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The Role of Epidermal Growth Factor in Skin Diseases
The Role of Epidermal Growth Factor in Skin Diseases BY LLOYD E. KING, JR., MD, PHD, DARREL L. ELLIS, MD, RONA~D E. GATES, PHD, WENDELYN H. INMAN, PHD, CHRISTA M. STOSCHECK, PHD, ROY A. FAVA, PHD, LILLIAN B. NANNEY, PHD
A
growth factor was defined more than three decades ago by its ability to stimulate cells to divide. I Purification of growth factors or mitogens whose effects are mediated by specific cell surface receptors led to a new era in developmental biology and. medicine. Numerous growth factors have been identified, and an abbreviated list is shown in Table 1. Since growth is a pleiotropic response, it is not surprising that growth factors affect a variety of cellular functions. In fact, growth factors may induce opposing effects on cells, depending on the cell type or state of differentiation of the cell. To illustrate some of the broader implications of gJ,'owth factor action, the role(s) of epidermal growth factor (EGF) and related molecules in normal and diseased human skin are discussed. EGF, a Prototypic Growth Factor
EGF, a small (53 amino acid) polypeptide, directly stimulates epidermal growth and differentiation. 2 Stanley Cohen reported that daily injections of submaxillary gland extracts into newborn mice accelerated eyelid opening and incisor eruption. Using this bioassay, a small polypeptide was isolated that produced the same changes when injected into newborn mice. 3 EGF was subsequently purified and the primary sequence determined. 2 In mice· the cDNA for EGF encodes a large precursor protein containing 976 amino acids. 4 The gene for EGF is on chromosome 4q (the same region as that for the T-cell growth factor interleukin-2). The physiologic role of EGF has been difficult to define because it is produced by multiple cell types in humans, stimulates the growth of a large variety of cells, and is found in platelets and all body fluids. In addition, the specific receptor for EGF is expressed on multiple cell types, including mesenchymal and epithelial cells, as well as in their transformed counterparts.5 •6 From Vanderbilt University and the Veterans Administration Medical Centers, Nashville, Tennessee. Reprint requests: Lloyd E. King, Jr., MD, PhD, Chairman of Dermatology, VA Medical Center, 1310 24th Avenue, South, Nashville, TN 37212.
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Mouse EGF has a primary structure similar to human urogastrone, a hormone that inhibits gastric acid secretion. 7 In vivo, EGF/urogastrone stimulates proliferation of skin, lung, corneal and tracheal epithelium, as well as ofthe epithelium of the gastrointestinal (GI) tract. B EGF/urogastrone is not destroyed by gastric acid, is absorbed intact, and retains its biological activity.2,7 These properties are important physiologically because the major growth promoting protein in human breast milk is EGF. 9 EGF and related peptide growth factors may therefore play a crucial role in developing mammalian tissues. Recent studies also have indicated an important role for EGF and related peptide growth factors in wound healing, another example of rapid tissue proliferation. lo ,l1 How EGF Works
All known effects of EGF are mediated by its specific membrane receptor, which contains an intrinsic tyrosine kinase. 4 EGF binding to the extracellular portion of its receptor induces a transmembrane signal that activates the intrinsic tyrosine kinase, causing the receptor to preferentially autophosphorylate specific tyrosine residues in its cytoplasmic portion. Adjacent receptors or other biologically active proteins inside the cell are phosphorylated less effectively4 (Table 2). The EGF receptor-kinase has been cloned and sequenced.4,12 The EGF receptor is a large single chain glycoprotein (approximately 180,000 daltons or 1,186 amino acids) containing several functional domains: an externally located EGF binding site; a small single chain transmembrane portion; and a cytoplasmic domain. The cytoplasmic domain contains: (1) the tyrosine autophosphorylation sites; (2) serine and threonine phosphorylation sites; and (3) the intrinsic EGF stimulated tyrosine kinase activity, a region that is 35% to 90% homologous with the transforming proteins from the src oncogene family. In fact, another oncogene, v-erb B, is homologous to a truncated version of the EGF receptor, which lacks the regulatory extracellular EGF binding site. EGF, like some other peptide hormones, binds to its specific extracellular receptors that cluster, interSeptember 1988 Volume 296 Number 3
King et 01
TABLE 1 Biologically and Structurally Well-characterized Growth Factors Epidermal growth factor Fibroblast growth factor. acidic Fibroblast growth factor. basic Insulin-like growth factor-I Insulin-like growth factor-Ii Colony stimulating factors
Insulin Nerve growth factor Platelet derived growth factor Transforming growth factor-a Transforming growth factor-{3 Interleukln-2
TABLE 3 Where Are High Levels of EGF Receptors Detected? Organ/System CNS
Liver Placenta Skin (epidermal)
nalize in clathrin coated pits, and become degraded within lysosomal vesicles. 4 Whether other substrates are phosphorylated and how such events relate to clustering, internalization, and mitogenic signaling is unclear not only for EGF but also for other tyrosine kinase associated growth factor receptors, such as insulin and PDGF. One possible candidate for a role in such processes is a recently identified calcium and phospholipid-binding protein termed p35 or lipocor-' tin 1. 13 It is phosphorylated directly by the EGF-receptor tyrosine kinase in vitro14 and in intact A431 cells (epithelioid carcinoma line).15 The kinetics of phosphorylation of p35 in intact cells following EGF exposure suggest a role in EGF internalization or accumulation in multivesicular bodies. What Does EGF Do in Skin?
