Epidermal structural proteins in skin disorders

JOURNALOF

Dermatological Science Journal of Dermatological Science 15 (1997) 143-165

Review article

Epidermal structural proteins in skin disorders Motomu Manabe *, Masayuki Mizoguchi, Hajime Suto, Hideoki Ogawa Department oj’ Dermatology,

Juntendo University School of Medicine, Hongo 3-l -3, Bunkyo-ku,

Tokyo 113, Japan

Received 11 November 1996; received in revised form 8 February 1997; accepted 8 February 1997

Keywords:

Epidermal structural proteins; Intermediatefilament proteins; Cornified envelopeproteins; Skin disorders

1. Introduction

Epidermal differentiation involves the progressive and orderly maturation of the major epiderma1 cell type (keratinocyte) from a basal cell with proliferative potential to lifeless, flattened, enucleated squames of the cornified cell (for a review, see [l]). During the course of differentiation, keratinocytes undergo a series of morphological and biochemical changes including the expression of large quantities of proteins which constitute cytoplasmic filamentous networks, keratohyalin granules and cornified envelopes. In the past few years, there has been a number of major scientific discoveries in the field of normal and abnormal epidermal differentiation due to the technological advances in molecular biology. We will review herein some important aspects of the biology of various epidermal structural proteins including intermediate filament proteins, intermediate filament-associated proteins and cornified envel* Corresponding author.

ope proteins, as well as highlighting recent advances in the elucidation of their roles in skin disorders (for reviews, see [2-61). 2. Intermediate filament proteins

Intermediate filament proteins (IF) are members of a large and evolutionary related group of proteins (for a review, see [7,8]). Recent studies have described a total of six distinctive types of IF classified on the basis of their amino acid sequence, gene structure and cell expression, i.e. type I, acidic keratins; type II, neutral-basic keratins; type III, vimentin, desmin, glial fibrillary acidic protein and peripherin; type IV, neurofilaments and a-internexin; type V, lamins; and type VI, nestin. All IF share a common, tripartite domain organization that consists of a central cc-helical rod domain of 310-350 amino acids, flanked by a non-helical amino-terminal head and carboxy-terminal tail domains of variable sizes. Keratins are the largest and most complex group

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of IF. Keratins form an elaborate cage-like network around the nucleus, spread like a spider-web throughout the cytoplasm, and make contact with specialized junctional apparatus such as desmosomes and hemidesmosomesat the cell periphery. The structural continuity of keratins in the cytoplasm and between cells implies a role for these filaments in the maintenance of the structural integrity of the entire epithelium. 2.1. Keratins 2.1.1. Keratin subfamilies Keratins have been divided into subfamilies type I and type II based on size, charge and gene structure (for a review, see[9,10]). Type 1 keratins are acidic (pKi = 4-6) and include 11 soft (epithelial) keratins, K9-K22. Type II keratins are neutral-basic (p& = 6-8) and include 8 soft (epithelial) keratins, Kl -K8 [7-lo]. Strict combinations of specific pairs of type I and type II keratins, the so-called ‘expression pairs’, are expressed in various epithelia according to the type of differentiation specific to the various stages of development [7-lo]. This division of keratins into two types is also seen at the gene level, where the type I keratin genes have 8 exons/7 introns and are located as a cluster on chromosome 17, while the type II keratin genes have 9 exons/8 introns and are located on chromosome 12 (for a review, see [7]). The keratin expression is tightly regulated with a balance between the proliferative and desquamation events (for a review, see [9,10]). For example, in normal epidermis, basal cells display a cytoplasmic network composed of the K5/K14 keratin pair. Once basal cells undergo a commitment to terminally differentiate, they downregulate the expression of the K5/K14 pair and express a new set of keratins, the Kl/KlO pair. In the late spinous-early granular cells, another keratin, K2e, is expressed, while the Kl/KlO expression is downregulated. The other keratins found in the epidermis and its appendages are the K6/ K16 pair, K17 and K9. The expression of keratin K9 is normally limited to the late spinous-early granular cells of the palmar and plantar epidermis. The expressions of the K6/K16 pair and K17

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are normally limited to the outer root sheath of the hair follicle, mucosal stratified squamous epithelia, and to the palmar and plantar epidermis, with K6/K16 in suprabasal cells and K17 in basal cells. However, during keratinocyte wounding, keratinocytes most likely respond to various growth factors and inflammatory cytokines, while the K6/K16 pair and K17 are expressed in hyperproliferative epidermis. Although the molecular mechanisms of keratin gene regulation are not fully understood, differential keratin expressions are considered to be related to the cellular functions of this multigene family. It is most probable that intermediate filaments composed of specific keratin pairs have distinct physical properties which may tailor intermediate filament networks to suit the tissue-specific structural requirements of tensile strength, flexibility and dynamics [7]. 2.1.2. Filament formation and structure of keratins Keratins have a characteristic protein domain structure consisting of a central a-helical rod domain that has a precisely conserved secondary structure, and a flanking non-helical sequence of amino- (head) and carboxy-terminal (tail) domains denoted as El, Vl, Hl and H2 (type II

Fig. 1. The structure of keratins (for reviews, see [4,7]). Keratins have a characteristic protein domain structure consisting of a central z-helical rod domain, and a flanking non-helical sequence of amino- (head) and carboxy-terminal (tail) domains denoted as El, Vl, Hl and H2 (type II keratins only), V2, and E2. The rod domain is subdivided into four discrete a-helical segments, lA, IB, 2A and 2B. These segments are interspersed by non-coiled-coil linkers, denoted as Ll, L12 and L2. The cc-helical rod domain is important for the assembly of keratins into a filamentous structure; while particularly important are regions at the beginning of rod IA and at the end of rod 2 which are referred to as helix initiation- and helix terminationmotifs, respectively. The mutations of keratin genes in keratin diseases are mainly clustered within the helix initiation and termination motifs, and the L12 linker region of the rod domain.

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Diseases

Mutations

in

Cause

Stratified

epithelial-type,

basal keratins

Skin-type,

supra-basal

Skin-type,

upper spinous

keratins

(KlIKlO)

keratin

supra-basal

Hyperproliferation-related,

basal keratin

Esophagus-type,

granular supra-basal

keratin

lchthyosis keratins

( K6/K16)

(K17)

(KQ)

keratins

Bullosa

Simplex

Epidermolytic Hyperkeratosis Diffuse NEPPK’

( K3e)

Hyperproliferation-related,

Palmoplantar-type,

Epidermolysis

(K6IK14)

(K4/K13)

Bullosa

of Siemens

Jadassohn-Lewandowsky Focal NEPPK” Jackson-Lawler

type of PC*

type of PC”

Epidermolytic

Palmoplantar

White Sponge

Nevus

l Nonspidermolytic Palmoplantar +* Pachyonychia Congenita

Keratoderma

Keratoderma

Fig. 2. Keratin mutations in genodermatoses (for a review, see [4]). It has been clarified that mutations in keratin genes cause a number of genodermatoses which are classified into so-called keratin diseases. The keratins which cause keratin diseases include K5/K14 in epidermolysis bullosa simplex, Kl/KlO mutation in epidermolytic hyperkeratosis or diffuse non-epidermolytic palmoplantar keratoderma, K2e mutation in ichthyosis bullosa of Siemens, K9 mutation in epidermolytic palmoplantar keratoderma, K6/K16 mutation in Jadassohn-Lewandowsky type of pachyonychia congenita or focal non-epidennolytic palmoplantar keratoderma, Kl7 mutation in Jackson-Lawler type of pachyonychia congenita, and K4/K13 mutation in white sponge nevus. These diseasesare mostly associated with keratin aggregation with/without cytolysis in the epidermis or oral mucous. Furthermore, the distribution of mutant keratins is identical with the phenotypes of these keratin diseases. For example, the expression of K9, of which the mutation causesepidermolytic palmoplantar keratoderma, is restricted to the palm and sole, and that of K4/K13 pair, of which the mutation causes white sponge nevus, is restricted to the mucous epithelia. Interestingly, the expression of each keratin which causes keratin diseasesoccurs in the location exactly corresponding to where the pathological change occurs. For example, in epidermolysis bullosa simplex, cytolysis occurs in basal cells because mutant K5/K14 are expressed in basal cells. Similarly, in ichthyosis bullosa of Siemens, epidermolytic degeneration occurs in upper spinous cells because mutant K2 is expressed in upper spinous cells.

keratins only), V2, and E2 (Fig. 1 and for reviews, see [l l-131). The a-helical structure of the rod domain is conferred by a repeating heptad amino acid motif of (a, b, c, d, e, f, g),, where the ‘a’ and ‘d’ residues are generally apolar and positioned on one face of the helix, with the potential to form a two-chain coiled-coil dimer molecule. The other residues alternate in charge and present a hydrophilic face that is important for higher order interactions. The hydrophobic interactions between the apolar faces of neighboring subunits drive the self-assembly process and stabilize the two-chain coiled-coil heterodimer. The rod domain is not continuous and is interspersed by non-coiled-coil linkers, denoted as Ll, L12, and L2, that subdivide into four discrete a-helical segments, 1A. lB, 2A and 2B (Fig. 1) and give flexibility to the cc-helical rod domains. The beginning and end of the rod domain is demarcated by

a region of about 15 amino acids, which are referred to as helix initiation- and helix termination-motifs, that are very highly conserved in all intermediate filament proteins (Fig. 1). Deletion mutagenesis studies indicated that the ends of the rod play a special role in filament assembly, with even subtle point mutations in these motifs having deleterious effects [ 14,151. 2.1.3. So-called ‘keratin diseases’ (Fig. 2)

Epidetmolysis bullosa simplex (EBS) is the first skin diseasein which keratin mutations were identified as being causative. In 1991, it was shown that transgenic mice expressing the truncated form of K14 exhibited keratin aggregation in basal cells and skin lesions which resemble the EBS phenotype [14]. Simultaneously, gene linkage data showed EBS as being mapped to keratin gene clusters on chromosome 17, with mutations

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being identified in K14 [15,16]. A number of other reports have confirmed the presence of mutations not only in K14 but also in its basic partner, K5, in patients with EBS (for a review, see [17]). Surprisingly, these studies revealed a striking correlation between the site of mutation and the severity of clinical symptoms [17]. Generally, with some exceptions, in severe generalized (DowlingMeara) type EBS, mutations of keratins are located in the helix initiation motif or in the helix termination motif at either end of the rod domain; while in either the milder generalized (Koebner) type or the localized (Weber-Cockayne) type EBS, mutations are located in the linker domain as well as the non-helical head or tail domain. After the identification of mutations in K5/K14 in patients with EBS, it became evident that mutations in other keratin genes should be causative in other genodermatoses. On the basis that the morphological abnormality of keratin appeared characteristic in diseases caused by keratin gene mutations as seen in EBS, other disorders which show clumped keratins as well as cell lysis have been studied. Subsequently, mutations in Kl or KlO genes were identified in epidermis that was suffering from an abnormally keratinized disorder called epidermolytic hyperkeratosis (EH) [ 18,191. Interestingly, the mutations were mainly clustered, with some exceptions, within the helix initiation and termination motifs as have been seen in EBS (for reviews, see [2-41). Ichthyosis bullosa of Siemens(IBS) shows striking clinical and morphologic similarities with EH. It was, therefore, speculated that IBS might be also a disorder of keratin which is expressedin the corresponding regions showing cytolytic degeneration. The mutations were indeed found to be mainly clustered within the helix initiation and the termination motifs of K2e which is a recently characterized keratin that is expressed in the upper suprabasal layers [20]. Furthermore, a hot spot for mutations was shown to occur at residue 117 (glutamic acid) of the 2B domain [21,22]. Similarly, K9, which has been shown to be expressed exclusively in the palmoplantar epidermis and outer root sheath epithelium, was subsequently studied as a possible candidate gene for epidermolytic palmoplantar keratoderma (EPPK),

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a genodermatosis which suffers palmoplantar epidermis. Mutations were indeed identified in the helix initiation motif of K9 in which the arginine at residue 10 was found to be a common target for mutation [4,23]. A subsequent study identified the isoleucine substitution for lysine in the amino-terminal Vl end domain of keratin 1 [24] in patients suffering from diffuse non-epidermolytic palmoplantar keratoderma (NEPPK) which has been termed the Unna-Thost type. This was an unexpected finding because the Kl is expressed throughout the whole body surface, and not confined to the palms and soles. However, interestingly, a recent study not only suggested that the Vl end domain of the head of K5 plays a role in binding with the desmosome component desmoplakin I, but also speculated that the isoleucine substitution for lysine in the Vl end domain of Kl might weaken the association between desmosomesand keratins, thereby contributing to the pathogenesis of NEPPK [25]. In contrast, a recent linkage analysis showed the causative defect in two pedigrees with a rare type of palmoplantar keratoderma called palmoplantar keratoderma tylosis, also termed HowelEvans type, and which was mapped to chromosome 17q24. Since the region, termed the tylosis esophageal cancer gene (TEC) locus, is distal to the type I keratin gene cluster, it excludes a keratin gene mutation as causal in the pathogenesis of this disease [26,27]. Pachyonychia congenita (PC) is a rare genodermatosis which has been subdivided into two major subtypes, the Jadassohn-Lewandowsky type and the Jackson-Lawler type. A recent study showed that the Jadassohn-Lewandowsky type is caused by a leucine to proline mutation in the helix initiation motif of K16 and that the Jackson-Lawler type is caused by an asparagine to aspartic acid mutation in the helix initiation motif of K17 [28]. It was further shown that a 3-bp deletion in exon 1 of the K6A gene, which removed a highly conserved asparagine residue from position 8 of the 1A helical domain, is also causative for the former type of PC [29]. The phenotypic differences between the two major subtypes of PC appears because K6/K16 and K17

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show slightly different tissue distributions and levels of expression [28]. Furthermore, interestingly, it was reported that focal non-epidermolytic palmoplantar keratoderma, whose clinical phenotypes are similar with PC, but which lacks the nail involvement, is also caused either by an arginine to cysteine mutation or by asparagine to serine mutation in the helix initiation motif of K16 [70]. White sponge nevus (WSN) is a rare genodermatosis involving abnormalities of the oral mucosa, tongue, esophagus and anogenital mucosa. The peculiar distribution and nature of lesions (leukokeratosis) in WSN suggested that the K4/ K13 keratin pair, which is the major differentiation specific keratin pair in the corresponding regions, represents a likely candidate gene. The sequenceanalysis showed a 3-base pair deletion in the helix initiation motif of K4 as well as a leucine to proline substitution in the helix initiation motif of K13 [30,31]. 3. Intermediate filament-associated proteins

At the terminal stage of epidermal differentiation, keratins are organized into an orderly, tightly packed aggregate which ultimately fills the cytoplasm of fully differentiated cornified cells. This aggregation is facilitated by a class of proteins that are generically classified as intermediate filament-associated proteins (for a review, see [32]). In recent years, much clearer images of the two intermediate filament-associated proteins, namely filaggrin and trichohyalin, have emerged and have contributed to our understanding of their roles in epidermal differentiation. Filaggrin is designated for its ability to cause keratin aggregation in vitro, Trichohyalin was previously considered to be unique to the trichohyalin granules in the inner root sheath of the hair follicle, but has since been shown to be expressed in the epidermis in some circumstances. 3.1. Filaggr in 3.1.1. Modulation

of jilaggrin

processing

Aggregation of keratins into a tight, orderly array involves filaggrin, a protein that is initially

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synthesized as a precursor protein designated as profilaggrin (for a review, see [32]). Filaggrin initially accumulates in the small granules in the lower granular cells which subsequently increase in size to form the large, mature keratohyalin granule in the upper granular cells (Fig. 3a and [50]). Filaggrin is uniformly distributed throughout the cytoplasm of lower cornified cells and gradually decreases in amount in the middle cornified cells becoming undetectable in the upper cornified cells (Fig. 3b and [50]). This finding is extremely important because the immunoreactivity of filaggrin in lower cornified cells correlated well with the aggregation of keratins, and the disappearance of filaggrin in upper cornified cells correlated precisely with the loosening of keratins. These data provided the strongest evidence available so far suggesting that filaggrin was indeed involved in promoting keratin aggregation in situ

POI.

Profilaggrin is expressed in the granular cells as a large precursor protein ( - 400 kDa in humans). During the terminal stagesof epidermal differentiation, profilaggrin undergoes a number of modifications such as phosphorylation, dephosphorylation and proteolysis (Fig. 4). Phosphorylation of profilaggrin is thought to prevent association with keratin, and may facilitate deposition in keratohyalin granules. Furthermore, it seemslikely that the dispersion of filaggrin from keratohyalin granules after dephosphorylation and proteolysis is tightly regulated in order to prevent premature collapse of the keratin network, although the regulatory mechanisms are not fully understood. In the upper cornified cells, filaggrin is degraded mostly into free amino acids, which are required for maintenance of epidermal osmolarity and flexibility. In the initial stages of processing, profilaggrin undergoes phosphorylation by several kinases at up to 21 sites in each filaggrin repeat [33]. Phosphorylated profilaggrin is insoluble and deposited in keratohyalin granules. Later, profilaggrin undergoes dephosphorylation by phosphatase 2A and at least one other phosphatase [34]. Subsequently, an initial proteolytic cleavage occurs at a hydrophobic site within a region referred to as the linker region, followed by independently regulated

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Fig. 3. Immunogold localization of filaggrin in normal and ichthyotic human epidermis. Filaggrin is initially detected in lower granular kells as small, irregular-shaped, keratohyalin granules which grow into large, stellate-shaped, keratohyalin granules in upper granular cells (a). Filaggrin is later distributed uniformly in the cytoplasm of lower cornified cells, but become progressively smaller in amount in middle cornified cells and finally become undetectable in upper comified cells (b). In ichthyotic epidermis, filaggrin can be detected in small, electron dense deposits (c) which correspond to the granules being in association with keratin bundles (d). (From [50], with permission of the publisher).

proteolytic processing to release the soluble filaggrin. The endoproteinase which acts at the first stage of profilaggrin proteolysis is a chymotrypsin-like serine protease and the second endoproteinase is a leupeptin-sensitive enzyme, most likely calpain [35,36]. Interestingly, two different primary sequences at the linker regions were found in mouse profilaggrin, in which these two variants differ by the presence or absence of an

FYPVY, insert [37]. The first stage of processing involves cleavage of only the one linker variant, followed by the second cleavage of the other linker variant. However, since there are no differences in the primary sequence of the linker regions in rat profilaggrin [38], some other explanation is required to account for the twostage processing observed in cultured rat keratinocytes [39].

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Fig. 4. Hybrid granules in mouse dorsal tongue epithelium. In the granular cells of mouse dorsal tongue epithelium, there exists morphologically heterogenous granules, many of which appear to be composed of discrete areas of distinct electron densities (a). Filaggrin and trichohyalin were both present in the hybrid granules (revealed by 15 nm and 40 nm immunogold particles, respectively) and that the more electron lucent regions contained filaggrin (arrow) while the electron dense regions contained trichohyalin (arrow head) (b).

3.1.2. Structure and gene organization offilaggrin Sequence analysis of the filaggrin repeats have shown that filaggrin is a histidine-rich, highly cationic protein, whose size, number and sequence varies between speciesas well as between individuals or strains [40--441(26 kD in mouse, 42 kD in rat and 37 kD in human, respectively). The structure of the human profilaggrin gene contains three exons interrupted by two introns [45,46]. The short S/-non-coding region (75 bp) is separated by the large first intron (9713 bp). The coding region begins in the second exon and continues in the third exon, which contains lo- 12 highly repetitive filaggrin sequences of 972 bp each. The complete DNA sequence revealed that profilaggrin also contains sequencesat the aminoand carboxy-termini, which share little similarity with the filaggrin repeat [45,46]. The amino-terminus can be divided into two distinct domains, A and B. Domain A is hydrophobic and contains two putative calcium-binding motifs similar to the EF-hands found in the S-100 family of calciumbinding proteins, whereas domain B is highly hydrophilic. It has recently been shown that the EF-hands of profilaggrin is functional and actually binds calcium [46,47], which suggested that

the EF-hands plays a role in profilaggrin processing and in other calcium-dependent events during the terminal stages of epidermal differentiation. Interestingly, profilaggrin has been mapped to human chromosome lq21 [41], where genes of a number of the S-100 family proteins, as well as a number of other epidermal structural proteins including loricrin, involucrin, the small prolinerich proteins and trichohyalin, have also been localized [48]. 3.1.3. Impaired regulation of filaggrin synthesis in ichthyotic epidermis Ichthyosis vulgaris is characterized by mild hyperkeratosis and a reduced presence or absence of keratohyalin granules in the epidermis. It has previously been shown that little profilaggrin is detectable in the skin of individuals affected with ichthyosis vulgaris [49]. In addition, keratohyalin granules were shown by immunoelectron microscopy to be structurally abnormal (Fig. 3c, d and [50]). Furthermore, a recent study incorporating the comparison of protein steady-state mRNA and transcriptional levels of profilaggrin showed that selectively impaired post-transcriptional regulation results in reduced profilaggrin mRNA and protein in ichthyosis vulgaris [51].

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3.1.4. Anti-jlaggrin autoantibody as a marker for rheumatoid arthritis Rheumatoid arthritis (RA) is characterized by a mononuclear cell infiltration of the synovium and by proliferation of the synovial cells, which leads to the destruction of articular cartilage. In RA, the presence of antibodies to the stratum corneum of rat esophagus epithelium has been widely reported [52-541. A recent study showed that the actual target of these autoantibodies was a neutral/acidic variant of filaggrin [55]. Nevertheless, their biological roles as well as their origin remains unclear, although the importance of antifilaggrin autoantibodies in diagnosis has been established [56-611. 3.2. Trichohyalin 3.2.1. Structure of and possible function of trichohyalin Trichohyalin is a high molecular weight (220 kD in humans), non-phosphorylated protein which is a major component of the trichohyalin granules in inner root sheath cells (for a review, see [5,62]). Recent studies elucidated that over 65% of sheep trichohyalin consists of two sets of tandem repeats which are based on two different consensus sequencesof both the central and carboxy-terminal repetitive regions [63]. The central repetitive region contains 16 tandem repeats which are based on a 28-amino acid consensus sequence, while the carboxy-terminal repetitive region contains 28 repeats based on the 23-amino acid consensus sequence. The two consensus sequences are considerably different with the only major similarity being the identity of two fiveamino acid stretches in each repeat. Further, the amino-terminus of trichohyalin contains two EFhand calcium-binding motifs typified by the SlOO family of small calcium-binding proteins; thus indicating that calcium plays a role in the modulation of function and/or conformational change of trichohyalin in the inner root sheath epithelium. In addition to sheep trichohyalin, the complete gene sequence of human trichohyalin has been determined resulting in further understanding of the structure of this protein [64]. Human trichohyalin consists of nine domains which possessat

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least three major functional properties: domain 1 contains a pair of calcium-binding domains of the EF-hand motifs; domain 6 constitutes the potential binding sites by ionic interactions with keratins; and domains 2-8 share significant sequence homology with the cornified envelope protein involucrin. The function of trichohyalin has been controversial. Our previous data established the presence of three immunologically distinguishable populations of trichohyalin protein which allowed us to define three distinct stagesof trichohyalin maturation ranging from its initial accumulation in small trichohyalin granules, through its release from the granule, and finally its association with the filaments in the inner root sheath [65]. Accordingly, such data strongly supported the role of trichohyalin as an intermediate filament-associated protein involved in the organization of keratin bundles in inner root sheath cells. 3.2.2. Existence of keratohyalin-trichohyalin hybrid granules in normal and abnormal epidermis It is well-known that rodent epidermis contains so-called ‘heterogenous keratohyalin granules’ which consist of at least two distinct components (Fig. 4a and for a review, see [66]). Our previous data showed the presence of hybrid granules in mouse tongue epithelium, in which filaggrin and trichohyalin were both present, but physically segregated in these granules (Figs. 4b, 6 and [67]). Consequently, it was proposed that these granules be referred to as keratohyalin-trichohyalin hybrid granules. The presence of hybrid granules was confirmed in epithelium other than mouse tongue epithelium. During the course of subsequent studies directed toward identifying epidermal diseases expressing trichohyalin, we found that the molluscum contagiosum virus (MCV)-affected epidermis elaborates abundant expression of trichohyalin and filaggrin [68]. Subsequent electron microscopic examination showed that the granular cells of MCV-affected epidermis contained morphologically heterogenous granules with distinct electron densities, in which the electron dense regions contained trichohyalin while the more electron lucent regions contained filaggrin (Fig. 5a and b), Fig. 6, Fig. 7. Furthermore, we found that trichohyalin

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Fig. 5. Hybrid granules in molluscum contagiosum virus (MCV)-affected epidermis. In the granular cells of MCV-affected epidermis, there exists numerous morphologically heterogenous granules, some of which are uniformly electron dense (arrow head), some of which are relatively less dense (arrow), and many of which are heterogenous (a). The dense and less dense regions of the heterogenous granules are composed of trichohyalin (arrow head) revealed by the large gold particles and filaggrin (arrow) revealed by the smaller gold particles, respectively (b). (From [68], with permission of the publisher).

was also expressed in the squamoid cells of a hair-related skin tumor called pilomatricoma, which were arranged in a circular configuration in the inner regions of the nest of basophilic

cells [69]. These findings suggested that a certain cell lineage of pilomatricoma undergoes the pathway of inner root sheath-type differentiation W91.

1.52

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The co-expression of trichohyalin and filaggrin in a number of normal and abnormal keratinocytes is particularly significant, since the genes for both profilaggrin and trichohyalin have been mapped to the same human genetic locus at lq21, a region which also contains genes for several other terminal differentiation-related proteins including involucrin and loricrin (described later). It is not precisely understood how the clustered chromosomal localization of these genes influences the regulation of the expression of these proteins. Understanding the detailed structural and functional interaction occurring between filaggrin and trichohyalin as well as their respective interactions with keratins should provide valuable insights into the terminal stages of both normal and abnormal epidermal differentiation. 4. Cornified envelope proteins Terminal differentiation in the epidermis involves the expression of cornified envelope (CE) proteins which deposit on the 15-nm-thick layer of cytoplasmic surface of the cell periphery. The

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extracellular surface SPRR loricrin &fin lnvolucrin cystatin A

Cornified \

cytoplasmic surface

\ Cornified Cell filaggrin trichohyaliF keratin -

Fig. 7. Diagrammatic representation of keratin pattern and cornified envelope in cornified cells. A number of proteins such as loricrin, cystatin A, SPRR, elafin and involucrin are accumulated into the cell periphery during the terminal stages of epidermal differentiation to form a thick inner cell membrane termed the cornified envelope (CE). CE assembly occurs as a complex but highly orchestrated sequence of events involving the sequential deposition of distinct proteins, in which the outer third of the CE consists mostly of loricrin, SPRRs (and keratins together with filaggrin); the middle third consists of elafin, loricrin and SPRRs; and the innermost third consists of involucrin and cystatin A with/without unknown proteins [2]. Simultaneously, keratins are aggregated with intermediate filament-associated proteins such as filaggrin and trichohyalin to form a densely packed filamentous structure which is known morphologically as a ‘keratin pattern’ in the cytoplasm of cornified cells.

Hvbrid Keratohvalin Granule cystatin A

loricrin

trichohyalin y<

profilaggrin

Granular Cells SPRR

keratin

&fin

Fig. 6. Diagrammatic representation of epidermal structural proteins in granular cells. The hybrid keratohyalin granules consist of distinct components including intermediate filamentassociated proteins and cornified envelope proteins. Profilaggrin, trichohyalin, loricrin and probably cystatin A are accumulated into keratohyalin granules in the granular cells being physically segregated in the areas of distinct electron densities. Other cornified envelope proteins such as SPRR, elafin and involucrin are distributed throughout the cytoplasm of granular cells.

CE proteins are rendered insoluble by cross-linking by E-(y-glutamyl)lysine isodipeptide bonds and disulfide bonds, serve as a physical barrier for the organism, and help to maintain the structural integrity of the epidermis. A number of proteins such as involucrin, loricrin, cystatin A (synonym keratolinin), small proline-rich proteins (SPRR), sciellin, have been implicated as CE precursor proteins [71]. CE assembly occurs as a complex but highly orchestrated sequenceof events involving the sequential deposition of distinct proteins, with a gradual progression of envelope thickness and rigidity. A recent report hypothesized on a model for the CE assembly, in which the outer (cytoplasmic) third of the CE consists mostly of loricrin, SPRRs and filaggrin; the middle third consists of elafin, loricrin and SPRRs; and the innermost third consists of involucrin and cystatin A with/without unknown proteins (Fig. 7 and [721).

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4.1. Loricrin 4.1.1. Regulation of loricrin expression

In situ hybridization has shown that loricrin transcripts are localized only in the upper spinous and granular layers of the epidermis [73]. By use of immunoelectron microscopy, loricrin was shown to be initially expressed in the granular layer and deposited as small spherical granules (L-granules), which were distinct from filaggrin immunoreactive granules, in new born mouse epidermis [74]. Through an unknown mechanism, loricrin is later released from the granular aggregates and disperses throughout the cytoplasm in transitional cells. In fully differentiated cornified cells, loricrin localizes exclusively on the inside of the cells at the cell periphery [74] and is then cross-linked by transglutaminases (TGases) as a dense CE (Figs. 6, 7 and [75]). However, in the case of human epidermis, the localization of loricrin within granular cells has been controversial. Yoneda et al. reported that loricrin colocalizes with profilaggrin in irregular-shaped keratohyalin granules [76], whereas Ishida-Yamamoto et al. reported that loricrin distributed throughout the cytoplasm [77,78]. Loricrin transcription has been shown to be upregulated by calcium, and repressed by retinoic acid in vitro [7.5]. Furthermore, it was recently shown that loricrin transcription in vitro requires binding of protein factors to an AP-1 consensus site in the proximal 60 base pairs of the promoter sequences [79]. Furthermore, transgenic mice bearing a P-galactosidase gene linked to 1.5 kb of S-flanking sequence and 2.2 kb of 3’-flanking sequencefrom the mouse loricrin gene revealed a strong expression of the transgene not only in the differentiated suprabasal cells but also in the undifferentiated basal cells in the epithelium normally expressing loricrin [79]. This finding indicated that additional cis-elements located outside the 6.5-kb fragment were required to restrict the expression of loricrin in these tissues. 4.1.2. Structure and gene organization of loricrin

Recently, the amino acid sequences of mouse [73] and human [75] loricrins have been found to have an unusual protein structure in which the

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bulk of their sequencesconsist of tandem quasirepeating peptides enriched in glycines and serines. This domain forms the novel glycine-loop configuration, which is flanked by lysine- and glutamine-rich terminal sequencesand interrupted by glutamine-rich domains that are involved in isodipeptide cross-links [80]. Furthermore, although only anecdotal data has been available on the nature of the TGase(s) responsible for crosslinking loricrins, a recent study showed that the TGase 3 reaction favored certain lysines and glutamines by forming mostly intra-chain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by inter-chain cross-links involving different lysines and glutamines [81]. Human loricrin has a structure comprising of a single intron of 1188 base pairs in the 5’-untranslated region with no introns in coding sequences, and has been mapped to chromosome location lq21 [76]. Human loricrin consists of two allelic size variants, due to sequence variations in its second glycine loop domain, which segregate in the human population by normal Mendelian mechanisms [76]. Furthermore, there are multiple variants within these two alleles due to various deletions of 12 base pairs in the glycine loop domain [76]. 4.1.3. Mutation syndrome

of loricrin gene in Vohwinkel’s

A defect in any of the genes involved in the correct formation of the CE structure might be involved in the pathology causing keratinization disorders by altering the barrier function of the epidermis [82]. Although no phenotype was found in mice over-expressing loricrin [83], a recent report, surprisingly, showed that the loricrin gene in Vohwinkel’s syndrome revealed an insertion that introduced frame shifts and a delayed termination codein in the C-terminal Gly- and Gln/Lys-rich domains [84]. The defective protein is predicted to contain a highly charged carboxy-terminal domain, very rich in arginine, and an additional 22 amino acids more than the wild-type protein, in place of its terminal 84 neutral-basic amino acids. It was speculated that the replacement of the fourth Gly-rich domain and the C-terminal Gin/ Lys-rich domain, which are conserved in evolu-

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tion and are critical for cross-linking by TGases, is likely to affect the function of CE and impair cornification by altering the interaction of loricrin with other CE components. 4.2. Cystatin A 4.2.1. Cystatin superfamily Various mammalian tissues contain a group of proteins, termed cystatins, that are potent inhibitors of the papain-like cysteine proteinases (for a review, see [85-871). Based upon functional and structural similarities, cystatins form a superfamily of sequentially homologous proteins subdivided into three major families plus newly discovered various proteins such as human histidine-rich glycoprotein and a2HSglycoprotein. Structural analysis of the members of three cystatin superfamilies revealed that they share extensive sequence homology, enough to be classified as members of one superfamily derived from an evolutionarily common ancestral gene [88,89]. The proteins in family I (type I cystatins) are proteins with - 11 kD which lack signal peptides, carbohydrates and internal disulfide bonds. The type I cystatins occur in multiple isoforms: cystatin A with pI values in the range 4.5-5.0, and cystatin B with pZ values in the range 5.9-6.5. Cystatin A is normally localized in the cell cytoplasm of various types of epithelial cells, polymorphonuclear leukocytes and lymphoid follicular dendritic cells, while cystatin B is found to be ubiquitously distributed among various cells and tissues. In contrast, those in family II (type II cystatins) are composed of - 115 amino acid residues with 130 kD, which contain two disulfide bonds near the carboxy-terminus. With the exception of rat cystatin C, the family II cystatins are not glycocylated. The type II cystatins contain the signal peptide necessary for protein secretion and are secreted to extracellular fluid after cleavage of the peptide. The chicken cystatin, the best known representative, was a mixture of two major isoelectric forms: a non-phosphorylated form with a pI of 6.5, and a phosphorylated form with a pI of 5.6. These proteins are found in many biological fluids such as cystatin C in seminal plasma, cerebrospinal fluid and synovial fluid. The family

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III cystatins (kininogens) are much larger than the first two families: high molecular weight kininogen with 88- 114 kD; low molecular weight kininogen with 50-68 kD; T-kininogen with 68 kD. Kininogens are acidic glycoproteins with pZ values for multiple isoelectric forms in the pH range 4.0-5.2. They are primarily synthesized in the liver with signal peptides, and are secreted into plasma and other secretions such as synovial fluids. Kininogens contain three repetitive segments with significant homology to family II cystatin, in which segment 1 is not inhibitory for cysteine proteinases although the other two segments retain inhibitory activity. 4.2.2. Structure and inhibitory activity of cystatins Cystatins do not form covalent bonds with cysteine proteinases, but instead cover the active site cleft of cysteine proteinases [90,91]. The molecular structure of cystatins consists of 10 amino acid residues in the amino-terminus, a first p-hairpin loop containing the highly conserved residues -QVVAG- and a second p-hairpin loop containing the less conserved residues Leu 102, His 104 in family I and Trp 104 in family II cystatins. The triparties form a hydrophobic wedge-shaped edge which is highly complementary to the active site cleft of serine proteinases and penetrates the active site in such a fashion so as to block the active site cysteine residue. Cystatins lost their inhibitory activity partially or completely by truncation of their amino-terminal segment, and, namely, cystatin a, which is a rat counterpart of cystatin A, was shown to be completely inactivated by amino-terminal truncation of 15 residues [92]. Furthermore, it was recently confirmed by successive truncation using genetic engineering that the truncations deleting the Nterminal residues of cystatin A such as Pro 3 and also beyond conservative Gly 4 and Gly 5 caused a remarkable decrease in the inhibitory activity, which further caused a conformational change in the reactive first reactive loop, -QVVAG- [93]. Human cystatin A and its rat counterpart, cystatin a, belong to family I (type I cystatins) and are found in high concentrations in various types of epithelial cells and in polymorphonuclear

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A inhibits cysteine leukocytes. Cystatin proteinases’ activities, of which the inhibition constant for papain is 1.9 x 1OW” M and that for cathepsin B is 7.3 x lo-’ M. Studies on the localization of cystatin tx in rat skin indicated that the more acidic form is phosphorylated caused by protein kinase C, and is localized in the keratohyalin granules of granular cells (Fig. 6 and [94,95]). Subsequent studies showed that phosphorylated cystatin c( is copolymerized by the action of epidermal transglutaminase with the linker regions of filaggrins forming E-(y-glutamyl)lysine cross-linking peptide bond, and is also localized in the cornified envelope of cornified cells (Fig. 7 and [96,97]). 4.2.3. Possible roles of cystatin A during abnormal epithelial differentiation

Reduced immunostaining of cystatin A has been observed in squamous cell carcinoma of skin as the tumor tissue becomes poorly differentiated [98]. Furthermore, more compellingly, it has been shown that the cystatin A mRNA level decreased during the progression of murine skin papillomas to carcinomas [99]. Collectively, these results suggest that cystatin A may be important for mainteepithelial cell nance of differentiation. Furthermore, the inhibitory activity of cystatin A from human sarcoma has been shown to be reduced against papain and cathepsin B, with the subsequent hypothesis that the reduced cystatin A activity might be due to a single point mutation as seen for cystatin C in the hereditary cystatin C amyloid angiopathy [ lOO,lOl]. Moreover, interestingly, the p21 ras proto-oncogene product has recently been suggested to be a member of the family I cystatin [ 102- 1051.Although it has been shown that cystatin plays a role as a bacteriostatic agent against group A streptococcus [106], further studies are necessary to elucidate the biological roles of cystatin A during normal and abnormal epidermal differentiation [107]. 4.3. Involucrin 4.3.1. Expression and processing of involucrin

Involucrin servesas one of the precursors of the cornified envelope (for a recent review, see [lOS]),

