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PubMed1989

Avian keratin genes. I. A molecular analysis of the structure and expression of a group of feather keratin genes.

The nucleotide sequence of the four complete chicken feather keratin genes A to D contained in the previously isolated recombinant lambda CFK1 has been determined. All four genes have a very similar structure; each gene encodes a polypeptide of 97 amino acid residues and contains an intron in the 5' non-coding region, 37 base-pairs from the cap site. Comparison of the previously determined feather keratin gene C sequence to genes A, B and D indicates that a high level of gene correction has occurred in the protein coding and 5' non-coding regions, which show more than 90% homology, whereas the intron and 3' non-coding regions are by contrast poorly conserved with one or two exceptions. The dramatic conservation of the 5' non-coding region between the feather keratin sequences and an unrelated but co-expressed gene encoding a histidine-rich protein suggests that this segment may play an important role in transcriptional regulation. In addition, both gene types contain an identically positioned intron in the 5' non-coding region. Northern blots performed using gene-specific probes show that the four characterized genes A to D plus gene E, which is partially contained in the recombinant lambda CFK1, are all expressed in feather tissue from 14-day old chick embryos. In addition, we report that a scale keratin gene (originally isolated from a scale complementary DNA library) is expressed at a low level in the embryonic feather.

Animals↗
PubMed1999

Epidermolytic palmoplantar keratoderma due to a novel type of keratin mutation, a 3-bp insertion in the keratin 9 helix termination motif.

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by diffuse keratoderma, typically with an erythematous border. Histologically, palmoplantar epidermis shows suprabasal cytolysis and ultrastructurally, tonofilament aggregation with overlying epidermolytic hyperkeratosis. Mutations in the KRT9 gene, encoding keratin 9 (K9), a cytoskeletal protein expressed exclusively in suprabasal keratinocytes of palmoplantar epidermis, have been reported to cause EPPK. To date, all KRT9 defects reported in EPPK have been missense mutations in exon 1, which encodes the start of the alpha-helical rod domain. However, based on studies of other keratin disorders, it was postulated that mutations at the other end of the rod domain might also produce the EPPK phenotype. Here, we report the first mutation in the 2B domain of KRT9, 1362ins3, leading to an insertion of histidine in the helix termination motif of the K9 polypeptide. Insertional mutations have not been previously described in keratins. The phenotype of this case is similar to EPPK caused by 1A domain mutations, demonstrating that mutations in either of the helix boundary motif sequences of K9 are detrimental to keratin function and keratinocyte structure.

Adult↗
PubMed1998

Patterns of keratins 8, 18 and 19 during gonadal differentiation in the mouse: sex- and time-dependent expression of keratin 19.

The acidic keratins K18 and K19 have been shown to display a sex-specific expression during gonadal differentiation in the rat. To extend these findings, we have undertaken a study of the expression of genes encoding for K18 and K19 and their basic partner K8 in the mouse from 10.5 days of gestation until adulthood, using immunofluorescence, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). In the urogenital ridge at 10.5 days of gestation, K18, K19, and K8 are present, in both sexes, in coelomic epithelium in the area of the prospective gonad. At 11 days and 10 h of gestation, they are detected in differentiating gonadal blastema. In male gonads at 11 days and 16 h of gestation the first Sertoli cells differentiate. They are stained for anti-Müllerian hormone by immunofluorescence and appear as dispersed cells throughout the blastema. Progressively, they adhere to each other and form differentiating seminiferous cords. K19 disappears as Sertoli cells differentiate. K18 and K8 continue to be detected in Sertoli cells during fetal life and after birth until 14 days postpartum. In the adult testis, no keratin is observed. In differentiating ovaries, the three keratins are present in somatic cells of the ovigerous cords during fetal life and in primordial follicles differentiating from 1-2 days postpartum. In the course of follicular development, K19 is no longer detected as primordial follicles differentiate into growing follicles. K18 and K18 are present in all stages of follicular development. These results show both differences and similarities with the results previously obtained in the rat. In the mouse, in contrast to the rat, keratins are detected in adult ovaries, and K18 is found in undifferentiated gonads and in ovaries. K18 is, thus, not specific to the testis in the mouse, as it is in the rat. In both species, K19 ceases to be expressed in male gonads as Sertoli cells differentiate and form seminiferous cords. The present observations confirm that downregulation of K19 gene expression in the fetal testis is one of the earliest molecular events attesting the commitment of the undifferentiated gonad to the male differentiative pathway.

