Expression of an epidermal keratin protein in liver of transgenic mice causes structural and functional abnormalities

To examine the role of keratin intermediate filament proteins in cell structure and function, transgenic mice were isolated that express a modified form of the human K14 keratin protein in liver hepatocytes. A modified K14 cDNA (K14.P) sequence was linked downstream of the mouse transthyretin (TTR) gene promoter and enhancer elements to achieve targeted expression in hepatocytes. Hepatocytes expressing high levels of the transgene were found to have abnormal keratin filament networks as detected by indirect immunofluorescence using an antibody specific for the transgene product. Light and electron microscopic level histological analysis of isolated liver tissue showed in many cases degenerative changes that included inflammatory infiltration, ballooning degeneration, an increase in fat containing vacuoles, and glycogen accumulation. These changes were most evident in older mice over four months of age. No indication of typical Mallory body structures were identified at either the light or electron microscopic level. To evaluate secretory function in transgenic livers, bile acid secretion rates were measured in isolated perfused liver and found to be approximately twofold lower than aged-matched controls. These findings indicate that expression of an abnormal keratin in liver epithelial cells in the in vivo setting can alter the structure and function of a tissue and suggest a role of the keratin network in cellular secretion.     

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis

The claw of lizards is largely composed of beta‐keratins, also referred to as keratin‐associated beta‐proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta‐keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha‐keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha‐keratins resembled that of beta‐keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles

The major histological components of the hair follicle are the hair cortex and cuticle. The hair cuticle cells encase and protect the cortex and undergo a different developmental program to that of the cortex. We report the molecular characterization of a set of evolutionarily conserved hair genes which are transcribed in the hair cuticle late in follicle development. Two genes were isolated and characterized, one expressed in the human follicle and one in the sheep follicle. Each gene encodes a small protein of 16 kD, containing greater than 50 cysteine residues, ranging from 31 to 36 mol% cysteine. Their high cysteine content and in vitro expression data identify them as ultra-high-sulfur (UHS) keratin proteins. The predicted proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Genomic blots reveal that the UHS keratin proteins are encoded by related multigene families in both the human and sheep genomes. Tissue in situ hybridization demonstrates that the expression of both genes is localized to the hair fiber cuticle and occurs at a late stage in fiber morphogenesis.     

Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8.

Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expression of a type I and a type II keratin was found to be both necessary and sufficient for formation of keratin filaments within fibroblasts. To determine whether a similar proteolytic system responsible for the degradation of K18 in fibroblasts also exists in simple epithelial cells which normally express a type I and a type II keratin, a mutant, truncated K18 protein missing the carboxy-terminal tail domain and a conserved region of the central, alpha-helical rod domain was expressed in mouse parietal endodermal cells. This resulted in destabilization of endogenous Endo A and Endo B and inhibition of the formation of typical keratin filament structures. Therefore, cells that normally express keratins contain a proteolytic system similar to that found in experimentally manipulated fibroblasts which degrades keratin proteins not found in their normal polymerized state.     

Trichohyalin mechanically strengthens the hair follicle: multiple cross-bridging roles in the inner root shealth

Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.     

'Hard' and 'soft' principles defining the structure, function and regulation of keratin intermediate filaments.

Keratins make up the largest subgroup of intermediate filament proteins and represent the most abundant proteins in epithelial cells. They exist as highly dynamic networks of cytoplasmic 10-12 nm filaments that are obligate heteropolymers involving type I and type II keratins. The primary function of keratins is to protect epithelial cells from mechanical and nonmechanical stresses that result in cell death. Other emerging functions include roles in cell signaling, the stress response and apoptosis, as well as unique roles that are keratin specific and tissue specific. The role of keratins in a number of human skin, hair, ocular, oral and liver diseases is now established and meshes well with the evidence gathered from transgenic mouse models. The phenotypes associated with defects in keratin proteins are subject to significant modulation by functional redundancy within the family and modifier genes as well. Keratin filaments undergo complex regulation involving post-translational modifications and interactions with self and with various classes of associated proteins.     

Cloning of human,murine, and marsupial keratin 7 and a survey of K7 expression in the mouse

Keratins are cytoplasmic intermediate filament proteins expressed by epithelial cells. Keratin 7 (K7) is expressed in a wide range of epithelial structures in humans. We have cloned and fully sequenced the human and mouse K7 genes and mRNAs, and the K7 mRNA from the marsupial Potorous tridactylis, from which the widely used simple epithelial cell lines PtK1 and PtK2 are derived. Percentage identity plots comparing the mouse and human genomic sequences revealed a number of conserved CpG islands associated with the K7 gene. There was considerable conservation of introns between the two species, which may indicate the presence of intronic regulatory elements. Only the most proximal 500bp of the promoter was conserved, although an additional conserved sequence island was found 2-3kb upstream. Protein sequence comparisons between the three species allowed identification of conserved regions of the keratin variable domains that may be candidates for protein-protein interactions and/or regulatory modification. From the mouse sequence, we generated a polyclonal rabbit antibody specific for murine K7. This antibody was used to perform a survey of K7 expression in the mouse. The expression pattern was similar to the reported human distribution, with substantial expression observed in lung, bladder, mesothelium, hair follicle, and ductal structures. We also noted previously unreported expression of K7 in the gastrointestinal tract and filiform papillae of the tongue and specific K7 expression in a range of "hard" epithelial tissues. The distribution of K7 in mouse and availability of genomic sequence from the 129/Sv mouse strain will allow the generation and analysis of transgenic mice expressing mutant forms of K7 and to predict the phenotype of human genetic disorders caused by mutations in this keratin.     

The human keratins: biology and pathology

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins--including numerous keratins characterized only recently--are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family.     

Expression of the Intermediate Filament Keratin Gene,K15,in the Basal Cell Layers of Epithelia and the Hair Follicle

The intermediate filament keratin, K15, is present in variable abundance in stratified epithelia. In this study we have isolated and characterized the sheepK15gene, focusing on its expression in the follicles of sheep and mice. We show thatK15is expressed throughout the hair cycle in the basal layer of the outer root sheath that envelops the follicle. Strikingly, however, in large medullated wool follicles, a small group of basal outer root sheath cells located in the region thought to contain hair follicle stem cells areK15-negative. In the follicle bulbK15is expressed in cells situated next to the dermal papilla but not in the inner bulb cells. Elsewhere,K15is expressed at a low, variable level in the basal layer of the epidermis and sebaceous gland, often in a punctate pattern. In the esophagus of the sheepK15expression is restricted to the basal layer, in contrast to human esophagus where it is expressed throughout the epithelium. Transgenic mouse lines established with a 15-kb sheepK15gene construct exhibited faithful expression and showed no phenotypic consequences ofK15overexpression. An investigation of transgene expression showed thatK15is continuously expressed in outer root sheath cells during the hair cycle. Given its expression in the mitotically active basal cell layers of diverse epithelia and the follicle,K15expression appears to signal an early stage in the pathway of keratinocyte differentiation that precedes the decision of a cell to become epidermal or hair-like.     

Sorting out IF networks: consequences of domain swapping on IF recognition and assembly

Vimentin and keratin are coexpressed in many cells, but they segregate into two distinct intermediate filament (IF) networks. To understand the molecular basis for the sorting out of these IF subunits, we genetically engineered cDNAs encoding hybrid IF proteins composed of part vimentin and part type I keratin. When these cDNAs were transiently expressed in cells containing vimentin, keratin, or both IFs, the hybrid IF proteins all recognized one or the other or both networks. The ability to distinguish networks was dependent upon which segments of IF proteins were present in each construct. Constructs containing sequences encoding either helix 1B or helix 2B seemed to be the most critical in conferring IF recognition. At least for type I keratins, recognition was exerted at the level of dimer formation with wild-type type II keratin, as demonstrated by anion exchange chromatography. Interestingly, despite the fact that swapping of helical domains was not as deleterious to IF structure/function as deletion of helical domains, keratin/vimentin hybrids still caused structural aberrations in one or more of the cytoplasmic IF network. Thus, sequence diversity among IF proteins seems to influence not only coiled-coil but also higher ordered associations leading to 10-nm filament formation and/or IF interactions with other cellular organelles/proteins.     

The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central “rod” region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.     

Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish

Keratins I and II form the largest subgroups of mammalian intermediate filament (IF) proteins and account as obligatory heteropolymers for the keratin filaments of epithelia. All human type I genes except for the K18 gene are clustered on chromosome 17q21, while all type II genes form a cluster on chromosome 12q13, that ends with the type I gene K18. Highly related keratin gene clusters are found in rat and mouse. Since fish seem to lack a keratin II cluster we screened the recently established draft genomes of a bird (chicken) and an amphibian (Xenopus). The results show that keratin I and II gene clusters are a feature of all terrestrial vertebrates. Because hair with its multiple hair keratins and inner root sheath keratins is a mammalian acquisition, the keratin gene clusters of chicken and Xenopus tropicalis have only about half the number of genes found in mammals. Within the type I clusters all genes have the same orientation. In type II clusters there is a rare gene of opposite orientation. Finally we show that the genes for keratins 8 and 18, which are the first expression pair in embryology, are not only adjacent in mammals, but also in Xenopus and three different fish. Thus neighboring K8 and K18 genes seem a feature shared by all vertebrates. In contrast to the two well defined keratin gene clusters of terrestrial vertebrates, three teleost fish show an excess of type I over type II genes, the lack of a keratin type II gene cluster and a striking dispersal of type I genes, that are probably the result of the teleost-specific whole genome duplication followed by a massive gene loss. This raises the question whether keratin gene clusters extend beyond the ancestral bony vertebrate to cartilage fish and lamprey. We also analyzed the complement of non-keratin IF genes of the chicken. Surprisingly, an additional nuclear lamin gene, previously overlooked by cDNA cloning, is documented on chromosome 10. The two splice variants closely resemble the lamin LIII a + b of amphibia and fish. This lamin gene is lost on the mammalian lineage.     

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.     

OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.     

The cDNA-deduced amino acid sequence for trichohyalin, a differentiation marker in the hair follicle, contains a 23 amino acid repeat

Trichohyalin is a highly expressed protein within the inner root sheath of hair follicles and is similar, or identical, to a protein present in the hair medulla. In situ hybridization studies have shown that trichohyalin is a very early differentiation marker in both tissues and that in each case the trichohyalin mRNA is expressed from the same single copy gene. A partial cDNA clone for sheep trichohyalin has been isolated and represents approximately 40% of the full-length trichohyalin mRNA. The carboxy-terminal 458 amino acids of trichohyalin are encoded, and the first 429 amino acids consist of full- or partial-length tandem repeats of a 23 amino acid sequence. These repeats are characterized by a high proportion of charged amino acids. Secondary structure analyses predict that the majority of the encoded protein could form alpha-helical structures that might form filamentous aggregates of intermediate filament dimensions, even though the heptad motif obligatory for the intermediate filament structure itself is absent. The alternative structural role of trichohyalin could be as an intermediate filament-associated protein, as proposed from other evidence.     

The complete sequence of the human intermediate filament chain keratin 10. Subdomainal divisions and model for folding of end domain sequences

We present the complete amino acid sequence of the human keratin 10 (type I) intermediate filament chain expressed in terminally differentiated epidermal cells. Comparisons of this sequence with its mouse and bovine counterparts allow us to describe structural features of the functional end domains. First, sections of their respective end domains are highly conserved and permit a redefinition of earlier models for their subdomainal organization. The amino-terminal end domain consists of El, the first 57-58 residues that are basic, glycine-rich, and have been highly conserved among the three species; V1, a region of well-defined quasi repeats of the motif aliphatic-serine/glycinen; and H1, a newly recognized short acidic sequence that has been conserved among the type I keratin family. The carboxyl-terminal end consists of V2 and E2 whose properties but not sequence resemble V1 and E1, respectively. Second, since the E1, H1, and E2 sequences have been highly conserved between the three species, we suggest they are critical elements in defining intermediate filament function. Third, we note that the E and V sequences of the keratin 10 (and other keratin) chains share many properties in common with protein chain turns found in globular proteins. We therefore propose a model in which these sequences form omega loop-like structures (Leszczynski, J. N. & Rose, G. D. (1986) Science 234, 849-855) on the surface of keratin intermediate filaments. This represents the first specific proposal for the end domain structure of any intermediate filament chain.