localization of rat fgf-5 protein in skin macrophage-like cells and fgf-5s protein in hair follicle:...

Localization of Rat FGF-5 Protein in Skin Macrophage-likeCells and FGF-5S Protein in Hair Follicle: Possible Involvementof two Fgf-5 Gene Products in Hair Growth Cycle Regulation

Satoshi Suzuki, Tomomi Kato, Hiroyuki Takimoto, Shigeki Masui, Hiroshi Oshima, Kazuo Ozawa,* Seigo Suzuki,*and Toru Imamura*POLA R&D Laboratories, Kashio-cho, Totsuka-ku, Yokohama, Japan; *Biosignaling Department, National Institute of Bioscience and Human Technology,Higashi, Tsukuba, Ibaragi, Japan

It has been reported that the gene for murine fibroblastgrowth factor-5 (Fgf-5) is expressed in the rat hair follicleand that this expression may be associated with catageninduction (Hebert et al, 1994). In this study, we analyzedthe Fgf-5 gene product in skin because the gene generatestwo mRNA that translate into the FGF-5 protein and ashort form of the FGF-5 protein (FGF-5S) as a result ofan alternative splicing (Hattori et al, 1996; Ozawa et al,1996). Indeed, we detected both types of FGF-5 mRNA inrat skin samples. Two monoclonal anti-FGF-5 antibodies,one (E723) being specific for FGF-5 long-form proteinand the other (B2B6) being reactive with both FGF-5and FGF-5S proteins, were used to locate these proteinsby immunohistochemistry. Staining of the rat skinrevealed that only the B2B6 antibody reacted with hairfollicles and that both antibodies reacted with macro-

In mammals, the hair growth cycle consists of three phases:anagen, when follicles grow and hair synthesis take place;catagen, when the follicles degenerate; and telogen, when thefollicles are at rest (Chase, 1954). In addition, anagen can bedivided into six short phases, anagen I to anagen VI (Chase,

1954). The hair growth is regulated by several cytokines, such as tumorgrowth factor α (Luetteke et al, 1993; Mann et al, 1993), epidermalgrowth factor (Green et al, 1984; Luetteke et al, 1994), bone morphog-enetic protein-2A (Lyons et al, 1990), insulin-like growth factor-I(Itami et al, 1995), and some of fibroblast growth factors (FGF) (duCros, 1993; Guo et al, 1993).

The FGF family consists of at least 13 members that have variousbiologic activities (Abraham et al, 1986; Jaye et al, 1986; Moore et al,1986; Delli-Bovi et al, 1988; Burgess and Maciag, 1989; Marics et al,1989; Rubin et al, 1989; Baird and Klagsbrun, 1991; Tanaka et al,1992, 1995; Miyamoto et al, 1993; Baird, 1994; Mason, 1994; Yamasakiet al, 1996; Hartung et al, 1997; Verdier et al, 1997). The Fgf-5 genewas originally reported as one of the human oncogenes (Zhan et al,1987), but then it was classified as belonging to the FGF family becauseof its high homology with acidic and basic FGF (Zhan et al, 1988).FGF-5 mRNA is expressed in the mouse embryo (Hebert et al, 1990,

Manuscript received November 22, 1997; revised August 19, 1998; acceptedfor publication August 23, 1998.

Reprint requests to: Satoshi Suzuki, POLA R&D Laboratories, 560 Kashio-cho, Totsuka-ku, Yokohama 244–0812, Japan.

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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phage-like round cells, suggesting that the product of theFgf-5 gene in the hair follicle is FGF-5S. The immunoreac-tivity of the FGF-5S protein increased during early anagenVI and decreased rapidly during catagen. The densityof FGF-5-positive macrophage-like cells in the dermisincreased during anagen and decreased during catagenand telogen, whereas the density of these cells in thepanniculus adiposus did not change during anagen andincreased during catagen and telogen. There was noapparent association between the density of FGF-5-positive macrophage-like cells and that of FGF-5-nega-tive, dendritic macrophage-like cells. Thus, the resultssuggest the possible involvement of FGF-5S in the hairfollicle in anagen VI and catagen development and thatthe density of FGF-5-positive macrophage-like cells mayalso be associated with the hair growth cycle. Key words:anagen VI/catagen. J Invest Dermatol 111:963–972, 1998

1991; Haub and Goldfarb, 1991; Ozawa et al, 1996) and in the centralnervous system of the adult mouse (Haub et al, 1990; Ozawa et al,1996). Hebert et al (1994) reported that FGF-5 mRNA is also expressedin the outer root sheath of mouse hair follicles during anagen VI, andthat anagen VI is abnormally prolonged in mice that lack exon 1 ofthe Fgf-5 gene. Furthermore, Rosenquist and Martin (1996) foundthat the FGF-5 receptor gene is expressed in the dermal papillae andseems to regulate the development and activity of the hair follicle.These findings suggest that the expression of the Fgf-5 gene inducesthe dermal papillae to finish anagen and to start catagen; however, itwas recently reported that the Fgf-5 gene produces a short form of theFGF-5 protein (FGF-5S) as well as the full-length FGF-5 protein(Hattori et al, 1996; Ozawa et al, 1996). The Fgf-5 gene consists ofthree exons; the FGF-5S lacks the region corresponding to exon 2and most of the region corresponding to exon 3 as a result of analternative splicing and a frame shift, respectively (Hattori et al, 1996).This finding gave rise to questions of which product of the Fgf-5 gene,FGF-5 or FGF-5S, is produced in hair follicles and whether these twoproducts are associated with the hair growth cycle.

In this study, we prepared an anti-FGF-5 monoclonal antibody usinga synthetic polypeptide-immunogen whose amino acid sequence iscontained in the FGF-5 protein but not in the FGF-5S protein. In aprevious study (Ozawa et al, manuscript in preparation), a monoclonalantibody against both FGF-5 and FGF-5S was also prepared. Usingthese antibodies, we carried out immunohistochemical studies of ratskin in order to localize FGF-5 and FGF-5S in the skin and to

964 SUZUKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 1. RT-PCR of rat FGF-5 and FGF-5S mRNA. Total RNA from 10 d old rat dorsalskin was reverse-transcribed using (dNTP)6primer and subjected to PCR using theprimer pair 59-CCTTCGGGGCGCCGGA-CCGGCA-39 (sense) and 59-AAGTTCCG-GTTGCTCGGACTGCTT-39 (anti-sense). (a)Schematic drawing of rat FGF-5 and FGF-5SmRNA, and their open reading frames. Exons(1, 2, 3) are indicated by boxes. Asterisksindicate the positions of the stop codon.Positions of the sense and anti-sense PCRprimers are indicated by arrows. (b) Rat FGF-5cDNA nucleotide sequence (lower case letters)and FGF-5 and FGF-5S amino acids (capitalletters). The primer annealing sites areunderlined. A broken line shows the positionof exon 2, which is truncated in FGF-5SmRNA and protein. Nucleotides are numberedfrom the first nucleotide of the ATG initiationcodon. Sequences are quoted from Hattori et al(1996).

investigate the association of Fgf-5 gene products with the hairgrowth cycle.

MATERIALS AND METHODS

Reverse transcriptase-polymerase chain reaction (RT-PCR) RNA from10 d old rat dorsal skins was prepared using Isogen solution (Nippon Gene,Tokyo, Japan) according to the manufacturer’s protocol (Chomczynski, 1993).RT-PCR was carried out as previously described (Ozawa et al, 1997). Theupper primer, 59-CCTTCGGGGCGCCGGACCGGCA-39, was set in exon1 and the lower primer, 59-AAGTTCCGGTTGCTCGGACTGCTT-39, wasset in exon 3 so both FGF-5 mRNA and FGF-5S mRNA were amplified, yetthe amplified fragments were distinguishable from each other (Fig 1). Theamplified fragments are 435 b for FGF-5 mRNA and 310 b for FGF-5SmRNA. PCR amplification was performed with a Perkin-Elmer ThermalCycler using 35 cycles as follows: 1 min at 94°C, 2 min at 55°C, and 1 min at72°C. The amplified DNA was separated by 1.3% agarose gel electrophoresisand photographed.

Preparation of FGF-5S recombinant protein The open reading frame ofhuman FGF-5S was subcloned into the pMAL-c2 vector (New England Biolabs,Beverly, MA). Transformed E. coli produced FGF-5S fused to maltose-bindingprotein. The fusion protein was purified by binding to amirose (New EnglandBiolabs), released with 10 mM maltose, and cleaved with specific proteasefactor Xa (New England Biolabs). The FGF-5S protein was separated frommaltose-binding protein by amirose column chromatography.

Preparation of antibodies To prepare a monoclonal antibody against FGF-5, a synthetic chimeric polypeptide with amino acid sequence CTYA-SAIHRTEKTG, named F5–1, was conjugated to keyhole limpet hemocyaninusing m-maleimidobenzoyl-N-hydroxysuccinimide ester and used as the immu-nogen. The F5–1 polypeptide contains a partial sequence of human FGF-5,TYASAIHRTEKTG, which is encoded by exon 3 (Zhan et al, 1988). Thissequence is not present in the FGF-5S protein (Hattori et al, 1996), but isshared by rat and mouse FGF-5 (Haub et al, 1990; Hattori et al, 1996) and noother members of the FGF family (Jaye et al, 1986; Abraham et al, 1986; Moore

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Figure 2. FGF-5 mRNA and FGF-5S mRNA are detectable in rat skin.RT-PCR experiments of RNA from 10 d old rat dorsal skin were carried outusing the primers that amplify both FGF-5 mRNA and FGF-5S mRNA, asshown in Fig 1. M, molecular weight marker ‘‘123 bp DNA ladder’’ (GibcoBRL). Lane 1, Rat-skin RNA; lane 2, negative control (no template).

Figure 3. E723 reacts with FGF-5 and B2B6 reacts with both FGF-5and FGF-5S. Recombinant human FGF-5, FGF-5S, FGF-6, and FGF-7 wereresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thentransferred to a polyvinylidene difluoride membrane. The membrane was treatedwith E723 antibody (a) or B2B6 antibody (b) and then with horseradishperoxide-conjugated goat anti-mouse Ig antibodies, followed by visualizationusing ECL.

et al, 1986; Delli-Bovi et al, 1988; Marics et al, 1989; Rubin et al, 1989; Tanakaet al, 1992, 1995). Amino acid C was required for conjugation to keyholelimpet hemocyanin. F5–1 was synthesized by Tana Laboratories (Houston, TX).

An emulsion of immunogen 100 µg and Freund’s complete adjuvant (Sigma,St. Louis, MO) was subcutaneously injected into BALB/C mice. Second andthird immunizations followed (at 1 wk intervals) with an emulsion of immunogen100 µg/Freund’s incomplete adjuvant (Sigma). Two weeks later, 50 µgimmunogen dissolved in 50 µl saline was injected intravenously as a boosterimmunization. After the booster immunization, spleen cells were fused withmyeloma P3 3 63Ag8.653 cells (Dainihon Seiyaku, Osaka, Japan), and

subsequent hybridoma selection was carried out in GIT medium (Wako, Osaka,Japan) containing hypoxanthine, aminopterin, and thymidine (Kshler andMilstein, 1975). Hybridomas producing the desired antibody were selectedby enzyme-linked immunosorbent assay using the polypeptide-immunogenconjugated to ovalbumin to eliminate antibodies that react with keyhole limpethemocyanin. The conjugation was carried out using the Activated ImmunogenConjugation Kit (Pierce, Rockford, IL). The antibodies were detected withhorseradish peroxidase-conjugated goat anti-mouse IgG (Tago, Bulingame,CA). One of these antibodies was named E723.

Anti-FGF-5/FGF-5S monoclonal antibody was generated essentially asdescribed above. The clone B2B6 was selected due to its reactivity to bothFGF-5 and FGF-5S proteins (Suzuki et al, manuscript in preparation).

The isotype of E723 was determined using a Mouse Monoclonal AntibodyIsotyping Kit (Amersham, Bucks, U.K.). The kit contains typing sticks carryinggoat antibodies specific for different types of mouse immunoglobulin chains. Aperoxidase-labeled antibody was used to detect the monoclonal antibody boundto the goat antibody on the stick.

Western blotting of FGF-5 and FGF-5S The reactivity of E723 antibodyand B2B6 antibody was examined by western blot analysis. Recombinanthuman FGF-5 protein (Sigma) or FGF-5S protein underwent sodium dodecylsulfate-polyacrylamide gel electrophoresis on a 5%–10% gradient gel andwas then transferred onto a polyvinylidene difluoride membrane (Pharmacia,Uppsala, Sweden). Recombinant human FGF-6 protein (Sigma) and FGF-7protein (Pepro Tech, Rocky Hill, NJ) also underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidenedifluoride membrane as negative controls. The amount of each protein wasadjusted to 0.1 µg per lane. Then, the polyvinylidene difluoride membranewas treated with E723 antibody (40 µg per ml) or B2B6 antibody (50-times diluted). Horseradish peroxide-conjugated goat anti-mouse Ig antibodies(Amersham) were used as a secondary antibody (5000-times diluted). Visualiza-tion and film exposure were performed using an ECL western blotting detectionkit (Amersham).

Immunofluorescence staining of NIH-3T3 cells transfected with FGF-5 cDNA Expression plasmid pMexNeo/FGF-5, which contains the full-length cDNA for human FGF-5, was mixed with Lipofect Amine (Gibco BRL,Paisley, U.K.) and added to NIH-3T3 mouse cell cultures. The cells wereincubated at 37°C for 23 h. The specificity of E723 antibody was assessed byimmunocytochemical staining of NIH-3T3 cells transfected with FGF-5 cDNA(NIH-3T3/FGF-5) and untransfected control. Cells were fixed in 10% formalinin phosphate-buffered saline (pH 7.2) for 20 min, and then treated with 0.5%Triton X-100 in phosphate-buffered saline for 5 min at room temperature. Thecells were incubated with E723 antibody (40 µg per ml) at room temperaturefor 30 min and then with 50-times diluted biotin-conjugated goat anti-mouseIgG (Chemicon, Temecula, CA) at room temperature for 30 min, followed byfurther incubation with 100-times diluted fluorescein-conjugated streptavidin(Biosource International, Camarillo, CA). After each treatment, cells werewashed with phosphate-buffered saline at room temperature (5 min, threetimes). The cells were observed under a fluorescence microscope (OlympusBH2) through a 515 nm absorption filter.

Immunohistochemical study of rat skin with anti-FGF-5 antibody andanti-FGF-5/FGF-5S antibody Dorsal skin samples were obtained fromWistar rats (2–26 d old; four rats of each age), fixed in 10% formalin at roomtemperature and embedded in paraffin. Sections (4 µm in thickness) weremounted on gelatin-coated glass slides (Matsunami Glass, Osaka, Japan). Threesections were prepared from each animal. The sections were deparaffinized inxylene, dehydrated through a series of ethanol solutions of increasing concentra-tion. The sections were immunostained using the avidin-biotin immunoperoxid-ase method. Sections were treated with 0.3% H2O2 in methanol and thenincubated in 10% goat serum (Nichirei, Tokyo, Japan) as blocking solution for10 min at room temperature. Then, in the following order, sections wereincubated with B2B6 (100-times diluted) or E723 (40 µg per ml) for 30 min,50-times diluted biotin-conjugated goat anti-mouse IgG (Chemicon) for 10 min,and horseradish peroxide-conjugated avidin solution (Nichirei) for 10 min. Allincubations were carried out at room temperature. Visualization by theperoxidase reaction using diaminobenzidine and hydrogen peroxide was per-formed using the SAB-PO (M) kit (Nichirei). After each treatment, sectionswere washed with phosphate-buffered saline at room temperature (5 min,three times).

At the same time, two 2 µm sections of 12 d old rat dorsal skin wereprepared. The sections were immunolabeled, one with B2B6 and the otherwith E723, as described above. They were then compared under microscopy.

Identification of E723-labeled cells Two 2 µm sections of 18 d old ratdorsal skin were prepared. The sections were immunolabeled, one with E723


Figure 4. The reaction of E723 is specific. NIH-3T3 cells transfected with an FGF-5 expression vector (NIH-3T3/FGF-5) and untransfected control cellswere labeled with E723 antibody followed by staining using the avidin-biotin method. (a) NIH-3T3/FGF-5 cells treated with E723 (dark field); (b) bright field of(a); (c) NIH-3T3 control cells treated with E723 (dark field); (d) bright field of (c). Scale bar: 20 µm.

and the other with a commercial anti-rat macrophage monoclonal antibody,40-times diluted ED2 (BMA, Rheinstrasse, Switzerland), as described above.They were then compared under microscopy. Normal mouse IgG (Chemicon)was used as the control.

Density of FGF-5-positive and FGF-5-negative macrophage-likecells Sections of dorsal skin (4 µm in thickness) were prepared from rats(three sections from each animal) of various ages (four rats of each age) andlabeled with E723 antibody as described above. The number of FGF-5-positivecells in the dermis and in the panniculus adiposus of each section were countedand then divided by the area of the dermis or panniculus adiposus to calculatethe density of FGF-5-positive cells. At the same time, the density of FGF-5-negative macrophage-like cells was also calculated by subtracting the density ofFGF-5-positive cells in the dermis or in the panniculus adiposus from the totalnumber of cells labeled with ED2 antibody. The results were statisticallyanalyzed by Student’s t test.

RESULTS

FGF-5 mRNA and FGF-5S mRNA are detectable in rat skin Asshown in Fig 1, the primers that amplify both FGF-5 and FGF-5SmRNA were used to detect these mRNA in skin by RT-PCR. Asshown in Fig 2, amplified products corresponding to both FGF-5(435 bp) and FGF-5S (310 bp) were detected in the 10 d old rat skinsample. Thus, it was confirmed that both FGF-5 mRNA and FGF-5SmRNA are expressed in anagen VI rat skin.

Isotype of E723 The isotype of E723 was identified as IgG1, havinga k light chain.

E723 reacts with FGF-5 and B2B6 reacts with both FGF-5 andFGF-5S In order to confirm the reactivity of the monoclonal


Figure 5. B2B6 antibody binds to the cortex of the hair follicle. Formalin-fixed, paraffin-embedded rat dorsal skin sections were incubated with B2B6 andstained by the avidin-biotin immunoperoxidase method. (a) Skin section of a 6 d old rat (anagen IV); (b) 12 d old rat (early anagen VI); (c) 18 d old rat (beginningof catagen); (d) 19 d old rat (catagen); (e) 20 d old rat (catagen); and (f) 25 d old rat (telogen). Negative control sections were treated with nonimmuno IgG (g).Scale bar: 20 µm.

antibodies used in this study, we performed western blot analysis usingrecombinant human FGF-5 and FGF-5S proteins. Recombinant humanFGF-6 and FGF-7 proteins were used as the negative controls. Asshown in Fig 3(a), E723 antibody reacted with FGF-5 but not withFGF-5S. B2B6 antibody reacted with both FGF-5 and FGF-5S(Fig 3b). These antibodies did not react with the other proteins used.

The reaction of E723 is specific Specificity of the E723 antibodyin the immunocytochemical study was further confirmed. E723 reactedwith NIH-3T3/FGF-5 cells (Fig 4a), but not with the control NIH-3T3 cells (Fig 4c). Thus, it was indicated that E723 antibody specificallyrecognizes the FGF-5 protein in immunocytochemical studies.

FGF-5S is present in the cortex of the hair follicle and FGF-5locates in skin round cells B2B6 stained the cortex of the hairfollicle (Fig 5) and skin round cells (Fig 6a). The cortex was stainedvery weakly during anagen IV (Fig 5a), and strongly during earlyanagen VI (Fig 5b). The immunoreactivity decreased a little butremained high at the beginning of catagen (Fig 5c), and decreasedrapidly during catagen (Fig 5d, e). During telogen, the immunoreactiv-ity was as low as during anagen IV (Fig 5f). The cortex was notstained in the negative controls (Fig 5g), indicating that the signalswere specific.

The round cells labeled by B2B6 (Fig 6a) were also labeled byE723 (Fig 6b). E723 bound only to these round cells and not to the


Figure 6. B2B6 antibody binds to cells that are also labeled with E723.Two 2 µm dorsal skin sections from 12 d old rats were prepared andimmunohistochemically labeled with B2B6 (a) and E723 (b). Arrows indicateround cells labeled with both antibodies. Scale bar: 20 µm.

hair follicle during all phases of the hair growth cycle, including anagenVI (days 9–17) and catagen (days 18–21) as shown in Fig 7(a–k).These cells were observed in the dermis and the panniculus adiposus,and the immunoreactivity of the cells remained constant throughoutthe hair cycle (Fig 7a–k). The diameter of most cells was between 10and 20 µm and their nuclei were egg- or kidney-shaped.

These results indicate that FGF-5S is present in the cortex of thehair follicle and FGF-5 locates in round cells in the skin.

FGF-5-positive cells are also labeled with anti-macrophageantibody To identify FGF-5-positive cells labeled with E723 anti-body, rat dorsal skin sections labeled with E723 or ED2 (monoclonalantibody against rat macrophages) were compared. Both ED2 andE723 labeled round cells (Fig 8a, b). ED 2 also labeled dendritic cells(Fig 8c) that were not labeled by E723 (Fig 8d). These results suggestthat there are two types of cell, namely the round shaped cell and thedendritic cell, which react with anti-macrophage antibody in the skinand E723 antibody binds only to the round-shaped cell. InFig 8(c, d), a portion of the outer layer of the hair follicle matrix is

slightly stained; however, this part is also stained in the negativecontrols (Fig 8e), indicating that the signal is nonspecific.

The density of FGF-5-positive and FGF-5-negative macro-phage-like cells change during the hair cycle When examiningthe association of FGF-5-positive macrophage-like cells with the hairgrowth cycle, changes in cell density (cell number per area) in thedermis and panniculus adiposus were analyzed separately. The densityof FGF-5-positive macrophage-like cells in the dermis increased duringanagen and decreased during catagen and telogen (Fig 9a), whereasthe density of these cells in the panniculus adiposus did not changeduring anagen and increased during catagen and telogen (Fig 9b).

There was no apparent association between the density of FGF-5-positive macrophage-like cells and that of FGF-5-negative, dendriticmacrophage-like cells. In the dermis, the density of FGF-5-negativemacrophage-like cells continued to increase as the hair cycle proceededfrom anagen to telogen (Fig 10a). In the panniculus adiposus, thedensity of these cells did not change during anagen or catagen butdecreased during telogen (Fig 10b).

DISCUSSION

Hebert et al (1994) reported that the Fgf-5 gene is expressed in theouter root sheath of mouse hair follicles during anagen VI. Similaritiesbetween the rat and mouse hair growth cycle with respect to thetiming of each phase and the concomitant morphologic changes inhair follicles (Randall, 1957), suggest that their hair growth cycles arecontrolled by the same system at a molecular level. If this is the case,then the Fgf-5 gene should also be expressed in the outer root sheathof the rat hair follicle; however, Hebert et al did not clarify whichmRNA, FGF-5 mRNA or FGF-5S mRNA, was expressed in the hairfollicle, because they used an anti-sense RNA probe for the full-lengthmRNA of FGF-5. Our RT-PCR result revealed that both FGF-5mRNA and FGF-5S mRNA are expressed in rat skin (Fig 2). Weherein report for the first time that not only FGF-5 mRNA (Petho-Schramn et al, 1996), but also FGF-5S mRNA is expressed in the skin.The immunohistochemistry with B2B6 antibody, which reacts withboth FGF-5 and FGF-5S, revealed that Fgf-5 gene products locate inthe cortex of the hair follicle and round cells of the skin (Figs 5, 6).Because E723 antibody reacts with FGF-5 bound only to round cellsand not to the hair follicle (Fig 7), it was concluded that the productof the Fgf-5 gene in the hair follicle is FGF-5S and that FGF-5 locatesin the round cells of the skin; however, FGF-5S located in the cortex,not in the outer root sheath. Some proteins are known to locate intissues different from their expression site (Hauguel-de Mouzon et al,1997; Kasai et al, 1997). Furthermore, the cortex is close to the outerroot sheath. Indeed, both FGF-5 and FGF-5S are secreted fromproducing cells after translation, due to the presence of a secretionsignal peptide in their primary structures (Ozawa et al, manuscript inpreparation). Therefore, the result of our immunohistochemistry doesnot necessarily contradict with the in situ hybridization study thatHebert et al performed. It remains unclear why Hebert et al did notdetect Fgf-5 gene expression in round cells. The immunoreactivity ofthese cells was, however, at least eight times weaker than the cortexof the hair follicle, as was indicated by the required concentrations ofB2B6 solution for immunostaining (data not shown). This indicatesthat the detection of Fgf-5 gene expression in round cells mightbe difficult.

The cortex of the hair follicle was stained very weakly with B2B6during anagen IV (Fig 5a), and strongly during early anagen VI(Fig 5b). The immunoreactivity decreased a little but remained highat the beginning of catagen (Fig 5c), and decreased rapidly duringcatagen (Fig 5d, e). The immunoreactivity measured by B2B6 in thehair follicle should reflect the concentrations of FGF-5S. AlthoughHebert et al (1994) reported that the Fgf-5 gene is expressed in thehair follicle only during early anagen VI, our data indicate that FGF-5S is weakly expressed even during anagen IV. The concentration ofthe FGF-5S protein, however, seems to be very low during anagenIV. This low expression level explains the ease with which one couldfail to detect the mRNA. Because the concentration of the FGF-5Sprotein seems to decrease rapidly during catagen, it is conceivable that


Figure 7. E723 labeled only round cells of rat skin during the hair cycle. Rat dorsal skin sections were incubated with E723 and stained by the avidin-biotin immunoperoxidase method. Skin section of a 6 d old rat (a), 12 d old rat (b), 13 d old rat (c), 14 d old rat (d), 15 d old rat (e), 16 day old rat (f), 17 d oldrat (g), 18 d old rat (h), 19 d old rat (i), 20 d old rat (j), and 21 d old rat (k). Arrows indicate E723-labeled cells. Scale bar: 20 µm.

the decrease of this protein could be in association with the progressof catagen. Petho-Schramn et al (1996) reported that FGF-5 mRNAwas detectable in the skin throughout the hair cycle by RT-PCR.Their primer included a part of the exon 2 fragment of FGF-5 mRNA.Therefore, it is considered that they detected only long-form FGF-5mRNA expression, and not FGF-5S mRNA expression. They alsoreported that FGF-5 mRNA expression level rises during anagen even

long before late anagen VI, and remains high after catagen initiation.Ozawa et al (manuscript in preparation) demonstrated that the functionsof the long-form FGF-5 protein, namely stimulation of fibroblastproliferation or morphologic change of nerve cells, are partiallyantagonized by FGF-5S in vitro. FGF-5S may also behave as anantagonist against FGF-5 in vivo. As described above, FGF-5S seemsto decrease rapidly during catagen. This gives rise to the idea that the


Figure 8. The FGF-5-positive cells are also labeled with anti-macrophage antibody. Two 2 µm dorsal skin sections from 18 d old rats were prepared andimmunolabeled with ED2 and E723. Round cells labeled with ED2 (a) or E723 (b). Dendritic cells labeled with ED2 (c) or E723 (d). Negative control sectiontreated with nonimmuno IgG (e). r, Round cells labeled with both ED2 and E723. Arrows indicate dendritic cells labeled only with ED2. Scale bar: 20 µm.

Figure 9. The density of FGF-5-positive macrophage-like cells changesduring the hair cycle. The density of the FGF-5-positive macrophages in thedermis (a) and in the panniculus adiposus (b) was determined. Data are pressedas mean values 1 SD. ** Statistically significant differences (p , 0.01) between12 d and 18 d old rats, and between 18 d and 26 d old rats by Student’s t test.NS, no significant difference. The hair growth cycle was adapted fromRandall (1957).

function of FGF-5 is in the induction of catagen and that FGF-5Smodifies the function of FGF-5, in other words the decrease of FGF-5S allows the action of FGF-5.

The diameter of most of the FGF-5-positive round cells was between10 and 25 µm and their nuclei were egg- or kidney-shaped (Figs 6,7). These features suggest that these cells are macrophages (van Furthet al, 1972). ED2 anti-rat macrophage antibody labeled the same typeof cells as E723 did (Fig 8a, b), although ED2 also labeled dendriticcells (Fig 8c), which E723 did not label (Fig 8d). Therefore, weconclude that there are two types of cell that react with anti-macrophageantibodies in rat skin, and that FGF-5 locates in only one of them,namely the round macrophage-like cell. We conducted immunohisto-chemical staining on rat thymus, spleen, kidney, and liver sectionsusing E723 and ED2. Although many macrophages were stained withED2 in these organs, none of them was stained with E723 (data notshown). This suggests that FGF-5 specifically locates in the round-shaped macrophage-like cells of the skin, rather than in macrophagesin general. Therefore, it is conceivable that FGF-5 expression isnot related to any generally known function of macrophages, e.g.,

Figure 10. The density of FGF-5-negative macrophage-like cells alsochanges, but differently from FGF-5-positive macrophage-like cells.The densities of FGF-5-positive and FGF-5-negative macrophage-like cells inthe dermis (a) and in the panniculus adiposus (b) were analyzed with 12 d old(anagen), 18 d old (catagen), and 26 d old (telogen) rat skin sections. The bargraph represents mean values 1 SD. *p , 0.05, ** p , 0.01, *** p , 0.001;statistically significant differences between samples as determined by Student’st test.

phagocytosis, antigen-presenting activities, and cytokine/proteasesecretion.

Immunohistochemical study using the E723 antibody revealed thatthe density of FGF-5-positive macrophage-like cells was associatedwith the hair cycle, and they behave differently in the dermis and thepanniculus adiposus. Therefore, we investigated the density of the cellsin both the dermis and the panniculus adiposus. The density of FGF-5-positive macrophage-like cells during anagen increased in the dermisbut stayed the same in the panniculus adiposus. During catagen andtelogen, the density of FGF-5-positive macrophage-like cells decreasedin the dermis but increased in the panniculus adiposus (Fig 9). Hairfollicles grow during anagen and then degenerate and move to thedermis from the panniculus adiposus during catagen and telogen (Chase,1954; Randall, 1957). The results suggested that the density of FGF-5-positive macrophage-like cells in the dermis or panniculus adiposusis also associated with the hair growth cycle, at least in the postnatalperiod. FGF-5-positive macrophage-like cells are possibly in control


of fibroblasts in the dermis and adipocytes in the panniculus adiposus.As described above, Petho-Schramn et al (1996) reported that FGF-5mRNA levels change during the hair cycle. Changes in the populationof FGF-5-positive macrophage-like cells, however, did not reflect theFGF-5 mRNA levels. A possible explanation for this contradiction isthat the expression level of FGF-5 mRNA in each cell may fluctuateduring the hair cycle, although it was unclear whether the immunoreac-tivity of E723 antibodies changed during the hair cycle. The densityof FGF-5-negative macrophage-like cells also changed during the hairgrowth cycle (Fig 10), but differently from those of FGF-5-positivemacrophage-like cells; however, dendritic macrophage-like cellinvolvement in the hair growth cycle has not been ruled out.

Hebert et al (1994) reported that mutant mice that lack exon 1 ofthe Fgf-5 gene exhibited prolonged anagen; however, since exon 1 isincluded in both FGF-5 and FGF-5S, their study left unclear thequestion of which protein, FGF-5 or FGF-5S, is associated with thehair growth cycle. Our results suggest that both FGF-5 and FGF-5Sare probably involved, at least in the postnatal hair cycle. It is notcertain whether our results are applicable to later cycles, although ithas been reported that the first hair growth cycle is similar to latercycles with respect to phase length and morphology (Moretti et al,1966); however, the most important purpose of this study was tolocalize Fgf-5 gene products, therefore we focused on the postnatalhair cycle in which Hebert et al detected Fgf-5 gene expression (1994).To further investigate the association of Fgf-5 gene products with thehair cycle, the direct effects of gene products on the hair cycle needto be investigated. E723 did not neutralize FGF-5 activity (data notshown), thus disabling the conduction of an inhibitory study. We arecurrently observing the effects of subcutaneous injection of FGF-5and/or FGF-5S on the entire hair growth cycle.

Recently, the regulation of the hair growth cycle, especially catageninduction, has been investigated at a protein and molecular level. Itwas reported that tumor growth factor (TGF)-β receptor I and IIstimulation (Paus et al, 1997) and parathyroid hormone (PTH)/PTHrelated peptide receptor stimulation (Schilli et al, 1997) are involvedin anagen VI–catagen transition. Apoptosis is thought to be related tocatagen induction. During catagen and telogen, the immunoreactivityof Bcl-2, a protooncogene product that provides protection againstapoptosis, decreases or disappears in the epithelial portion of the follicle,whereas the dermal papilla is constantly Bcl-2 positive throughout thehair cycle (Stenn et al, 1994). Seiberg et al (1995) reported thatexpression of several genes – TGF-β gene, tumor necrosis factor-βgene, c-myc, c-myb, and c-jun – are involved in follicular apoptosisand, thus, in the regulation of catagen. Furthermore, the initiation ofcatagen was characterized by a significant and rapid decrease ofmelanogenesis in hair bulb melanocytes (Slominski et al, 1994).Chemical compounds that affect catagen induction are also studied.Dexamethasone induces catagen (Paus et al, 1994), whereas someimmunophilin ligands, cyclosporine A and FK506, inhibit dexametha-sone-induced catagen development (Maurer et al, 1997). CyclosporineA prolongs the anagen hair growth of human hair follicles in vitro (Tayloret al, 1993) and inhibits spontaneous murine catagen development in vivo(Paus et al, 1993). These studies suggest that many factors are involvedin hair cycle regulation. To further investigate the association of FGF-5 and FGF-5S with the hair growth cycle, interactions with theseother factors must be taken into consideration.

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localization of rat fgf-5 protein in skin macrophage-like cells and fgf-5s protein in hair follicle:...

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