Localization of Rat FGF-5 Protein in Skin Macrophage-like Cells and FGF-5S Protein in Hair Follicle: Possible Involvement of twoFgf-5 Gene Products in.

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Localization of Rat FGF-5 Protein in Skin Macrophage-like Cells and FGF-5S Protein in Hair Follicle: Possible Involvement of twoFgf-5 Gene Products in Hair Growth Cycle Regulation Satoshi Suzuki, Tomomi Kato, Hiroyuki Takimoto, Shigeki Masui, Hiroshi Oshima Journal of Investigative Dermatology Volume 111, Issue 6, Pages 963-972 (December 1998) DOI: 10.1046/j.1523-1747.1998.00427.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 RT-PCR of rat FGF-5 and FGF-5S mRNA. Total RNA from 10 d old rat dorsal skin was reverse-transcribed using (dNTP)6 primer and subjected to PCR using the primer pair 5′-CCTTCGGGGCGCCGGA- CCGGCA-3′ (sense) and 5′-AAGTTCCG- GTTGCTCGGACTGCTT-3′ (anti-sense). (a) Schematic drawing of rat FGF-5 and FGF-5S mRNA, and their open reading frames. Exons (1, 2, 3) are indicated by boxes. Asterisks indicate the positions of the stop codon. Positions of the sense and anti-sense PCR primers are indicated byarrows. (b) Rat FGF-5 cDNA nucleotide sequence (lower case letters) and FGF-5 and FGF-5S amino acids (capital letters). The primer annealing sites are underlined. A broken line shows the position of exon 2, which is truncated in FGF-5S mRNA and protein. Nucleotides are numbered from the first nucleotide of the ATG initiation codon. Sequences are quoted fromHattoriet al. (1996). Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

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 out using the primers that amplify both FGF-5 mRNA and FGF-5S mRNA, as shown in Figure 1. M, molecular weight marker “123 bp DNA ladder” (Gibco BRL).Lane 1, Rat-skin RNA;lane 2, negative control (no template). Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 E723 reacts with FGF-5 and B2B6 reacts with both FGF-5 and FGF-5S. Recombinant human FGF-5, FGF-5S, FGF-6, and FGF-7 were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. The membrane was treated with E723 antibody (a) or B2B6 antibody (b) and then with horseradish peroxide-conjugated goat anti-mouse Ig antibodies, followed by visualization using ECL. Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

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 cells were 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. Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 B2B6 antibody binds to the cortex of the hair follicle. Formalin-fixed, paraffin-embedded rat dorsal skin sections were incubated with B2B6 and stained 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 (beginning of 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. Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

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 and immunohistochemically labeled with B2B6 (a) and E723 (b).Arrows indicate round cells labeled with both antibodies.Scale bar: 20 μm. Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

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 old rat (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. Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

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 and immunolabeled with ED2 and E723. Round cells labeled with ED2 (a) or E723 (b). Dendritic cells labeled with ED2 (c) or E723 (d). Negative control section treated 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. Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 The density of FGF-5-positive macrophage-like cells changes during the hair cycle. The density of the FGF-5-positive macrophages in the dermis (a) and in the panniculus adiposus (b) was determined. Data are pressed as mean values + SD. **Statistically significant differences (p < 0.01) between 12 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). Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 The density of FGF-5-negative macrophage-like cells also changes, but differently from FGF-5-positive macrophage-like cells. The densities of FGF-5-positive and FGF-5-negative macrophage-like cells in the 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 bar graph represents mean values + SD. *p < 0.05, **p < 0.01, ***p < 0.001; statistically significant differences between samples as determined by Student’s t test. Journal of Investigative Dermatology 1998 111, 963-972DOI: (10.1046/j.1523-1747.1998.00427.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions