The EGF receptor – an essential regulator of multiple epidermal functions

ARTICLE

The term "epidermal growth factor" was coined in the early sixties by Stanley Cohen to describe a polypeptide purified from mouse submaxillary glands that affects epidermal growth and differentiation in the newborn mouse [1, 2]. Specifically, EGF causes premature eyelid opening and incisor eruption reflecting the accelerated differentiation of these structures during neonatal development. Shortly after its discovery, EGF was found to stimulate proliferation of epidermal keratinocytes [3] leading to the characterization of a cell surface receptor specific for EGF [4, 5]. During the last 15 years multiple EGF-related growth factors and at least four different EGF receptor-like receptors (c-ErbBs) have been identified (Table 1) and these ligand-receptor systems have been shown to affect development, regeneration, differentiation and transformation of cells derived from multiple tissues. The analysis of EGFR-dependent events is complicated not only by the presence of multiple ligands that bind to either one or more members of a family of EGFR-like receptors but also by the capacity of the EGFR to synergize with other EGFR family members by dimerization and cross-phosphorylation.

In this review, we will first provide a brief overview of the EGFR family of tyrosine kinases and their ligands. We will then focus on functional contributions of the EGFR to different aspects of keratinocyte biology including proliferation, differentiation, migration, and survival of normal keratinocytes. Observations in cultured keratinocytes will be placed in the context of findings in genetically engineered mice in which EGF receptor function has either been ablated (knock-out mice) or enhanced (transgenic mice). Due to space limitations only a fraction of the relevant literature is cited; we apologize to those whose work could not be included here.

The EGF receptor family and its ligands

The EGFR is a type-1 tyrosine kinase characterized by an extracellular ligand binding domain, a short transmembrane region, and an intracellular signal transduction module consisting of a tyrosine kinase moiety and several autophosphorylation sites [6] (Fig. 1). Upon phosphorylation these tyrosines serve as docking stations for signaling molecules including phospholipase(PL)-Cgamma, phosphoinositol-3-kinase (PI-3K), and various adaptor molecules (for example Shc, Grb2) which facilitate additional downstream signaling events. Based on their sequence homology to the EGF receptor a series of related molecules (termed c-erbB2, 3, and 4) were identified (Table 1). The acronym "erbB" refers to a viral oncogene (v-erbB) derived from the avian erythroblastosis virus, which is homologous to the intracellular portion of the EGFR [6-8]. Interestingly, only the EGFR (ErbB1) and ErbB4 contain all three functional domains necessary for signal transduction in the absence of other receptors of the same family. By contrast, ErbB2 has been called an "orphan" receptor because no ligand has been identified as yet that would bind to its extracellular domain. Thus, signal transduction by ErbB2 is thought to be triggered through heterodimerization with other EGFR family members. ErbB3 contains an extracellular ligand-binding domain but lacks an intracellular tyrosine kinase moiety. For more details of the ErbB family of receptors please refer to recent excellent reviews [9, 10].

Mature, bioactive EGF and EGF-like ligands are characterized by a triple loop structure held in place by 3 intramolecular cysteine bridges [11]. The majority of these ligands (i.e. EGF, heparin binding (HB)-EGF, amphiregulin (AR), transforming growth factor (TGF)-alpha, and epiregulin (EPI)) are exclusive EGFR ligands whereas betacellulin (BC) can bind to both the EGFR and ErbB4. The heregulins/neuregulins bind to ErbB3 and/or ErbB4 but not to the EGFR.

Upon ligand binding members of the ErbB family dimerize and their tyrosine kinase domains are activated. Importantly, extensive heteromerization occurs between different receptors of this family (for example the EGFR and ErbB2; see Table 1) if they are coexpressed by a particular cell type. The close proximity of the dimerization partners allows cross-phosphorylation on tyrosine residues, an important prerequisite for activation of ErbB2 which has no known ligand but can be activated when it is paired with either the EGFR or ErbB4.

In conclusion, in excess of 10 ligands can interact with either one or more erbB family members. Depending on the availability of dimerization partners these receptors will then transmit quantitatively or qualitatively different signals in different cell types. Epidermal keratinocytes express at least three erbB family members in vitro and in situ, i.e., the EGFR [12-14], ErbB2 [15] and ErbB3 [16]. By contrast, ErbB4 is not expressed in cultured mouse keratinocytes [15, 16] although two studies reported weak expression of ErbB4 mRNA [17] and protein [18] in human skin.

EGFR-dependent signal transduction pathways

As outlined above, activation of the EGFR leads to dimerization and autophosphorylation providing docking sites for a variety of intracellular signal transducers. All of the known second messengers activated by either the EGFR or its relatives are also activated by other growth factor receptors and even adhesion receptors. This has been described for major pathways including the RAS-RAF-MEK-MAPK, the PLC-gamma/PKC, and the PI-3K/AKT cascades. It also applies to additional EGFR-stimulatable pathways leading to STAT or NFkappaB activation. These observations pose the question how EGFR activation triggers specific changes in cellular phenotype (see below). It is likely that, rather than eliciting any specific signal EGFR activation affects the balance of multiple signal transducers shared with other receptors present on the same cell. Thus, phenotypic changes seen after EGFR activation are contingent upon the cooperation of an array of cooperating receptors unique to each cell type and differentiation state.

Keratinocyte proliferation

Soon after its discovery EGF was found to increase epidermal thickness and cellularity and to stimulate proliferation of epidermal keratinocytes [2, 3]. More recent studies demonstrated that EGFR activation is indispensable for DNA synthesis [19] and cell cycle progression from the G1 to S phase [20] in cultured keratinocytes. Interestingly, keratinocytes in vitro and in situ can produce several EGFR ligands that contribute to keratinocyte proliferation [21-24]. Autocrine keratinocyte-derived EGFR ligands include TGF-alpha [22, 25, 26], amphiregulin [23, 24, 27], heparin-binding EGF-like growth factor [24] and betacellulin [24]. In several studies, inhibitors of EGFR activation have been used to confirm an essential role of EGFR activation in keratinocyte proliferation. These inhibitors include antagonistic antibodies which bind to the extracellular domain of the EGFR [23, 28] and small molecular weight inhibitors of the tyrosine kinase moiety of the EGFR [28-30].

The development of mice with targeted disruptions of the EGFR locus allowed the examination of EGFR-dependent skin phenotypes induced by autocrine and exogenous EGFR ligands in the intact animal [31-33]. Inactivation of the EGFR by homologous recombination results in at least three different phenotypes in different mouse strains ranging from peri-implantation lethality to postnatal lethality. This complicates interpretation of the results as any phenotype may or may not be relevant to the outbred human population. Only the mildest phenotype allows limited postnatal development and this form is associated with epithelial hypoplasia manifesting as thin skin.

Epidermal differentiation

The first biological activity of EGF to be recognized was its ability to accelerate epidermal differentiation when administered in vivo, causing premature eyelid opening in newborn mice [1] and increased epidermal thickness [2]. These findings are contrasted by studies in cultured human keratinocytes which showed that addition of EGF to keratinocyte cultures markedly extends their life-span in culture and inhibits expression of several differentiation markers [34]. Simlarly, Peus et al. [35] demonstrated that EGF treatment inhibits expression of early molecular markers of terminal differentiation (keratin 1 and keratin 10) whereas inhibition of the EGFR in keratinocyte cultures induces keratin 1/10 expression. EGF treatment of transformed keratinocytes was also found to inhibit late markers of epidermal differentiation including cornified envelope formation and type 1 transglutaminase expression [36]. In skin reconstructs, EGF treatment depressed several indicators of differentiation including expression of profilaggrin/filaggrin and led to nuclear retention in the upper layers of the differentiated epidermal sheet [37]. However, in the absence of extracellular matrix adhesion, EGF appears to promote expression in normal keratinocytes of markers of late terminal differentiation including profilaggrin and type 1 transglutaminase expression, and cornified envelope formation [38].

The studies cited above focused primarily on the effects of exogenous EGFR ligands on keratinocyte differentiation but did not address the contribution of endogenous, keratinocyte-derived EGFR ligands to this phenomenon. By contrast, EGFR knock-out mice enabled the examination of epidermal differentiation in the absence of any EGFR signaling. The skin in these mice reveals only subtle and strain-specific changes in differentiation. Specifically, Miettinen et al. [39] described disturbed terminal differentiation in EGFR^­/­ mice whereas Hansen et al. [33] described normal expression and localization of markers of epidermal differentiation. It is possible that compensatory effects exerted by other erbB family members mask the phenotypic manifestation of EGFR-dependent epidermal differentiation.

Taken together, these studies suggest complex and sometimes opposite regulation of keratinocyte differentiation by exogenous and endogenous EGFR ligands depending on experimental conditions.

Keratinocyte migration

Barrandon and Green first recognized the ability of EGF and TGF-alpha to enhance migration of normal keratinocytes on tissue culture-treated surfaces [40]. Concurrently, both EGFR ligands were found to accelerate wound healing in mice [41], an effect which may be partially attributed to enhanced wound closure by lateral migration of keratinocytes during re-epithelialization [42]. Subsequently, activation of the EGFR was found to be also required for directional migration of keratinocytes in electric fields of physiological strength [43]. As cell migration is an obligatory step not only in physiological processes including development and wound healing but also in pathological states such as metastasis formation, the signaling pathways required for EGFR-dependent migration are of great interest.

Cell survival

In normal, self-renewing epithelia, including epidermis, a tight balance must be maintained between proliferation of cells in the basal layer and loss of terminally differentiated, dead cells from the surface. During the later stages of terminal differentiation (i.e. the conversion of granular layer cells to cornified cells), keratinocytes exhibit some features characteristically associated with programmed cell death or apoptosis. Specifically, DNA nicking, nuclear fragmentation and activation of transglutaminase have been described [44]. It is of great importance to epidermal homeostasis that keratinocyte apoptosis be tightly regulated. If it is initiated too early, the epidermis would likely be thin and compromised with regard to its barrier function. Conversely, the loss of ability to initiate apoptosis has been correlated with the development of neoplasia (reviewed in [45]).

Specific growth factors have been implicated in regulation of cell survival. For example, fibroblasts depend on IGF-1 [46, 47] and certain hemopoietic cell on interleukin-3 [48-50] for survival in culture; removal of these cytokines or blockade of their receptors leads to cell death in the respective target cells. Recent studies by us [51-53] and others [54] demonstrated that the EGFR similarly regulates programmed cell death in human keratinocytes. If the EGFR is blocked either by antagonistic antibodies or by EGFR-selective inhibitors of the tyrphostin class, keratinocytes become highly susceptible to induction of cell death by cell stress, for example stress imposed by ultraviolet irradiation (Jost, Gasparro and Rodeck, unpublished observation). This apoptosis-prone phenotype is associated with downregulation of a protector against apoptotic cell death, Bcl-xL [52-54]. The signal transduction pathways which lead from the EGFR to Bcl-xL expression or survival, represent an area of intense investigation. This is partly due to the realization that inappropriate signaling from the EGFR and other ErbB family members is frequent in epithelial neoplasms including squamous cell carcinomas (see next paragraph) and is likely to provide a worthwhile target for improving the sensitivity of such tumors to chemotherapeutic attack.

The EGF-R in skin diseases

Activation of the epidermal growth factor receptor (EGF-R) has been shown to stimulate cell cycle progression of normal epidermal keratinocytes through autocrine and paracrine mechanisms [23, 25, 27, 34]. In normal skin, the EGF-R is most strongly, although not exclusively, expressed in the basal layer of the epidermis [13, 55], consistent with the involvement of the EGF-R in epidermal growth control. Several observations indicate that abnormalities in expression of the EGF-R and/or its ligands TGF-alpha and AR are common features of hyperproliferative and neoplastic epithelia. For example, in psoriatic epidermis, the EGF-R is overexpressed not just in the basal layer, but in all nucleated strata of the epidermis [56-58], consistent with the suprabasal proliferation that occurs in this disease. Furthermore, both TGF-alpha and AR are found at elevated levels throughout the nucleated layers of psoriatic epidermis [59-62]. In squamous carcinomas, overexpression of the EGFR is commonly observed [63-67] consistent with the view that EGFR signaling is upregulated and constitutive in such tumors. Furthermore, epithelial neoplasms frequently coexpress high levels not only of the EGF-R but also of its ligands, TGF-alpha [68] or AR [69, 70], thereby creating constitutive autocrine loops dependent on the EGF-R. Direct support for a role of EGFR activation in the development of skin tumors comes from studies in transgenic mice, in which overexpression of TGF-alpha targeted to the epidermis elicits hyperplasia, hyperkeratosis, papillomas, and squamous cell carcinomas [71-74].

Abbreviations

AR Amphiregulin
BC Betacellulin
EGFR Epidermal growth factor receptor
EPI Epiregulin
HB Heparin binding
IGF Insulin-like growth factor
NF Nuclear factor
PI-3K Phosphoinositol-3-kinase
PLC Phospholipase
PK Protein kinase
STAT Signal transducer and activator of transcription
TGF Transforming growth factor

CONCLUSION

The EGFR family has been found to control multiple aspects of epithelial biology including cell survival, proliferation, migration, and differentiation. Any of these may serve distinct functions in the establishment of a hyperproliferative or neoplastic epidermal phenotype. Current investigations focus on the relative contribution and integration of EGFR-dependent intracellular signals as they relate to the malignant phenotype in keratinocytes. Insights from these studies are likely to have wider implications for other epithelial neoplasms which, like squamous cell carcinomas, are characterized by deregulated EGFR-dependent signaling pathways.

Accepted on 15/5/00

REFERENCES

1. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem 1962; 237: 1555-62.

2. Cohen S, Elliott GA. The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J Invest Dermatol 1963; 40: 1-5.

3. Cohen S. The stimulation of epidermal proliferation by a specific protein (EGF). Dev Biology (Orlando) 1965; 12: 394-407.

4. Carpenter G, Lembach KJ, Morrison MM, Cohen S. Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts. J Biol Chem 1975; 250: 4297-304.

5. Buhrow SA, Cohen S, Staros JV. Affinity labeling of the protein kinase associated with the epidermal growth factor receptor in membrane vesicles from A431 cells. J Biol Chem 1982; 257: 4019-22.

6. Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 1984; 309: 418-25.

7. Sergeant A, Saule S, Leprince D, Begue A, Rommens C, Stehelin D. Molecular cloning and characterization of the chicken DNA locus related to the oncogene erbB of avian erythroblastosis virus. EMBO Journal 1982; 1: 237-42.

8. Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J, Waterfield MD. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 1984; 307: 521-7.

9. Alroy I, Yarden Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Letters 1997; 410: 83-6.

10. Klapper LN, Kirschbaum MH, Sela M, Yarden Y. Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv Cancer Res 2000; 77: 25-79.

11. Prestrelski SJ, Arakawa T, Wu CS, O'Neal KD, Westcott KR, Narhi LO. Solution structure and dynamics of epidermal growth factor and transforming growth factor alpha. J Biol Chem 1992; 267: 319-22.

12. O'Keefe E, Battin T, Payne R Jr. Epidermal growth factor receptor in human epidermal cells: direct demonstration in cultured cells. J Invest Dermatol 1982; 78: 482-7.

13. Nanney LB, McKanna JA, Stoscheck CM, Carpenter G, King LE. Visualization of epidermal growth factor receptors in human epidermis. J Invest Dermatol 1984; 82: 165-9.

14. King LE Jr, Gates RE, Stoscheck CM, Nanney LB. The EGF/TGF alpha receptor in skin. J Invest Dermatol 1990; 94: 164S-70S.

15. Xian W, Rosenberg MP, DiGiovanni J. Activation of erbB2 and c-src in phorbol ester-treated mouse epidermis: possible role in mouse skin tumor promotion. Oncogene 1997; 14: 1435-44.

16. Marikovsky M, Lavi S, Pinkas-Kramarski R, Karunagaran D, Liu N, Wen D, Yarden Y. ErbB-3 mediates differential mitogenic effects of NDF/heregulin isoforms on mouse keratinocytes. Oncogene 1995; 10: 1403-11.

17. Srinivasan R, Poulsom R, Hurst HC, Gullick WJ. Expression of the c-erbB-4/HER4 protein and mRNA in normal human fetal and adult tissues and in a survey of nine solid tumour types. J Pathol 1998; 185: 236-45.

18. Ibrahim SO, Vasstrand EN, Liavaag PG, Johannessen AC and Lillehaug JR. Expression of c-erbB proto-oncogene family members in squamous cell carcinoma of the head and neck. Anticancer Res 1997; 17: 4539-46.

19. Coffey RJ Jr, Sipes NJ, Bascom CC, Graves-Deal R, Pennington CY, Weissman BE, Moses HL. Growth modulation of mouse keratinocytes by transforming growth factors. Cancer Res 1988; 48: 1596-602.

20. Kobayashi T, Hashimoto K, Okumura H, Asada H, Yoshikawa K. Endogenous EGF-family growth factors are necessary for the progression from the G1 to S phase in human keratinocytes. J Invest Dermatol 1998; 111: 616-20.

21. Cook PW, Pittelkow MR, Shipley GD. Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors. J Cell Physiology 1991; 146: 277-89.

22. Pittelkow MR, Cook PW, Shipley GD, Derynck R, Coffey R Jr. Autonomous growth of human keratinocytes requires epidermal growth factor receptor occupancy. Cell Growth Differ 1993; 4: 513-21.

23. Vardy DA, Kari C, Lazarus GS, Jensen PJ, Zilberstein A, Plowman GD, Rodeck U. Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1. J Cell Physiol 1995; 163:257-65.

24. Dlugosz AA, Cheng C, Williams EK, Darwiche N, Dempsey PJ, Mann B, Dunn AR, Coffey R Jr,Yuspa SH. Autocrine transforming growth factor alpha is dispensible for v-rasHa-induced epidermal neoplasia: potential involvement of alternate epidermal growth factor receptor ligands. Cancer Res 1995; 55: 1883-93.

25. Coffey RJ Jr, Derynck R, Wilcox JN, Bringman TS, Goustin AS, Moses HL, Pittelkow MR. Production and auto-induction of transforming growth factor-alpha in human keratinocytes. Nature 1987; 328: 817-20.

26. Coffey R Jr, Graves-Deal R, Dempsey PJ, Whitehead RH, Pittelkow MR. Differential regulation of transforming growth factor alpha autoinduction in a nontransformed and transformed epithelial cell. Cell Growth Differ 1992; 3: 347-54.

27. Cook PW, Mattox PA, Keeble WW, Pittelkow MR, Plowman GD, Shoyab M, Adelman JP, Shipley GD. A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin. Mol Cell Biol 1991; 11: 2547-57.

28. Stoll S, Garner W, Elder J. Heparin-binding ligands mediate autocrine epidermal growth factor receptor activation In skin organ culture. J Clin Invest 1997; 100: 1271-81.

29. Dvir A, Milner Y, Chomsky O, Gilon C, Gazit A, Levitzki A. The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers. J Cell Biol 1991; 113: 857-65.

30. Ben-Bassat H, Rosenbaum-Mitrani S, Hartzstark Z, Shlomai Z, Kleinberger-Doron N, Gazit A, Plowman G, Levitzki R, Tsvieli R, Levitzki A. Inhibitors of epidermal growth factor receptor kinase and of cyclin-dependent kinase 2 activation induce growth arrest, differentiation, and apoptosis of human papilloma virus 16-immortalized human keratinocytes. Cancer Res 1997; 57: 3741-50.

31. Sibilia M, Wagner EF. Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 1995; 269: 234-8.

32. Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D, LaMantia C, Mourton T, Herrup K, Harris RC, et al. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 1995; 269: 230-4.

33. Hansen LA, Alexander N, Hogan ME, Sundberg JP, Dlugosz A, Threadgill DW, Magnuson T, Yuspa SH. Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development. Am J Pathol 1997; 150: 1959-75.

34. Rheinwald JG, Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 1977; 265: 421-4.

35. Peus D, Hamacher L, Pittelkow MR. EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation. J Invest Dermatol 1997; 109: 751-6.

36. Monzon RI, McWilliams N, Hudson LG. Suppression of cornified envelope formation and type 1 transglutaminase by epidermal growth factor in neoplastic keratinocytes. Endocrinology 1996; 137: 1727-34.

