The cutaneous epidermal growth factor network: Can it be translated clinically to stimulate hair growth?
1. Georgetown Dermatology, Washington, DC. email@example.com
The influences exerted by the epidermal growth factor receptor (EGFR) on the skin act at multiple levels, which involve compartments that normally express EGFR. These include the basal and suprabasal layers of the epidermis, sebaceous glands, and the outer root sheath of the hair follicles. The physiological roles of EGFR ensure epidermal renewal and integrity, along with a gatekeeping and function and hair growth stimulation functions. Important cellular functions that are altered during EGF receptor blocking therapy consist of epidermal differentiation, proliferation, apoptosis, and migration, with an overall dominating effect of inducing growth arrest and terminal differentiation of the keratinocytes in the basal layers. The effects of EGFR blockage on the hair cycle include terminal differentiation of the hair follicle, which in certain cases may be associated with trichomegaly. Trichomegaly of the eyelashes may occur as an isolated occurrence or, frequently, as part of a generalized phenomenon that may be associated with the use of the EGFR inhibitors. Molecular changes associated with EGFR blockage are discussed, relevant to their association with hair growth. Modulation of Akt, AP2α, CDK4, Notch-1, p27KIP1, and Hedgehog expression are involved in the initiation of the hair cycle and inducement of the anagen phase, followed by proliferation and differentiation of the hair follicles. Epidermal growth factor receptor inhibitors have been developed as therapeutic molecules directed against cancer; in these regimens the knowledge of EGF receptor signaling functions has been translated into significant clinical results. However, among their various collateral effects on the skin, hair growth is observed to occur in certain patients. A particular "wavy" hair phenotype is observed during the pharmacological EGFR receptor blockade, just as in murine transgenic models that carry loss of function of TGF-α or EGFR genes. A better characterization of the individual roles pertaining to the EGF family ligands and receptors, has the potential provide new strategies for the management of hair loss.
Discovery of a new hair phenotype
The first observations of an altered hair phenotype consisting of wavy hairs were by Francis Crew and Clyde Keeler, who in 1933 and 1935, respectively, observed two mouse strains having these abnormalities, currently known as waved-1 and waved-2 [1, 2]. The phenotypical alterations in the second strain were even more pronounced, consisting of "marcelled hairs" and curled vibrissae. A mixed strain between the two types of "waved" had a normal coat; at the time this created the impression that "two genes are involved" in the phenomenon. Indeed, the mutations inherited in a dominant fashion by these mice have recently being identified in perpetuated strains to pertain to loss of function of transformed growth factor alpha (TGF-α) and the epidermal growth factor receptor (egfr) gene, respectively [3, 4]. Not surprisingly, both alterations belong to the EGFR network, one of the most developed biological signal systems connecting the intercellular environment to cell response mechanisms.
The upside of trichomegaly: it has more causes than alopecia
Various causes of trichomegaly have been identified and include idiopathic and inflammatory causes, infections, and drugs (Table 1). It is probable that the pathogenetic mechanisms are different, at least at the level of the initiation of the cellular chain of events. However, main players are likely to consist of a facilitating genetic background, along with the presence of growth factors and cytokine mediators of inflammation and cell growth and differentiation. A distinct mediation of alopecia and hair growth through androgenic receptors is well known, and will not be discussed in this review, although a cross-talk between the androgen receptor and the epidermal growth factor has been demonstrated in a neoplastic setting, leading to effects on cellular mediators of cellular proliferation and cycle progression . Recent work has revealed a potential role for the transcription factor NFATc1, in conjunction with the CDK4 circuitry in mediating the hair growth produced by cyclosporine . While the role of the EGF-EGFR system in the morphogenesis and regulation of the hair cell cycle is well established, we will discuss the alterations produced in the biology of hair growth by this key mechanism and review the effects of its pharmacological manipulation with the class of EGFR inhibitor drugs. Infections and pro-inflammatory states may also act on hair cell regulation, at least in part, through a down-regulation of EGF, which will be discussed in the next paragraphs. Hypertrichosis has been reported with all the EGFR inhibitors in current wide clinical use: gefitinib (Iressa®), erlotinib (Tarceva®), and cetuximab (Erbitux®).
The EGFR network
The four receptors of the EGFR family (EGFR or HER1, ERBB2 or HER2, ERBBS or HER3, and ERBB4 or HER4) are stimulated by seven known ligands (such as EGF, HBEGF, TGF-α, Amphiregulin [AREG], Epigen, Epiregulin, Betacellulin [BTC]), with resultant homo- and heterodimerization and activation of adaptor mediators. This complex pathway of stimulation generates the transcription of complex factors responsible for skin proliferation, differentiation, apoptosis, and carcinogenesis  (Fig. 1).
EGFR network has broad effects on the skin
Specific patterns of EGF binding are fundamental for the normal growth and differentiation of the skin, as well as the abnormal growth and differentiation associated with pathological conditions like psoriasis, neoplasms, paraneoplastic syndromes, and viral infections . Epidermal growth factor receptor is mainly expressed in the basal layer of the skin, where it enhances epidermal growth and wound healing and mediates an inhibition of differentiation. The results of EGFR inhibition consist of a decrease in the growth and migration of keratinocytes, which is occasionally accompanied by the occurrence of a mixed inflammatory infiltrate in the upper part of the dermis .
EGFR and the hair follicle homeostasis
The effects of EGF on the homeostasis of the hair follicle were demonstrated over thirty years ago, when EGF injection in sheep skin produced hair loss and epidermal thickening . Conversely, EGFR down-regulation has been linked with hair follicle formation [11, 12]. Similar effects are produced in human skin. Epithelial growth factor (EGF) was shown to retard hair growth ; the EGF receptor mediates the termination of the anagen stage .
Deciphering the exceedingly complicated EGF-EGFR network produced nothing short of surprises in regard to its effects on hair growth and structure. A normal level of EGFR stimulation appears to participate in the physiological growth of the proliferating skin compartment, the basal keratinocytes and the outer root sheath cells, which strongly express this receptor. It is notable that relatively small decreases in the EGFR activity, such as those produced by an incomplete egfr +/- phenotype, do not result in evident morphological alteration of the hair. However, more significant positive or negative variations of the EGFR signaling intensity produce changes consisting of wavy hairs and curly whiskers. Therefore, either dominant-negative EGFR or constitutive activated mutants result in the expression of a wavy coat, demonstrating that a balance in the paracrine and autocrine influences is required for maintenance of the natural appearance of the hair in rodents .
However, the influence of one particular ligand is less likely to influence the system, as redundancy ensures that another stimulus can take over the initiation of the signaling pathway. The effect of up to three missing factors (EGF, TGF-α, and Amphiregulin) have been shown to be compensated by other EGF family ligands, as shown by the presence of an identical hair phenotype for triple null mice when compared with the mice that are just TGF-α null. Thus, EGF, which produces hyperproliferation of the basal cell layers and arrest of the hair follicle development, competes for the same receptors with other ligands, such as ERBB2, which induces hyperplasia of keratinocytes in the basal layers (with a particular high intensity in the hair follicles) . Yet, EGFR appears to be necessary for the development and health of the hair unit, which is demonstrated by the failure of hair growth (absence of progression into the follicle cycle and occurrence of follicular necrosis) when EGFR is totally absent, as in an egfr -/- mutation. Therefore, manipulation of EGF receptors (and in particular of EGFR which is important not only as the most frequent single path of signaling, but also as a heterodimerization partner for the other receptors) may be more efficient in controlling streamed signaling than interference with the particular molecular ligands.
The effects of the EGFR cascade are not limited to influences on the induction or maintenance of a proliferating fraction of the hair progenitors. Cycling of the hair follicles also appears to be influenced by several of the ligands. Negative influences are exerted, by which BTC retards the induction of the hair cycle . Although important in embryonic follicle morphogenesis, EGF resumes later roles in the initiation of hair growth in the first hair cycle through EGFR/ErbB2 signaling and in the subsequent maintenance of hair morphogenesis by activation at the entry into anagen and silencing before the entry into telogen and catagen.
EGF may therefore be a more important factor than other EGFR agonists for initiation of hair growth; the lack of its EGF receptor competitor TGF-α, for example, appears to be associated with a faster hair regrowth after plucking .
Molecular effects downstream of EGFR blockade and synergy with other cellular effectors
Cooperation of EGF with other molecules appears to be necessary, such as with the downregulation of TGF-α, but synergism with other effectors may be also required. For example, a long hair phenotype can be seen with a null FGF-5 (fibroblast growth factor 5) , whereas TGF-α repression, along with producing a wavy phenotype, stimulates the growth of hair. Prompting of hair growth by EGFR blockage is likely to represent a complex interplay that involves modulation of several other cellular mediators (Fig. 2).
The cyclin-dependent kinase (CDK) inhibitor, p27KIP1, is also a marker for growth arrest. This protein is increased in gefitinib, erlotinib, and in cetuximab-treated patients [18, 19, 20]. A twofold increase of cytoplasmic labeling for p27KIP1 was observed in epidermal keratinocytes in patients treated with cetuximab compared to untreated skin and this was most pronounced in the basal and suprabasilar keratinocytes . The end results associated with its activation are concordant with the physiological activities of EGFR to regulate keratinocyte proliferation and to facilitate late terminal differentiation of the hair follicle keratinocytes (a decrease of the Ki67 labeling index, suppressed MAPK activation, and reduced thickness of the corneal layers) [22, 23]. In-situ hybridization for p27(kip1) showed its localization in the cortex and the upper half of the hair bulb. Higher expression of p27 levels are manifest during anagen than during telogen, correlating with mRNA expression of two hair differentiation markers: type I hair keratin (Ha3) and high sulfur protein B2. Thus, this CDK inhibitor, p27(kip1), appears to be involved in the differentiation of follicular epithelial cells  and mediates the effects of EGFR blockers on the epidermal and follicular homeostasis .
Activator protein 2 (AP-2) represents a family of large DNA binding transcription factors mediating ERGF transcription. AP-2 represses the EGFR gene expression and is involved in controlling the balance between growth and differentiation in the epidermis , which was also demonstrated using a conditional knockout model . Such a role in controlling skin proliferative homeostasis is supported by its demonstrated over-expression in the basal cells that are not actively cycling . The lack of epidermal AP-2 produces an over-expression of EGFR in the differentiating layers, which produces PI3k/Akt activation and keratinocyte hyperproliferation upon receptor activation . Conversely, AP-2alpha directly represses EGFR. By assuming a hitherto unrecognized repressive role in regulating EGFR gene transcription, elevated AP-2alpha levels are commonly associated with terminal differentiation . An AP-2alpha upregulation in the epithelial and mesenchymal follicle compartments appeared to be associated with the induction of major remodeling processes. Thus, in the follicular papilla, AP-2alpha is weakly expressed in telogen, then consistently upregulated in early anagen (during the initiation of hair shaft formation and active follicle downward growth), followed by a gradual decline, and a final upregulation in middle catagen. AP-2alpha expression was also found in the inner root sheaths, mostly expressed during early and middle anagen and during middle catagen . Furthermore, an AP-2 role in regulating the cyclic transformations of the HF is supported by the similarity of the hair follicle ectodermal-mesodermal interaction system with the patterns of embryonic follicle development, in which it plays an important role . Accordingly, during the early stages of hair follicle morphogenesis, AP-2alpha is over-expressed in the epidermal embrional tissue, in the basal keratinocytes of the hair follicle bud, and subsequently in the inner root sheath cells . The overexpression of AP-2 in anagen is consistent with the known EGFR signal suppression, which allows the stage to be set for active cellular proliferation and differentiation, whereas its weak expression in telogen is compatible with the need for a brief EGF induction of entry into anagen. The roles of AP-2 are involved in important cellular functions that span effects from terminal differentiation to premalignant transformation [16, 27].
The hedgehog (HH)/GLI family of intercellular signaling proteins is required for hair follicle morphogenesis during embryogenesis and for the subsequent regulation of follicular cycling and growth . This data is supported by the action of a topically applied Hh-agonist, which appeared to facilitate the transition from telogen to the anagen phase in murine hair . EGFR signaling modulates the target gene expression profile of GLI transcription factors in epidermal cells and is critical for the GLI-induced cell cycle progression in epidermal cells. The synergism occurs through a convergence of EGFR and HH/GLI signaling at the level of promoters of several GLI target genes. In favor of a synergistic role in anagen hair follicles in-between the EGFR and HH pathways is the co-expression of EFGR, activated ERK1/2, and the GLI/EGF, target IL1R2, in human hair. This inter-pathway interaction appears to be important in specifying the fate of ORS cells . The significance of EGFR blockage appears to consist in a positive effect on the GLI-mediated induction of epidermal stem cell marker expression, as the administration of EGF treatment seems to neutralize the process .
