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Dual-Mode Regulation of Hair Growth Cycle by Two Fgf-5 Gene Products

Satoshi Suzuki, Yutaka Ota, Kazuo Ozawa,* and Toru Imamura* POLA R&D Laboratories, Totsuka-ku Yokohama, Japan; *Biosignaling Department, National Institute of Bioscience and Human Technology, Tsukuba, Ibaragi, Japan

As the result of alternative mRNA splicing, Fgf-5, catagen-promoting activity of ®broblast growth the gene encoding ®broblast growth factor-5, trans- factor-5 when the two proteins were injected simul- lates to both long and short forms of the protein, taneously. Because neither ®broblast growth factor-5 respectively, designated ®broblast growth factor-5 nor ®broblast growth factor-5S affected skin thick- and ®broblast growth factor-5S. We previously ness, it is postulated that changes in skin thickness showed that localization of ®broblast growth factor-5 during hair cycle are separately regulated by factors and the level of ®broblast growth factor-5S in other than those regulating hair and follicle growth. murine skin are hair-cycle dependent. In this study, The present results, together with our earlier ®ndings we examined the effect of ®broblast growth factor-5 that ®broblast growth factor-5-producing cells gather and ®broblast growth factor-5S on the hair growth around dermal papillae during catagen, whereas cycle in mice. Once the anagen phase of the hair ®broblast growth factor-5S is abundantly expressed growth cycle was induced in the dorsal skin by depil- in the hair follicles only during the latter half of ation during telogen, and effects of subcutaneous anagen, suggests that the mouse hair growth cycle is injection of ®broblast growth factor-5 and ®broblast regulated by the two Fgf-5 gene products acting in growth factor-5S into the affected region were ana- concert: ®broblast growth factor-5 induces catagen, lyzed. We found that ®broblast growth factor-5 whereas ®broblast growth factor-5S antagonizes this inhibited hair growth during anagen and promoted activity during anagen. Key words: anagen/catagen/ the transition from anagen to catagen. Interestingly, ®broblast growth factor-5/®broblast growth factor-5S. whereas ®broblast growth factor-5S alone exerted no J Invest Dermatol 114:456±463, 2000 effect on hair growth, it signi®cantly inhibited the

n mammals, the hair growth cycle consists of three phases: Among these, Fgf-5, the gene encoding FGF-5, was originally (i) anagen, when follicles grow and hair synthesis takes place reported to be one of the human oncogenes (Zhan et al, 1987), but and skin thickness increases; (ii) catagen, when the follicles was later classi®ed as belonging to the FGF family because of its Iregress and skin becomes thin; and (iii) telogen, when the high homology with acidic and basic FGF (Zhan et al, 1988). follicles and skin are at rest (Chase, 1954). In addition, FGF-5 mRNA is expressed in the mouse embryo (HeÂbert et al, anagen can be divided into six short phases: anagen I±VI (Chase, 1990, 1991; Haub and Goldfarb, 1991; Ozawa et al, 1996) and in 1954). Hair growth is regulated by several cytokines, including the central nervous system of the adult mouse (Haub et al, 1990; tumor growth factor a (Luetteke et al, 1993; Mann et al, 1993), Ozawa et al, 1996). epidermal growth factor (Green et al, 1984; Luetteke et al, 1994), HeÂbert et al (1994) observed that in mice, deletion of Fgf-5 bone morphogenetic protein-2A (Lyons et al, 1990), insulin-like abnormally prolonged anagen VI. Furthermore, Rosenquist and growth factor-1 (Itami et al, 1995), and certain ®broblast growth Martin (1996) found that Fgfr1, the gene encoding the mouse factors (FGF) (du Cros, 1993; Guo et al, 1993). FGF-5 receptor, is expressed in the dermal papillae and seems to The FGF family consists of at least 19 members, exhibiting a regulate the development and activity of the hair follicle, suggesting variety of biologic activities (Abraham et al, 1986; Jaye et al, 1986; that expression of Fgf-5 induces dermal papillae to ®nish anagen and Moore et al, 1986; Delli-Bovi et al, 1988; Burgess and Maciag, to begin catagen. 1989; Marics et al, 1989; Rubin et al, 1989; Baird and Klagsbrun, It was recently shown that Fgf-5 translates to both long and short 1991; Tanaka et al, 1992, 1995; Miyamoto et al, 1993; Baird, 1994; forms of the protein: FGF-5 and FGF-5S, respectively (Ozawa et al, Mason, 1994; Yamasaki et al, 1996; Hartung et al, 1997; McWhirter 1998). Fgf-5 consists of three exons; as a result of alternative splicing et al, 1997; Verdier et al, 1997; Hoshikawa et al, 1998; Hu et al, and a frameshift, respectively, FGF-5S lacks the region correspond- 1998; Miyake et al, 1998; Ohbayashi et al, 1998; Yamamoto et al, ing to exon 2 and most of the region corresponding to exon 3 1998; Danilenko et al, 1999; Hu et al, 1999; Nishimura et al, 1999). (Hattori et al, 1996; Ozawa et al, 1998). It remains unclear which Fgf-5 gene product, FGF-5 or FGF-5S, induces catagen. Manuscript received July 26, 1999; revised December 10, 1999; accepted We previously reported that both FGF-5 mRNA and FGF-5S for publication December 13, 1999. mRNA are expressed in murine skin, with FGF-5 located in Reprint requests to: Mr. Satoshi Suzuki, POLA R&D Laboratories, 560 round, macrophage-like cells and FGF-5S located in the hair Kashio-cho, Totsuka-ku Yokohama 244-0812, Japan. follicle (Suzuki et al, 1998). In that report, we con®rmed that the

0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 456 VOL. 114, NO. 3 MARCH 2000 DUAL-MODE REGULATION OF HAIR GROWTH CYCLE BY TWO FGF-5 GENE PRODUCTS 457 location of FGF-5-positive cells and the level of FGF-5S in the hair Evaluation The effect of local injection of FGF-5 and/or FGF-5S on the follicle varies during the course of the hair growth cycle, suggesting hair growth cycle was analyzed photometrically and histologically. In each that both FGF-5 and FGF-5S are in some way linked to the cycle. case, statistical analysis was performed with ®ve animals: animals exhibiting In this study, therefore, we investigated the effects of FGF-5 and the maximum and minimum values in each groupwere excluded from the analysis. In the ®rst experiment, the luminance (*L)-value at the injection FGF-5S on the mouse hair growth cycle. Because it has not been site was measured three times using a chroma meter CR-200 (Minolta, clear whether or not hair follicle growth and the corresponding Tokyo, Japan). *L-values quantitated whiteness and darkness and therefore skin thickening were regulated by the same factor, we also corresponded to the relative pigmentation. Because melanin synthesis in investigated the effect of these two proteins on changes in skin follicular melanocytes is strictly coupled to hair follicle growth (Slominski thickness during the hair growth cycle. et al, 1994), skin pigmentation was considered to be an index of hair follicle growth. Indeed, it was previously shown that *L-values correlate well with mouse hair growth (Inaoka et al, 1994). MATERIALS AND METHODS In all three experiments, skin samples were obtained from the injection Preparation of recombinant FGF-5 and FGF-5S proteins in sites, ®xed in 10% formalin, and embedded in paraf®n. Four 4 mm thick Escherichia coli Recombinant FGF-5 and FGF-5S proteins were sections were prepared from each animal, stained with hematoxylin and obtained as previously reported (Ozawa et al, 1998). Full length FGF-5 eosin, and analyzed histologically under a microscope. Hair follicle length protein was produced using a bacterial expression vector system, pET-3c was de®ned as the distance from the end of the dermal papilla to the pore in (Studier et al, 1990). The FGF-5 cDNA fragment was ampli®ed with the the skin. Measurement was carried out for all hair follicles in each section. primer set, 5¢-CGGAA TTCCA TATGG GTGAA AAGCG TCTCG Thickness of the dermis and panniculus adiposus, which is known to CCCCC AAA-3¢ (sense) and 5¢-CGCCA TATGT TTATC CAAAG correspond to hair follicle length (Chase et al, 1953; Moffat, 1968; Hansen CGAAA CTT-3¢ (anti-sense), using a pLTR122 template containing the et al, 1984), was also measured at ®ve points in each section (a total of 20 entire open reading frame for human FGF-5 (Zhan et al, 1988); an points in each animal). N-terminal hydrophobic signal sequence for the original FGF-5 protein was not included in this construct (Clements et al, 1993). The ampli®ed RESULTS fragment was cloned into pBluescript SK+, sequenced as described above, digested with restriction enzyme NdeI(underlined), and inserted into the FGF-5 inhibited hair growth in mice during anagen In the NdeI site of the pET-3c vector. The recombinant plasmid was maintained ®rst experiment, mice were subcutaneously injected according the in E. coli (strain BL21(DE3)pLysS) (Studier et al, 1990), and the desired protocol described in Materials and Methods, and skin pigmentation protein was expressed and extracted as described previously (Clements et al, was measured to assess hair growth (Slominski et al, 1994). During 1993). the 7 d observation period, the skin of control (PBS-injected) mice FGF-5S polypeptide was produced from an FGF-5S/malE fusion protein (Guan et al, 1988). The open reading frame of the FGF-5S cDNA was became black due to the formation of hair melanin (Fig 1a), ampli®ed by the polymerase chain reaction using the primer set, 5¢- whereas at the administration sites of FGF-5-injected mice, the skin TCAGA ATTCG GGGAG AAGCG TCTCG CC-3¢ (sense) and 5¢-CGC- remained unpigmented (Fig 1b). Consistent with earlier ®ndings GG ATCCC TTTAT CTGTG AACTT GGCTT AACA- (Inaoka et al, 1994), the *L-values of the injection sites correlated T ATTGG CTTCG TGcGA TCCAT-3¢ (anti-sense). EcoRI and BamHI well with hair growth (Table I, Fig 1). The *L-value in FGF-5- recognition sites were attached to the sense and anti-sense primers, injected mice was signi®cantly higher than in controls (Table I), respectively (underlined), and a silent mutation (G to ``c'') was introduced meaning their skin was whiter and thus hair growth diminished. to eliminate a BamHI site within the open reading frame. After Changes in skin pigmentation in FGF-5S-injected mice were the ampli®cation with this primer set and with pLTR122 DNA as a template, same as in the controls (Fig 1c), as were the corresponding the ampli®ed fragment was cloned into pBluescript SK+ and sequenced. The inserts were then cut out using EcoRI and BamHI and introduced into *L-values (Table I). a pMAL-c2 plasmid vector (New England Biolabs, Beverly, MA), Histologic analysis of the injection sites showed that hair follicles previously digested with the same two restriction enzymes. The resultant in control mice were long and thick, and were morphologically chimeric plasmid encodes a fusion protein comprised of maltose binding determined to be in anagen V or early anagen VI (Fig 2a). Hair protein (MalE) plus FGF-5S. As described above, an N-terminal follicles in FGF-5-injected mice, by contrast, were short and thin hydrophobic signal sequence was not included. E. coli (strain BL21) and morphologically in anagen II±IV (Fig 2b). Hair follicle size and (Studier and Moffatt, 1986) harboring the chimeric plasmid were grown in shape in FGF-5S-injected mice were the same as in control mice L broth. The recombinant fusion protein was then induced, puri®ed in an (Fig 2c). Quantitative analysis revealed hair follicles in FGF-5- amylose resin (New England Biolabs) af®nity column and cleaved using injected mice to be signi®cantly shorter than in control mice protease factor Xa (New England Biolabs), as previously described (Maina et al, 1988). The FGF-5S protein was separated from MBP by amylose (Fig 3a). On the other hand, the thicknesses of the dermis (Fig 3b) column chromatography. and panniculus adiposus (Fig 3c) were the same in all three groups. FGF-5 and FGF-5S proteins were dissolved to concentrations of 100 or FGF-5 promoted the involution of anagen hair follicles 200 mg per ml in phosphate-buffered saline (PBS) at pH 7.5 and stored at ±20°C until use. The second experiment began on the ninth day after the induction of anagen, with mice being injected daily for 8 d. During this Animals and treatment Seven week old male C3H/He mice were period, hair follicles in control (PBS-injected) mice appeared large, procured from Japan SLC (Hamamatsu, Japan) and maintained on a and were morphologically determined to be in late anagen VI standard laboratory diet and water ad libitum. One week after procurement, (Fig 4a). Although there was little difference between FGF-5- the anagen phase of the hair growth cycle was induced in the mice by injected (Fig 4b) and control mice with respect to hair follicle depilation of their dorsal hair shafts, and three experiments were carried shape, the length of hair follicles in FGF-5-injected mice was out; in each case, the mice were divided into three groups, with each group signi®cantly shorter than in control mice (Fig 5a). The thicknesses consisting of seven mice. of the dermis (Fig 5b) and panniculus adiposus (Fig 5c) were In the ®rst experiment, mice were subcutaneously injected in their dorsal unaffected by FGF-5. In FGF-5S-injected mice, hair follicle shape regions with 50 ml PBS alone (control), or with 5 mg FGF-5 or 5 mg FGF- (Fig 4c), size (Fig 5a), and the thicknesses of the dermis (Fig 5b) 5S, once daily for 7 d, beginning on the ®rst day after the induction of anagen (during anagen I±anagen V or early anagen VI in control mice). In and panniculus adiposus (Fig 5c) were all the same as in control the second experiment, mice were similarly injected with PBS, FGF-5, or mice. FGF-5S once daily for 8 d, beginning on the ninth day after the induction FGF-5S had an antagonistic effect on FGF-5 Although of anagen (during anagen VI in control mice). In the ®nal experiment, mice were injected with either PBS or FGF-5, whereas the third groupwas FGF-5 protein appears to be expressed in a variety tissues, abundant administered both 5 mg FGF-5 and 5 mg FGF-5S once daily for 8 d, expression of FGF-5S is limited to a few, which include the central beginning on the ninth day after the induction of anagen (during anagen VI nervous system (Ozawa et al, 1998) and skin (Suzuki et al, 1998). in control mice). The growth phase at the time of injection was decided on Evidence suggests that FGF-5S functions as a partial antagonist/ the basis of the size and shape of hair follicles observed in skin sections. All agonist for FGF-5-evoked neurotrophic activity (Ozawa et al, mice were killed 1 d after the last injection. 1998). We therefore examined the extent to which FGF-5S also 458 SUZUKI ETAL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY affects FGF-5 activity in the regulation of hair growth cycle. Two morphology placed them in late anagen VI (Fig 6a), and whereas groups of mice were subcutaneously injected daily for 8 d with follicle shape differed little in FGF-5-injected (Fig 6b) and control either PBS or FGF-5, beginning on the ninth day after the mice, follicle length was signi®cantly shorter in FGF-5-injected induction of anagen (during anagen VI), whereas a third groupwas mice (Fig 7a). Hair follicles in mice injected with both FGF-5 and injected with both FGF-5 and FGF-5S. As in the second FGF-5S also appeared to be in late anagen VI (Fig 6c), but experiment, hair follicles in control mice were large, their interestingly, follicle length in these mice was the same as in control mice, which was signi®cantly longer than in mice injected with

Figure 1. FGF-5 inhibited skin pigmentation in mice during anagen. Mice were subcutaneously injected in the dorsal region with PBS (a), FGF-5 (b), or FGF-5S (c) once daily for 7 d beginning 1 d after the induction of anagen (anagen I±anagen V or early anagen VI). The mice were killed on day 8, and skin pigmentation, considered to be an index of hair growth, was observed. Arrows indicate the injection sites.

Table I. The injection sites in FGF-5-injected mice remained unpigmented during anagena

Group *L-value

Control mice 54.04 6 0.68 Figure 2. FGF-5 inhibited hair growth in mice during anagen. Mice FGF-5-injected mice 58.53 6 0.96b were subcutaneously injected in the dorsal region with PBS (a), FGF-5 (b), FGF-5S-injected mice 54.33 6 0.72 or FGF-5S (c) once daily for 7 d beginning 1 d after the induction of anagen (anagen I±anagen V or early anagen VI), and killed on day 8, as described in aThe injection sites shown in Fig 1 were photometrically analyzed as described Fig 1. Shown are histologic sections representative of the data summarized in Materials and Methods. Data are expressed as the mean 6 SD of ®ve mice. in Fig 1. Scale bar: 100 mm. D, dermis; PA, panniculus adiposus; hf, hair bp< 0.01 versus control (Dannett's multiple comparison). follicle; dp, dermal papilla. VOL. 114, NO. 3 MARCH 2000 DUAL-MODE REGULATION OF HAIR GROWTH CYCLE BY TWO FGF-5 GENE PRODUCTS 459

Figure 3. FGF-5 inhibited the growth of hair follicles but did not affect skin thickness in mice during anagen. The experiment began 1 d after the induction of anagen, with mice being injected daily for 7 d, as described in Fig 2. After preparation of the skin sections from the injection sites shown in Fig 2, hair follicle length (a) and the thicknesses of the dermis (b) and panniculus adiposus (c) were measured. Data are expressed as the mean 6 SD of ®ve mice; **p< 0.01 versus control (Dannett's multiple comparison). Figure 4. Representative histologic sections from mice injected FGF-5 alone (Fig 7a). The thicknesses of the dermis (Fig 7b) and with PBS, FGF-5, or FGF-5S during anagen VI. Mice were panniculus adiposus (Fig 7c) were the same in all three groups. subcutaneously injected with PBS (a), FGF-5 (b), or FGF-5S (c) once daily for 8 d beginning on the ninth day after the induction of anagen DISCUSSION (during anagen VI). The mice were killed 1 d after the last injection, and skin sections from the injection sites were observed. Scale bar: 100 mm. D, The results of this study provide new information about Fgf-5 gene dermis; PA, panniculus adiposus; hf, hair follicle; dp, dermal papilla. products and their involvement in hair cycle regulation. Our earlier immunohistochemical study revealed that FGF-5 is located in (HeÂbert et al, 1994), those results suggested that both FGF-5 and round macrophage-like cells in the skin, whereas FGF-5S is located FGF-5S are involved in the regulation of hair growth cycle. This in hair follicles, and that the location of FGF-5-positive cells and study was conducted to gain further insight into this issue. We the level of FGF-5S in the hair follicle varies during the course of observed that: (i) injection of FGF-5 during anagen VI produced the hair growth cycle (Suzuki et al, 1998). Together with the hair follicles that were shorter than the control, whereas follicle ®nding that knocking out Fgf-5 resulted in prolonged anagen length was unaffected by FGF-5S; (ii) by itself, FGF-5S had no 460 SUZUKI ETAL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 5. FGF-5 promoted involution of anagen hair follicles but did not affect skin thickness. The experiment began on the ninth day after the induction of anagen, with mice being injected daily for 8 d, as described in Fig 4. After preparation of the skin sections from the injection Figure 6. Representative histologic sections from mice injected sites shown in Fig 4, hair follicle length (a), the thicknesses of the dermis with PBS, FGF-5, or both FGF-5 and FGF-5S during anagen VI. (b), and panniculus adiposus (c) were measured. Data are expressed as the Mice were subcutaneously injected with PBS (a) or FGF-5 (b), or with mean 6 SD of ®ve mice; **p< 0.01 versus control (Dannett's multiple both FGF-5 and FGF-5S (c), once daily for 8 d starting on the ninth day comparison). after the induction of anagen (during anagen VI). The mice were killed 1 d after the last injection, and skin sections from the injection sites were observed. Scale bar: 100 mm. D, dermis; PA, panniculus adiposus; hf, hair effect on hair growth, but it neutralized the capacity of FGF-5 to follicle; dp, dermal papilla. induce catagen; and (iii) neither FGF-5 nor FGF-5S affected skin thickness during the hair growth cycle. morphology, we cannot be certain whether the follicles in the The ®nding that whereas injection of FGF-5 during anagen VI FGF-5-injected mice shown in Fig 4(b) were in catagen or not. caused hair follicles to be shorter than the control, FGF-5S had no Nonetheless, hair follicle shape at the beginning of catagen is effect on follicle length (Figs 4 and 5a) is notable, because follicle identical with that in late anagen VI (Straile et al, 1961), making it length reaches a maximum during anagen III and remains stable possible that the regressing hair follicles in FGF-5-injected mice during the latter half of anagen (Chase, 1954). Furthermore, hair were in early catagen (Fig 4b). Therefore, it was concluded that follicles in this study appeared to reach maturity prior to the time FGF-5, but not FGF-5S, induces the transition from anagen to injections were begun on the ninth day after the induction of catagen. anagen (data not shown). This means that FGF-5 must have Rosenquist and Martin (1996) showed that Fgfr1, a gene promoted the involution of mature hair follicles. From their encoding mouse FGF-5 receptor FGFR1, is expressed in the VOL. 114, NO. 3 MARCH 2000 DUAL-MODE REGULATION OF HAIR GROWTH CYCLE BY TWO FGF-5 GENE PRODUCTS 461

Figure 7. FGF-5S antagonized the effect of FGF-5. Two groups of Figure 8. Schematic summary of the supposed function of FGF-5- mice were subcutaneously injected daily for 8 d with either PBS or FGF-5, producing macrophage-like cells and FGF-5S in the hair growth beginning on the ninth day after the induction of anagen (during anagen cycle. (a) During early anagen, the number of FGF-5-producing VI), whereas a third groupwas injected with both FGF-5 and FGF-5S, as macrophage-like cells is small, and little FGF-5S is produced in the hair shown in Fig 6. After preparation of the skin sections of the injection site follicles. (b) During late anagen, the number of FGF-5-producing cells shown in Fig 6, hair follicle length (a) and the thicknesses of the dermis (b) increases in the dermis and FGF-5S, which is now produced in quantity, and panniculus adiposus (c) were measured. Data are expressed as the antagonizes FGF-5. (c) During catagen, FGF-5-producing cells migrate to mean 6 SD of ®ve mice. *p< 0.05, **p< 0.01 (t test). the panniculus adiposus, gather around dermal papillae and release FGF-5; production of FGF-5S rapidly declines, allowing FGF-5 to bind to its receptor expressed in the dermal papillae. Thereafter, the hair follicles dermal papillae. We previously found that the macrophage-like, degenerate. D, dermis; PA, panniculus adiposus; hf, hair follicle; dp, dermal FGF-5-producing cells exist in the skin in small numbers at the papilla; fmc, FGF-5-producing macrophage-like cell. beginning of anagen, increase in number only in the dermis during the later half of anagen, and then migrate from the dermis to the panniculus adiposus during catagen and telogen (Suzuki et al, 1998). receptors are expressed at low levels, which are nonetheless This suggests that FGF-5-producing cells gather around FGFR1- suf®cient to mediate FGF-5 stimulation of dermal papillae. Another expressing dermal papillae in the panniculus adiposus to induce possibility is that FGFR1 is synthesized during anagen and then involution of the hair follicles during catagen (Fig 8). remains functional until the end of catagen. It has yet to be On the other hand, Rosenquist and Martin (1996) also reported determined why Fgfr1 is expressed during anagen. A possible that expression of FGFR1 is only detectable during anagen, and it explanation is that FGFR1 serves as a receptor for other FGF family remains to be determined how FGF-5 stimulates dermal papillae members (Dikov et al, 1998; Giri et al, 1999), and these members during catagen. One possibility is that during catagen, FGF-5 also have an effect on hair growth during anagen. 462 SUZUKI ETAL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

As mentioned above, FGF-5-producing cells increased in the regulation of the hair growth cycle, including the skin organ, is dermis during the latter half of anagen (Suzuki et al, 1998), although apparently more intricate than previously considered. functionally, they are believed to induce the involution of hair It has been reported that stimulation of tumor growth factor-b follicles during catagen (Fig 8). If these cells did not begin receptor I and II (Paus et al, 1997) as well as stimulation of increasing in number until catagen, that phase would be very parathyroid hormone/parathyroid hormone-related peptide recep- much prolonged. Consequently, it seems likely that FGF-5- tor (Schilli et al, 1997) are involved in the transition from anagen VI producing cells must begin increasing before catagen and do so in to catagen. On the other hand, it appears that apoptosis plays a part the dermis, a region where dermal papillae are absent. in the induction of catagen. For instance, during catagen and Although FGF-5 inhibited hair and follicle growth during early telogen, the immunoreactivity of Bcl-2, a protooncogene product anagen (Figs 1, 2, and 3a, Table I), this effect is not considered to that provides protection against apoptosis, decreases or disappears in be physiologic in nature because follicle growth and hair synthesis the epithelial portion of the follicle, whereas the dermal papilla are the norm for anagen. In fact, the inhibitory activity by FGF-5 remains Bcl-2 positive throughout the hair cycle (Stenn et al, 1994). more likely relates to the induction of catagen, during which Further, Seiberg et al (1995) reported that expression of several follicles regress. The report that the morphology of hair follicle genes, including the genes encoding tumor growth factor-b and continues to mature during the latter half of anagen when hair tumor necrosis factor-b, as well as c-myc,c-myb, and c-jun, are follicle length remains stable (Randall, 1957) gave rise to an idea involved in follicular apoptosis and thus regulation of catagen. that the morphologic maturation of hair follicle is also inhibited by Chemical compounds that affect catagen induction have also FGF-5 via the same mechanism that inhibits follicle extension, and been studied. Dexamethasone is known to induce catagen (Paus this inhibition of maturation, in turn, induces the onset of catagen. et al, 1994), whereas some immunophilin ligands (e.g., cyclosporine Whereas FGF-5S did not itself affect hair growth, it did A and FK506) inhibit dexamethasone-induced catagen develop- neutralize the catagen-inducing activity of FGF-5 (Figs 6 and 7a). ment (Maurer et al, 1997). In vitro, cyclosporine A prolongs anagen FGF-5S is known to bind to FGF-5 receptors and thus antagonize hair growth in human hair follicles (Taylor et al, 1993) and in vivo,it FGF-5-evoked neurotrophic activity (Ozawa et al, 1998), making it inhibits spontaneous murine catagen development (Paus et al, likely that FGF-5S exerted an analogous inhibitory effect on FGF-5 1993). during the hair growth cycle. We did not investigate whether HeÂbert et al (1994) reported that anagen is abnormally prolonged FGF-5S antagonized the FGF-5-induced inhibition of hair growth (about 1 y) in Fgf-5-de®cient mice, which is indicative of the during early anagen because as already mentioned, that effect was central part played by FGF-5 and FGF-5S in the induction of deemed unphysiologic. By binding to the FGF-5 receptor, catagen. Nevertheless, they also reported that catagen ensues after however, FGF-5S would be expected to antagonize the effect of prolonged anagen, even in the absence of Fgf-5. Those investigators FGF-5 on hair growth at any time during the hair growth cycle, suggested that some other signaling molecule, capable of inducing catagen, acts in the follicle when anagen VI is abnormally including early anagen. We previously con®rmed that the small prolonged due to an absence of Fgf-5. It is clear that to understand quantities of FGF-5S protein are produced in the hair follicles fully the hair growth cycle, the involvement of other factors, in during early anagen, that FGF-5S production increases signi®cantly addition to FGF-5 and FGF-5S, will have to be taken into during anagen VI, and that production then rapidly declines during consideration. catagen (Suzuki et al, 1998). Based on this sequence of events, it is hypothesized that the changes FGF-5S production take place to REFERENCES regulate the FGF-5 activity. FGF-5-producing cells increase mainly in the dermis during anagen, and few of these cells are located in Abraham JA, Whang JL, Tumolo A, Mergia A, Friedman J, Gospodarowicz D, Fiddes JC: Human basic ®broblast growth factor: nucleotide sequence and the panniculus adiposus where dermal papillae are situated (Suzuki genomic organization. EMBO J 5:2523±2528, 1986 et al, 1998). Moreover, as the density of FGF-5-producing cells in Baird A: Fibroblast growth factors: activities and signi®cance of non-neurotropin the dermis is very high during the latter part of anagen, synthesis of neurotropic growth factors. Curr Opin Neurobiol 4:78±86, 1994 FGF-5 there should be high, resulting in substantial diffusion of Baird A, Klagsbrun M: The ®broblast growth factor family. Cancer Cells 3:239±243, 1991 FGF-5 from the dermis to the panniculus adiposus. The binding of Burgess WH, Maciag T: The heparin-binding (®broblast) growth factor family of FGF-5 to its receptor in the dermal papillae during anagen, proteins. Annu Rev Biochem 58:575±606, 1989 however, would result in the abnormal progress of this phase, Chase HB: Growth of the hair. Physiol Rev 34:113±126, 1954 leading to slow hair growth and premature induction of catagen. Chase H, Montagna W, Malone J: Changes in the skin in relation to the hair growth cycle. Anat Rec 116:75±82, 1953 The results of this study suggest that FGF-5S is produced in Clements DA, Wang JK, Dionne CA, Goldfarb M: Activation of ®broblast growth quantity during anagen VI in order to suppress FGF-5 activity. factor (FGF) receptors by recombinant human FGF-5. Oncogene 8:1311±1316, Then at the appropriate time, FGF-5S levels rapidly decline, 1993 allowing FGF-5 to bind to its receptor during catagen (Fig 8). du Cros DL: Fibroblast growth factor in¯uences the development and cycling of murine hair follicles. Dev Biol 156:444±453, 1993 It appears to us that endogenous FGF-5S synthesized in the skin Danilenko DM, Montestruque S, Philo JS, et al: Recombinant rat ®broblast growth may have affected our data. Injection of FGF-5 during early anagen factor-16: structure and biological activity. Arch Biochem Biophys 361:34±46, inhibited hair growth by more than 50% (Fig 3a), and that effect 1999 persisted for at least 24 h. Interestingly, the effect of injecting Delli-Bovi P, Curatola AM, Newman KM, et al: Processing, secretion, and biological properties of a novel growth factor of the ®broblast growth factor family with FGF-5 during anagen VI (Figs 5a and 7a) was much smaller than oncogenic potential. Mol Cell Biol 8:2933±2941, 1988 the effect seen during early anagen (Fig 3a). Because the amount of Dikov MM, Reich MB, Dworkin L, Thomas JW, Miller GG: A functional ®broblast FGF-5S in the skin was higher during anagen VI than during early growth factor-1 immunoglobulin fusion protein. J Biol Chem 273:15811± anagen, we believe that FGF-5 injected during anagen VI was 15817, 1998 antagonized by endogenous FGF-5S produced in the mouse hair Giri D, Ropiquet F, Ittmann M: Alterations in expression of basic ®broblast growth factor (FGF) 2 and its receptor FGFR-1 in human prostate cancer. Clin Cancer follicles. Res 5:1063±1071, 1999 It is known that during the spontaneous hair growth cycle, the Green