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Fibroblast growth factor and epidermal growth factor in hair development
Fibroblast Growth Factor and Epidermal Growth Factor in Hair Development Diana Lee du Cros Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington, U.S.A.
Hair follicles arise in developing skin as a result of a complex of interactions that are likely to be mediated by diffusible,
cell- and matrix-bound factors. Growth factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF) have been implicated in the control of epidermal and mesenchymal cell function, and it is likely that they also affect proliferation and differentiation of the cells of the cutament. Immunolocalization neous appendages during develo in developing of basic FGF adjacent to areas o 4 roliferation and in mature follicles suggests t R at this factor may regulate the mitotic activity of epithelially-derived cells; acidic FGF, cells of the on the other hand, ap ears in the differentiating in the formation of follicle bulb and may t K erefore participate structural corn onents of the follicle or of the fiber. EGF has been identifie B as a potent modulator of cellular growth and is also present during follicle differentiation. These factors may act through autocrine and paracrine mechanisms be-
T
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he hair follicles in mammals form as a consequence of interactions between the epidermis and mesenchyme
during fetal development [l]. Follicle initiation and development begin with the condensation of dermal fibroblasts and epidermal keratinocytes at initiation sites. The cells form aggregations adjacent to each other but on opposite sides of the basement membrane. This close association is maintained during subsequent follicle development as the epiderma1 cells begin to proliferate and penetrate the dermis as plugs. As each follicle differentiates, the epidermally derived cells surround the dermal condensation and incorporate it into a pocket at the base. The dermally derived structure is now called the dermal papilla. Grafting studies have shown that it is essential for normal hair follicle function and fiber production. The first follicles to a pear during embryonic development in the mouse are the vibrissa Pollicles. These are initiated in rows on the snout at approximately 12 d of fetal age [2] and are highly vascular [3]. Pelage or coat follicles, on the other hand, are relatively avascular. Up to 30% of these follicles are initiated before birth and the remainder in the first few days following birth [4,5]. Following maturation, the follicles undergo cycles of activity and rest of approximately 4 weeks duration [4]. The cycle has three phases: anagen, the growth stage when fiber production takes place; catagen, the transition stage when follicle activity declines; and telogen, the resting phase when the follicles have contracted towards the surface of the skin and fiber production has ceased. These phases are accomReprint re uests to: Dr. Diana L. du Cros, School of Medicine SM-20, University o 9 Washmgton, Seattle, WA 98195. Abbreviations: aFGF, acidic fibroblast growth factor; FGFR, fibroblast growth factor receptor.
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cause their receptors are also found on epidermally derived and mesenchymal structures in the skin. We have studied the effects of these growth factors on hair follicle development in the newborn mouse. Daily injections for 1 week after birth resulted in significant changes in the morphogenesis of the hair follicle population. Histologic examination of skin of FGF-treated mice suggested that the growth factor had affected hair follicle initiation and development, which resulted in a significant delay in the first and subsequent hair cycles when compared to control animals. Because aFGF and bFGF are not readily diffusible, these effects remained confined to the area of treatment. In contrast, EGF affected the whole body coat of the treated animals, induced hyperkeratinization of the skin, and caused a significant delay in hair follicle development. J Invest Dermatol 1993
panied by changes in skin thickness. This is at a maximum during anagen with a thickened dermis overlying a deep adipose layer and the epidermis is at its thinnest. During catagen, the dermis and adipose layers begin to thin and the epidermis thickens slightly. The skin is thinnest in telogen. GROWTH
IN SKIN
Throughout follicle morphogenesis and in mature skin, a close association is maintained between the mesenchymal and epithelially derived cells of the hair follicles, indicating that interactions between the two cell populations are essential for the development and growth to occur. It is becoming increasingly clear that these interactions are mediated in many instances by a multiplicity of factors that are diffusible or bound to the cell surfaces and the surrounding matrices [6 - 81. Growth factors have been implicated in the control of a number of the complex of events taking place during embryonic development. These proteins appear to exert their effects via autocrine or paracrine pathways between the cell types. Most of the major growth factor families and their receptors have been implicated in the control of skin cell function. These include epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-CX) [9- 131; fibroblast growth factor (FGF) [14181; transforming growth factor-beta and bone morphogenetic protein-4 [19-211; and insulin-like growth factor-l [22,23]. Data strongly suggest that EGF and two members of the FGF family, acidic and basic (aFGF and bFGF) play important roles in the processes of hair follicle morphogenesis. In vitro studies have shown that aFGF, bFGF, and EGF are mitogens for skin-derived cells [24,25] and all have been localized in skin prior to and during hair follicle initiation and development [17,26-281. The roles of these
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%elow. FIBROBLAST
GROWTH
FACTOR
The fibroblast growth factor family comprises seven closely related proteins remarkable for their mitogenic effects on cells of mesodermal, ectodermal, and neuroectodermal origin, and for their affinity for he arin-like molecules [29,30]. The most widely known and studie s members are aFGF and bFGF, which share approximately 55% structural homology [31]. The latest FGF family member identified is keratinocyte growth factor (KGF), distinguishable from the others by its specificity for epithelially derived cells [32-341. Effects on Skin Cells In Vitro The FGFs were initially recognized for their effects on fibroblastic cells. They are mitogenic for cultured dermal fibroblasts and dermal components of hair follicles [15,25,35]. A bFGF-l’k1 e molecule has been detected in the medium of cultured human dermal fibroblasts [14] and was found to stimulate cell growth, indicating an autocrine function in vitro. The growth factor can also act in a paracrine fashion [16]. Basic FGF, a mitogen for melanocytes [36], was found to be produced by keratinocytes in co-culture with melanocytes [16]. A close association between the two cell types was found to be essential because keratinocyte-conditioned medium alone lacked growth-promoting activity. Although aFGF and bFGF were initially regarded as having no effect on epithelial cells [37,38], more recent studies have shown that they do stimulate proliferation of keratinocyte cultures [15,24,39-411. H owever, the different forms of FGF display some cell specificity. Acidic FGF appears to be a more potent mitogen than bFGF for epidermal keratinocytes [15,24], whereas the growth of dermal fibroblasts is stimulated more by bFGF than aFGF [15]. Basic FGF has been thought to be a more potent mitogen than aFGF [29], unless aFGF is added in the presence of heparin, which reportedly stabilizes the molecule [42,43] and prolongs its half-life [44]. FGF and Embryonic Development Studies have shown that bFGF is involved in various developmental events such as angiogenesis in mammals [45] and establishment of anterio-posterior polarity and early mesodermal induction in amphibia [46,47]. Other members of the FGF family also ap ear to be involved in mammalian embryogenesis as shown by di g erential expression patterns in develo ment [48]. In the rat embryo, immunoreactive aFGF was identi Ked in mesodermal tissues throughout development but, at later stages, the growth factor was confined to mesenchymal tissues whereas fully differentiated tissues undergoing differentiation, showed no immunoreactivity [49]. In contrast, aFGF mRNA transcripts are widely distributed in the mouse at all stages of development [48]. FGF and its receptors have been implicated in the control of epidermal and mesenchymal cell function [17,27,50 - 521. Evidence of a role for aFGF in skin differentiation and follicle morphogenesis comes from immunolocalization studies [27]. The growth factor was polarized to the upper and lower surfaces of basal keratinocytes during follicle initiation and was found in both epidermal and mesenchymal components during follicle development. In mature follicles, strong immunoreactivity was observed in bulb cells in the area of the zone of keratinization suggesting that aFGF is involved in the formation of the inner root sheath, one of the structural components of the follicle, or in early hair fiber differentiation. In fetal rodent and adult human skin, bFGF and its mRNA are primarily epidermis-associated, although the basement membrane and adjacent mesenchymal cells also appeared to contain the growth factor and its transcri ts [17,50]. During follicle morphogenesis in the sheep, bFGF was !?ound throughout the epidermis prior to follicle initiation, and at the basement membrane zone of the follicle plugs during early follicle formation [27]. The presence of the growth factor in epidermally derived cells during follicle induction suggested that bFGF might act as a signal molecule for cell migration and proliferation. As follicles matured, bFGF was associated
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with cells of the outer root sheath and was localized at the interface between the bulb matrix and dermal papilla. The presence of bFGF adjacent to the proliferative zone of mature follicles suggests that it . regulating mitotic activity in the follicle bulb may be involved m cells. PGF Receptors in the Skin Both aFGF and bFGF probably act as autocrine or paracrine factors in skin because their receptors are also found on epidermally derived and mesenchymal cells [51,52]. However, FGF is not ty ical of secreted proteins because it lacks a signal peptide, but it has ! een found to have very strong affinities for heparin and heparin sulfate molecules. This may account for its specific localization to basement membranes and extracellular matrix (ECM) in vivo and mechanisms involving degradative enzymes, such as heparanases, have been proposed recently for its release from cells and ECM [30]. During hair follicle morphogenesis, bFGF is found in regions that are known to be rich in he arin sulfate proteoglycans (HSPG) [53]. It appears that low-a P nity receptors for bFGF are cell-associated heparin-like molecules, syndecan being one possible candidate [54 - 561. The binding of bFGF to low-aflinity receptors may be a prerequisite for binding to its high-affinity receptor [57]. At least five receptors have been identified for the FGF family [58]. T wo high-affinity receptors, designated FGFRl and FGFR2, both bind aFGF and bFGF, whereas an isoform of FGFR2 binds KGF [52]. In situ hybridization techniques have revealed that genes for these receptors are expressed in the skin during development [18,52]. FGFRl is confined to the mesenchyme just below the dermal-epidermal junction and to the dermal papillae of hair follicles in fetal mouse skin [52]. In contrast, FGFR2 is expressed both in follicle bulb cells and dermal papillae. These findings clearly implicate the various forms of FGF in follicle morphogenesis although their specific functions remain to be defined. FGF and Mouse Hair Follicle Development To obtain more information on the roles of aFGF and bFGF during skin development, a study was undertaken to examine their effects in uivo [59]. Daily subcutaneous injections of either aFGF or bFGF given at a dose rate of 1 pg/g body weight for 7 d after birth induced significant changes in hair follicle development. In the presence of bFGF, pale areas were apparent on all animals in the region of the injection site by the fourth day; areas of control animals injected with bovine serum albumin (BSA) did not differ in appearance from the rest of the body. Skin sections from control animals revealed follicles at various stages of development. Those initiated before birth had produced fibers that were present in the pilary canals. Follicles initiated after birth were still at the peg stage (Fig 1.4). In contrast, the skin of bFGF-injected mice contained follicles that were not as advanced as those of the controls, and hair-peg stage follicles were absent (Fig 1B). This suggested that the development of follicles initiated after birth had been inhibited. In addition, the thickness of the connective tissue underlying the panniculus carnosus had greatly increased in the bFGF-injected areas. This tissue contained tendon-like arrays of fibroblasts and collagen, and remained thickened until about 2 d after bFGF injections ceased. By days 7 to 9, areas of hairless, unpigmented skin persisted at the sites treated with bFGF (Fig 2). The skin of both control and bFGF-injected animals had thickened, indicating rapid follicle growth, but there were fewer follicles in the bFGF-affected regions than in control sites. The same trend was apparent by the time the skin was at maximum thickness around ages 7 - 14 d. Emergence of hair in the affected patches was first seen at 14 - 16 d of age and hair growth continued in these regions until they were mostly covered about 4 d later. At 21 d of age, the coats of all mice appeared similar, but follicles of control mice had entered telogen (Fig 1C). In contrast, the skin of bFGF-injected mice was still in late anagen (Fig 1D) and was not in the telogen phase until around 30 d, by which stage the hair follicles of control animals were entering the anagen phase of the second hair cycle. This could also be observed when the mice were shaved: pale areas were readily apparent at the sites affected by bFGF, whereas control mice were an evenly pigmented dark color. Sites injected with BSA only were indistinguishable from the surround-
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e 1. Skin sections from mice given subcutaneous injections of BSA alone or bFGF and BSA daily for 7 d from birth: controls injected with BSA (A) 3- ay-old mouse and (C) 21-day-old mouse; mice injected with bFGF and BSA (B) 3-day-old mouse skin showing a large build-up of connective tissue in response to bFGF (arrour), and (0) 21-day-old mouse.
T
ing hairy skin at all ages. Furthermore, skin sam les taken from the control animals injected with BSA alone showe 8. similar patterns of follicle develo ment and activity to non-injected mice. Thus, it appeared that tK e growth factor had affected hair follicle initiation and development, which resulted in a significant delay in the first and subsequent hair cycles when compared to control animals. As a consequence of suppressing follicle development, the normal hair cycle was delayed over the 7-d period during which bFGF was administered, but re-entered the normal cycle when exogenous bFGF was no longer present. This had an overall effect on the hair cycle such that bFGF-injection sites were out of synchrony by 7 d compared to adjacent sites on the animals. Fifteen weeks after the injection regime, telogen follicles were observed at the growth factor injection site surrounded by follicles in anagen. Preliminary results obtained by injecting aFGF into neonatal mice show a similar phenomenon, that is, delay in follicle initiation and development. However, the effects on the hair cycle remain to be fully evaluated. The mechanism of action of FGF on the hair follicles is not yet known. However, interactions between the growth factor, its highaffinity rece tors, and heparin-like molecules in the skin matrices and hair fol Picles are most likely involved. Such interactions can explain the observed interference with follicle morphogenesis at the initiation stage and, later, during differentiation. Follicle initiation is dependent on interactions between epidermal keratinocytes and the mesenchymal cells that form dermal condensations at the initiation sites. Basic FGF is expressed by epidermal keratinocytes and is mitogenic for mesenchymal cells [24,25], and thus could be important in the cellular interactions that take lace between these two cell ty es during initiation. However, at g.igh concentrations, the growt R factor clearly inhibits these associations, presumably by oc-
cupying all available high-affinity cell and matrix receptors. This could interfere with the condensation process by blocking cellular communication, thus delaying initiation of those follicles formed after birth by interfering with cell migration and aggregation. Studies are now being conducted to test this possibility. Later in follicle development, bFGF is localized to the basement membrane region between the dermal papilla and the epithelialderived cells of the follicle bulb [27]. The ECM of the dermal papilla contains basement-membrane-type components such as HSPG [60] and the papilla itself is rich in FGF receptors [51,52]. Large amounts ofexogenous FGF might be expected to bind to and downregulate these receptors, thus altering the interaction between the papilla and follicle bulb. This could influence follicle growth rate and, ultimately, the hair cycle. EPIDERMAL
GROWTH
FACTOR
EGF and TGFd are the most extensively studied members of EGF family of growth factors. They are structurally related bind to the same receptor. Isolated by Cohen in 1962 [61], EGF since been shown to be a potent modulator of the growth differentiation of a wide variety of cell types.
the and has and
Effects on Skin Cellsh Vitro A wide variety of cultured cells of ectodermal and mesodermal origin respond to EGF [37,62]. The growth factor is a mitogen for human foreskin fibroblasts regardless of cell density [63]. However, as with FGF, cell-specific responses have been observed. In a study of the effects of EGF on fibroblasts derived from the skin and vibrissa follicles of sheep, Pisansarakit et al [25] showed that not all fibroblasts are res onsive to the growth factor. EGF receptors are found on cultured t!broblasts [64] and EGF is secreted by human foreskin fibroblasts in culture [l 11.AlI of these
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FGF AND EGF IN HAIR DEVELOPMENT
Figure 2. Appearance of g-day-old mice following daily injections of BSA alone (leffl or bFGF and BSA (right). Mice were injected in the dorsal region near ehe tail (arrows) for 7 d from birth.
results indicate that the rowth factor probably acts as both an autocrine and paracrine e 4 ector. This concept is further supported by evidence that the closely-related molecule, TGF-ar, is auto-induced in cultured human keratinocytes [13]. EGF also acts as a mitogen for keratinocytes in culture [24], which is consistent with its role as a potent proliferative agent for epidermal cells in skin-organ cultures [9], and both TGF-c~ and EGF romote cell migration, an essential as ect of keratinocyte growth P651. Furthermore, EGF can dramatica Ply increase the longevity of these cells in culture [38], implying that it delays terminal differentiation. EGF and Its Receptors In Viva EGF was first isolated from the submaxillary glands of male mice [61] and has been found to accumulate in rodent skin [66,67]. The presence of EGF receptors in the epidermally derived components of the follicle [68-701 and an EGF-like molecule in extracts of isolated vibrissae capsules [25], are strong evidence of a role for EGF in hair follicle function. In ovine skin, the distribution of the growth factor has been studied immunohistochemically during wool follicle development [28]. EGF immunoreactivity was localized in the intermediate and peridermal layers of the epidermis during the early stages of follicle formation, then in the outer root sheath cells and differentiating cells of the sebaceous glands as the follicles matured. No reaction was found in the proliferative layers of the epidermis and follicle bulb. The specific distribution of EGF in the e idermis and follicles suggested that the growth factor regulates s ifferentiation rather than proliferation in this species. EGF rece tors are distributed at sites reported for EGF immunoreactivity alt Kough the receptors appear to have a much wider distribution in skin [68,69,71,72]. However, these data contrast to those of Green et al [12], who suggested that the distribution and number of receptors in rat skin cells were correlated with proliferation. The findings of Nanney and co-workers [69,70] for human skin also support the hypothesis that EGF receptors are correlated with roliferation, although receptor expression was not limited to pro11J erating tissues. Adamson and Meek [73], who studied EGF receptors in the developing mouse, suggested that EGF initially
stimulates proliferation in embryonic cells then has a role in differentiation as tissues mature. An association between receptor distribution and cellular expression of EGF may indicate regulation of cell turnover via an autocrine mechanism such as that suggested for TGF-cx [13]. The abundance of EGF receptors in the skin suggests that EGF and the related TGF-a may have wound healing functions in this organ [74,75]. Effects of EGF on the Skin Besides hyperproliferation of the epidermis when injected into juvenile and adult mice [76-781, EGF also induces keratinization and an overall thickening of rodent skin [lo]. Furthermore, injection of EGF into mouse skin has rofound effects on hair follicles, delaying development [76,77], an B decreasing the rate of hair growth and hair diameter. We have examined the effects of EGF on the B6C3-based strain of mouse. Newborn animals were injected for 7 d from the day of birth with 1 pg/ body weight EGF in 0.15 M saline solution. The most dramatic e P ect seen early in the regime was on body growth rate. EGF-treated mice were noticeably smaller by 3 d of age than their control littermates, which were injected with saline solution alone. Unlike mice injected with bFGF, which showed only local effect, the skin and hair follicles of EGF-injected animals were affected over the whole of the body surface. Examination of skin sections from 3-day-old mice revealed fewer follicles in skin treated with EGF than in control skin. At 5 d of age, the skinofEGF mice was pale and had begun to flake, indicating that substantial keratinization was occurring. This was confirmed by examination of skin sections, wherein the stratum corneum was thicker than in control skin and was separating from the epidermis, which was also thickened (Fig 3A,B). Unlike FGF-treated skin, however, the overall skin thickness was significantly reduced compared to controls at this age, although there a peared to be a similar number of follicles. The data indicated that t Re hair follicles had initiated but that EGF was delaying their development. Hence, the experimental animals were paler than controls because few hairs had penetrated the skin surface. By 8 d of age, the skin of EGF-injected mice was still signifi-
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Figure 3. Skin sections from mice given subcutaneous injections of saline or EGF daily for and (C) 21-day-old mouse; EGF-injected mice (B) 5 d old and (0) 21 d old.
