Malignant transformation of white sponge nevus: a case report of a novel keratin 4 mutation.

BACKGROUND: White Sponge Nevus (WSN) is traditionally considered a benign genetic disorder affecting the oral mucosa, primarily caused by pathogenic mutations in keratin 4 (KRT4) or keratin 13 (KRT13). Despite its benign nature, recent evidence has begun to question the malignant potential of WSN. CASE PRESENTATION: We report a case involving a 70-year-old man who presented with a white lesion on the right floor of his mouth. Initial diagnostic evaluations confirmed the lesion as WSN. Over a one-year follow-up, the lesion underwent malignant transformation, evolving into local epithelial moderate-to-severe dysplasia. Exome sequencing identified a novel insertion mutation in exon 1 of the KRT4 gene, resulting in a deletion-insertion amino acid mutation involving glycine. Single-cell RNA sequencing further revealed altered epithelial proliferation and differentiation dynamics within the lesion. CONCLUSIONS: This case not only expands the known genetic spectrum of KRT4 mutations associated with WSN but also provides preliminary evidence suggesting the malignant potential of WSN. The novel pathogenic mutation in KRT4 is postulated to alter epithelial proliferation and differentiation, thereby raising concerns about the malignant transformation of WSN. Further studies are warranted to confirm these findings.

Identification of overlay differentially expressed genes in both rats and goats with blast lung injury through comparative transcriptomics.

PURPOSE: To identify the potential target genes of blast lung injury (BLI) for the diagnosis and treatment. METHODS: This is an experimental study. The BLI models in rats and goats were established by conducting a fuel-air explosive power test in an unobstructed environment, which was subsequently validated through hematoxylin-eosin staining. Transcriptome sequencing was performed on lung tissues from both goats and rats. Differentially expressed genes were identified using the criteria of q ≤ 0.05 and |log2 fold change| ≥ 1. Following that, enrichment analyses were conducted for gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathways. The potential target genes were further confirmed through quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay. RESULTS: Observations through microscopy unveiled the presence of reddish edema fluid, erythrocytes, and instances of focal or patchy bleeding within the alveolar cavity. Transcriptome sequencing analysis identified a total of 83 differentially expressed genes in both rats and goats. Notably, 49 genes exhibited a consistent expression pattern, with 38 genes displaying up-regulation and 11 genes demonstrating down-regulation. Enrichment analysis highlighted the potential involvement of the interleukin-17 signaling pathway and vascular smooth muscle contraction pathway in the underlying mechanism of BLI. Furthermore, the experimental findings in both goats and rats demonstrated a strong association between BLI and several key genes, including anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4, which exhibited up-regulation. CONCLUSIONS: Anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4 hold potential as target genes for the prognosis, diagnosis, and treatment of BLI.

Identification of overlay differentially expressed genes in both rats and goats with blast lung injury through comparative transcriptomics / 中华创伤杂志(英文版)

PURPOSE@#To identify the potential target genes of blast lung injury (BLI) for the diagnosis and treatment.@*METHODS@#This is an experimental study. The BLI models in rats and goats were established by conducting a fuel-air explosive power test in an unobstructed environment, which was subsequently validated through hematoxylin-eosin staining. Transcriptome sequencing was performed on lung tissues from both goats and rats. Differentially expressed genes were identified using the criteria of q ≤ 0.05 and |log2 fold change| ≥ 1. Following that, enrichment analyses were conducted for gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathways. The potential target genes were further confirmed through quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay.@*RESULTS@#Observations through microscopy unveiled the presence of reddish edema fluid, erythrocytes, and instances of focal or patchy bleeding within the alveolar cavity. Transcriptome sequencing analysis identified a total of 83 differentially expressed genes in both rats and goats. Notably, 49 genes exhibited a consistent expression pattern, with 38 genes displaying up-regulation and 11 genes demonstrating down-regulation. Enrichment analysis highlighted the potential involvement of the interleukin-17 signaling pathway and vascular smooth muscle contraction pathway in the underlying mechanism of BLI. Furthermore, the experimental findings in both goats and rats demonstrated a strong association between BLI and several key genes, including anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4, which exhibited up-regulation.@*CONCLUSIONS@#Anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4 hold potential as target genes for the prognosis, diagnosis, and treatment of BLI.

Splice site m6A methylation prevents binding of DGCR8 to suppress KRT4 pre-mRNA splicing in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the 11th most prevalent tumor worldwide. Despite advantages of therapeutic approaches, the 5-year survival rate of patients with OSCC is less than 50%. It is urgent to elucidate mechanisms underlying OSCC progression for developing novel treatment strategies. Our recent study has revealed that Keratin 4 (KRT4) suppresses OSCC development, which is downregulated in OSCC. Nevertheless, the mechanism downregulating KRT4 in OSCC remains unknown. In this study, touchdown PCR was utilized to detect KRT4 pre-mRNA splicing, while m6A RNA methylation was identified by methylated RNA immunoprecipitation (MeRIP). Besides, RNA immunoprecipitation (RIP) was used to determine RNA-protein interaction. Herein, this study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m6A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. Besides, m6A methylation suppressed the binding of splice factor DGCR8 microprocessor complex subunit (DGCR8) to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC. These findings revealed the mechanism downregulating KRT4 in OSCC and provided potential therapeutic targets for OSCC.

White Sponge Nevus Caused by Keratin 4 Gene Mutation: A Case Report.

White sponge nevus (WSN) is a rare autosomal dominant disease with a family history, often caused by mutations of the keratin 4 (K4) and keratin 13 (K13) genes in patients. It is characterized by frequently occurred white corrugated folds in the bilateral buccal mucosa with soft texture. On histopathological examination, hyperkeratosis of epithelial cells, edema, and vacuolar changes in the spinous cells are observed in the lesions, despite a normal layer of basal cells. WSN should be differentiated from other oral white spot diseases, mainly oral lichen planus, oral candidiasis, oral white edema, and Heck's disease, to reduce misdiagnosis and unnecessary treatment. At present, there is no specific treatment method. The purpose of this study was to report the clinical data of four WSN patients of the same family with the K4 gene mutation. The occurrence of WSN in a pair of monozygotic twins with very similar clinical presentations was identified for the first time. The gene sequencing results showed that there was a heterozygous deletion (C. 438_440delCAA) in exon 1 of the K4 gene, resulting in an aspartic acid loss in both the proband and his father. Finally, the etiology, pathogenesis, pathological manifestations, clinical manifestations, diagnosis, differential diagnosis, and related treatment methods are discussed to provide a reference for clinical treatment of the disease.

[A boy with white sponge naevus of the buccal mucosa]. / Een jongen met witte afwijkingen op het mondslijmvlies.

White sponge naevus (WSN) is a rare, autosomal dominant disorder that causes various complaints WSN is most commonly found on the buccal mucosa. Clinically, the white, slightly elevated lesions of WSN may be confused with other disorders on oral mucosa. We report a case of WSN in a 14-year-old boy who had complaints for a considerable period of time. WSN is caused by mutations in KRT4 and KRT13.

CD24 and CK4 are upregulated by SIM2, and are predictive biomarkers for chemoradiotherapy and surgery in esophageal cancer.

Definitive chemoradiotherapy (CRT) is a less invasive therapy compared with surgery for some types of cancer; however, the 5­year survival rate of patients with stages II­III esophageal squamous cell carcinoma (ESCC) is only 37%. Therefore, prediction of CRT responders is necessary. Unfortunately, no definitive biomarker exists that is useful to predict survival outcome following CRT. From our previous microarray study, CD24 and keratin 4 (KRT4), which encodes cytokeratin 4 (CK4), were overexpressed in the favorable prognostic epithelial subtype with SIM bHLH transcription factor 2 (SIM2) expression. This study investigated the association between their mRNA and protein expression levels, and clinicopathological characteristics, and also investigated the functions of CD24 in SIM2­mediated tumor differentiation and CRT sensitivity. High CD24 and KRT4 mRNA expression was associated with a favorable prognosis following CRT. Multivariate analyses revealed that high CD24 and CK4 protein expression, as determined by immunohistochemistry, and differentiated type were independent factors for predicting a favorable prognosis in response to CRT. Notably, in cases with low CD24 or CK4, surgery was suggested to be a good therapeutic modality compared with CRT. CD24 and KRT4 were expressed preferentially in differentiated layers of the normal esophageal mucosa, and their mRNA expression in 3D cultured ESCC cells was induced by SIM2 transfection, thus suggesting that CD24 and KRT4 were downstream differentiation markers of SIM2. Furthermore, CD24 small interfering RNA increased the mRNA expression levels of superoxide dismutase 2 and enhanced H2O2 resistance, thus indicating the involvement of CD24 in the radiosensitivity of patients with ESCC; however, it had no effect on cisplatin sensitivity. In conclusion, the two markers CD24 and CK4 may be considered predictive biomarkers for definitive CRT.

Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4.

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. Recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. In the current study, tissues of eight dromedaries receiving inoculation of MERS-Coronavirus (MERS-CoV) after recombinant Modified-Vaccinia-Virus-Ankara (MVA-S)-vaccination (n = 4), MVA-vaccination (mock vaccination, n = 2) and PBS application (mock vaccination, n = 2), respectively, were investigated. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. MERS-CoV infection in mock-vaccinated dromedaries revealed high numbers of MERS-CoV-nucleocapsid positive cells, T cells, and macrophages within nasal turbinates and trachea at day four post infection. Double immunolabeling demonstrated cytokeratin (CK) 18 expressing epithelial cells to be the prevailing target cell of MERS-CoV, while CK5/6 and CK14 expressing cells did not co-localize with virus. In addition, virus was occasionally detected in macrophages. The acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase 4 (DPP4), known to mediate virus entry. DPP4 was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. The present study underlines significant species-specific manifestations of MERS and highlights ciliary loss as an important finding in dromedaries. The obtained results promote a better understanding of coronavirus infections, which pose major health challenges.

Keratin 4 regulates the development of human white sponge nevus.

BACKGROUND: The aim of this study was to investigate the roles of keratin 4 (KRT4) gene in the development of human white sponge nevus (WSN). METHODS: Transgenic mice were created using the microinjection method with pcDNA3.1 vectors expressing KRT4 wild-type (WT) gene and E520K mutation. Polymerase chain reaction (PCR) and Western blotting were used to identify the genotype of transgenic founders and their filial generations. Expression of KRT4 in mouse oral mucosa was characterized by immunohistochemistry (IHC), and the whole epithelium layer of transgenic mice was observed using transmission electron microscope (TEM). RESULTS: The positive rate of KRT4 transgenic mice in F1 generation was 45.5%. Expression level of KRT4 protein was significantly higher in 2-month-old transgenic mice than WT mice. Furthermore, all the epithelial lamina of 3-month-old transgenic mice showed reduced staining of KRT4. The surface and spinous layers were full of hyalocytes and bubble cells, which are similar to the clinical symptoms of WSN. For the ultrastructure, both tonofilaments and Odland bodies increased. CONCLUSIONS: Our study indicated the mutated KRT4 gene may play important roles in the pathogenesis of WSN.

Clinical features and molecular genetic analysis in a Turkish family with oral white sponge nevus.

BACKGROUND: Oral white sponge nevus (WSN) is a rare autosomal dominant benign condition, characterized by asymptomatic spongy white plaques. Mutations in Keratin 4 (KRT4) and 13 (KRT13) have been shown to cause WSN. Familial cases are uncommon due to irregular penetrance. Thus, the aim of the study was: a) to demonstrate the clinical and histopathological features of a three-generation Turkish family with oral WSN b) to determine whether KRT4 or KRT13 gene mutation was the molecular basis of WSN. MATERIAL AND METHODS: Out of twenty members of the family ten were available for assessment. Venous blood samples from six affected and five unaffected members and 48 healthy controls were obtained for genetic mutational analysis. Polymerase chain reaction was used to amplify all exons within KRT4 and KRT13 genes. These products were sequenced and the data was examined for mutations and polymorphisms. RESULTS: Varying presentation and severity of clinical features were observed. Analysis of the KRT13 gene revealed the sequence variant Y118D as the disease-causing mutation. One patient revealed several previously unreported polymorphisms including a novel mutation in exon 1 of the KRT13 gene and a heterozygous deletion in exon 1 of KRT4. This deletion in the KRT4 gene was found to be a common polymorphism reflecting a high allele frequency of 31.25% in the Turkish population. CONCLUSIONS: Oral WSN may manifest variable clinical features. The novel mutation found in the KRT13 gene is believed to add evidence for a mutational hotspot in the mucosal keratins. Molecular genetic analysis is required to establish correct diagnosis and appropriate genetic consultation.

Mutations in the genes for keratin-4 and keratin-13 in Swedish patients with white sponge nevus.

BACKGROUND: White sponge nevus is a rare autosomal dominant disorder that affects the non-keratinised stratified squamous epithelium. Mutations in the genes that encode mucosa-specific keratin-4 and keratin-13 are strongly linked to the manifestation of white sponge nevus. This study involved mutational analysis of the genes encoding keratin-4 and keratin-13 in two Swedish families with white sponge nevus. METHODS: The diagnosis of white sponge nevus was based on disease history, clinical characteristics of the lesions and, in the majority of the cases, histopathological examination. Samples were collected from the affected buccal mucosa using buccal swabs. DNA was subsequently extracted and amplified using touchdown-PCR. The keratin-4 and keratin-13 genes were sequenced, and a genetic analysis was performed. RESULTS: A novel heterozygous missense mutation was identified in exon 1A of the keratin-4 gene in Family 2. In addition, previously reported heterozygous missense mutations were identified in the keratin-4 (E449K, A72V, Q156R, R208H) and keratin-13 (L115P) genes in both families. CONCLUSION: We describe a novel heterozygous missense mutation in the keratin-4 gene of a Swedish family with white sponge nevus. Our results support the notion that mutations in keratin-4 and keratin-13 are the underlying cause of white sponge nevus.

A rare castration-resistant progenitor cell population is highly enriched in Pten-null prostate tumours.

Castration-resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild-type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSCmed according to their FACS profile (Lin- /Sca-1+ /CD49fmed ). Here, we investigated the prevalence and castration resistance of LSCmed in various mouse models of prostate tumourigenesis (Pb-PRL, Ptenpc-/- , and Hi-Myc mice). LSCmed prevalence is low (∼8%, similar to WT) in Hi-Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Ptenpc-/- prostates. LSCmed tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSCmed from Ptenpc-/- mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSCmed represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSCmed tolerate androgen deprivation. This also illuminates why Ptenpc-/- tumours are castration-resistant since LSCmed represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSCmed on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSCmed in various mouse prostate specimens. In castrated Ptenpc-/- prostates, there was significant proliferation of CK4+ cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSCmed as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate-resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The alcohol-sensitive period during early octavolateral organ development in zebrafish (Danio rerio).

Fetal alcohol exposure can cause Fetal Alcohol Spectrum Disorders (FASD), completely preventable developmental disabilities characterized by permanent birth defects. However, specific gestational timing when developing organs are most sensitive to alcohol exposure is unclear. In this study, we examined the temporal effects of embryonic alcohol exposure on octavolateral organs in zebrafish (Danio rerio), including inner ears and lateral line neuromasts that function in hearing, balance, and hydrodynamic detection, respectively. To determine an alcohol-sensitive period in the first 24 hours post fertilization (hpf), Et(krt4:EGFP)sqet4 zebrafish that express green fluorescent protein in sensory hair cells were treated in 2% alcohol for 2, 3, and 5-hours. Octavolateral organs of control and alcohol-exposed larvae were examined at 3, 5, and 7 days post fertilization (dpf). Using confocal and light microscopy, we found that alcohol-exposed larvae had significantly smaller otic vesicles and saccular otoliths than control larvae at 3 dpf. Only alcohol-exposed larvae from 12-17 hpf had smaller otic vesicles at 5 dpf, smaller saccular otoliths at 7 dpf and fewer saccular hair cells, neuromasts and hair cells per neuromast at 3 dpf. In addition, auditory function was assessed by microphonic potential recordings from inner ear hair cells in response to 200-Hz stimulation. Hearing sensitivity was reduced for alcohol-exposed larvae from 7-12 and 12-17 hpf. Our results show that 12-17 hpf is an alcohol-sensitive time window when morphology and function of zebrafish octavolateral organs are most vulnerable to alcohol exposure. This study implies that embryonic alcohol exposure timing during early development can influence severity of hearing deficits. © 2017 Wiley Periodicals, Inc.

A unique lineage gives rise to the meibomian gland.

PURPOSE: To identify the lineage that contributes to the morphogenesis of the meibomian gland. METHODS: To examine which cell lineage gives rise to the meibomian gland, the expression of Pax6 as well as that of various cytokeratin markers, including keratin 14 (Krt14), Krt15, Krt4, and Krt10, was examined with immunofluorescent staining of C57BL/6J mouse eyelids from P2 to P11 pups and adult mice. RESULTS: Pax6 was localized to the cytoplasm within the acinar region of the meibomian glands during morphogenesis but was absent in the fully developed gland. Keratin 14 was expressed throughout the gland at all stages whereas keratin 15 was absent at all stages. Keratin 4, a marker of mucosal lineage, was present throughout the gland and was colocalized with keratin 10 (epidermal lineage marker) in the developing duct at P4. This colocalization region decreased as the gland developed becoming restricted to the central duct near the opening to the acini in the fully developed gland. CONCLUSIONS: We identified a unique cell lineage that expresses markers characteristic of mucosal and epidermal epithelia during meibomian gland morphogenesis. This unique group of cells was located in the central duct with a concentration near the ductule orifice. The expression of these cells reduced during meibomian gland morphogenesis and may play a role in the development and homeostasis of the gland.

Ex vivo construction of a novel model of bioengineered bladder mucosa: A preliminary study.

OBJECTIVE: To generate and to evaluate ex vivo a novel model of bioengineered human bladder mucosa based on fibrin-agarose biomaterials. METHODS: We first established primary cultures of stromal and epithelial cells from small biopsies of the human bladder using enzymatic digestion and selective cell culture media. Then, a bioengineered substitute of the bladder lamina propria was generated using cultured stromal cells and fibrin-agarose scaffolds, and the epithelial cells were then subcultured on top to generate a complete bladder mucosa substitute. Evaluation of this substitute was carried out by cell viability and histological analyses, immunohistochemistry for key epithelial markers and transmission electron microscopy. RESULTS: The results show a well-configured stroma substitute with a single-layer epithelium on top. This substitute was equivalent to the control bladder mucosa. After 7 days of ex vivo development, the epithelial layer expressed pancytokeratin, and cytokeratins CK7, CK8 and CK13, as well as filaggrin and ZO-2, with negative expression of CK4 and uroplakin III. A reduction of the expression of CK8, filaggrin and ZO-2 was found at day 14 of development. An immature basement membrane was detected at the transition between the epithelium and the lamina propria, with the presence of epithelial hemidesmosomes, interdigitations and immature desmosomes. CONCLUSIONS: The present results suggest that this model of bioengineered human bladder mucosa shared structural and functional similarities with the native bladder mucosa, although the epithelial cells were not fully differentiated ex vivo. We hypothesize that this bladder mucosa substitute could have potential clinical usefulness after in vivo implantation.

Reconstruction of conjunctival epithelium-like tissue using a temperature-responsive culture dish.

PURPOSE: To study the feasibility of engineering conjunctival epithelial cell sheets on a temperature-responsive culture dish for ocular surface reconstruction. METHODS: Rabbit conjunctival epithelial cells (rCjECs) were cultured in DMEM/F-12 (1:1) medium. The morphology and phenotype of the rCjECs were confirmed with phalloidin staining, periodic acid-Schiff (PAS) staining, and immunocytochemistry. The rCjECs cultured on a temperature-responsive culture dish for 10 days produced confluent conjunctival epithelial cell sheets. Then, the phenotype, structure, and function of the conjunctival epithelial cell sheets were examined. RESULTS: The conjunctival epithelial cells were compact, uniform, and cobblestone shape. All cultured conjunctival epithelial cells were harvested as intact cell sheets by reducing the culture temperature to 20 °C. Conjunctival epithelial cells were stratified in four to five cell layers similar to the conjunctival epithelium. CCK-8 analysis, 5-bromo-2'-deoxyuridine (BrdU) staining, and the live and dead viability assay confirmed that viable proliferation cells were retained in the cell sheets. Immunohistochemistry for CK4, CK19, and MUC5AC showed the cell sheets still maintained characteristics of the conjunctival epithelium. CONCLUSIONS: A temperature-responsive culture dish enables fabrication of viable conjunctival epithelial cell sheets with goblet cells and proliferative cells. Conjunctival epithelial cell sheets will be promising for reconstruction of the conjunctival epithelium.

Latent transforming growth factor-ß binding proteins (LTBP-1 and LTBP-2) and gingiva keratinization.

