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Comprehensive Analysis of FGF and FGFR Expression in Skin: FGF18 Is Highly Expressed in Hair Follicles and Capable of Inducing Anagen from Telogen Stage Hair Follicles

2005; Elsevier BV; Volume: 124; Issue: 5 Linguagem: Inglês

10.1111/j.0022-202x.2005.23693.x

1523-1747

Mitsuko Kawano, Akiko Komi-Kuramochi, Masahiro Asada, Masashi Suzuki, Junko Oki, Ju Jiang, Toru Imamura,

Wnt/β-catenin signaling in development and cancer

We quantified the mRNA expression of all 22 fibroblast growth factor family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair growth cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, 2, 5, 7, 10, 13, and 22), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair growth cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and 22 at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal keratinocytes and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair growth was observed. Our findings suggest that FGF18 is important for the regulation of hair growth and the maintenance of skin in adult mice. We quantified the mRNA expression of all 22 fibroblast growth factor family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair growth cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, 2, 5, 7, 10, 13, and 22), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair growth cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and 22 at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal keratinocytes and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair growth was observed. Our findings suggest that FGF18 is important for the regulation of hair growth and the maintenance of skin in adult mice. fibroblast growth factor FGF receptor human dermal fibroblasts human hair follicle dermal papilla cells human epidermal keratinocytes human outer root sheath cells human umbilical vein endothelial cells phosphate-buffered saline A variety of polypeptide growth factors, including various members of the fibroblast growth factor (FGF) family, are expressed in skin. In both mice and humans, FGF are encoded by twenty-two distinct genes (Ornitz and Itoh, 2001Ornitz D.M. Itoh N. Fibroblast growth factors.Genome Biol. 2001; 2 (REVIEWS3005): 1-12Crossref Google Scholar), whereas four genes encode transmembrane FGF receptor (FGFR) tyrosine kinases (Jaye et al., 1992Jaye M. Schlessinger J. Dionne C.A. Fibroblast growth factor receptor tyrosine kinases: Molecular analysis and signal transduction.Biochim Biophys Acta. 1992; 1135: 185-199Crossref PubMed Scopus (597) Google Scholar). Among them, FGF1, 2, 5, 7, 10, 13, and 22, are known to be expressed in dermal and hair follicular cells and to regulate hair growth and skin regeneration (du Cros, 1993du Cros D.L. Fibroblast growth factor and epidermal growth factor in hair development.J Invest Dermatol. 1993; 101: 106S-113SAbstract Full Text PDF PubMed Google Scholar; du Cros et al., 1993du Cros D.L. Isaacs K. Moore G.P. Distribution of acidic and basic fibroblast growth factorsin ovine skin during follicle morphogenesis.J Cell Sci. 1993; 105: 667-674PubMed Google Scholar; Hebert et al., 1994Hebert J.M. Rosenquist T. Gotz J. Martin G.R. FGF5 as a regulator of the hair growth cycle: Evidence from targeted and spontaneous mutations.Cell. 1994; 78: 1017-1025Abstract Full Text PDF PubMed Scopus (489) Google Scholar; Danilenko et al., 1995Danilenko D.M. Ring B.D. Yanagihara D. Benson W. Wiemann B. Starnes C.O. Pierce G.F. Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development, and differentiation.Am J Pathol. 1995; 147: 145-154PubMed Google Scholar; Marchese et al., 1995Marchese C. Chedid M. Dirsch O.R. et al.Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin.J Exp Med. 1995; 182: 1369-1376Crossref PubMed Scopus (147) Google Scholar; Guo et al., 1996Guo L. Degenstein L. Fuchs E. Keratinocyte growth factor is required for hair development but not for wound healing.Genes Dev. 1996; 10: 165-175Crossref PubMed Scopus (464) Google Scholar; Petho-Schramm et al., 1996Petho-Schramm A. Muller H.J. Paus R. FGF5 and the murine hair cycle.Arch Dermatol Res. 1996; 288: 264-266Crossref PubMed Scopus (26) Google Scholar; Rosenquist and Martin, 1996Rosenquist T.A. Martin G.R. Fibroblast growth factor signalling in the hair growth cycle: Expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle.Dev Dyn. 1996; 205: 379-386Crossref PubMed Scopus (155) Google Scholar; Mitsui et al., 1997Mitsui S. Ohuchi A. Hotta M. Tsuboi R. Ogawa H. Genes for a range of growth factors and cyclin-dependent kinase inhibitors are expressed by isolated human hair follicles.Br J Dermatol. 1997; 137: 693-698Crossref PubMed Scopus (31) Google Scholar; Ortega et al., 1998Ortega S. Ittmann M. Tsang S.H. Ehrlich M. Basilico C. Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor-2.Proc Natl Acad Sci USA. 1998; 95: 5672-5677Crossref PubMed Scopus (483) Google Scholar; Suzuki et al., 1998Suzuki S. Kato T. Takimoto H. et al.Localization of rat FGF-5 protein in skin macrophage-like cells and FGF-5S protein in hair follicle: Possible involvement of two Fgf-5 gene products in hair growth cycle regulation.J Invest Dermatol. 1998; 111: 963-972Crossref PubMed Scopus (53) Google Scholar, Suzuki et al., 2000Suzuki S. Ota Y. Ozawa K. Imamura T. Dual-mode regulation of hair growth cycle by two Fgf-5 gene products.J Invest Dermatol. 2000; 114: 456-463Crossref PubMed Scopus (76) Google Scholar; Nakatake et al., 2001Nakatake Y. Hoshikawa M. Asaki T. Kassai Y. Itoh N. Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle.Biochem Biophys Acta. 2001; 1517: 460-463Crossref PubMed Scopus (64) Google Scholar; Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1119) Google Scholar; Beyer et al., 2003Beyer T.A. Werner S. Dickson C. Grose R. Fibroblast growth factor 22 and its potential role during skin development and repair.Exp Cell Res. 2003; 287: 228-236Crossref PubMed Scopus (44) Google Scholar; Kawano et al., 2004Kawano M. Suzuki S. Suzuki M. Oki J. Imamura T. Bulge-and basal layer-specific expression of fibroblast growth factor 13 (FHF-2) in mouse skin.J Invest Dermatol. 2004; 122: 1084-1090Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). For instance, earlier experiments revealed the crucial role played by FGF5 in regulating the onset of catagen during the mouse hair cycle; that Fgf5 translates into two proteins (FGF5 and FGF5S) through generation of alternatively spliced mRNA (Ozawa et al., 1998Ozawa K. Suzuki S. Asada M. et al.An alternatively spliced fibroblast growth factor (FGF)-5 mRNA is abundant in brain and translates into a partial agonist/antagonist for FGF-5 neurotrophic activity.J Biol Chem. 1998; 273: 29262-29271Crossref PubMed Scopus (32) Google Scholar); and that FGF5S antagonizes the catagen-inducing activity of FGF5 (Suzuki et al., 2000Suzuki S. Ota Y. Ozawa K. Imamura T. Dual-mode regulation of hair growth cycle by two Fgf-5 gene products.J Invest Dermatol. 2000; 114: 456-463Crossref PubMed Scopus (76) Google Scholar). In addition, FGF1, 2, and 7 reportedly promote hair growth, FGF13 (FHF2) is highly expressed in the bulge region of hair follicles, and FGF7 and 10 function in concert via FGFR2 IIIb to stimulate keratinocyte proliferation in normal and wounded skin (Werner et al., 1994Werner S. Smola H. Liao X. Longaker M.T. Krieg T. Hofschneider P.H. Williams L.T. The function of KGF in epithelial morphogenesis and wound re-epithelialisation.Science. 1994; 266: 819-822Crossref PubMed Scopus (514) Google Scholar; Marchese et al., 1995Marchese C. Chedid M. Dirsch O.R. et al.Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin.J Exp Med. 1995; 182: 1369-1376Crossref PubMed Scopus (147) Google Scholar; Igarashi et al., 1998Igarashi M. Finch P.W. Aaronson S.A. Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7).J Biol Chem. 1998; 273: 13230-13235Crossref PubMed Scopus (270) Google Scholar; Ohuchi et al., 2000Ohuchi H. Hori Y. Yamasaki M. Harada H. Sekine K. Kato S. Itoh N. FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development.Biochem Biophys Res Commun. 2000; 277: 643-649Crossref PubMed Scopus (512) Google Scholar; Kawano et al., 2004Kawano M. Suzuki S. Suzuki M. Oki J. Imamura T. Bulge-and basal layer-specific expression of fibroblast growth factor 13 (FHF-2) in mouse skin.J Invest Dermatol. 2004; 122: 1084-1090Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Although these studies suggest the importance of the concerted activities of FGF in skin, the expression of all FGF family members in skin have never been systematically investigated in a single study. We therefore established an experimental system with which to quantify the expression of FGF and FGFR mRNA in skin, and comprehensively profiled the dynamic changes in their expression over the course of the hair growth cycle. We initially established an experimental system to quantify the expression of all Fgf and Fgfr family members (described in Materials and Methods). The level of expression of each FGF and FGFR mRNA at the indicated stage in the hair cycle is shown in Figure 1 and Figure 2, respectively. In addition, the highest expression levels of all the mRNA and the phases of the hair cycle at which they occurred are summarized in Table I. The most strongly expressed genes were Fgf1, 5, 7, 10, 13, 18, and 22 (Figure 1a and Table I). Within this group, the levels of FGF7 and FGF10 mRNA (or keratinocyte growth factor (KGF) 1 and KGF 2, respectively) exhibited similar variation throughout the hair cycle, with the highest expression levels occurring at anagen V (day 8; Figure 1a). The highest levels of FGF5 and 22 mRNA occurred at anagen VI (day 18: Figure 1a). Expression of FGF1 mRNA (or acidic FGF) was comparatively constant, with maximum levels attained at telogen (day 0; Figure 1a). At their highest levels, the expression of some FGF (e.g., FGF7 and 18) was approximately 1 × 104 copies per ng mRNA. This level of expression was about 60-fold lower than that of β-actin (5.6 × 105 copies per ng mRNA at anagen V; Table I), one of the abundant structural proteins in skin, and was similar to that observed for cytokine tumor necrosis factor-alpha in some cell lines (5 × 103–1 × 104 copies per ng mRNA in U937 and HL60 cells;Takahashi et al., 2001Takahashi M. Funato T. Kumura-Ishii K. Kaku M. Sasaki T. Measurement of tumor necrosis factor-alpha messenger RNA in synovial fibroblasts by real-time quantitative reverse transcriptase-polymerase chain reaction.J Lab Clin Med. 2001; 137: 101-106Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar).Figure 2Expression of fibroblast growth factor receptor (FGFR) family genes in skin throughout the hair cycle. A skin cDNA was prepared, amplified with specific primers for the indicated FGFR, and then analyzed as described in the legend to Figure 1. Symbols represent means±SE obtained with cDNA from five mice for each stage. All quantification experiments were carried out at least twice in each set of animal experiment to confirm the results. Moreover, the entire set of animal experiments were performed three times, and the results obtained were very similar. T, telogen; A, anagen; C, catagen. The numbers in the parentheses indicate the days after depilation.View Large Image Figure ViewerDownload (PPT)Table IExpression of FGF and FGFR mRNA in adult mouse skinHighest mRNA expressionGeneCopy number (copies per ng mRNA)Phase of the hair cycle at which the highest mRNA expression was attained (day after depilation)Fgf12900 ± 152Telogen (0)Fgf2751 ± 51Telogen (0)Fgf3667 ± 87Anagen VI (18)Fgf4(−)(−)Fgf54307 ± 578Anagen VI (18)Fgf6542 ± 94Telogen (0)Fgf77467 ± 475Anagen V (8)Fgf8(−)(−)Fgf9306 ± 34Telogen (22)Fgf104389 ± 341Anagen V (8)Fgf111422 ± 84Telogen (0)Fgf12188 ± 24Anagen V (8)Fgf134945 ± 87Telogen (0)Fgf14(−)(−)Fgf15(−)(−)Fgf16171 ± 34Anagen VI (18)Fgf17179 ± 7Anagen VI (18)Fgf189822 ± 1,875Telogen (0)Fgf20465 ± 50Telogen (0)Fgf21898 ± 113Anagen VI (18)Fgf222685 ± 307Anagen VI (18)Fgf23(−)(−)Fgfr147,108 ± 3088Catagen (20)Fgfr210,414 ± 1179Anagen VI (18)Fgfr328,540 ± 2253Anagen VI (18)Fgfr41028 ± 214Catagen (20)β-actin562,240 ± 40,910Anagen V (8)The absolute mRNA copy number for each FGF (Fgf) and FGF receptor (Fgfr) gene was determined as described in the text; each mRNA copy number is indicated with the hair cycle phase at which mRNA expression was maximal. The values are means ± SE of five replicate samples.(−), genes with low copy numbers (<100 copies per ng mRNA); FGF, fibroblast growth factor; FGFR, FGF receptor. Open table in a new tab The absolute mRNA copy number for each FGF (Fgf) and FGF receptor (Fgfr) gene was determined as described in the text; each mRNA copy number is indicated with the hair cycle phase at which mRNA expression was maximal. The values are means ± SE of five replicate samples. (−), genes with low copy numbers (<100 copies per ng mRNA); FGF, fibroblast growth factor; FGFR, FGF receptor. It is noteworthy that even compared to those genes already known to be expressed in skin (i.e., FGF1, 2, 5, 7,10, 13, and 22), the expression of FGF18 mRNA, which peaked during telogen (days 0 and 22), was quite strong (Figure 1a). Indeed, at most stages, the level of FGF18 expression was much higher than that of the well-studied FGF2 (or basic FGF; Figure 1b), and the other FGF as well (Figure 1a). Moderate levels of FGF3, 6, 9, 11, 20, and 21 mRNA were also expressed (Figure 1a and b), but little or no expression of FGF4, 8, 14, or 15 mRNA was detected at any stage of the hair cycle (<100 copies per ng mRNA; Figure 1b and Table I). With respect to FGFR, the mRNA encoding both FGFR1 and FGFR3 were abundantly expressed (a maximum of ∼47,000 copies per ng mRNA for FGFR1 or ∼28,000 copies per ng mRNA for FGFR3; Figure 2). The strongest expression of the former occurred at anagen VI-catagen (days 18 and 20), whereas that of the latter peaked at anagen VI (day 18; Figure 2). The level of expression of FGFR2 mRNA, including its IIIb subclass, which is specific for epithelial cells, was lower than those of either FGFR1 or -3 mRNA (Figure 2). The level of FGFR4 mRNA was the lowest among the four receptors (Figure 2). This study addresses the expression and function of FGF18 in skin, so we initially determined the distribution of its mRNA using in situ hybridization. We found that at anagen V (day 8), anagen VI (day 18), and catagen (day 20), FGF18 mRNA was expressed virtually exclusively in the transient portion of the inner root sheath cells of the hair follicles, close to the hair bulb (Figure 3a, c, d; purple signals). At telogen, FGF18 mRNA signals were detected in the bulge region (Figure 3e). The sense probe produced no signal (Figure 3b). Although FGF18 mRNA was most abundant at telogen (Figure 1a), we discovered that mouse telogen skin contains high levels of a putative, non-specific inhibitor that strongly suppressed in situ detection of even abundantly expressed mRNA such as β-actin. 1Kawano and Imamura, manuscript in preparation. Consequently, an extended period of time was required to detect the rather weak FGF18 mRNA signals. To examine the target cell specificity of FGF18 in skin, we examined its mitogenic effect on human hair follicle dermal papilla cells (HDPC), human outer root sheath cells (HORSC), human dermal fibroblasts (HDF), human epidermal keratinocytes (HEK), and human umbilical vein endothelial cells (HUVEC). We found that at relatively high concentrations (100 and 1000 ng per mL) FGF18 induced DNA synthesis in HDPC, HDF, HEK, and HUVEC (Figure 4a, c, d, and e, respectively), which is in contrast to FGF7 (KGF), the well-characterized keratinocyte mitogen that stimulates DNA synthesis in HORSC and HEK (Figure 4b and d, respectively), but very weakly in HDPC (Figure 4a), HDF (Figure 4c), and HUVEC (Figure 4e). The in vivo effect of FGF18 on telogen hair follicles was investigated in C3H/HeN mice. In one set of experiments (Figure 5) carried out, 21 d after subcutaneous administration of FGF18 protein adsorbed onto Sepharose beads, four of five mice exhibited hair follicle growth. The exterior surface of the dorsal skin of one mouse exhibited vigorous hair growth throughout the entire test area, whereas three others exhibited hair growth and/or pigmentation at various parts of the test area (photographs not shown). Examination of the reverse side of the skin revealed extensive growth of anagen hair follicles in the mice with pigmentation and/or