MR, Phil D, Couchman JR: Distribution of epidermal growth factor thickness of mouse skin corresponds to hair follicle length (Chase receptors in rat tissue during embryonic skin development, hair formation, and et al, 1953; Moffat, 1968; Hansen et al, 1984), but it has not been the adult hair growth cycle. J Invest Dermatol 83:118±123, 1984 Guan C-d, Li P, Riggs PD, Inouye H: Vectors that facilitate the expression and clear whether hair follicle growth and skin thickening are puri®cation of foreign peptides in Escherichia coli by fusion to maltose-binding dissociable. The present ®ndings show that FGF-5 selectively protein. Gene 67:21±30, 1988 affected the hair follicle without affecting the thickness of the Guo L, Yu QC, Fuchs E: Targeting expression of keratinocyte growth factor to dermis (Figs 3b, 5b, and 7b) or the panniculus adiposus (Figs 3c, 5c, keratinocytes elicit striking changes in epithelial differentiation in transgenic mice. EMBO J 12:973±986, 1993 and 7c). This suggests that skin thickness is regulated separately by Hansen LS, Coggle JE, Wells J, Charles MW: The in¯uence of the hair cycle on the factors other than those regulating hair and follicle growth. Thus, thickness of mouse skin. Anat Rec 210:569±573, 1984 VOL. 114, NO. 3 MARCH 2000 DUAL-MODE REGULATION OF HAIR GROWTH CYCLE BY TWO FGF-5 GENE PRODUCTS 463

Hartung H, Feldman B, Lovec H, Coulier F, Birnbaum D, Goldfarb M: Murine topography and protein coding potential of mouse int-2: a putative oncogene FGF-12 and FGF-13: expression in embryonic nervous system, connective activated by mouse mammary tumour virus. EMBO J 5:919±924, 1986 tissue and heart. Mech Dev 64:31±39, 1997 Nishimura T, Utsunomiya Y, Hoshikawa M, Ohuchi H, Itoh N: Structure and Hattori Y, Yamasaki M, Itoh N: The rat FGF-5 mRNA variant generated by expression of a novel human FGF, FGF-19, expressed in the fetal brain. alternative splicing encodes a novel truncated form of FGF-5. Biochim Biophys Biochem Biophys Acta 1444:148±151, 1999 Acta 1306:31±33, 1996 Ohbayashi N, Hoshikawa M, Kimura S, Yamasaki M, Fukui S, Itoh N: Structure and Haub O, Goldfarb M: Expression of the ®broblast growth factor-5 gene in the mouse expression of the mRNA encoding a novel ®broblast growth factor, FGF-18. J embryo. Development 112:397±406, 1991 Biol Chem 273:18161±18164, 1998 Haub O, Drucker B, Goldfarb M: Expression of the murine ®broblast growth factor Ozawa K, Urano T, Miyakawa K, Seo M, Imamura T: Expression of the ®broblast 5 gene in the adult central nervous system. Proc Natl Acad Sci USA 87:8022± growth factor family and their receptor family genes during mouse brain 8026, 1990 development. Mol Brain Res 41:279±288, 1996 HeÂbert JM, Basilico C, Goldfarb M, Haub O, Martin GR: Isolation of cDNAs Ozawa K, Suzuki S, Asada M, et al: An alternatively-spliced FGF-5 mRNA is encoding four mouse FGF family members and characterization of their abundant in brain and translates into a partial agonist/antagonist for FGF-5 expression patterns during embryogenesis. Dev Biol 138:454±463, 1990 neurotrophic activity. J Biol Chem 273:29262±29271, 1998 HeÂbert JM, Boyle M, Martin GR: mRNA localization studies suggest that murine Paus R, Bottge JA, Czarnetzki BM: Cyclosporin A, PSC 833 and FK506, but not FGF-5 plays a role in gastrulation. Development 112:407±415, 1991 cyclosporin H and rapamycin induce anagen and inhibit catagen in murine HeÂbert JM, Rosenquist T, Gotz J, Martin GR: FGF5 as a regulator of the hair growth skin. J Invest Dermatol 101:420a, 1993 cycle: Evidence from targeted and spontaneous mutations. Cell 78:1017±1025, Paus R, Handjiski B, Czarnetzki BM, Eichmuller S: A murine model for inducing 1994 and manipulating hair follicle regression (catagen). Effects dexamethasone Hoshikawa M, Ohbayashi N, Yonamine A, Konishi M, Ozaki K, Fukui S, Itoh N: cyclosporin A. J Invest Dermatol 103:143±147, 1994 Structure and expression of a novel ®broblast growth factor, FGF-17, Paus R, Foitzik K, Welker P, Bulfone-Paus S, Eichmuller S: Transforming growth preferentially expressed in the embryonic brain. Biochem Biophys Res Commun factor-b receptor type I and type II expression during murine hair follicle 244:187±191, 1998 development and cycling. J Invest Dermatol 109:518±526, 1997 Hu MC, Qui WR, Wang YP, et al: FGF-18, a novel member of the ®broblast Randall P: Discussion of paper in ``Skin homografts in rat (Ballantyne and growth factor family, stimulates hepatic and intestinal proliferation. Mol Cell Converse)''. Ann N Y Acad Sci 64:963±966, 1957 Biol 18:6063±6074, 1998 Rosenquist TA, Martin GR: Fibroblast growth factor signaling in the hair growth Hu MC, Wang YP, Qui WR: Human ®broblast growth factor-18 stimulates cycle: expression of the ®broblast growth factor receptor and ligand genes in ®broblast cell proliferation and is mapped to chromosome 14p11. Oncogene the murine hair follicle. Dev Dynam 205:379±386, 1996 18:2635±2642, 1999 Rubin JF, Osada H, Finch PW, Taylor WG, Rudikoff S, Aaronson SA: Puri®cation Inaoka Y, Shakuya A, Fukuzawa H, et al: Studies on active substances in herbs used and characterization of a newly identi®ed growth factor speci®c for epithelial for hair treatment. I. Effects of herb extracts on hair growth and isolation of an cells. Proc Natl Acad Sci USA 86:802±806, 1989 active substance from polyporus umbellatus F. Chem Pharm Bull 42:530±533, Schilli MB, Ray S, Paus R, Obi-Tabot E, Holick MF: Control of hair growth with 1994 parathyroid hormone (7±34). J Invest Dermatol 108:928±932, 1997 Itami S, Kurata S, Takayasu S: Androgen induction of follicular epithelial cell growth Seiberg M, Marthinuss J, Stenn KS: Changes in expression of apoptosis-associated is mediated via insulin-like growth factor-I from dermal papilla cells. Bichem genes in skin mark early catagen. J Invest Dermatol 104:78±82, 1995 Biophys Res Commun 212:988±994, 1995 Slominski A, Paus R, Plonka P, Chakraborty A, Maurer M, Pruski D, Lukiewicz: Jaye M, Howk R, Burgess W, et al: Human endothelial cell growth factor: cloning, Melanogenesis during the anagen-catagen-telogen transformation of the nucleotide sequence, and chromosome location. Science 233:541±545, 1986 murine hair cycle. J Invest Dermatol 102:862±869, 1994 Luetteke NC, Qui TH, Peiffer RL, Oliver O, Smithies O, Lee DC: TGFa de®ciency results in hair follicle and eye abnormalities in targeted and waved-1 mice. Cell Stenn KS, Lawrence L, Veis D, Korsmeyer S, Seiberg M: Expression of the bcl-2 73:263±278, 1993 protooncogene in the cycling adult mouse hair follicle. J Invest Dermatol Luetteke NC, Phillips HK, Qiu TH, Opeland NG, Earp HS, Jenkins NA, Lee DC: 103:107±111, 1994 The mouse waved-2 phenotype results from a point mutation in the EGF Straile WE, Chase HB, Arsenault C: Growth and differentiation of hair follicles receptor tyrosine kinase. Genes Dev 8:399±413, 1994 between periods of activity and quiescence. J Exp Zool 148:205±221, 1961 Lyons KM, Pelton RW, Hogan BLM: Organogenesis and pattern formation in the Studier FW, Moffatt BA: Use of bacteriophage T7 RNA polymerase to direct mouse: RNA distribution patterns suggest a role for Bone Morphogenetic selective high-level expression of cloned genes. J Mol Biol 189:113±130, 1986 Protein-2A (BMP-2A). Development 109:833±844, 1990 Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase Maina CV, Riggs PD, Grandea AG, III, et al:AnEscherichia coli vector to express and to direct expression of cloned genes. Methods Enzymol 185:60±89, 1990 purify foreign proteins by fusion to and separation from maltose-binding Suzuki S, Kato T, Takimoto H, et al: Localization of rat FGF-5 protein in skin protein. Gene 74:365±373, 1988 macrophage-like cells and FGF-5S in hair follicle: possible involvement of two Mann GB, Fowler KJ, Gabriel A, Nice EC, Williams RL, Dunn AR: Mice with a Fgf-5 gene products in hair growth cycle regulation. J Invest Dermatol 111:963± null mutation of the TGFa gene have abnormal skin architecture, wavy hair, 972, 1998 and curly whiskers and often developcorneal in¯ammation. Cell 73:249±261, Tanaka A, Miyamoto K, Minamino N, Takeda M, Sato B, Matsuo H, Matsumoto K: 1993 Cloning and characterization of an androgen-induced growth factor essential Marics I, Adelaide J, Raybaud F, et al: Characterization of the HST-related FGF 6 for the. androgen-dependent growth of mouse mammary carcinoma cells. Proc gene, a new member of the ®broblast growth factor gene family. Oncogene Natl Acad Sci USA 89:8928±8932, 1992 4:335±340, 1989 Tanaka A, Miyamoto K, Matsuo H, Matsumoto K, Yoshida H: Human androgen- Mason IJ: The ins and outs of ®broblast growth factors. Cell 78:547±552, 1994 induced growth factor in prostate and breast cancer cells: its molecular cloning Maurer M, Handjiski B, Paus R: Hair growth modulation by topical immunophilin and growth properties. FEBS Lett 363:226±230, 1995 ligands. Am J Pathol 150:1433±1441, 1997 Taylor M, Ashcroft ATT, Messenger AG: Cyclosporin A prolongs human hair McWhirter JR, Goulding M, Weiner JA, Chun J, Murre C: A novel ®broblast growth in vitro. J Invest Dermatol 100:237±239, 1993 growth factor gene expressed in the developing nervous system is a Verdier AS, Mattei MG, Lovec H, Hartung H, Goldfarb M, Birnbaum D, Coulier F: downstream target of the chimeric homeodomain oncoprotein E2A-Pbx 1. Chromosomal mapping of two novel human FGF gene, FGF-11 and FGF-12. Development 124:3221±3232, 1997 Genomics 40:151±154, 1997 Miyake A, Konishi M, Martin FH, et al: Structure and expression of a novel member, Yamamoto S, Mikami T, Ohbayashi N, Ohta M, Itoh N: Structure and expression of FGF-16, on the ®broblast growth factor family. Biochem Biophys Res Commun a novel isoform of mouse FGF homologous factor (FHF)-4. Biochem Biophys 243:148±152, 1998 Acta 1398:38±41, 1998 Miyamoto M, Naruo K, Seko C, Matsumoto S, Kondo T, Kurokawa T: Molecular Yamasaki M, Miyake A, Tagashira S, Itoh N: Structure and expression of the rat cloning of a novel cytokine cDNA encoding the ninth member of the mRNA encoding a novel member of the ®broblast growth factor family. J Biol ®broblast growth factor family, which has a unique secretion property. Mol Cell Chem 271:15918±15921, 1996 Biol 13:4251±4259, 1993 Zhan X, Culpepper A, Reddy M, Loveless J, Goldfarb M: Human oncogenes Moffat GH: The growth of hair follicles and its relation to the adjacent dermal detected by a de®ned medium culture assay. Oncogene 1:369±376, 1987 structures. J Anat 102:527±540, 1968 Zhan X, Bates B, Hu X, Goldfarb M: The human FGF-5 oncogene encodes a novel Moore R, Casey G, Brookes S, Dixon M, Peters G, Dickson C: Sequence, protein related to ®broblast growth factors. Mol Cell Biol 8:3487±3495, 1988