cantly thinner than that of controls, although the follicles of both treated and control mice were obviously in anagen. The coats of treated animals were shorter than those of controls reflecting the retardation in development, and were noticeably duller due to the presence of curved hair fibers as shown previously [76]. At 21 d, the EGF-treated mice had thicker skin than the controls because hair follicles of the latter were in telogen (Fig 3C,D). By 30 d, differences in the skin and hair follicles of experimental and control mice were still noticeable. This is similar to treatment with bFGF, whereby bFGF-affected skin and follicles remained out of synchrony with surrounding untreated areas. However, the effect on the animals was not as great as that observed for FGF. In addition, treated mice remained smaller than controls although the differences decreased with age (Fig 4). Moore et al [76] compared EGFtreated mice with underfed animals and concluded that the effects of EGF on hair development were not attributable solely to changes in body weight. The main difference between the study described here and previous work [76,77] is the effect on the hair cycle. Previously, retardation of the first cycle was only noted in early anagen and the cycle had recovered to its normal stage by 21 d [77]. Here, the effects carried through to the second cycle. The differences diminished with time and the periodicity of the EGF-treated follicles eventually became synchronized with follicles of the pelages of control littermates. The response to EGF is linked to the time of injection. Application of the growth factor in the first few days after birth elicits a greater res onse than after this period [79]. Infusion of EGF into pregnant sReep at late stages of gestation results in hypertrophy of the skin of the fetus as well as degenerative changes in the developing wool follicles, leading to shedding of fetal wool fibers (801. In older, mature rodents, EGF induces keratinization of tail and footpad epidermis but not dorsal skin, and changes in weight were not observed [lo]. In contrast, injection of EGF antiserum to neonatal mice resulted in a delay of eyelid opening and tooth eruption, although a delay in weight gain, as with EGF, was noted [8 11. Interestingly, hair-growth rates in these animals was accelerated, indicating that EGF does indeed have a normal physiologic role in hair follicle growth and differentiation. There are several possible mechanisms by which EGF modifies
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d from birth: controls injected with saline (A) 5-day-old moose
hair follicle development and fiber growth. For example, by maintaining proliferation and differentiation, as seen by an increase in the thickness of the epidermis and by hyperkeratinization of the skin after birth, respectively, EGF could have profound effects on follicle development. Furthermore, although EGF is initially involved in cell proliferation, Moore er al [76] suggested that the factor acts as a mitotic inhibitor of follicle bulb cells; this would lead to the observed decline in hair fiber growth rate and decrease in fiber diameter. Similar results have been obtained by infusing sheep with EGF [82]. It has been proposed that, following the initial stimulation by EGF, down-regulation of EGF receptors occurs, resulting in the inhibitory response [80,83]. Adamson and Warshaw [83] suggested that, in embryonic tissues, maximal stimulation by endogenous EGF was already occurring so that exogenous EGF would lead to receptor down-regulation. Carpenter and Cohen [62] suggested that, following binding of EGF to its receptor, the decrease in receptor activity that occurs is due to internalization of the EGF/receptor complex without the accompanying production of new rece tors. This process could remove the stimulus by saturating, and su t: sequently down-regulating, receptors already present. Comparative Effects of bPGF and EGP There are several differences in the effects of bFGF and EGF on the skin. One of the most striking effects is in the area of skin affected. Administration of EGF affects the whole body coat, resumably by entering the circulatory system. In contrast, skin a P ected by bFGF remains confined to a relatively small area surrounding the injection sites on the backs of the animals. This is expected because exogenous FGF is likely to be sequested by matrix-associated HSPG before it is able to enter the circulation. Another difference between administration of EGF and bFGF concerns the long-term effects produced by each. Although EGF produces effects on fiber characteristics for up to two or three hair cycles, only the first hair cycle is partly asynchronous. Basic FGF has been shown to have a dramatic effect on the hair cycle whereby suppression of the cycle and its subsequent asynchrony is maintained. Finally, injection of EGF into neonatal mice produces premature eyelid opening and incisor eruption as well as retarding growth rate [61,76], whereas bFGF does not affect any of these processes.
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Administration of EGF to neonatal mice resulted in hyperkeratinization of the skin, initial epidermal thickening, and delay in hair-follicle development. Furthermore, the effects were widespread over the bod y, unlike bFGF with which the areas affected by the growth factor were very localized. The EGF-treated animals were also significantly smaller than their control littermates. Data indicated that hair follicles were initiating during the EGF-treatment regime but that their development was retarded. In addition, the hair fibers produced by the follicles were not as long and were curved at their tips. Injection of EGF also resulted in delay of the first hair cycle but this was not as dramatic as the effect produced by bFGF and was not prolonged much beyond the first cycle. The findings presented here suggest important roles for both FGF and EGF in follicle initiation and development. Although the mechanisms of action of these growth factors are not yet known, future studies encompassing receptor binding and cell behavior, such as migration and proliferation, in response to these potent molecules should prove enlightening in further understanding the processes of hair follicle morphogenesis.
I thank Dr. K. A. Holbrook for her valued support and helpful discussions; Dr. G.P.M. Moorefor critical reading ofthe manuscript; Ms.]. Fangmanfor cutting and staining the tissue sections; Mr. R.A. Underwoodfor photographic assistance; and Dr. L.S. Cousens and Chiron Corporarionfor Be& of bFGF. Thepnancial support of the National Ins&&s of Health (HD 176641, the Dermatology Foundation, and Dermik Laboratories is gratefully acknowledged.
REFERENCES
Pigure
mice injected with saline (l&j or EGF (right) 4. Eight-day-old daily for 7 d from birth. At this age, there was a considerable difference in the growth rates of the two animals. Note also the flaking skin of the EGFtreated animal.
SUMMARY nents of the epidermis and mesenbetween corn ollicles during fetal development and at thyme give rise to the hair p” birth. This complex of interactions is likely to be mediated in part by growth factors that are diffusible or cell and matrix bound. Immunolocalization studies indicate that aFGF and bFGF are directly involved in the process of hair follicle mo hogenesis: aFGF appears to have a role in epithelial differentiation ‘g 0th in the early stages of follicle develo ment and at maturity; bFGF, located adjacent to areas of proli Peration, may be regulating the mitotic activity of epithelial-derived cells. The specific distribution of EGF in the skin and throughout follicle morphogenesis suggests that this growth factor has a more important role in differentiation than in proliferation. Both bFGF and EGF have profound and dramatic effects on the develo ing skin when given at critical stages of growth. Administrain tion o 4 bFGF during the eriod of hair follicle morphogenesis mouse skin appeared to in Kibit the initiation and development process. Follicles in bFGF-treated areas appeared able to form and develop normally but at a much slower rate. This suppressed state continued until the influence of exogenous FGF was removed, whereupon the normal rate of development resumed and the full hair coat was formed. As a consequence of suppressing follicle development, the normal hair cycle was delayed over the period during which bFGF was administered, but also resumed normal activity when the exogenous bFGF was removed. However, the effects on the hair cycle appeared to be long-term because evidence of the disruption to the cycle was still visible 4 months after the animals were injected with the growth factor. Interactions
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Moscatelli D: High and low affinity binding sites for basic fibroblast growth factor on cultured cells: absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells. J Cell Physiol 131:123- 130, 1987
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Bernfield M, Sanderson RD: Syndecan, a developmentally regulated cell surface proteoglycan that binds extracellular matrix and growth factors. Phil Trans R Sot Land B 327:171- 186, 1990
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Moore GPM, Panaretto BA, Robertson D: Epidermal growth factor delays the develo ment of the epidermis and hair follicles of mice during growth o P the first coat. Anat Ret 205:47-55, 1983
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Hollis DE, Chapman RE, Panaretto BA, Moore GPM: MO hological changes in the skin and wool fibres of merino sheeo in3: sed with . mouse epidennal growth factor. Aust J Biol Sci 3634’19-434, 1983
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growth factor. J
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1
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Nanney LB, Stoscheck CM, King LE. Underwood RA, Holbrook KA: Immunolocalization of epidermal growth factor receptors in normal developing human skin. J Invest Dermatol94:742-748, 1990 Nanney LB, Magid M, Stoscheck CM, King LE: Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J Invest Dermatol 83:385 - 393.1984 Green MR, Couchman JR: Distribution of epidermal growth factor receptors in rat tissues during embryonic skin development, hair formation, and the adult hair growth cycle. J Invest Dermatol 83:118-123.1984 Green MR, Couchman
JR: Differences
in human
skin between
the
rat with epidermal growth facresponse. Pediatr Res 20:468-
serum on newborn
mice.
