Elastin Peptides Induce Migration and Terminal Differentiation of Cultured Keratinocytes Via 67 kDa Elastin Receptor in Vitro: 67 kDa Elastin Receptor is Expressed in the Keratinocytes Eliminating Elastic Materials in Elastosis Perforans Serpiginosa  Norihiro Fujimoto, Shingo Tajima, Akira Ishibashi  Journal of Investigative Dermatology  Volume 115, Issue 4, Pages 633-639 (October 2000) DOI: 10.1046/j.1523-1747.2000.00117.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Migration of cultured keratinocytes in response to elastin peptides VGVAPG. The peptides were added to the lower compartments of the chambers at the concentrations indicated and migration from the upper compartments was measured. Values are mean ± SD from quadruplicate assays. Journal of Investigative Dermatology 2000 115, 633-639DOI: (10.1046/j.1523-1747.2000.00117.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Suppression of cell proliferation of cultured keratinocytes by tropoelastin or synthetic elastin peptide VGVAPG. (a) Keratinocytes were plated at a density of 450 per mm2 in a 24-well microplate. On day 2 in culture, cells were treated with tropoelastin or synthetic elastin peptides (VGVAPG or VGVPG) for 48 h at the doses indicated. At the termination of the treatment, cell number was counted. (b) Cells were treated with elastin peptide (10−6 M) alone for 48 h or in a combination of elastin peptide (10−6 M) and anti-elastin receptor antibody (anti-S-Gal antibody) (10 ng per ml) for 48 h. Values are mean ± SD from triplicate assays. Journal of Investigative Dermatology 2000 115, 633-639DOI: (10.1046/j.1523-1747.2000.00117.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effects of elastin peptide on involucrin or TGase-1 expression in cultured keratinocytes. Cells were treated for 48 h with 0 (lane 1), 10−6 M (lane 2) and 10−5 M (lane 3) elastin hexapeptides in the absence (left) or presence of the anti-elastin receptor antibody (10 ng per ml) (right panel). RNA was isolated from the cells and fractionated on 1% agarose gel then blotted on to nylon membranes. Involucrin (Invol), TGase-1, and glyceraldehyde-3-phosphate-dehydrogenase probes were labeled by random priming, then hybridized with the blots. The blots were then autoradiographed. Journal of Investigative Dermatology 2000 115, 633-639DOI: (10.1046/j.1523-1747.2000.00117.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression of the 67 kDa elastin receptor in cultured keratinocytes. (a) Reverse transcription–polymerase chain reaction was performed with keratinocyte RNA and oligoprimers corresponding to exons 2 and 5 of β-galactosidase cDNA under the condition described in Materials and Methods. Two DNA fragments with 342 and 127 bp were detected; the former indicates unspliced β-galactosidase mRNA and the latter represents spliced variant arising from the deletion of exons 3 and 4 of β-galactosidase mRNA. The left lane indicates molecular markers with 1057, 770, 612, 495, 392, 341, 297, 210, and 162 bp. (b) Cells were extracted with 50 mM Tris–HCl, pH 7.4, 0.1% Noniodet P-40. Solubilized fraction was applied on the elastin affinity column and eluted with 0.1 M lactose. The eluates were resolved on 2/15% SDS–PAGE, then visualized with silver staining (lane 1), or blotted on to membranes and stained with anti-S-Gal antibody (lane 2). (c) Cells were incubated without or with elastin peptides (10−6 M) for 24 h. Proteins of cell layers were extracted with 50 mM Tris–HCl, pH 7.4, containing 1% SDS. Resulting supernatant was directly resolved on 2/15% SDS–PAGE. The proteins were transfer blotted on to the membrane, then stained with anti-S-Gal antibody. Lanes 1 and 2 indicate without and with elastin peptides, respectively. (d) Cells were plated on the glass chamber slides and incubated without (upper panel) or with 10−6 M elastin peptides for 24 h (lower panel). Cells were fixed with acetone and stained with anti-67 kDa elastin receptor antibody (BCZ-67). Bound antibody was visualized with fluorescein-conjugated rabbit anti-mouse immunoglobulins. (original magnification × 200). Journal of Investigative Dermatology 2000 115, 633-639DOI: (10.1046/j.1523-1747.2000.00117.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effects of elastin on the cell number of involucrin-producing cells. Cells were treated with the hexapeptide VGVAPG (10−6 or 10−5 M) for 48 h in the absence or presence of anti-67 kDa elastin receptor antibody (anti-S-Gal antibody) (10 ng per ml), then stained with anti-involucrin antibody. (A) Indicated are the involucrin-positive cells treated with 0 (a), 10−6 M (b), and 10−5 M (c) VGVAPG in the absence of anti-elastin receptor antibody, or 10−5 M VGVAPG in the presence of anti-elastin receptor antibody (d). (B) Percentage of the cells stained with involucrin antibody was calculated from the observation of random, nonoverlapping 100 cells. Values are mean ± ranges from triplicate assays. (*) indicates statistical significance between untreated (0 M) and treated (10−6 or 10−5 M) cells at p < 0.01. Journal of Investigative Dermatology 2000 115, 633-639DOI: (10.1046/j.1523-1747.2000.00117.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of the 67 kDa elastin receptor in the patient with elastosis perforans serpiginosa. The paraffin-embedded sections of the lesional skin specimens taken from case 1 (a), case 2 (b), and the patient with psoriasis (c) were stained with anti-67 kDa elastin receptor antibody (anti-S-Gal antibody) at 1:1000 for 24 h. The sections were incubated with anti-rabbit immunoglobulin antibody at 1:600 for 2 h. The antigen–antibody complex was visualized by the avidin–biotin complex. The sections were counterstained with hematoxylin (original magnification × 200). Note the immunoreactivity of the epidermis at the site of elimination with the anti-67 kDa elastin receptor antibody (a,b) and no specific immunoreactivity of the epidermis at other sites (arrows in b). (*) indicates degenerated elastic fibers undergoing transepidermal elimination. Skin specimens from the patient with psoriasis showed no specific immunoreactivity with the antibody (c). Antibody absorbed twice with 10 μg synthetic peptide antigen showed no positive stain in EPS skin (case 1) (d). Journal of Investigative Dermatology 2000 115, 633-639DOI: (10.1046/j.1523-1747.2000.00117.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions