growth factor receptors in normal developing human skin.

The embryogenesis of normal human skin is a complex process involving multiple cell types and developmentally regulated growth factors. The immunohistochemical localization of epidermal growth factor receptors (EGF-R) was studied in human fetal skin because this receptor modulates all known actions of EGF and TGF-alpha. EGF-R are present in developing skin as early as the 42nd day of gestation. Immunoreactive EGF-R are present in keratinocytes, endothelial, and skeletal muscle cells. In contrast to normal adult human skin in which the EGF-R are primarily restricted to the basal and immediately suprabasal keratinocytes, the fetal epidermis showed a persistent expression of EGF-R in all cell layers. The absence of EGF-R on the outer, apical surface of periderm cells that are exposed to amniotic fluid was unexpected and may reflect down-regulation of EGF-R by EGF/TGF-alpha or related fetal growth factors present in amniotic fluid. The complex regulation of EGF-R in embryonic hair follicles and sebaceous glands indicates an active and perhaps regulatory role for EGF/TGF-alpha in the development and function of pilosebaceous glands as well as mammalian skin in general.     

Epidermal growth factor stimulates phosphorylation of pig epidermal keratin protein

Endogenous protein phosphorylation of pig epidermis by epidermal growth factor (EGF) was studied to elucidate biologic roles of EGF on epidermal cells. EGF stimulated phosphorylation of keratin proteins (Mr: 65,000, 60,000, 56,000, and 51,000) identified by the Ouchterlony immunodiffusion analysis, a low Mr protein (16,000 dalton) of the urea-SDS-mercaptoethanol soluble fraction, and a 30,000 dalton Tris-HCl soluble protein. The phosphorylated epidermal proteins such as keratin proteins and a 30,000 dalton protein of the Tris-HCl soluble fraction were slightly dephosphorylated following the addition of unlabeled phosphate. Anti-EGF serum eliminated the EGF-stimulated phosphorylation of keratin proteins, a low Mr protein, and a 30,000 dalton Tris-HCl soluble protein. The overall results indicate that EGF specifically stimulated phosphorylation of several epidermal proteins, one of which was keratin protein.     

Increased epidermal growth factor receptors in melanocytic lesions.

BACKGROUND: Epidermal growth factor receptors (EGF/R) have been reported to be absent in melanomas or, in contrast, to be markers for potential malignancy in melanocytic lesions. OBJECTIVE: Our purpose was to evaluate the literature discrepancies regarding the presence of EGF/R in melanocytic lesions and to determine whether EGF/R presence correlates with the potential for malignancy of melanocytic lesions. METHODS: An EGF/R-specific polyclonal antibody was used to study melanomas, dysplastic nevi, congenital nevi, and nevocellular nevi. RESULTS: All melanocytic cell types (nevus and melanoma cells) in the lesions studied had immunoreactive EGF/R. EGF/R immunoreactivity was also observed throughout the epidermal basal to granular cell layers overlying the melanocytic lesions, although dermal fibroblasts were negative. CONCLUSION: The pattern of increased immunoreactive EGF/R in both benign and malignant nevocellular lesions suggests that although EGF/R are not a specific marker of potential malignancy in melanocytic lesions, they may mediate or coordinate growth of keratinocytes and nevus cells.     

Epidermal and dermal effects of epidermal growth factor during wound repair

Epidermal growth factor (EGF), a well-characterized peptide that stimulates in vitro cell proliferation, has now been shown to enhance in vivo resurfacing of porcine wounds. Topical formulations containing either recombinant EGF or placebo were applied daily to partial-thickness wounds along the dorsal surface of pigs. Following full-thickness removal of these wounds, tissues were sectioned and stained, and histologic sections were subjected to computerized morphometric analysis. A significant acceleration of epithelialization across the wound surface was noted following daily EGF treatments. EGF delivered in a variety of topical formulations also produced a marked increase in the cellularity and thickness in the neodermis. A dose-responsive increase in the thickness of the granulation tissue was also observed. In conclusion, topical application of EGF stimulates epithelialization of partial-thickness wounds and produces a positive impact on the underlying dermis during the early phases of wound repair.     

Abnormal expression of epidermal growth factor receptor in cutaneous epithelial tumours

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are important keratinocyte mitogens. Their effects are mediated by a cell membrane receptor (EGFR), quantitative and qualitative abnormalities of which may be responsible for deranged keratinocyte proliferation and differentiation. We have therefore examined EGFR expression immunohistochemically in a variety of benign and malignant epithelial neoplasms using monoclonal antibodies to the extracellular and intracellular receptor domains. In benign tumours (virus wart, seborrhoeic keratosis, keratoacanthoma), there was an ordered pattern of EGFR expression. In malignant tumours (basal and squamous cell carcinoma), there was loss of membrane labelling and cytoplasmic accumulation of the receptor. In premalignant proliferations, there was loss of membrane receptor with either absent cytoplasmic EGFR (actinic keratosis) or cytoplasmic receptor accumulation (Bowen's disease). Evidence of truncated receptors was not found. We suggest that dysregulation of the EGFR may be important in the development of cutaneous epithelial malignancies but that grossly abnormal forms of the receptor do not occur.     