Hair follicles arise in developing skin as a result of a complex of interactions that are likely to be mediated by diffusible,
cell- and matrix-bound factors. Growth factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF) have been implicated in the control of epidermal and mesenchymal cell function, and it is likely that they also affect proliferation and differentiation of the cells of the cutament. Immunolocalization neous appendages during develo in developing of basic FGF adjacent to areas o 4 roliferation and in mature follicles suggests t R at this factor may regulate the mitotic activity of epithelially-derived cells; acidic FGF, cells of the on the other hand, ap ears in the differentiating in the formation of follicle bulb and may t K erefore participate structural corn onents of the follicle or of the fiber. EGF has been identifie B as a potent modulator of cellular growth and is also present during follicle differentiation. These factors may act through autocrine and paracrine mechanisms be-
T
101:104s-113s,
he hair follicles in mammals form as a consequence of interactions between the epidermis and mesenchyme
during fetal development [l]. Follicle initiation and development begin with the condensation of dermal fibroblasts and epidermal keratinocytes at initiation sites. The cells form aggregations adjacent to each other but on opposite sides of the basement membrane. This close association is maintained during subsequent follicle development as the epiderma1 cells begin to proliferate and penetrate the dermis as plugs. As each follicle differentiates, the epidermally derived cells surround the dermal condensation and incorporate it into a pocket at the base. The dermally derived structure is now called the dermal papilla. Grafting studies have shown that it is essential for normal hair follicle function and fiber production. The first follicles to a pear during embryonic development in the mouse are the vibrissa Pollicles. These are initiated in rows on the snout at approximately 12 d of fetal age [2] and are highly vascular [3]. Pelage or coat follicles, on the other hand, are relatively avascular. Up to 30% of these follicles are initiated before birth and the remainder in the first few days following birth [4,5]. Following maturation, the follicles undergo cycles of activity and rest of approximately 4 weeks duration [4]. The cycle has three phases: anagen, the growth stage when fiber production takes place; catagen, the transition stage when follicle activity declines; and telogen, the resting phase when the follicles have contracted towards the surface of the skin and fiber production has ceased. These phases are accomReprint re uests to: Dr. Diana L. du Cros, School of Medicine SM-20, University o 9 Washmgton, Seattle, WA 98195. Abbreviations: aFGF, acidic fibroblast growth factor; FGFR, fibroblast growth factor receptor.
0022-202X/93/$06.00
Copyright
cause their receptors are also found on epidermally derived and mesenchymal structures in the skin. We have studied the effects of these growth factors on hair follicle development in the newborn mouse. Daily injections for 1 week after birth resulted in significant changes in the morphogenesis of the hair follicle population. Histologic examination of skin of FGF-treated mice suggested that the growth factor had affected hair follicle initiation and development, which resulted in a significant delay in the first and subsequent hair cycles when compared to control animals. Because aFGF and bFGF are not readily diffusible, these effects remained confined to the area of treatment. In contrast, EGF affected the whole body coat of the treated animals, induced hyperkeratinization of the skin, and caused a significant delay in hair follicle development. J Invest Dermatol 1993
panied by changes in skin thickness. This is at a maximum during anagen with a thickened dermis overlying a deep adipose layer and the epidermis is at its thinnest. During catagen, the dermis and adipose layers begin to thin and the epidermis thickens slightly. The skin is thinnest in telogen. GROWTH
IN SKIN
Throughout follicle morphogenesis and in mature skin, a close association is maintained between the mesenchymal and epithelially derived cells of the hair follicles, indicating that interactions between the two cell populations are essential for the development and growth to occur. It is becoming increasingly clear that these interactions are mediated in many instances by a multiplicity of factors that are diffusible or bound to the cell surfaces and the surrounding matrices [6 - 81. Growth factors have been implicated in the control of a number of the complex of events taking place during embryonic development. These proteins appear to exert their effects via autocrine or paracrine pathways between the cell types. Most of the major growth factor families and their receptors have been implicated in the control of skin cell function. These include epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-CX) [9- 131; fibroblast growth factor (FGF) [14181; transforming growth factor-beta and bone morphogenetic protein-4 [19-211; and insulin-like growth factor-l [22,23]. Data strongly suggest that EGF and two members of the FGF family, acidic and basic (aFGF and bFGF) play important roles in the processes of hair follicle morphogenesis. In vitro studies have shown that aFGF, bFGF, and EGF are mitogens for skin-derived cells [24,25] and all have been localized in skin prior to and during hair follicle initiation and development [17,26-281. The roles of these
0 1993 by The Society for Investigative
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FACTORS
Dermatology,
Inc.
VOL. 101, NO. 1, SUPPLEMENT.
