promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phorbol ester ... changes in mouse epidermal keratin expression following a single topical application of ...
Carcinogenesis vol .8 no.9 ppi193-1I99, 1987
Alterations in the expression of specific epidermal keratin markers in the hairless mouse by the topical application of the tumor promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phorbol ester 12-0-tetradecanoylpborbol-13-acetate
Christopher J.MoIIoy, Michael A.GaHo and Jeffrey D.Laskin' Department of Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey—Robert Wood Johnson Medical School and the Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ 08854, USA 'To whom reprint requests should be sent
The potent toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes chloracne and acts as a tumor promoter in the hairless IIRS/J mouse model. In the present study we characterized changes in mouse epidermal keratin expression following a single topical application of TCDD to the skin of hairless (hr/hr) and haired (hr/ +) HRS/J mice. Morphologic changes and alterations in keratin biosynthesis following TCDD treatment were compared with those induced by the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). Both TPA and TCDD induced dose-dependent epidermal hyperplasia in hr/hr and hr/ + mice and this was associated with altered keratin subunit expression. In hr/hr mice TCDD caused a pattern of keratin expression that was similar to TPA, characterized by a marked decrease in the synthsis of the Mr 67 000 (basic) and 59 000 (acidic) keratins that are specific markers for suprabasal differentiation in the epidermis. In addition, the synthesis of an acidic keratin of Mr 48 000 and a basic keratin of M 62 000 was also decreased. Concomitantly, TCDD caused an increase in the synthesis of a basic keratin of Mr 60 000 and acidic keratins of Mr 54 000,52 000 and 49 000 that are normally observed in proliferating basal cells and primary epidermal cell cultures. In contrast, while TPA induced similar changes in keratinization in both the hr/+ and hr/hr mice, TCDD-induced hyperplasia in hr/+ mice was only associated with changes in keratin synthesis reflecting increased basal cell proliferation. These results demonstrate that a single application of TCDD to the skin alters the normal pattern of epidermal differentiation in the hr/hr mouse. Molecular events influencing the expression of the keratin genes associated with this process may be linked to the strain- and/or species-specific toxicity of TCDD. Introduction The halogenated aromatic hydrocarbon, 2,3,7,8-tetrachiorodibenzo-p-dioxin (TCDD*) is one of the most toxic synthetic chemical compounds known (for structure, see Figure 1). Among the many biological effects of this toxin in humans is the development of epidermal hyperkeratimzation which culminates in chloracne, a characteristic persistent skin condition (1,2). Chloracne, which only occurs in a few mammalian species, has been reproduced experimentally in several animal models, in-
cluding the hairless mouse (HRS/J, hr/hr; for a review, see ref. 3). Exposure of HRS/J mice to TCDD results in epidermal hyperplasia, hyperkeratosis and sebaceous gland metaplasia (4). Interestingly, these effects are not observed in congenic HRS/J mice, the hr/ + haired phenotype, which are heterozygous at the hr locus (5). In addition, Poland et al. (6) have reported that TCDD acts as a tumor-promoting agent in the classic mouse skin two-stage initiation —promotion carcinogenesis model using the hr/hr but not the hr/-I- mouse. Thus these inbred mice, which differ only by an allele on the chromosome 14 hr locus (7), provide an excellent animal model to study the epidermal proliferation and differentiation-modifying effects of TCDD, processes that may also be related to tumor promotion. Normal epidermal keratinization is a complex process involving the sequential expression of several keratin genes that accompanies epidermal differentiation (8,9). During epidermal replenishment, stem cells from the basal layer divide and undergo a program of morphologic and biochemical changes as they ascend to form the tough, cornified barrier cells of the stratum corneum. Throughout this process, the differentiating keratinocytes synthesize distinct acidic and basic keratin proteins which serve to form the intermediate filaments of the cytoskeleton (10). The stages of epidermal cell differentiation are marked by the sequential expression of specific keratin subunits which are the products of distinct genes (8,11,12). These proteins include markers for differentiation (Mr 67 000 basic and 59 000 acidic keratins) and proliferation (Mr 60 000 basic and 54 000, 52 000 and 49 000 acidic keratins) (13, Molloy and Laskin, in preparation). Concomitant with an increase of proliferation keratins, 'heterodirner' complexes, composed of both acidic and basic keratin subunits, are produced that are additional markers for epidermal cell hyperproliferation (14, Molloy and Laskin, in preparation). We have previously shown that topical application of the tumorpromoting phorbol esters 12-0-tetradecanoylphorbol-1 3-acetate (TPA; Figure 1), to mouse skin dramatically alters the pattern OR' OR2 HO
0 OH / CH2OH R1 =C0-(CH2),2CH3 R2 COCH,
TPA Cl _-
0
I
C1
I
TCDD TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TPA, 12-0-tetradecanoylphorbol- 13-acetate; SDS, sodium dodecyl sulfate; 1FF, isoelectric focusing; NEPHGE, non-equilibrium pH gradient electrophoresis. IRL Press Limited, Oxford, England
Fig. 1. Structures of 12-0-tetradecanoylphorbol-13-acetate (TPA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
1193
