Acta Derm Venereol (Stockh) 1999; 79: 122^126 Expression of Trichohyalin in Dermatological Disorders: a Comparative Study with Involucrin and Filaggrin by Immunohistochemical Staining SEUNG-CHUL LEE1,2, JEE-BUM LEE1, JAE-JEONG SEO1 and YOUNG PIO KIM1 1Department of Dermatology, Chonnam University Medical School and 2Research Institute of Medical Sciences, Chonnam University, Kwangju, South Korea To investigate the function of trichohyalin during terminal In the present study, an attempt was made to delineate the di¡erentiation of the skin, immunohistochemical studies were role of trichohyalin as a component of the CE during terminal performed on trichohyalin and its related proteins, ¢laggrin di¡erentiation by screening its expression in various skin disor- and involucrin, the components of the corni¢ed cell envelope. ders. We also evaluated the expression of trichohyalin in com- In skin disorders unrelated to tumours, weak trichohyalin parison with that of ¢laggrin and involucrin to clarify the expression was found in a few granular cells or in the horny functional relationship among them as precursor proteins of layer of psoriasis, ichthyosis, keratosis pilaris, porokeratosis, the CE. chronic dermatitis and callus. Similar trichohyalin expression was found in epidermal tumours, such as seborrheic keratosis, actinic keratosis, Bowen's disease and well-di¡erentiated squamous cell carcinoma. In follicular tumours, trichohyalin MATERIALS AND METHODS expression was positive in trichoepithelioma, keratotic basal Materials cell epithelioma, proliferating trichilemmal tumour, trichi- Formalin-¢xed, para¤n-embedded samples were selected after evalua- lemmoma, pilomatricoma and keratoacanthoma. From com- tion of their histopathological features with haematoxylin and eosin parative studies with ¢laggrin and involucrin, trichohyalin staining. In the present study, 49 samples of skin disorders unrelated expression was co-localized with them in molluscum contagio- to tumours, 36 samples of epidermal tumours and 25 samples of sum, keratoacanthoma and pilomatricoma. From this study, follicular tumours were examined. trichohyalin is revealed to have close functional relationship with other markers of terminal di¡erentiation as a precursor Antibodies of the corni¢ed cell envelope of the skin. Key words: terminal A polyclonal anti-trichohyalin antibody was elicited from rabbits with di¡erentiation; corni¢ed cell envelope; precursor. recombinant human trichohyalin of 1,250 ^ 1,849 residues (domain 8) (Accepted September 7, 1998.) located in the carboxy-terminus (16). Antibodies to involucrin and Acta Derm Venereol (Stockh) 1999; 79: 122^126. ¢laggrin were purchased from Biomedical Technologies Inc. (MA, USA). The polyclonal anti-involucrin antibody was raised from rabbits Seung-Chul Lee, Department of Dermatology, Chonnam with puri¢ed cultured human epidermal cells (17). The monoclonal University Medical School, 8 Hak-dong, Tong-gu, Kwangju anti-¢laggrin antibody was elicited from mice with partially puri¢ed 501 ^ 190, South Korea. ¢laggrin of the newborn human epidermis (18). Immunohistochemical staining of trichohyalin, involucrin and During terminal di¡erentiation of the skin, a characteristic ¢laggrin structure called the corni¢ed cell envelope (CE) is formed in Immunohistochemical procedures were carried out according to the the inner surface of the cell periphery of the granular layer. It protocol described in the LSAB kit (DAKO, CA, USA) with minor is a rigid, complex structure composed of many precursor pro- modi¢cations. Samples 5 mm thick were depara¤nized, rehydrated teins of transglutaminase (TGase) which catalyses e-(c-gluta- and pre-treated with 3% hydrogen peroxide for 10 min to block endo- myl)lysine cross-linking (1 ^ 7). Trichohyalin is one of genous peroxidase activity, followed by incubation in the blocking substrate proteins of TGase, and it can bind with keratin ¢la- reagent for 10 min. Then the sections were incubated with the primary ments as intermediate ¢lament associated protein (IFAP) (8, antibodies under the following conditions: anti-trichohyalin at 1 : 50 9). Trichohyalin is found mainly in the inner root sheath dilution for 1 h; anti-involucrin at 1 : 10 dilution for 30 min; and anti- (IRS) and medulla of the hair follicle where it gives a structural ¢laggrin at 1 : 150 dilution for 30 min at room temperature. For tricho- rigidity, but its possible function as a precursor of the CE has hyalin staining, sectioned samples were pre-treated in the autoclave at been proposed previously (8). In a recent report (10), tricho- 121³C, 15 lbs/inch2 for 5 min before reaction with the primary anti- hyalin is found as a cross-linked component of mouse foresto- body to retrieve antigenicity. Then the sections were