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the confirmation of which was attained using chemical cleavage methods to release involucrin fragments from highly purified cornified envelopes as well as by immunoelectron microscopy (Fig. 7, and [109]). Involucrin is initially synthesized as a soluble cytosolic protein in the suprabasal cell layers of the esophagus, epidermis, conjunctiva, cervix, and other stratifying tissues including epidermis (Fig. 6, and [l lo- 1131).During the terminal stages of epidermal differentiation, glutamine residues of involucrin become covalently crosslinked to other envelope precursors via covalent E-(y-glutamyl)lysine bonds which are catalyzed by transglutaminase ([log] and references therein). During this processing, involucrin disappears from the soluble phase into the particulate fraction following calcium-dependent activation of TGase [ 110,114- 1161.A recent study using proteolytic enzyme cleavage indicated that involucrin is likely to be deposited at the inner side of the plasma membrane at the earliest stage of the cornified envelope assembly [72]. 4.3.2. Structure and cross-linking

of involucrin

Involucrin has a molecular weight of 6.8 kD with a high percentage of glutamine and glutamate residues (25 mol% and 20 mol%, respectively) [ 1171. The molecule consists of a central segment composed of 39 tandem repeats, each consisting of 10 amino acids and having the consensus sequence QEGQLKHLEQ [117]. The central segment is flanked by 153 amino acid N-terminal and 45 amino acid C-terminal segments that contain three and four b-sheet domains, respectively, with each b-sheet segment separated by a short random coil segment [118]. The central a-helical region is hydrophilic, whereas the flanking amino and carboxy terminal, b-sheet regions are relatively hydrophobic [ 1181.A recent study suggested that involucrin is an extended, flexible, rod-shaped molecule that has a mean contour length of 460 A, an axial ratio (i.e. 1ength:width) of 3O:l and possessesbetween 50 and 75% x-helical content [118]. Three glutamine residues, on average, are circumferentially distributed along the length of each of the central segment repeats, which allows glutamyl side chains of involucrin to interact with the lysine

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residues of other cornified envelope proteins in the formation of e-(y-glutamyl)lysine bonds [118]. Furthermore, since the repeating structure of the central segment appears to be conserved across species, involucrin is well characterized as a marker of molecular evolution [119-1211. 4.3.3. Involucrin in human diseases At present, no information is available concerning the possible involvement of involucrin in human disease pathogenicity. Studies using transgenic mice which express human involucrin has shown that the epidermal keratinocytes in these mice exhibit scaling and an abnormal hair phenotype [122]. The most likely explanation for these abnormal findings is that the barrier function of the epidermis was impaired by the presence of an excess amount of involucrin and/or that of exogenous human involucrin, the structure of which differs from mouse involucrin. 4.4. Small proline-rich proteins 4.4,l. SPRR family Small proline-rich proteins (SPRR) were initially identified as UV- or TPA-induced genes in human keratinocyte cultures [123,124]. SPRR were given the name SPRR (small proline-rich proteins) due to their small molecular mass of less than 20 kD and their exceptionally high proline content [125]. SPRR are subdivided into three distinct members of a very similar primary structure designated SPRRl, SPRR2 and SPRR3. Cornifin CIand cornifin j’, which are cornified envelope precursors of the rabbit squamous cell [126- 1281, appear to be rabbit homologues of SPRRl and SPRR3, respectively. Furthermore, it has recently been reported that a 14.9 kD protein of the pancornulin family, which was previously identified by anti-cornified envelope monoclonal antibody [129], is identical to the SPRRl [130]. In addition to the 14.9 kD pancornulin, the 16.9 kD and 22 kD pancornulins were also shown to belong to the SPRR family [130]. 4.4.2. Expression of SPRR in normal epithelium In normal inter-follicular epidermis, SPRRl has been found to be strongly expressed in supra-

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basal cells in newborn skin [ 1311, but absent in adult skin [132]. In follicular epithelium, SPRRl is expressed in the upper spinous and granular cells of the infundibulum as well as differentiated cells of the inner root sheath [132- 1341.SPRRl is also expressedin the focal areas of the palmoplantar and foreskin epidermis [132]. In non-keratinized epithelium, SPRRl is expressed in the upper cells of the epithelium of the tongue, oral mucosa and vagina [132,133]. In normal airway epithelium, the expression of SPRRl is very low, although, it can be rapidly induced by the addition of phorbol ester or by a vitamin A deficiency condition which induces airway epithelial cells to undergo squamous cell metaplasia [135]. In normal epidermis, SPRR2 is expressed in the upper spinous and granular cells of inter-follicular epidermis, infundibulum of hair follicles and palmoplantar epidermis [132]. SPRR2 is also expressed in focal areas of the foreskin epidermis [132]. In normal upper digestive tracts, SPRR2 is expressed in the upper cells of the dorsal side of tongue epithelium, but is absent in the abdominal side of the tongue epithelium and the esophageal epithelium [132]. In contrast to SPRRl and SPRR2, SPRR3 is absent in normal epidermis, but is expressed in the suprabasal epithelium of the tongue, esophagus and vagina [132,133]. All members of the SPRR family are preferentially localized at the cell periphery [127,132], become covalently cross-linked by the actions of transglutaminase [ 127,129,130] and show a structural resemblance to known cornified envelope proteins such as loricrin and involucrin, thus suggesting that the SPRR family constitutes a new class of cornified envelope precursor proteins (Fig. 7). 4.4.3. Regulation of SPRR gene expression The regulatory mechanisms of the SPRRl gene are well-characterized. It has been demonstrated that the SPRRl mRNA level is increased by either phorbol ester or dibutyryl cyclic AMP, which suggests that both protein kinase C and protein kinase A play important roles in regulating the transcription of the SPRRl gene [136]. Subsequently, it was shown that an AP-1 binding site was indeed present at - 142, and a putative

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cyclic AMP-responsive element at - 597 base pairs upstream of the transcription start site of SPRRl gene, and that these elements are probably involved in reg,ulating the expression of the SPRRl gene [136]. Furthermore, a recent study showed that the SPRRl expression was able to be induced by physiologic doses of UV irradiation in vivo within 24 h [131], although it was previously reported that SPRRl is not induced by UV in vitro [137]. A subsequent study showed that the SPRRl expression is upregulated by two inflammatory cell-derived cytokines such as interleukin1 and interleukin-3, and is elevated in the epidermis after UV irradiation, while it was downregulated by TGF-a which reduces the interleukin- 1 receptor in keratinocytes [ 1311. The distinct SPRR genes show a diversity in regulation in response to various treatments. SPRRl and SPRR2 are both UV- and TPA-inducible [131,134,138], though SPRR3 is only UV inducible [137]. Furthermore, the addition of 107 M retinoic acid to cultured differentiating keratinocytes has been shown to significantly downregulate the expression of SPRR2 and SPRR3 transcripts, whereas it slightly decreased that of SPRRl [132]. 4.4.4. Structure and gene organization of the SPRR family All members of the SPRR family of proteins have a characteristic structure of central segments that are built up from a variable number of tandemly repeated units with a common sequence motif xKxPEPxx [137]: the consensus octamer PKVPEPCQ is found 6 times in SPRRl, the nonamer PKCPEPCPP 3 times in SPRR2, and the octamer TKVPEPGC 14 times in SPRR3. The N- and C-terminal domains of these proteins show strong sequence homology with the corresponding domains of loricrin and involucrin [139]. The gene structure of the SPRR family is similar to that of loricrin and involucrin, consisting of two exons and a single intron located within the 5’-untranslated region with the second exon containing a complete open reading frame [137]. SPRR genes are clustered within a 300 kb DNA segment on chromosome lq21 [137], a close region where loricrin and involucrin genes have

