• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

Epidermal Differentiation: The Role of Proteases and Their Inhibitors

Posted on: Saturday, 26 February 2005, 03:00 CST

Cystatin M/E; Legumain; Tranaglutaminases; Mouse mutation; Cysteine proteinases; Skin; Stratum corneum; Hair follicle; Cornification

Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency in furless mice, targeted ablation of the serine protease Matriptase/MT-SP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent findings on the role of cystatin M/E and legumain as a functional dyad in skin and hair follicle cornification, a paradigm example of the regulatory functions exerted by epidermal proteases, will be discussed.

The cornified cell envelope

Terminally differentiating stratified squamous epithelial cells assemble a complex of cross-linked proteins and lipids on their periphery termed the cornified cell envelope (CE) (Reichert et al., 1993; Roop, 1995). The CE is an insoluble, approximately 10-nm thick and of uniform density proteinaceous layer (Jarnik et al., 1998) deposited subjacent to the plasma membrane. In the specialized case of the epidermis, a 5-nm thick layer of ceramide lipids is covalently bound to the proteins (Elias and Menon, 1991; Marekov and Steinert, 1998; Swartzendrubcr et al., 1987), which organize extracellular lipids into orderly lamellae. The insolubility of this proteinaceous layer on the surface of epidermal and hair corneocytes was found to be the result of both disulfide bonding, and extensive cross-linking by transglutaminases (TGases) that catalyze the formation of N^sup &947;-glutamyl)lysine isopeplide bonds (Polakowska and Goldsmith, 1991; Rice and Green, 1977, 1978). Together, the protein envelope and the lipid envelope are essential for effective physical and water barrier function in the skin. Over the last decade a large number of studies have reported the identification of the major protein and lipid constituents of CEs, and the role of TGases in the process of protein cross-linking. In addition, the discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier.

Table 1. Cornified cell envelope precursor proteins.

To date, cell biological, biochemical, and protein sequencing studies have shown that at least 20 proteins (summarized in Table 1) are used to assemble the CE (Kalinin et al., 2002; Ncmes and Steinert, 1999; Steinert and Marekov, 1999). It is known that genes for many human CE proteins map to a 2.5-Mbp cluster termed the "epidermal differentiation complex" (EDC, MIM#601588) located on chromosome 1q21. Members of the EDC all share significant sequence similarities, for example glutamine- and lysine-rich tracts that are usually involved in intrachain and interchain cross-linking by TGases.

Involucrin is a major structural protein of the CE that was firstly identified by Rice and Green (1979). Expression of involucrin appears at the onset of terminal differentiation in epithelia (Eckert et al., 1993; Watt, 1983), and is eventually incorporated into CEs in a cross-linking reaction catalyzed by the membrane-associated TGase 1 (Nemes et al., 1999b; Simon and Green, 1984; Steven and Steinert, 1994). Sequencing of CE-derived peptides has revealed that it is cross-linked to almost all other CE proteins, and it is a principal substrate for attachment of ceramides to form the lipid envelope (Marekov and Steinert, 1998).

Loricrin expression occurs very late during the terminal differentiation program of keratinocytes in the granular cells of the epidermis, where it accumulates in sulphur-rich keratohyalin granules (L-granules) (Hohl et al., 1991; Hohl and Roop, 1993; Steven et al., 1990). It functions as the major CE component constituting for more than 70% of total cross-linked protein (Hohl and Roop, 1993; Steinert and Marekov, 1995; Steven and Steinert, 1994). Immunoreactivity of loricrin is also observed in parts of the hair follicle (the inner root sheath cells, hair canals, and inner cells of the outer root sheath in the region of the isthmus), in harmony with involucrin and keratinocyte TGases (Akiyama et al., 2002). This is not surprising as the molecular events of CE development between the hair canal and interfollicular epidermis are thought to be almost identical (Dale et al., 1985; Holbrook et al., 1993).

The amino- and carboxyl-terminal domains of small proline-rich proteins (SPRs) both contain several glutamine and lysine residues that participate in TGase-catalyzed cross-linking to other protein constituents of the CE (Candi et al., 1999; Robinson et al., 1997; Steinert et al., 1998). In vitro studies have shown that SPR1, SPR2, and SPR3 proteins are cross-linked primarily by the TGase 3 enzyme, and to a minor extent by TGase 1 (Candi et al., 1999; Steinert et al., 1999; Tarcsa et al., 1998). It is thought that the TGase 3 enzyme first cross-links the SPRs into short oligomers that are later connected to the CE by the TGase 1 enzyme (Steinert et al., 1998).

Profilaggrin is a polyprotein consisting of numerous filaggrin units flanked by distinctive amino- and carboxyl-terminal domains (Presland et al., 1992), and contains two functional Ca^sup 2+^- binding domains known as "EF hands" (Markova et al., 1993; Presland et al., 1995). During terminal differentiation, profilaggrin undergoes dephosphorylation and proteolysis to form filaggrin, which then acts as a glue-like protein that facilitates dense packing of keratin intermediate filaments into macrofibrils typically seen in corneocytes (Dale et al., 1978, 1993; Presland and Dale, 2000). Enzymes thought to be involved in profilaggrin processing include protein phosphatase PP2A (Kam et al., 1993), PEP1, a chymotrypsin- like protease (Resing et al., 1995), calpain, a Ca^sup 2+^- dependent protease (Resing et al., 1993; Yamazaki et al., 1997), and the serine protease furin (Pearton et al., 2001). Some filaggrin is eventually incorporated into CE proteins (Simon et al., 1996; Steinert and Marekov, 1995), presumably together with and at the same time as the keratins.

Keratins are the predominant cytoskeletal component of stratified keratinizing epithelia, and some of them become cross-linked to the peripheral CE during terminal differentiation (Dale et al., 1978; Nemes and Steinert, 1999; Steinert and Marekov, 1995). Keratin intermediate filaments appear to be an integral part of a structural network that also includes hemidesmosomes, desmosomes, and the basement membrane, which provides mechanical stability throughout the epidermis (Garrod, 1993; Smack et al., 1994). The connection of keratin intermediate filaments to desmosomes occurs through several clesmosomal proteins, including desmoplakin and envoplakin (Green and Jones, 1996; Jamora and Fuchs, 2002). At the final stage of terminal differentiation, the cell integrity collapses, thereby destroying most cell organelles as well the structural integrity of desmosomes. During this process, the keratin intermediate filaments become integrated into the CE and form a stable insoluble structure for the corneocyte.

Desmoplakin, envoplakin, and periplakin are three sequence- related proteins of the plakin family that localize to intermediate filaments and filament attachment sites at the plasma membrane (DiColandrea et al., 2000; Karashima and Watt, 2002; Ruhrberg et al., 1996, 1997; Ruhrberg and Watt, 1997). By connecting cytoskeletal elements to each other and to junctional complexes, the plakin family of "cytolinkers" plays a crucial r\ole in orchestrating cellular development and maintaining tissue integrity. Envoplakin expression is restricted to terminally differentiating epithelial cells, while periplakin is more widely expressed. Both proteins, together with involucrin, are recruited to the cell periphery during the earliest stages of CE assembly, where they become cross-linked components of mature CEs (Steinert and Marekov, 1997).

S100 proteins are a family of EF-hand-containing Ca^sup 2+^- binding proteins that function to transmit Ca^sup 2+^-dependent cell regulatory signals (Heizmann et al., 2002). Members of this protein family have been shown to interact with several effector proteins within cells thereby regulating enzyme activities, the dynamics of cytoskeleton constituents, cell growth and differentiation, and calcium homeostasis (Donato, 2003). S100 calcium-binding proteins such as S100A7 (psoriasin), S100A10 (calpactin), and S100A11 (calgizzarin) are expressed in terminally differentiated cells (Robinson et al., 1997; Ruse et al., 2001), and could serve as TGase substrates in vitro. It is thought that this feature could account for the formation of S100 protein-containing complexes in vivo, which may be a general mechanism that terminates S100 protein action. One possibility is that S100 proteins localize at the plasma membrane to achieve specific goals related to, e.g., cell signaling/ membrane transport, and that, once these functions are fulfilled, they become incorporated as CE components (Broome et al., 2003; Ruse et al., 2001).

Trichohyalin is an intermediate filament-associated protein that associates with keratin intermediate filaments of the inner root sheath cells of the hair follicle and the granular layer of the epidermis and is a known substrate of TGases (Lee et al., 1993; Steinert et al., 2003). The presence of Ca^sup 2+^-binding domains in these intermediate filament-associated proteins has led to the hypothesis that calcium binding may be required for granule formation or for activation, regulation, and/or coordination of posttranslational modification events during differentiation (Dale et al., 1993).

LEP/XP-5 proteins encode proteins that incorporate into the CE late in development and late in the process of envelope maturation during epidermal differentiation (Marshall et al., 2001; Zhao and Elder, 1997). LEP/XP-5 proteins contain shared characteristics of loricrin, SPRs, and involucrin, with regard to their amino terminal sequences, which may serve as key sites for TGase-mediated cross- linking.

Other CE components include the protease inhibitors cystatin A and SKALP/elafin. Cystatin A is a cysteine protease inhibitor (Jarvinen et al., 1987) and is identical to a TGase substrate termed keratolinin (Takahashi et al., 1997). Cystatin A has been identified as a minor cross-linked component of CEs in a phosphorylated form (Takahashi et al., 1992; Zettergren et al., 1984), which is proposed to be relevant for the antimicrobial properties of the skin (Takahashi et al., 1994a, b). SKALP/elafin is a potent inhibitor of the leukocytic serine proteases elastase and protease-3 (Schalkwijk et al., 1991; Wiedow et al., 1991), and is expressed in several human stratifying squamous epithelia, except for epidermis where it is only expressed in the context of inflammation, such as psoriasis or wound healing (Pfundt et al., 1996; Schalkwijk et al., 1993).

Transglutaminases

The characteristic insolubility of the CE is largely due to cross- linking of its components catalyzed by TGases (EC 2.3.2.13). The TGase cross-linking reaction is based on a Ca^sup 2+^-dependent exchange of primary amines for ammonia at the γ-carboxamide group of glutamine residues. Pepticle-bound lysine residues or polyamines serve as the primary amines to form either ε- (γ-glulamyl)lysine or (γ-glutamyl)polyamine bonds between proteins (Folk and Finlayson, 1977), which are highly resistant to chemical and enzymatic degradation (Folk, 1983). To date, nine members of the TGase family have been identified in the human genome (Grenard et al., 2001), however, only six have hitherto been isolated and characterized at the protein level (Griffin et al., 2002; Lorand and Graham, 2003). Four of these, TGase 1, TGase 2, TGase 3, and TGase 5, are commonly expressed in epithelia (Aeschlimann et al., 1998; Kim et al., 1991), although only TGases 1, 3, and 5, have proven to be involved in CE assembly (Candi et al., 1995, 1999, 2001; Kalinin et al., 2002; Steinert et al., 1999; Steinert and Marekov, 1997; Tarcsa et al., 1998). These studies have demonstrated tight substrate specificities and, in particular, preferred reaction of specific glutamines and lysines on substrates by the different enzymes.

TGase 1 is involved in the formation of the CE in epidermal keratinocytes, and is detected in proliferating cells but is more abundantly expressed in differentiating cells (Reichert et al., 1993). TGase 1 enzyme activity is largely found near the cytosolic site of the plasma membrane, where it is anchored by fatty acid esterification (Phillips et al., 1993; Rice et al., 1990; Sleinert et al., 1996b). Besides its cross-linking capacities towards constituent proteins of CEs, TGase 1 could also catalyze the cross- linking of ω-hydroxy-ceramides to involucrin via lipid ester linkages (Nemes et al., 1999a), which contribute to a hydrophobic surface of the corneocytes. During terminal differentiation some of the full-length TGase 1 enzyme molecules undergo proteolytic cleavage that results in ~100 to 1000-fold higher specific activity, as shown by standard TGase assays in vitro (Kimet al., 1995; Steinert et al., 1996a). It is well established that intracellular TGase activities are switched on by increasing Ca^sup 2+^- concentrations, in both in vitro assays and in vivo (Folk and Finlayson, 1977; Melino et al., 2000). There is some evidence that calpain, a Ca^sup 2+^-dependent protease, could activate TGase 1 in vivo. The details of this calpain-mediated activation processes remains to be elucidated (Ando et al., 1988; Kim and Bae, 1998) since in vitro recombinant TGase 1 was shown to be susceptible for limited proteolysis by calpain, but this did not induce elevation of the specific enzyme activity (Hitomi et al., 2000). Very recently it has been reported that cathepsin D, an aspartate protease, is involved in the regulation of TGase 1 (Egberts el al., 2004). In vitro experiments have shown that TGase 1 activity is stimulated by exogenous cathepsin D in cultured keratinocytes; in cathepsin D- deficient mice a reduced TGase 1 enzymatic activity and defective TGase 1 processing was observed.

TGase 3 is, like TGase 1, essential for skin maturation and integrity, and its non-redundancy is indicated by preliminary evidence that mice with a null mutation in the TGase 3 gene show an embryonic-lethal phenotype (Kim et al., 2002). No human diseases have yet been linked to mutations in the TGase 3 gene, which could be due to non-viability as observed in the knockout mice. Although TGase 3 accounts for more than 75% of the total TGase activity in the epidermis (Kim et al., 1990), it cannot compensate for CE formation in patients with lamellar ichthyosis in which TGase 1 activity is absent (Candi et al., 1998; Jeon et al., 1998; Matsuki et al., 1998). TGase 3 protein expression is located in the cytoplasm of the upper differentiating layers of lhe epidermis (Hitomi et al., 2003) and in the hair follicles (Martinet et al., 1988). Activation of TGase 3 during keralinocyte differentiation involves cleavage of a 77-kDa zymogen by an unknown prolease resulting in the release of 30-kDa and 47-kDa fragments, which then associate and form the active enzyme (Ahvazi et al., 2002; Kim et al., 1990, 2001). Recently, it was found that binding of Ca^sup 2+^ ions at specific sites could induce structural changes in the enzyme that subsequently affect its activity (Ahvazi et al., 2002; Ahvazi and Steinert, 2003). These in vitro experiments were performed using recombinant TGase 3 that was proteolyzed by dispase, a bacterial enzyme, which is commonly used to activate TGase 3 in vitro (Kim et al., 1990). Until now, the proteolytic activation of TGase 3 in vivo, and the responsible protease(s) remain to be elucidated.

The last TGase known to be involved in CE assembly, TGase 5, has been found to be induced by calcium treatment of keratinocytes in culture (Aeschlimann et al., 1998). Furthermore, a biochemical study has demonstrated high affinity for TGase 5 in using loricrin, involucrin, and SPRs as specific epidermal substrates. Loricrin and SPR3 cross-linking products catalyzed by the action of TGase 1 and TGase 5 were more similar to each other than to loricrin and SPR3 cross-linking products formed by TGase 3 (Candi et al., 2001).

Disorders of cornification and animal models

Discovery of the mechanisms that underlie the development and differentiation of the epidermis of the skin, will pave the way for new insights into not only the biology of the skin, but also its genetic diseases. The disorders of cornification form a heterogeneous group of diseases characterized by abnormal differentiation of the epidermis. These diseases are manifested as ichthyosiform dermatoses and palmoplantar keratodermas (PPKs). The mutations found in human disorders of cornification have provided valuable new information on the functions of different CE components and the proteins that are involved in its assembly. For example, mutations that destroy TGase 1 enzyme activity cause lamellar ichthyosis (Huber et al., 1995; Russell et al., 1995). Affected individuals are born embedded in hyperkeratotic skin (collodion- baby), which later cracks and exposes the underlying tissue, leading to life-threatening dehydration and systemic infections. If the newborn survives the first months of life, the disease eventually develops into large brown plate-like scales, which cover th\e entire body surface. Lamellar ichthyosis affects only the epidermis and its derivates, while other internal epithelia, which also abundantly express TGase 1 but do not form a lipid envelope, are not notably affected. This observation led to the conclusion that in these tissues other TGases possibly can replace the lost protein cross- linking activity of TGase 1, but that in skin membrane-bound TGase 1 is indispensable for ceramide attachment to proteins by ester formation. Considering that a knockout mouse model for TGase 1 also displays loss of water barrier function (Kuramoto et al., 2002; Matsuki et al., 1998), lamellar ichthyosis may be primarily caused by a failed lipid envelope assembly. In addition, there are other forms of autosomal ichthyosis clinically similar to lamellar ichthyosis that are not linked to TGase 1 enzyme deficiency. It is thought that some of these forms might be caused by mutations in genes whose products are necessary for the processing of the TGase 1 zymogen into its active form (Kim et al., 2002). It is also conceivable that mutations in other TGases (TGase 3 and 5) are responsible for these ichthyosiform pathologies. However, TGase 3 knockout mice have revealed embryonic lethality (Kim et al., 2002), which could mean that mutations in this gene probably will not be discovered in humans due to non-viability. Another option is that significantly elevated TGase activity is responsible for some cases of ichthyosis, but this still remains to be investigated (Kim et al., 2002).

Recent analysis of several mouse mutants with defects in structural components of the CE, catalyzing enzymes, lipid processing, transcriptional regulators and the intercellular junctions have highlighted their essential function in establishing the epidermal permeability barrier. However, there is growing evidence for considerable redundancy and/or compensatory mechanisms in CE assembly (Steinert, 2000). Distinct mutations in the major CE protein loricrin cause some unique human genodermatoses that are collectively designated as loricrin keratoderma. Transgenic mice expressing a mutant form of loricrin, due to insertions affecting the carboxyl terminus of the coding sequence, indeed have skin manifestations that are very similar to the clinical presentation of loricrin keratoderma patients (Suga et al., 2000). Surprisingly, the generation of loricrin knockout mice has revealed that there is almost no phenotype (Koch et al., 2000). However, an apparently upregulation of certain members of the SPR family and repetin was noticed. Thus, these and perhaps other proteins seem to have compensated for the absence of loricrin. The lack of a phenotype was not only seen in loricrin knockout mice, since other experiments in which major CE constituents were knocked out gave the same remarkable results. For example, involucrin knockout mice (Djian et al., 2000), and envoplakin knockout mice (Maatta et al., 2001) gave no discernable phenotype, which implies that compensatory mechanisms exist in epithelia that can overcome the absence of these CE proteins. Whereas the absence of some structural CE proteins appears to be well tolerated, the mutation or absence of desmosomal or cytoskeletal proteins in differentiated kcratinocytes often leads to severe pathology and disturbance of barrier function. This is witnessed in mouse models by targeted mutation and/or ablation of desmoplakin (Vasioukhin et al., 2001), desmoglein (Koch et al., 1997), desmocollin-1 (Chidgey et al., 2001), keratin KlO (Porter et al., 1996), keratin K2e (Pitch et al., 2003), and keratin K1 (Bickenbach et al., 1996). Recent experiments have also demonstrated an essential role for the intercellular tight junction components (cell-to-cell adhesion molecules) in epidermal barrier function as is concluded from the severe skin phenotype of claudin-1 knockout mice (Furuse et al., 2002), and an essential function in barrier acquisition for transcriptional regulators was shown by targeted ablation of the transcription factor kruppel-like factor 4 (Klf4) (Segre et al., 1999). Finally, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle, and this will be the topic of the remainder of this review.

Regulated proteolysis in epithelia

Proleascs were long considered to be primarily responsible for intralysosomal protein degradation, mediating "housekeeping" functions in the cell. However, nowadays regulation of proteolytic enzyme activity is well recognized to be essential for cell and tissue homeostasis. Investigations over the last decade have shown that cathepsins, which are lysosomal cystcine proteases, are involved in a variety of physiologic processes such as proenzyme activation, enzyme activation, antigen presentation, hormone maturation, tissue remodeling, and bone matrix resorption (Dickinson, 2002). Recent studies also suggest that cathepsins act as mediators of programmed cell death, and have demonstrated the existence of lysosomal cathepsin-dependent apoptosis pathways (Foghsgaard et al., 2001; Stoka et al., 2001; Turk et al., 2002a). Collectively, cathepsins take part in multiple host systems in both health and disease. Their implication in numerous vital processes and pathologies make them highly attractive targets for drug design, and this class of proteases is regarded as a "druggable" target by leading innovative pharmaceutic industries (Hopkins and Groom, 2002). The regulation of activity of cysteine proteases for correct functioning is a fragile balance of many factors, one of the most crucial being their cognate protease inhibitors. Disturbance of this protease-antiprotease balance at a wrong time and location may have disastrous consequences. The importance of regulated proteolysis in epithelia is well demonstrated by the discovery of the SPINK5 serine protease inhibitor as the defective gene in Netherton syndrome (Chavanas et al., 2000), cathepsin C mutations in Papillon-Lefevre syndrome (Toomes et al., 1999), cathepsin L deficiency in furless mice (Roth et al., 2000), targeted ablation of the serine protease Matriptase/MT-SP1 (List et al., 2002), targeted ablation of the aspartate protease cathepsin D (Egbertset al.,2004), and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice (Hansson et al., 2002). The severe phenotype of cystatin M/E-deficient ichq mice provides another example of a disturbed protease-antiprotease balance that causes faulty differentiation processes in the epidermis and hair follicle (see section "Cystatin M/E").

The serine protease inhibitor Kazal-type 5 (SPINK5) was recently identified as the defective gene in Netherton syndrome (NS, MIM#256500). It was shown that the proteolytic processing and distribution of the protein product of SPINK5, LEKT1, is disturbed in NS patients (Bitoun et al., 2002; Chavanas et al., 2000). NS is a congenital ichthyosis associated with erythroderma, a specific hair shaft defect, and atopic features. It was hypothesized that defective inhibitory regulation by LEKT1 results in increased protease activity in the stratum corneum, accelerated degradation of desmoglein-1, and overdesquamation of corneocytes (Komatsu et al., 2002). Colocalization of LEKT1 transcripts with stratum corneum serine proteases (stratum corneum tryptic enzyme (SCTE) and stratum corneum chymotryptic enzyme (SCCE)) in hair follicles suggested that the regulation of the activity of these proteases by LEKT1 might also affect hair growth and morphogenesis.

Targeted epidermal overexpression of SCCE (also named kallikrein 7) results in pathologic skin changes (Hansson et al., 2002), which suggest that increased activity of proteases present in the skin may indeed play a significant part in skin pathophysiology. Several studies have already shown that SCCE is probably involved in the desquamation process of cornified cells by means of degrading intercellular parts of desmosomes (Egelrud, 2000), and this molecule has shown to play a role in the regulation of hair growth and/or cycling (Ekholm and Egelrud, 1998).

Papillon-Lefevre syndrome (PLS, MIM#24500) is a palmoplantar keratoderma (PPK) with the characteristic clinical features of palmoplantar hyperkeratosis and periodontal destruction. The phenotype of this rare autosomal recessive disorder shows severe early onset periodonlitis, premature tooth loss, and thickening and scaling of the skin of palms and soles. It was found that mutations in the cathepsin C gene are responsible for PLS (Hart et al., 1999; Toomes et al., 1999). Furthermore, cathepsin C mutations were found to he responsible for Haim-Munk syndrome (HMS, MIM#245010), an allelic variant of PLS with some other clinical features (e.g. nail deformities, acroosteolysis) that is only found among members of a small community of Jews from Cochin, India (Hart et al., 2000). Cathepsin C (CTSC) is a lysosomal cysteine protease that has a role in intracellular degradation of proteins and appears to be important for activation of serine proleases in immune/inflammatory cells (Rao et al., 1997). CTSC knockout mice have revealed that cathepsin C plays an essential role in processing and activation of granzymes A and B, which are required for T-cell-mediated cell killing (Pham and Ley, 1999). Therefore, it was suggested that loss-of-function mutations in the CTSC gene could result in an altered immune response to infection in PLS and HMS. However, these CTSC-deficient mice do not resemble the human disorders mentioned above; they develop normally, appeared healthy, and were fertile. Moreover, cathepsin C was proposed to be essential for establishing and maintaining the structural organization of the epidermis of the extremities and the integrity of the tissues surrounding the teeth, and may indirectly contribute to the processing of protei\ns such as keratins (Nuckolls and Slavkin, 1999). The aberrant differentiation of the junctional epithelium that binds the gingival to the tooth surface could possibly be responsible for weakening the mechanical barrier to periodontal pathogens (Toomes et al., 1999).

Cathepsin L (CTSL) is another lysosomal cysteine protease that has shown to be involved in epidermal homeostasis. Cathepsin L- deficient mice develop epidermal hyperplasia, acanthosis, hyperkeratosis, and periodic hair loss (Roth et al., 2000). The phenotype of CTSL-deficient mice is reminiscent of the spontaneous mouse mutants furless (fs) (Green, 1954) and nackt (nkt) (Benavides et al., 1999). Mutation analysis of the ctsl gene of fs mice by Roth et al (2000) revealed that a single missense mutation abolishes the enzymatic activity of CTSL. Mutation analysis of the ctsl gene of nkt mice showed that a deletion in this gene was the cause for CTSL deficiency (Benavides et al., 2001). A study to the effect of CTSL deficiency on hair follicle development and cycling in CTSL knockout mice revealed that this protease is involved in stages of hair follicle morphogenesis associated with kcratinization, cornification, and desquamation. Given the prominent inner root sheet phenotype in this mutant mouse, it was suggested that CTSL might play a role in the processing of trichohyalin, a structural protein of the inner root sheet of the hair follicle (Tobin et al., 2002). Another study has revealed that the processing of profilaggrin to filaggrin and the final steps in filaggrin degradation seem to be impaired in nkt skin, which lead to a delay in transit from stratum granulosum to stratum corneum (Benavides et al., 2002). This group reported that the CTSL-deficient nkt mice display some characteristics of PLS in human patients, suggesting complementary functions for cathepsin L and cathepsin C.

Targeted deletion of the type II transmembrane serine protease Matriptase/MT-SP1 in mice has demonstrated that this protease is required for postnatal survival and has pleiotropic functions in epidermal differentiation, hair follicle development, and thymic homeostasis (List et al., 2002). The loss of epidermal barrier function in these Matriptase/MT-SP1-deficient mice causes neonatal death due to dehydration. Subsequently, it was shown that Matriptase/ MT-SP1 deficiency disturbs lipid matrix formation, CE morphogenesis, and stratum corneum desquamation (List et al., 2003). These observations were linked to defective proteolylic processing of profilaggrin. Proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed a loss of mature filaggrin monomer and filaggrin S-100 protein, whereas no alterations in appearance of a range of other epidermal proteins, or proleolytic processing of epidermal TGases were detected. It was suggested that pericellular, organelle- associated, and cytoplasmic proteases, are all potentially participants in profilaggrin processing during terminal differentiation of keratinocytes. Grafting studies showed that prolonged exposure of Matriptase/MT-SP1-deficient skin to dehydration leads to severe ichthyosis (List et al., 2003). It was concluded that Matriptasc/MT-SP1 plays as a key enzyme in terminal epidermal differentiation.

Cathepsin D (CSTD) is the main aspartatic protease of endolysosomes, and in skin this protease is thought to be associated with the final stage of desquamation (Horikoshi et al., 1999). Recently, it was demonstrated that CTSD is crucially involved in the activation of TGase 1 and in the regulation of CE protein expression during epidermal differentiation (Egberts et al., 2004). CTSD- deficient mice revealed reduced TGase 1 activity and reduced protein levels of the CE proteins involucrin and loricrin. Structural changes in the stratum corneum and the transition of stratum granulosum to stratum corneum were also noticed in these CTSD- deficient mice. The observed similarities between the skin of CTSD- deficient mice, the skin of TGase 1-deficient mice and the human skin disease lamellar ichthyosis were explained by the diminished TGase 1 expression in the skin of CTSD-deficient mice. It was suggested that the skin symptoms (e.g. epidermal hyperkeratosis) are the result of a diminished ability of corneocytes to bind intercellular lipids, caused by the reduced expression of involucrin and loricrin in the stratum corneum.

Regulation of epidermal homcoslasis has been a major topic of our group for the past decade. More in particular, we have studied the role of proteases and their inhibitors, in barrier function and host defense. We have previously cloned and characterized an epithelial- specific protease inhibitor (SKALP/elafin) (Alkemade et al., 1994; Molhuizen et al., 1993; Schalkwijk et al., 1991), which was shown to regulate phagocyte-derived protease activity under inflammatory conditions (Pfundt et al., 1996). Both SKALP/elafin and SLPI, a related protease inhibitor, were found to possess antimicrobial activity and are part of the cutaneous host defense system (Hiemstra et al., 1996; Sallenave, 2002; Simpson et al., 1999; Wicdow et al., 1998; Wingens et al., 1998). In addition to regulation of inflammation and infection, these protease inhibitors (notably SLPI) are probably also involved in maintaining the integrity of dermal connective tissue structures (Kramps et al., 1989; Wingens et al., 1998). These small dual-function proteins appear to be specific for epithelia (Schalkwijk et al., 1999) and have considerably diversified in other species (Zeeuwen et al., 1997).

In order to obtain a systematic and quantitative overview of genes expressed by human epidermal cells we have recently established transcriplomes of human cultured keratinocytes and epidermis via Serial Analysis of Gene Expression (SAGE) (Vclculescu et al., 1995). This technique can be used for quantitative analysis of gene expression, and is not restricted to detection of known genes (as for example the DNA-chip technology). These SAGE libraries (accessible at the NCBI site; www.ncbi.nlm.nih.gov/SAGE) revealed quantitative expression data on numerous proteases and protease inhibitors (JansEn et al., 2001; van RuissEn et al., 2002a, b). Members of the cystatin family were among the most highly expressed genes in human keratinocytes. One of the unidentified high-abundant transcripts of our SAGE libraries was shortly thereafter cloned by two other groups and was named cystatin M or cystatin E, respectively (Ni et al., 1997; Sotiropoulou et al., 1997).

Cystatin M/E and legumain

Cystatins are natural and specific inhibitors of endogenous mammalian lysosomal cysteine proteases (Abrahamson et al., 2003; Turk and Bode, 1991), and exogenous microbial cysteine proteases (Bjorck, 1990). Several studies have indicated that cystatins provide important regulatory and protective functions against uncontrolled proteolysis by cysteine proteases from host, bacterial and viral origin (Bobek and Levine, 1992). A disturbed balance between proteinases and their inhibitors can lead to irreversible damage as found in chronic inflammatory reactions (Henskens et al., 1996) and tumor metastasis (Calkins and Sloane, 1995). In addition to their protease inhibitory activity, some of the cystatins were shown to have antimicrobial activity against bacteria and viruses (Bjorck et al., 1989,1990; Blankenvoorde et al., 1998; Korant et al., 1986). In general, cystatins are competitive, reversible, tight- binding proteins that inhibit cysteine proteases in a M to pM range (Turk et al., 1997). Cystatins are viewed as "emergency" inhibitors, i.e. they rapidly trap a protease without delay and keep it in a stable complex, preventing any additional proteolysis (Bode and Huber, 2000; Turk et al., 2002b). In normal situations such an inhibitor would have a physiological concentration in large excess over the putative protease target(s). These inhibitors are in general physically separated from their target proteases and primarily act on escaped endogenous proteases or exogenous proteases of invading microorganisms.

Cystatin M was initially identified by differential display as a down-regulated mRNA in metastatic breast tumour cells when compared to normal and primary breast tumour cells (Sotiropoulou et al., 1997). Independently, the same molecule was found by others via expressed sequence tag sequencing in cDNA libraries derived from epithelial cells, and was designated cystatin E (Ni et al., 1997). We found that cystatin M/E has a tissue-restricted expression pattern in humans, which is largely limited to cutaneous epithelia (Zeeuwen et al., 2001, 2002a). Cystatin M/E is a 14-kDa secreted protein that shares only 35% homology with the human family-2 cystatins. It has a similar overall structure, such as a signal peptide and two intrachain disulfide bonds, but possesses the unusual characteristic of being a glycoprotein. This protein is only distantly related to the other known family members as reflected by the position of the CST6 gene. It resides on chromosome 11q13 (Stenman et al., 1997), whereas all other family-2 cystatin genes are clustered in a narrow region on chromosome 20p11.2 (Schnittger et al., 1993). We reported that a null mutation in the mouse cystatin M/E gene (Cst6) causes the murine ichq phenotype, which is characterized by neonatal lethality and abnormalities in cornification and desquamation, demonstrating an essential role for cystatin M/E in the final stages of epidermal differentiation (Zeeuwen et al., 2002b). The ichq mouse phenotype includes hypcrkeratosis, abnormally large mitochondria, absence of lamellar granules, and a characteristic keratin expression pattern in the interfollicular epidermis; some of these morphological and biochemical features are similar to abnormalities found in some human heriditable skin disorders, suggesting that the ichq mouse might be a good model for a human ichthyosis (Sundberg et al., 1997). We hav\e cloned and characterized the human cystatin M/E gene and could exclude cystatin M/E as a major gene in a lethal form of ichthyosis in humans (Zeeuwen et al., 2003). Recently, we have shown that cystatin M/E functions in the epidermis as a physiological inhibitor of the novel asparaginyl endopeptidase legumain (Zeeuwen et al., 2004).

Asparaginyl endopeptidase (AEP, EC 3.4.22.34), also called legumain, belongs to clan CD of the cysteine proteases, and has a strict specificity for hydrolysis of asparaginyl bonds. Initially, legumain was only known from plants, as a cysteine protease responsible for the maturation of seed storage proteins and designated vacuolar processing enzyme (VPE) (Hara-Nishimura et al., 1991 ; Kembhavi et al., 1993), and from Schistosoma mansoni (Dallon et al., 1995), but recently it has been described in mammals (human, pig and mouse) (Chen et al., 1997,1998). Legumain was found at low levels in many tissues, but was particularly abundant in kidney. This lysosomal protease is a major enzyme involved in processing a microbial antigen, tetanus toxin, for class II MHC presentation by macrophages (Manoury et al., 1998). Legumain was also shown to be involved in the proteolytic processing of human myelin basic protein, an autoantigen in the pathogenesis of the MHC class II- associated autoimmune disease multiple sclerosis (Beck et al., 2001). Both studies have revealed a (non-redundant) role for legumain in class II MHC-restricted antigen processing (Watts et al., 2003). Biochemical data from other labs have shown that the novel asparaginyl endopeptidase legumain binds to cystatin M/E with high affinity and could possibly represent a physiological target enzyme, although no biological evidence has been provided so far (Abrahamson et al., 2003; Alvarez-Fernandez et al., 1999).

We have recently reported evidence for the mechanism by which deficiency of cystatin M/E leads to disturbed cornification, impaired barrier function and dehydration (Zeeuwen et al., 2004). We have shown that absence of cystatin M/E causes unrestricted activity of its target protease legumain in hair follicles and epidermis, which is the exact location where cystatin M/E is normally expressed. Morphological and functional analysis indicated a faulty stratum corneum and aberrant cornification of the hair follicles in cystatin M/E-deficient mice (Sundberg et al., 1997; Zeeuwen et al., 2002b). We therefore have investigated the expression and distribution of two major components of the stratum corneum, involucrin and loricrin. We demonstrated that soluble monomers and dimers of loricrin, the most abundant stratum corneum protein, are strongly diminished in the epidermis of cystatin M/E-deficient mice. We hypothesized that abnormal cross-linking of loricrin molecules in cystatin M/E-deficient mice is due to aberrant TGase 3 activity and we wondered if the observed unrestricted legumain activity could be involved in TGase 3 processing. Legumain was found to indirectly mediate the activation of TGase 3 zymogen via lysosomal cysteine proteases in vitro, and in cyslatin M/E-deficient mice we found premature processing of TGase 3. Our findings have provided evidence that disturbed cornification in cystatin M/E-deficient mice is caused by abnormalities in loricrin processing, which may be the result of abnormal activation of TGase 3 by cathepsins, the activities of which in turn could be regulated by legumain. We conclude that cystatin M/E and legumain form a functional dyad in skin that regulates correct TGase-dependent formation of the cornified layer.

The cystatin M/E-legumain dyad in epidermal cornification: unresolved issues

Despite the conceptually satisfying nature of the discovery of free (unrestricted) legumain activity at sites where cystatin M/E would normally be present in ichq mice, this fails to address the issue of compartmentalization. Protease inhibitors are often present in relative excess in the extracellular space, to capture activated proteases that have been produced locally or have escaped from the cell. Legumain is present in the late endosomes and has thus far not been demonstrated extracellularly. Cystatin M/E is secreted in the extracellular space, but probably also has a role within the cell. Probably it is present in secretory vesicles but possibly in other compartments as well (lamellar bodies), which has also been shown for the serine protease inhibitor SKALP/elafin (Nakane et al., 2002; Pfundt et al., 1996). As lamellar bodies both contain lipid membranes and acid hydrolases (and possibly proteases and protease inhibitors) intended for extrusion into the intercellular space (Elias et al., 1998; Menon et al., 1992; Rassner et al., 1999), the lamellar body is something of a cross between a secretory granule and a lysosome. When CE assembly is initiated these granules are unable to fuse with the plasma membrane and disintegrate, hereby releasing their ingredients into the cytoplasm. At this moment the cytoplasm of the cell suddenly contains acid hydrolases, and probably proteases and protease inhibitors. Our data on the cystatin M/E knockout mouse show that the absence of cystatin M/E causes free legumain activity intracellularly in the epidermis and hair follicle. This suggests multiple routes of cystatin M/E in the cell and suggests that cystatin M/E could be active both intra- and extracellularly.

Finally, I would like to speculate how cystalin M/E regulates legumain activity in skin, resulting in TGasc 3 processing, as these proteins are not found in the same cellular compartments. A model for the role of legumain, cystatin M/E and lysosomal cysteine proteases in the proteolytic activation of TGase 3 is presented in Figure 1. Steps 1, 2, and 4 in this Figure are adapted from the model for CE assembly proposed by the group of Peter Steinert (Kalinin et al., 2001), which proceeds in three principal stages: i) initiation of CE assembly; ii) formation of the corneocyte lipid envelope; and iii) reinforcement of the CE. At this time we propose that in normal healthy skin, post-translational proteolytic processing of TGase 3 is completed at the final stage of the granular cell layer when cell integrity is lost and proteases are released leading to progressively more TGase 3 enzyme activity. Despite the apparent loss of cell integrity, organization and compartmentalization in the terminally differentiating keratinocyte, this must be a highly ordered and well-orchestrated process. Recent studies have reported increasing evidence that lysosomal proteases play important roles in physiological processes not restricted to lysosomes only (Turk et al., 2002a). Protease activity and regulation outside lysosomes (and possibly lamellar bodies) potentially contributes to propagation of apoptosis, a process that is distinct from terminal differentiation of the epidermis but nevertheless shares some molecular and cellular features, in epidermis there is a balanced regulation of protease activity that, when disturbed, could lead to faulty cornification processes in the epidermis and upper part of the hair follicle. The molecular consequences of misregulated proteolysis in skin diseases have not been identified for most of the disease-causing genes identified sofar (SPINK, cathepsin L, cathepsin C). The evidence that we provide on the role of the legumain-cystatin M/E dyad in the formation of the skin barrier could serve as a paradigm for the molecular pathology that results from disturbance of the protease- antiprolease balance in human skin.

Fig. 1. A model for the role of legumain, cystatin M/E and lysosomal cysteine proteases in the proteolytic activation of TGase3. Step 1,2 and 4 are adapted from a model proposed by Kalinin et al (2001). Step 1: As intracellular Ca^sup 2+^-concentrations rise in suprabasal cells, CE assembly starts by TGase 1-mediated cross-linking of involucrin to itself, envoplakin and pcriplakin, to form a scaffold along the inner surface of the plasma membrane. Step 2: Enrichment of the plasma membrane with ω-hydroxy-ceramides and other barrier lipids, via membrane fusion of lamellar bodies, and TGase 1-catalyzed esterification of ceramide to scaffold proteins (not shown in this figure). Step 3: Coalescence and/or degradation of lysosomal and endosomal compartments; decrease of intracellular pH; limited activation of procathepsins by legumain followed by activation of TGase 3. Step 4: Activated TGase 3 subsequently catalyzes the homodimerization of loricrin and the heterotrimerization of loricrin and SPRs. These are then translocated to the cell periphery where they are cross-linked to the preexisting scaffold by membrane-anchored TGase 1. Deficiency of cystatin M/E, as in the ichq mouse, allows unrestricted legumain activity that indirectly leads to premature or excessive TGase 3 activation, thereby disturbing normal loricrin cross-linking and skin barrier formation. SC, stratum eorneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale.

Acknowledgements. Joost Schalkwijk and Wiljan Hendriks are acknowledged for critical reading of this manuscript, and for their suggestions and contribution related to my PhD project. I thank Ivonne van Vlijmen-Willems and Diana Olthuis for their outstanding technical assistance. This work was financially supported by grant 902.11.092 from the Netherlands Organization for Scientific Research (NWO).

References

Abrahamson, M., Alvarez-Fernandez, M., Nathanson, C. M., 2003. Cystatins. Biochem. Soc. Symp. 70,179-199.

Aeschlimann, D., Koeller, M. K., Allen-Hoffmann, B. L., Mosher, D. F., 1998. Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers. J. Biol. Chem. 273, 3452-3460.

Ahvazi, B., Kim, H. C., Kce, S. H., Neme\s, Z., Steinert, P. M., 2002. Three-dimensional structure of the human transglutaminase 3 enzyme: binding of calcium ions changes structure for activation. EMBO J. 21,2055-2067.

Ahvazi, B., Steinert, P. M., 2003. A model for the reaction mechanism of the transglutaminase 3 enzyme. Exp. Mol. Med. 35, 228- 242.

Akiyama, M., Matsuo, I., Shimizu, H., 2002. Formation of cornified cell envelope in human hair follicle development. Br. J. Dermatol. 146, 968-976.

Alkemade, J. A., Molhuizen, H. O., Ponec, M., Kempenaar, J. ?., Zeeuwen, P. L., de Jongh, G. X, Vlijmen-Willems, I. M., van Erp, P. E., Van de Kerkhof, P. C., Schalkwijk, J., 1994. SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes. J. Cell Sci. 107, 2335-2342.

Alvarez-Fernandez, M., Barrett, A. J., Gerhartz, B., Dando, P. M., Ni, J., Abrahamson, M., 1999. Inhibition of mammalian legumain by some cyslatins is clue to a novel second reactive site. J. Biol. Chem. 274, 19195-19203.

Ando, Y., Imamura, S., Murachi,T., Kannagi, R., 1988. Calpain activates two transglutaminases from porcine skin. Arch. Dermatol. Res. 280, 380-384.

Beck, H.,Schwarz, G., Schroter, C. J., Deeg, M., Baier, D., Stevanovic, S., Weber, E., Driesscn, C, Kalbacher, H., 2001. Cathepsin S and an asparagine-specific cndoproteasc dominate the proteolytic processing of human myclin basic protein in vitro. Eur. J. Immunol. 31, 3726-3736.

Benavides, E, Giordano, M., Fiette, L., Bueno Brunialti, A. L., Martin, P. N., Vanzulli, S., Baldi, P., Schmidt, R., Dosne, P. C, Guenet, J., 1999. Nackt (nkt), a new hair loss mutation of the mouse with associated CD4 deficiency. Immunogenetics 49, 413-419.

Benavides, F., Starost, M. E, Flores, M., Gimenez-Conti, I. B., Guenet, J. L., Conti, C. J., 2002. Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice, a cathepsin L-deficient mutation. Am. J. Pathol. 161, 693-703.

Benavides, F., Venables, A., Poetschke, K. H., Glasscock, E., Rudensky, A., Gomez, M., Martin, P. N., Guenet, J. L., Richie, E. R., Conti, C. X, 2001. The CD4 T cell-deficient mouse mutation nackt (nkt) involves a deletion in the cathepsin E (CtsI) gene. Immunogenetics 53, 233-242.

Bickenbach, J. R., Eongley, M. A., Bundman, D. S., Dominey, A. M., Bowden, P. E., Rothnagel, J. A., Roop, D. R., 1996. A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease cpidermolytic hyperkcratosis. Differentiation 61, 129-139.

Bitoun, E., Chavanas, S., Irvine, A. D., Eonie, E., Bodemer, C., Paradisi, M., Hamel-Teillac, D., Ansai, S., Mitsuhashi, Y., Taieb, A., de Prost, Y., Zambruno, G., Harper, J. I., Hovnanian, A., 2002. Netherton syndrome: disease expression and spectrum of SPINK5 mutations in 21 families. J. Invest. Dermatol. 118, 352-361.

Bjorck, E. 1990. Proteinase inhibition, immunoglobulin-binding proteins and a novel antimicrobial principle. Mol. Microbiol. 4, 1439-1442.

Bjorck, E., Akesson, P., Bohus, M., Trojnar, J., Abrahamson, M., Olafsson, I., Grubb, A., 1989. Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor. Nature 337, 385-386.

Bjorck, L., Grubb, A., Kjellen, E., 1990. Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus. J. Virol. 64,941-943.

Blankenvoorde, M. F., van't Hof, W., Walgreen-Weterings, E., van Steenbergen, T. J., Brand, H. S., Veerman, E. C., Nieuw Amerongen, A. V., 1998. Cystatin and cystatin-derived peptides have antibacterial activity against the pathogen Porphyromonas gingivalis. Biol. Chem. 379, 1371-1375.

Bobek, E. A., Eevine, M. J., 1992. Cystatins - inhibitors of cysteine proteinases. Crit Rev. Oral Biol. Med. 3, 307-332.

Bode, W., Huber, R., 2000. Structural basis of the endoproteinase- protein inhibitor interaction. Biochim. Biophys. Acta 1477, 241 - 252.

Broome, A. M., Ryan, D., Eckert, R. E., 2003. S100 protein subcellular localization during epidermal differentiation and psoriasis. J. Histochem. Cytochem. 51, 675-685.

Calkins, C. C., Sloane, B. F., 1995. Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression. Biol. Chem. Hoppe Seyler 376, 71-80.

Candi, E., Melino, G., Lahm, A., Ceci, R., Rossi, A., Kim, I. G., Ciani, B., Steinerl, P.M., 1998. Transglutaminase 1 mutations in lamellar ichthyosis. Loss of activity due to failure of activation by proteolytic processing. J. Biol. Chem. 273, 13693-13702.

Candi, E., Melino, G., Mei, G., Tarcsa, E., Chung, S. E, Marekov, E. N., Steinert, P. M., 1995. Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein. J. Biol. Chem. 270, 26382- 26390.

Candi, E., Oddi, S., Terrinoni, A., Paradisi, A., Ranalli, M., Finazzi-Agro, A., Melino, G., 2001. Transglutaminase 5 cross-links loricrin, involucrin, and small proline-rich proteins in vitro. J. Biol. Chem. 276, 35014-35023.

Candi, E., Tarcsa, E., Idler, W. W., Kartasova, T., Marekov, L. N., Steinert, P. M., 1999. Transglutaminase cross-linking properties of the small proline-rich 1 family of corntfied cell envelope proteins. Integration with Ioricrin. J. Biol. Chem. 274, 7226-7237.

Chavanas, S., Bodemer, C, Rochat, A., Hamel-Teillac, D., Ali, M., Irvine, A. D., Bonafe, J. L., Wilkinson, J., Taieh, A., Barrandon, Y., Harper, J. I., de Prost, Y., Hovnanian, A., 2000. Mutations in SPINK5, encoding a serine proteasc inhibitor, cause Netherton syndrome. Nat. Genet. 25, 141-142.

Chen, J. M., Dando, P. M., Rawlings, N. D., Brown, M. A., Young, N. E., Stevens, R. A., Hewitt, E., Watts, C, Barren, A. J., 1997. Cloning, isolation, and characterization of mammalian legumain, an asparaginyl cndopeptidase, J. Biol. Chem. 272, 8090-8098.

Chen, J. M., Dando, P. M., Stevens, R. A., Fortunate, M., Barren, A. J., 1998. Cloning and expression of mouse legumain, a lysosomal endopeptidase. Biochem. J. 335, 111-117.

Chidgey, M., Brakebusch, C, Gustafsson, E., Cruchley, A., Hail, C., Kirk, S., Merritt, A., North, A., Tselepis, C, Hewitt, J., Byrne, C, Fassler, R., Garrod, D., 2001. Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation. J. Cell Biol. 155, 821-832.

Dale, B. A., Holbrook, K. A., Kimball, J. R., Hoff, M., Sun, T. T., 1985. Expression of epidermal keratins and filaggrin during human fetal skin development. J. Cell Biol. 101, 1257-1269.

Dale, B. A., Holbrook, K. A., Steinert, P. M., 1978. Assembly of stratum corneum basic protein and keratin filaments in macrofibrils. Nature 276, 729-731.

Dale, B. A., Presland, R. B., Fleckman, P., Kam, E., Resing, K. A. 1993. Phenotypic expression and processing of filaggrin in epidermal differentiation, in: Darmon, M., Blumenberg, M. (Eds.), Molecular Biology of the Skin. The Keratinocyle. Academic Press, Inc., San Diego, pp. 79-106.

Dalton, J. P., Hola-Jamriska, L., Brindley, P. J., 1995. Asparaginyl endopeptidase activity in adult Schistosoma mansoni. Parasitology 111, 575-580.

Dickinson, D. P. 2002. Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease. Crit. Rev. Oral Biol. Med. 13, 238- 275.

DiColandrea, T., Karashima, T., Maatla, A., Watt, F. M., 2000. Subcellular distribution of envoplakin and periplakin: insights into their role as precursors of the epidermal cornified envelope. J. Cell Biol. 151, 573-586.

Djian, P., Easley, K., Green, H., 2000. Targeted ablation of the murine involucrin gene. J. Cell Biol. 151, 381-388.

Donato, R. 2003. Intracellular and extracellular roles of S100 proteins. Microsc. Res. Tech. 60, 540-551.

Eckert, R. L., Yaffe, M. B., Crish, J. E, Murthy, S., Rorke, E. A., Welter, J. F., 1993. Involucrin - structure and role in envelope assembly. J. Invest. Dermatol. 100, 613-617.

Egberts, F., Heinrich, M., Jensen, J. M., Winoto-Morbach, S., Pfeiffer, S., Wickel, M., Schunck, M., Steude, J., Saftig, P., Proksch, E., Schutze, S., 2004. Cathepsin D is involved in the regulation of transglutaminase 1 and epidermal differentiation. J. Cell Sci. 117, 2295-2307.

Egelrud, T. 2000. Desquamation in the stratum corneum. Acta Derm. Venereol. Suppl. (Stockh.) 208, 44-45.

Ekholm, E., Egclrud, T., 1998. The expression of stratum corneum chymotryptic enzyme in human anagen hair follicles: further evidence for its involvement in desquamation-like processes. Br. J. Dermatol. 139, 585-590.

Elias, P. M., Cullander, C., Mauro, T., Rassner, U., Komuves, L., Brown, B. E., Menon, G. K., 1998. The secretory granular cell: the outermost granular cell as a specialized secretory cell. J. Invest. Dermatol. Symp. Proc. 3, 87-100.

Elias, P.M., Menon, O.K., 1991. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv. Lipid Res. 24, 1-26.

Fitch, K. R., McGowan, K. ., van Raamsdonk, C. D., Fuchs, H., Lee, D., Puech, A., Herault, Y., Threadgill, D. W., Hrabe, D. A., Barsh, G. S., 2003. Genetics of dark skin in mice. Genes Dev. 17, 214-228.

Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M., Jaattela, M., 2001. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J. Cell Biol. 153, 999-1010.

Folk, J. E. 1983. Mechanism and basis for specificity of transglutaminase-catalyzed epsilon-(gamma-glutamyl)lysine bond formation. Adv. Enzymol. Relat. Areas Mol. Biol. 54, 1-56.

Folk, J. E., Finlayson, J. S., 1977. The epsilon-(gamma- glutamyl)lysine crosslink and the catalytic role of transglutaminases. Adv. Protein Chem. 31, 1-133.

Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A., Sugitani, Y., Noda, T., Kubo, A., Tsukita, S., 2002. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1 -deficient mice. J. Cell Biol. 156, 1099- 1111.

Garrod, D. R. 1993. Desmosomes and \hemidesmosomes. Curr. Opin. Cell Biol. 5, 30-40.

Green, E. L. 1954. The genetics of a new hair deficiency, furless, in the house mouse. J. Hered. 45, 115-118.

Green, K. J., Jones, J. C., 1996. Desmosomes and hemidesmosomes: structure and function of molecular components. FASEB J. 10, 871- 881.

Grenard, P., Bates, M. K., Aeschlimann, D., 2001. Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z. J. Biol. Chem. 276, 33066-33078.

Griffin, M., Casadio, R., Bergamini, C. M., 2002. Transglutaminases: nature's biological glues. Biochem. J. 368, 377- 396.

Hansson, L., Backman, A., Ny, A., Edlund, M., Ekholm, E., Ekstrand, H. B., Tornell, J., Wallbrandt, P., Wennbo, H., Egelrud, T., 2002. Epidermal overexpression of stratum corneum chymotryptic enzyme in mice: a model for chronic itchy dermatitis. J. Invest. Dermatol. 118, 444-449.

Hara-Nishimura, I., Inouc, K., Nishimura, M., 1991. A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Lett. 294, 89-93.

Hart, T. C., Hart, P. S., Bowden, D. W., Michalec, M. D., Callison, S. A., Walker, S. J., Zhang, Y., Firatli, E., 1999. Mutations of the cathepsin C gene are responsible for Papillon- Lefevre syndrome. J. Med. Genet. 36, 881-887.

Hart, T. C, Hart, P. S., Michalec, M. D., Zhang, Y, Firatli, E., Van Dyke, T. E., Stabholz, A., Zlorogorski, A., Shapira, L., Soskolne, W. A., 2000. Haim-Munk syndrome and Papillon-Lefevre syndrome are allelic mutations in cathepsin C. J. Med. Genet. 37, 88- 94.

Heizmann, C. W., Fritz, G., Schafer, B. W., 2002. S100 proteins: structure, functions and pathology. Front. Biosci. 7, d1356-d1368.

Henskens, Y. M., Veerman, E. C, Nieuw Amerongen, A. V., 1996. Cystatins in health and disease. Biol. Chcm. Hoppe Seylcr 377, 71- 86.

Hiemstra, P. S., Maassen, R. J., Stolk, J., Heinzel-Wieland, R., Stcffens, G. J., Dijkman, J. H., 1996. Antibacterial activity of antilcukoprotcasc. Infect. Immun. 64, 4520-4524.

Hitomi, K., Presland, R. B., Nakayama, T., FJeckman, P., Dale, B. A., Maki, M., 2003. Analysis of epidermal-type transglutaminase (transglutaminase 3) in human stratified epithelia and cultured keratinocytes using monoclonal antibodies. J. Dermatol. Sci. 32, 95- 103.

Hitomi, K., Yamagiwa, Y, Ikura, K., Yamanishi, K., Maki, M., 2000. Characterization of human recombinant transglutaminase 1 purified from baculovirus-infectcd insect cells. Biosci. Biotechnol. Biochem. 64, 2128-2137.

Hohl, D., Mehrel, T., Lichti, U., Turner, M. L., Roop, D. R., Steinert, P. M., 1991. Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins. J. Biol. Chem. 266, 6626-6636.

Hohl, D., Roop, D. 1993. Loricrin, in: Darmon, M., Blumenberg, M. (Eds.), Molecular biology of the skin. The kcratinocyte, Academic Press, Inc., San Diego, pp. 151-179.

Holbrook, K. A., Smith, L. T., Kaplan, E. D., Minami, S. A., Hebert, G.P., Underwood, R. A., 1993. Expression of morphogens during human follicle development in vivo and a model for studying follicle morphogenesis in vitro. J. Invest. Dermatol. 101, 39S-49S.

Hopkins, A. L., Groom, C. R., 2002. The druggable genome. Nat. Rev. Drug Discov. 1, 727-730.

Horikoshi, T., Igarashi, S., Uchiwa, H., Brysk, H., Brysk, M. M., 1999. Role of endogenous cathepsin D-like and chymotrypsin-like proteolysis in human epidermal desquamation. Br. J. Dermatol. 141, 453-459.

Huber, M., Refiler, I., Bernasconi, K., Frenk, E., Lavrijsen, S. P., Ponec, M., Bon, A., Lautenschlager, S., Schordcret, D. F., Hohl, D., 1995. Mutations of keratinocyte lransglutaminase in lamellar ichthyosis. Science 267, 525-528.

Jamora, C., Fuchs, E., 2002. Intercellular adhesion, signalling and the cytoskeleton. Nat. Cell Biol. 4, E101-E108.

Jansen, B. J., van Ruissen, F., de Jongh, G., Zeeuwen, P. L., Schalkwijk, J., 2001. Serial analysis of gene expression in differentiated cultures of human epidermal keratinocytes. J. Invest. Dermatol. 116, 12-22.

Jarnik, M., Simon, M. N., Steven, A. C, 1998. Cornified cell envelope assembly: a model based on electron microscopic determinations of thickness and projected density. J. Cell Sci. 11, 1051-1060.

Jarvincn, M., Rinne, A., Hopsu-Havu, V. K., 1987. Human cystatins in normal and diseased tissues - a review. Acta Histochem. 82, 5 - 18.

Jeon, S., Djian, P., Green, H., 1998. Inability of keratinocytes lacking their specific transglutaminase to form cross-linked envelopes: absence of envelopes as a simple diagnostic test for lamellar ichthyosis. Proc. Natl. Acad. Sci. USA 95, 687-690.

Kalinin, A., Marekov, L. N., Steinert, P. M., 2001. Assembly of the epidermal cornified cell envelope. J. Cell Sci. 114, 3069-3070.

Kalinin, A. E., Kajava, A. V., Steinert, P. M., 2002. Epithelial barrier function: assembly and structural features of the cornified cell envelope. Bioessays 24, 789-800.

Kam,E.,Resing,K. A.,Lim, S. K.,Dale, B. A., 1993. Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2A family. J. Cell Sci. 106, 219-226.

Karashima, T., Watt, F. M., 2002. Interaction of periplakin and envoplakin with intermediate filaments. J. Cell Sei. 115, 5027- 5037.

Kembhavi, A. A., Buttle, D. J., Knight, C. G., Barrett, A. J., 1993. The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays. Arch. Biochem. Biophys. 303, 208-213.

Kirn, H. C., Idler, W. W., Kirn, I. G., Han, J. H., Chung, S. I., Steinert, P.M., 1991. The complete amino acid sequence of the human transglutaminase K enzyme deduced from the nucleic acid sequences of cDNA clones. J. Biol

Source: European Journal of Cell Biology

More News in this Category