The best studied effects of EGF are its ability to increase proliferation, differentiation, and repair of . epithelial and nonepithelial tissues. 5 Several of these
TABLE 2 Biologically Active Proteins Phosphorylated by EGF Receptor In Vitro Contractile proteins Structural proteins Enzymes Membrane receptors Hormones Proteins with undefined functions
Actin. myosin. tubulin Intermediate filaments. keratins. ezrin Ribonuclease. ATP citrate lyase. enolase Insulin receptor Gastrin. growth hormone. angiotensin p35 (Lipocortin I)
See Reference 4 for detailed list.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Skin (dermal)
Celitype ependymal celis neurons purkinje celis pyramidal cells hepatocytes syncytial trophoblasts "decidual celis" amnion epithelial celis basal keratinocytes eccrine sweat duct celis hair folilcle outer root sheath celis basal sebocytes apocrine myoepithelial cells vascular smooth muscle celis arrector pllorum muscle cells
effects may be directly related to the observation that EGF increases the migration of both fibroblasts 16 and keratinocytes 17 in culture. The target cells for the effects of EGF in the skin generally are deduced from the distribution of the EGF receptor and of the receptor-kinase substrates. As indicated in Table 3 EGF receptors are located on multiple cell types. I~ normal human epidermis, EGF receptors are present in highest concentration in the basal cell layer and decrease in number as the keratinocytes differentiate. 18.19 Aside from basal keratinocytes, EGF receptors in normal human skin are found primarily on sebocytes, arrector pilorum muscles, myoepithelial cells, and vascular smooth muscle. 18.19 The EGF receptor substrate, p35, also is detectable in all of these cell types as well as dermal fibroblasts. 2o As is true for the EGF receptor,18,19 p35 is found in highest concentration in the epithelium of eccrine sweat duct cells, normally a slowly dividing cell population that actively transports ions. 20 EGF receptors also are found in high concentration on other epithelial cells that actively transport ions, such as the choroid plexus and ependyma in the nervous system and cells in the G I tract. 21 Regulation of Growth Factor Activity
Cellular responses to EGF require the presence of EGF cell surface receptors. The number of EGF cell surface receptors does not directly correlate with the magnitude of the cell's response to EGF, eg, cells with high levels of EGF receptors often are growth inhibited by EGF. 22 ,23 Growth factor receptors may be regulated by binding their ligands or structurally related
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EGF In Skin
growth factors, which causes down-regulation or down-modulation,2,4 eg, TGF-a to the EGF receptor; insulin to the IGF-l receptor, by binding of a growth factor to a related receptor (receptor cross-talk, or transmodulation, eg, insulin and IGF-2 receptor 24 ). Growth factor receptors also can be regulated by cytoplasmic factors that alter the affinity ofthe receptor for its ligand or affect the receptor kinase activity, eg, phorbol esters and protein kinase C (PKC) activation. 4 Natural factors such as mitogenic lectins, inflammatory mediators such as arachidonic acid, and immune modulators such as interleukin I may partially mimic a growth factor's mitogenic or nonmitogenic effects. Stringent criteria for defining growth factors include the ability to bind to, interact with and modulate specific growth factor receptors. For example, a partially purified substance from a tumor extract that was termed sarcoma growth factor (SGF) was found to interact with EGF receptors because it contained two distinct growth factors, TGF-a and TGF_,B.25-28 These studies led to the recognition of a growth factor (TGF-a) that is structurally related to EGF and binds to the EG F receptor 25-27 and another growth factor (TGF-{3) with diverse functions that include modulation of the activity ofEGF. 25,26,28 As more growth factors are purified, it is clear that the combined effects of multiple growth factors coupled with their chronologic sequence of presentation may influence the precise pattern of cellular responses. The binding of EGF to its receptor increases the transport of calcium and the turnover of phosphatidyl inositol. 29 Furthermore the affinity of EGF for its receptor and the activity of the receptor-kinase are reduced following PKC mediated phosphorylation of the receptor on serine and threonine residues. 4 An obvious question is how these interactions affect proliferation and differentiation ofthe epidermis. One clear link is that EGF receptor distribution is abnormal in psoriasis30 and PKC levels are decreased. 31 Other calcium related enzymes such as calmodulin also are abnormally regulated in psoriasis. 31 It is unclear whether these are epiphenomena or pathogenic events. Protein kinases A and G can affect calcium metabolism, receptor-linked PI turnover, and PKC related events,32 but specific in vivo regulation is not clearly defined. Studies with tumor-promoting phorbol esters and retinoids used as antipromoters may shed more light on EGF, PKC, and retinoid interaction in vivo. Retinoic acid increases by as much as sevenfold the number of EGF receptors expressed on some cell lines without altering their affinity for EGF. 33 EGF stimulates terminal differentiation (like phorbol esters34 ), whereas retinoids tend to inhibit terminal differentiation}4,35 No consistent effect of retinoids on EGF receptor metabolism has been defined, although this possible interaction clearly is of interest. 156
Growth Factors Modulating EGF Activity In the Skin
TGF-a is a 50 amino acid peptide, originally purified from a sarcoma tumor extract, that has a limited overall homology to EGF but is antigenically related and has sufficient structural homology to bind to the EGF receptor. Although its effects are presumed to be mediated through the EGF receptor, in some systems TGF-a has more persistent effects than EGF. 26,27 TGF -a initially was thought to be a fetal growth factor originating from fetal or transformed cells, but this concept recently has been challenged by the observations that its mRNA has been detected in normal adult and neonatal skin, normal keratinocytes in culture,36 and in mouse maternal decidua,37 Its role in fetal development as well as in normal adult skin remains to be determined. TGF-{3 is a dimeric growth factor comprised of two 112 amino acid subunits. TGF-,B also was purified from a sarcoma extract, as well as 'from many other noncancerous sources such as platelets. 28 It has its own separate receptors, unrelated to the EGF recep tor, and is antigenic ally and structurally unrelated to EGF.25 TGF-{3 acts synergistically with either EGF or TGF-a to cause the growth of fibroblasts in soft agar. The property of fibroblasts to grow in soft agar is highly correlated with tumorgenicity and is, therefore, a hallmark of the transformed phenotype. Hence, the simultaneous secretion of TGF-a and TGF-,B by tumor-derived cells in culture has been interpreted as an example of an autocrine regulatory mechanism by which growth factors contribute to the expression of the transformed phenotype. TGF-,B alone does not stimulate fibroblast growth in soft agar, but it does, in some instances, stimulate the growth of fibroblasts attached to plastic. However, its primary effect on epithelial cells is thought to be growth inhibition, as it potently inhibits proliferation of keratinocytes in vitro.28 TGF-,B recently has been shown to accelerate wound healing in rats 38 and may prove to be clinically useful in human wound healing. EGF binding and receptor activity also is affected by other growth factors such as platelet derived growth factor (PDGF), the tumor promoting phorbol esters (including TPA), extracellular calcium levels and virally produced EGF-related growth factors. 4 Since interleukin-l, also termed epidermal thymocyte activating factor (ETAF), and basic fibroblast growth factor also stimulate keratinocyte growth in culture24 (Table 4), these and related factors may potentially affect EGF metabolism in vivo. The Role of EGF in Skin Diseases
In skin diseases in which keratinocytes abnormally differentiate, the distribution and number of EGF receptors change. For example, in psoriasis vulgaris, the number ofEGF receptors is increased twofold to fourfold in active lesions because the EGF receptors persistently are expressed in the abnormally differenti September 1988 Votume 296 Number 3
King etal
TABLE 4 Regulation of Epithelial Cell Growth stimulators Epidermal growth factor Transforming growth factor ex Insulin Insulin-like growth factor I, II Fibroblast growth factor, acidic Fibroblast growth factor, basic Interleukin I/ETAF
Inhibitors transforming growth factor {J1 transforming growth factor {J2 Interferon cd{J-1 Interferon {J-2 Interferon 'Y tumor necrosis factor
ating stratum spinosum and corneum. 2B Similarly in epithelial cancers at other body sites such as squamous cell carcinomas of the lung,39 epidermoid carcinoma of the vulva (A-431 cells22 ,23), and even in gliomas 40 the number ofEGF receptors is increased. The mRNAs for the EGF receptor and transforming growth factors alpha (TGF-O') and beta (TGF-f3) are present in cancers derived from epidermal cells, including melanomas. 5 ,6 EGF and related molecules also are found in mesenchymal tumors such as sarcomas, which produce SGF. Growth factor interactions may play a controlling role in skin cancers and related paraneoplastic syndromes. We noted41 that the sign of Leser-Trelat, malignant acanthosis nigricans, and eruptive acrochordons were associated. with abnormalities in EGF /TGF -0' metabolism in a patient with malignant melanoma. When the melanoma was removed, the concentration of urinary TGF-ex decreased, and the acanthosis nigricans almost disappeared. The EGF receptor levels in the acanthosis nigricans skin initially were increased but normalized after surgery, indicating a possible dynamic clinical interaction of TG F -0' with other local factors. Viral diseases of the skin are common and affect the EGF receptor distribution. In verruca vulgaris the number of EGF receptors appear to be decreased and abnormally distributed. 42 In molluscum contagiosum, a pox virus, the EGF receptors also are decreased. 42 Pox viruses produce an interesting EGF -like molecule termed vaccina virus growth factor, which is structurally similar to EGF and binds to the EGF receptor. 43 Conclusion
EGF and related growth factors have been cloned and are available for clinical trials. Therefore, the field of peptide growth factors now is of general clinical interest. Because TGF-O' may be excreted into body fluids in detectable concentrations when transformed epithelial cells are present, assays for TGF-O' and related growth factors may become useful in the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