37. Chen CS, Lavker RM, Rodeck U, Risse B, Jensen PJ. Use of a serum-free epidermal culture model to show deleterious effects of epidermal growth factor on morphogenesis and differentiation. J Invest Dermatol 1995; 104: 107-12.

38. Wakita N, Takigawa M. Activation of epidermal growth factor receptor promotes late terminal differentiation of cell-matrix interaction-disrupted keratinocytes. J Biol Chem 1999; 274: 37285-91.

39. Miettinen PJ, Berger JE, Meneses J, Phung Y, Pedersen RA, Werb Z, Derynck R. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 1995; 376: 337-41.

40. Barrandon Y, Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-alpha and epidermal growth factor. Cell 1987; 50: 1131-7.

41. Schultz GS, White M, Mitchell R, Brown G, Lynch J, Twardzik DR, Todaro GJ. Epithelial wound healing enhanced by transforming growth factor-alpha and vaccinia growth factor. Science 1987; 235: 350-2.

42. Ando Y, Jensen PJ. Epidermal growth factor and insulin-like growth factor I enhance keratinocyte migration. J Invest Dermatol 1993; 100: 633-9.

43. Fang KS, Ionides E, Oster G, Nuccitelli R, Isseroff RR. Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields. J Cell Science 1999; 112: 1967-78.

44. Polakowska RR, Piacentini M, Bartlett R, Goldsmith LA, Haake AR. Apoptosis in human skin development: morphogenesis, periderm, and stem cells. Dev Dyn 1994; 199: 176-88.

45. White E. Life, death, and the pursuit of apoptosis. Genes Dev 1996: 101.

46. Harrington EA, Bennett MR, Fanidi A, Evan GI. c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines. Embo Journal 1994; 13: 3286-95.

47. Kulik G, Klippel A, Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase and Akt. Mol Cell Biol 1997; 17: 1595-606.

48. Baffy G, Miyashita T, Williamson JR, Reed JC. Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J Biol Chem 1993; 268: 6511-9.

49. Mekori YA, Oh CK, Metcalfe DD. IL-3-dependent murine mast cells undergo apoptosis on removal of IL-3. Prevention of apoptosis by c-kit ligand. J Immunol 1993; 151: 3775-84.

50. Kinoshita T, Yokota T, Arai K, Miyajima A. Suppression of apoptotic death in hematopoietic cells by signalling through the IL-3/GM-CSF receptors. Embo J 1995; 14: 266-75.

51. Rodeck U, Jost M, Kari C, Shih DT, Lavker RM, Ewert DL, Jensen PJ. EGF-R dependent regulation of keratinocyte survival. J Cell Science 1997; 110: 113-21.

52. Rodeck U, Jost M, DuHadaway J, Kari C, Jensen PJ, Risse B, Ewert DL. Regulation of Bcl-xL expression in human keratinocytes by cell-substratum adhesion and the epidermal growth factor receptor. Proc National Academy of Sciences of the United States of America 1997; 94: 5067-72.

53. Jost M, Class R, Kari C, Jensen PJ, Rodeck U. A central role of Bcl-X(L) in the regulation of keratinocyte survival by autocrine EGFR ligands. J Invest Dermatol 1999; 112: 443-9.

54. Stoll SW, Benedict M, Mitra R, Hiniker A, Elder JT, Nunez G. EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL. Oncogene 1998; 16: 1493-9.

55. Nanney LB, Stoscheck CM, King L Jr, Underwood RA, Holbrook KA. Immunolocalization of epidermal growth factor receptors in normal developing human skin. J Invest Dermatol 1990; 94: 742-8.

56. Nanney LB, Stoscheck CM, Magid M, King L Jr. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis. J Invest Dermatol 1986; 86: 260-5.

57. King L Jr, Gates RE, Stoscheck CM, Nanney LB. Epidermal growth factor/transforming growth factor alpha receptors and psoriasis. J Invest Dermatol 1990; 95: 10S-2S.

58. Nanney LB, Yates RA, King LE Jr. Modulation of epidermal growth factor receptors in psoriatic lesions during treatment with topical EGF. J Invest Dermatol 1992; 98: 296-301.

59. Gottlieb AB, Chang CK, Posnett DN, Fanelli B, Tam JP. Detection of transforming growth factor alpha in normal, malignant and hyperproliferative human keratinocytes. J Exp Med 1988; 167: 670-5.

60. Elder JT, Fisher GJ, Lindquist PB, Bennett GL, Pittelkow MR, Coffey RJ Jr, Ellingsworth L, Derynck R, Voorhees JJ. Overexpression of transforming growth factor alpha in psoriatic epidermis. Science 1989; 243: 811-4.

61. Finzi E, Harkins R, Horn T. TGF-alpha is widely expressed in differentiated as well as hyperproliferative skin epithelium. J Invest Dermatol 1991; 96: 328-32.

62. Cook PW, Pittelkow MR, Keeble WW, Graves-Deal R, Coffey RJ Jr, Shipley GD. Amphiregulin messenger RNA is elevated in psoriatic epidermis and gastrointestinal carcinomas. Cancer Res 1992; 52: 3224-7.

63. Ozawa S, Ueda M, Ando N, Shimizu N, Abe O. Prognostic significance of epidermal growth factor receptor in esophageal squamous cell carcinomas. Cancer 1989; 63: 2169-73.

64. Mukaida H, Toi M, Hirai T, Yamashita Y, Toge T. Clinical significance of the expression of epidermal growth factor and its receptor in esophageal cancer. Cancer 1991; 68: 142-8.

65. Scambia G, Panici PB, Battaglia F, Ferrandina G, Almadori G, Paludetti G, Maurizi M, Mancuso S. Receptors for epidermal growth factor and steroid hormones in primary laryngeal tumors. Cancer 1991; 67: 1347-51.

66. Shirasuna K, Hayashido Y, Sugiyama M, Yoshioka H, Matsuya T. Immunohistochemical localization of epidermal growth factor (EGF) and EGF receptor in human oral mucosa and its malignancy. Virchows Arch A Pathol Anat Histopathol 1991; 418: 349-53.

67. Maurizi M, Scambia G, Benedetti Panici P, Ferrandina G, Almadori G, Paludetti G, De Vincenzo R, Distefano M, Brinchi D, Cadoni G, et al. EGF receptor expression in primary laryngeal cancer: correlation with clinico-pathological features and prognostic significance. Int J Cancer 1992; 52: 862-6.

68. Derynck R, Goeddel DV, Ullrich A, Gutterman JU, Williams RD, Bringman TS, Berger WH. Synthesis of messenger RNAs for transforming growth factors alpha and beta and the epidermal growth factor receptor by human tumors. Cancer Res 1987; 47: 707-12.

69. Le Jeune S, Leek R, Horak E, Plowman G, Greenall M, Harris AL. Amphiregulin, epidermal growth factor receptor and estrogen receptor expression in human primary breast cancer. Cancer Res 1993; 53: 3597-602.

70. Barnard JA, Graves-Deal R, Pittelkow MR, DuBois R, Cook P, Ramsey GW, Bishop PR, Damstrup L, Coffey RJ. Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family. J Biol Chem 1994; 269: 22817-22.

71. Vassar R, Fuchs E. Transgenic mice provide new insights into the role of TGF-alpha during epidermal development and differentiation. Genes Dev 1991; 5: 714-27.

72. Vassar R, Hutton ME, Fuchs E. Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis. Mol Cell Biol 1992; 12: 4643-53.

73. Dominey AM, Wang XJ, King L Jr, Nanney LB, Gagne TA, Sellheyer K, Bundman DS, Longley MA, Rothnagel JA, Greenhalgh DA, et al. Targeted overexpression of transforming growth factor alpha in the epidermis of transgenic mice elicits hyperplasia, hyperkeratosis, and spontaneous, squamous papillomas. Cell Growth Differ 1993; 4: 1071-82.

74. Amundadottir LT, Nass SJ, Berchem GJ, Johnson MD, Dickson RB. Cooperation of TGF alpha and c-Myc in mouse mammary tumorigenesis: coordinated stimulation of growth and suppression of apoptosis. Oncogene 1996; 13: 757-65.