rowth
factors,
JULY
EGF and bFGF
1993
FGF AND EGF IN HAIR DEVELOPMENT
in particular,
will be discussed
%elow. FIBROBLAST
GROWTH
FACTOR
The fibroblast growth factor family comprises seven closely related proteins remarkable for their mitogenic effects on cells of mesodermal, ectodermal, and neuroectodermal origin, and for their affinity for he arin-like molecules [29,30]. The most widely known and studie s members are aFGF and bFGF, which share approximately 55% structural homology [31]. The latest FGF family member identified is keratinocyte growth factor (KGF), distinguishable from the others by its specificity for epithelially derived cells [32-341. Effects on Skin Cells In Vitro The FGFs were initially recognized for their effects on fibroblastic cells. They are mitogenic for cultured dermal fibroblasts and dermal components of hair follicles [15,25,35]. A bFGF-l’k1 e molecule has been detected in the medium of cultured human dermal fibroblasts [14] and was found to stimulate cell growth, indicating an autocrine function in vitro. The growth factor can also act in a paracrine fashion [16]. Basic FGF, a mitogen for melanocytes [36], was found to be produced by keratinocytes in co-culture with melanocytes [16]. A close association between the two cell types was found to be essential because keratinocyte-conditioned medium alone lacked growth-promoting activity. Although aFGF and bFGF were initially regarded as having no effect on epithelial cells [37,38], more recent studies have shown that they do stimulate proliferation of keratinocyte cultures [15,24,39-411. H owever, the different forms of FGF display some cell specificity. Acidic FGF appears to be a more potent mitogen than bFGF for epidermal keratinocytes [15,24], whereas the growth of dermal fibroblasts is stimulated more by bFGF than aFGF [15]. Basic FGF has been thought to be a more potent mitogen than aFGF [29], unless aFGF is added in the presence of heparin, which reportedly stabilizes the molecule [42,43] and prolongs its half-life [44]. FGF and Embryonic Development Studies have shown that bFGF is involved in various developmental events such as angiogenesis in mammals [45] and establishment of anterio-posterior polarity and early mesodermal induction in amphibia [46,47]. Other members of the FGF family also ap ear to be involved in mammalian embryogenesis as shown by di g erential expression patterns in develo ment [48]. In the rat embryo, immunoreactive aFGF was identi Ked in mesodermal tissues throughout development but, at later stages, the growth factor was confined to mesenchymal tissues whereas fully differentiated tissues undergoing differentiation, showed no immunoreactivity [49]. In contrast, aFGF mRNA transcripts are widely distributed in the mouse at all stages of development [48]. FGF and its receptors have been implicated in the control of epidermal and mesenchymal cell function [17,27,50 - 521. Evidence of a role for aFGF in skin differentiation and follicle morphogenesis comes from immunolocalization studies [27]. The growth factor was polarized to the upper and lower surfaces of basal keratinocytes during follicle initiation and was found in both epidermal and mesenchymal components during follicle development. In mature follicles, strong immunoreactivity was observed in bulb cells in the area of the zone of keratinization suggesting that aFGF is involved in the formation of the inner root sheath, one of the structural components of the follicle, or in early hair fiber differentiation. In fetal rodent and adult human skin, bFGF and its mRNA are primarily epidermis-associated, although the basement membrane and adjacent mesenchymal cells also appeared to contain the growth factor and its transcri ts [17,50]. During follicle morphogenesis in the sheep, bFGF was !?ound throughout the epidermis prior to follicle initiation, and at the basement membrane zone of the follicle plugs during early follicle formation [27]. The presence of the growth factor in epidermally derived cells during follicle induction suggested that bFGF might act as a signal molecule for cell migration and proliferation. As follicles matured, bFGF was associated
107s
with cells of the outer root sheath and was localized at the interface between the bulb matrix and dermal papilla. The presence of bFGF adjacent to the proliferative zone of mature follicles suggests that it . regulating mitotic activity in the follicle bulb may be involved m cells. PGF Receptors in the Skin Both aFGF and bFGF probably act as autocrine or paracrine factors in skin because their receptors are also found on epidermally derived and mesenchymal cells [51,52]. However, FGF is not ty ical of secreted proteins because it lacks a signal peptide, but it has ! een found to have very strong affinities for heparin and heparin sulfate molecules. This may account for its specific localization to basement membranes and extracellular matrix (ECM) in vivo and mechanisms involving degradative enzymes, such as heparanases, have been proposed recently for its release from cells and ECM [30]. During hair follicle morphogenesis, bFGF is found in regions that are known to be rich in he arin sulfate proteoglycans (HSPG) [53]. It appears that low-a P nity receptors for bFGF are cell-associated heparin-like molecules, syndecan being one possible candidate [54 - 561. The binding of bFGF to low-aflinity receptors may be a prerequisite for binding to its high-affinity receptor [57]. At least five receptors have been identified for the FGF family [58]. T wo high-affinity receptors, designated FGFRl and FGFR2, both bind aFGF and bFGF, whereas an isoform of FGFR2 binds KGF [52]. In situ hybridization techniques have revealed that genes for these receptors are expressed in the skin during development [18,52]. FGFRl is confined to the mesenchyme just below the dermal-epidermal junction and to the dermal papillae of hair follicles in fetal mouse skin [52]. In contrast, FGFR2 is expressed both in follicle bulb cells and dermal papillae. These findings clearly implicate the various forms of FGF in follicle morphogenesis although their specific functions remain to be defined. FGF and Mouse Hair Follicle Development To obtain more information on the roles of aFGF and bFGF during skin development, a study was undertaken to examine their effects in uivo [59]. Daily subcutaneous injections of either aFGF or bFGF given at a dose rate of 1 pg/g body weight for 7 d after birth induced significant changes in hair follicle development. In the presence of bFGF, pale areas were apparent on all animals in the region of the injection site by the fourth day; areas of control animals injected with bovine serum albumin (BSA) did not differ in appearance from the rest of the body. Skin sections from control animals revealed follicles at various stages of development. Those initiated before birth had produced fibers that were present in the pilary canals. Follicles initiated after birth were still at the peg stage (Fig 1.4). In contrast, the skin of bFGF-injected mice contained follicles that were not as advanced as those of the controls, and hair-peg stage follicles were absent (Fig 1B). This suggested that the development of follicles initiated after birth had been inhibited. In addition, the thickness of the connective tissue underlying the panniculus carnosus had greatly increased in the bFGF-injected areas. This tissue contained tendon-like arrays of fibroblasts and collagen, and remained thickened until about 2 d after bFGF injections ceased. By days 7 to 9, areas of hairless, unpigmented skin persisted at the sites treated with bFGF (Fig 2). The skin of both control and bFGF-injected animals had thickened, indicating rapid follicle growth, but there were fewer follicles in the bFGF-affected regions than in control sites. The same trend was apparent by the time the skin was at maximum thickness around ages 7 - 14 d. Emergence of hair in the affected patches was first seen at 14 - 16 d of age and hair growth continued in these regions until they were mostly covered about 4 d later. At 21 d of age, the coats of all mice appeared similar, but follicles of control mice had entered telogen (Fig 1C). In contrast, the skin of bFGF-injected mice was still in late anagen (Fig 1D) and was not in the telogen phase until around 30 d, by which stage the hair follicles of control animals were entering the anagen phase of the second hair cycle. This could also be observed when the mice were shaved: pale areas were readily apparent at the sites affected by bFGF, whereas control mice were an evenly pigmented dark color. Sites injected with BSA only were indistinguishable from the surround-
108s du CROS
THE JOURNAL
OF INVESTIGATIVE
DERMATOLOGY
e 1. Skin sections from mice given subcutaneous injections of BSA alone or bFGF and BSA daily for 7 d from birth: controls injected with BSA (A) 3- ay-old mouse and (C) 21-day-old mouse; mice injected with bFGF and BSA (B) 3-day-old mouse skin showing a large build-up of connective tissue in response to bFGF (arrour), and (0) 21-day-old mouse.
