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Keratins are the major structural fibrous proteins of hair, feathers, wool, and nail. Because keratin is protein based, cheap, and biocompatible, it has found applications from tissue engineering to textile industry. Simultaneous UV-reactive electrospinning technique is used to fabricate nanofiber scaffolds with 4-vinyl benzene boronic acid–hydroxyapatite/poly(vinyl alcohol) composite containing different amounts of keratin. Human hair as keratin supports the scaffolds for cell culture applications in our study. Our aim was to obtain nanofiber scaffolds which were designed to be nontoxic. The structure and the morphology of electrospun membranes were investigated by scanning electron microscopy and Fourier transform infrared spectroscopy technique. For the cell culture applications, endothelium (ECV 304) and sarcoma osteogenic (SAOS) cells were seeded on the electrospun fibrous scaffolds. Nanofiber scaffolds were found to have an average diameter of 350 ± 20 nm. These scaffolds provided a medium for cells to grow. POLYM. COMPOS., 2015. © 2015 Society of Plastics Engineers

White sponge nevus (WSN) is an autosomal dominant hereditary disease. Keratin 4 (KRT4) and Keratin 13 (KRT13) gene mutations were involved in the WSN. We recruited two WSN Chinese families, and oral lesion biopsy with hematoxylin and eosin staining showed that patients had significant pathological characteristics. The mutations of KRT4 and KRT13 gene were detected by PCR and direct sequencing. The multiple alignments of KRT13 from 23 diverse species homology analyses were performed by the ClustalW program. The KRT13 expression was measured by Real-Time RT-PCR and Western blot analysis. Sequencing analysis revealed two mutations of KRT13 gene: one mutation was 332T>C and amino acid change was Leu111Pro. Another mutation was 340C>T and amino acid change was Arg114Cys. The sequence of KRT13 was highly conserved. Real-Time RT-PCR and Western blot analysis results show that KRT13 expression level is lower in patient but keep almost no change in mRNA level. When cells were treated with MG132, KRT13 protein level was increased and kept almost the same in normal and patient cells. We identified two heritable mutations in the KRT13 gene, which were associated with the development of WSN. The abnormal degradation of KRT13 protein of WSN may probably associate with the abnormal ubiquitination process.

The structure and characterization of the oligopeptide crystals formed from the feather keratin solution obtained by superheated water treatment are reported. The FTIR spectra and 1H and 13C solid-state NMR results indicate that the peptides, arranged mostly in a beta-sheet structure in feather, reorganize into a mainly alpha-helix and less beta-sheet mixed secondary structure, when self-assemble from the solution at room temperature. MALDI-ToF-ToF spectra confirm that the most primary sequence with the mass 1884 come from the feather keratin 4, KRFA_CHICK of Gallus gallus. The largely preservation of all but cystine amino acid species and the increase of hydrophobic amino acids content in the oligopeptide crystals are proved by the amino acid analysis.

Disorders of keratinization are often treated with vitamin A derivatives (retinoids) which affect keratinocyte differentiation, including keratin (KRT) gene expression. In vivo, suprabasal keratinocytes normally express only keratin (K) 1, K2 and K10, but after topical application of all-trans retinoic acid (ATRA), the granular cells will additionally express K4 and K13, i.e. keratins normally present in oral mucosa and in cultured epidermal keratinocytes. To learn more about the retinoid regulation of keratin expression under in vivo-like conditions, we cultured keratinocytes on de-epidermized dermis in only 0.5% serum. These cells produce a normal-looking epidermis that expresses high mRNA levels of KRT1, KRT2 and KRT10, but minimal amounts of KRT4 and KRT13. Addition of ATRA to the medium for 48 h caused a dose-dependent increase in KRT4/KRT13 and a down-regulation of KRT2 mRNA. An increase in K4 protein was also found. The response was greater than the up-regulation of another retinoid-regulated gene, CRABPII. By studying 10 retinoids with different affinities for the retinoic acid receptors (RAR) and retinoid X receptors (RXR) isoforms, the reciprocal expression of KRT2 and KRT4/KRT13 could be connected with agonists for RARalpha. Two of these agonists, CD336/Am580 and CD2081, altered the expression profile with similar potency as the pan-RAR agonists ATRA and CD367. Co-addition of a pan-RAR antagonist (CD3106/AGN193109) markedly inhibited the induction of KRT4/KRT13 expression, whereas the down-regulation of KRT2 was less affected. In conclusion, RARalpha agonists elicit a reciprocal modulation of KRT2 and KRT4/KRT13 expression in human epidermis, but whether or not the keratin genes also possess RARalpha-specific regulatory elements is still unclear.

White sponge nevus (WSN) in the oral mucosa is a rare autosomal dominant genetic disease. The involved mucosa is white or greyish, thickened, folded and spongy. The genes associated with WSN include mutant cytokeratin keratin 4 (KRT4) and keratin 13 (KRT13). In recent years, new cases of WSN and associated mutations have been reported. Here, we summarise the recent progress in our understanding of WSN, including clinical reports, genetics, animal models, treatment, pathogenic mechanisms and future directions. Gene-based diagnosis and gene therapy for WSN may become available in the near future and could provide a reference and instruction for treating other KRT-associated diseases.

To identify differentially expressed genes during the development of oral malignancy, differential display, northern blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses were undertaken using oral squamous cell carcinoma (OSCC) and leukoplakia tissues. Significantly higher levels of keratin (Ker)-14 and -17 mRNAs, combined with lower levels of Ker-4, Ker-13 and transglutaminase 3 (TG-3) transcripts, were observed in OSCC and severely dysplastic tissues, whereas this expression profile was reversed in hyperplasia and in mild to moderate dysplasia. The expression of Ker-4 and Ker-13 was elevated in density-arrested OSCC cell lines (Ca9-22, HSC-2, -3 and -4) but the expression of Ker-17 mRNA was elevated in these cells, regardless of the growth conditions. In addition, Ker-4 and Ker-13 proteins were predominantly expressed in moderate dysplasia and hyperplasia, whereas Ker-17 was markedly expressed in OSCC tissues. The expression patterns of these genes could therefore be an important determinant of the manifestation of oral malignancy.

We established two gingival epithelial cell lines (GE1 and GE6), originating from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GE1 and GE6 grew at a permissive temperature (33 degrees C) in a pavement arrangement and solely formed multilayers that exhibited morphological features similar to those of the stratified oral epithelium, with neither the use of stromal equivalents nor feeder layers. Both GE cells underwent apoptosis at a non-permissive temperature (39 degrees C). Characteristic keratin peptides, keratin 4 and 13, for mucosal epithelium were obviously expressed in the suprabasal cells, and keratohyalin granules and involucrin were present in the surface flat cells in the multilayered culture. Keratin 10 (one of the markers for higher keratinized gingival epithelium) was rarely found in some uppermost cells, and filaggrin (a component of keratohyalin granules) appeared sparsely in uppermost desquamating cells in the older cultures. These observations indicated that GE1 and GE6 cells exhibited the phenotype characterizing nonkeratinized sulcular epithelium, which possessed the potency undergoing keratinization in such highly stratified cultures as oral gingival epithelium. GE cells increased the expression levels of mRNA of interleukin-1beta and tumor necrosis factor alpha by the stimulation of lipopolysaccharide and extracellular substances of oral streptococci. The GE cell lines thus could serve as an excellent experimental system for further studies on the physiology of gingival epithelium and corresponding diseases, such as periodontal disease, epithelial hyperplasia, and gingival tumors.

The ets family of transcription factors comprises many members which contribute to diverse cellular functions that vary depending upon the cell- and tissue-type context. Recently, different groups have identified a novel member of the ets family that is epithelial-specific. Variably called ESE-1, ERT, jen, ESX, this gene is designated currently as ELF3. In order to understand transcriptional regulatory mechanisms mediated by ELF3, we investigated its effect on the human keratin 4 gene promoter based upon the role of keratin 4 in early differentiation of the esophageal squamous epithelium. Interestingly, ELF3 suppressed basal keratin 4 promoter activity in both esophageal and cervical epithelial cancer cell lines, a novel result, while simultaneously activating the late-differentiation linked SPRR2A promoter. Furthermore, serial deletion constructs of the keratin 4 promoter continued to be suppressed by ELF3, a phenomenon that was only partially rescued by ELF3 ets domain mutants, but completely abrogated by deletion of the ELF3 pointed domain. These results suggest that ELF3 may have dual functions in the transcriptional regulation of genes involved in squamous epithelial differentiation. One of these functions may not be exclusively mediated through DNA binding in the context of transcriptional suppression of the keratin 4 promoter.

Retinoids affect keratinocyte differentiation and modulate the expression of many epidermal proteins, among them cellular retinoic acid-binding protein II and the family of cytokeratins. The upregulation of the former protein is a well-known phenomenon, whereas the retinoid-induced regulation of epidermal keratin expression is more complex and only partially understood. We studied the effect of topical retinoids on the expression in healthy skin of cellular retinoic acid-binding protein II, tazarotene-induced genes 1 and 2, several epidermal keratins (K1, K2e, and K10), and two mucous keratins (K4 and K13) known to appear in epidermis under certain abnormal conditions. Reverse transcription–polymerase chain reaction experiments showed that the K4 expression was the one most overtly induced by 2 wk of open treatment with 0.05% of retinoic acid and tazarotene. Using real-time quantitative polymerase chain reaction (TaqMan) and normalization of the mRNA values to β-actin, the increase in K4 was found to be 100–1000-fold. In comparison, the expression of K13 and cellular retinoic acid-binding protein II was increased 10–50-fold, the K1 and K10 mRNA levels remained unchanged, and the K2e level decreased by a factor of 100–1000. In parallel biopsies, immunohistochemistry showed no change in K1, K2e, or K10 staining, but a strong de novo appearance of K4 in the granular layer after retinoid treatment. In a separate study, occlusive application of 0.025% retinoic acid in four healthy subjects produced a maximal K4 mRNA signal after 48 h and strong K4 staining after 80 h. Finally, a dose–response study showed that the de novo appearance of K4 can be utilized as a sensitive test for retinoid bioactivity in epidermis in vivo.

The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between -163 and -140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors.

Keratins are intermediate filaments of epithelial cells. Mutations in keratin genes expressed in skin lead to human disorders, including epidermolysis bullosa simplex and epidermolytic hyperkeratosis. We examined the role of keratin 4 (K4) in maintaining the integrity of internal epithelial linings by using gene targeting to generate mice containing a null mutation in the epithelial K4 gene. Homozygous mice that do not express K4 develop a spectrum of phenotypes that affect several organs which express K4 including the esophagus, tongue, and cornea. The cellular phenotypes include basal hyperplasia, lack of maturation, hyperkeratosis, atypical nuclei, perinuclear clearing, and cell degeneration. These results are consistent with the notion that K4 is required for internal epithelial cell integrity. As mutations in K4 in humans lead to a disorder called white sponge nevus, the K4-deficient mice may serve as models for white sponge nevus and for understanding the role of K4 in cellular proliferation and differentiation.

Complex nanomicelles were made from casein and keratin through electrostatic self-assembly and transglutaminase fixation that was proved to be harmless and green. The complex nanomicelles were characterized by dynamic light scattering, scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and steady-state florescence. The results show that the complex nanomcelles acquired at the neutral pH in the mass ratio of casein to keratin 4 : 1 exhibit an anomalous sphere shape with uniform size which the diameter is about 40–70 nm. The complex nanomicelles in solution possess excellent dilution and storage stability due to the fixation and their highζ-potential (22.8 mV). The complex nanomicelles are relatively hydrophilic and have a good potential for industrial application.

Liarozole, a novel imidazole derivative, inhibits the cytochrome P450-dependent 4-hydroxylation of retinoic acid, resulting in increased tissue levels of retinoic acid. Twelve male patients with ichthyosis were given oral liarozole, 150 mg twice daily, in an open study for 12 weeks. Immunohistochemical parameters of inflammation, epidermal proliferation and differentiation were assessed before and after treatment. Extent and severity of the skin lesions was markedly reduced in all patients. Clinical side-effects were reminiscent of those with synthetic retinoids. No relevant changes were found in the haematological, urinary and biochemical parameters. Immunohistochemical assessment showed a statistically significant induction of keratin 4 after liarozole treatment in 10 of 12 patients. In two of these patients keratin 13 was induced. This open study showed that oral liarozole treatment was efficacious and well tolerated in the treatment of different types of ichthyosis. The immunohistochemical results suggest a retinoid mechanism as the mode of action.

