A Novel Human Type II Cytokeratin, K6hf, Specifically Expressed in the Companion Layer of the Hair Follicle  Hermelita Winter, Martina Jacobs, Michael A. Rogers, Jürgen Schweizer  Journal of Investigative Dermatology  Volume 111, Issue 6, Pages 955-962 (December 1998) DOI: 10.1046/j.1523-1747.1998.00456.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Nucleotide sequence and derived amino acid sequence of the new human type II cytokeratin.Arrowheads indicate the central α-helix, whose subdomains are marked by bentarrows. The polyadenylation signal is underlined. The sequence is available from the EMBL database (accession number Y17282). Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Amino acid sequence comparison of the new cytokeratin (upper lines) with type II cytokeratins K6b and K5. The α-helical rod domain are indicated byarrowheads. Note the highly conserved nonhelical H1 and H2 subdomains adjacent to the α-helix of the three cytokeratins. K6b and K5 amino acid sequences are fromTakahashiet al. (1995) andLersch & Fuchs (1988), respectively. Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 One-dimensional SDS-PAGE and western blot analysis. (a) Keratin extracts from intact anagen follicles (lane 1), naked follicles (lane 2), and footsole epidermis (lane 3) were run on a 10% SDS-polyacrylamide gel, transferred to a nylon membrane that was stained with Coomassie blue. (b) Western blot of (a) with the K6-specific antibody K6.KA12; and (c) western blot of (a) with an anti-serum raised against the new cytokeratin. Cytokeratins inlane 1 and 3 are designated according toMollet al. (1982), hair keratins are collectively abbreviated HKII (type II hair keratins) and HKI (type I hair keratins). The lane in (a), marked by anasterisk, contains the following molecular weight markers (from top to bottom): bovine serum albumin, 66 kDa; ovalbumin, 43 kDa; GA-3P-DH, 36 kDa; carbonic anhydrase, 29 kDa; trypsinogen, 24 kDa. Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Two-dimensional SDS-PAGE and western blot analysis. (a) Keratin extracts obtained by gentle homogenization of intact anagen follicles were resolved by two-dimensional gel electrophoresis, transferred to a nylon membrane that was stained with Coomassie blue. Cytokeratins are designated according toMollet al. (1982). Weakly visible type I hair keratins are marked by anarrowhead. Asterisks indicate the isoelectric mobilities of tetrameric complexes of type I and II cytokeratins not dissociated in the first dimension. Isoelectric marker proteins are bovine serum albumin (B), actin (A), and phosphoglycerokinase (P). (b) Western blot of (a) with K6 antibody KS6.KA12. The specific reaction of this antibody with the basic portion of the extended protein spot at 60 kDa is clearly visible due to the arti-factual inclusion of an air bubble at this position of the gel [arrows in (a) and (b)]. Note also the positive reaction of the antibody with complexed K6 [asterisk in (b)]. (c) Western blot of (a) with the anti-serum raised against the new cytokeratin. The dotted line indicates the position of the K6 reactive part of the extended 60 kDa protein spot. Note that the new cytokeratin does not show up within complexed cytokeratins. Proteins were separated by nonequilibrium pH gradient gel electrophoresis in the first dimension (N) and by SDS-PAGE (10% gel) in the second dimension (S). Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Demonstration of K6hf mRNA expression in human hair follicles. (A)In situ hybridization with a specific cRNA probe of K6hf on three differently sectioned anagen hair follicles. (B) Higher magnification of part of the sections shown in (A).Red triangles in (A) and (B) indicate onset and cessation of K6hf mRNA expression;white arrows in (A) and (B) denote the site of horizontal ORS expansion;black arrows in (B) indicate nonlabeled ORS cells in the bulb region. ORS, outer root sheath; IRS, inner root sheath; dp, dermal papilla.Scale bars: (A) 100 μm; (B) 200 μm. Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Demonstration of K6hf protein synthesis in the hair follicle. Shown is a composed section of a hair follicle that has been reacted with the K6hf anti-serum. Because of the slightly oblique angle of section, companion cells above the middle cortex show up as a gradually enlarging cell band. Thewhite arrows indicate the contours of the follicle. Due to a rupture and distortion of the scalp sample from which the section has been obtained, the uppermost part of the follicle cannot be demonstrated. There was, however, evidence for only a small region of about three to four cell layers of diffuse and weak staining above the level shown here. dp, dermal papilla.Scale bar: 250 μm. Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Comparative expression study of K6hf and the cuticular hair keratin hHa2 in hair folicles. (A) Doublein situ hybridization with K6hf- and hHa2-specific cRNA probes; (B) double label immunofluorescence with the K6hf anti-serum (green color) and a monoclonal antibody to hHa2 (red color).Red triangles, hair cuticle;white arrows, inner root sheath;green triangles, companion layer.Scale bar: 100 μm. Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Comparative expression study of K6hf and the type I cytokeratins K16 and K17 in hair follicles. (A, C) Longitudinal sections of the lower part of the follicle, (B, D) cross-sections at the height of the lower to middle isthmus of the follicle. (A, B) Double label immunofluorescence with the K6hf anti-serum (red) and the K16 antibody K16.1275 (green). Yellow-stained cells indicate coexpression of K6hf and K16.Arrows in (A) denote earliest signs of K6 expression in the companion layer. The dashed line in (A) marks the contours of the follicle.Small white arrowheads in (B) indicate basal ORS cells;white arrows in (B) denote companion cells that show up due to the oblique angle of section; thelarge white arrowhead in (B) points to a portion of K16-positive sebaceous glands. (C, D) IIF with the K17 antibody KS17.E3.White arrows in (C) indicate basal ORS cells revealed by DAPI staining; thewhite arrowhead in (D) points to a portion of K17-positive sebaceous gland cells.Open arrowheads in (B) and (D) denote signs of detachment of companion cells from ORS cells. CL, companion layer; ORS, outer root sheath; IRS, inner root sheath.Scale bars: 100 μm. Journal of Investigative Dermatology 1998 111, 955-962DOI: (10.1046/j.1523-1747.1998.00456.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions