[Frontiers in Bioscience 17, 1517-1532, January 1, 2012]

Update on the epidermal differentiation complex

Julie Henry1,2,3, Eve Toulza1,2,3,5, Chiung-Yueh Hsu1,2,3, Laurence Pellerin1,2,3, Stefana Balica1,2,3,4, Juliette Mazereeuw-Hautier1,2,3,4, Carle Paul1,2,3,4, Guy Serre1,2,3, Nathalie Jonca1,2,3, Michel Simon1,2,3

1UMR5165 CNRS, Toulouse, France, 2U1056 INSERM, Toulouse, France, 3University of Toulouse, Toulouse, France, 4Department of Dermatology, University Hospital of Toulouse, Toulouse, France, 5Laboratoire Ecosystèmes Lagunaires, UMR5119 CNRS, UM2, IFREMER, IRD, University of Montpellier 2, Montpellier, France

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Discovery of the epidermal differentiation complex
4. Small proline rich and late cornified envelope proteins
4.1. SPRRs
4.2. LCE proteins
5. Family of the S100-fused type proteins
5.1. Profilaggrin
5.2. Hornerin
5.3. Filaggrin2
5.4. Trichohyalin
5.5. Trichohyalin-like 1
5.6. Repetin
6. Conclusions
7. Acknowledgments
8. References

1. ABSTRACT

On human chromosome 1q21, a 2-Mb region called the epidermal differentiation complex comprises many genes encoding structural and regulatory proteins that are of crucial importance for keratinocyte differentiation and stratum corneum properties. Apart from those for involucrin and loricrin, most of the genes are organized in four families: the genes encoding EF-hand calcium-binding proteins of the S100A family, the genes encoding the small proline rich proteins (SPRRs) and the late cornified envelope (LCE) proteins, two families of cornified cell envelope components, and the genes encoding the S100-fused type proteins (SFTPs). This review focuses on the SPRRs, LCE proteins and SFTPs. It describes their structures, their specific functions and, when known, the mechanisms involved in the regulation of their expression. It also highlights their possible involvement in skin diseases.

2. INTRODUCTION

The skin is the largest human organ (~1.8 m� in the adult). At the interface between the body and its outside environment, it provides mechanical protection and defends against the invasion of pathogens, the entry of undesirable chemicals such as allergens or toxins, the disastrous effects of oxidative reagents and ultraviolet (UV) radiation, and uncontrolled loss of water and solutes (Figure 1). These functions, collectively called "barrier functions", are largely carried out by the epidermis, a stratified squamous epithelium, and particularly by its outermost layer, the stratum corneum (also known as the cornified or horny layer) (1-6). Premature newborns with incomplete acquisition of the epidermal barrier functions (2), common inflammatory skin diseases, such as psoriasis and atopic dermatitis (AD), with decreased barrier functions (6-8), and animal models underscore the essential role of the stratum corneum (9, 10).

The stratum corneum is a highly organized and resilient layer approximately 20 �m thick. It is formed by the stacking of the so-called corneocytes, the end-products of keratinocyte terminal differentiation (4). The keratinocyte differentiation is an oriented program of gene expression starting in the basal layer and ending at the surface of the epidermis. During their journey through the spinous and granular layers, the cells turn specific genes on and off, and undergo a series of metabolic and structural changes in sequence (1). For instance, the expression of KRT1 and KRT10, encoding keratin 1 and 10, starts in the spinous layer, whereas filaggrin is detected in the granular layer and lower stratum corneum (11). Finally, the granular keratinocytes undergo a specialized form of programmed cell death called cornification.

Cornification is characterized by the elimination of all organelles and of the nucleus, by the aggregation of intermediate filaments to form an intracellular fibrous matrix, and by the assembly of a resistant protein-shell at the keratinocyte periphery, the cornified cell envelope (CE) (12). At the same time, desmosomes, the intercellular junctional structures, are transformed into corneodesmosomes after the addition of corneodesmosin (13). The CE is a 15-nm thick, highly insoluble, complex protein network, with a monolayer of ω-hydroxyceramides attached to its extracellular surface (12). CE replaces the plasma membrane of terminally differentiated keratinocytes. It results from the formation of very stable ε-(γ-glutamyl)lysine isopeptide bonds between various precursors of a proteinaceous nature, including involucrin and loricrin. This reaction is catalyzed by calcium-dependent enzymes called transglutaminases (EC 2.3.2.13). The CE, together with corneodesmosomes, is critical for the stratum corneum barrier functions since it confers resistance on the layer (12, 13). In addition, it is involved in the structural organization of lipids filling the inter-corneocyte spaces in the form of lamellae after their secretion by lamellar bodies. This extracellular hydrophobic matrix, enriched in cholesterol, ceramides and free fatty acids, plays a major role in the watertightness of the horny layer (14).

A 2-Mb region on the locus 1q21 comprises sixty genes, many of them encoding structural and regulatory proteins that are of crucial importance for keratinocyte differentiation and stratum corneum properties (Figure 2), i.e. intermediate filament-associated proteins (abbreviated IFAP), calcium-binding proteins and proteins involved in the CE formation. The region is known as the "epidermal differentiation complex" (EDC) (15-17).

3. DISCOVERY OF THE EPIDERMAL DIFFERENTIATION COMPLEX

The end of the eighties saw the publication of the first localization in human chromosome 1q21, by fluorescence in-situ hybridization, of genes involved in the late steps of epidermal differentiation. These are the genes encoding loricrin (18), involucrin (19), profilaggrin (20) and trichohyalin (21). From 1992 came the idea that the genes of CE precursors, including loricrin, involucrin and members of the small proline-rich protein (SPRR, formerly SPR) family, may derive from a common gene ancestor (22). The term "functional cluster" of epidermal differentiation genes was then used (23). The name "EDC" was suggested for the first time in 1996 to define this large region of human chromosome 1 and its region of synteny in mouse chromosome 3F1.F2 (15, 24). Since the beginning of the second millennium, the entire sequence of the human genome has been known. This has provided an accurate and definitive view of all the genes clustered in 1q21. Apart from the genes for involucrin and loricrin, most are organized in four families, (a) two families of genes encoding proteins of the CE, namely the late cornified envelope (LCE) proteins and the SPRRs, (b) genes encoding EF-hand calcium-binding proteins of the S100A family, and (c) genes encoding the seven known S100-fused type proteins (SFTPs): profilaggrin, hornerin, filaggrin-2 (also known as ifapsoriasin), repetin, cornulin, trichohyalin and trichohyalin-like 1. Analysis of the GenBank and Ensembl databases has revealed the existence of the EDC throughout evolution, in rodents, marsupials and birds but not in fishes (25). The identification of involucrin, cornulin and SPRR genes in Gallus Gallus indicates that they were present in a common ancestor of birds and mammals >300 million years ago (16). Since the function and properties of involucrin (26), loricrin (12) and S100A proteins (27) are well known, this review will focus on the LCEs, SPRRs and SFTPs, the more recently identified and less well characterized protein families.

4. SMALL PROLINE-RICH AND LATE CORNIFIED ENVELOPE PROTEINS

4.1. SPRRs

The SPRRs form a class of ten keratinocyte-specific closely related human proteins, the expression of which is strongly induced during differentiation and after UV or phorbol ester treatments (28-30). Four groups have been defined on the basis of their amino acid sequences and tandem repetitions (Figure 3): SPRR1A and 1B (also known as cornifin A and B, SPRK and 19 kDa pancornulins), SPRR2 (six different proteins, SPRR2A-2G, SPRR2C being a pseudogene), SPRR3 (a single member formerly called esophagin, cornifin-beta or 22 kDa pancornulin) and SPRR4. These are all short polypeptides (72-89 amino acids long, 8-10 kDa predicted molecular mass) except for SPRR3 (169 amino acids, 18.2 kDa). They all consist of a conserved NH2-terminal domain, three to sixteen 9-8 amino acids long tandemly repeated units that are highly enriched in proline residues, and a conserved COOH-terminal domain (Figure 2) (28-30). They seem to have little or no secondary structure in solution, the central domain consisting of b-turns, and being flexible (31). The head and tail regions of SPRRs are essential for incorporation into the CE, the residues used by transglutaminases 1 and 3 both in vitro and in vivo for crosslinking being mainly localized in these domains. For example in SPRR3, only Gln4, Lys6, Gln17 and Gln25, and Glu158, Lys161 and Lys163 are used (32-34). SPRRs are mainly cross-linked to loricrin, the ratio of loricrin to SPRRs in the CE varying in the epidermis from one body site to another or from the epidermis to another epithelium. This ratio is suspected to be involved in the alteration of the physicochemical properties of the envelopes, including their mechanical properties (12, 34, 35 and references therein). On the other hand, it has been proposed that the central repetitive domain interacts with and coils around the helical solenoid formed by the repetitive central domain of involucrin (31).

The expression of SPRR genes has been carefully analyzed by semi-quantitative RT-PCR experiments (30). All of them except SPRR2F are induced during calcium-mediated in vitro keratinocyte differentiation. In the stratified epithelia tested (from skin, oesophagus, cervix, uterus and ovary), differential expression was observed. The expression of most of them is low or null in the non-squamous epithelia. All but SPRR4 are expressed in the oesophagus and cervix, and all but SPRR3 are expressed in the skin, in either the inter- or intra-follicular epidermis. SPRR4 expression has been essentially detected in the granular keratinocytes. In addition, individual SPRR genes have been shown to respond selectively to in vitro UV irradiation of keratinocytes: SPRR4 and SPRR2G, the most specific genes for epidermis, are also the only ones to be strongly induced by UV light (30). SPRR4 in particular is strongly induced in normal human epidermis after chronic UV exposure and incorporated into fragile CEs, concomitant with a thickening of the stratum corneum (36). An overexpression of SPRR2 also occurs in both bronchial and intestinal epithelium during allergic inflammation (37). These data suggest that SPRRs are part of an adaptive tissue response to environmental stress. In favor of this hypothesis, SPRRs, both as isolated proteins and in purified CEs, seem to have a role in the quenching of reactive oxygen species, resulting in inter- and intramolecular S-S bound formation (38). Again in agreement, some SPRR genes are upregulated in psoriatic lesions (SPRR2B, SPRR2G and SPRR3) and in the epidermis of loricrin-deficient mice (Sprr2a and 2h) (39, 40). This result is evocative of a compensatory mechanism to maintain the skin barrier function.

It should be noted that the Sprr2 group contains four additional proteins in the mouse: Sprr2h-k (Sprr2j gene is probably a pseudogene; compared to other members of the family it presents a frameshift mutation after the codon encoding amino acid 20). Twelve of the fourteen mouse Sprr genes are upregulated in mice with a target ablation of the transcription factor Kruppel-like factor 4. The sequences upstream of the transcription start site of the Sprr2 genes contain common putative binding sites for Fos/Jun (AP-1), Oct, Ets and Kruppel-like zinc-finger transcription factors. These regulatory elements are conserved in human SPRR2 genes (41). The clustering of these genes and their pattern of expression suggest that they may be held together in a single locus to allow coordinated regulation. Interestingly, a conserved non-coding sequence serves as a long-range strong enhancer of the Sprr genes in stress conditions only (42).

In addition, LELP1 (also referenced as late cornified envelope-like proline-rich protein 1) encoded by a gene located on the telomeric side of SPRR2G contains five repeats related to those of SPRR2s but an unrelated head domain and no tail domain (our unpublished observations). Its function is completely unknown.

4.2. LCE proteins

The eighteen genes of this family of proteins span over 320 kb within the EDC, where they are located between the involucrin gene and those encoding the SFTPs (43). The "late cornified envelope" (LCE) generic name resumes the various terms used in the past: LEP for "late envelope protein", SPRL for "small proline-rich like" and xp for "skin specific protein". The LCE genes can be divided into three groups designated LCE1 (seven different LCE1A-F genes), LCE2 (LCE2A-D) and LCE3 (LCE3A-E), based on chromosomal position and protein homology. The LCE1, LCE2 and LCE3 clusters are interspersed with three individual LCE genes (LCE4A, LCE5A and LCE6A), four LCE pseudogenes (LCEP1-4) and several non-LCE genes (NICE-1 or C1orf42, xp33 or C1orf46, xp32 or LEP7 or C1orf68 and hKPRP or C1orf45). The LCE genes generally comprise three exons, the first one being non-coding. The LCE proteins, 80-118 amino acids long (Figure 4), are serine-, glycine- and cysteine-rich polypeptides and, unlike SPRRs, do not contain any tandemly repeated sequences. Most of the LCE genes are expressed late during keratinocyte differentiation, in the granular keratinocytes. They are among the last CE components to be cross-linked to the structure. Using real-time PCR, Jackson et al. (43) found that the LCE1 and LCE2 genes are expressed in the epidermis, particularly LCE1C, LCE2A and LCE2B, whereas the LCE3 genes show low-level, tissue-specific expression in stratified non-cornified epithelia, including those of the oesophagus and tongue. LCE6A expression is highly upregulated during keratinocyte differentiation (44). When the expression of most LCE genes was analyzed in a large panel of human tissues (45), most samples have demonstrated very low to undetectable mRNA levels. Moderate expression has been observed in some oral epithelia and trunk skin. High expression level, in particular of LCE6A, has been noted in plantar skin. The LCE genes responded in a group-specific manner to calcium and UV light, with calcium causing upregulation of LCE2, and UV light causing upregulation of LCE1 and LCE2 (43).

In the mouse, there are only three groups of Lce proteins: eleven Lce1 proteins (Lce1a1, Lce1a2, Lce1b-j, Lce1l and Lce1m), four Lce3 proteins (Lce3a-c and Lce3f) and a unique Lce6a. Lce and Sprr genes are differentially regulated in mice with a target ablation of the Grainyhead-like 3 transcription factor, e.g. Sprr2a, Sprr2d and Lce3c are up regulated whereas Sprr1a and Lce1m are down regulated (46).

Among the several psoriasis susceptibility loci identified in genome-wide search analyses, PSORS4 has been shown to comprise the EDC (47). More recently, a genome-wide scan for copy number variants showed that a deletion of LCE3B and LCE3C (LCE3C_LCE3B-del) was significantly associated with susceptibility to the disease in a cohort of 557 patients from Spain (48). This finding has been replicated in additional cohorts from several European and North American countries (a total of 2,831 cases and a combined p value of 1.38 x 10-8), and also in a family-based study of 2,473 individuals (p=5.4 x 10-4) (48). In addition, a single-nucleotide polymorphism (SNP) in strong linkage disequilibrium with LCE genes has been linked to psoriasis susceptibility in a Chinese cohort (49), suggesting that LCE gene deletion is a general risk factor independent of the ethnic origin. However, this deletion is not associated with AD susceptibility (50). Quantitative expression studies using RT-PCR have shown that the expression of LCE genes is differentially regulated in psoriatic epidermis: all members of the LCE1, 2, 5 and 6 groups are down-regulated whereas all genes of the LCE3 group are induced. After tape stripping, the LCE genes are similarly either down- or up-regulated (45). A reconstructed skin model has been used to show that a combination of the psoriasis-associated proinflammatory cytokines TNF-alpha, IL-1alpha and IL6 induce the expression of LCE3 genes (45). It has been speculated that LCE3 proteins may have a role in repairing a defective and stressed CE (45). In psoriasis, deletion of LCE3B and LCE3C could potentially affect the epidermal barrier repair in response to skin injury. In consequence, environmental antigens may penetrate through the altered stratum corneum, leading to activation of innate immune mechanisms in predisposed individuals.

5. FAMILY OF THE S100-FUSED TYPE PROTEINS

The genes encoding the SFTPs are clustered in a region spanning ~330 kb. They all display the same intron/exon organization, with a small 5' non-coding exon, a small second exon containing the translation start site, and an unusually large third exon encoding the major part of the protein (Figure 5A). The SFTPs also share the same structural organization at the protein level. They all contain an N-terminal domain homologous to S100A proteins which is fused to a large central domain consisting of multiple tandem repeats of one or more sequences, and to a C-terminal region. The latter is specific to each SFTP (Figure 5B). Except for the S100 domain (Figure 5C), the amino acid sequences of the SFTPs are not really related. Like S100A proteins, the N-terminal region of SFTPs comprises two EF-hand calcium-binding domains. In vitro overlay assays with radioactive calcium have been performed to test for the functionality of profilaggrin, trichohyalin, repetin, and cornulin EF-hands sites. The results clearly demonstrate that they bind calcium in a reversible manner (51, 52). This property has also been shown for the S100 domain of mouse filaggrin-2 (53). The precise function of this N-terminal region is as yet unknown, although its involvement in the regulation of intracellular calcium levels and terminal differentiation program may be suspected. The central repetitive domain is believed to impart their specific functions to each of the proteins. However, the repetitive domain of hornerin and filaggrin-2 exhibit the highest sequence homology with filaggrin (41.5% and 42% respectively). In addition, the amino acid compositions of the three proteins are well conserved, with high levels of serine, glycine, arginine, histidine and glutamine, these 5 amino acids accounting for more than 70% of the global composition of the three proteins (Table 1). This suggests that their functions may also be conserved (see 4.1 to 4.3). Finally, the role if any of the C-terminal domain of SFTPs remains to be discovered. Another common feature of the SFTPs is their specific expression in the stratified cornified epithelia and/or the hair follicles.

5.1. Profilaggrin

Profilaggrin is the best characterized and the most studied of the SFTPs. Since many excellent reviews have already been published, e.g. see (11, 54-56), we only mention the major points concerning this protein here. Profilaggrin (400 kDa in human) consists of 10-12 tandem repeats of filaggrin joined by a short hydrophobic linker peptide (7 amino acids long), and flanked by two truncated repeats and by NH2- and COOH-unique terminal domains (Figure 5B) (57). It is synthesized by granular keratinocytes, where it is stored in the cytoplasmic keratohyalin granules as a phosphorylated precursor. Several kinases including casein kinase II are suspected to be involved in profilaggrin phosphorylation (54). In rodents, profilaggrin is formed by at least twenty identical filaggrin subunits (58). During cornification, profilaggrin is dephosphorylated and proteolytically processed into mature basic filaggrin monomers (324 amino acids; 37 kDa in human). The S100-related amino-terminal domain released is translocated to the nucleus of transitional cells, thanks to a functional bipartite nuclear localization signal (59). At the same time, filaggrin associates with intermediate filaments to promote the formation of macrofibrils and the fibrous matrix (60). For this reason, filaggrin has been classified as an intermediate filament associated protein. In the lower stratum corneum, deimination of filaggrin arginyl-residues by peptidylarginine deiminases types 1 and 3 (PAD1 and PAD3) results in the dissociation of filaggrin from the matrix (61-63) and promotes degradation of the protein into free amino acids. These are important for the production of the so-called natural moisturizing factor, for retaining water in the stratum corneum (in particular pyrollidone carboxylic acid derived from glutamine), and for UV protection (in particular urocanic acid derived from histidine) (64, 65). Several proteases, mainly caspase 14, calpain 1 and bleomycin hydrolase, are involved in this breakdown (66, 67). Surprisingly enough, the amino acid sequences of filaggrin subunits may display up to 40% variation; for example, when the full-length cDNA of a single individual was sequenced for the first time, 100 of 324 (31%) amino acids were shown to be variable (57). Many of the mutations are conservative but 33% induce changes in charge. This partly explains the multiple isoelectric variants of filaggrin observed after 2D-gel electrophoresis (68). This also shows that a precisely defined sequence of amino acids is not necessary for the functions of filaggrin. It is the global composition of the protein that is important. Indeed, a regular distribution of positively charged amino acids is necessary for the interactions of filaggrin with keratins, and the composition is in relation to the behavior of filaggrin, giving rise to a mixture of free amino acids and their derivatives. In addition, part of filaggrin is also incorporated in CEs, together with some molecules of keratins (69, 70). Therefore the fibrous corneocyte matrix and CE are linked together by covalent linkages. This may contribute to the high mechanical resistance of corneocytes and the cornified layer.

The importance of filaggrin in skin homeostasis was recently demonstrated when nonsense mutations of its gene (FLG) were shown to be responsible for ichthyosis vulgaris and to be a major risk factor for the development of AD, asthma associated with AD as well as systemic allergies (71-73, for reviews see 55, 56, 74, 75 and references therein). Filaggrin deficiency in the patient epidermis leads to dry and scaly skin inducing a high trans-epidermal water loss and entry of allergens into the epidermis where they trigger the inflammatory response. This has been a sort of revolution in our understanding of atopic diseases, and induced a new paradigm for therapy, aiming to repair the epidermal barrier.

5.2. Hornerin

Human HRNR gene encodes hornerin (also known as S100A18), a protein of 2850 amino acids with a predicted molecular mass of 282 kDa and a predicted isoelectric point ≈10. The hornerin large central domain consists of six tandem repeated basic subunits (468�2 amino acids), each being divided into 3 segments A, B and C (Figure 5B) (76).

Honerin has been first identified in the mouse where it was shown to be expressed in the epidermis and the cornified epithelia, including those of the tongue, forestomach and esophagus (77, 78). In humans, although conflicting results were first published, it is now established that HRNR gene is expressed in healthy epidermis. Hornerin has been detected in the granular and cornified layers of the epidermis in skin biopsies from different body sites, including head, trunk, leg, hands and feet (79, 80). In the stratum granulosum, hornerin is present in the cytoplasm of the upper keratinocytes at the periphery of keratohyalin granules, surrounding filaggrin molecules uniformly distributed within these aggregates (78-80). This suggests that hornerin is synthesized later than profilaggrin during keratinocyte differentiation. In agreement with this, the lowest granular keratinocytes have been shown to contain profilaggrin but not hornerin. In the stratum corneum, while filaggrin is mainly detected in the intracorneocyte fibrous matrix of only the lower corneocytes, hornerin is detected at the periphery of all corneocytes, from the lowest to the most superficial ones (80). Human epidermal hornerin appears not to be very soluble, since it is extracted only in denaturing and/or reducing conditions. It has been detected as numerous immunoreactive bands corresponding to the full-length protein and to smaller fragments down to 45 kDa, indicating that hornerin, like filaggrin, is produced as a large precursor and then processed by proteolysis during cornification (80).

Based on its localization at the periphery of corneocytes, as highlighted by immunoelectron microscopy, and on its high content in glutamine residues (9.5%), hornerin has been suspected to be a component of CE (80). Immunoelectron microscopy analysis of purified plantar envelopes confirmed this hypothesis and revealed that hornerin was located on the internal face of the structures. The presence of hornerin in CEs has been further evidenced by western blot analysis of the purified envelopes digested with protease V8. Among the three different transglutaminases involved in CE formation, in vitro experiments have suggested that transglutaminase 3 is the isoform responsible for hornerin cross-linking. It has also been shown that hornerin and involucrin colocalize at the periphery of in vitro differentiated primary keratinocytes, although hornerin accumulates there several days after involucrin, suggesting that hornerin is cross-linked to the protein scaffold at a late step in CE formation (80). Hornerin function is probably to reinforce the envelopes and to contribute to the mechanical resistance of the stratum corneum. A role in antimicrobial defense has also been proposed for some hornerin-derived peptides (81). Additional experiments are needed to accurately assess the role of hornerin in the epidermal barrier functions.

Immunofluorescence experiments have revealed a decreased level of hornerin in the cornified layer of involved and uninvolved epidermis of AD patients (n = 5). A reduced expression of hornerin has also been underlined by western blot analysis of epidermal extracts of the same patients (80). Interestingly, a SNP located 6.5 kb downstream of the hornerin-encoding gene has been reported as a susceptibility variant in AD (82). Whether this polymorphism could modify the transcription or stability of HRNR mRNA has not been tested yet. Although these results need to be confirmed using a large cohort of patients and controls, they suggest that abnormalities in hornerin expression could contribute to alterations in the CE properties and therefore to the epidermal barrier defects associated with AD. This hypothesis deserves to be experimentally tested.

In a study published in 2006, HRNR was identified as the candidate gene involved in a case of acute myeloid leukemia with t(1;2) (q21;q37) chromosome translocation. Inappropriate expression of hornerin was suspected to play a role in the development of human malignant diseases (83).

5.3. Filaggrin-2

The human FLG2 gene encodes filaggrin-2, a protein of 2391 amino acids (with a predicted molecular mass of 248 kDa and a predicted pI of 8.45). Filaggrin-2 large central domain contains two types of tandem repeats, all of them being 75-77 amino acids long (Figure 5B). The nine A-type repeats are highly homologous to hornerin subunits (50-77% identity), while the 14 B-type repeats are closer to filaggrin monomers (28-39% identity) (84). FLG2 is mainly expressed in skin from different body sites (44, 84). Similarly to FLG, FLG2 gene expression is probably regulated at the transcriptional level since the level of the corresponding mRNA is dramatically (800 fold) increased in granular compared to basal keratinocytes (44). In agreement, FLG2 expression at the mRNA level is highly increased (~200 fold) in response to calcium-induced keratinocyte differentiation after four days of culture, as well as FLG and HRNR (84). Filaggrin-2 is mainly detected in the granular and lower cornified layers of the epidermis (84, 85). Immunofluorescence double staining has shown that filaggrin-2 is expressed slightly later than profilaggrin during keratinocyte differentiation but nevertheless colocalizes with profilaggrin in keratohyalin granules of the upper granular keratinocytes. In the stratum corneum, filaggrin-2 and filaggrin are detected in the intracorneocyte fibrous matrix of the lower corneocytes, both proteins concomitantly disappearing in the upper cornified layer (84, 85).

Western blot analysis of epidermal protein extracts indicates that filaggrin-2 is synthesized as a large precursor with an apparent molecular weight of 250,000 in SDS-gels. This form probably accumulates in keratohyalin granules since it requires either urea or SDS to be extracted. Filaggrin-2 is then proteolytically processed. Fragments of 130, 45 and 15 kDa have been detected. They correspond to the entire B-type repeat domain, part of this domain, and to part of the N-terminus spacer, respectively (84, 85).

The colocalization of filaggrin and filaggrin-2 in the epidermis, their simultaneous disappearance in the upper stratum corneum, and their similar physicochemical properties and proteolytic processing suggest that filaggrin-2 may be modified by one or several of the enzymes involved in filaggrin catabolism, e.g. PADs, caspase 14 and calpain 1. We have demonstrated that B-type repeats of filaggrin-2 are proteolyzed in vitro by calpain 1 into several peptides. We have further shown that deimination of filaggrin-2 promotes its proteolysis by calpain 1 into numerous small peptides and probably into free amino acids. Calpain 1 cleavage sites have been identified in all B-type repeats of filaggrin-2 but not in the A-type repeats, suggesting a different metabolism for the two domains (85).

The role of filaggrin-2 is still unclear. Related molecular structure and amino acid composition of filaggrin, hornerin and filaggrin-2 (Table 1) indicate that these proteins have similar or complementary functions in the epidermis. First, filaggrin-2 B-type domain is rich in histidine (15%) and glutamine (8%) two essential components of the natural moisturizing factor, and second it may be completely proteolyzed into free amino acids in the upper stratum corneum. Thus filaggrin-2 could contribute, along with filaggrin, to stratum corneum hydration and photoprotection properties. As stated above, another role of filaggrin in the lower cornified layer is the aggregation of intermediate filaments to form the intracellular corneocyte matrix. This is performed through ionic interactions between positive charges of filaggrin and negative charges distributed along the rod domain of keratins (86). The high percentage of basic amino acids (histidine + arginine = 23.4%) in the filaggrin-2 B-type repeat domain suggests this protein is able to associate with intermediate filaments. In agreement with this hypothesis, a recombinant fragment of mouse filaggrin-2 repeats have been shown, in vitro, to interact with and bundle keratin filaments isolated from bovine muzzle epidermis (53). The high homology between the A-type repeats of filaggrin-2 and hornerin subunits and their high content in glutamine (14.9%) suggest they may be component of CEs. Consistently with this hypothesis, two peptides derived from filaggrin-2 spacer-1 have been identified in purified CEs (38). Finally, whether the N-terminal S100-like domain of filaggrin-2 is translocated to the nucleus remains to be tested. Interestingly, this domain contains a potential monopartite nuclear localization signal (residues 100-104).

Comparative proteomic profiling of superficial extracts of the epidermis of atopic patients has revealed that filaggrin-2 is expressed at significantly lower levels in lesional versus non lesional skin, suggesting exacerbation of barrier defects and water loss (87). Since disruption of lipid rafts in keratinocytes evokes typical features of AD, it is interesting in this context to note that FLG2 has been identified as one of the mostly downregulated genes (fold change ~-17; p < 2x10-4) in a transcription profiling analysis after cholesterol depletion (88).

5.4. Trichohyalin

Human trichohyalin (220 kDa) comprises a highly charged central domain that consists of tandem repeated sequences of 23 amino acids. Barely detected in sparce keratinocytes in the granular and cornified layers of the epidermis, and in the filiform papillae of the tongue epithelium, trichohyalin is most abundantly expressed in the inner root sheath cells and in the medulla of the hair follicle (89-91).

Like profilaggrin, trichohyalin initially accumulates as large cytoplasmic electron-dense granules termed trichohyalin granules. Upon terminal differentiation, trichohyalin is deiminated by PADs, which induces granule solubilization (92, 93). After deimination, the fate of trichohyalin appears to be site-dependent. In the medulla, it forms unorganized amorphous deposits. In the inner root sheath, it acts as an intermediate filament-associated protein (92, 94). Its central region, predicted to form a flexible single-stranded α-helical domain, associates with keratin filaments in regular arrays, to produce a rigid structure allowing the reinforcement of the inner root sheath which supports the hair shaft growth (95). More accessible to transglutaminases, the deiminated trichohyalin contributes to CE reinforcement, either by direct homo-oligomerization, or by cross-linking with other envelope precursors, or as an intermediate protein necessary for keratin filament binding to this structure (92, 94). Finally, it is interesting to note that unlike the three SFTPs described above, trichohyalin is not proteolyzed during keratinocyte differentiation (95, 96).

5.5. Trichohyalin-like 1

Human trichohyalin-like 1 (904 amino acids, 99 kDa predicted mass) is the most recently identified member of the SFTP family, and is not much characterized. It is predominantly expressed in the inner root sheath of the hair follicle (98). In vitro assays have shown that trichohyalin-like 1 is a substrate of transglutaminase 1, suggesting its incorporation into CEs. This is consistent with its high glutamine residue content (9.1%). Because a recombinant fragment of trichohyalin-like 1 appeared to display in vitro killing activity against E. coli, it has also been proposed that trichohyalin-like 1 contributes to the antimicrobial properties of the cornified layer (99).

5.6. Repetin

Human repetin is a protein of 784 amino acids with a predicted molecular weight of 91,000. Its central domain contains 28 repeats of 12 amino acids and is very rich in glutamine residues (23.8%) (51). Its murine ortholog is a 1130 amino acid protein with a calculated molecular mass of 130 kDa and pI of 7.7. The central segment of mouse repetin consists of 49 tandem repeats of 12 amino acids (99). Repetin is expressed at very low levels in the interfollicular epidermis, where it is detected closely associated with keratohyalin granules of granular keratinocytes and diffusely distributed in the cytoplasm of transitional cells, but is not detected in the upper corneocytes. Repetin is also detected in the inner root sheath of hair follicles and in the filiform papillae of the tongue. By immunoblot analysis of foreskin extracts, repetin is detected as a double band with an apparent molecular mass of 100 kDa, suggesting that repetin most likely undergoes unknown post-translational modifications but no proteolytic processing during epidermal differentiation (51, 99).

Some biochemical data have demonstrated that repetin is cross-linked in vivo to CE precursors, including trichohyalin, involucrin, SPRR1 and SPRR2, in the inner root sheath of mouse hair follicles (96). Cross-links between repetin and loricrin, SPRR1 and SPRR2 have also been detected in human foreskin (51). This evidences a role for repetin probably as a cross-bridging protein involved in CE formation and in hair follicle integrity. These data are corroborated by its late expression, its high glutamine residue content, and its overexpression in the epidermis of loricrin-deficient mice (39). A strong induction of epidermal repetin expression has been reported in kruppel-like factor 4-null mice (100). This indicates that repetin can be strongly upregulated upon alteration of the epidermal barrier, and suggests that repetin overexpression is involved, like LCE3 and SPRR proteins, in the compensatory mechanisms allowing the organism to repair an altered epidermal barrier.

5.7. Cornulin

Human cornulin gene encodes a protein (also called SEP53) of 495 amino acids, whose central domain consists of 2 repeats of 60 amino acids. Initially described as an oesophagus-specific and cancer-associated protein (101), cornulin expression has also been found in normal scalp and foreskin epidermis. It has been detected in the granular layer of the inter-follicular epidermis and in the inner root sheath of the hair follicles (102). At the ultrastructural level, cornulin is located at the periphery of granular keratinocytes and lower corneocytes, suggesting its involvement in CE formation although its function is still poorly understood. This involvement is supported by the fact that it is a glutamine-rich (14.1%) protein. Interestingly it may be involved in the protection from apoptosis since it attenuates deoxycholic acid-induced cell death implicated in oesophageal reflux disease. It is up-regulated by heat-shock and other stresses. When overexpressed in oral squamous carcinoma cell lines, cornulin negatively regulates cell proliferation by the induction of G1 arrest (103, 104).

Western blot analysis of protein extracts from differentiated primary keratinocytes has revealed an aberrant migration of cornulin in SDS-gels with an apparent molecular mass of 70 kDa (instead of a predicted 54 kDa), suggesting it undergoes post-translational modifications, e.g. N-glycosylation (102). This is in agreement with the in silico prediction of N-glycosylation sites. To date, there is no evidence of proteolytic processing of cornulin in vivo.

In addition, cornulin mRNA level in the skin is decreased in eczema, both in a Der-p2 induced mouse model and in AD patients (105). However, further studies are needed to test whether this contributes to the disease.

6. CONCLUSIONS

Among the 60 different genes of the EDC, many encode components of the CEs: involucrin, loricrin, SPRRs, LCE proteins and some of the S100 family but also most of the SFTPs, i.e. hornerin, trichohyalin, repetin, cornulin and, to a lesser extend, filaggrin. Involucrin and loricrin are the major components and are involved in the initiation of CE assembly and their maturation, respectively. The function of the other proteins is probably to reinforce the envelopes. Their specific expression depends on the tissue type and environmental stimuli. In particular many of them seem to be induced when the epidermal barrier is impaired. Coordinate expression of the EDC genes during embryonic skin development and adult epidermal differentiation suggests a genomic mechanism and common regulatory elements to control their transcription. Non-coding sequences conserved during the evolution and shown to display keratinocyte- and differentiation-specific enhancer activities have been identified throughout the locus (106). Recently, many studies have provided evidence that proteins encoded by genes of the EDC are associated with several common skin disorders. In particular, a deletion of the genes LCE3B and LCE3C has been associated with psoriasis, and loss-of-function mutations in FLG have been shown to cause ichthyosis vulgaris and to predispose to AD, and very recently an extra octapeptide repeat of SPRR3 was identified as a risk factor for eczema (107). A possible pathophysiological implication in skin diseases including ichthyosis of the other proteins encoded by the EDC genes remains to be studied.

7. ACKNOWLEDGMENTS

We would like to apologize to our colleagues for omitting some of their work in this review because of lack of space. The authors were supported in part by the CNRS, the Toulouse III University, the INSERM, the "Société de Recherche Dermatologique", the French Society for Dermatology ("Société Française de Dermatologie"), and Pierre Fabre Dermo-Cosmétique. We acknowledge the support of the COST BM0903 action (skin barrier in atopic dermatitis, SkinBAD).

8. REFERENCES

1. E. Fuchs and S. Raghavan: Getting under the skin of epidermal morphogenesis. Nat Rev Genet 3, 199-210 (2002) 
doi:10.1038/nrg758

2. K. C. Madison: Barrier function of the skin. J Invest Dermatol 121, 231-241 (2003) 
doi:10.1046/j.1523-1747.2003.12359.x

3. P. M. Elias: The epidermal permeability barrier: from the early days at Harvard to emerging concepts. J Invest Dermatol 122, 36-39 (2004) 
doi:10.1046/j.0022-202X.2004.22233.x

4. C. R. Harding: The stratum corneum: structure and function in health and disease. Dermatologic Therapy 17, 6-15 (2004) 
doi:10.1111/j.1396-0296.2004.04S1001.x

5. E. Proksch, J. Brandner and J.-M. Jensen: The skin: an indispensable barrier. Exp Dermatol 17, 1063-1072 (2008) 
doi:10.1111/j.1600-0625.2008.00786.x

6. P. M. Elias: Skin barrier function. Curr Allergy Asthma Rep 8, 299-305 (2008) 
doi:10.1007/s11882-008-0048-0

7. S. Chavanas, C. Bodemer, A. Rochat, D. Hamel-Teillac, M. Ali, A. D. Irvine, J. L. Bonafe, J. Wilkinson, A. Taieb, Y. Barrandon, J. I. Harper, Y. de Prost and A. Hovnanian: Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome. Nat Genet 25, 141-142 (2000) 
doi:10.1038/75977

8. J. Segre: Epidermal barrier formation and recovery in skin disorders. J Clin Invest 116, 1150-1158 (2006) 
doi:10.1172/JCI28521

9. E. Leclerc, A. Huchenq, N. Mattiuzzo, D. Metzger, P. Chambon, N. Ghyselinck, G. Serre, N. Jonca and M. Guerrin: Corneodesmosin gene ablation induces lethal skin barrier disruption and hair follicle degeneration related to desmosome dysfunction. J Cell Sci 122, 2699-2709 (2009) 
doi:10.1242/jcs.050302

10. P. L. J. M. Zeeuwen, T. Cheng and J. Schalkwijk: The biology of cystatin M/E and its cognate target proteases. J Invest Dermatol 129, 1327-1338 (2009) 
doi:10.1038/jid.2009.40

11. R. B. Presland and B. A. Dale: Epithelial structural proteins of the skin and oral cavity: function in health and disease. Crit Rev Oral Biol Med 11, 383-408 (2000) 
doi:10.1177/10454411000110040101

12. E. Candi, R. Schmidt and G. Melino: The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 6, 328-40 (2005) 
doi:10.1038/nrm1619

13. N. Jonca, E. A. Leclerc, C. Caubet, M. Simon, M. Guerrin and G. Serre: Corneodesmosomes and corneodesmosin. Eur J Dermatol 21, 35-42 (2011)

14. J. A. Bouwstra, P. L. Honeywell-Nguyen, G. S. Gooris and M. Ponec: Structure of the skin barrier and its modulation by vesicular formulations. Prog Lipid Res 42, 1-36 (2003) 
doi:10.1016/S0163-7827(02)00028-0

15. D. Mischke, B. P. Korge, I. Marenholz, A. Volz and A. Ziegler: Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ("epidermal differentiation complex") on human chromosome 1q21. J Invest Dermatol 106, 989-992 (1996) 
doi:10.1111/1523-1747.ep12338501

16. D. Marshall, M. J. Hardman, K. M. Nield and C. Byrne: Differentially expressed late constituents of the epidermal cornified envelope. Proc Natl Acad Sci USA 98, 13031-13036 (2001) 
doi:10.1073/pnas.231489198

17. I. Marenholz, M. Zirra, D. F. Fischer, C. Backendorf, A. Ziegler and D. Mischke: Identification of human epidermal differentiation complex (EDC)-encoded genes by substratctive hybridization of entire YACs to a gridded keratinocytes cDNA library. Genome Res 11, 341-355 (2001) 
doi:10.1101/gr.114801

18. K. Yoneda, D. Hohl, O. W. McBride, M. Wang, K. U. Cehrs, W. W. Idler and P. M. Steinert: The human loricrin gene. J Biol Chem 267, 18060-18066 (1992)

19. M. Simon, M. Phillips, H. Green, H. Stroh, K. Glatt, G. Burns and S. A. Latt: Absence of a single repeat from the coding region of the human involucrin gene leading to RFLP. Am J Hum Genet 45, 910-916 (1989)

20. P. V. Haydock and B. A. Dale: The repetitive structure of the profilaggrin gene as demonstrated using epidermal profilaggrin cDNA. J Biol Chem 261, 12520-12525 (1986)

21. J. A. Rothnagel and G.E. Rogers: Trichohyalin, an intermediate filament associated protein of the hair follicle. J Cell Biol 102, 1419-1429 (1986) 
doi:10.1083/jcb.102.4.1419

22. C. Backendorf and D. Hohl: A common origin for cornified envelope proteins? Nat Genet 2, 91 (1992) 
doi:10.1038/ng1092-91

23. A. Volz, B. P. Korge, J. G. Compton, A. Ziegler, P. M. Steinert and D. Mischke: Physical mapping of a functional cluster of epidermal differentiation genes on chromosome 1q21. Genomics 18, 92-99 (1993) 
doi:10.1006/geno.1993.1430

24. I. Marenholz, A. Volz, A. Ziegler, A. Davies, I. Ragoussis, B. P. Korge and D. Mischke: Genetic analysis of the epidermal differentiation complex (EDC) on human chromosome 1q21: chromosomal orientation, new markers, and a 6-Mb YAC contig. Genomics 37, 295-302 (1996) 
doi:10.1006/geno.1996.0563

25. A. Vanhoutteghem, P. Djian and H. Green: Ancient origin of the gene encoding involucrin, a precursor of the cross-linked envelope of epidermis and related epithelia. Proc Natl Acad Sci USA 105, 15481-15486 (2008) 
doi:10.1073/pnas.0807643105

26. R. L. Eckert, J. F. Crish, T. Efimova, S. R. Dashti, A. Deucher, F. Bone, G. Adhikary, G. Huang, R. Gopalakrishnan and S. Balasubramanian: Regulation of involucrin gene expression. J Invest Dermatol 123, 13-22 (2004) 
doi:10.1111/j.0022-202X.2004.22723.x

27. R. L. Eckert, A. M. Broome, M. Ruse, N. Robinson, D. Ryan and K. Lee: S100 proteins in the epidermis. J Invest Dermatol 123, 23-33 (2004) 
doi:10.1111/j.0022-202X.2004.22719.x

28. S. Gibbs, R. Fijneman, J. Wiegant, A. G. van Kessel, P. van De Putte and C. Backendorf: Molecular characterization and evolution of the SPRR family of keratinocyte differentiation markers encoding small proline-rich proteins. Genomics 16, 630-637 (1993) 
doi:10.1006/geno.1993.1240

29. D. Hohl, P. A. de Viragh, F. Amiguet-Barras, S. Gibbs, C. Backendorf and M. Huber: The small proline-rich proteins constitute a multigene family of differentially regulated cornified cell envelope precursor proteins. J Invest Dermatol 104, 902-909 (1995) 
doi:10.1111/1523-1747.ep12606176

30. A. Cabral, P. Voskamp, A. M. Cleton-Jansen, A. South, D. Nizetic and C. Backendorf: Structural organization and regulation of the small proline-rich family of cornified envelope precursors suggest a role in adaptive barrier function. J Biol Chem 276, 19231-19237 (2001) 
doi:10.1074/jbc.M100336200

31. A. E. Kalinin, A. V. Kajava and P. M. Steinert: Epithelial barrier function: assembly and structural features of the cornified cell envelope. Bioessays 24, 789-800 (2002) 
doi:10.1002/bies.10144

32. E. Tarcsa, E. Candi, T. Kartasova, W. W. Idler, L. N. Marekov and P. M. Steinert: Structural and transglutaminase substrate properties of the small proline-rich 2 family of cornified cell envelope proteins. J Biol Chem 273, 23297-23303 (1998) 
doi:10.1074/jbc.273.36.23297

33. P. M. Steinert, E. Candi, E. Tarcsa, L. N. Marekov, M. Sette, M. Paci, B. Ciani, P. Guerrieri and G. Melino: Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein. Cell Death Differ 6, 916-930 (1999) 
doi:10.1038/sj.cdd.4400568

34. E. Candi, E. Tarcsa, W. W. Idler, T. Kartasova, L. N. Marekov and P. M. Steinert: Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins. Integration with loricrin. J Biol Chem 274, 7226-7237 (1999) 
doi:10.1074/jbc.274.11.7226

35. P. M. Steinert, E. Candi, T. Kartasova and L. Marekov: Small proline-rich proteins are cross-bridging proteins in the cornified cell envelopes of stratified squamous epithelia. J Struct Biol 122, 76-85 (1998) 
doi:10.1006/jsbi.1998.3957

36. A. Cabral, A. Sayin, S. de Winter, D. F. Fischer, S. Pavel and C. Backendorf: SPRR4, a novel cornified envelope precursor. J Cell Sci 114, 3837-3843 (2001)

37. N. Zimmermann, M. P. Doepker, D. P. Witte, K. F. Stringer, P. C. Fulkerson, S. M. Pope, E. B. Brandt, A. Mishra, N. E. King, N. M. Nikolaidis, M. Wills-Karp, F. D. Finkelman and M. E. Rothenberg: Expression and regulation of small proline-rich protein 2 in allergic inflammation. Am J Respir Cell Mol Biol 32, 428-435 (2005) 
doi:10.1165/rcmb.2004-0269OC

38. W. P. Vermeij, A. Alia and C. Backendorf: ROS Quenching Potential of the Epidermal Cornified Cell Envelope. J Invest Dermatol 131, 1435-41 (2011) 
doi:10.1038/jid.2010.433

39. P. J. Koch, P. A. de Viragh, E. Scharer, D. Bundman, M. A. Longley, J. Bickenbach, Y. Kawachi, Y. Suga, Z. Zhou, M. Huber, D. Hohl, T. Kartasova, M. Jarnik, A. C. Steven and D. R. Roop: Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein. J Cell Biol 151, 389-400 (2000) 
doi:10.1083/jcb.151.2.389

40. X. Zhou, J. G.Krueger, M. C. Kao, E. Lee, F. Du, A. Menter, W. H. Wong and A. M. Bowcock: Novel mechanisms of T-cell and dendritic cell activation revealed by profiling of psoriasis on the 63,100-element oligonucleotide array. Physiol Genomics 13, 69-78 (2003)

41. S. Patel, T. Kartasova and J. A. Segre: Mouse Sprr locus: a tandem array of coordinately regulated genes. Mamm Genome 14, 140-148 (2003) 
doi:10.1007/s00335-002-2205-4

42. N. Martin, S. Patel and J. A. Segre: Long-range comparison of human and mouse Sprr loci to identify conserved noncoding sequences involved in coordinate regulation. Genome Res 14, 2430-2438 (2004) 
doi:10.1101/gr.2709404

43. B. Jackson, C. M. Tilli, M. J. Hardman, A. A. Avilion, M. C. MacLeod, G. S. Ashcroft and C. Byrne: Late cornified envelope family in differentiating epithelia--response to calcium and ultraviolet irradiation. J Invest Dermatol 124, 1062-1070 (2005) 
doi:10.1111/j.0022-202X.2005.23699.x

44. E. Toulza, N. R. Mattiuzzo, M. F. Galliano, N. Jonca, C. Dossat, D. Jacob, A. de Daruvar, P. Wincker, G. Serre and M. Guerrin: Large-scale identification of human genes implicated in epidermal barrier function. Genome Biol 8(6):R107 (2007) 
doi:10.1186/gb-2007-8-6-r107

45. J. G. Bergboer, G. S. Tjabringa, M. Kamsteeg, I. M. van Vlijmen-Willems, D. Rodijk-Olthuis, P. A. Jansen, J. Y. Thuret, M. Narita, A. Ishida-Yamamoto, P. L. Zeeuwen and J. Schalkwijk: Psoriasis Risk Genes of the Late Cornified Envelope-3 Group Are Distinctly Expressed Compared with Genes of Other LCE Groups. Am J Pathol 178, 1470-1477 (2011) 
doi:10.1016/j.ajpath.2010.12.017

46. Z. Yu, K. K. Lin, A. Bhandari, J. A. Spencer, X. Xu, N. Wang, Z. Lu, G. N. Gill, D. R. Roop, P. Wertz and B. Andersen: The Grainyhead-like epithelial transactivator Get-1/Grhl3 regulates epidermal terminal differentiation and interacts functionally with LMO4. Dev Biol 1, 122-136 (2006) 
doi:10.1016/j.ydbio.2006.07.015

47. F. Capon, S. Semprini, B. Dallapiccola and G. Novelli: Evidence for interaction between psoriasis-susceptibility loci on chromosomes 6p21 and 1q21. Am J Hum Genet 65, 1798-1800 (1999) 
doi:10.1086/302653

48. R. de Cid, E. Riveira-Munoz, P. L. Zeeuwen, J. Robarge, W. Liao, E. N. Dannhauser, E. Giardina, P. E. Stuart, R. Nair, C. Helms, G. Escaramis, E. Ballana, G. Martín-Ezquerra, M. den Heijer, M. Kamsteeg, I. Joosten, E. E. Eichler, C. Lazaro, R. M. Pujol, L. Armengol, G. Abecasis, J. T. Elder, G. Novelli, J. A. Armour, P. Y. Kwok, A. Bowcock, J. Schalkwijk and X. Estivill: Deletion of the late cornified envelope LCE3B and LCE3C genes as a susceptibility factor for psoriasis. Nat Genet 41, 211-215 (2009) 
doi:10.1038/ng.313

49. X. J. Zhang, W. Huang, S. Yang, L. D. Sun, F. Y. Zhang, Q. X. Zhu, F. R. Zhang, C. Zhang, W. H. Du, X. M. Pu, H. Li, F. L. Xiao, Z. X. Wang, Y. Cui, F. Hao, J. Zheng, X. Q. Yang, H. Cheng, C. D. He, X. M. Liu, L. M. Xu, H. F. Zheng, S M. Zhang, J. Z. Zhang, H. Y. Wang, Y. L. Cheng, B. H. Ji, Q. Y. Fang, Y. Z. Li, F. S. Zhou, J. W. Han, C. Quan, B. Chen, J. L. Liu, D. Lin, L. Fan, A. P. Zhang, S. X. Liu, C. J. Yang, P.G. Wang, W. M. Zhou, G. S. Lin, W. D. Wu, X. Fan, M. Gao, B. Q. Yang, W. S. Lu, Z. Zhang, K. J. Zhu, S. K. Shen, M. Li, X. Y. Zhang, T. T. Cao, W. Ren, X. Zhang, J. He, X. F. Tang, S. Lu, J. Q. Yang, L. Zhang, D. N. Wang, F. Yuan, X. Y. Yin, H. J. Huang, H. F Wang, X. Y. Lin and J. J. Liu: Psoriasis genome-wide association study identifies susceptibility variants within LCE gene cluster at 1q21. Nat Genet 41, 205-210 (2009) 
doi:10.1038/ng.310

50. J. G. Bergboer, P. L. Zeeuwen, A. D. Irvine, S. Weidinger, E. Giardina, G. Novelli, M. Den Heijer, E. Rodriguez, T. Illig, E. Riveira-Munoz, L. E. Campbell, J. Tyson, E. N. Dannhauser, G. M. O'Regan, E. Galli, N. Klopp, G. H. Koppelman, N. Novak, X. Estivill, W. H. McLean, D. S. Postma, J. A. Armour and J. Schalkwijk: Deletion of Late Cornified Envelope 3B and 3C genes is not associated with atopic dermatitis. J Invest Dermatol 130, 2057-2061 (2010) 
doi:10.1038/jid.2010.88

51. M. Huber, G. Siegenthaler, N. Mirancea, I. Marenholz, D. Nizetic, D. Breitkreutz, D. Mischke and D. Hohl: Isolation and characterization of human repetin, a member of the fused gene family of the epidermal differentiation complex. J Invest Dermatol 124, 998-1007 (2005) 
doi:10.1111/j.0022-202X.2005.23675.x

52. R. Contzler, B. Favre, M. Huber and D. Hohl: Cornulin, a new member of the "fused gene" family, is expressed during epidermal differentiation. J Invest Dermatol 124, 990-997 (2005) 
doi:10.1111/j.0022-202X.2005.23694.x

53. R. B. Presland, J. A. Rothnagel and O.T. Lawrence: Profilaggrin and the fused S100 family of calcium binding proteins. In Skin Barrier, P. M. Elias and K. R. Feingold (Eds). Taylor and Francis, New York, 111-140 (2006)

54. B. A. Dale, K. A. Resing and R. B. Presland: Keratohyalin granule proteins. In The Keratinocyte Handbook, I. Leigh, B. Lane and F. Watt (Eds). Cambridge University Press, London, 323-350 (1994)

55. J. A. McGrath: Filaggrin and the great epidermal barrier grief. Australasian J Dermatol 49, 67-74 (2008) 
doi:10.1111/j.1440-0960.2008.00443.x

56. S. J. Brown and A. D. Irvine: Atopic eczema and the filaggrin story. Semin Cutan Med Surg 27, 128-137 (2008) 
doi:10.1016/j.sder.2008.04.001

57. S.-Q. Gan, W. McBride, W. W. Idler, N. Markova and P. M. Steinert: Organization, structure, and polymorphisms of the human profilaggrin gene. Biochem 29, 9432-9440 (1990) 
doi:10.1021/bi00492a018

58. B. A. Dale, K. A. Resing and J. D. Lonsdale-Eccles: Filaggrin: a keratin filament associated protein. Ann N Y Acad Sci 455, 330-342 (1985) 
doi:10.1111/j.1749-6632.1985.tb50420.x

59. D. J. Pearton, B. A. Dale and R. B. Presland: Functional analysis of the profilaggrin N-terminal peptide: identification of domains that regulate nuclear and cytoplasmic distribution. J Invest Dermatol 119, 661-669 (2002) 
doi:10.1046/j.1523-1747.2002.01831.x

60. P. M. Steinert, J. S. Cantieri, D. C. Teller, J. D. Lonsdale-Eccles and B. A. Dale : Characterization of a class of cationic proteins that specifically interact with intermediate filaments. Proc Natl Acad Sci U S A 78, 4097-4101 (1981) 
doi:10.1073/pnas.78.7.4097

61. R. Nachat, M.-C. Mechin, H. Takahara, S. Chavanas, M. Charveron, G. Serre and M. Simon: Peptidylarginine deiminase isoforms 1-3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin. J Invest Dermatol 124, 384-393 (2005) 
doi:10.1111/j.0022-202X.2004.23568.x

62. M.-C. Mechin, M. Enji, R. Nachat, S. Chavanas, M. Charveron, A. Ishida-Yamamoto, G. Serre, H. Takahara and M. Simon: The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations. Cell Mol Life Sci 62, 1984-1995 (2005) 
doi:10.1007/s00018-005-5196-y

63. M.-C. Mechin, M. Sebbag, J. Arnaud, R. Nachat, C. Foulquier, V. Adoue, F. Coudane, H. Duplan, A.-M. Schmitt, S. Chavanas, M. Guerrin, G. Serre and M. Simon: Update on peptidylarginine deiminases and deimination in skin physiology and severe human diseases. Intern J Cosmetic Sci 29, 147-168 (2007) 
doi:10.1111/j.1467-2494.2007.00377.x

64. A. V. Rawlings and C.R. Harding: Moisturization and skin barrier function. Dermatol Ther 17, Suppl 1, 43-48 (2004) 
doi:10.1111/j.1396-0296.2004.04S1005.x

65. C. Barresi, C. Stremnitzer, V. Mlitz, S. Kezic, A. Kammeyer, M. Ghannadan, K. Posa-Markaryan, C. Selden, E. Tschachler and L. Eckhart: Increased sensitivity of histidinemic mice to UVB radiation suggests a crucial role of endogenous urocanic acid in photoprotection. J Invest Dermatol 131, 188-194 (2011) 
doi:10.1038/jid.2010.231

66. G. Denecker, E. Hoste, B. Gilbert, T. Hochepied, P. Ovaere, S. Lippens, C. Van den Broecke, P. Van Damme, K. D'Herde, J. P. Hachem, G. Borgonie, R. B. Presland, L. Schoonjans, C. Libert, J. Vandekerckhove, K. Gevaert, P. Vandenabeele and W. Declercq: Caspase-14 protects against epidermal UVB photodamage and water loss. Nat Cell Biol 9, 666-674 (2007) 
doi:10.1038/ncb1597

67. Y. Kamata, A. Taniguchi, M. Yamamoto, J. Nomura, K. Ishihara, H. Takahara, T. Hibino and A. Takeda: A Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids. J Biol Chem 284, 12829-12836 (2009) 
doi:10.1074/jbc.M807908200

68. C. R. Harding and I. R. Scott: Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation. J Mol Biol 170, 651-73 (1983) 
doi:10.1016/S0022-2836(83)80126-0

69. P. M. Steinert and L. N. Marekov: The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope. J Biol Chem 270, 17702-17711 (1995) 
doi:10.1074/jbc.270.30.17702

70. M. Simon, M. Haftek, M. Sebbag, M. Montezin, E. Girbal-Neuhauser, D. Schmitt and G. Serre: Evidence that filaggrin is a component of cornified cell envelopes in human plantar epidermis. Biochem J 317, 173-177 (1996)

71. F. J. Smith, A. D. Irvine, A. Terron-Kwiatkowski, A. Sandilands, L. E. Campbell, Y. Zhao, H. Liao, A. T. Evans, D. R. Goudie, S. Lewis-Jones, G. Arseculeratne, C. S. Munro, A. Sergeant, G. O'Regan, S. J. Bale, J. G. Compton, J. J. DiGiovanna, R. B. Presland, P. Fleckman and I. W. H. McLean: Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nat Genet 38, 337-342 (2006) 
doi:10.1038/ng1743

72. C. N. Palmer, A. D. Irvine, A. Terron-Kwiatkowski, Y. Zhao, H. Liao, S. P. Lee, D. R. Goudie, A. Sandilands, L. E. Campbell, F. J. Smith, G. M. O'Regan, R. M. Watson, J. E. Cecil, S. J. Bale, J. G. Compton, J. J. DiGiovanna, P. Fleckman, S. Lewis-Jones, G. Arseculeratne, A. Sergeant, C. S. Munro, B. El Houate, K. McElreavey, L. B. Halkjaer, H. Bisgaard, S. Mukhopadhyay and W. H. McLean: Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 38, 441-446 (2006) 
doi:10.1038/ng1767

73. A. Sandilands, G. M. O'Regan, H. Liao, Y. Zhao, A. Terron-Kwiatkowski, R. M. Watson, A. J. Cassidy, D. R. Goudie, F. J. Smith, I. W. H. McLean and A. D. Irvine: Prevalent and rare mutations in the gene encoding filaggrin cause ichthyosis vulgaris and predispose individuals to atopic dermatitis. J Invest Dermatol 126, 1770-1775 (2006) 
doi:10.1038/sj.jid.5700459

74. W. H. McLean: The allergy gene: how a mutation in a skin protein revealed a link between eczema and asthma. F1000 Med Rep 3, 2 (2011)

75. M. J. Cork, S. G. Danby, Y. Vasilopoulos, J. Hadgraft, M. E. Lane, M. Moustafa, R. H. Guy, A. L. Macgowan, R. Tazi-Ahnini and S. J. Ward: Epidermal barrier dysfunction in atopic dermatitis. J Invest Dermatol 129, 1892-1908 (2009) 
doi:10.1038/jid.2009.133

76. M. Takaishi, T. Makino, M. Morohashi and N. H. Huh: Identification of human hornerin and its expression in regenerating and psoriatic skin. J Biol Chem 280, 4696-4703 (2005) 
doi:10.1074/jbc.M409026200

77. T Makino, M. Takaishi, M. Morohashi and N. H. Huh: Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin. J Biol Chem 276, 47445-52 (2001) 
doi:10.1074/jbc.M107512200

78. T. Makino, M. Takaishi, M. Toyoda, M. Morohashi and N. H. Huh: Expression of hornerin in stratified squamous epithelium in the mouse: a comparative analysis with profilaggrin. J Histochem Cytochem 51, 485-92 (2003) 
doi:10.1177/002215540305100410

79. Z. Wu, U. Meyer-Hoffert, K. Reithmayer, R. Paus, B. Hansmann, Y. He, J. Bartels, R. Glaser, J. Harder and J. M. Schroder: Highly complex peptide aggregates of the S100 fused-type protein hornerin are present in human skin. J Invest Dermatol 129, 1446-58 (2009) 
doi:10.1038/jid.2008.370

80. J. Henry, C.-Y. Hsu, M. Haftek, R. Nachat, H. D. de Koning, I. Gardinal-Galera, K. Hitomi, S. Balica, C. Jean-Decoster, A.-M. Schmitt, C. Paul, G. Serre and M. Simon: Hornerin is a component of the epidermal cornified cell envelopes. Faseb J 25, 1567-1576 (2011) 
doi:10.1096/fj.10-168658

81. Z. H. Wu, U. Meyer-Hoffert, J. Bartels, Y. He, J. Harder and J. M. Schroder: Antimicrobially active hornerin peptides as protective factors of healthy skin. J Invest Dermatol 127 Suppl. 2, S2-S2 (2007)

82. J. Esparza-Gordillo, S. Weidinger, R. Fölster-Holst, A. Bauerfeind, F. Ruschendorf, G. Patone, K. Rohde, I. Marenholz, F. Schulz, T. Kerscher, N. Hubner, U. Wahn, S. Schreiber, A. Franke, R. Vogler, S. Heath, H. Baurecht, N. Novak, E. Rodriguez, T. Illig, M. A. Lee-Kirsch, A. Ciechanowicz, M. Kurek, T. Piskackova, M. Macek, Y. A.Lee and A. Ruether: A common variant on chromosome 11q13 is associated with atopic dermatitis. Nat Genet 41, 596-601 (2009) 
doi:10.1038/ng.347

83. L. Wang, Y. Y. Wang, Q. Cao, Z. Chen and S. J. Chen: Hornerin gene was involved in a case of acute myeloid leukemia transformed from myelodysplastic syndrome with t(1;2)(q21;q37). Leukemia 20, 2184-2187 (2006) 
doi:10.1038/sj.leu.2404436

84. Z. Wu, B. Hansmann, U. Meyer-Hoffert, R. Gläser and J. M. Schröder: Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family. PLoS One 4, e5227 (2009) 
doi:10.1371/journal.pone.0005227

85. C.-Y. Hsu, J. Henry, A.-A. Raymond, M.-C. Mechin, V. Pendaries, D. Nassar, B. Hansmann, S. Balica, O. Burlet-Schiltz, A.-M. Schmitt, H. Takahara, C. Paul, G. Serre and M. Simon: Deimination of human filaggrin-2 promotes its proteolysis by calpain 1. J Biol Chem in press (2011)  
doi:10.1074/jbc.M110.197400

86. J. W. Mack, A. C. Steven and P. M. Steinert: The mechanism of interaction of filaggrin with intermediate filaments. The ionic zipper hypothesis. J Mol Biol 232, 50-66 (1993) 
doi:10.1006/jmbi.1993.1369

87. C. J. Broccardo, S. Mahaffey, J. Schwarz, L. Wruck, G. David, P. M. Schlievert, N. A. Reisdorph and D. Y. Leung: Comparative proteomic profiling of patients with atopic dermatitis based on history of eczema herpeticum infection and Staphylococcus aureus colonization. J Allergy Clin Immunol 127, 186-193 (2011) 
doi:10.1016/j.jaci.2010.10.033

88. C. Mathay, M. Pierre, M. R. Pittelkow, E. Depiereux, A. F. Nikkels, A. Colige and Y. Poumay: Transcriptional profiling after lipid raft disruption in keratinocytes identifies critical mediators of atopic dermatitis pathways. J Invest Dermatol 131, 46-58 (2011) 
doi:10.1038/jid.2010.272

89. G. E. Rogers, H. W. Harding and I. J. Llewellyn-Smith: The origin of citrulline-containing proteins in the hair follicle and the chemical nature of trichohyalin, an intracellular precursor. Biochim Biophys Acta 495, 159-175 (1977)

90. E. H. Hamilton, R. E. Jr Payne and E. J. O'Keefe: Trichohyalin: presence in the granular layer and stratum corneum of normal human epidermis. J Invest Dermatol 96, 666-672 (1991) 
doi:10.1111/1523-1747.ep12470590

91. E. J. O'Keefe, E. H. Hamilton, S. C. Lee and P. Steinert: Trichohyalin: a structural protein of hair, tongue, nail, and epidermis. J Invest Dermatol 101(Suppl 1):65S-71S (1993) 
doi:10.1111/1523-1747.ep12362866

92. E. Tarcsa, L. N. Marekov, J. Andreoli, W. W. Idler, E. Candi, S. I. Chung and P. M. Steinert: The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases. J Biol Chem 272, 27893-27901 (1997) 
doi:10.1074/jbc.272.44.27893

93. R. Nachat, M.-C. Mechin, M. Charveron, G. Serre, J. Constans and M. Simon: Peptidylarginine deiminase isoforms are differentially expressed in the anagen hair follicles and other human skin appendages. J Invest Dermatol 125, 34-41 (2005) 
doi:10.1111/j.0022-202X.2005.23763.x

94. P. M. Steinert, D. A. Parry and L. N. Marekov: Trichohyalin mechanically strengthens the hair follicle: multiple cross-bridging roles in the inner root shealth. J Biol Chem 278, 41409-41419 (2003) 
doi:10.1074/jbc.M302037200

95. S. C. Lee, I. G. Kim, L. N. Marekov, E. J. O'Keefe, D. A. Parry and P. M. Steinert: The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein. J Biol Chem 268, 12164-12176 (1993)

96. P. M. Steinert, T. Kartasova and L. N. Marekov: Biochemical evidence that small proline-rich proteins and trichohyalin function in epithelia by modulation of the biomechanical properties of their cornified cell envelopes. J Biol Chem 273, 11758-11769 (1998) 
doi:10.1074/jbc.273.19.11758

97. Z. Wu, T. Latendorf, U. Meyer-Hoffert and J.-M. Schroder: Identification of trichohyalin-like 1, an S100 fused-type protein selectively expressed in hair follicles. J Invest Dermatol (2011) doi:10.1038/jid.2011.71

98. Z. H. Wu, T. Latendorf, U. Meyer-Hoffert, B. Hansmann and J.-M. Schroeder: Trichohyalin-like 1, a novel human hair follicle-specific S100 fused-type protein. J Invest Dermatol 130 Suppl. 2, S91 (2010)

99. P. Krieg, M. Schuppler, R. Koesters, A. Mincheva, P. Lichter and F. Marks: Repetin (Rptn), a epidermal differentiation protein. Genomics 43, 339-348 (1997) 
doi:10.1006/geno.1997.4818

100. J. A. Segre, C. Bauer and E. Fuchs: Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22, 356-360 (1999) 
doi:10.1038/11926

101. Z. Xu, M.-R.Wang, X. Xu, Y. Cai, Y.-L. Han, K.-M. Wu, J. Wang, B.-S. Chen, X.-Q. Wang and M. Wu: Novel human esophagus-specific gene C1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer. Genomics 69, 322-330 (2000) 
doi:10.1006/geno.2000.6344

102. R. Contzler, B. Favre, M. Huber and D. Hohl: Cornulin, a new member of the "fused gene" family, is expressed during epidermal differentiation. J Invest Dermatol 124, 990-997 (2005) 
doi:10.1111/j.0022-202X.2005.23694.x

103. J. Darragh, M. Hunter, E. Pohler, L. Nelson, J. F. Dillon, R. Nenutil, B. Vojtesek, P. E. Ross, N. Kernohan and T. R. Hupp: The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid-mediated injury. FEBS J 273, 1930-1947 (2006) 
doi:10.1111/j.1742-4658.2006.05206.x

104. L. Nelson, S. Anderson, A. L. Archibald, S. Rhind, Z. H. Lu, A. Condie, N. McIntyre, J. Thompson, R. Nenutil, B. Vojtesek, C. B. Whitelaw, T. J. Little and T. Hupp: An animal model to evaluate the function and regulation of the adaptively evolving stress protein SEP53 in oesophageal bile damage responses. Cell Stress Chaperones 13, 375-385 (2008) 
doi:10.1007/s12192-008-0037-1

105. A. Lieden, E. Ekelund, I. C. Kuo, I. Kockum, C. H. Huang, L. Mallbris, S. P. Lee, L. K. Seng, G. Y. Chin, C. F. Wahlgren, C. N. Palmer, B. Bjorksten, M. Stahle, M. Nordenskjold, M. Bradley, K. Y. Chua and M. D'Amato: Cornulin, a marker of late epidermal differentiation, is down-regulated in eczema. Allergy 64, 304-311 (2009) 
doi:10.1111/j.1398-9995.2008.01856.x

106. C. de Guzman Strong, S. Conlan, C. B. Deming, J. Cheng, K. E. Sears and J. A. Segre: A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis. Hum Mol Genet 19, 1453-1460 (2010) 
doi:10.1093/hmg/ddq019

107. I. Marenholz, V. A. Gimenez Rivera, J. Esparza-Gordillo, A. Bauerfeind, M.-A. Lee-Kirsch, A. Ciechanowicz, M. Kurek, T. Piskackova, M. Macek and Y.-A. Lee: Association screening in the epidermal differentiation complex (EDC) identifies an SPRR3 repeat number variant as a risk factor for eczema. J Invest Dermatol (2011) doi: 10.1038/jid.2011.90

Abbreviations: AD, atopic dermatitis; CE, cornified cell envelope; EDC, epidermal differentiation complex; LCE, late cornified envelope protein; SFTP, S100-fused type proteins; SNP, single nucleotide polymorphism; SPRR, small proline rich proteins; UV, ultraviolet

Key Words: Keratinocytes, Differentiation, Filaggrin, Atopic Dermatitis, Psoriasis, Epidermis, Cornified Cell Envelopes, Review

Send correspondence to: Michel Simon, UMR5165 CNRS, Toulouse, France, Tel: 335-6115-8427, Fax: 335-6149-9036, E-mail:michel.simon@udear.cnrs.fr