The molecular basis for cross-reactive antibody binding to human T cell leukemia virus type I (HTLV-I) p19 core protein and human thymic epithelium has been defined with two monoclonal antibodies (mAbs), 12/1-2 and 13B12, raised to HTLV-I p19. The mAb 12/1-2 has previously been shown to react with HTLV-I p19, HTLV-II p22, and antigens of normal human thymic epithelium, placenta, and foreskin, whereas mAb 13B12 binds only to the carboxyl terminus of HTLV-I p19. In the present study, mAb 12/1-2 bound to a subset of Triton X-100-insoluble intermediate filaments in human thymic epithelium also recognized by antikeratin antibodies AE1 and AE3. The mAb 12/1-2 also reacted in Western blot assays with proteins of 54, 46, and 40 kDa present in extracts of human thymic epithelium and with hexameric peptides containing overlapping sequences of HTLV-I p19 with the amino acids IPP (amino acids 117-119). In contrast, the HTLV-I-specific mAb 13B12 did not bind to human thymic epithelium and reacted with a single hexameric peptide containing the carboxy-terminal HTLV-I p19 sequence IPPPYV (amino acids 117-122). Binding of mAb 12/1-2 to thymic epithelium could be inhibited by adsorption with peptide SP-79 containing a C-terminal sequence (amino acids 112-125) of p19. The crossreactive IPP site is within a region of p19 that has been previously shown to be highly immunogenic in HTLV-I-infected individuals and that is also encoded by genes or mRNA of human cytokeratin 17, keratin 4, epidermal cytokeratin 2, and 50-kDa type I epidermal keratin. Thus, our studies define the sequence of a cross-reactive antigen on HTLV-I p19 that is also associated with keratin intermediate filaments from human thymic epithelium and other normal human tissues and that could serve as a focus of an autoimmune response during HTLV-I infection.

White sponge nevus (WSN) is a rare autosomal dominant disorder characterized by white plaques of oral mucosa; it is benign condition with no effective treatment. The disorder usually manifests during early childhood or adolescence. Mutations of keratin 4 or 13 gene have been identified as causing WSN. The aim of this study is to determine whether keratin 4 or 13 gene mutation was the molecular basis of WSN in a Japanese family. The proband in this family was an 11-year-old boy, with three other people affected by WSN. Genomic DNA was extracted from two affected members and an unaffected member. Segments of keratin 4 and 13 genes were amplified by PCR, and direct DNA sequencing was carried out. Sequence analysis revealed a heterozygous 3bp deletion (N160Del) localized in the helix-initiation motif at the beginning of alpha-helical domain 1A of keratin 4 gene from affected members. One member lacking the phenotype was genetically tested normal. The authors identified a mutation of the keratin 4 gene recurrent in a family affected by WSN. Further investigation of the multifunctional role of keratin genes is warranted in the group of inherited epithelial disorders that may result in identification of effective treatment for this genetic disease.

Human sebocytes maintained in medium containing delipidized serum were studied for ultrastructural characteristics, cell proliferation, lipid synthesis, immunophenotype, and keratin expression before and after the addition of the synthetic retinoids isotretinoin and acitretin (10(-8)-10(-5) M). Compared to the properties of sebocytes cultured in normal sebocyte medium (1-2 x 10(-7) M vitamin A), the use of delipidized serum (undetectable amounts of vitamin A) resulted in prominent decrease of i) proliferation; ii) number of intracellular lipid droplets and synthesis of total lipids, especially triglycerides, squalene, and wax esters; and iii) labeling with monoclonal antibodies identifying progressive and late-stage sebocyte differentiation. Intercellular spaces narrowed and cell-to-cell contacts were established by abundant desmosomes. Lanosterol was induced. Keratins 14, 16, 17, and 18 were upregulated and the keratin 16: keratin 4 ratio, negatively correlating with sebocyte differentiation, increased. Addition of isotretinoin and acitretin exerted a biphasic effect. At concentrationsor = 10(-7) M, both compounds enhanced sebocyte proliferation and synthesis of total lipids, especially triglycerides and cholesterol, and decreased lanosterol, keratin 16, and the keratin 16:keratin 4 ratio. In contrast, retinoid concentrations10(-7) M inhibited sebocyte proliferation in a dose-dependent manner. Our findings indicate that vitamin A is essential for proliferation, synthetic activity, and differentiation of human sebocytes in vitro. Synthetic retinoids partially reinstate the altered functions of sebocytes maintained in medium containing delipidized serum. In contrast to the previously shown isotretinoin-specific response of cultured sebocytes in the presence of vitamin A, similar effects of isotretinoin and acitretin were obtained in its absence. This suggests different interactions of synthetic retinoids with vitamin A, possibly influencing their efficacy on the sebaceous gland.

