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PubMed2003

Structural changes in trichocyte keratin intermediate filaments during keratinization.

The so-called hard alpha-keratins, such as quill and hair, have a composite structure in which intermediate filaments (IF) are embedded in a sulfur-rich matrix. Recent studies of these trichocyte keratin IF have revealed that substantial changes in the molecular architecture take place when oxidation of the cysteine residues occurs as part of the terminal differentiation/keratinization process. Recent cryoelectron microscope studies suggest that the IF has a tubular structure prior to keratinization, but transmission electron micrographs of thin sections of fully keratinized fibers exhibit a "ring-core" structure. In the present contribution we develop a generic model for the IF in the reduced state based on cross-linking studies and discuss two possibilities for the way in which this structure may be modified during the keratinization process.

Animals↗
PubMed2001

New mutations in keratin 1 that cause bullous congenital ichthyosiform erythroderma and keratin 2e that cause ichthyosis bullosa of Siemens.

The intermediate filaments of epithelial cells are formed by keratins, a family of structurally related proteins, which are expressed in pairs of acidic (type I) and basic (type II) polypeptides in a tissue- and differentiation-specific manner. Mutations in the genes encoding several keratins have been implicated in the pathogenesis of diseases of keratinization. We report molecular analysis of two patients with the rare autosomal dominant disorders bullous congenital ichthyosiform erythroderma (BCIE) and ichthyosis bullosa of Siemens (IBS). Previous studies have shown that these genodermatoses are due to mutations in the KRT1 and KRT2E genes, respectively. We report a new amino acid substitution mutation in codon 155 of KRT1 (valine to aspartic acid) in the conserved H1 domain of the protein in the patient with BCIE. We also report a novel amino acid substitution mutation in codon 192 of KRT2E (asparagine to lysine) in the conserved 1A helix initiation peptide of the protein in the patient with IBS. Our results demonstrate that these mutations are deleterious to keratin filament network stability and lead to specific clinical inherited disorders of keratinization.

Adult↗
PubMed1997

A proline residue in the alpha-helical rod domain of type I keratin 16 destabilizes keratin heterotetramers.

The type I keratins 14 (K14) and 16 (K16) are distinct in their assembly properties and their expression pattern despite a high degree of sequence identity. Understanding K16 function and regulation is of interest, given its strong induction in keratinocytes located at the wound edge after injury to stratified epithelia. We reported previously that, compared with K14, K16 forms unstable heterotetramers with either K5 or K6 as the type II keratin pairing partner (Paladini, R. D., Takahashi, K., Bravo, N. S., and Coulombe, P. A. (1996) J. Cell Biol. 132, 381-397). We show here that yet another related type I keratin, K17, forms stable heterotetramers with a variety of type II keratins, further accentuating the unique nature of K16. Analysis of chimeric K14-K16 proteins in a heterotetramer formation assay indicated that the instability determinant resides in a 220-amino acid segment within the alpha-helical rod domain of K16. Site-directed mutagenesis revealed that Pro188, an amino acid residue located in subdomain 1B of the rod, accounts quantitatively for the instability of K16-containing heterotetramers under denaturing conditions. In vitro polymerization studies suggest that the presence of Pro188 correlates with a reduction in assembly efficiency. In addition to their implications for the stable conformation of the keratin heterotetramers, these findings suggest that the tetramer-forming properties of K16 may influence its partitioning between the soluble and polymer pools, and hence contribute to its regulation in epithelial cells under resting and wound repair conditions.

Amino Acid Sequence↗
PubMed2004

Determination of keratin degradation by fungi using keratin azure.

Azure dye-impregnated sheep's wool keratin (keratin azure) was incorporated in a high pH medium and overlaid on a keratin-free basal medium. The release and diffusion of the azure dye into the lower layer indicated production of keratinase. Fifty-eight fungal taxa, including 49 members of the Arthrodermataceae, Gymnoascaceae and Onygenaceae (Order Onygenales), were assessed for keratin degradation using this method. The results were comparable to measures of keratin utilization reported in studies using tests based on the perforation or erosion of human hair in vitro.

Animals↗
PubMed1993

Mouse differentiation-specific keratins 1 and 10 require a preexisting keratin scaffold to form a filament network.

Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.

3T3 Cells↗
PubMed1991

Hair-specific keratins: characterization and expression of a mouse type I keratin gene.

A genomic clone for a member of the mouse type I hair keratin protein family has been isolated and analyzed in order to study the regulation of this keratin during the hair growth cycle. The coding sequence is divided into seven exons. The gene structure is typical of keratins in particular and intermediate filaments in general in that the intron-exon borders are not located at the domain borders of the protein. Comparison with a sheep wool keratin gene shows that the splice sites in the two hair keratin genes are found at identical locations relative to the amino acid sequence of the proteins. Similarly, comparison of the promoter areas of these genes shows several areas of nucleotide sequence conservation, including the area around the TATA box and an SV40 core enhancer sequence. In addition, a high degree of sequence identity exists in the fourth intron. In situ hybridization shows that transcripts of this gene are first found in the relatively undifferentiated proximal cortex area in the keratogenous zone of mouse vibrissae.

Amino Acid Sequence↗
PubMed1987

Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins.

Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes. These cell types were selected by growth in medium containing 0.05 mM Ca2+ (basal cell) or 1.4 mM Ca2+ (suprabasal cell). The cultured basal cells expressed K6 and K14, but less than 1% expressed K1 and K10. Within a few hours after being placed in 1.4 mM Ca2+, K1 expression was observed, and by 24 h, 10%-17% of the cells expressed K1. K10 expression appeared to lag behind K1 expression, with only 5%-10% of cells in 1.4 mM Ca2+ exhibiting K10 immunoreactivity. Double-labeling studies indicated that virtually all K10-positive cells also expressed K1, while only about one-half of the K1-positive cells expressed K10. The treatment of basal cells with retinoic acid at pharmacological concentrations prevented the expression of K1 and K10 when cells were challenged by 1.4 mM Ca2+. Similarly, the introduction of the v-rasH oncogene into basal cells by a defective retroviral vector prevented the expression of suprabasal keratins in 1.4 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Cloning of a type I keratin from goldfish optic nerve: differential expression of keratins during regeneration.

We report the cDNA sequence and predicted amino acid sequence of a novel type I keratin, designated as GK50, and show that keratin expression in the goldfish optic nerve is highly complex. The GK50 protein is one of at least three type I keratins expressed in goldfish optic nerve based on both antibody reactivity and blot-binding to the type II keratin ON3. After optic nerve crush in situ hybridization shows a localized increase in GK50 mRNA expression in the crush zone. This is in contrast to ON3 mRNA which shows a localized increase that is limited to the proximal and distal margins of the crush zone, suggesting a diversity of keratin expression in different cell types of the goldfish optic nerve.

Amino Acid Sequence↗
PubMed1997

Mutational analysis of the human papillomavirus type 16 E1--E4 protein shows that the C terminus is dispensable for keratin cytoskeleton association but is involved in inducing disruption of the keratin filaments.

