Verrucous carcinoma in epidermolysis bullosa simplex is possibly associated with a novel mutation in the keratin 5 gene.

Epidermolysis bullosa simplex (EBS) is mainly caused by mutations in the KRT5 and KRT14 genes. Squamous cell carcinoma (SCC) represents the second most frequent skin neoplasia with complex aetiology. The molecular events disrupting the orchestrated interplay between the cytoskeleton, cell adhesion molecules and signalling proteins are ill understood in SCC. We describe the molecular background and the unusual course of the disease in a patient with EBS Dowling-Meara, severe keratoderma and a massive verrucous carcinoma. Skin and tumour samples from the patient were analysed using light microscopy, immunohistochemistry and immunofluorescence mapping. Mutation analysis of the KRT5 and KRT14 genes identified the novel KRT5 mutation p.E477D. Invasive tumour areas were characterized by downregulation of keratins 5 and 14, reduced and irregular desmocollin-2 expression and increased expression of keratins 6, 16 and 17. Levels of Ki-67 were increased and levels of E-cadherin strongly reduced in the tumour tissue. In this case a novel KRT5 mutation led to increased fragility of keratinocytes. Desmosome and adherens junctions were destabilized, which may trigger keratinocyte-mediated inflammation, possibly via p120-catenin-dependent signalling, suggesting a link between a keratin mutation and SCC, which adds weight to the hypothesis that disturbance of the cytoskeleton represents a major cause in the appearance of the malignant phenotype. Some individuals with EBS may be at risk of developing secondary SCC.

Keratin 18 provides resistance to Fas-mediated liver failure in mice.

BACKGROUND: Keratins are intermediate filament proteins of epithelial cells with pivotal functions for cell integrity. They comprise keratins 18 [K18] and 8 [K8] in hepatocytes. Keratins are of major importance for an intact cellular microarchitecture and have protective functions in human liver diseases. In mice, K8 has been demonstrated to protect against Fas-antibody-induced liver failure by direct interaction with apoptotic regulators, while the role of K18 remains unresolved. MATERIALS AND METHODS: We analysed effects of K18 deficiency on Fas-induced liver failure in mice. We determined survival and analysed induction of apoptosis after injection of the agonistic Fas antibody Jo2 into K18(-/-) and wild-type control mice by TUNEL assay and fluorometrically analysed caspase-3, -8 and -9 activities 1, 2 and 3 h after Jo2 injection. RESULTS: In K18(-/-) mice, survival of Fas-antibody treated mice was significantly shorter than that of wild-type controls (P = 0.02). However, shortened survival of K18(-/-) mice was caused by increased hepatic damage but was not correlated to enhanced induction of apoptotic pathways, as neither numbers of TUNEL positive apoptotic cells nor activities of caspases-3, -8 and -9 differed between K18(-/-) and K18(+/+) mice at any point of time. CONCLUSION: K18(-/-) mice are significantly more susceptible to Fas-antibody-induced liver failure. The cytoprotective effect of K18 is not explained by a differential activation of caspases-3, -8 and -9, suggesting that K18 does not directly interfere with apoptotic regulators. Importantly, however, K18 exerts significant protective functions by other mechanisms.

Keratins: a structural scaffold with emerging functions.

Intermediate filament proteins form an essential part of the cytoskeleton and provide topological order to cells and tissues. These features result from their intrinsic property of self-organization and their response to extrinsic cues. Keratins represent the largest subgroup among all intermediate filament proteins and are differentially expressed as pairs of type I and type II intermediate filament proteins in epithelia. Their primary function is to impart mechanical strength to cells. This function is illustrated by patients with keratin mutations and by gene-deficient mice. Additional functions include their participation in the response to stress, cell signalling and apoptosis, and thus the keratin cytoskeleton appears far more dynamic than previously anticipated. This may result from hyperphosphorylation and possibly from interaction with associated proteins. How signalling networks affect keratin organization, turnover and function and vice versa will be a major challenge for future investigations.

Disturbances in hepatic cell-cycle regulation in mice with assembly-deficient keratins 8/18.

Simple epithelial tissues such as liver and pancreas express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. K8 and K18 null mice and transgenic mice that express mutant K18 (K18C) manifest several hepatocyte abnormalities and demonstrate that K8/18 are important in maintaining liver tissue and cell integrity, although other potential functions remain uncharacterized. Here, we report an additional abnormal liver phenotype, which is similar in K8 null, K18 null, and K18C mouse models. Liver histologic examination showed large polynuclear areas that lacked cell membranes, desmosomal structures, and filamentous actin. Similar, but less prominent, areas were observed in the pancreas. The parenchyma outside the polynuclear areas displayed irregular sinusoidal structures and markedly enlarged nuclei. Most K8 null hepatocytes were positive for the proliferating cell nuclear antigen (PCNA) with a doubled DNA content in comparison with the predominantly PCNA-negative wild-type hepatocytes. The distribution of the 14-3-3zeta protein was also altered in K8 null mice. Taken together, our results indicate that absence of keratin filaments causes disturbances in cell-cycle regulation, driving cells into the S-G2 phase and causing aberrant cytokinesis. These effects could stem from disturbed functions of K8/18-dependent cell-cycle regulators, such as the signaling integrator, 14-3-3.

Genes for intermediate filament proteins and the draft sequence of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18.

We screened the draft sequence of the human genome for genes that encode intermediate filament (IF) proteins in general, and keratins in particular. The draft covers nearly all previously established IF genes including the recent cDNA and gene additions, such as pancreatic keratin 23, synemin and the novel muscle protein syncoilin. In the draft, seven novel type II keratins were identified, presumably expressed in the hair follicle/epidermal appendages. In summary, 65 IF genes were detected, placing IF among the 100 largest gene families in humans. All functional keratin genes map to the two known keratin clusters on chromosomes 12 (type II plus keratin 18) and 17 (type I), whereas other IF genes are not clustered. Of the 208 keratin-related DNA sequences, only 49 reflect true keratin genes, whereas the majority describe inactive gene fragments and processed pseudogenes. Surprisingly, nearly 90% of these inactive genes relate specifically to the genes of keratins 8 and 18. Other keratin genes, as well as those that encode non-keratin IF proteins, lack either gene fragments/pseudogenes or have only a few derivatives. As parasitic derivatives of mature mRNAs, the processed pseudogenes of keratins 8 and 18 have invaded most chromosomes, often at several positions. We describe the limits of our analysis and discuss the striking unevenness of pseudogene derivation in the IF multigene family. Finally, we propose to extend the nomenclature of Moll and colleagues to any novel keratin.

Formation of a normal epidermis supported by increased stability of keratins 5 and 14 in keratin 10 null mice.

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10(-/-) mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10(-/-) mice suggests that there is a considerable redundancy in the keratin gene family.

Complete cytolysis and neonatal lethality in keratin 5 knockout mice reveal its fundamental role in skin integrity and in epidermolysis bullosa simplex.

In human patients, a wide range of mutations in keratin (K) 5 or K14 lead to the blistering skin disorder epidermolysis bullosa simplex. Given that K14 deficiency does not lead to the ablation of a basal cell cytoskeleton because of a compensatory role of K15, we have investigated the requirement for the keratin cytoskeleton in basal cells by inactivating the K5 gene in mice. We report that the K5(-/-) mice die shortly after birth, lack keratin filaments in the basal epidermis, and are more severely affected than K14(-/-) mice. In contrast to the K14(-/-) mice, we detected a strong induction of the wound-healing keratin K6 in the suprabasal epidermis of cytolyzed areas of postnatal K5(-/-) mice. In addition, K5 and K14 mice differed with respect to tongue lesions. Moreover, we show that in the absence of K5 and other type II keratins, residual K14 and K15 aggregated along hemidesmosomes, demonstrating that individual keratins without a partner are stable in vivo. Our data indicate that K5 may be the natural partner of K15 and K17. We suggest that K5 null mutations may be lethal in human epidermolysis bullosa simplex patients.

Skin-specific expression of ank-3(93), a novel ankyrin-3 splice variant.

Ankyrins represent a protein family whose members are associated with membrane proteins and the actin cytoskeleton. The principal ankyrin domain structure comprises an amino-terminal membrane-binding, a spectrin-binding, and a regulatory domain, and can be modulated by alternative splicing. In order to investigate the role of ankyrin-3 in skin, we have isolated three complete ankyrin-3 cDNA clones of 5.8 kb, 5.2 kb, and 2.5 kb by reverse transcription-polymerase chain reaction of mouse skin RNA. DNA sequencing confirmed the isolated clones to be splice variants of ankyrin-3. Of these, the smallest cDNA represents a novel ankyrin named ankyrin-3(93). Surprisingly, this novel ankyrin subtype lacks not only all ankyrin repeats, but also the first 75 amino acids of the spectrin-binding domain. Immuno-fluorescence analysis of mouse skin showed that ankyrin-3 is expressed in all living layers of mouse epidermis. Here, it predominates along the basal and lateral membranes of the basal layer in addition to an even cytoplasmic distribution. In primary mouse keratinocytes grown at elevated Ca2+ levels, ankyrin-3(93) was localized along the plasma membrane and throughout the cell in a Golgi-like fashion. Depending on fixation conditions, nuclear staining became apparent in many cells. In agreement with previous data, northern blotting revealed a widespread distribution of the two larger ankyrin splice variants. In contrast, the mRNA coding for ankyrin-3(93)was restricted to mouse skin. Reverse transcription-polymerase chain reaction of mouse skin RNA strongly suggested additional ankyrin isoforms in skin. Our data on ankyrin-3(93), which lacks a part of the spectrin-binding domain that regulates the affinity to spectrin, suggests a new function for this member of the ankyrin family.

Supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes.

Mutations in keratin genes give rise to a number of inherited skin fragility disorders, demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder caused by mutations in keratins K5 or K14, which are expressed in the basal layer of stratified epithelia. Using a keratinocyte cell line established from an EBS patient, we investigated whether the muscle-specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes. We show that in stably transfected EBS cells, desmin forms an extended keratin-independent cytoskeleton. Immunogold-EM analysis demonstrated that in the presence of numerous keratin filaments attached to desmosomes, desmin could nevertheless interact with desmosomes in the same cell, indicating the dynamic nature of the filament-desmosome association. When desmin-transfected cells were subjected to heat shock, the mutant keratin filaments showed a transient collapse while desmin filaments were maintained. Thus the defective keratin filaments and the wild-type desmin filaments appear to coexist in cells without interference. Expression of a type III intermediate filament protein like desmin may offer a strategy for the treatment of patients suffering from epidermal keratin mutations.

Targeted deletion of keratins 18 and 19 leads to trophoblast fragility and early embryonic lethality.

It has been reported previously that keratin 8 (K8)-deficient mice of one strain die from a liver defect at around E12.5, while those of another strain suffer from colorectal hyperplasia. These findings have generated considerable confusion about the function of K8, K18 and K19 that are co-expressed in the mouse blastocyst and internal epithelia. To resolve this issue, we produced mice doubly deficient for K18 and K19 leading to complete loss of keratin filaments in early mouse development. These embryos died at around day E9.5 with 100% penetrance. The absence of keratins caused cytolysis restricted to trophoblast giant cells, followed by haematomas in the trophoblast layer. Up to that stage, embryonic development proceeded unaffected in the absence of keratin filaments. K18/19-deficient mouse embryos die earlier than any other intermediate filament knockouts reported so far, suggesting that keratins, in analogy to their well established role in epidermis, are essential for the integrity of a specialized embryonic epithelium. Our data also offer a rationale to explore the involvement of keratin mutations in early abortions during human pregnancies.

Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(-)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(-) and K18(-) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.

General or cell type-specific deletion and replacement of connexin-coding DNA in the mouse.

Here we describe several gene targeting approaches currently used in our laboratory for the generation of deletion or replacement mutants of connexin genes in the mouse and discuss the advantage of the double-replacement strategy for the generation of conditional mutants. For the analysis of complementary functions of connexins, it will be necessary to generate mice with mutations in several connexin genes. We also report how this can be effectively accomplished. The replacement of targeted connexin-coding DNA with a reporter gene, to mimic expression of the deleted gene product, is currently being used in several laboratories. The use of different reporter genes or their differently localized gene products could allow distinction of promoter activity in double or triple connexin mutant mice.

Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle.