incubated with a mach CE, and it is thought to modulate the biomechanical biotin-labelled anti-immunoglobulin antiserum for 15 min, followed by peroxidase-conjugated streptoavidin for 15 min. As the chromogen, properties of the CE. In the skin, however, trichohyalin is £eet- 3-amino-9-ethylcarbazole, was applied, red-coloured precipitates ingly expressed in the neonatal foreskin, and it is not detected appeared at the antigen site. During immunohistochemical staining, in the adult epidermis (11, 12). A few scattered observations each sample was washed with phosphate-bu¡ered saline (pH 7.4). Sec- reported that it is aberrantly expressed only in a number of der- tions were counterstained with Meyer's haematoxylin and mounted matoses, such as psoriasis, molluscum contagiosum, actinic with Universal Mount (Research Genetics, AL, USA). As a negative keratosis, trichofolliculoma, epidermolytic hyperkeratosis control, sections were incubated with non-immunized serum instead and pilomatricoma (13 ^ 15). of the primary antibodies. Acta Derm Venereol (Stockh) 79 # 1999 Scandinavian University Press. ISSN 0001-5555 Trichohyalin expression in dermatological disorders 123 Fig. 1. Localization of trichohyalin in (a) ichthyosis vulgaris (6160), (b) Bowen's disease (6200), (c) trichoepithelioma (6200) and (d) keratotic basal cell epithelioma (6200). Arrows and arrowheads indicate positive expression of trichohyalin. RESULTS keratosis and SCC, several dyskeratotic cells were also positive in trichohyalin expression. No immunoreactivity was found in Trichohyalin expression in skin disorders unrelated to malignant tumours of non-keratotic basal cell carcinoma tumours (BCC). In depara¤nized autoclaved sections, trichohyalin expression was not detected in the interfollicular epidermis of normal adult skin, but it was strongly expressed in the IRS and medulla Trichohyalin expression in follicular tumours of hair follicles (data not shown). Such immunoreactivity was Immunoreactivity of trichohyalin was found in trichoepithe- not detected in the sections without pre-treatment by autoclav- lioma (Fig. 1c), keratotic BCC (Fig. 1d), proliferating tri- ing. Among samples, weak immunoreactivity of trichohyalin chilemmal tumour, trichilemmoma, pilomatricoma and was localized to a few granular cells or to the horny layer of keratoacanthoma. In trichoepithelioma and keratotic BCC, ichthyosis (Fig. 1a), psoriasis, keratosis pilaris, porokeratosis, immunoreactivity was found around horn cysts, but there was chronic dermatitis and callus. Strong immunreactivity of tri- contrast between them; major immunoreactivity was localized chohyalin was found from the horny to spinous layers of mol- around cysts showing a palisading pattern in trichoepithe- luscum contagiosum (Fig. 2b). lioma, while positive immunoreactivity was localized to cystic contents in keratotic BCC. In pilomatricoma, immunoreactiv- ity of trichohyalin was localized mainly to the junction between Trichohyalin expression in epidermal tumours basophilic and shadow cells (Fig. 2h). The expression pattern in several epidermal tumours, such as seborrheic keratosis, actinic keratosis, Bowen's disease Sequential expression of trichohyalin, ¢laggrin and involucrin (Fig. 1b) and squamous cell carcinoma (SCC), was similar to in molluscum contagiosum, keratoacanthoma and that of skin disorders unrelated to tumours, as described pilomatricoma before. In particular, all samples of seborrheic keratosis showed positive expression of trichohyalin in the granular In a comparative study of trichohyalin with ¢laggrin and and horny layers, and in the lining cells of horn cysts. In actinic involucrin, we found a sequential order among them in 3 Acta Derm Venereol (Stockh) 79 124 S.-C. Lee et al. Fig. 2. Co-localization of (b, e, h) trichohyalin with (c, f, i) ¢laggrin and (a, d, g) involucrin in molluscum contagiosum (a ^ c, 6100), keratoa- canthoma (d ^ f, 6132) and pilomatricoma (g ^ i, 680). dermatoses. In molluscum contagiosum, major immunoreac- and follicular origins. Such morphological evidence strongly tivity of involucrin was localized to the upper or mid-spinous suggests a functional role for trichohyalin as a precursor of layer, while that of ¢laggrin was con¢ned to the horny layer of the CE in the keratinizing process of the skin. Notably, tricho- molluscum body (Fig. 2a, c). Immunoreactivity of trichohyalin hyalin was co-localized with the other precursors of the CE, was scattered to whole layers, from the horny to upper spinous involucrin and ¢laggrin, in molluscum contagiosum and in fol- layers, overlapping with the other precursors (Fig. 2b). A simi- licular-originating tumours, such as keratoacanthoma and lar pattern of immunoreactivity was found in the follicular- pilomatricoma. The overlapped