hair growth (Figure 5a and b, the reverse side of the pigmented area is shown; black spots indicate anagen hair follicles). We also noted that the skin was thicker in these areas than was the skin from control mice (Figure 5c and e), just as normal skin with physiological anagen hair follicles is thicker. The skin from one of the five mice that received FGF18 showed no apparent changes, whereas none of the skin samples from the five control mice exhibited hair growth or strong pigmentation. Examination of the reverse side of control skin revealed little or no anagen hair follicles, as is indicated by their white color (Figure 5d). Finally, these results were further confirmed by histological examination of skin sections (Figure 5c and e; representative results are shown). Anagen follicles were often observed in, but not limited to, the approximate area of the implanted beads, suggesting that FGF18 protein was not tightly bound to the Sepharose beads. When a similar set of experiments in which mice were kept for 24 d was carried out, five of eight mice that received FGF18 showed apparent induction of anagen hair follicles, as judged by pigmentation on the reverse side of the resected skin. By contrast, only two of seven mice that received phosphate-buffered saline (PBS) did so. Finally, we carried out another in vivo experiment, entailing examination of larger numbers of mice for a longer period of time (57 d) after injection of FGF18. In this experiment, all of the FGF18-injected mice showed vigorous hair growth by the end of the observation period, as did some of the PBS-injected mice (Figure 6). For comparison, the ratio of the hairy area to the total test area in each mouse was compared between the FGF18-and PBS-administrated mice. Statistical analysis showed this ratio to be significantly larger in FGF18-injected than PBS-injected mice [0.424±0.260 (n=15) vs 0.174±0.124 (n=11); p<0.01 (ANOVA)]. Among the many polypeptide growth factor systems involved in the dynamics of dermal function, the FGF system appears to play multiple roles in such physiological processes as wound healing and hair growth. No earlier studies, however, have presented a comprehensive or quantitative analysis of Fgf or Fgfr expression in skin and its appendages. We have now carried out such a study, and our results clearly show that a number of Fgf genes are active in mouse skin and that their expression changes dynamically and in differing patterns during the hair growth cycle. This suggests that they play distinct roles in the regulation of hair growth and related events. FGFR2 IIIb and its ligands, FGF7, 10 and, presumably, 22, comprise an important cell-type-specific system for regulating keratinocyte proliferation. Although FGF7 and FGF10 are expressed in dermal fibroblasts and papilla cells and stimulate proliferation of keratinocytes, FGF22 is expressed in epidermal and hair follicle keratinocytes and also stimulates keratinocyte proliferation (Nakatake et al., 2001Nakatake Y. Hoshikawa M. Asaki T. Kassai Y. Itoh N. Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle.Biochem Biophys Acta. 2001; 1517: 460-463Crossref PubMed Scopus (64) Google Scholar; Beyer et al., 2003Beyer T.A. Werner S. Dickson C. Grose R. Fibroblast growth factor 22 and its potential role during skin development and repair.Exp Cell Res. 2003; 287: 228-236Crossref PubMed Scopus (44) Google Scholar). Thus, this FGF system stimulates keratinocyte proliferation via both an epithelial–mesenchymal interaction and epithelial autocrine/paracrine stimulation. The present results confirmed that all three of these previously characterized keratinocyte mitogens are indeed expressed at high levels in skin. Furthermore, we noted remarkable variation in their mRNA expression throughout the hair cycle. It is interesting that both FGF7 and FGF10 are maximally expressed at anagen V (day 8), when hair grows vigorously, whereas expression of FGF22, a presumed autocrine keratinocyte growth factor, is strongly expressed at anagen VI (day 18), when hair follicle length reaches its maximum. FGF1, a wide-specificity prototype FGF ligand with the capacity to stimulate keratinocytes, is expressed in inner root sheath cells and other follicular cells (Kawano et al., 2004Kawano M. Suzuki S. Suzuki M. Oki J. Imamura T. Bulge-and basal layer-specific expression of fibroblast growth factor 13 (FHF-2) in mouse skin.J Invest Dermatol. 2004; 122: 1084-1090Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) and appears to induce dermal papilla cells to secrete growth promoting factors affecting the outer root sheath cells (Ota et al., 2002Ota Y. Saitoh Y. Suzuki S. Ozawa K. Kawano M. Imamura T. Fibroblast growth factor 5 inhibits hair growth by blocking dermal papilla cell activation.Biochem Biophys Res Commun. 2002; 290: 169-176Crossref PubMed Scopus (51) Google Scholar). Thus, although FGF1, 7, 10, and 22 are all capable of inducing cellular proliferation of the keratinocyte lineage, they differ in their expressing cells, mode of action, and target populations. A gene knockout study revealed that FGF5 is responsible for inducing catagen during the hair cycle, presumably by inhibiting activation of dermal papilla cells (Ota et al., 2002Ota Y. Saitoh Y. Suzuki S. Ozawa K. Kawano M. Imamura T. Fibroblast growth factor 5 inhibits hair growth by blocking dermal papilla cell activation.Biochem Biophys Res Commun. 2002; 290: 169-176Crossref PubMed Scopus (51) Google Scholar). We found that there was little expression of FGF5 mRNA at telogen (Figure 1a; days 0 and 22), but that the level was 20-fold higher at anagen VI (day 18), which is consistent with its ability to induce catagen. Other FGF also showed variation in their expression that was tied to hair cycle progression. Among strongly expressed FGF, FGF13 is expressed in hair follicle bulge cells and some basal layer keratinocytes (Kawano et al., 2004Kawano M. Suzuki S. Suzuki M. Oki J. Imamura T. Bulge-and basal layer-specific expression of fibroblast growth factor 13 (FHF-2) in mouse skin.J Invest Dermatol. 2004; 122: 1084-1090Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Although its activity related to stem cell function is not yet understood, the pattern of its mRNA expression observed in this study suggests a role during telogen. We showed here for the first time that FGF18 mRNA is strongly expressed in hair follicles. Our in situ hybridization showed that FGF18 mRNA was expressed in the bulge region during telogen, and that the site of expression moves to the transient portion of the inner root sheath with progression of hair follicle growth. We also found that FGF18 stimulates DNA synthesis in dermal fibroblasts and papilla cells, vascular endothelial cells, and also epidermal keratinocytes (Figure 4). This was somewhat unexpected, as FGF18 is known to activate FGFR2 IIIc and FGFR3 IIIc, but activity via FGFR2 IIIb, the keratinocyte-specific receptor, has never been reported. Still, one earlier study showed that administration of recombinant FGF18 to mice induces proliferation of hepatic parenchymal cells and pancreatic ductal epithelium (Hu et al., 1998Hu M.C. Qiu W.R. Wang Y.P. et al.FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation.Mol Cell Biol. 1998; 18: 6063-6074Crossref PubMed Scopus (108) Google Scholar), illustrating FGF18's capacity to exert effects on a wide range of cell types. FGF18 is also reportedly expressed in the developing brain (Hoshikawa et al., 2002Hoshikawa M. Yonamine A. Konishi M. Itoh N. FGF-18 is a neuron-derived glial cell growth factor expressed in the rat brain during early postnatal development.Brain Res Mol Brain Res. 2002; 105: 60-66Crossref PubMed Scopus (27) Google Scholar), pancreas (Dichmann et al., 2003Dichmann D.S. Miller C.P. Jensen J. Scott Heller R. Serup P. Expression and misexpression of members of the FGF and TGFbeta families of growth factors in the developing mouse pancreas.Dev Dyn. 2003; 226: 663-674Crossref PubMed Scopus (92) Google Scholar), and adipose tissue (Gabrielsson et al., 2002Gabrielsson B.G. Johansson J.M. Jennische E. et al.Depot-specific expression of fibroblast growth factors in human adipose tissue.Obes Res. 2002; 10: 608-616Crossref PubMed Scopus (74) Google Scholar). It also reportedly stimulates proliferation of osteoblasts and chondrocytes (Shimoaka et al., 2002Shimoaka T. Ogasawara T. Yonamine A. et al.Regulation of osteoblast, chondrocyte, and osteoclast functions by fibroblast growth factor (FGF)-18 in comparison with FGF-2 and FGF-10.J Biol Chem. 2002; 277: 7493-7500Crossref PubMed Scopus (133) Google Scholar), influences lung morphogenesis (Whitsett et al., 2002Whitsett J.A. Clark J.C. Picard L. et al.Fibroblast growth factor 18 influences proximal programming during lung morphogenesis.J Biol Chem. 2002; 277: 22743-22749Crossref PubMed Scopus (68) Google Scholar) and improves learning and memory (Cavallaro et al., 2002Cavallaro S. D'Agata V. Manickam P. Dufour F. Alkon D.L. Memory-specific temporal profiles of gene expression in the hippocampus.Proc Natl Acad Sci USA. 2002; 99: 16279-16284Crossref PubMed Scopus (151) Google Scholar). FGFR-2 and FGFR-3 are expressed in hair follicle matrix cells (Widelitz et al., 1997Widelitz R.B. Jiang T.X. Noveen A. Ting-Berreth S.A. Yin E. Jung H.S. Chuong C.M. Molecular histology in skin appendage morphogenesis.Microsc Res Tech. 1997; 38: 452-465Crossref PubMed Scopus (47) Google Scholar), suggesting that these cells may be the targets of FGF 18 activity. Interestingly, it was recently reported that Fgf18 is a target gene of Wnt/β-catenin (Shimokawa et al., 2003Shimokawa T. Furukawa Y. Sakai M. Li M. Miwa N. Lin Y.M. Nakamura Y. Involvement of the FGF18 gene in colorectal carcinogenesis, as a novel downstream target of the beta-catenin/T-cell factor complex.Cancer Res. 2003; 63: 6116-6120PubMed Google Scholar), which is an essential signaling pathway for skin and hair formation (Van Mater et al., 2003Van Mater D. Kolligs F.T. Dlugosz A.A. Fearon E.R. Transient activation of beta-catenin signaling in cutaneous keratinocytes is sufficient to trigger the active growth phase of the hair cycle in mice.Genes Dev. 2003; 17: 1219-1224Crossref PubMed Scopus (222) Google Scholar). FGF18 may thus be an important modulator of skin and hair formation. These results prompted us to investigate the activity of FGF18 on hair growth in vivo. Administration to mice with dorsal telogen phase hair follicles revealed that FGF18 is capable of inducing the anagen growth state, which is consistent with its involvement in modulating hair growth. It is presently unclear whether FGF18 directly promotes the proliferation of hair follicle keratinocytes or does so indirectly. Given that, in vitro, FGF18 induced DNA synthesis in HDPC and HUVEC but not HORSC, even at a high concentration, it is unlikely that its ability to potently promote hair growth in vivo involves direct mitogenic activity. Instead, the observation that it takes 3 wk or more to induce hair growth suggests that FGF18 acts indirectly to modulate the hair growth cycle. FGF18 may induce dermal papilla cells to secrete growth factors affecting hair follicle cells, or it may affect blood circulation by acting on vascular endothelial cells. On the other hand, FGF18 exerted a mitogenic effect on dermal fibroblasts (Figure 4) and may be involved in the growth of adipocytes (Gabrielsson et al., 2002Gabrielsson B.G. Johansson J.M. Jennische E. et al.Depot-specific expression of fibroblast growth factors in human adipose tissue.Obes Res. 2002; 10: 608-616Crossref PubMed Scopus (74) Google Scholar), suggesting that the increased skin mass seen in FGF18-treated mice may be attributable to a direct effect on those cells. In sum, the results suggest that by acting on various cell types under specific sets of conditions, FGF dynamically regulate dermal system function. Direct administration of FGF18 and/or modulation of its expression may thus be an effective approach to stimulating hair growth. Throughout this study, all animals received humane care that was in accordance with the guidelines of National Institute of Advanced Industrial Science and Technology (AIST). All protocols were approved by the animal experiment committee of AIST Central 6. For hair cycle experiments, 25 seven-wk-old male C3H/HeN mice (Tlr4+/+)