Plectin is a multifunctional cytoskeletal linker protein with an intermediate filament-binding site and sequence elements with high homology to actin-binding domains. Mutations of the human plectin gene as well as the targeted inactivation of its murine analog cause a generalized blistering skin disorder and muscular dystrophy, thus implying its essential role in cells that are exposed to mechanical stress. In the present study we report the characterization of two new domain-specific plectin antibodies as well as ultrastructural localization of plectin in normal human skeletal muscle. Using immunogold electron microscopy, we localized plectin at three prominent sites: 1) Plectin is found at regularly spaced intervals along the cytoplasmic face of the plasma membrane. 2) It is distinctly localized at filamentous bridges between Z-lines of peripheral myofibrils and the sarcolemma and 3) at structures forming the intermyofibrillar scaffold. At the latter two locations, plectin and desmin were found to colocalize. Our ultrastructural analysis suggests that plectin may have a central role in the structural and functional organization of the intermediate filament cytoskeleton in mature human skeletal muscle.

Plectin in the human central nervous system: predominant expression at pia/glia and endothelia/glia interfaces.

Plectin is a high molecular weight protein that serves as a versatile cytoskeletal cross-linker molecule. Mutations of the human plectin gene have recently been identified to cause the autosomal recessive disorder epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). A subgroup of EBS-MD patients display signs of a neurodegenerative disorder suggesting that the expression of defective plectin may also interfere with the structural and functional integrity of the human central nervous system. However, the expression pattern of plectin in the human brain is still unknown. We therefore analyzed the immunohistochemical distribution of plectin in normal hippocampal specimens obtained at autopsy and in neocortical and hippocampal tissue of patients who had undergone epilepsy surgery. In general, plectin-immunoreactive cells were identified as capillary endothelia and astrocytes. A striking feature seen in all specimens was the accentuated plectin immunoreactivity of astrocytic end feet abutting on blood vessels and on the pial surface. Furthermore, the analysis of hippocampal tissue of epilepsy patients with Ammon's horn sclerosis (AHS) revealed a strong plectin labeling of reactive astrocytes. The latter finding suggests that the up-regulation of plectin, which parallels the increase of glial fibrillary acidic protein, may be a general feature of reactive astroglia. The predominant expression of plectin at pia/glia and endothelia/glia interfaces in the human brain indicates that plectin may have an integral role in the structural organization of the blood-brain barrier and the leptomeninges.

The relationship between hyperproliferation and epidermal thickening in a mouse model for BCIE.

Epidermal thickening is a phenomenon common to many genodermatoses but little is known about the underlying causes. We have recently created a mouse model for the human skin disease bullous congenital ichthyosiform erythroderma by gene targeting. Mice heterozygous for a truncated keratin 10 gene exhibit acanthosis and hyperkeratosis as seen in the human disease. The degree of epidermal thickening is highly variable, offering a novel opportunity to investigate how epidermal homeostasis is modulated in keratin disorders by comparing epidermis from different body regions. We have performed bromodeoxyuridine labeling experiments and detected proliferation antigens by immunohistochemical means to compare proliferation in the epidermis of wild-type and heterozygous mice. These results have been compared with the expression of epidermal differentiation markers and of the "hyperproliferation associated" keratins K6 and K16. These experiments indicated that hyperproliferation is only partly responsible for the morphologic changes and that other mechanisms such as decreased desquamation are likely to be involved.

Lessons from keratin 18 knockout mice: formation of novel keratin filaments, secondary loss of keratin 7 and accumulation of liver-specific keratin 8-positive aggregates.

Here, we report on the analysis of keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of keratin filaments. Nevertheless, typical desmosomes were formed and maintained. Old K18 null mice, however, developed a distinctive liver pathology with abnormal hepatocytes containing K8-positive aggregates. These stained positively for ubiquitin and MM120-1 and were identified as Mallory bodies, one hallmark of human alcoholic hepatitis. This is the first demonstration that the ablation of one keratin leads to the accumulation of its single partner. Another striking finding was the absence or drastic down regulation of K7 in several tissues despite its ongoing transcription. Moreover, K18 null mice revealed new insights in the filament-forming capacity of the tail-less K19 in vivo. Due to the unexpected secondary loss of K7, only K8/19 are expressed in the uterine epithelium of K18 null mice. Immunoelectron microscopy of this tissue demonstrated the presence of typical K8/19 IF, thus highlighting in vivo that K19 is a fully competent partner for K8.