management of neoplastic diseases. Also of clinical interest are preliminary data that EGF is useful in the therapy of superficial wounds such as burns and may be important in the pathophysiology of psoriasis and viral diseases. References 1. Levi-Montalcini R: The nerve growth factor 35 years later. Science 237:1154-1162, 1987. 2. Carpenter G, Cohen S: Epidermal growth factor. Annu Rev Biochem 48:193-216, 1979. 3. Cohen S: Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J Bioi Chem 237:1555-1562, 1962. 4. Stoscheck CM, King LE Jr: Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins. J Cell Biochem 31:135-152,1986. 5. King LE Jr, Carpenter GF: Epidermal growth factor, in Goldsmith LA (ed): Biochemistry and Physiology of the Skin. Oxford, Oxford University Press, 1983, pp 269-281. 6. Derynck R, Goeddel DV, Ul1rich A, Gutterman JU, Williams RD, Bringman TS, Berger WH: Synthesis of messenger RNAs for transforming growth factors ('( and {3 and the epidermal growth factor receptor by human tumors. Cancer Res 47:707712,1987. 7. Gregory H: Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257:325-327, 1975. 8. Carpenter G: Epidermal growth factor, in Baserga R (ed): Handbook of Experimental Pharmacology. Berlin, SpringerVerlag, 1981, pp 89-132. 9. Carpenter G: Epidermal growth factor is a major growth-promoting agent in human milk. Science 210:198-199,1980. 10. Buckley A, Davidson JM, Kamerath CD, Wolt TB, Woodward SC: Sustained release of epidermal growth factor accelerates wound repair. Proc Natl Acad Sci USA 82:7340-7344, 1985. 11. Schultz GS, White M, Mitchel1 R, Brown G, Lynch J, Twardzik DR, Todaro GJ: Epithelial wound healing enhanced by transforming growth factor-a and vaccinia growth factor. Science 235:350-353, 1987. 12. Carpenter G, Zendegui JG: Epidermal growth factor, its receptor, and related proteins. Exp Cell Res 164:1-10,1986. 13. Brugge JS: The p35-p36 substrates of protein-tyrosine kinases as inhibitors of phospholipase A z. Cell 46:149-150, 1986. 14. Fava RA, Cohen S: Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cel1s. J Bioi Chem 259:2636-2645, 1984. 15. Sawyer ST, Cohen S: Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cel1s. J Bioi Chem 260(14):8233-8236, 1985. 16. Westermark B, Bloomquist E: Stimulation of fibroblast migration by epidermal growth factor. Cell Bioi Int Rep 4:649-654, 1980. 17. Barrandon Y, Green H: Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-a and epidermal growth factor. Cell 50:11311137,1987. 18. Nanney LB, Magid M, Stoscheck CM, King LE Jr: Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J Invest DermatoI83:385-393, 1984. 19. King LE Jr, Gates RE, Nanney LB, Stoscheck CM: Epidermal growth factor (EGF) regulates non-mitogenic functions of differentiated mammalian tissues, in Bernstein lA, Hirone T (eds): Processes in Cutaneous Epidermal Differentiation. New York, Praeger Scientific, 1987, pp 233-247. 20. Fava RA, Nanney LB, King LE Jr: Immunolocalization ofp35 (Lipocortin-I) in human skin. Clin Res 36(l):84A, 1988. 21. Werner MH, Nanney LB, Stoscheck CM, King LE Jr: Local-
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ization of immunoreactive epidermal growth factor receptors in human nervous system. J Histochem Cytochem 36:81-86, 1988. 22. Buss JE, Kudlow JE, Lazar CS, Gill GN: Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF. Proc Natl Acad Sci USA 79:2574-2578, 1982. 23. Stoscheck CM, Carpenter G: Biology of the A-431 cell: a useful organism for hormone research. J Cell Biochem 23:191-202, 1983. 24. Sporn MB, Roberts AB: Peptide growth factors are multifunctional. Nature 332:217-219, 1988. 25. Stoscheck CM, King LE Jr: The role of epidermal growth factor in carcinogenesis. Cancer Res 46:1030-1037,1986. 26. Roberts AB, Frolik CA, Anzano MA, Sporn MB: Transforming growth factors from neoplastic and nonneoplastic tissues. Fed Proc 42(9):2621-2626, 1983. 27. Derynck R: Transforming growth factor-a: structure and biological activities. J Cell Biochem 32:293-304, 1986. 28. Sporn MB, Roberts AB, Wakefield LM, deCrombrugghe B: Mini-review: some recent advances in the chemistry and biology oftransforming growth factor-(j. JCell Biol105:1039-1045, 1987. 29. Sawyer ST, Cohen S: Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells. Biochemistry 20:6280-6286, 1981. 30. Nanney LB, Stoscheck CM, Magid M, King LE Jr: Altered p25I]epidermal growth factor binding and receptor distribution in psoriasis. J Invest Dermatol86:260-265, 1986. 31. Horn F, Marks F, Fisher GJ, Marcelo CL, Voorhees JJ: Decreased protein kinase C activity in psoriatic ve~sus normal epidermis. J Invest Dermatol88:220, 1987. 32. Berridge MJ: The molecular ba.sis of communication within the cell. Sci Am 253(4):142-152, 1985. . 33. Jetten AM: Retinoids specifically enhance the number of epidermal growth factor receptors. Nature 284:626-629, 1980.
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34. Lichti U, Yuspa SH: Inhibition of epidermal terminal differentiation and tumour promotion by retinoids. Ciba Foundation Symposium 113, in Retinoids, Differentiation and Disease. London, Pitman, 1985, pp 77-89. 35. Fuchs E, Green H: Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell 25:617-'-625, 1981. 36. Coffey RJ Jr, Derynck R, Wilcox IN, Bringman TS, Goustin AS, Moses HL, Pittelkow MR: Production and auto-induction of transforming growth factor-a in human keratinocytes. Nature 328:817-819,1987. 37. Han VKM, Hunter ES III, Pratt RM, Zendegui JG, Lee DC: Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua. Mol Cell Biol7(7):2335-2343, 1987. 38. Mustoe TA, Pierce GF, Thomason A, Gramates P, Sporn MB, Deuel TF: Accelerated healing of incisional wounds in rats induced by transforming growth factor-(j. Science 237:13331336,1987. 39. Hendler FL, Ozanne BW: Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest 74:647-651, 1984. 40. Libermann TA, Bartal AD, Yarden Y,.Schlessinger J, Soreq H: Expression of epidermal growth factor receptors in human brain tumors. Cancer Res 44:753-760,1984. 41. Ellis DL, Kafka SP, Chow JC, Nanney LB, Inman WH, McCadden ME, King LE Jr: Melanoma, growth factors, acanthosis nigricans, the sign of Leser-Trelat, and multiple acrochordons. N Engl J Med 317(25):1582-1586,1987. 42. Nanney LB, Ellis DL, Dale B, Stoscheck CM, Holbrook K, King LE Jr: Epidermal growth factor receptors (EGF-R) in idiopathic and virally induced hyperproliferative skin diseases. J Invest Dermatol90:592, 1988. 43. Brown JP, Twardzik DR, Marquardt H, Todaro GJ: Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor. Nature (London) 313: 491-492,1985.
September 1988 Volume 296 Number 3
A
growth factor was defined more than three decades ago by its ability to stimulate cells to divide. I Purification of growth factors or mitogens whose effects are mediated by specific cell surface receptors led to a new era in developmental biology and. medicine. Numerous growth factors have been identified, and an abbreviated list is shown in Table 1. Since growth is a pleiotropic response, it is not surprising that growth factors affect a variety of cellular functions. In fact, growth factors may induce opposing effects on cells, depending on the cell type or state of differentiation of the cell. To illustrate some of the broader implications of gJ,'owth factor action, the role(s) of epidermal growth factor (EGF) and related molecules in normal and diseased human skin are discussed. EGF, a Prototypic Growth Factor
EGF, a small (53 amino acid) polypeptide, directly stimulates epidermal growth and differentiation. 2 Stanley Cohen reported that daily injections of submaxillary gland extracts into newborn mice accelerated eyelid opening and incisor eruption. Using this bioassay, a small polypeptide was isolated that produced the same changes when injected into newborn mice. 3 EGF was subsequently purified and the primary sequence determined. 2 In mice· the cDNA for EGF encodes a large precursor protein containing 976 amino acids. 4 The gene for EGF is on chromosome 4q (the same region as that for the T-cell growth factor interleukin-2). The physiologic role of EGF has been difficult to define because it is produced by multiple cell types in humans, stimulates the growth of a large variety of cells, and is found in platelets and all body fluids. In addition, the specific receptor for EGF is expressed on multiple cell types, including mesenchymal and epithelial cells, as well as in their transformed counterparts.5 •6 From Vanderbilt University and the Veterans Administration Medical Centers, Nashville, Tennessee. Reprint requests: Lloyd E. King, Jr., MD, PhD, Chairman of Dermatology, VA Medical Center, 1310 24th Avenue, South, Nashville, TN 37212.
154
Mouse EGF has a primary structure similar to human urogastrone, a hormone that inhibits gastric acid secretion. 7 In vivo, EGF/urogastrone stimulates proliferation of skin, lung, corneal and tracheal epithelium, as well as ofthe epithelium of the gastrointestinal (GI) tract. B EGF/urogastrone is not destroyed by gastric acid, is absorbed intact, and retains its biological activity.2,7 These properties are important physiologically because the major growth promoting protein in human breast milk is EGF. 9 EGF and related peptide growth factors may therefore play a crucial role in developing mammalian tissues. Recent studies also have indicated an important role for EGF and related peptide growth factors in wound healing, another example of rapid tissue proliferation. lo ,l1 How EGF Works
All known effects of EGF are mediated by its specific membrane receptor, which contains an intrinsic tyrosine kinase. 4 EGF binding to the extracellular portion of its receptor induces a transmembrane signal that activates the intrinsic tyrosine kinase, causing the receptor to preferentially autophosphorylate specific tyrosine residues in its cytoplasmic portion. Adjacent receptors or other biologically active proteins inside the cell are phosphorylated less effectively4 (Table 2). The EGF receptor-kinase has been cloned and sequenced.4,12 The EGF receptor is a large single chain glycoprotein (approximately 180,000 daltons or 1,186 amino acids) containing several functional domains: an externally located EGF binding site; a small single chain transmembrane portion; and a cytoplasmic domain. The cytoplasmic domain contains: (1) the tyrosine autophosphorylation sites; (2) serine and threonine phosphorylation sites; and (3) the intrinsic EGF stimulated tyrosine kinase activity, a region that is 35% to 90% homologous with the transforming proteins from the src oncogene family. In fact, another oncogene, v-erb B, is homologous to a truncated version of the EGF receptor, which lacks the regulatory extracellular EGF binding site. EGF, like some other peptide hormones, binds to its specific extracellular receptors that cluster, interSeptember 1988 Volume 296 Number 3
King et 01
TABLE 1 Biologically and Structurally Well-characterized Growth Factors Epidermal growth factor Fibroblast growth factor. acidic Fibroblast growth factor. basic Insulin-like growth factor-I Insulin-like growth factor-Ii Colony stimulating factors
Insulin Nerve growth factor Platelet derived growth factor Transforming growth factor-a Transforming growth factor-{3 Interleukln-2
TABLE 3 Where Are High Levels of EGF Receptors Detected? Organ/System CNS
Liver Placenta Skin (epidermal)
nalize in clathrin coated pits, and become degraded within lysosomal vesicles. 4 Whether other substrates are phosphorylated and how such events relate to clustering, internalization, and mitogenic signaling is unclear not only for EGF but also for other tyrosine kinase associated growth factor receptors, such as insulin and PDGF. One possible candidate for a role in such processes is a recently identified calcium and phospholipid-binding protein termed p35 or lipocor-' tin 1. 13 It is phosphorylated directly by the EGF-receptor tyrosine kinase in vitro14 and in intact A431 cells (epithelioid carcinoma line).15 The kinetics of phosphorylation of p35 in intact cells following EGF exposure suggest a role in EGF internalization or accumulation in multivesicular bodies. What Does EGF Do in Skin?