β-catenin signaling has been shown to play a fundamental role in embryonic development and tumorigenesis. In this study, we investigated the role of β-catenin (Ctnnb1) in corneal homeostasis and tumorigenesis. Conditional expression of a murine Ctnnb1 gain-of-function mutation alone caused corneal neoplasia and neovascularization, resembling human ocular surface squamous neoplasia (OSSN). These corneas displayed an upregulation of cell proliferative markers (PCNA and p63), while presenting downregulation of both the Pax-6 transcription factor and the corneal differentiation marker cytokeratin 12. In addition, the expression of limbal-type keratin 15 ectopically extended to cornea, but the pattern of conjunctival keratin 4 and epidermal keratin 10 were unchanged. Moreover, epithelial E-cadherin and laminins decreased concomitantly with elevated levels of MMP-7. We also noticed a dramatic upregulation of pro-angiogenic factors (Vegf-A, Vegfr1) and angiopoietins in these corneas. Interestingly, all human OSSN specimens examined revealed nuclear β-catenin immunoreactivity. Taken together, these results argue that β-catenin activation is a crucial step during OSSN pathogenesis. Thus, inhibition of β-catenin might be beneficial for treating this disease.

Human sebocytes obtained as explants after in vitro culture of isolated sebaceous glands were recently shown to maintain in part a sebocytic differentiation. The aim of this study was to further identify markers of sebocytic differentiation in vitro. Therefore, the morphology of cultured human sebocytes, and their differentiation with lipid storing and expression of cellular proteins were investigated by microscopy, electron microscopy, study of cell kinetics, cytochemistry and immunocytochemistry, and were compared to cultured human keratinocytes obtained from the same skin specimens. At first, sebocytes in all stages of sebocytic differentiation were detected in vitro. Abundant cytoplasmic lipids and the absence of desmosomes were identified as their ultrastructural characteristics. Secondly, an increasing number of sebocytes storing lipids was detected during cell proliferation. Sebocytes contained up to 4 times more lipids than keratinocytes in vitro. Squalene and increased quantities of wax/sterol esters could be extracted from secondary sebocyte cultures. Thirdly, the monoclonal antibodies 6B10 (keratin 4), RPN1162 (keratin 7), and OM-1 labeled only sebocytes in vitro. Furthermore, sebocytes presented a marked expression of keratin 19 in comparison to keratinocytes, as detected with CK 4.62, and a lack of RPN1161 (keratins 1 and 2) expression, which was typically found to be expressed in cultured keratinocytes. The culture of human sebocytes possessing several characteristics of sebocytic differentiation in vivo offers unique possibilities in investigating direct effects on sebaceous cell growth, differentiation and their regulation.

Objective: We investigated white sponge nevus (WSN) in a Chinese family, and tried to find new mutation and demonstrated that this mutation is the causative mutation for WSN in this family and this condition affects a functionally important segment of the keratin 4 protein. Materials and methods: We studied the affected family with the 32-year-old female patient, her mother, her younger sister and her daughter. Pathologic examinations were performed. DNA was extracted from peripheral blood lymphocytes, K4 and K13 genes were amplified by polymerase chain reaction (PCR) and sequenced. Results: Direct sequencing of PCR products revealed two new mutations in the keratin 4 gene, the heterozygous missense mutation 1829G→A in exon 2B, and 2324A→G in non-coding region. No any mutation was found in the keratin 13 gene. Conclusions: We found two new mutations in the keratin 4, which may be related with the development of WSN.

Streptococcus agalactiae is frequently the cause of bacterial sepsis and meningitis in neonates. In addition, it is a commensal bacterium that colonizes the mammalian gastrointestinal tract. During its commensal and pathogenic lifestyles, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present study, the serine-rich repeat protein Srr-1 from S. agalactiae was functionally investigated. Immunofluorescence microscopy showed that Srr-1 was localized on the surface of streptococcal cells. The Srr-1 protein was shown to interact with a 62-kDa protein in human saliva, which was identified by matrix-assisted laser desorption ionization-time-of-flight analysis as human keratin 4 (K4). Immunoblot and enzyme-linked immunosorbent assay experiments allowed us to narrow down the K4 binding domain in Srr-1 to a region of 157 amino acids (aa). Furthermore, the Srr-1 binding domain of K4 was identified in the C-terminal 255 aa of human K4. Deletion of the srr-1 gene in the genome of S. agalactiae revealed that this gene plays a role in bacterial binding to human K4 and that it is involved in adherence to epithelial HEp-2 cells. Binding to immobilized K4 and adherence to HEp-2 cells were restored by introducing the srr-1 gene on a shuttle plasmid into the srr-1 mutant. Furthermore, incubation of HEp-2 cells with the K4 binding domain of Srr-1 blocked S. agalactiae adherence to epithelial cells in a dose-dependent fashion. This is the first report describing the interaction of a bacterial protein with human K4.

