The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14

Keratin 5 and keratin 14 have been touted as the hallmarks of the basal keratin networks of all stratified squamous epithelia. Absence of K14 gives rise to epidermolysis bullosa simplex, a human blistering skin disorder involving cytolysis in the basal layer of epidermis. To address the puzzling question of why this disease is primarily manifested in skin rather than other stratified squamous epithelia, we ablated the K14 gene in mice and examined various tissues expressing this gene. We show that a key factor is the presence of another keratin, K15, which was hitherto unappreciated as a basal cell component. We show that the levels of K15 relative to K14 vary dramatically among stratified squamous epithelial tissues, and with neonatal development. In the absence of K14, K15 makes a bona fide, but ultrastructurally distinct, keratin filament network with K5. In the epidermis of neonatal mutant mice, K15 levels are low and do not compensate for the loss of K14. In contrast, the esophagus is unaffected in the neonatal mutant mice, but does appear to be fragile in the adult. Parallel to this phenomenon is that esophageal K14 is expressed at extremely low levels in the neonate, but rises in postnatal development. Finally, despite previous conclusions that the formation of suprabasal keratin filaments might depend upon K5/K14, we find that a wide variety of suprabasal networks composed of different keratins can form in the absence of K14 in the basal layer.     

Claudin-based barrier in simple and stratified cellular sheets

For homeostasis in multicellular organisms, isolation and compartmentalisation of the internal environment are essential, and are established by various cellular sheets. For these cellular sheets to function as barriers, the intercellular route must be sealed. Recent advances reveal that claudins - major cell adhesion molecules in tight junctions - are directly involved in this intercellular sealing, not only in simple but also in stratified cellular sheets in vertebrates.     

Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin

Injury to epidermis and other stratified epithelia triggers profound but transient changes in the pattern of keratin expression. In postmitotic cells located at the wound edge, a strong induction of K6, K16, and K17 synthesis occurs at the expense of the keratins produced under the normal situation. The functional significance of these alterations in keratin expression is not known. Here, we report that overexpression of a wild-type human K16 gene in a tissue-specific fashion in transgenic mice causes aberrant keratinization of the hair follicle outer root sheath and proximal epidermis, and it leads to hyperproliferation and increased thickness of the living layers (acanthosis), as well as cornified layers (hyperkeratosis). The pathogenesis of lesions in transgenic mouse skin begins with a reorganization of keratin filaments in postmitotic keratinocytes, and it progresses in a transgene level-dependent fashion to include disruption of keratinocyte cytoarchitecture and structural alterations in desmosomes at the cell surface. No evidence of cell lysis could be found at the ultrastructural level. These results demonstrate that the disruption of the normal keratin profile caused by increased K16 expression interferes with the program of terminal differentiation in outer root sheath and epidermis. They further suggest that when present at sufficiently high intracellular levels, K16, along with K6 and K17, appear capable of inducing a reorganization of keratin filaments in the cytoplasm of skin epithelial cells.     

Syncoilin is required for generating maximum isometric stress in skeletal muscle but dispensable for muscle cytoarchitecture

Syncoilin is a striated muscle-specific intermediate filament-like protein, which is part of the dystrophin-associated protein complex (DPC) at the sarcolemma and provides a link between the extracellular matrix and the cytoskeleton through its interaction with alpha-dystrobrevin and desmin. Its upregulation in various neuromuscular diseases suggests that syncoilin may play a role in human myopathies. To study the functional role of syncoilin in cardiac and skeletal muscle in vivo, we generated syncoilin-deficient (syncoilin-/-) mice. Our detailed analysis of these mice up to 2 yr of age revealed that syncoilin is entirely dispensable for cardiac and skeletal muscle development and maintenance of cellular structure but is required for efficient lateral force transmission during skeletal muscle contraction. Notably, syncoilin-/- skeletal muscle generates less maximal isometric stress than wild-type (WT) muscle but is as equally susceptible to eccentric contraction-induced injury as WT muscle. This suggests that syncoilin may play a supportive role for desmin in the efficient coupling of mechanical stress between the myofibril and fiber exterior. It is possible that the reduction in isometric stress production may predispose the syncoilin skeletal muscle to a dystrophic condition.     

A histological method for the visualization of the intercellular permeability barrier in mammalian stratified squamous epithelia

Summary Mammalian epidermis and oral epithelia possess an intercellular permeability barrier which is located in the superficial region of the tissue. This study reports a staining reaction which appears to demonstrate a histological correlate of this functional property. Specimens of ear skin, palate, buccal and oesophageal mucosa and of cornea and bladder were obtained from adult rabbits and rats, bisected and either incubatedin vitro with 2.5% horseradish peroxidase as a tracer or fixed and processed for light microscopy and stained with a modification of Hart's elastin stain. Examination of specimens prepared by each procedure showed a complementary staining pattern in the intercellular spaces of the stratum corneum or in the superficial region of the non-keratinized tissue. In the epidermis and oral and oesophageal epithelia, the region which excluded the tracer stained with the modified elastin stain. In contrast, the corneal and bladder epithelia neither excluded the tracer nor showed intercellular staining. This relationship between staining of the intercellular space and the exclusion of tracer suggests that the intercellular material in the superficial region of epithelia may be chemically altered to form a barrier substance, possibly as the result of the discharge of the contents of the membrane-coating granules which are present in all the epithelia examined except the cornea and bladder.     

The keratin polypeptide patterns in heterotypically recombined epithelia of skin and mucosa of adult mouse

Epithelial-mesenchymal interactions were investigated considering both morphologic criteria and keratin polypeptide expression in homotypic and heterotypic recombinants of adult mouse skin and oral mucosa. Two series of cross-recombinants of epithelia with different morphology and keratin patterns were chosen: (a) footpad epidermis/ear dermis and ear epidermis/footpad dermis; (b) palate epithelium/cheek connective tissue and cheek epithelium/palate connective tissue. Homotypic and heterotypic recombinants were prepared after EDTA-separation of the original tissues and then grown on syngeneic mice in subcutaneously prepared protected graft chambers. EDTA-separation is especially suited to completely separate the epidermal-dermal union, and the transplantation procedure used strictly prevents contamination with host epithelium. Five weeks after implantation keratins were analyzed by one and two-dimensional gel electrophoresis and peptide mapping. In both series, homotypic recombination of the tissues did not alter the original morphology and keratin polypeptide composition of the individual epithelial components. Ear epidermis displayed no significant changes in structure or keratin pattern in heterotypic recombinants. Recombined with ear dermis, footpad epidermis showed acquisition of some morphologic features typical for ear epidermis and slight changes in keratin composition which were, however, difficult to interpret due to the normal similarities of footpad keratin with that of ear. In contrast, the heterorecombinants of the palate/cheek series exhibited considerable alterations in their keratin patterns. Either epithelium showed suppression of distinct keratin subunits and de novo expression of subunits characteristic of the epithelium normally associated with the connective tissue component. The keratin patterns of both matches closely resembled each other and represented patterns intermediate between the normal patterns. This partial, however, significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors.     

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.     

Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeast

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.     

Murine mCLCA5 is expressed in granular layer keratinocytes of stratified epithelia

CLCA proteins represent a large family of proteins widely expressed in mammalian tissues with a unique expression pattern for each family member analyzed so far. However, their functions in normal and diseased tissues are poorly understood. Here, we present the cellular expression pattern of mCLCA5 in murine tissues using immunohistochemistry, confocal laser scanning microscopy and immune electron microscopy with specific antibodies and RT-qPCR following laser-capture microdissection. The mCLCA5 protein was localized to granular layer keratinocytes of virtually all stratified squamous epithelia of the body. Biochemical protein characterizations revealed that the amino-terminal cleavage product is fully secreted by the cell, while the carboxy-terminal cleavage product remains associated with the cell. The results imply that mCLCA5 may play a role in maturation and keratinization of squamous epithelial cells.     

Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.     

Septate junctions are required for ommatidial integrity and blood-eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.     

The imd gene is required for local Cecropin expression in Drosophila barrier epithelia

Surfaces of higher eukaryotes are normally covered with microorganisms but are usually not infected by them. Innate immunity and the expression of gene-encoded antimicrobial peptides play important roles in the first line of defence in higher animals. The immune response in Drosophila promotes systemic expression of antimicrobial peptides in response to microbial infection. We now demonstrate that the epidermal cells underlying the cuticle of larvae respond to infected wounds by local expression of the genes for the antimicrobial peptide cecropin A. Thus, the Drosophila epidermis plays an active role in the innate defence against microorganisms. The immune deficiency (imd) gene was found to be a crucial component of the signal-induced epidermal expression in both embryos and larvae. In contrast, melanization, which is part of the wound healing process, is not dependent on the imd gene, indicating that the signalling pathways promoting melanization and antimicrobial peptide gene expression can be uncoupled.     

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.     

Roles for microtubules in the proliferative and differentiated cells of stratified epithelia

While microtubule dynamics and organization have been extensively studied in vitro, both biochemically and in cultured cells, recent work has begun to extend this into tissues ex vivo and organisms in vivo. Advances in genetic tools and imaging technology have allowed studies on the dynamics, function, and organization of microtubules in the stratified epithelia of the epidermis. Here, we discuss recent work that highlights the varied roles that microtubules play in supporting epidermal function. These findings demonstrate that studying microtubules in tissues has revealed not only novel aspects of epidermal biology but also new principles of microtubule regulation.     

Peroxiredoxin 6 is required for blood vessel integrity in wounded skin

Peroxiredoxin 6 (Prdx6) is a cytoprotective enzyme with largely unknown in vivo functions. Here, we use Prdx6 knockout mice to determine its role in UV protection and wound healing. UV-mediated keratinocyte apoptosis is enhanced in Prdx6-deficient mice. Upon skin injury, we observe a severe hemorrhage in the granulation tissue of knockout animals, which correlates with the extent of oxidative stress. At the ultrastructural level endothelial cells appear highly damaged, and their rate of apoptosis is enhanced. Knock-down of Prdx6 in cultured endothelial cells also increases their susceptibility to oxidative stress, thus confirming the sensitivity of this cell type to loss of Prdx6. Wound healing studies in bone marrow chimeric mice demonstrate that Prdx6-deficient inflammatory and endothelial cells contribute to the hemorrhage phenotype. These results provide insight into the cross-talk between hematopoietic and resident cells at the wound site and the role of reactive oxygen species in this interplay.     

Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing

Alternative splicing in the extracellular domain is a characteristic feature of members of the fibroblast growth factor receptor (FGFR) family. This splicing event generates receptor variants, which differ in their ligand binding specificities. A poorly characterized splice variant is FGFR1-IIIb, recently found to be a functional FGF receptor predominantly expressed in the skin. Here we show that FGFR1-IIIb is expressed in normal and wounded mouse skin. Reduced expression of this type of receptor was found in wounds of healing-impaired genetically diabetic mice, suggesting that downregulation of FGFR1-IIIb is associated with wound healing defects. To address this possibility, we deleted the IIIb exon of FGFR1 in mice. The lack of FGFR-IIIb did not alter the expression of either FGFR1-IIIc, other FGF receptor genes or of FGFR1-IIIb ligands in normal and wounded skin. Histological analysis of the skin of FGFR1-IIIb knockout animals did not reveal any obvious abnormalities. Furthermore, full-thickness excisional skin wounds in these mice healed normally and no defects could be observed at the macroscopic or histological level. Finally, several genes that encode key players in wound repair were normally expressed in these animals. These data demonstrate that FGFR1-IIIb is dispensable for skin development and wound repair.