The best studied effects of EGF are its ability to increase proliferation, differentiation, and repair of . epithelial and nonepithelial tissues. 5 Several of these
TABLE 2 Biologically Active Proteins Phosphorylated by EGF Receptor In Vitro Contractile proteins Structural proteins Enzymes Membrane receptors Hormones Proteins with undefined functions
Actin. myosin. tubulin Intermediate filaments. keratins. ezrin Ribonuclease. ATP citrate lyase. enolase Insulin receptor Gastrin. growth hormone. angiotensin p35 (Lipocortin I)
See Reference 4 for detailed list.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Skin (dermal)
Celitype ependymal celis neurons purkinje celis pyramidal cells hepatocytes syncytial trophoblasts "decidual celis" amnion epithelial celis basal keratinocytes eccrine sweat duct celis hair folilcle outer root sheath celis basal sebocytes apocrine myoepithelial cells vascular smooth muscle celis arrector pllorum muscle cells
effects may be directly related to the observation that EGF increases the migration of both fibroblasts 16 and keratinocytes 17 in culture. The target cells for the effects of EGF in the skin generally are deduced from the distribution of the EGF receptor and of the receptor-kinase substrates. As indicated in Table 3 EGF receptors are located on multiple cell types. I~ normal human epidermis, EGF receptors are present in highest concentration in the basal cell layer and decrease in number as the keratinocytes differentiate. 18.19 Aside from basal keratinocytes, EGF receptors in normal human skin are found primarily on sebocytes, arrector pilorum muscles, myoepithelial cells, and vascular smooth muscle. 18.19 The EGF receptor substrate, p35, also is detectable in all of these cell types as well as dermal fibroblasts. 2o As is true for the EGF receptor,18,19 p35 is found in highest concentration in the epithelium of eccrine sweat duct cells, normally a slowly dividing cell population that actively transports ions. 20 EGF receptors also are found in high concentration on other epithelial cells that actively transport ions, such as the choroid plexus and ependyma in the nervous system and cells in the G I tract. 21 Regulation of Growth Factor Activity
Cellular responses to EGF require the presence of EGF cell surface receptors. The number of EGF cell surface receptors does not directly correlate with the magnitude of the cell's response to EGF, eg, cells with high levels of EGF receptors often are growth inhibited by EGF. 22 ,23 Growth factor receptors may be regulated by binding their ligands or structurally related
155
EGF In Skin
growth factors, which causes down-regulation or down-modulation,2,4 eg, TGF-a to the EGF receptor; insulin to the IGF-l receptor, by binding of a growth factor to a related receptor (receptor cross-talk, or transmodulation, eg, insulin and IGF-2 receptor 24 ). Growth factor receptors also can be regulated by cytoplasmic factors that alter the affinity ofthe receptor for its ligand or affect the receptor kinase activity, eg, phorbol esters and protein kinase C (PKC) activation. 4 Natural factors such as mitogenic lectins, inflammatory mediators such as arachidonic acid, and immune modulators such as interleukin I may partially mimic a growth factor's mitogenic or nonmitogenic effects. Stringent criteria for defining growth factors include the ability to bind to, interact with and modulate specific growth factor receptors. For example, a partially purified substance from a tumor extract that was termed sarcoma growth factor (SGF) was found to interact with EGF receptors because it contained two distinct growth factors, TGF-a and TGF_,B.25-28 These studies led to the recognition of a growth factor (TGF-a) that is structurally related to EGF and binds to the EG F receptor 25-27 and another growth factor (TGF-{3) with diverse functions that include modulation of the activity ofEGF. 25,26,28 As more growth factors are purified, it is clear that the combined effects of multiple growth factors coupled with their chronologic sequence of presentation may influence the precise pattern of cellular responses. The binding of EGF to its receptor increases the transport of calcium and the turnover of phosphatidyl inositol. 29 Furthermore the affinity of EGF for its receptor and the activity of the receptor-kinase are reduced following PKC mediated phosphorylation of the receptor on serine and threonine residues. 4 An obvious question is how these interactions affect proliferation and differentiation ofthe epidermis. One clear link is that EGF receptor distribution is abnormal in psoriasis30 and PKC levels are decreased. 31 Other calcium related enzymes such as calmodulin also are abnormally regulated in psoriasis. 31 It is unclear whether these are epiphenomena or pathogenic events. Protein kinases A and G can affect calcium metabolism, receptor-linked PI turnover, and PKC related events,32 but specific in vivo regulation is not clearly defined. Studies with tumor-promoting phorbol esters and retinoids used as antipromoters may shed more light on EGF, PKC, and retinoid interaction in vivo. Retinoic acid increases by as much as sevenfold the number of EGF receptors expressed on some cell lines without altering their affinity for EGF. 33 EGF stimulates terminal differentiation (like phorbol esters34 ), whereas retinoids tend to inhibit terminal differentiation}4,35 No consistent effect of retinoids on EGF receptor metabolism has been defined, although this possible interaction clearly is of interest. 156
Growth Factors Modulating EGF Activity In the Skin
TGF-a is a 50 amino acid peptide, originally purified from a sarcoma tumor extract, that has a limited overall homology to EGF but is antigenically related and has sufficient structural homology to bind to the EGF receptor. Although its effects are presumed to be mediated through the EGF receptor, in some systems TGF-a has more persistent effects than EGF. 26,27 TGF -a initially was thought to be a fetal growth factor originating from fetal or transformed cells, but this concept recently has been challenged by the observations that its mRNA has been detected in normal adult and neonatal skin, normal keratinocytes in culture,36 and in mouse maternal decidua,37 Its role in fetal development as well as in normal adult skin remains to be determined. TGF-{3 is a dimeric growth factor comprised of two 112 amino acid subunits. TGF-,B also was purified from a sarcoma extract, as well as 'from many other noncancerous sources such as platelets. 28 It has its own separate receptors, unrelated to the EGF recep tor, and is antigenic ally and structurally unrelated to EGF.25 TGF-{3 acts synergistically with either EGF or TGF-a to cause the growth of fibroblasts in soft agar. The property of fibroblasts to grow in soft agar is highly correlated with tumorgenicity and is, therefore, a hallmark of the transformed phenotype. Hence, the simultaneous secretion of TGF-a and TGF-,B by tumor-derived cells in culture has been interpreted as an example of an autocrine regulatory mechanism by which growth factors contribute to the expression of the transformed phenotype. TGF-,B alone does not stimulate fibroblast growth in soft agar, but it does, in some instances, stimulate the growth of fibroblasts attached to plastic. However, its primary effect on epithelial cells is thought to be growth inhibition, as it potently inhibits proliferation of keratinocytes in vitro.28 TGF-,B recently has been shown to accelerate wound healing in rats 38 and may prove to be clinically useful in human wound healing. EGF binding and receptor activity also is affected by other growth factors such as platelet derived growth factor (PDGF), the tumor promoting phorbol esters (including TPA), extracellular calcium levels and virally produced EGF-related growth factors. 4 Since interleukin-l, also termed epidermal thymocyte activating factor (ETAF), and basic fibroblast growth factor also stimulate keratinocyte growth in culture24 (Table 4), these and related factors may potentially affect EGF metabolism in vivo. The Role of EGF in Skin Diseases
In skin diseases in which keratinocytes abnormally differentiate, the distribution and number of EGF receptors change. For example, in psoriasis vulgaris, the number ofEGF receptors is increased twofold to fourfold in active lesions because the EGF receptors persistently are expressed in the abnormally differenti September 1988 Votume 296 Number 3
King etal
TABLE 4 Regulation of Epithelial Cell Growth stimulators Epidermal growth factor Transforming growth factor ex Insulin Insulin-like growth factor I, II Fibroblast growth factor, acidic Fibroblast growth factor, basic Interleukin I/ETAF
Inhibitors transforming growth factor {J1 transforming growth factor {J2 Interferon cd{J-1 Interferon {J-2 Interferon 'Y tumor necrosis factor
ating stratum spinosum and corneum. 2B Similarly in epithelial cancers at other body sites such as squamous cell carcinomas of the lung,39 epidermoid carcinoma of the vulva (A-431 cells22 ,23), and even in gliomas 40 the number ofEGF receptors is increased. The mRNAs for the EGF receptor and transforming growth factors alpha (TGF-O') and beta (TGF-f3) are present in cancers derived from epidermal cells, including melanomas. 5 ,6 EGF and related molecules also are found in mesenchymal tumors such as sarcomas, which produce SGF. Growth factor interactions may play a controlling role in skin cancers and related paraneoplastic syndromes. We noted41 that the sign of Leser-Trelat, malignant acanthosis nigricans, and eruptive acrochordons were associated. with abnormalities in EGF /TGF -0' metabolism in a patient with malignant melanoma. When the melanoma was removed, the concentration of urinary TGF-ex decreased, and the acanthosis nigricans almost disappeared. The EGF receptor levels in the acanthosis nigricans skin initially were increased but normalized after surgery, indicating a possible dynamic clinical interaction of TG F -0' with other local factors. Viral diseases of the skin are common and affect the EGF receptor distribution. In verruca vulgaris the number of EGF receptors appear to be decreased and abnormally distributed. 42 In molluscum contagiosum, a pox virus, the EGF receptors also are decreased. 42 Pox viruses produce an interesting EGF -like molecule termed vaccina virus growth factor, which is structurally similar to EGF and binds to the EGF receptor. 43 Conclusion
EGF and related growth factors have been cloned and are available for clinical trials. Therefore, the field of peptide growth factors now is of general clinical interest. Because TGF-O' may be excreted into body fluids in detectable concentrations when transformed epithelial cells are present, assays for TGF-O' and related growth factors may become useful in the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
