E. Birgitte Lane

New consensus nomenclature for mammalian keratins

Keratins are intermediate filament–forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species.

INTERMEDIATE FILAMENTS IN DISEASE

Intermediate filaments are major structural proteins encoded by a large multigene family. Their tissue-specific expression makes them important in studies of development, differentiation and pathology. Most intermediate filaments are keratins; recent discoveries of keratin mutations in a range of genetic skin disorders have clarified their role as providing essential structural support for cells in different physical settings.

Phenotypes, genotypes and their contribution to understanding keratin function

A large number of mutations in keratin genes underlie inherited tissue fragility disorders of epithelia. The genotype-phenotype correlations emerging from these studies provide a rich source of information about the function of keratins that would have taken decades to achieve by a purely transgenic approach. Human disease studies are being supplemented by engineered mouse mutant studies, which give access to the effects of genetic alterations unlikely to occur naturally. Evidence is emerging that the great diversity of keratins might be required to enable cells to adapt their structure in response to different signalling pathways.

Keratin expression in basal cell carcinomas.

The keratin phenotype of 15 cases of basal cell carcinoma was assayed immunohistochemically using a panel of monospecific antibodies to single keratin polypeptides. Whilst tumour tissue strongly expressed primary keratins 5 and 14 (normally synthesized in basal keratinocytes) no expression of secondary keratins 1 and 10 (characteristic of skin‐type differentiation) was detected. Keratin 17, characteristic of the outer hair root sheath, was strongly expressed in all tumours. Keratin 19 was also normally expressed in parts of the hair follicle and was detected in four cases. The ‘high cell turnover’ keratin 16 was frequently induced in the overlying epidermis, but was rare within tumour tissue. No expression of simple epithelial keratins 8 and 18 was detected. Whilst the keratin phenotype of tumour cells is similar to that of basal cells within part of the hair root sheath, in keeping with suggestions of a follicular origin for basal cell carcinomas, the findings are also compatible with an origin from interfollicular pluripotent stem cells differentiating towards follicular structures.

‘See-saw’ expression of microRNA-198 and FSTL1 from a single transcript in wound healing

Post-transcriptional switches are flexible effectors of dynamic changes in gene expression. Here we report a new post-transcriptional switch that dictates the spatiotemporal and mutually exclusive expression of two alternative gene products from a single transcript. Expression of primate-specific exonic microRNA-198 (miR-198), located in the 3′-untranslated region of follistatin-like 1 (FSTL1) messenger RNA, switches to expression of the linked open reading frame of FSTL1 upon wounding in a human ex vivo organ culture system. We show that binding of a KH-type splicing regulatory protein (KSRP, also known as KHSRP) to the primary transcript determines the fate of the transcript and is essential for the processing of miR-198: transforming growth factor-β signalling switches off miR-198 expression by downregulating KSRP, and promotes FSTL1 protein expression. We also show that FSTL1 expression promotes keratinocyte migration, whereas miR-198 expression has the opposite effect by targeting and inhibiting DIAPH1, PLAU and LAMC2. A clear inverse correlation between the expression pattern of FSTL1 (pro-migratory) and miR-198 (anti-migratory) highlights the importance of this regulatory switch in controlling context-specific gene expression to orchestrate wound re-epithelialization. The deleterious effect of failure of this switch is apparent in non-healing chronic diabetic ulcers, in which expression of miR-198 persists, FSTL1 is absent, and keratinocyte migration, re-epithelialization and wound healing all fail to occur.

Multiple self-healing squamous epithelioma is caused by a disease-specific spectrum of mutations in TGFBR1

Multiple self-healing squamous epithelioma (MSSE), also known as Ferguson-Smith disease (FSD), is an autosomal-dominant skin cancer condition characterized by multiple squamous-carcinoma–like locally invasive skin tumors that grow rapidly for a few weeks before spontaneously regressing, leaving scars. High-throughput genomic sequencing of a conservative estimate (24.2 Mb) of the disease locus on chromosome 9 using exon array capture identified independent mutations in TGFBR1 in three unrelated families. Subsequent dideoxy sequencing of TGFBR1 identified 11 distinct monoallelic mutations in 18 affected families, firmly establishing TGFBR1 as the causative gene. The nature of the sequence variants, which include mutations in the extracellular ligand-binding domain and a series of truncating mutations in the kinase domain, indicates a clear genotype-phenotype correlation between loss-of-function TGFBR1 mutations and MSSE. This distinguishes MSSE from the Marfan syndrome–related disorders in which missense mutations in TGFBR1 lead to developmental defects with vascular involvement but no reported predisposition to cancer.

Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock

The intermediate filament cytoskeleton is thought to confer physical resilience on tissue cells, on the basis of extrapolations from the phenotype of cell fragility that results from mutations in skin keratins. There is a need for functional cell assays in which the impact of stress on intermediate filaments can be induced and analyzed. Using osmotic shock, we have induced cytoskeleton changes that suggest protective functions for actin and intermediate filament systems. Induction of the resulting stress response has been monitored in keratinocyte cells lines carrying K5 or K14 mutations, which are associated with varying severity of epidermolysis bullosa simplex. Cells with severe mutations were more sensitive to osmotic stress and took longer to recover from it. Their stress-activated response pathways were induced faster, as seen by early activation of JNK, ATF-2 and c-Jun. We demonstrate that the speed of a cells response to hypotonic stress, by activation of the SAPK/JNK pathway, is correlated with the clinical severity of the mutation carried. The response to hypo-osmotic shock constitutes a discriminating stress assay to distinguish between the effects of different keratin mutations and is a potentially valuable tool in developing therapeutic strategies for keratin-based skin fragility disorders.

Mechanical stress induces profound remodelling of keratin filaments and cell junctions in epidermolysis bullosa simplex keratinocytes.

The outer epidermal layer of the skin is an epithelium with remarkable protective barrier functions, which is subject to pronounced physical stress in its day-to-day function. A major candidate component for absorbing this stress is the K5/K14 keratin intermediate filament network. To investigate the part played by keratins in stress resilience, keratinocyte cell lines were subjected to mechanical stress. Repeated stretch and relaxation cycles over increasing time produced reproducible changes in the configuration of the keratin network. When wild-type cells were compared with cells carrying a keratin mutation associated with severe epidermolysis bullosa simplex-type skin fragility, the mutant keratin filaments were unable to withstand the mechanical stress and progressively fragmented yielding aggregates and novel ring structures. The cell junctions into which the keratin filaments are normally anchored also progressively disassembled, with all components tested of the cytoplasmic plaques becoming relocated away from the membrane and onto the keratin rings, while integral membrane receptors integrins and cadherins remained at the plasma membrane. The results suggest that maintenance of desmosomes and hemidesmosomes may require some tension, normally mediated by keratin attachments.

Effects on normal fibroblasts and neuroblastoma cells of the activation of the p53 response by the nuclear export inhibitor leptomycin B

p53 tumour suppressor protein levels and p53-dependent transcriptional activity have been recently shown to increase in cells treated with leptomycin B (LMB), an inhibitor of nuclear export. Experiments presented here show that LMB treatment leads to growth arrest and a senescence-like phenotype in human normal fibroblast cultures. This effect is reversible after removal of the drug and further passage by trypsinization. Instead, LMB has a strong cytotoxic effect on human neuroblastoma cell lines even at nanomolar concentrations. In both these cell types the effects of LMB are attenuated when the activity of the endogenous wild type p53 protein is abrogated by overexpression of a dominant negative p53 mutant. We conclude that the induction of the p53 response by LMB plays an important role in the effects of this drug on cultured cells.

The development of epithelial phenotypes in the human fetal and infant breast

In order to explain the molecular events that contribute to benign and malignant breast disease, it is essential to understand the cellular context in which these are occurring. This study describes a detailed analysis of the epithelial phenotypes in the human fetal and infant breast and provides a starting point for such consideration. Using methacarn‐fixed, paraffin sections from ten fetal and 45 infant breast, immunostained with a panel of antibodies to cytoskeletal proteins and κ‐casein, it has been possible to define in detail the chronological evolution of the major cell types in the human breast from 16 weeks of intrauterine life to 2 years of age, in both sexes. Cells at the tips of the lobular buds and terminal end buds have a characteristic cytoskeletal protein profile, suggesting that they may have the capacity to generate both basal cells and luminal cells. Based on the expression of cytoskeletal proteins in the developing fetal and infant breast, a model system has been proposed for mammary epithelial differentiation.