Genetic disorders of keratin: are scarring alopecias a sub-set?

JOURNALOF

Dermatological Science ELSEVIER

Journal

of Dermatological

Science 7 (Suppl.)

(1994) S164-S169

Genetic disorders of keratin: are scarring alopecias a sub-set? Joseph A. Rothnagel *a*b Mary A. Langley”, Rhanda A. Holder”, Donnie S. Bundmana, Toshihiko iek?, Jackie R. Bickenbach”, Dennis R. Roo~“,~ “Department of Cell Biology, hDepartment of Dermatology, Baylor College of Medicine, Houston, TX 77030, USA “Shiseido Research Center, Yokohama, Japan

Abstract Recent advances have challenged the prevailing view that keratins are merely passive bystanders of keratinocyte biology. With the exciting discovery that three autosomal dominant genetic skin disorders, epidermolysis bullosa simplex (EBS), epidermolytic hyperkeratosis (EHK) and palmoplantar keratoderma (PPK), are in fact disorders of keratins comes the realization that the integrity of the keratin filament network is crucial to the structural integrity of the skin. Since it has been recently established that mutations in keratins K5/K14, Kl/KlO and K9 are causative for these keratinocyte disorders, it is very likely that mutations in K6 or in its obligate partner, K16 will result in disease. In order to test this we have produced tiansgenic mice that express a mutant K6 gene. These mice develop a progressive scarring alopecia at about 6 months of age. Later, the denuded areas developed a keratosis which was prone to infection. Ultrastructural analysis suggests that hair loss is due to the destruction of the outer root sheath. We believe that these mice are models of another keratin disorder. Key words:

Genetics; Keratins; Mutations;

Hair follicle; Disease

1. Epidermal keratinocytes

The keratinocyte is the most abundant cell type in the epidermis. The mammalian epidermis is comprised of 4 histologically defined layers, each representing a distinct stage in the differentiation of the keratinocyte [l-3]. Keratinocytes arise from stem cells that line the basement membrane and together with their daughter cells make up the basal layer. In response to as yet unidentified signals, these cells cease their mitotic activity and embark on a terminal differen* Corresponding

author.

0923-I 811/94/$07.00 0 1994 Elsevier SSDlO923- 18 1I (94)00302-U

Science Ireland

tiation pathway, during the course of which they migrate from the basal layer and pass through the spinous and granular layers and finally terminate as the flattened squames of the stratum corneum to be eventually sloughed off into the environment. Epidermal keratinocytes also populate the outer root sheath, a band of several cell layers that surrounds the hair follicle and is contiguous with the epidermis [3]. Keratinocytes express a distinct subset of genes characteristic of their location and degree of differentiation [ 1,2,4]. Keratinocytes in the basal cell layer express keratins K5 and K14 as their major products [5,6]. Upon maturation to

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a spinous layer cell, the genes for K5 and K14 are down-regulated and the genes for Kl and KlO are induced [7-91. As these cells differentiate into granular layer cells, they cease expression of Kl and KlO and induce the expression of the late differentiation markers. The most predominant of these are filaggrin and loricrin [lo131. The other major keratins found in the epidermis are K6 and its partner K16. The expression of K6 is normally limited to the outer root sheath of the hair follicle [ 141 however, when the epidermis is stressed, as occurs in certain skin ,disorders such as psoriasis, skin cancers or wounding, K6 expression is induced in the inter-follicular epidermis [ 151. In these situations the K6/K16 keratin filament network replaces the pre-existing Kl/KlO network. The functional significance for the expression different keratin gene pairs in proliferating, differentiating and hyperproliferating keratinocytes is not yet known. 2. Keratin intermediate filaments Keratins are the major gene products of the keratinocyte ,and belong to a multi-gene family of structural proteins called intermediate filaments [ 16,171. Two types of keratin subunits have been described based on their physio-chemical properties. In general, type I subunits are smaller, with an acidic pI and include keratins K9 through K21, while type II subunits are Ionger and more basic and include keratins Kl through KS [ IS]. Functional genes for the keratins have been found clustered with the type I keratins located on chromosome 17 and the type II keratins on chromosome 12 [ 191. Keratin intermediate filaments are obligatory heterodimers and one member from each type is required to form the two chain coiled-coil which is the basic building block of the filament [20,21]. Intermediate filaments comprise one of the three components of the cytoskeleton and are so named because their diameter of 8-10 nm falls intermediate between that of actin filaments and microtubules. The basic structural characteristic of intermediate filaments is the a-helix which was first determined from X-ray crystallographic

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analysis f22]. The a-helix is conferred by a repeating unit of 7 amino acids denoted as (a,b,c,d,e,f,g), which make up the rod domain of individual keratin subunits [ 161. The ‘a’ and ‘d’ positions are generally filled by apolar residues such as leucine and interactions between these residues and those of neighboring protein chains drives the self-assembly process and stabilizes the two chain coiled-coil heterodimer. The u-helical domain is not continuous and is interrupted by three linker regions which are denoted as Ll, L12 and L2 and separate the rod domain into four segments termed, lA, lB, 2A and 2B. Within the 2B segment the periodicity of the heptad repeats is also interrupted. This discontinuity is termed the ‘stutter’ and is common to all intermediate filaments. The ends of the rod domain are demarcated by a region of highly conserved residues that are relatively invariant between all intermediate filament types. These regions are about 15 residues in length and have been referred to as helix initiation and helix termination motifs [23] although it is not known if they actually live up to their moniker. However, there is evidence from both in vitro studies and from the identification of keratin mutations in patients with epidermolysis bullosa simplex (EBS), epidermolytic hyperkeratosis (EHK) and palmoplantar keratoderma (PPK) that these regions are important for stabilizing the cohesive interactions between the end of the 2B region of one molecule and the beginning of the 1A region of a neighboring molecule [24 and references therein]. As such, these regions contribute to the stability and assembly of the keratin intermediate filament. While the sequences of the rod domain are largely conserved between all intermediate filaments, the globular, non-helical sequences of the N and C termini exhibit wide variations [ 161. Although, for a given intermediate filament protein, these sequences are highly conserved between species, suggesting that they have evolved to mediate specialized functions. Moreover, their position at the ends of the rod domain allows them to readily interact with intermediate filament associated proteins and other non-intermediate filament proteins. In the

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epidermis, for example, it has been postulated that the end domains of keratins Kl and KlO interact with the glycine-serine loops of the loricrin molecule, thus integrating the filament network with the cornified envelope [24]. It was generally thought that the non-a-helical end domains have little to do with filament assembly since crude filaments could be formed from intermediate filament subunits in which these regions had been removed proteolytically and transfection studies with ‘tailless’ and ‘headless’ keratin subunits into cultured cells, showed that these mutant keratins could incorporate into the pre-existing network without perturbation of the cytoskeleton [25,26]. However, the non-helical end domains of keratins show areas of significant sequence conservation near the beginning of the rod domain of both keratin types (the Hl region) and at the end of the rod domain for type II keratins (the H2 region). Recent data suggest that these regions do indeed participate in filament assembly and that amino acid substitutions in these sequences can result in a perturbed filament network [27-301. 3. In vitro and in vivo models of keratin dysfunction In vitro studies have provided insights into the contribution of the different domains of the keratin subunit to filament biology. Expression of truncated human keratins in cultured cells showed that deletions of the variable end domains were well tolerated, however deletions of the highly conserved rod domain resulted in disruption of the intermediate filament network [25,26]. Subsequent studies in which point mutations were introduced along the rod domain of these keratins have shown that the terminal regions of the rod domains are more sensitive to mutations, in terms of filament disruption, than more centrally located regions [31]. These in vitro studies showed that even small amounts of a mutant keratin can cause dominant phenotypic changes and thus foreshadowed the expression of mutant keratins in transgenic mice. The first transgenic experiment employed a mutant keratin K14 gene, in which most of the 2B segment,

including the ‘stutter’ and all of the variable end domain sequences were deleted from the carboxy end of the protein. Expression of the mutant protein in mice produced a phenotype resembling the human disease EBS [32]. In subsequent experiments, expression of essentially the same construct, but under the control of the KlO promoter in order to direct expression to the suprabasal cells of the epidermis, resulted in a phenotype similar to EHK [33]. In our own laboratory we have produced transgenic mice expressing a mutant keratin Kl which is lacking most of the 2B coiled-coil region but with the C-terminal end domain sequences intact. Expression of the mutant Kl protein in these mice resulted in a phenotype that closely resembles the disease of EHK [34]. The generation of transgenic mice with phenotypes resembling the human disorders of EBS and EHK intensified the search for mutations in the keratin genes in these patients. Indeed, shortly after, investigators discovered mutations in the coding regions of the genes for K5 and K14 in patients with EBS [23,28,35-421 and in Kl and KlO in patients with EHK [27,29,4347]. More recently point mutations have been identified in the highly conserved helix initiation region of K9 in patients with PPK [48,49]. To date, mutations within the hyperproliferative keratins, K6 and K16 have not been shown to be associated with a specific disease. 4. Transgenic expression of a mutant keratin K6 gene In order to identify a likely candidate skin disorder in which mutations in keratins K6/K16 are causative, we introduced a mutant K6 gene into the germline of mice. Interestingly, transgenie animals were completely normal at birth and indistinguishable from their non-transgenic litter mates in terms of appearance and gross morphology. Their epidermis and hair follicles were unremarkable for the first few months of life. However, at about 6 months some of the transgenic animals showed signs of alopecia. The alopecia always developed on the dorsal side and first occurred at the nape of the neck and

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steadily progressed towards the tail. The denuded areas then developed a keratosis which was prone to infection. These areas often filled with exudate, never properly healed and could progress to involve the entire skin surface of the mouse requiring the animal to be sacrificed. The presentation of this phenotype appears to be independent of transgene expression levels as it developed in all lines but with a delayed onset in the low expressor lines. In fact, in mice homozygous for the transgene, the phenotype was evident by 4 weeks of age and in the highest homozygous expressors, seen at birth and nearly always fatal. Histological analysis confirmed areas of infection, however treatment with antibiotics did not reverse the course of the disease. Ultrastructural analysis suggests that hair loss is due to the destruction of the outer root sheath with the keratosis, subsequent infiltrate and infection occurring as secondary events. Our preliminary data suggests that the outer root sheath cells, where K6 is normally expressed, have a weakened filament network that compromises the structural integrity of these cells, leading to cytolysis when these cells are stressed. We believe that these mice are models of another disorder of keratin, after EBS, EHK and PPK. Potential candidate disorders which resemble the phenotype of these mice include one or more of the various forms of cicatricial alopecias [50]. Cicatricial alopecia is a collective term that encompasses those disorders that lead to the irreversible destruction of the hair follicle with the formation of scar tissue. A number of these disorders have a hereditary component, including Darier’s disease (dyskeratosis follicularis) and scarring follicular keratosis (keratosis pilaris atophicans). The latter term encompasses three disorders that may or may not be distinct entities. One of them, keratosis pilaris decalvans (keratosis follicularis spinulosa decalvans) has been recently mapped to human chromosome Xp21.2-~22.2, effectively ruling out the keratins as candidate genes for this disorder [ 5 I]. Darier’s disease is an autosomal dominant inherited disease that has severe follicular involvement. Histologically, it is characterized by hyperkeratosis, parakeratosis and acanthosis, with suprabasal

lacunae caused by proliferation of the hair follicle cells. However, Darier’s disease has recently been mapped to chromosome 12q24 but outside the type II keratin gene cluster, again suggesting that keratin K6 is not a candidate gene for this disorder [52,53]. This still leaves many other scarring alopecias to be investigated and a systematic ultrastructural study of these disorders, focusing on the morphology of the tonofilaments of the outer root sheath would seem to be warranted. In the meantime, we have initiated studies using a direct gene sequencing approach to determine whether patients with similar clinical findings to our transgenic mice bear mutations in their K6 or K16 genes. 5. Acknowledgements We thank Janelle Laminack for her help in the preparation of this manuscript. This work was supported by an NIH research grant HD25479 to DRR. JAR is a recipient of a Dermatology Foundation Career Development Award sponsored by Ortho Pharmaceutical Corporation. TS was a visiting research associate sponsored by Shiseido Co., Ltd. 6. References 1 Roop

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