management of neoplastic diseases. Also of clinical interest are preliminary data that EGF is useful in the therapy of superficial wounds such as burns and may be important in the pathophysiology of psoriasis and viral diseases. References 1. Levi-Montalcini R: The nerve growth factor 35 years later. Science 237:1154-1162, 1987. 2. Carpenter G, Cohen S: Epidermal growth factor. Annu Rev Biochem 48:193-216, 1979. 3. Cohen S: Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J Bioi Chem 237:1555-1562, 1962. 4. Stoscheck CM, King LE Jr: Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins. J Cell Biochem 31:135-152,1986. 5. King LE Jr, Carpenter GF: Epidermal growth factor, in Goldsmith LA (ed): Biochemistry and Physiology of the Skin. Oxford, Oxford University Press, 1983, pp 269-281. 6. Derynck R, Goeddel DV, Ul1rich A, Gutterman JU, Williams RD, Bringman TS, Berger WH: Synthesis of messenger RNAs for transforming growth factors ('( and {3 and the epidermal growth factor receptor by human tumors. Cancer Res 47:707712,1987. 7. Gregory H: Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257:325-327, 1975. 8. Carpenter G: Epidermal growth factor, in Baserga R (ed): Handbook of Experimental Pharmacology. Berlin, SpringerVerlag, 1981, pp 89-132. 9. Carpenter G: Epidermal growth factor is a major growth-promoting agent in human milk. Science 210:198-199,1980. 10. Buckley A, Davidson JM, Kamerath CD, Wolt TB, Woodward SC: Sustained release of epidermal growth factor accelerates wound repair. Proc Natl Acad Sci USA 82:7340-7344, 1985. 11. Schultz GS, White M, Mitchel1 R, Brown G, Lynch J, Twardzik DR, Todaro GJ: Epithelial wound healing enhanced by transforming growth factor-a and vaccinia growth factor. Science 235:350-353, 1987. 12. Carpenter G, Zendegui JG: Epidermal growth factor, its receptor, and related proteins. Exp Cell Res 164:1-10,1986. 13. Brugge JS: The p35-p36 substrates of protein-tyrosine kinases as inhibitors of phospholipase A z. Cell 46:149-150, 1986. 14. Fava RA, Cohen S: Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cel1s. J Bioi Chem 259:2636-2645, 1984. 15. Sawyer ST, Cohen S: Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cel1s. J Bioi Chem 260(14):8233-8236, 1985. 16. Westermark B, Bloomquist E: Stimulation of fibroblast migration by epidermal growth factor. Cell Bioi Int Rep 4:649-654, 1980. 17. Barrandon Y, Green H: Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-a and epidermal growth factor. Cell 50:11311137,1987. 18. Nanney LB, Magid M, Stoscheck CM, King LE Jr: Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J Invest DermatoI83:385-393, 1984. 19. King LE Jr, Gates RE, Nanney LB, Stoscheck CM: Epidermal growth factor (EGF) regulates non-mitogenic functions of differentiated mammalian tissues, in Bernstein lA, Hirone T (eds): Processes in Cutaneous Epidermal Differentiation. New York, Praeger Scientific, 1987, pp 233-247. 20. Fava RA, Nanney LB, King LE Jr: Immunolocalization ofp35 (Lipocortin-I) in human skin. Clin Res 36(l):84A, 1988. 21. Werner MH, Nanney LB, Stoscheck CM, King LE Jr: Local-
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ization of immunoreactive epidermal growth factor receptors in human nervous system. J Histochem Cytochem 36:81-86, 1988. 22. Buss JE, Kudlow JE, Lazar CS, Gill GN: Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF. Proc Natl Acad Sci USA 79:2574-2578, 1982. 23. Stoscheck CM, Carpenter G: Biology of the A-431 cell: a useful organism for hormone research. J Cell Biochem 23:191-202, 1983. 24. Sporn MB, Roberts AB: Peptide growth factors are multifunctional. Nature 332:217-219, 1988. 25. Stoscheck CM, King LE Jr: The role of epidermal growth factor in carcinogenesis. Cancer Res 46:1030-1037,1986. 26. Roberts AB, Frolik CA, Anzano MA, Sporn MB: Transforming growth factors from neoplastic and nonneoplastic tissues. Fed Proc 42(9):2621-2626, 1983. 27. Derynck R: Transforming growth factor-a: structure and biological activities. J Cell Biochem 32:293-304, 1986. 28. Sporn MB, Roberts AB, Wakefield LM, deCrombrugghe B: Mini-review: some recent advances in the chemistry and biology oftransforming growth factor-(j. JCell Biol105:1039-1045, 1987. 29. Sawyer ST, Cohen S: Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells. Biochemistry 20:6280-6286, 1981. 30. Nanney LB, Stoscheck CM, Magid M, King LE Jr: Altered p25I]epidermal growth factor binding and receptor distribution in psoriasis. J Invest Dermatol86:260-265, 1986. 31. Horn F, Marks F, Fisher GJ, Marcelo CL, Voorhees JJ: Decreased protein kinase C activity in psoriatic ve~sus normal epidermis. J Invest Dermatol88:220, 1987. 32. Berridge MJ: The molecular ba.sis of communication within the cell. Sci Am 253(4):142-152, 1985. . 33. Jetten AM: Retinoids specifically enhance the number of epidermal growth factor receptors. Nature 284:626-629, 1980.
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34. Lichti U, Yuspa SH: Inhibition of epidermal terminal differentiation and tumour promotion by retinoids. Ciba Foundation Symposium 113, in Retinoids, Differentiation and Disease. London, Pitman, 1985, pp 77-89. 35. Fuchs E, Green H: Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell 25:617-'-625, 1981. 36. Coffey RJ Jr, Derynck R, Wilcox IN, Bringman TS, Goustin AS, Moses HL, Pittelkow MR: Production and auto-induction of transforming growth factor-a in human keratinocytes. Nature 328:817-819,1987. 37. Han VKM, Hunter ES III, Pratt RM, Zendegui JG, Lee DC: Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua. Mol Cell Biol7(7):2335-2343, 1987. 38. Mustoe TA, Pierce GF, Thomason A, Gramates P, Sporn MB, Deuel TF: Accelerated healing of incisional wounds in rats induced by transforming growth factor-(j. Science 237:13331336,1987. 39. Hendler FL, Ozanne BW: Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest 74:647-651, 1984. 40. Libermann TA, Bartal AD, Yarden Y,.Schlessinger J, Soreq H: Expression of epidermal growth factor receptors in human brain tumors. Cancer Res 44:753-760,1984. 41. Ellis DL, Kafka SP, Chow JC, Nanney LB, Inman WH, McCadden ME, King LE Jr: Melanoma, growth factors, acanthosis nigricans, the sign of Leser-Trelat, and multiple acrochordons. N Engl J Med 317(25):1582-1586,1987. 42. Nanney LB, Ellis DL, Dale B, Stoscheck CM, Holbrook K, King LE Jr: Epidermal growth factor receptors (EGF-R) in idiopathic and virally induced hyperproliferative skin diseases. J Invest Dermatol90:592, 1988. 43. Brown JP, Twardzik DR, Marquardt H, Todaro GJ: Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor. Nature (London) 313: 491-492,1985.
September 1988 Volume 296 Number 3