T
ing hairy skin at all ages. Furthermore, skin sam les taken from the control animals injected with BSA alone showe 8. similar patterns of follicle develo ment and activity to non-injected mice. Thus, it appeared that tK e growth factor had affected hair follicle initiation and development, which resulted in a significant delay in the first and subsequent hair cycles when compared to control animals. As a consequence of suppressing follicle development, the normal hair cycle was delayed over the 7-d period during which bFGF was administered, but re-entered the normal cycle when exogenous bFGF was no longer present. This had an overall effect on the hair cycle such that bFGF-injection sites were out of synchrony by 7 d compared to adjacent sites on the animals. Fifteen weeks after the injection regime, telogen follicles were observed at the growth factor injection site surrounded by follicles in anagen. Preliminary results obtained by injecting aFGF into neonatal mice show a similar phenomenon, that is, delay in follicle initiation and development. However, the effects on the hair cycle remain to be fully evaluated. The mechanism of action of FGF on the hair follicles is not yet known. However, interactions between the growth factor, its highaffinity rece tors, and heparin-like molecules in the skin matrices and hair fol Picles are most likely involved. Such interactions can explain the observed interference with follicle morphogenesis at the initiation stage and, later, during differentiation. Follicle initiation is dependent on interactions between epidermal keratinocytes and the mesenchymal cells that form dermal condensations at the initiation sites. Basic FGF is expressed by epidermal keratinocytes and is mitogenic for mesenchymal cells [24,25], and thus could be important in the cellular interactions that take lace between these two cell ty es during initiation. However, at g.igh concentrations, the growt R factor clearly inhibits these associations, presumably by oc-
cupying all available high-affinity cell and matrix receptors. This could interfere with the condensation process by blocking cellular communication, thus delaying initiation of those follicles formed after birth by interfering with cell migration and aggregation. Studies are now being conducted to test this possibility. Later in follicle development, bFGF is localized to the basement membrane region between the dermal papilla and the epithelialderived cells of the follicle bulb [27]. The ECM of the dermal papilla contains basement-membrane-type components such as HSPG [60] and the papilla itself is rich in FGF receptors [51,52]. Large amounts ofexogenous FGF might be expected to bind to and downregulate these receptors, thus altering the interaction between the papilla and follicle bulb. This could influence follicle growth rate and, ultimately, the hair cycle. EPIDERMAL
GROWTH
FACTOR
EGF and TGFd are the most extensively studied members of EGF family of growth factors. They are structurally related bind to the same receptor. Isolated by Cohen in 1962 [61], EGF since been shown to be a potent modulator of the growth differentiation of a wide variety of cell types.
the and has and
Effects on Skin Cellsh Vitro A wide variety of cultured cells of ectodermal and mesodermal origin respond to EGF [37,62]. The growth factor is a mitogen for human foreskin fibroblasts regardless of cell density [63]. However, as with FGF, cell-specific responses have been observed. In a study of the effects of EGF on fibroblasts derived from the skin and vibrissa follicles of sheep, Pisansarakit et al [25] showed that not all fibroblasts are res onsive to the growth factor. EGF receptors are found on cultured t!broblasts [64] and EGF is secreted by human foreskin fibroblasts in culture [l 11.AlI of these
VOL. 101, NO. 1, SUPPLEMENT,
JULY
1993
FGF AND EGF IN HAIR DEVELOPMENT
Figure 2. Appearance of g-day-old mice following daily injections of BSA alone (leffl or bFGF and BSA (right). Mice were injected in the dorsal region near ehe tail (arrows) for 7 d from birth.
results indicate that the rowth factor probably acts as both an autocrine and paracrine e 4 ector. This concept is further supported by evidence that the closely-related molecule, TGF-ar, is auto-induced in cultured human keratinocytes [13]. EGF also acts as a mitogen for keratinocytes in culture [24], which is consistent with its role as a potent proliferative agent for epidermal cells in skin-organ cultures [9], and both TGF-c~ and EGF romote cell migration, an essential as ect of keratinocyte growth P651. Furthermore, EGF can dramatica Ply increase the longevity of these cells in culture [38], implying that it delays terminal differentiation. EGF and Its Receptors In Viva EGF was first isolated from the submaxillary glands of male mice [61] and has been found to accumulate in rodent skin [66,67]. The presence of EGF receptors in the epidermally derived components of the follicle [68-701 and an EGF-like molecule in extracts of isolated vibrissae capsules [25], are strong evidence of a role for EGF in hair follicle function. In ovine skin, the distribution of the growth factor has been studied immunohistochemically during wool follicle development [28]. EGF immunoreactivity was localized in the intermediate and peridermal layers of the epidermis during the early stages of follicle formation, then in the outer root sheath cells and differentiating cells of the sebaceous glands as the follicles matured. No reaction was found in the proliferative layers of the epidermis and follicle bulb. The specific distribution of EGF in the e idermis and follicles suggested that the growth factor regulates s ifferentiation rather than proliferation in this species. EGF rece tors are distributed at sites reported for EGF immunoreactivity alt Kough the receptors appear to have a much wider distribution in skin [68,69,71,72]. However, these data contrast to those of Green et al [12], who suggested that the distribution and number of receptors in rat skin cells were correlated with proliferation. The findings of Nanney and co-workers [69,70] for human skin also support the hypothesis that EGF receptors are correlated with roliferation, although receptor expression was not limited to pro11J erating tissues. Adamson and Meek [73], who studied EGF receptors in the developing mouse, suggested that EGF initially
stimulates proliferation in embryonic cells then has a role in differentiation as tissues mature. An association between receptor distribution and cellular expression of EGF may indicate regulation of cell turnover via an autocrine mechanism such as that suggested for TGF-cx [13]. The abundance of EGF receptors in the skin suggests that EGF and the related TGF-a may have wound healing functions in this organ [74,75]. Effects of EGF on the Skin Besides hyperproliferation of the epidermis when injected into juvenile and adult mice [76-781, EGF also induces keratinization and an overall thickening of rodent skin [lo]. Furthermore, injection of EGF into mouse skin has rofound effects on hair follicles, delaying development [76,77], an B decreasing the rate of hair growth and hair diameter. We have examined the effects of EGF on the B6C3-based strain of mouse. Newborn animals were injected for 7 d from the day of birth with 1 pg/ body weight EGF in 0.15 M saline solution. The most dramatic e P ect seen early in the regime was on body growth rate. EGF-treated mice were noticeably smaller by 3 d of age than their control littermates, which were injected with saline solution alone. Unlike mice injected with bFGF, which showed only local effect, the skin and hair follicles of EGF-injected animals were affected over the whole of the body surface. Examination of skin sections from 3-day-old mice revealed fewer follicles in skin treated with EGF than in control skin. At 5 d of age, the skinofEGF mice was pale and had begun to flake, indicating that substantial keratinization was occurring. This was confirmed by examination of skin sections, wherein the stratum corneum was thicker than in control skin and was separating from the epidermis, which was also thickened (Fig 3A,B). Unlike FGF-treated skin, however, the overall skin thickness was significantly reduced compared to controls at this age, although there a peared to be a similar number of follicles. The data indicated that t Re hair follicles had initiated but that EGF was delaying their development. Hence, the experimental animals were paler than controls because few hairs had penetrated the skin surface. By 8 d of age, the skin of EGF-injected mice was still signifi-
THE JOURNAL
Figure 3. Skin sections from mice given subcutaneous injections of saline or EGF daily for and (C) 21-day-old mouse; EGF-injected mice (B) 5 d old and (0) 21 d old.
