Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.

We have isolated and characterized a full-length cDNA clone encoding human loricrin. Curiously, this protein displays major differences from the recently described mouse loricrin (Mehrel, T., Hohl, D., Nakazawa, H., Rothnagel, J.A., Longley, M.A., Bundman, D., Cheng, C.K., Lichti, U., Bisher, M.E., Steven, A. C., Steinert, P.M., Yuspa, S.H., and Roop, D.R. (1990) Cell 61, 1103-1112). Although both proteins are glycine-serine-cysteine-rich, the sequences have not been conserved. However, analysis of the sequences reveals a common motif of quasi-peptide repeats of an aliphatic or aromatic amino acid residue followed by several glycine and/or serine and cysteine residues. These sequences are interspersed and flanked by short glutamine- or glutamine/lysine-rich peptides. Thus loricrins consist of a family of cell envelope proteins of highly variable sequences that nevertheless retain common structural elements. We show that unlike all other putative protein components of the cell envelope, loricrins are highly insoluble, due at least in part to cross-linking by disulfide bonds. Furthermore, we have isolated four peptides from purified human cell envelopes that contain recognizable loricrin sequences and which are cross-linked by the N epsilon-(gamma-glutamyl)lysine isodipeptide bond. The presence of such bonds thus affords an explanation for the extraordinary insolubility of loricrin by cross-linking to the cell envelope and can also explain the low steady-state levels of monomeric loricrin in cytoskeletal extracts of epidermis. This study represents the first report of this isodipeptide cross-link in a protein component of the cornified cell envelope. We propose a model for the structure of loricrin in which (i) the unusual glycine-serine-rich sequences adopt a flexible loop conformation, indexed on the recurrent aliphatic residues; (ii) inter- or intramolecular isodipeptide and disulfide cross-links induce or stabilize folding of loricrin so as to form a more compact rosette-like structure; and (iii) the presence of the flexible glycine-rich loops necessarily will impact a flexible character to the cell envelope and entire epithelium.

Identification of a major keratinocyte cell envelope protein, loricrin.

During epidermal cell cornification, the deposition of a layer of covalently cross-linked protein on the cytoplasmic face of the plasma membrane forms the cell envelope. We have isolated and characterized cDNA clones encoding a major differentiation product of mouse epidermal cells, which has an amino acid composition similar to that of purified cell envelopes. Transcripts of this gene are restricted to the granular layer and are as abundant as the differentiation-specific keratins, K1 and K10. An antiserum against a C-terminal peptide localizes this protein in discrete granules in the stratum granulosum and subsequently at the periphery of stratum corneum cells. Immunofluorescence and immunoelectron microscopy detect this epitope only on the inner surface of purified cell envelopes. Taken together, these results suggest that it is a major component of cell envelopes. On the basis of its presumed function, this protein is named loricrin.

Differential keratin gene expression in developing, differentiating, preneoplastic, and neoplastic mouse mammary epithelium.

Two keratins whose expression has been associated with proliferation (K14) and hyperproliferation (K6) in mouse epithelia were detected in normal, preneoplastic, and neoplastic mouse mammary tissues. K6 and K14 keratins were independently expressed in distinct epithelial cell populations in developing mammary anlage. K6 was confined to a small number of mammary epithelial cells associated with the growing end buds and among the proximal luminal epithelium, whereas K14 expression appeared in basally located fusiform cells that correspond to the location of mammary myoepithelial cells. This pattern was maintained in mature glands and through full functional differentiation with the exception that K6-positive cells were only rarely detectable. During lobuloalveolar growth in early pregnancy, K6 and K6/K14 coexpressing cells were observed among the luminal and suprabasal cells in the expanding lobular epithelium. This K6/K14 coexpressing epithelial subset persisted throughout pregnancy, lactation, and involution, albeit in much smaller numbers than observed in early pregnancy. Two patterns of K6 and K14 expression in preneoplastic and neoplastic lesions of mouse mammary glands were induced by various carcinogenic stimuli. In one, increased numbers of K6- or K14-positive cells were present in distinct cellular populations; in the other, coexpression of K6/K14 was found in a large subpopulation of both preneoplastic and neoplastic mammary epithelium. These observations suggest that expression of K6 and K14 keratins in the mouse mammary gland is associated with growth and expansion of specific mammary epithelial cell populations, and as such these keratins may be useful probes with which to identify mammary epithelium-specific primordial cells. In agreement with this possibility, K6/K14 expression was demonstrated within a distinct subset of morphologically distinct luminal mammary epithelial cells that have been reported to possess kinetic properties in vitro consistent with those expected of latent mammogenic stem cells.

Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis.

