The epidermal cornified cell envelope (CE) is a complex protein–lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing ∼70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor−/− mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4–5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor−/− mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of “small proline rich proteins”, and repetin, a member of the “fused gene” subgroup of the S100 gene family.

The cornified cell envelope (CE) is a specialized structure involved in barrier function in stratified squamous epithelia, and is assembled by transglutaminase crosslinking of several proteins. Murine forestomach epithelium undergoes particularly rigorous mechanical trauma, and these CEs contain the highest known content of small proline-rich proteins (SPRs). Sequencing analyses of these CEs revealed that SPRs function as cross-bridgers by joining other proteins by use of multiple adjacent glutamines and lysines on only the amino and carboxyl termini and in functionally non-polar ways. Forestomach CEs also use trichohyalin as a novel cross-bridging protein. We performed mathematical modeling of amino acid compositions of the CEs of mouse and human epidermis of different body sites. Although the sum of loricrin ؉ SPRs was conserved, the amount of SPRs varied in relation to the presumed physical requirements of the tissues. Our data suggest that SPRs could serve as modifiers of a composite CE material composed of mostly loricrin; we propose that increasing amounts of cross-bridging SPRs modify the structure of the CE, just as cross-linking proteins strengthen other types of tissues. In this way, different epithelia may use varying amounts of the cross-bridging SPRs to alter the biomechanical properties of the tissue in accordance with specific physical requirements and functions.

By screening of a cDNA library made on mRNA isolated from UV-irradiated human epidermal keratinocytes for sequences whose relative concentration increases in the cytoplasm after irradiation, we have isolated 40 cDNA clones (T. Kartasova, B. J. C. Cornelissen, P. Belt, and P. van de Putte, Nucleic Acids Res. 15:5945-5962, 1987). Here we describe two distinct groups of cDNA clones which do not cross-hybridize to each other but nevertheless encode proteins of very similar primary structure. These polypeptides are small (8 to 10 kilodaltons) and exceptionally rich in proline, cysteine, and glutamine and have similar repeating elements not found elsewhere. The new proteins were designated sprl and sprIl (small, proline rich). The presence of prolines and cysteines suggests that they may be either structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins. Southern blot and sequence analyses of the cDNAs indicate that at least the sprII group of clones represents a family of related genes. The nucleotide sequence of both groups seems to be conserved upon evolution. The level of mRNAs corresponding to the two groups of cDNAs is increased in the cytoplasm of human epidermal keratinocytes after both UV irradiation and treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate. UV light is well known for its carcinogenic effect on human skin. Photoproducts which appear in DNA after UV exposure may be mutagenic and can lead to skin malignancy via mutation induction. To gain a better understanding of the interference of UV light with normal cellular functions and of the cellular response to a challenge with UV light, we have initiated a study of the UV-inducible regulation of gene expression in cultured human epidermal keratinocytes by cloning genes which are induced upon UV irradiation. Keratinocytes derived from the skin epidermis are functionally specialized to protect the organism against the damaging effects of different external agents, including UV light. Therefore, they represent an attractive system to study the influence of UV irradiation on gene expression.A cDNA library was constructed from poly(A)+ RNA isolated after UV irradiation. This library was differentially screened with cDNA probes synthesized on poly(A)+ RNA isolated from either UV-irradiated or nonirradiated cells (9). Here we describe two groups of clones of which the deduced proteins show a striking homology at the level of both their amino acid composition and structural organization. For both groups a relative increase in the cytoplasmic mRNA level was detected on Northern (RNA) blots after UV irradiation and treatment with either 4-nitroquinoline 1-oxide (4-NQO) or the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). MATERIALS AND METHODSCell culture and treatment with UV, TPA, and 4-NQO. A primary culture of epidermal keratinocytes was established from human foreskin by the method of Rheinwald and Green (21) with some modifications described by Ponec et al. (20 Kerat...

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no ␣ or ␤ structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.A large body of recent amino acid sequencing data has demonstrated that members of the three known classes of small proline-rich (SPR) 1 proteins serve as constituents of the cornified cell envelope (CE) of stratified squamous epithelia (1-5), which is a specialized structure essential for barrier function (6 -9). The SPR1 (two members), SPR2 (8 -11 members) and SPR3 (one member) are assembled from a common plan (10 -14). Their amino (head) and carboxyl (tail) domains are enriched in Gln and Lys residues and consist of sequences that have been conserved between each member of an SPR class but differ between classes. These flank a central domain consisting of a series of Pro-rich peptide repeats of sequences that likewise have been conserved between members of a class but vary between classes. The SPRs become cross-linked to themselves and other CE structural protein constituents by both disulfide bonds and N ⑀ -(␥-glutamyl)lysine or N 1 ,N 8 -bis(␥-glutamyl)-spermidine isopeptide bonds formed by transglutaminases (TGases), resulting in an insoluble macromolecular protein complex ideal for barrier function (6 -9). Moreover, for reasons that are not yet understood, individual SPR family members are differentially expressed in highly variable ways in many different types of epithelia (10 -31).Examination of the way in which the SPRs were cross-linked to protein partners in human and mouse CE preparations revealed several novel features of their properties and functions (1-5). First, only head and tail domain Gln and Lys residues were used for cross-linking. Indeed, multiple ad...