With the goal of increasing the number of genetic entry points for studying physiologic processes and human disease, large-scale, systematic, chemical mutagenesis projects in mice have been initiated in several different centers. We have been studying mouse mutants that exhibit dominantly inherited defects in either skin and/or hair color. Here, we describe a bright coat color mutant, Bright coat color 1 (Bcc1), which develops light-colored hair at 4 weeks of age, and when homozygous exhibits oral leukoplakia and blistering, and growth retardation. We identified a missense mutation in mutant animals that predicts an N154S amino-acid substitution in the 1A domain of Keratin 4 (encoded by the Krt2-4 gene), a region known to be mutated in human patients with white sponge nevus (WSN). Bcc1 recapitulates the gross pathologic, histologic, and genetic aspects of the human disorder, WSN. © 2006 The Society for Investigative Dermatology.

Although pathogenic keratin mutations have been well characterized in inherited epidermal disorders, analogous defects in keratins expressed in non-epidermal epithelia have yet to be described. White sponge nevus (WSN) is a rare autosomal dominant disorder of non-cornifying squamous epithelial differentiation that presents clinically as bilateral white, soft, thick plaques of the oral mucosa. Less frequently the mucous membranes of the nose, esophagus, genitalia and rectum are involved1,2. Histopathological features, including epithelial thickening, parakeratosis, extensive vacuolization of the suprabasal keratinocytes2 and compact aggregates of keratin intermediate filaments (KIF) in the upper spinous layers3,4, resemble those found in epidermal disorders due to keratin defects. We analysed a multigenerational family with WSN and found cosegregation of the disease with the keratin gene cluster on chromosome 17. We identified a missense mutation in one allele of keratin 13 that leads to proline substitution for a conserved leucine. The mutation occurred within the conserved 1A region of the helical rod domain, which is critical for KIF stability and is the site of most pathogenic keratin mutations. This mutation enlarges the spectrum of keratins with disease-causing defects to include mucosally expressed keratin 13, and extends the known keratin diseases to disorders of non-cornifying stratified squamous epithelia.

Objective We sought to investigate a novel mutation in the keratin genes assumed to be responsible for a familial case of oral white sponge nevus. Patients and methods The affected family consisted of a 36-year-old woman, her 17-year-old daughter, and her 14-year-old son. Keratin 4 and 13 genes extracted from venous blood lymphocytes were amplified by using the polymerase chain reaction and directly sequenced. Results Sequencing analysis of the 3 patients revealed the presence of a novel heterozygous T-to-C transition mutation in exon 1 of the keratin 13 gene, with no abnormalities detected in the keratin 4 gene. Conclusion We identified a novel heterozygous missense mutation at 332T>C in the keratin 13 gene believed to be related to the development of white sponge nevus.

SummaryBackground White sponge naevus (WSN) is a rare, autosomal dominant disorder that predominantly affects noncornified stratified squamous epithelia, most commonly the buccal mucosa. Clinically, WSN manifests as thickened spongy mucosa with a white opalescent tint in the mouth and may be confused with other disorders that cause white lesions on oral mucosa. Recent studies have identified pathogenic mutations in KRT4 and KRT13, the genes encoding mucosa-specific keratins, in WSN. Objectives To search for possible mutations in KRT4 and KRT13. Methods We report a case of WSN in a young man who presented with diffuse irregular whitish plaques involving the buccal and gingival mucosae and the tongue. Results Pathologically, the affected mucosa showed epithelial thickening, parakeratosis and extensive vacuolization of the suprabasal keratinocytes. Mutation analysis revealed a heterozygous missense mutation 1345GA in KRT4, predicting an amino acid change, E449K, in the 2B domain of the K4 polypeptide. Conclusions We report the first mutation analysis of a Taiwanese patient with WSN. Potentially this novel mutation could disrupt the stability of keratin filaments and result in WSN.