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been mapped [140]. This cluster constitutes a multigene family containing two SPRRl genes, 8 SPRR2 genes and a single SPRR3 gene [137]. Their clustered chromosomal organization together with sequence homology suggest that this family of genes has evolved from a single progenitor gene by multiple intra- and intergenic duplications [ 1371. 4.4.5. The expression of SPRR in psoriasis In psoriatic epidermis and well-differentiated squamous cell carcinoma, SPRRl has been found to be expressed at much higher levels than in normal epidermis [132,133,141]. However, the biological roles of the SPRR family in normal and abnormal epidermis are still unknown. Further studies are required to clarify whether the regulatory mechanisms of the SPRR gene expression relate to hyperproliferative conditions. 4.5. Elajin 4.5.1. Expression patterns of elafin in various normal and abnormal epitheliurn It has been reported that an elastase inhibiting activity is induced in normal human skin as a response to injury as well as in several scaling skin disorders [ 142,143]. The related inhibitor was later purified from psoriatic epidermis, cultured human keratinocytes and bronchial secretions, and was termed elafin, also known as skin-derived antileukoproteinase (SKALP) and elastase-specific inhibitor (ESI) [144-1471. Elafin is a very heat stable and pH stable molecule [144,146], and is very cationic with a calculated isoelectric point of 8.84 [148]. Elafin is a potent, fully reversible inhibitor of human leukocyte elastase, porcine pancreatic elastase and proteinase 3 with dissociation constants (K,) of 6 x 1OW’” M to 2 x lo-” M, 1 x lop9 M for [144,149,150] and 9.5 x 10W9M [151,152], respectively, but does not inhibit the serine proteinases cathepsin G, trypsin, chymotrypsin or plasmin [ 145,146,149,151,152]. A recent review reported that elafin is present in hair follicles, tongue, vagina, pharynx, epiglottis, esophagus, uterine cervix, gingiva, palate/lingual tonsil and vocal fold, but is absent in lung alveoli, bronchus, liver, kidney, colon, duodenum, ure-

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thra, brain, cornea, connective tissue, blood cells, cartilage and bone [153]. Furthermore, elafin is present in the cytoplasm of upper spinous cells and near the plasma membrane in cornified cells in psoriatic epidermis [154,155]. However, it was recently shown that elafin mRNA is present in normal skin, whereas elafin is absent at protein level [156]. Elafin was also present in well-differentiated squamous cell carcinomas, but was absent in poorly-differentiated squamous cell carcinomas and in basal cell carcinomas [157]. Based on the expression in normal and abnormal keratinization, the expression of elafin in epiderma1 keratinocytes appears to be associated not with differentiation programs but with hyperproliferation conditions such as psoriasis [ 154,155], tape stripping epidermis [ 1581, cultured keratinocytes [146] and tumor cells [157]. Although the expression of elafin in simple epithelial cells has been shown to be regulated at the translational level [159], it is not clear whether the synthesis of elafin in epidermal keratinocytes is regulated in the same manner. 4.5.2. Structure and gene organization of elajin

The complete sequence of a 6 kDa elafin fragment from psoriatic scales [144], a partial internal sequence of SKALP [146] and a partial amino acid sequence of ES1 purified from sputum [160] suggested that these three inhibitors were the same molecule. It has been previously reported that an 18 kDa elastase inhibitor, of which the biochemical and immunochemical properties were indistinguishable from those of elafin in psoriatic scales, has been found in cultured human keratinocytes [146]. The cloned cDNA showed that the 18 kDa elastase inhibitor synthesized in cultured human keratinocytes is a larger precursor molecule of the low molecular (6 kDa) form found in psoriatic scales [148]. Results showed that elafin is initially synthesized as a precursor molecule, consisting of 117 amino acids including a hydrophobic signal peptide of 22 amino acids, with later cleavage of this signal peptide resulting in a mature protein of 95 amino acids with a molecular mass of 9.9 kDa [148]. The amino-terminal part of the mature elafin was found to contain four repeats, which are

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highly homologous to a consensus sequencebeing able to act as a putative substrate domain for epidermal transglutaminase [148]. A subsequent study showed that elafin can indeed become crosslinked by transglutaminase to proteins extracted from psoriatic scales, thus suggesting that elafin is able to exist both as a free low molecular (6 kD) form and as an immobilized high molecular (9.9 kD) form covalently attached to cornified envelopes by transglutaminase cross-linking (Fig. 1 and [148]). The carboxyl-terminal part of elafin contains a characteristic eight cysteine motif which is highly conserved in small proteins called ‘four-disulfide core’ proteins [161]. It is speculated that the elastase inhibiting activity can be obtained by the formation of four disulfide bonds from these eight cysteine residues [149]. The elafin gene is N 1.7 kb long and contains three exons and two introns [162]. The first exon encodes the 5’-non-coding region and the hydrophobic signal sequence as well as the first four amino acids of the mature protein. The second exon encodes the remainder of the protein and the first nucleotide of the 3’-non-coding region, while the third exon encodes the remainder of the 3’non-coding region. The elafin gene was mapped at human chromosome 20, region q12-13 [163]. Interestingly, a cluster of other proteinase inhibitors, the family II cystatins, have been mapped to the same chromosomal region [164], although no structural homology was found between elafin and the cystatins. 4.5.3. Possible roles of elajin in hyperproliferative epidermis

Although no skin disease has so far been mapped to chromosome 2Oq12-q13 where the elafin gene is localized as has been indicated above, elafin is expressed in a number of hyperproliferative epidermis in which it is inducible by inflammation (psoriasis), mechanical trauma (tape stripping), some growth factors (cultured keratinocytes) or oncogene activation (squamous cell carcinoma). Elafin has also been found to be present at high levels in not only urine but also serum from psoriatic patients [165- 1671. It is likely that elafin is able to protect tissue from damage by elastase-mediated proteolysis in psori-

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atic epidermis and to block tumor cell invasion either directly or indirectly through interference with elastolytic activity.

Acknowledgements

We thank Drs. Kazumi Ishidoh (Juntendo University, Japan), Joseph A. Rothnagel (University of Queensland, Australia) and Daniel Hohl (Centre Hospitalieu Universitaire Vaudois, Switzerland) for their critical evaluation of this manuscript.

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