Activation of quiescent follicular stem cells that leads to proliferation and differentiation was shown to result from a downregulation of the transcription factor NFATc1 (nuclear factor of activated T cells c1), which is preferentially expressed in the follicular bulge and embryonic hair buds compared to the normal epidermis . This downregulation consequently suppresses cyclin-dependent kinase 4 (CDK4) inhibition, which relieves its physiologic stimulatory activity on G1/S progression of the quiescent stem cells . CDK4 is also expressed in the basal follicular bulge, in the developing hair germ, and in the matrix of the anagen hair bulb [Horsley V 2008]. CDK upregulation, with its positive effects on cell cycle progression, was observed during the transition from telogen to the anagen phase . NFATc1 inhibition was associated with hair growth during cyclosporine therapy or after conditional ablation of the NFATc1 gene . EGFR signal downregulation, by escape from the stimulatory effect of integrins, is one of the components that relieves suppression of CDK4 in epithelial cells, therefore allowing for stem cell activation and cellular cycle progression .
Activation of Notch signaling, was shown to serve complex functions in the hair follicle development, such as the regulation of hair follicle ORS and stem cell fate . Notch-1 and its ligands, Jagged -1 and Jagged-2, are expressed in the ectodermal-derived cells of the follicle and in the suprabasilar cells of the outer root sheath . The expression of Notch-1 in the upper follicle bulb overlaps Jagged-1 expression, correlating with bulb cell differentiation into hair shaft cortical and cuticle keratinocytes . EGFR signaling was identified as a key negative regulator of Notch1 gene expression in primary human keratinocytes, which appears to involve a mechanism relying on transcriptional suppression of p53 by the EGFR effector c-Jun .
Akt, a marker for cellular survival and proliferation was found to be increased during treatment with the EGFR inhibitor, erlotinib, in patients with metastatic breast cancer . Activation of Akt produces hyperplasia of the interfollicular epidermis and the hair follicles, probably caused by the proliferation of keratinocytes in the interfollicular epidermis and the outer root sheath of hair follicles . The PI3k/Akt activation was able to increase the number and induce proliferation of the epidermal progenitors in the outer root sheath of hair follicles; new hair growth was observed in mice after Akt signaling activation. A self-renewal of stem cells rather that the generation of committed progenitors appears to be responsible for the process. The entry of the hair follicles into the anagen phase of the hair cycle was determined by Akt activation of the resting follicles . A contrary effect of retardation of hair follicle development was seen by deletion of Akt1 .
Collateral skin effects of EGFR inhibitors
Epidermal growth factor receptor (EGFR) inhibitors have become an essential integral part of the research efforts and of the standard treatment employed for therapy for many types of cancers, such as colorectal, head and neck, lung, breast, pancreatic, sarcoma, ovarian, esophageal, and renal carcinoma.
Epidermal growth factor receptor inhibition is associated with new and puzzling dermatologic side effects. The most frequently encountered adverse effect entails a non-severe skin eruption that consists of sterile follicular pustules that commonly responds to treatment. However, significant discomfort for the patients occurs as a result of using this treatment modality. Skin toxicities include the classical "acneiform" pustular rash, xerosis, pruritus, paronychia, and hair abnormalities . Effects on the eyes occur in about one third of patients and have been classified into 3 groups: changes in the tear film (dysfunctional tear syndrome), changes in the eyelids (trichomegaly, blepharitis, meibomitis), and others (corneal ulcerations, iridocyclitis) .
Clinical applicability: the story of anthralin
Anthralin, an EGFR signaling inhibitor exerts inhibitory effects that are independent of the number of EGFR receptors; they do not act specifically on the EGF-R pathway . In a clinical study of alopecia areata patients comparing anthralin (dithranol) to azelaic acid, the former was able to produce complete responses in 56.2 percent of cases and an average terminal hair re-growth score (RGS) of 1.37 (score of 0 meaning inadequate response and 2 signifying a complete response). This showed that a topical treatment that inhibits the EGFR pathway can be clinically useful to stimulate hair re-growth in patchy alopecia areata (AA) .
The feasibility of using a topical medication to modulate the EGF-EGFR system is suggested by a murine biological system in which EGF-rich mouse saliva, which is passed by the animals onto their ventral chin area, was able to produce a robust proliferative response . Topical delivery of an EGFR inhibitor may, by analogy, locally influence the epidermal growth signaling homeostasis in the opposite direction. A more effective local delivery of the drug should be able to induce greater variations in the cutaneous EGF signaling than a systemic manipulation, possibly also at the expense of less systemic toxicity.
Trichomegaly in the setting of cancer treatment
Development of trichomegaly during the treatment with the EGFR inhibitors cetuximab, erlotinib, and gefitinib is well known [41-48] (Fig. 3). However, it is interesting to remark that in all cases published to date, trichomegaly occurred in association with the development of a tumor response, which varied from stabilization of the disease to the induction of significant tumor shrinkage [41-48]. The presence of sustained increases in hair length suggests that EFGR inhibitor use for the purpose of stimulating hair growth may be a realistic application.
Trichomegaly in inflammatory eye conditions: presence of EGF. Why are some specific sites affected more by trichomegaly?
The concentration of EGF was found to be higher in healthy subjects than in patients with mild inactive trachoma, with even progressively lower levels in cases of moderate and severe disease (1748, 750, 543, and 466 pg/ml, respectively, p<0.0001) . It then appears interesting to note that such inflammatory conditions of the eye, are associated with trichomegaly; one study quantified an increase over normal from 8.73 to 9.66 mm for the upper eyelid and from 5.64 to 6.32 mm in patients with uveitis .
Although the causes for this association are not entirely clear, it is possible that a contributing factor is the low EGF encountered in the inflammatory disorders of the eye. From this perspective, it is important to elucidate why not all hairs on the body surface share the same responsiveness to EGF signal ablation. As with many other physiological phenomena, we can speculate that along with the concentration of the EGF ligand, the density of the receptors (EGFR) and the variation in the agonistic activity may be contributors to the growth signal escape.
Whereas the EGF serum level remains constant, 328 +/– 21 pg/ml , much higher concentrations appear to be expressed in the tear fluid; measurements of hEGF of (8466 pg/ml)  to 6589 pg/ml, or 1784 pg/ml as determined by Satici et al, have been recorded. . However, during corneal disease the concentration of EGF in tear fluid considerably decreases to levels even lower than those found during short term stimulation of reflex tearing (mean 2762 pg/ml) . Similar differences are found also for the EGFR concentrations between the skin and lacrimal tissue . Trichomegaly may therefore be a result of the sudden drop in the epidermal growth pathway signaling, which appears to be an important homeostatic mechanism of the eye . Such a possibility is raised by the presence of an elevated physiologic concentration of EGF in the tears, which would sustain a higher variation in the transmitted signal during the EGFR blockade.
In summary, current data support the fact that EGF is central in the regulation of hair morphogenesis, with its cyclical on/off switch being important for the progression of the hair cycle. Cooperation with other molecules appears to be necessary; the downregulation of some effectors (TGF-α), but synergism with others (FGF-5) result in a longer hair phenotype . On the other hand, continuous expression of EGF, or TGF-α, although producing a wavy phenotype, impedes the growth of hair. Therefore, cyclic variations in the level of EGFR, which is a key intermediate in signal transmission, may result in hair growth and produce new hair formation. Conversely, continuous EGFR blockage would interfere with new hair formation and a severe decrement in its function can even result in hair destruction. The principle of cycling and maintenance of a low level of stimulation has proven biological relevance. It was demonstrated as a paradigm by the results of studying the stimulation/inhibition for androgenic blockade in prostate cancer through using a LHRH agonist administered continuously and by ensuring contraception through providing uniform levels of the hormones that otherwise produce ovulation when subjected to brisk variations.
A better understanding of the individual roles of the EGF family ligands and receptors, as well as their interplay and physiological phases, has the potential to produce a revolution in the management of hair loss. Availability of topical EGFR blockers and the development of more specific molecules that will stimulate the hair growth pathways will build on the fact that EGFR blockade can produce long-term hair growth. An apparent lack of tolerance to the hair growth effect after long term treatment, as recently seen, contrasts with other systemic and cutaneous toxicities of medications from the EGFR blocking class, thus creating the possibility of clinically relevant long-lasting effects.
Furthermore, it is possible that particular and yet unidentified subclasses of EGF receptors are responsible for hair growth and other effects on the skin. Of substantial importance is avoidance of the pro-inflammatory effect of EGFR inhibition on the skin, which may cause secondary alopecia, or otherwise interfere with the growth rate and formation of the hair. Characterization of the EGF pathway and its cell surface receptors may therefore lead to a revolution in the management of hair growth disorders.
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