Animals↗
PubMed2002

Interferon-gamma, a strong suppressor of cell proliferation, induces upregulation of keratin K6, one of the inflammatory- and proliferation-associated keratins.

Keratin K6 is known as an inflammatory and hyperproliferative keratin, and is induced by an inflammatory and hyperproliferative agent. In this study, we demonstrated that interferon-gamma, an antiproliferative agent, also induces keratin K6. We used normal human ex vivo skin, normal human cultured keratinocytes, HaCaT keratinocytes, and DJM cells to examine the induction of K6 by interferon-gamma, by immunohistochemical staining, Western blot analysis, promoter chloramphenicol acetyl transferase assay, and reverse transcriptase polymerase chain reaction of mRNA. We succeeded in demonstrating the induction of keratin K6 by interferon-gamma in ex vivo human skin and HaCaT keratinocytes at the protein and message level, and in cultured normal human keratinocytes at the promoter level. The inhibition of the signal transducing activator of transcription 1 pathway by a dominant-negative transfer gene caused the inhibition of K6 induction by interferon-gamma, and the blocking of nuclear factor kappaB using antisense oligonucleotides also inhibited the K6 induction. We also blocked the released interleukin-1alpha from keratinocytes after stimulation with interferon-gamma by neutralizing antibodies, which showed a decrease in the K6 induction. Our results suggest that a small amount of interleukin-1alpha, which cannot induce K6 by itself, is secreted upon stimulation by interferon-gamma, and that the induction of K6 occurs through the synergistic effect of the interferon-gamma/signal transducing activator of transcription 1 and interleukin-1alpha/nuclear factor kappaB pathways. This is the first report to describe K6 induction in epidermal keratinocytes by interferon-gamma and indicate a probable signal transduction pathway, and demonstrates that K6 is a possible partner of K17 in the inflammatory process.

Blotting, Western↗
PubMed1986

Distinct keratin patterns demonstrated by immunoperoxidase staining of adenocarcinomas, carcinoids, and mesotheliomas using polyclonal and monoclonal anti-keratin antibodies.

The authors assessed whether distinct patterns for keratin could be demonstrated in 10 adenocarcinomas, 10 carcinoids, and 4 mesotheliomas by an immunoperoxidase reaction using 3 polyclonal and 3 monoclonal antibodies to keratin. When color development in diaminobenzidine (DAB) was allowed to proceed for less than 2 minutes, distinct patterns for keratin could be demonstrated using two polyclonal and two monoclonal antibodies; these were plasma membrane and/or web-like in the adenocarcinomas, punctate or crescentic in the carcinoids, and perinuclear in the mesotheliomas consisting of tumor cells with abundant cytoplasm. Immunoelectron microscopy using protein A colloidal gold confirmed these results. When color development in DAB was allowed to proceed for more than 2 minutes, only diffuse staining was seen. The distinct patterns of immunostaining for keratin obtained with the shorter color development were helpful in differentiating adenocarcinomas, carcinoids, and mesotheliomas.

Adenocarcinoma↗
PubMed1988

Inactivation of human keratin genes: the spectrum of mutations in the sequence of an acidic keratin pseudogene.

Keratins are cytoskeletal proteins encoded by a multigene family. We have identified the first human keratin pseudogene and determined its complete nucleotide sequence. Sequence comparisons indicate that the pseudogene arose from a very recent duplication of the 50-kd keratin (K14) gene. The coding and the intron sequences of the two genes are 95% and 93% identical, respectively. Although the sequence of the regulatory region in the pseudogene is virtually identical to that in the 50-kd functional gene, several deleterious mutations have been identified in the pseudogene. There are three frameshifts in the coding regions, one of which is a perfect 8-bp duplication. A single-base-pair deletion in the first exon and a single-base-pair insertion in the penultimate exon also result in frameshifts. The three remaining deleterious mutations interfere with the mRNA processing signals: two alter the intron/exon boundaries, and the third disrupts the polyadenylation signal. These mutations clearly identify the sequence as a human keratin pseudogene.

Base Sequence↗
PubMed1991

Epidermolysis bullosa simplex (Dowling-Meara type) is a genetic disease characterized by an abnormal keratin-filament network involving keratins K5 and K14.

The distribution and morphology of tonofilament (TF) clumps were examined by light and electron microscopy in skin samples from a total of 17 patients with the Dowling-Meara (DM) form of epidermolysis bullosa simplex (EBS). TF clumps extending from the basal to the upper-spinous epidermal layer were seen in all lesional skin samples and in the majority of peri-lesional and non-lesional skin samples. TF clumps were also noted in adnexal epithelia, including outer hair root sheaths, sweat ducts, and sebaceous glands. Cultured keratinocytes from two patients also demonstrated characteristic TF clumps. All these epithelial cells have in common their expression of the keratin pair K5 and K14. Post-embedding immunogold electron microscopy using antibodies to K5, K14, and K10 showed similar expressed keratins in DM-EBS skin from four patients compared with normal skin, with K5 and K14 predominantly in the basal cell layer and K10 in the suprabasal layers. The clumped TF in DM-EBS samples were labeled strongly with anti-K5 and K14 antibodies in the basal and suprabasal layers. In contrast, the suprabasal clumps were only slightly reactive with anti-K10 antibodies and labeling was usually restricted to the periphery of the clumps. We conclude that DM-EBS is associated with an intrinsic abnormality of the keratin-filament network involving the K5 and K14 pair that is likely to result in impaired resistance of basal epidermal cells to external shearing forces, leading to the characteristic intraepidermal blisters. DM-EBS may become the first genetic skin disease to be recognized as having a specific keratin abnormality.

Adolescent↗
PubMed1995

Relative amounts of keratin 17 are higher than those of keratin 16 in hair-follicle-derived tumors in comparison with nonfollicular epithelial skin tumors.

Specimens of trichilemmal cyst, malignant trichilemmoma, keratoacanthoma, and epidermal cyst were examined to characterize keratin peptides in hair-follicle-derived tumors. Keratins were extracted from the specimens and analyzed by two-dimensional gel electrophoresis and densitometry; the results were then compared with those for normal epidermis, the outer root sheath of hair follicles, psoriatic epidermis, and various nonfollicular cutaneous epithelial tumors. The specific nonfollicular tumors examined were squamous cell carcinoma, Bowen disease, actinic keratosis, eccrine porocarcinoma, and sebaceous carcinoma. Immunohistochemistry also was performed with a few anti-keratin monoclonal antibodies. As a general rule, K6 and K16 were expressed in hyperproliferative conditions, such as epidermal tumors, and K17 was coexpressed in the same lesions. The ratio of K16 to K17 in many epithelial skin tumors has been unclear until now. K17 content exceeded K16 content in most follicular tumors, whereas in almost all the nonfollicular tumors and the psoriatic epidermis, K17 levels were less than or about equal to K16 levels. There was a significant difference in the ratio of K16 to K17 between follicular and nonfollicular skin tumors. These results indicate that alterations in the content of these keratins may be associated with follicular differentiation.

Epidermis↗
PubMed1991

Variable expression of retinoic acid receptor (RAR beta) mRNA in human oral and epidermal keratinocytes; relation to keratin 19 expression and keratinization potential.

Previous studies have revealed that the cells that form the different regions of the oral and epidermal stratified squamous epithelia represent a number of intrinsically distinct keratinocyte subtypes, each of which is developmentally programmed to preferentially express a particular pattern of keratins and type of suprabasal histology. Retinoic acid (RA) is known to modulate stratified squamous epithelial differentiation, including expression of the basal cell keratin K19 and the suprabasal keratins K1/K10 and K4/K13. We have found that all keratinocyte subtypes are similar in their steady state levels of RAR alpha and RAR gamma mRNAs in culture and that these levels are only minimally affected by RA. In contrast, RAR beta mRNA expression varies greatly among keratinocyte subtypes and, in eight of ten cell strains examined, directly correlated with their levels of K19 mRNA. Exposure to 10(-6) M RA increases the levels of RAR beta and K19 mRNA; conversely, complete removal of RA from the medium results in reduced levels of these messages. RA does not coordinately induce RAR beta and K19 messages in nonkeratinocyte cell types: fibroblasts cultured in the presence of 10(-6) M RA express very high levels of RAR beta mRNA but do not express detectable K19, and mesothelial cells decrease their levels of RAR beta and K19 mRNA in response to 10(-6) M RA. The correlation between RAR beta and K19 mRNA levels in most keratinocyte subtypes suggests a role for RAR beta in specifying patterns of keratin expression and suprabasal differentiation in stratified squamous epithelia.

Adult↗
PubMed1995

Epitope mapping of monoclonal antibodies to keratin 19 using keratin fragments, synthetic peptides and phage peptide libraries.

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.

Amino Acid Sequence↗
PubMed1999

Spiny keratoderma--a demonstration of hair keratin and hair type keratinization.

Six cases of spiny keratoderma were analyzed with hair specific antikeratin antibodies (AE13, AE14) and by electron microscopy. The keratotic column exhibited a different keratin birefringence and the underlying viable epidermis was less eosinophilic than the surrounding epidermis. AE13, which is specific for hair cortex, was positive in the lower column and variably positive in the viable epidermis, often beyond the columnar lesion. AE14 was negative in the lesion. Electron microscopy demonstrated features of keratinization of normal hair cortex, i.e. by the accretion of keratin filaments without production of keratohyalin or trichohyalin granules. Cementsomes (lamellar granules) and marginal bands were not produced as they are not formed in normal cortical keratinization. It was suggested that spiny keratoderma represents an ectopic hair formation of palms and soles.

Aged↗
PubMed1986

Three tightly linked genes encoding human type I keratins: conservation of sequence in the 5'-untranslated leader and 5'-upstream regions of coexpressed keratin genes.

We have isolated and subcloned three separate segments of human DNA which share strong sequence homology with a previously sequenced gene encoding a type I keratin, K14 (50 kilodaltons). Restriction endonuclease mapping has demonstrated that these three genes are tightly linked chromosomally, whereas the K14 gene appears to be separate. As judged by positive hybridization-translation and Northern blot analyses, the central linked gene encodes a keratin, K17, which is expressed in abundance with K14 and two other type I keratins in cultured human epidermal cells. None of these other epidermal keratin mRNAs appears to be generated from the K17 gene through differential splicing of its transcript. The sequence of the K17 gene reveals striking homologies not only with the coding portions and intron positions of the K14 gene, but also with its 5'-noncoding and 5'-upstream sequences. These similarities may provide an important clue in elucidating the molecular mechanisms underlying the coexpression of the two genes.

Base Sequence↗
PubMed2003

Familial footpad hyperkeratosis and inheritance of keratin 2, keratin 9, and desmoglein 1 in two pedigrees of Irish Terriers.

OBJECTIVE: To investigate the possibility that variants in the acidic or basic keratin genes or in desmoglein 1 may cause the clinical manifestation of familial footpad hyperkeratosis in Irish Terriers. ANIMALS: 11 dogs belonging to 2 related affected pedigrees of Irish Terriers. PROCEDURE: Genomic DNA was extracted from blood samples obtained from each dog. The DNA markers linked to the genes keratin 2, keratin 9, and desmoglein 1 were amplified by use of a polymerase chain reaction technique, and length of the products was determined by use of an automatic DNA analyzer. RESULTS: All tested markers yielded information. None of the markers (genotype) cosegregated with the clinical status of the dogs (phenotype) in the 2 pedigrees. CONCLUSIONS AND CLINICAL RELEVANCE: Mutations in the genes encoding keratin 2 and 9 as well as desmoglein 1 are highly unlikely to be the primary cause of familial footpad hyperkeratosis in Irish Terriers.

Animals↗
PubMed1989

Stepwise modifications of keratin polypeptides during keratinization in palmar-plantar epidermis.

Precursor-product relationships among keratin polypeptides (cytokeratins) were studied in living and cornified layers of palmar-plantar epidermis. Serial 10 microns horizontal freeze-cut sections of punch biopsies were analysed by means of light microscopy and one-dimensional electrophoresis in a system that permitted identification of all major keratin polypeptides with apparent molecular weights over 60 kDa. Peptides that appeared to be related were compared by means of peptide mapping after partial proteolysis. The results suggest that both the basic-neutral (type II) cytokeratin no. 1 and the acidic (type I) cytokeratin no. 9 undergo two distinct modification steps with subsequent decreases in apparent molecular weight during keratinization. For both polypeptides the first modification appeared to take place and run to completion in close relation to the transition between the uppermost living epidermal layers and the lowest cornified layers. The second conversions of cytokeratins nos. 1 and 9 both appeared to take place within the stratum corneum but differed in two respects: 1) they appeared to start at different tissue sites; 2) whereas the second modification step appeared to comprise all cytokeratin 1 molecules, only a fraction of the cytokeratin 9 molecules passed through this step. These variations suggest that the different modification steps may be produced by different mechanisms that are regulated separately. It is concluded that the processing of cytokeratins during keratinization may be more complex than has previously been realized.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1979

Histochemical and electron microscopical investigations on the calcified keratin in the horn pearls of a glans carcinoma (calcified keratin).

The calcified keratin in the horn pearls of a glans carcinoma has been studied by histochemical and ultrastructural methods. The former have shown that the calcified areas contain glycoproteins and acid proteoglycans and have a concentration of sulphydryl groups greater than that of the surrounding, uncalcified keratin. Electron microscopy has shown the presence of intracellular needle- and filament-like crystals closely related to the keratinocyte filaments and similar to those found in other calcified tissues. Besides the close relationship discovered between crystals and keratinocyte filaments, the other main conclusions are that keratin calcification is an intracellular process and that keratin molecules are responsible for the induction and regulation of the process.

Aged↗
PubMed1975

Surface characteristics of cells from different layers of keratinized and non-keratinized oral epithelia.

Portions of clinically healthy non-keratinized and keratinized oral epithelia were removed from adult vervet monkeys (Cercopithecus aethiops). The epithelium was separated from the underlying lamina propria by trypsin digestion, following which the epithelial cells were separated from the various epithelial layers. These were examined with the light and scanning electron microscope. Cells from non-keratinized epithelia have surface microplications while those from keratinized epithelia show microvilli and pits. Two distinct types of cell are therefore present. It is suggested that the different surface appearances are related to different types of mechanical adhesion between the cells.

Animals↗
PubMed2003

Recessive epidermolysis bullosa simplex phenotype reproduced in vitro: ablation of keratin 14 is partially compensated by keratin 17.

Recessive epidermolysis bullosa simplex (REBS) is characterized by generalized cutaneous blistering in response to mechanical trauma. This results from fragility of the basal keratinocytes that lack keratin tonofilaments because of homozygote null mutation in the keratin 14 gene. REBS patients display in addition focal dyskeratotic skin lesions with histology of epidermolytic hyperkeratosis (EHK) and tonofilament clumping in the suprabasal layers of the epidermis. In this study we examined whether it is possible to mimic in vitro the bullous and dyskeratotic cellular phenotype. For this purpose, fibroblasts from nondyskeratotic (K14-/-) and dyskeratotic (K14-/-) skin of a REBS patient and fibroblasts from a healthy donor (K14+/+) were isolated and incorporated into collagen matrices. Subsequently, fresh biopsies originating from the nondyskeratotic and dyskeratotic skin of the patient and from a healthy donor were placed onto the collagen matrices and cultured at the air-liquid interface. Epidermal morphogenesis was evaluated on the basis of tissue morphology and the expression of a series of keratins. The results of the present study indicate that basal cell vacuolization in REBS can be mimicked in vitro but not the EHK. Fibroblasts seem to play an important regulatory role in establishing the REBS phenotype. These findings suggest that wild-type fibroblasts may enhance the stability of K14-/- keratinocytes in vitro.

Biopsy↗
PubMed2002

Keratin 1 and keratin 10 mutations causing epidermolytic hyperkeratosis in Chinese patients.

Epidermolytic hyperkeratosis (EHK) is a rare dominantly inherited skin disorder with erythroderma and hyperkeratosis. Mutations have been found in keratin 1 (K1) or keratin 10 (K10) gene. In the present study, we reported three sporadic and one familial Chinese EHK patients with their mutation findings. All the mutations turned out to be single heterozygous point substitutions. A novel mutation designated as E477K of K1 was identified in one patient, and previous reported mutations in codon 156 of K10, i.e. R156S, R156P, R156H were found in other patients. This is the first report of the keratin mutations in Chinese kindreds. The results showed that the possible correlation between the genotype and phenotype in these patients was complex, not only depended on the position of the mutation but also on the actual amino acid substitution. And palmoplantar keratoderma (PPKD) can be an accompanied symptom caused by either K1 or K10 mutation.

Adult↗