White Sponge Nevus (WSN) is a rare, autosomal dominant disorder that predominantly affects noncornified stratified squamous epithelia. Clinically, it is characterized by the presence of soft, white, and ‘‘spongy’’ plaques in the oral mucosa. The characteristic histopathologic features are epithelial thickening, parakeratosis, and vacuolization of the suprabasal layer of oral epithelial keratinocytes. Mutations in keratin 4 (K4) and keratin 13 (K13) genes have already been demonstrated to be responsible for WSN; the identification of new keratin mutations in a stratified squamous epithelia closely related to epidermis is of relevance for the understanding of the biochemistry of intermediate filaments, and for genotype phenotype correlations. In this study we investigated a 27-y-old, female Italian patient, affected by white asymptomatic oral plaques. Sequence analysis revealed a 3 bp (ACA) heterozygous insertion localized in the helix initiation motif of the 1A alpha helical domain of K4. We report this new K4 gene mutation and describe an amino acid insertion, in the 1A domain, responsible for a keratin disease.

The environmental contaminant dioxin exerts most of its effects by activating the aryl hydrocarbon receptor (AhR). The AhR is considered to play not only a role in the regulation of xenobiotic metabolism, but also for development, growth, and differentiation. The transcript levels of the AhR and its associated translocator protein (ARNT) were found to increase with ongoing differentiation in the human keratinocyte cell line HaCaT. Correspondingly, in situ hybridization studies in normal human skin revealed an absence of AhR-expression in proliferating basal cells and increasing transcript levels in upper cell layers, in dependence of keratinocyte differentiation. AhR expression in differentiation-deficient hyperproliferative psoriatic skin was markedly decreased. When keratinocytes were continuously treated with 1 microM retinoic acid (RA), the upregulation of AhR- and ARNT-mRNA levels was inhibited as was keratin 4-expression, a marker of HaCaT-keratinocyte differentiation. In contrast, treatment of already differentiated cells with RA did not down-regulate these transcript levels. The mRNA levels of the prevalent retinoic acid receptors in keratinocytes, RAR gamma and RXR alpha, were not influenced by the process of differentiation or by addition of RA. Our data suggest that the regulation of AhR-, ARNT- and keratin 4-expression by RA is indirect and mediated by a yet to be identified factor.

White sponge nevus (WSN) is a benign autosomal dominant disorder which affects non-cornifying stratified squamous epithelia (MIM 193900) (ref. 1). Phenotypically it presents as white 'spongy' plaques (oral leukokeratoses), most commonly in the mouth but also reported in the esophagus and anogenital mucosa. Histologically, the plaques show evidence of hyperproliferation, acanthosis and tonofilament aggregation. These types of pathogenic changes are characteristic of many of the epidermal keratin disorders. Keratins are expressed in pairs by epithelial cells in a tissue and cell specific manner. The major differentiation specific keratins of the buccal mucosa, nasal, esophageal and anogenital epithelia are K4 and K13 (ref. 7). The tissue distribution and nature of the lesions in patients affected by WSN suggested that mutations in K4 and/or K13 might be responsible for this disorder. We have now confirmed this hypothesis and report here a three base-pair (bp) deletion in the helix initiation peptide of K4 in affected members from two families with this condition.

Autologous oral mucosal epithelial cell sheets have been used for treating epithelial defects such as cornea and esophagus. The cell source of patients' oral mucosal epithelial cell sheet should be examined in normality because it has individual difference. In this study, oral mucosal epithelial cells were less invasively collected by brush biopsy from the buccal, gingival, labial, and palate mucosa of four healthy volunteer donors without anesthesia, and analyzed the keratin expressions by western blotting and the obtained results were compared with those by immunohistochemistry of each of the native tissues. All of the oral mucosal epithelial cells expressed keratin 4 (K4) and K13, which were mucosal stratified squamous epithelial cell markers. K1 and K10, keratinized epithelial cell markers, were also detected in keratinized tissues such as gingival and palate mucosa. The markers of epithelial basal cells such as p63 and K15 were not detected by brush biopsy-western blotting. Although this method does not include basal layers of oral mucosa, protein expressions of upper layer of lesion area are different from normal. Therefore, brush biopsy-western blotting was extremely less invasive and would contribute to quality control of the fabrication of autologous oral mucosal epithelial cell sheets., Highlights • Cell sheet source of patient oral mucosal condition has individual difference. • A new less invasive method for quality check of human oral mucosal epithelial cells. • Keratin expressions were examined by brush biopsy western blot.