Mary A. Longley

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.

Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is a hereditary skin disorder characterized by blistering and a marked thickening of the stratum corneum. In one family, affected individuals exhibited a mutation in the highly conserved carboxyl terminal of the rod domain of keratin 1. In two other families, affected individuals had mutations in the highly conserved amino terminal of the rod domain of keratin 10. Structural analysis of these mutations predicts that heterodimer formation would be unaffected, although filament assembly and elongation would be severely compromised. These data imply that an intact keratin intermediate filament network is required for the maintenance of both cellular and tissue integrity.

Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b

The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b−/− mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, ∼25% of MK6a/b−/− mice survive to adulthood. Remarkably, the surviving MK6a/b−/− mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b−/− mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b−/− follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b−/− animals.

Inducible Activation of Oncogenic K-ras Results in Tumor Formation in the Oral Cavity

Mouse models for cancer represent powerful tools to analyze the causal role of genetic alterations in cancer development. We have developed a novel mouse model that allows the focal activation of mutations in stratified epithelia. Using this system, we demonstrate that activation of an oncogenic K-rasG12D allele in the oral cavity of the mouse induces oral tumor formation. The lesions that develop in these mice are classified as benign squamous papillomas. Interestingly, these tumors exhibit changes in the expression pattern of keratins similar to those observed in human premalignant oral tumors, which are reflective of early stages of tumorigenesis. These results demonstrate a causal role for oncogenic K-ras in oral tumor development. The inducible nature of this model also makes it an ideal system to study cooperative interactions between mutations in oncogenes and/or tumor suppressor genes that are similar to those observed in human tumors. To our knowledge, this is the first reported inducible mouse model for oral cancer.

Keratin 6 is not essential for mammary gland development

IntroductionKeratin 6 (K6) has previously been identified as a marker of early mammary gland development and has also been proposed to be a marker of mammary gland progenitor cells. However, the function of K6 in the mammary gland was not known, so we examined the expression pattern of the protein during both embryonic and postnatal mammary development, as well as the mammary gland phenotype of mice that were null for both K6a and K6b isoforms.MethodImmunostaining was performed to determine the expression pattern of K6a throughout mammary gland development, from the embryonic mammary bud to lactation. Double immunofluorescence was used to co-localize K6 with known markers of mammary gland development. Wild-type and K6ab-null mammary tissues were transplanted into the cleared fat pads of nude mice and the outgrowths were analyzed for morphology by whole-mount staining and for markers of mammary epithelium by immunostaining. Finally, progesterone receptor (PR) and bromodeoxyuridine co-localization was quantified by double immunofluorescence in wild-type and K6ab-null mammary outgrowths.ResultsHere we report that K6 is expressed earlier than described previously, by embryonic day 16.5. K6a is the predominant isoform expressed in the mammary gland, localized in the body cells and luminal epithelial cells but not in the cap cells or myoepithelial cells. Co-localization studies showed that most K6a-positive cells express steroid receptors but do not proliferate. When both the K6a and K6b genes are deleted, mammary gland development appears normal, with similar expression of most molecular markers examined in both the pubertal gland and the mature gland. Loss of K6a and K6b, however, leads to an increase in the number of steroid-receptor-positive cells, and increased co-localization of steroid receptor expression and proliferation was observed.ConclusionAlthough K6a was not essential for mammary gland development, loss of both K6a and K6b resulted in an increase in PR-positive mammary epithelial cells and decreased proliferation after exposure to steroid hormones. There was also increased co-localization of PR and bromodeoxyuridine, suggesting alterations in patterning events important for normal lobuloalveolar development.

Prenatal diagnosis for keratin mutations to exclude transmission of epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma) is an autosomal dominant skin disorder caused by mutations in keratins 1 and 10. We have used direct gene sequencing to ascertain the status of a 15 week fetus of parents whose first child was affected with this disorder. The parents show no clinical signs of epidermolytic hyperkeratosis but were concerned about the possibility of transmitting the disorder due to germline mosaicism. Molecular analysis of the affected son revealed a G to A mutation in codon 156 of keratin 10, resulting in an arginine to histidine substitution within the highly conserved 1A region. Codon 156 has been previously identified as a mutational hot spot and substitutions of this arginine residue are very common in epidermolytic hyperkeratosis patients. Analysis of genomic DNA isolated from amniotic cells showed that the fetus did not harbour this mutation and a healthy infant was eventually born that was unaffected by this disorder.

An asparagine to threonine substitution in the 1A domain of keratin 1: a novel mutation that causes epidermolytic hyperkeratosis

Abstract: Epidermolytic hyperkeratosis (EHK) is a congenital, autosomal dominant disorder of cornification characterized by hyperkeratosis and blister formation. The clinical manifestations are heterogeneous, with respect to the extent of body surface involvement, palmar and plantar hyperkeratosis and the presence of erythroderma. Point mutations in the genes encoding the suprabasal‐specific keratins, keratins 1 and 10 have been identified in EHK patients. The inappropriate amino acid substitutions cause a collapse of the keratin filament network, resulting in cytolysis of the involved keratinocytes. We report a severe case of EHK with a single base pair mutation that causes a threonine for asparagine substitution in residue 8 (N8T) of the 1A region of the keratin 1 protein. This is the region involved in molecular overlaps between neighboring keratin heterodimers. These findings suggest that even conservative amino acid substitutions in overlap regions can cause tonofilament clumping.

Identification of a novel mutation in keratin 1 in a family with epidermolytic hyperkeratosis

Abstract: Epidermolytic hyperkeratosis (EHK) is a hereditary skin disorder typified by blistering due to cytolysis. One in 100,000 individuals is affected by this autosomal‐dominant disease. The onset of the disease phenotype is typically at birth. Histological and ultrastructural examination of the epidermis shows a thickened stratum corneum and tonofilament clumping around the nucleus of suprabasal keratinocytes. Linkage studies localized the disease genes on chromosomes 12q and 17q which contain the type II and type I keratin gene clusters. Recently, several point mutations in the genes encoding the suprabasal keratins, K1 and K10, have been reported in EHK patients. We have investigated a large kindred affected by EHK and identified a new point mutation in the 2B region of keratin 1 (I107T), resulting from a T to C transition in codon 478.

Genetic disorders of keratin: are scarring alopecias a sub-set?

Recent advances have challenged the prevailing view that keratins are merely passive bystanders of keratinocyte biology. With the exciting discovery that three autosomal dominant genetic skin disorders, epidermolysis bullosa simplex (EBS), epidermolytic hyperkeratosis (EHK) and palmoplantar keratoderma (PPK), are in fact disorders of keratins comes the realization that the integrity of the keratin filament network is crucial to the structural integrity of the skin. Since it has been recently established that mutations in keratins K5/K14, K1/K10 and K9 are causative for these keratinocyte disorders, it is very likely that mutations in K6 or in its obligate partner, K16 will result in disease. In order to test this we have produced transgenic mice that express a mutant K6 gene. These mice develop a progressive scarring alopecia at about 6 months of age. Later, the denuded areas developed a keratosis which was prone to infection. Ultrastructural analysis suggests that hair loss is due to the destruction of the outer root sheath. We believe that these mice are models of another keratin disorder.