Keratin Intermediate Filaments Research Articles

Characterization of human epiplakin: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks

Epiplakin is a member of the plakin family with multiple copies of the plakin repeat domain (PRD). We studied the subcellular distribution and interactions of human epiplakin by immunostaining, overlay assays and RNAi knockdown. Epiplakin decorated the keratin intermediate filaments (IF) network and partially that of vimentin. In the binding assays, the repeat unit (PRD plus linker) showed strong binding and preferentially associated with assembled IF over keratin monomers. Epiplakin knockdown revealed disruption of IF networks in simple epithelial but not in epidermal cells. In rescue experiments, the repeat unit was necessary to prevent the collapse of IF networks in transient knockdown; however, it could only partially restore the keratin but not the vimentin IF network in stably knocked down HeLa cells. We suggest that epiplakin is a cytolinker involved in maintaining the integrity of IF networks in simple epithelial cells. Furthermore, we observed an increase of epiplakin expression in keratinocytes after the calcium switch, suggesting the involvement of epiplakin in the process of keratinocyte differentiation.

Dissection of keratin dynamics: different contributions of the actin and microtubule systems

It has only recently been recognized that intermediate filaments (IFs) and their assembly intermediates are highly motile cytoskeletal components with cell-type- and isotype-specific characteristics. To elucidate the cell-type-independent contribution of actin filaments and microtubules to these motile properties, fluorescent epithelial IF keratin polypeptides were introduced into non-epithelial, adrenal cortex-derived SW13 cells. Time-lapse fluorescence microscopy of stably transfected SW13 cell lines synthesizing fluorescent human keratin 8 and 18 chimeras HK8-CFP and HK18-YFP revealed extended filament networks that are entirely composed of transgene products and exhibit the same dynamic features as keratin systems in epithelial cells. Detailed analyses identified two distinct types of keratin motility: (I) Slow (∼0.23 μm/min), inward-directed, continuous transport of keratin filament precursor particles from the plasma membrane towards the cell interior, which is most pronounced in lamellipodia. (II) Fast (∼17 μm/min), bidirectional and intermittent transport of keratin particles in axonal-type cell processes. Disruption of actin filaments inhibited type I motility while type II motility remained. Conversely, microtubule disruption inhibited transport mode II while mode I continued. Combining the two treatments resulted in a complete block of keratin motility. We therefore conclude that keratin motility relies both on intact actin filaments and microtubules and is not dependent on epithelium-specific cellular factors.

Defining the properties of the nonhelical tail domain in type II keratin 5: insight from a bullous disease-causing mutation.

Inherited mutations in the intermediate filament (IF) proteins keratin 5 (K5) or keratin 14 (K14) cause epidermolysis bullosa simplex (EBS), in which basal layer keratinocytes rupture upon trauma to the epidermis. Most mutations are missense alleles affecting amino acids located in the central alpha-helical rod domain of K5 and K14. Here, we study the properties of an unusual EBS-causing mutation in which a nucleotide deletion (1649delG) alters the last 41 amino acids and adds 35 residues to the C terminus of K5. Relative to wild type, filaments coassembled in vitro from purified K5-1649delG and K14 proteins are shorter and exhibit weak viscoelastic properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is purely a function of the K5-1649delG tail domain; in contrast, the cloned 109 residue-long tail domain from wild type K5 is distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5's assembly properties.

The formation of wrinkles caused by transition of keratin intermediate filaments after repetitive UVB exposure

It has been reported that the formation of wrinkles involves changes in the elastic properties of the dermis due to the denaturation of elastic fibers. Several studies have shown that the hydration condition of the stratum corneum is also important in wrinkle formation. It is, however, still unclear how the stratum corneum contributes to wrinkle formation. Here we investigated the relationship between the formation of wrinkles and changes in the physical properties and condition of the skin after repetitive ultraviolet B (UVB) irradiation of hairless mice (HR/ICR). Repetitive UVB irradiation caused wrinkles on the dorsal skin of the mice. The elasticity (E') of the stratum corneum of UVB-irradiated mice was significantly lower than that of age-matched control (unirradiated) mice. UVB exposure also caused a deterioration of the fibrous ultrastructure of keratin intermediate filaments (KIFs) in the skin. We conclude that the deterioration of KIFs in the stratum corneum caused by repetitive UVB irradiation decreases the elastic properties of the stratum corneum, resulting in the formation of wrinkles.

Krtap16, Characterization of a New Hair Keratin-associated Protein (KAP) Gene Complex on Mouse Chromosome 16 and Evidence for Regulation by Hoxc13

Intermediate filament (IF) keratins and keratin-associated proteins (KAPs) are principal structural components of hair and encoded by members of multiple gene families. The severe hair growth defects observed upon aberrant expression of certain keratin and KAP genes in both mouse and man suggest that proper hair growth requires their spatio-temporally coordinated activation. An essential prerequisite for studying these cis-regulatory mechanisms is to define corresponding gene families, their genomic organization, and expression patterns. This work characterizes eight recently identified high glycine/tyrosine (HGT)-type KAP genes collectively designated Krtap16-n. These genes are shown to be integrated into a larger KAP gene domain on mouse chromosome 16 (MMU16) that is orthologous to a recently described HGT- and high sulfur (HS)-type KAP gene complex on human chromosome 21q22.11. All Krtap16 genes exhibit strong expression in a narrowly defined pattern restricted to the lower and middle cortical region of the hair shaft in both developing and cycling hair. During hair follicle regression (catagen), expression levels decrease until expression is no longer detectable in follicles at resting stage (telogen). Since isolation of the Krtap16 genes was based on their differential expression in transgenic mice overexpressing the Hoxc13 transcriptional regulator in hair, we examined whether bona fide Hoxc13 binding sites associated with these genes might be functionally relevant by performing electrophoretic mobility shift assays (EMSAs). The data provide evidence for sequence-specific interaction between Hoxc13 and Krtap16 genes, thus supporting the concept of a regulatory relationship between Hoxc13 and these KAP genes.

Mallory bodies and liver diseases

Abstract Mallory bodies are cytoplasmic inclusions in hepatocytes consisting of abnormal keratins, ubiquitin and several proteins (p62, heat shock proteins) involved in the unfolded protein response. They are morphologic hallmarks of alcoholic and nonalcoholic steatohepatitis but may also be associated with metabolic and toxic liver cell injury and hepatocellular neoplasms. Mallory bodies can be experimentally produced in mouse liver by chronic intoxication with griseofulvin or 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC). Mallory body formation is associated with derangement of the keratin intermediate filament cytoskeleton of the hepatocyte. The analysis of Mallory body composition and particularly their experimental induction in animal models and in tissue culture cells disclosed a major role of oxidative stress in the underlying liver cell injury.

FOXN1 Is Critical for Onycholemmal Terminal Differentiation in Nude (Foxn1nu) Mice

Nude mice have a mutation in the transcription factor Foxn1(nu), resulting in downregulation of hair keratins. Although hair follicles develop normally, the hair fibers become structurally weak, curl, and break off at the surface. Nails in nude mice are deformed, based on alterations of the onychocyte differentiation process. Elemental microanalysis of the nail plate reveals marked decreases in sulfur concentrations in the nude mouse nail plates. Immunohistochemistry shows a lack of keratin 1 expression in terminally differentiating keratinocytes of the nail matrix. Instead, the typical differentiation process of the matrix is altered toward an epidermis-like differentiation pattern, comprising the production of filaggrin-containing keratohyalin granules in cells resembling those of the stratum granulosum, which are never observed in normally haired mice. The nail plate has diffuse basophilic stippling. It is thinner than normal, weak, and in most Foxn1(nu)/Foxn1(nu) mice breaks where it separates from the hyponychium. These studies indicate that the Foxn1(nu) mutated gene has effects beyond downregulating keratin expression, including changes in filaggrin expression, and is critical for normal onycholemmal differentiation. The nails of nude mice provide new insights into the molecular controls of onychocyte differentiation, and they offer a useful model to investigate the pathogenesis of nail hypergranulosis, a common feature in human nail diseases.

Plakin proteins are coordinately cleaved during apoptosis but preferentially through the action of different caspases

In epithelial cells, cell-cell and cell-matrix junctions, desmosomes and hemidesmosomes, provide anchorage sites for the keratin-intermediate filaments. The plakin proteins desmoplakin (DP), plectin, and periplakin represent intracellular constituents of these adhesion junctions. In staurosporine-treated apoptotic HaCaT cells, DP, plectin, and periplakin became cleaved coordinately with the elimination of keratins 10 and 14, while involucrin, actin, and keratin 18 displayed considerable stability. The caspase inhibitor zVAD-fmk prevented both the cell detachment and protein cleavage, indicating the function of caspases in these events. Closer examination in vitro revealed that while caspases 2 and 4 most efficiently cleaved DP, and plectin served as a target for caspases 3 and 7, periplakin as well as keratins were cleaved by caspase 6. The involvement of multiple caspases in the destruction of epithelial cell integrity ensures the efficient elimination of cytoskeleton, but also provides specificity for selectively targeting individual adhesion molecules.

The keratin cytoskeleton in liver diseases

The keratin intermediate filament (IF) cytoskeleton of hepatocytes has continuously gained medical relevance over the last two decades. Originally it was mainly recognized as a differentiation marker for diagnostic purposes in pathology. However, keratin IFs were soon identified as major cellular structures to be affected in a variety of chronic liver diseases, such as alcoholic and non-alcoholic steatohepatitis (ASH, NASH), copper toxicosis, and cholestasis. Based on observations in keratin gene knock-out mice, the insight into the functional role of keratins was extended from a mere structural role providing mechanical stability to hepatocytes, to an additional role as target and modulator of toxic stress and apoptosis. The functional relevance of keratins in human diseases has recently been underlined by the identification of mutations in keratin genes in patients with liver cirrhosis.

Stratum Corneum Keratin Structure, Function, and Formation: The Cubic Rod-Packing and Membrane Templating Model

A new model for stratum corneum keratin structure, function, and formation is presented. The structural and functional part of the model, which hereafter is referred to as "the cubic rod-packing model", postulates that stratum corneum keratin intermediate filaments are arranged according to a cubic-like rod-packing symmetry with or without the presence of an intracellular lipid membrane with cubic-like symmetry enveloping each individual filament. The new model could account for (i) the cryo-electron density pattern of the native corneocyte keratin matrix, (ii) the X-ray diffraction patterns, (iii) the swelling behavior, and (iv) the mechanical properties of mammalian stratum corneum. The morphogenetic part of the model, which hereafter is referred to as "the membrane templating model", postulates the presence in cellular space of a highly dynamic small lattice parameter (<30 nm) membrane structure with cubic-like symmetry, to which keratin is associated. It further proposes that membrane templating, rather than spontaneous self-assembly, is responsible for keratin intermediate filament formation and dynamics. The new model could account for (i) the cryo-electron density patterns of the native keratinocyte cytoplasmic space, (ii) the characteristic features of the keratin network formation process, (iii) the dynamic properties of keratin intermediate filaments, (iv) the close lipid association of keratin, (v) the insolubility in non-denaturating buffers and pronounced polymorphism of keratin assembled in vitro, and (vi) the measured reduction in cell volume and hydration level between the stratum granulosum and stratum corneum. Further, using cryo-transmission electron microscopy on native, fully hydrated, vitreous epidermis we show that the subfilametous keratin electron density pattern consists, both in corneocytes and in viable keratinocytes, of one axial subfilament surrounded by an undetermined number of peripheral subfilaments forming filaments with a diameter of approximately 8 nm.

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.

The keratins and their disorders.

Diseases caused by mutations in gene encoding keratin intermediate filaments (IF) are characterized by a loss of structural integrity in the cells expressing those keratins in vivo. This is manifested as cell fragility, compensatory epidermal hyperkeratosis, and keratin filament aggregation in some affected tissues. Keratin disorders are a novel molecular category including quite different phenotypes such as epidermolysis bullosa simplex (EBS), bullous congenital ichthyosiform erthroderma (BCIE), pachyonychia congenital (PC), steatocystoma multiplex, ichthyosis bullosa of Siemens (IBS), and white sponge nevus (WSN) of the orogenital mucosa.

Striate palmoplantar keratoderma arising from desmoplakin and desmoglein 1 mutations is associated with contrasting perturbations of desmosomes and the keratin filament network.

Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients. To investigate the effects of heterozygous nonsense mutations in desmoplakin (Dp) and desmoglein (Dsg) 1 which cause the autosomal dominant disorder striate palmoplantar keratoderma (SPPK), focusing on changes in desmosome structure and composition and the associated keratin intermediate filament (KIF) network in palm skin, and in cultured keratinocytes generated from the same site. We analysed palm and nonpalm skin sections from four SPPK patients with Dp mutations and one patient with a Dsg1 mutation with respect to tissue and subcellular morphologies, and correlated the in vivo and in vitro findings. Using electron microscopy, we found abnormalities of desmosomes and cell-cell adhesion in the suprabasal layers in the epidermis from patients with both Dsg1- and Dp-associated SPPK. These changes were more advanced in skin from patients with Dp mutations. Both Dp and Dsg1 mutations were accompanied by significantly reduced numbers of desmosomes in the suprabasal layers, while decreased desmosome size was evident only in Dsg1-associated SPPK. Confocal microscopy analysis showed marked differences in the expression of keratins and of desmosome components, both between the two types of SPPK, and between SPPK and normal skin. The expression of keratins K5, K14 and K10 was reduced in Dsg1-associated SPPK skin, whereas perinuclear aggregation of keratin filaments was more evident in Dp-associated SPPK. In both types of SPPK upregulation of K16 was pronounced and involucrin labelling was abnormal. Mutations in Dp and Dsg1 genes causing SPPK may be associated with perturbations in epidermal differentiation accompanied by a marked disruption of several components of the epidermal scaffold including desmosomes and the KIF network.

Conformational changes in the rod domain of human keratin 8 following heterotypic association with keratin 18 and its implication for filament stability.

Keratin intermediate filaments are heteropolymers of type I and type II polypeptides that constitute the bulk of the epithelial cytoskeleton. We microinjected seven keratin monoclonal antibodies into human epithelial cells, and two of them, only A45-B/B3 and LP3K, caused the formation of keratin aggregates. The keratin filaments in human epithelial cells were also disrupted by a monovalent A45-B/B3 Fab fragment, suggesting that the binding of the antibody, rather than cross-linking, collapses the filaments. Immunoblotting and ELISA experiments suggested that the antibody reacted weakly with recombinant K8 but did not react with recombinant K18 at all. However, the antibody reactivity increased substantially when a mixture of the two keratin polypeptides, either recombinant or derived from MCF-7, was used. The epitopes of 15 monoclonal antibodies recognizing human K8 were characterized by their reactivity with recombinant fragments of K8. Reactivity of antibody A45-B/B3 with fragments of K8 in the presence of K18 revealed that the antibody recognizes an epitope in the rod domain of K8, between residues 313 and 332, on the amino-terminal side of the stutter in helix 2B, which is involved in heterotypic association. The data suggest that this region of K8 undergoes a conformational change following interaction with the complementary K18 either to expose the epitope or to increase its affinity for the antibody. Taken together, the data highlight the role of this epitope in heterotypic association and in filament stabilization.

Great promises yet to be fulfilled: Defining keratin intermediate filament function in vivo

Keratins are abundant proteins in epithelial cells, in which they occur as a cytoplasmic network of 10 - 12 nm wide intermediate filaments (IFs). They are encoded by a large family of conserved genes in mammals, with more than 50 individual members partitioned into two sequence types. A strict requirement for the heteropolymerization of type I and type II keratin proteins during filament formation underlies the pairwise transcriptional regulation of keratin genes. In addition, individual pairs are regulated in a tissue-type and differentiation-specific manner. Elucidating the rationale behind the diversity and differential distribution of keratin proteins offers the promise of novel insight into epithelial biology. At present, we know that keratin IFs act as resilient yet pliable scaffolds that endow epithelial cells with the ability to sustain mechanical and non-mechanical stresses. Accordingly, inherited mutations altering the coding sequence of keratins underlie several epithelial fragility disorders. In addition, keratin IFs influence the cellular response to pro-apoptotic signals in specific settings, and the routing of membrane proteins in polarized epithelia. Here we review studies focused on a subset of keratin genes, K6, K16 and K17, showing a complex regulation in vivo, including a widely known upregulation during wound repair and in diseased skin. Progress in defining the function of these and other keratins through gene manipulation in mice has been hampered by functional redundancy within the family. Still, detailed studies of the phenotype exhibited by K6 and K17 null mice yielded novel insight into the properties and function of keratin IFs in vivo.

Roads to the Edge of the Cell: Intracellular Transport of Cornified Envelope Precursors

Roads to the Edge of the Cell: Intracellular Transport of Cornified Envelope Precursors

Keratinocyte Heal Thyself: A New Form of “Natural Gene Therapy”

In little more than a decade, the biologic importance and clinical significance of keratin intermediate filaments in epithelial cells has become very evident. Indeed, several autosomal dominant (and less commonly, recessive) human diseases involving mutated keratin genes have been described (Irvine and McLean, 1999;Smith, 2003). From such studies, the accumulating mutation databases provide useful clinical information for predicting prognosis, optimizing genetic counseling, and, if clinically and ethically appropriate, undertaking DNA-based prenatal diagnosis. Delineation of the exact nature of the pathogenic mutations also provides a basis for the development of suitable and perhaps optimal approaches to gene therapy.

Functional analysis of the human papillomavirus type 16 E1=E4 protein provides a mechanism for in vivo and in vitro keratin filament reorganization.

High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer. The HPV16 E1=E4 protein associates with keratin intermediate filaments and causes network collapse when expressed in epithelial cells in vitro. Here, we show that keratin association and network reorganization also occur in vivo in low-grade cervical neoplasia caused by HPV16. The 16E1=E4 protein binds to keratins directly and interacts strongly with keratin 18, a member of the type I intermediate-filament family. By contrast, 16E1=E4 bound only weakly to keratin 8, a type II intermediate-filament protein, and showed no detectable affinity for the type III protein, vimentin. The N-terminal 16 amino acids of the 16E1=E4 protein, which contains the YPLLXLL motif that is conserved among supergroup A viruses, were sufficient to target green fluorescent protein to the keratin network. When expressed in the SiHa cervical epithelial cell line, the full-length 16E1=E4 protein caused an almost total inhibition of keratin dynamics, despite the phosphorylation of keratin 18 at serine 33, which normally leads to 14-3-3-mediated keratin solubilization. Mutant 16E1=E4 proteins which lack the LLKLL motif, or which have lost amino acids from their C termini, and which were compromised in the ability to associate with keratins did not disturb normal keratin dynamics. 16E1=E4 was found to exist as dimers and hexamers, whereas a C-terminal deletion mutant (16E1=E4Delta87-92) existed as monomers and formed multimeric structures only poorly. Considered together, our results suggest that by associating with keratins through its N terminus, and by associating with itself through its C terminus, 16E1=E4 may act as a keratin cross-linker and prevent the movement of keratins between the soluble and insoluble compartments. The increase in avidity associated with multimeric binding may contribute to the ability of 16E1=E4 to sequester its cellular targets in the cytoplasm.

Keratin 9 gene mutations in five Korean families with epidermolytic palmoplantar keratoderma

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant disease characterized clinically by localized palmoplantar thickening and histopathologically by granular degeneration of the epidermis. Recent molecular biological studies have revealed that EPPK is caused by mutations of the keratin 9 gene in sequences mainly encoding the highly conserved 1 A rod domain. Here we demonstrate a novel mutation of N160H (position 8 of the 1 A domain) and two other previously reported mutations, R162W and N160S, in five unrelated Korean families with EPPK. The three-dimensional structure of the 1 A domain of the related vimentin intermediate filament protein chain is now known. Based on its likely similarity to the keratin 9 chain, we predict that inappropriate amino acid substitutions in position 10 of 1 A will likely interfere with coiled-coil dimer stability, and those in position 8 will interfere with tetramer stability. Accordingly, these mutations compromise the structural integrity of the keratin intermediate filaments leading to the pathology of EPPK.

Glycogen synthase kinase-3 regulates cytoskeleton and translocation of Rac1 in long cellular extensions of human keratinocytes

Wound keratinocytes form long cellular extensions that facilitate their migration from the wound edge into provisional matrix. We have previously shown that similar extensions can be induced by a long-term exposure to EGF or rapidly by staurosporine in cultured cells. This morphological change depends on the activity of glycogen synthase kinase-3 (GSK-3). Here, we have characterized the cytoskeletal changes involved in formation of these extended lamellipodia (E-lam) in human HaCaT keratinocytes. E-lams contained actin filaments, stable microtubules and keratin intermediate filaments. E-lam formation was prevented by cytochalasin D, colchicine and low concentrations of taxol and nocodazole, suggesting that actin and microtubule organization and dynamics are essential for E-lam formation. Staurosporine induced recruitment of filamentous actin (F-actin), cortactin, filamin, Arp2/3 complex, Rac1 GTPase and phospholipase C-γ1 (PLC-γ1) to lamellipodia. Treatment of cells with the GSK-3 inhibitors SB-415286 and LiCl2 inhibited E-lam formation and prevented the accumulation of Rac1 and Arp2/3 complex at lamellipodia. The formation of E-lams was dependent on fibronectin-binding integrins and normally regulated Rac1, and expression of either dominant-negative or constitutively active forms of Rac1 prevented E-lam formation. Overexpression of either RhoA or Cdc42 GTPases suppressed E-lam formation. We conclude that extended lamellipodia formation in keratinocytes requires actin and tubulin assembly at the leading edge, and this process is regulated by Rac1 downstream of GSK-3.