OBJECTIVE: Transforming growth factor-beta (TGF-ß) proteins are involved in epithelial keratinization. The major function of latent TGF-ß binding proteins (LTBPs) is modulating TGF-ß activity. However, whether LTBP-1 and LTBP-2 play roles in gingiva keratinization remains unclear. MATERIALS AND METHODS: Human keratinized gingiva and non-keratinized alveolar mucosa were processed for LTBP-1, LTBP-2, cytokeratin-1 (K1), cytokeratin-4 (K4), and TGF-ß immunohistochemical (IHC) staining. Porcine heterotopically transplanted connective tissues and newly grown epithelia were harvested for IHC staining. The expression levels of LTBP-1 and LTBP-2 were compared between differentiated and undifferentiated human normal oral keratinocytes (hNOK). The expression of LTBP-1 and LTBP-2 was knocked down in a cell line (OEC-M1) to evaluate the effects on the expression of K1, K4, and involucrin (INV). RESULTS: In human and porcine specimens, LTBP-2 expression patterns distinguished keratinized and non-keratinized oral epithelia. Western blotting results showed that K1, LTBP-1, and INV proteins were upregulated in differentiated hNOK. In OEC-M1 cells, LTBP-2 knockdown resulted in upregulated the expression of K1 and INV and downregulated the expression of K4. LTBP-1 knockdown resulted in opposite effects. CONCLUSION: The expression patterns of LTBP-2 differ in keratinized gingiva and non-keratinized mucosa. LTBP-1 and LTBP-2 are involved in the keratinization of oral epithelium; however, the underlying mechanism remains to be elucidated.

Structure of KRT4 binding domain of Srr-1 from Streptococcus agalactiae reveals a novel ß-sheet complementation.

The serine rich repeat protein-1 (Srr-1) is an adhesive protein of Streptococcus agalactiae. It is the first bacterial protein identified to interact with human keratin 4 (K4 or KRT4). Within Srr-1, the residues 311-641 constitute the non-repeat ligand binding region (Srr-1-BR(311-641)). The C-terminal part of Srr-1-BR(311-641), comprising of residues 485-642 (termed Srr-1-K4BD), have been identified to bind to K4. Here we report the crystal structure of recombinant Srr-1-K4BD(485-642) and its possible mode of interaction with K4 through docking studies. The dimeric structure of Srr-1-K4BD(485-642) reveals a novel two way "slide lock" parallel ß-sheet complementation where the C-terminal strand of one monomer is positioned anti-parallel to the N-terminal strand of the adjacent monomer and this arrangement is not seen so far in any of the homologous structures. The dimerization of Srr-1-K4BD(485-642) observed both in the crystal structure and in solution suggests that similar domain association could also be possible in in vivo and we propose this association would likely generate a new binding site for another host molecule. It is likely that the adhesin can recognize multiple ligands using its ligand binding sub-domains through their intra and inter domain association with one another.

Early dental epithelial transcription factors distinguish ameloblastoma from keratocystic odontogenic tumor.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from 27 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 12 sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into 2 distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX2, MSX2, DLX2, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive growth, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX2 was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription factors. In contrast, gene expression of KCOT indicates differentiation toward keratinocytes.

Transcriptional regulation of genes involved in keratinocyte differentiation by human papillomavirus 16 oncoproteins.

The life cycle of human papillomaviruses (HPVs) is strictly linked to the differentiation of their natural host cells. The HPV E6 and E7 oncoproteins can delay the normal differentiation program of keratinocytes; however, the exact mechanisms responsible for this have not yet been identified. The goal of this study was to investigate the effects of HPV16 oncoproteins on the expression of genes involved in keratinocyte differentiation. Primary human keratinocytes transduced by LXSN (control) retroviruses or virus vectors expressing HPV16 E6, E7 or E6/E7 genes were subjected to gene expression profiling. The results of microarray analysis showed that HPV 16 E6 and E7 have the capacity to downregulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm the microarray data. To investigate the effects of the HPV oncoproteins on the promoters of selected keratinocyte differentiation genes, luciferase reporter assays were performed. Our results suggest that the HPV 16 E6 and/or E7 oncogenes are able to downregulate the expression of several genes involved in keratinocyte differentiation (such as desmocollin 1, keratin 4, S100 calcium-binding protein A8 and small proline-rich protein 1A), at least partially by downregulating their promoter activity. This activity of the HPV oncoproteins may have a role in the productive virus life cycle, and also in virus-induced carcinogenesis.