cantly thinner than that of controls, although the follicles of both treated and control mice were obviously in anagen. The coats of treated animals were shorter than those of controls reflecting the retardation in development, and were noticeably duller due to the presence of curved hair fibers as shown previously [76]. At 21 d, the EGF-treated mice had thicker skin than the controls because hair follicles of the latter were in telogen (Fig 3C,D). By 30 d, differences in the skin and hair follicles of experimental and control mice were still noticeable. This is similar to treatment with bFGF, whereby bFGF-affected skin and follicles remained out of synchrony with surrounding untreated areas. However, the effect on the animals was not as great as that observed for FGF. In addition, treated mice remained smaller than controls although the differences decreased with age (Fig 4). Moore et al [76] compared EGFtreated mice with underfed animals and concluded that the effects of EGF on hair development were not attributable solely to changes in body weight. The main difference between the study described here and previous work [76,77] is the effect on the hair cycle. Previously, retardation of the first cycle was only noted in early anagen and the cycle had recovered to its normal stage by 21 d [77]. Here, the effects carried through to the second cycle. The differences diminished with time and the periodicity of the EGF-treated follicles eventually became synchronized with follicles of the pelages of control littermates. The response to EGF is linked to the time of injection. Application of the growth factor in the first few days after birth elicits a greater res onse than after this period [79]. Infusion of EGF into pregnant sReep at late stages of gestation results in hypertrophy of the skin of the fetus as well as degenerative changes in the developing wool follicles, leading to shedding of fetal wool fibers (801. In older, mature rodents, EGF induces keratinization of tail and footpad epidermis but not dorsal skin, and changes in weight were not observed [lo]. In contrast, injection of EGF antiserum to neonatal mice resulted in a delay of eyelid opening and tooth eruption, although a delay in weight gain, as with EGF, was noted [8 11. Interestingly, hair-growth rates in these animals was accelerated, indicating that EGF does indeed have a normal physiologic role in hair follicle growth and differentiation. There are several possible mechanisms by which EGF modifies
7
OF INVESTIGATIVE
DERMATOLOGY
d from birth: controls injected with saline (A) 5-day-old moose
hair follicle development and fiber growth. For example, by maintaining proliferation and differentiation, as seen by an increase in the thickness of the epidermis and by hyperkeratinization of the skin after birth, respectively, EGF could have profound effects on follicle development. Furthermore, although EGF is initially involved in cell proliferation, Moore er al [76] suggested that the factor acts as a mitotic inhibitor of follicle bulb cells; this would lead to the observed decline in hair fiber growth rate and decrease in fiber diameter. Similar results have been obtained by infusing sheep with EGF [82]. It has been proposed that, following the initial stimulation by EGF, down-regulation of EGF receptors occurs, resulting in the inhibitory response [80,83]. Adamson and Warshaw [83] suggested that, in embryonic tissues, maximal stimulation by endogenous EGF was already occurring so that exogenous EGF would lead to receptor down-regulation. Carpenter and Cohen [62] suggested that, following binding of EGF to its receptor, the decrease in receptor activity that occurs is due to internalization of the EGF/receptor complex without the accompanying production of new rece tors. This process could remove the stimulus by saturating, and su t: sequently down-regulating, receptors already present. Comparative Effects of bPGF and EGP There are several differences in the effects of bFGF and EGF on the skin. One of the most striking effects is in the area of skin affected. Administration of EGF affects the whole body coat, resumably by entering the circulatory system. In contrast, skin a P ected by bFGF remains confined to a relatively small area surrounding the injection sites on the backs of the animals. This is expected because exogenous FGF is likely to be sequested by matrix-associated HSPG before it is able to enter the circulation. Another difference between administration of EGF and bFGF concerns the long-term effects produced by each. Although EGF produces effects on fiber characteristics for up to two or three hair cycles, only the first hair cycle is partly asynchronous. Basic FGF has been shown to have a dramatic effect on the hair cycle whereby suppression of the cycle and its subsequent asynchrony is maintained. Finally, injection of EGF into neonatal mice produces premature eyelid opening and incisor eruption as well as retarding growth rate [61,76], whereas bFGF does not affect any of these processes.
VOL. 101, NO. 1, SUPPLEMENT,
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1993
FGF AND EGF IN HAIR DEVELOPMENT
111s
Administration of EGF to neonatal mice resulted in hyperkeratinization of the skin, initial epidermal thickening, and delay in hair-follicle development. Furthermore, the effects were widespread over the bod y, unlike bFGF with which the areas affected by the growth factor were very localized. The EGF-treated animals were also significantly smaller than their control littermates. Data indicated that hair follicles were initiating during the EGF-treatment regime but that their development was retarded. In addition, the hair fibers produced by the follicles were not as long and were curved at their tips. Injection of EGF also resulted in delay of the first hair cycle but this was not as dramatic as the effect produced by bFGF and was not prolonged much beyond the first cycle. The findings presented here suggest important roles for both FGF and EGF in follicle initiation and development. Although the mechanisms of action of these growth factors are not yet known, future studies encompassing receptor binding and cell behavior, such as migration and proliferation, in response to these potent molecules should prove enlightening in further understanding the processes of hair follicle morphogenesis.
I thank Dr. K. A. Holbrook for her valued support and helpful discussions; Dr. G.P.M. Moorefor critical reading ofthe manuscript; Ms.]. Fangmanfor cutting and staining the tissue sections; Mr. R.A. Underwoodfor photographic assistance; and Dr. L.S. Cousens and Chiron Corporarionfor Be& of bFGF. Thepnancial support of the National Ins&&s of Health (HD 176641, the Dermatology Foundation, and Dermik Laboratories is gratefully acknowledged.
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Pigure
mice injected with saline (l&j or EGF (right) 4. Eight-day-old daily for 7 d from birth. At this age, there was a considerable difference in the growth rates of the two animals. Note also the flaking skin of the EGFtreated animal.
SUMMARY nents of the epidermis and mesenbetween corn ollicles during fetal development and at thyme give rise to the hair p” birth. This complex of interactions is likely to be mediated in part by growth factors that are diffusible or cell and matrix bound. Immunolocalization studies indicate that aFGF and bFGF are directly involved in the process of hair follicle mo hogenesis: aFGF appears to have a role in epithelial differentiation ‘g 0th in the early stages of follicle develo ment and at maturity; bFGF, located adjacent to areas of proli Peration, may be regulating the mitotic activity of epithelial-derived cells. The specific distribution of EGF in the skin and throughout follicle morphogenesis suggests that this growth factor has a more important role in differentiation than in proliferation. Both bFGF and EGF have profound and dramatic effects on the develo ing skin when given at critical stages of growth. Administrain tion o 4 bFGF during the eriod of hair follicle morphogenesis mouse skin appeared to in Kibit the initiation and development process. Follicles in bFGF-treated areas appeared able to form and develop normally but at a much slower rate. This suppressed state continued until the influence of exogenous FGF was removed, whereupon the normal rate of development resumed and the full hair coat was formed. As a consequence of suppressing follicle development, the normal hair cycle was delayed over the period during which bFGF was administered, but also resumed normal activity when the exogenous bFGF was removed. However, the effects on the hair cycle appeared to be long-term because evidence of the disruption to the cycle was still visible 4 months after the animals were injected with the growth factor. Interactions
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