The function of the human papillomavirus (HPV) E4 proteins is unknown. In cultured epithelial cells the proteins associate with the keratin intermediate filaments (IFs) and, for some E4 types, e.g., HPV type 16 (HPV-16), induce collapse of the keratin networks. An N-terminal leucine-rich motif (LLXLL) is a conserved feature of many E4 proteins. In a previous study we showed that deletion of this region from the HPV-1 and -16 E4 proteins abrogated the localization of the mutant proteins to the keratin cytoskeleton in a simian virus 40-transformed human keratinocyte cell line (S. Roberts, I. Ashmole, L. J. Gibson, S. M. Rookes, G. J. Barton, and P. H. Gallimore, J. Virol. 68:6432-6445, 1994). The E4 proteins of HPV-1 and -16 have little sequence homology except at the N terminus. Therefore, to establish the role of sequences other than those at the N terminus, we have performed a mutational analysis of the HPV-16 E4 protein. The results of the analysis were as follows: (i) similar to findings for the HPV-1 protein, no mutation of HPV-16 E4 sequences (other than the N-terminal leucine motif) results in a mutant protein which fails to colocalize to the keratin IFs; (ii) the C-terminal domain (residues 61 to 92) is not essential for association with the cytoskeleton; and (iii) deletion of C-terminal sequences (residues 84 to 92; LTVIVTLHP) corresponding to part of a domain conserved between mucosal E4 proteins affects the ability of the mutant protein to induce cytoskeletal collapse, despite colocalization with the keratin IFs. Further analysis of this region showed that conserved hydrophobic residues valines 86 and 88 are important. In addition, we show that the HPV-16 E4 protein is detergent insoluble and exists as several disulfide-linked, high-molecular-weight complexes which could represent homo-oligomers. The C-terminal sequences (residues 84 to 92), in particular valines 86 and 88, are important in the formation of these insoluble complexes. The results of this study support our postulate that the E4 proteins include functional domains at the N terminus and the C terminus, with the intervening sequences possibly acting as a flexible hinge.

Amino Acid Sequence↗
PubMed2004

Differential expression of anti-apoptotic protein Bcl-2 in keratinizing versus non-keratinizing squamous cell carcinoma of the anus.

BACKGROUND: Histologically, tumors of the anal region are either keratinizing (K) or non-keratinizing (NK) squamous cell carcinomas (SCCA). We hypothesized that these two variants might represent, not only morphologically, but also biologically, dissimilar malignancies. The present study was undertaken to compare the expression of apoptosis-regulating proteins Bcl-2 and p53 in K versus NK SCCA. METHODS: We performed an immunohistochemical analysis on 98 pre-treatment biopsies of patients with anal canal cancers. Tissue sections were examined immunohistochemically for expression of proteins Bcl-2 (clone 124, DAKO, 1:100) and p53 (clone DO7, DAKO, 1:200). Expression of p53 and Bcl-2 was considered positive when >5% of tumor cells were stained. Tumor histology was correlated with protein expression as well as with other clinical variables. RESULTS: There were 64 NK and 34 K SCC. The proportion of Bcl-2 positive tumors was statistically higher in NK carcinomas (51.5 vs. 23.5%, p=0.009). In addition, women were more likely than men to present with NK carcinomas (71 vs. 45%, p=0.03) as well as with Bcl-2 positive tumors (47 vs. 29%, p=0.05). The more distal the tumor is (anal margin), the more frequently the keratinizing subtype is observed (87 vs. 23%, p=0.0002). By contrast, there was no correlation between p53 and tumor histology (p=0.83). CONCLUSIONS: Our data demonstrate that non-keratinizing and keratinizing SCCA differ in their Bcl-2 expression. In addition, significant differences were observed in the distribution of these two histological subtypes according to gender and tumor sublocation. These findings may indicate possible differences in the carcinogenesis process of these two histological subtypes.

Adult↗
PubMed1999

Ontogeny and regional variability of keratin 2e (K2e) in developing human fetal skin: a unique spatial and temporal pattern of keratin expression in development.

Keratin 2e (K2e) is expressed in the upper spinous and granular cells of adult epidermis. A highly specific polyclonal antibody was made against a C-terminal peptide of K2e and used to observe K2e expression at different developmental stages. At 12.5 weeks estimated gestational age (EGA) K2e was detected in trunk skin in scattered cells in the intermediate layer. At 13.5 weeks EGA, greater numbers of intermediate cells were stained with variable intensity, and staining in this pattern increased with age. Epidermal sheets from 14 weeks EGA showed that K2e + cells were excluded from developing hair follicles. At 135 days EGA, the following regional patterns were observed: in cheek, trunk, dorsal and ventral knee, elbow and dorsal hand there was moderate to intense staining of upper intermediate keratinocytes excluding cells of the hair canals and sweat ducts. The periumbilical region distinctly lacked K2e staining, while more distal areas showed increasing numbers of K2e + cells. The earliest expression of K2e was at 10 weeks EGA in the presumptive nail bed of developing digits. By 13.5 weeks EGA this pattern had shifted to the proximal nail fold, and K2e was absent in the nail bed. K2e was excluded from developing sweat glands and ducts and from developing hair follicles at the hair germ and early peg stages. By 15 weeks EGA in the fetal hair follicle small groups of cells were K2e + and by 19 weeks K2e + cells were seen at the level of the matrix. Some overlap in staining was detected for K2e with K10, and in palmar skin with K9; however, mostly the filamentous staining patterns for these keratins were distinctive. This study shows that the complex patterns of temporal and regional expression of K2e differ from known patterns for other epidermal keratins and suggest different regulation and function for this epidermal keratin.

Embryonic and Fetal Development↗
PubMed2001

Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21.

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank(TM) data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3'-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.

Amino Acid Sequence↗
PubMed2001

Keratin 23 (K23), a novel acidic keratin, is highly induced by histone deacetylase inhibitors during differentiation of pancreatic cancer cells.

Sodium butyrate (NaBu) was shown to induce differentiation and apoptosis in the human pancreatic cancer cell line AsPC-1. A suppression subtractive hybridization-based technique was used to identify genes induced by NaBu. A novel cDNA was found to be highly up-regulated in AsPC-1 cells in response to NaBu. The gene expresses a 1.65-kb mRNA encoding a protein with an open reading frame of 422 amino acids. It has an intermediate filament signature sequence and extensive homology to type I keratins. Sequence comparison with known keratins indicated that the gene shares 42-46% amino acid identity and 60-65% similarity within the alpha-helical rod domain. The gene is named K23 (for human type I Keratin 23, KRT23). K23 mRNA was highly induced by NaBu in different pancreatic cancer cells. Trichostatin A (TSA), a potent and specific inhibitor of histone deacetylase, similarly induced K23 mRNA expression. Treatment with either actinomycin D or cycloheximide efficiently blocked the induction of K23 mRNA by NaBu/TSA. These results indicate that K23 mRNA induction by NaBu or TSA is a downstream event of histone hyperacetylation. We also demonstrated that expression of p21(WAF1/CIP1) antisense RNA efficiently blocked the induction of K23 mRNA induced by NaBu. Our results suggest that K23 is a novel member of the acidic keratin family that is induced in pancreatic cancer cells undergoing differentiation by a mechanism involving histone hyperacetylation. p21(WAF1/CIP1) serves as an important mediator during the induction process of K23 by NaBu.

Acetylation↗
PubMed1993

Concurrence between the molecular overlap regions in keratin intermediate filaments and the locations of keratin mutations in genodermatoses.

By analysis of the existing available data, we have found that the locations of disease-causing mutations in epidermal keratin genes are distributed in a non-random manner. Most occur in exons 1 and 7 which encode the highly conserved 1A and 2B rod domain sequence regions of the keratin chains. Recent structural studies have suggested these sequences define an important overlap between neighboring molecules in keratin intermediate filaments. In order to better map the extent of these overlap sequences and concurrently to identify those sequences likely to be sensitive to mutations, we have used a series of synthetic peptides in an established filament disassembly assay. Thus residue positions 7-16 of the 1A and positions 107-117 of the 2B rod domain segments describe the extent of the molecular overlap window wherein mutations in keratin (and perhaps other) intermediate filaments are most likely to alter filament stability and lead to abnormalities.

Amino Acid Sequence↗
PubMed1993

Keratin specificity analyses of eight anti-keratin monoclonal antibodies, and their immunostaining patterns in normal skin using formalin-fixed and paraffin-embedded tissue specimens.

Keratin specificity analyses of eight anti-keratin antibodies (34 beta B4 (K1), 35 beta H11 (K8), Ks 13.1 (K13), Ks 19.1 (K19), PKK1, LP34 (CK1), KL1 and AE1) using keratin protein derived from normal thigh epidermis, normal parotid gland and a human squamous cell carcinoma cell line (HSC-5) were performed, and compared with those described in the data sheets. The reactivities of LP34, KL1 and PKK1 were markedly different from those mentioned in the data sheets. The immunostaining pattern of these antibodies in normal skin using formalin-fixed and paraffin-embedded tissue specimens was also examined. The staining patterns of suprabasal keratinocytes (K1, K13, CK1 and KL1 positive), basal cells of the epidermis (PKK1 and AE1 positive), inner cells of the ducts (K8, K13, CK1, KL1 and AE1 positive) and secretory cells of sweat glands (K8, K19, PKK1, KL1 and AE1 positive), mature cells (K8 and KL1 positive) and peripheral cells (CK1, KL1 and AE1 positive) of sebaceous glands and outer root sheaths (PKK1, CK1, KL1 and AE1 positive) were specific. Thus, we conclude that the differentiation of epidermis and skin appendages is possible by immunostaining with these eight anti-keratin antibodies using formalin-fixed and paraffin-embedded tissue specimens with proper protease pretreatment.

Adolescent↗
PubMed2001

Differential expression of human beta-defensin 2 in keratinized and non-keratinized oral epithelial lesions; immunohistochemistry and in situ hybridization.

Human beta-defensin(hBD)-2, an antimicrobial peptide, is produced by various epithelial cells. Because hBD-2 expression in the oral epithelium has not been assessed, we investigated its localization in normal oral epithelium and epithelial lesions. hBD-2 expression was studied using immunohistochemistry and in situ hybridization on formalin-fixed, paraffin-embedded tissue sections from 30 cases of squamous cell carcinoma and 6 cases of leukoplakia. Immunostaining for hBD-2 was more intense in hyperkeratinized than in ortho- or non-keratinized epithelium. In contrast, signals for hBD-2 mRNA were frequently stronger in non-keratinized epithelium than in hyper- or ortho-keratinized epithelium. The results suggest that keratinization in oral epithelium plays an important role in the biological function of hBD-2 both at the mRNA level and in the retention of the peptide in the epithelium.

Carcinoma, Squamous Cell↗
PubMed1982

Maturation of human gingival keratinocytes cultured with fibroblasts from keratinizing and non-keratinizing epithelia.

When keratinocytes from epidermis, gingiva and buccal mucosa are cultured in vitro they form a stratified squamous epithelium that lacks evidence of orthokeratinization or parakeratinization. We attempted to induce orthokeratinization or parakeratinization in cultured gingival keratinocytes by co-cultivation with fibroblasts from human skin, gingiva and buccal mucosa. Keratinization was defined by the morphological appearance of the cultured cells and by the presence of large molecular weight keratin proteins (63,000 and 67,000 mol. wt). Using these criteria, the chosen fibroblasts failed to induce any alteration in the pattern of keratinization. We conclude that under our present culture conditions, fibroblasts alone cannot induce keratinization in cultured keratinocytes.

Cell Differentiation↗
PubMed1984

Demonstration of anti-keratin antibodies by ELISA using keratin or thiol-containing compounds in urea as antigens.

During the development of an enzyme-linked immunosorbent assay (ELISA) for the demonstration of anti-keratin antibodies (AKA) it was observed that rabbit anti-keratin antisera also reacted with polystyrene surfaces treated with beta-mercaptoethanol in 8 M urea (ME-urea). Sera from non-immunized rabbits or rabbits immunized with antigens unrelated to keratin failed to react. The specificity of the reaction was further assessed by absorption experiments and by testing affinity-purified AKA. IgM activity against ME-urea could be demonstrated in 62.5% of sera from patients with infectious mononucleosis and in 37.5% of sera from patients with rheumatoid arthritis, and there was a good correlation to the presence of AKA. Coating of the solid phase with compounds containing free SH groups in 4-8 M urea generated the antigen of this ELISA. The exact molecular configuration of this presumptive synthetic antigen is obscure, but the ME-urea ELISA seems to provide a simple way to detect anti-keratin antibodies of a certain specificity.

Animals↗