Type II Collagen Accumulation in Overlying Dermo-Epidermal Junction of Pilomatricoma Is Mediated by Bone Morphogenetic Protein 2 and 4 Hideki Mieno,

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Type II Collagen Accumulation in Overlying Dermo-Epidermal Junction of Pilomatricoma Is Mediated by Bone Morphogenetic Protein 2 and 4 Hideki Mieno, Kei Kuroda, Hiroshi Shinkai, Hidekatsu Yoshioka, Shingo Tajima Journal of Investigative Dermatology Volume 122, Issue 4, Pages 878-884 (April 2004) DOI: 10.1111/j.0022-202X.2004.22417.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunohistochemical staining of the overlying skin of pilomatricoma with monospecific anti-type II collagen antibody. Overlying skins of case 1 (a) and case 2 (b) of pilomatricoma and normal skin (c) were incubated with anti-type II collagen antibody at 1:40 dilution, then with biotin-conjugated anti-mouse immunoglobulin antibody. Localization of antigen was visualized with avidin–biotin complex (original magnification × 100). Journal of Investigative Dermatology 2004 122, 878-884DOI: (10.1111/j.0022-202X.2004.22417.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunohistochemical detection of bone morphogenetic protein 2 or 4 (BMP2 or BMP4) in pilomatricoma. Skin specimens obtained from cases 1 and 2 were incubated with anti-BMP2/4 antibody for 24 h at 4°C, then incubated with biotin-conjugated anti-goat Ig antibody at 1:500 dilution for 2 h at room temperature. Antigen–antibody complex was reacted with avidin–biotin complex. Original magnification is × 100. S, shadow cell. Journal of Investigative Dermatology 2004 122, 878-884DOI: (10.1111/j.0022-202X.2004.22417.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Proα1(II) collagen transcript is induced in cultured human keratinocytes by the homogenate prepared from pilomatricoma, or by recombinant human bone morphogenetic protein 2 and 4 (BMP2 and BMP4). Cultured keratinocytes and fibroblasts were treated with the homogenate prepared from pilomatricoma (a), or cultured keratinocytes were treated with recombinant BMP2 and BMP4 (b), or treated with the combination of recombinant BMP2 and recombinant noggin (c). RNA was isolated from the cultured keratinocytes. RT-PCR was performed using proα1(II) (upper panel) and β-actin (lower panel) primer pairs. PCR products were analyzed by an electrophoresis on agarose gel. (a) Keratinocytes were treated for 24 h without (lane 1) or with (lane 2) the homogenate isolated from pilomatricoma. Fibroblasts were treated for 24 h with the homogenate (lane 3). (b) Keratinocytes were treated for 24 h with 0 (lane 1), 25 (lane 2), 100 (lane 3), and 250 ng per mL (lane 4) BMP2, or treated with 0 (lane 5), 25(lane 6), 100 (lane 7), and 250 (lane 8) ng per mL BMP4. (c) Keratinocytes were treated for 24 h with 100 ng per mL BMP2 alone (lane 1) or with the combination of 100 ng per mL BMP2 and 1.6 ng per mL noggin (lane2). M indicates molecular markers of 770, 612, 495, 392, 341, 297, 210 bp. M′ indicates molecular markers of 1360, 1107, 926, 658, 489, 267 bp. Journal of Investigative Dermatology 2004 122, 878-884DOI: (10.1111/j.0022-202X.2004.22417.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Alternative splicing of exon 2 of type II collagen primary transcript expressed by cultured keratinocytes in the presence of bone morphogenetic protein 2 or 4 (BMP2 or BMP4). (a) Keratinocytes were treated without (lanes 1 and 3) or with either BMP2 (250 ng per mL) (lane 2) or BMP4 (250 ng per mL) (lane 4) for 48 h. RNA was isolated and RT-PCR was performed using upstream (exon 1) and downstream (exon 5) primers of proα1(II) (upper panel) and β-actin (lower panel). PCR product was analyzed by agarose gel electrophosesis. Positive control assays were done using the RNA isolated from ear cartilage (lane 5). M indicates molecular markers of 770, 612, 495, 392, 341, 297, 210 bp. (b) RT-PCR products were blotted onto filters and hybridized with the radiolabeled oligonucleotide probe spanning 3′ end of exon 1 and 5′ end of exon 2. The filters were washed and subjected to autoradiography. Journal of Investigative Dermatology 2004 122, 878-884DOI: (10.1111/j.0022-202X.2004.22417.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Cell proliferation of cultured keratinocytes is suppressed in type II collagen-coated dishes. Human keratinocytes were plated at an intial density of 1 × 103 per dish on type I(open circle), type II (open triangle), type IV collagen-coated (closed triangle) or plain (closed circle) dishes and maintained in KGM over 14 d. Cell number was counted every 2 d after trypsin treatment. Values are mean±SEM from two sets of assays done in triplicate. Statistical significance at p<0.01 is indicated by * and **. Journal of Investigative Dermatology 2004 122, 878-884DOI: (10.1111/j.0022-202X.2004.22417.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions