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.     

Keratin 20 helps maintain intermediate filament organization in intestinal epithelia

Of the >20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80-->His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia.     

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.     

Isolation and Chromosomal Localization of a Cornea-Specific Human Keratin 12 Gene and Detection of Four Mutations in Meesmann Corneal Epithelial Dystrophy

Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin.     

The immunomodulatory protein B7-H4 is overexpressed in breast and ovarian cancers and promotes epithelial cell transformation

B7-H4 protein is expressed on the surface of a variety of immune cells and functions as a negative regulator of T cell responses. We independently identified B7-H4 (DD-O110) through a genomic effort to discover genes upregulated in tumors and here we describe a new functional role for B7-H4 protein in cancer. We show that B7-H4 mRNA and protein are overexpressed in human serous ovarian cancers and breast cancers with relatively little or no expression in normal tissues. B7-H4 protein is extensively glycosylated and displayed on the surface of tumor cells and we provide the first demonstration of a direct role for B7-H4 in promoting malignant transformation of epithelial cells. Overexpression of B7-H4 in a human ovarian cancer cell line with little endogenous B7-H4 expression increased tumor formation in SCID mice. Whereas overexpression of B7-H4 protected epithelial cells from anoikis, siRNA-mediated knockdown of B7-H4 mRNA and protein expression in a breast cancer cell line increased caspase activity and apoptosis. The restricted normal tissue distribution of B7-H4, its overexpression in a majority of breast and ovarian cancers and functional activity in transformation validate this cell surface protein as a new target for therapeutic intervention. A therapeutic antibody strategy aimed at B7-H4 could offer an exciting opportunity to inhibit the growth and progression of human ovarian and breast cancers.     

Ovine keratome: identification,localisation and genomic organisation of keratin and keratin‐associated proteins

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin‐associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST‐derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.     

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.     

Tissue distribution of keratin 7 as monitored by a monoclonal antibody

Monoclonal antibody (RCK 105) directed against keratin 7 was obtained after immunization of BALB/c mice with cytoskeletal preparations from T24 cells and characterized by one- (1D) and two-dimensional (2D) immunoblotting. In cultured epithelial cells, known from gel electrophoretic studies to contain keratin 7, this antibody gives a typical keratin intermediate filament staining pattern, comparable to that obtained with polyclonal rabbit antisera to skin keratins or with other monoclonal antibodies, recognizing for example keratins 5 and 8 or keratin 18. Using RCK 105, the distribution of keratin 7 throughout human epithelial tissues was examined and correlated with expression patterns of other keratins. Keratin 7 was found to occur in the columnar and glandular epithelium of the lung, cervix, breast, in bile ducts, collecting ducts in the kidney and in mesothelium, but to be absent from gastrointestinal epithelium, hepatocytes, proximal and distal tubules of the kidney and myoepithelium. Nor could it be detected in the stratified epithelia of the skin, tongue, esophagus, or cervix but strongly stained all cell layers of the urinary bladder transitional epithelium. When applied to carcinomas derived from these different tissue types it became obvious that an antibody to keratin 7 may allow an immunohistochemical distinction between certain types of adenocarcinomas.     

Mouse keratin 19: complete amino acid sequence and gene expression during development

The complete amino acid sequence of the mouse keratin 19 (K19) was determined from a partial sequence of cDNA isolated from a mouse (day 10.5) embryo library and an amplified genomic fragment. Analysis of the sequence reveals strong evolutionary conservation with other K19s. Examination of the expression of the gene encoding K19 (K19) during development using an RNase protection assay reveals it is expressed in extra-embryonic tissues by day 8.5 and in the embryo proper by at least day 9.5. Furthermore, the K19 gene is induced in differentiating F9 embryonal carcinoma cells. These results indicate that K19 is another keratin, in addition to the K8-K18 pair, which is synthesized early during mouse development. Finally, Southern analysis of the K19 gene reveals that it is found as a unique copy in the mouse genome, in contrast to what is found in humans, which have at least one processed pseudogene.     

Cloning, mapping, and expression of two novel actin genes, actin-like-7A (ACTL7A) and actin-like-7B (ACTL7B), from the familial dysautonomia candidate region on 9q31.

Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45. 2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a "head-to-head" orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.     

A single human keratin 18 gene is expressed in diverse epithelial cells of transgenic mice

The expression of keratin 18 (K18) is restricted in humans primarily to a variety of single layered or simple epithelia. However, direct introduction of a cloned K18 gene into cultured, somatic cells by DNA transfection has been shown to result in the promiscuous expression of K18 even while the endogenous mouse form of K18 (Endo B) remains silent. To determine if the cloned K18 genomic DNA fragment contains sufficient information to be regulated appropriately when subjected to a normal developmental environment, and to determine if the cloned gene is expressed in diverse epithelia, the K18 gene, including 2.5 kb of 5' flanking sequence and 3.5 kb of 3' flanking sequence, has been introduced into the germ line of mice. Mice from all three resulting K18 transgenic lines express the gene in an appropriate tissue-specific pattern that includes hepatocytes, simple epithelia of the intestinal tract, ductal cells of several glands and epithelial cells of the thymus. No expression of K18 was found in muscle, heart, or in most of the brain even in mice carrying 18 copies of the K18 gene. In most tissues, the level of K18 RNA was directly proportional to copy number and was as efficiently expressed as the endogenous Endo B gene. The K18 protein was identified by both protein blotting methods and indirect immunofluorescence staining. No pathological consequences of overexpression of the K18 gene were observed. The cloned K18 gene appears to contain all cis-acting DNA sequences necessary for appropriate expression. In addition, diverse epithelial cell types are able to express this single human gene.     

Homology between a 173-kb Region from Mouse Chromosome 10, Telomeric to the Ifng Locus, and Human Chromosome 12q15

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).