Epidermal growth factor stimulates tyrosine phosphorylation of pig epidermal fibrous keratin

Epidermal growth factor (EGF) stimulated phosphorylation of pig epidermal proteins, one of which was pig epidermal keratin. In order to further characterize phosphorylated proteins and specify the EGF-dependent protein phosphorylation, we attempted to identify phosphorylated keratin proteins and to analyze phosphorylated phosphoamino acids of keratin proteins stimulated by EGF. Four major polypeptide bands of pig epidermal keratin were immunoprecipitated by antihuman callus keratin antibody which reacted with fine networks of fibrous keratin of pig epidermal cells grown in vitro. Four major polypeptide bands were greatly phosphorylated by EGF in a dose-dependent manner. The analysis of phosphorylated phosphoamino acids revealed that EGF stimulated tyrosine phosphorylation of pig epidermal fibrous keratin.     

Immunohistochemical localization by monoclonal antibody of human epidermal growth factor in mixed tumours of the skin

Immunohistochemical distribution of human epidermal growth factor (hEGF) was described in 17 cases of mixed tumour of the skin with monoclonal antibody. In normal sweat glands, epithelial cells in the secretory portion and in the transitional area between secretory portion and duct showed prominent staining for hEGF. In the salivary pleomorphic adenoma type of mixed tumour of the skin, luminal tumour cells of tubular and duct-like structures gave a very characteristic hEGF staining reaction. The tumour cells showing strong staining for hEGF were scattered throughout the solid foci in this type of mixed tumour. Tubular epithelial cells in the clear cell adenoma type also displayed a positive hEGF reaction. And apocrine mixed tumours strong staining for hEGF occurred on the apical side of tubular and ductal tumour cells. In view of the immunohistochemical staining patterns for hEGF, the histologic origin of mixed tumours of the skin is suggested to be cells in the secretory portion and those in the transitional portion between secretory portion and duct of the sweat gland.     

Surface iodination of epidermal growth factor receptors in intact cells

The membranes from epidermoid carcinoma cells (A-431) that were surface iodinated while intact using catalysis by lactoperoxidase and 125I as iodide contain one major labeled protein of Mr = 180,000. This protein is clearly iodinated on the outside of the intact cell because it is not the major protein labeled when isolated membranes are iodinated. This major surface-iodinated protein is almost certainly the epidermal growth factor (EGF) receptor, since both have the same Mr and have identical sensitivity to proteases. Both are nearly quantitatively converted from an Mr = 180,000 form to an Mr = 160,000 form by an endogenous calcium-activated neutral protease when cells are broken in the presence of calcium. Both are degraded by trypsin only if trypsin has access to the inside of the cell. This latter finding implies that the surface-iodinated EGF receptor spans the plasma membrane. Since the EGF receptor is an autophosphorylating kinase whose activity is enhanced in the presence of EGF, the receptor was labeled and identified using [gamma-32P] ATP. While both iodination and EGF-enhanced phosphorylation occur on tyrosine residues, peptide mapping of the iodinated or phosphorylated Mr = 180,000 band showed that different peptides were being labeled. Since the EGF receptor-kinase spans the plasma membrane, the peptide iodinated on the surface of the intact cell must be different from the peptides that are probably autophosphorylated on the cytoplasmic side of the membrane.     

Epidermal hyperplasia and wool follicle regression in sheep infused with epidermal growth factor

The proliferative activities of germinative cells of the wool follicles and the epidermis have been determined in sheep treated with epidermal growth factor (EGF). Infusions of 0.17-0.72 mg EGF/kg metabolic body weight (MBW) for 28 h resulted in marked declines in the mitotic indices (MI) of the follicle bulb cell populations 24 h after the beginning of treatment, the lowest values being recorded at 48 h. Follicular activity subsequently recovered and the MI returned to preinfusion levels after 3-8 days. The inhibition of fiber production resulting from the decline in bulb cell division caused the development of a break in the fleece. By contrast, the MI of the peripheral cells of the sebaceous gland acini and the basal cells of the epidermis increased after EGF treatment, reaching peaks 48-72 h after the beginning of infusion. The degree to which all of these responses were observed appeared to be approximately correlated with the amount of EGF administered.     

Pachydermoperiostosis: study of epidermal growth factor and steroid receptors

Pachydermoperiostosis is a rare osteo-cutaneous disease characterized by hypertrophy of bones and surrounding soft tissues. The cutaneous manifestations include coarsening of facial features, cutisverticis gyrata, digital clubbing, hyperhidrosis and scborrhoea. The pathogenetic mechanism of the disease is still debated, and proposed aetiological factors include genetic influences, anomalies in fibroblast activity, or alteration in peripheral blood flow. We studied a patient with the incomplete form of Pachydermoperiostosis. assessing epidermal growth factor receptor (EGF-R) and sex hormone steroid receptors (SR) in the affected skin, and also evaluating the urinary excretion of EGF. The results showed high levels of nuclear steroid receptors, increased cytosolic oestrogen receptors, and no detectable progesterone and androgen cytosolic receptors. EGF-R was also undetectable, and the urinary excretion of EGF was elevated. These findings suggest that the increased tissue sensitivity to circulating sex-steroids could induce enhanced tissue EGF/transforming growth factor α (TGF-α) production and utilization. The SR-EGF-R system could therefore be involved in determining hypertrophy of the affected tissues.