Monospecific antikeratin antisera and specific complementary DNA probes were used to analyze expression of keratin genes in newborn mouse skin and skin papillomas and carcinomas by indirect immunofluorescence, immunoblotting, and in situ hybridization. Tumors were induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate. Type I epidermal keratin K14 protein (Mr 55,000) is found in all living layers of the newborn skin but is most abundant in the lower strata. K1 (Mr 67,000) and K10 (Mr 59,000) proteins are predominantly suprabasal and K1 is processed in the stratum corneum. Transcripts for K14 were confined largely to the basal cell layer by in situ hybridization. Transcripts for K1 and K10 were highly expressed in suprabasal cells including the granular cell layer. In benign tumors, distribution of K14 protein is similar to that in newborn skin, while the abundance of K1 and K10 appears to be somewhat reduced although the tissue distribution remains suprabasal. Transcription of K14 is aberrant in benign tumors and transcripts persist throughout much of the suprabasal cell layers. Transcripts of K1 and K10 are normally distributed in papillomas but grain density is less intense than in newborn epidermis. Keratin expression in carcinomas is highly disturbed. K14 protein and transcripts are highly expressed in all strata in carcinomas while protein and transcripts for K1 and K10 are essentially absent. These results suggest that papilloma cells fail to respond to or generate signals to regulate K14 expression in the differentiating suprabasal cell layers and may not fully express their suprabasal cell keratins. Carcinomas fail to express suprabasal cell keratins and this is regulated at the transcriptional level. The loss of suprabasal keratin expression may provide a marker for malignant conversion in the mouse skin carcinogenesis model.

Aberrant expression during two-stage mouse skin carcinogenesis of a type I 47-kDa keratin, K13, normally associated with terminal differentiation of internal stratified epithelia.

Specific keratin cDNA probes and monospecific antikeratin antisera were used to analyze mouse epidermis and epidermal tumors for the expression of a type I 47-kDa keratin, K13, normally associated with terminal differentiation of internal stratified epithelia. We demonstrated that this keratin was virtually absent from the entire body epidermis at various stages of development. Also, it was not detected in various forms of acute and chronic epidermal hyperproliferation or in epidermal cells cultured under conditions that favored either cell proliferation or in vitro differentiation. In contrast, K13 was consistently expressed in squamous cell carcinomas of the skin induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate (TPA), whereas papillomas obtained by the same two-stage protocol were distinctly heterogeneous with regard to the expression of this keratin. These findings were true for two different strains of mice (NMRI and Sencar). Papillomas collected from Sencar mice after 12 wk or from NMRI mice after 15 wk of promotion with TPA were either negative for K13 or elicited variable amounts of this keratin. In all cases of positive expression of K13 in tumors, as in normal stratified internal epithelia, both the keratin protein and its mRNA invariably occurred in the differentiating cell compartments. In contrast to what we found in internal stratified epithelia, however, K13 was expressed without its commonly encountered type II 57-kDa partner, K4. Papillomas negative for the K13 protein were also devoid of K13 transcripts. This indicates that the aberrant K13 expression in tumors is regulated at the level of transcription. Our results suggest that K13 may provide a marker for malignant conversion in the mouse two-stage skin carcinogenesis model and may be especially suited for studies of gene expression regulation.

The gene for mouse epidermal filaggrin precursor. Its partial characterization, expression, and sequence of a repeating filaggrin unit.

Filaggrin is an important keratin intermediate filament-associated protein of terminally differentiated mammalian epidermis. Its aberrant expression has been implicated in a number of keratinizing disorders. We have isolated and sequenced a cDNA clone to mouse filaggrin, of 1.479 kilobase pairs, which represents less than 10% of the full-length mRNA estimated by Northern blot analysis to be 17 kilobases long. The cDNA clone delineates a 744-base pair repeat. This encodes a protein of 248 amino acids or 26,330 Da, which is almost identical to the known properties of mouse filaggrin in size, amino acid composition, and charge. Total mouse genomic DNA and the filaggrin gene isolated from a cosmid library were found to contain a super-stoichiometric repeat of the same size. These data support the hypothesis (Haydock, P.V., and Dale, B.A. (1986) J. Biol. Chem. 261, 12520-12525) that filaggrin is initially synthesized as a polyprotein precursor containing many tandem copies. However, our data suggest that the repeating filaggrin units of the precursor are not separated by "large linker" peptides as suggested by these authors. In situ hybridization was used to show that the filaggrin precursor mRNA is located precisely over the granular layer of the epidermis, indicating that expression of this gene is regulated at the transcriptional level as for the differentiation-specific keratin 1 protein. These probes will now permit detailed studies on the regulation of expression of the filaggrin gene.