Pattern-Specific Loss of Desmoplakin I and II Immunoreactivity in Erythema Multiforme and its Variants: A Possible Aid in Histologic Diagnosis.

Erythema multiforme (EM), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN) comprise a family of mucocutaneous diseases associated with significant morbidity and mortality. Previous studies have confirmed the presence of autoantibodies to desmoplakin (Dp) I and II in patients with EM, SJS, and TEN. Truncated Dp production leads to characteristic changes visible on light microscopy: perinuclear clumping of keratin filaments and dyskeratotic keratinocyte. Based on these observations, the question arises as to whether a loss of Dp immunoreactivity in skin biopsies could serve as a diagnostic marker of EM, SJS, and TEN. This study analyzed Dp immunostaining patterns in 20 patients with EM or SJS/TEN. To assess the specificity of this approach, Dp immunostaining was also performed on specimens from patients with 5 potential histologic mimics of EM, SJS, and TEN. All of the samples from patients with EM, SJS, and TEN demonstrated absent or markedly diminished staining for Dp. A χ test demonstrated a statistically significant difference between the staining patterns in EM, SJS, and TEN and each of the other diagnostic groups that were investigated. This is the first report demonstrating statistically significant specificity of Dp staining patterns in EM/SJS/TEN as compared with other interface dermatitides.

Novel Role for γ-Catenin in the Regulation of Cancer Cell Migration via the Induction of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1).

γ-Catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors.

High motility of triple-negative breast cancer cells is due to repression of plakoglobin gene by metastasis modulator protein SLUG.

One of highly pathogenic breast cancer cell types are the triple negative (negative in the expression of estrogen, progesterone, and ERBB2 receptors) breast cancer cells. These cells are highly motile and metastatic and are characterized by high levels of the metastasis regulator protein SLUG. Using isogenic breast cancer cell systems we have shown here that high motility of these cells is directly correlated with the levels of the SLUG in these cells. Because epithelial/mesenchymal cell motility is known to be negatively regulated by the catenin protein plakoglobin, we postulated that the transcriptional repressor protein SLUG increases the motility of the aggressive breast cancer cells through the knockdown of the transcription of the plakoglobin gene. We found that SLUG inhibits the expression of plakoglobin gene directly in these cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Blocking of SLUG function with a double-stranded DNA decoy that competes with the E2-box binding of SLUG also increased the levels of plakoglobin mRNA, protein, and promoter activity in the SLUG-high triple negative breast cancer cells. Overexpression of SLUG in the SLUG-deficient cells elevated the motility of these cells. Knockdown of plakoglobin in these low motility non-invasive breast cancer cells rearranged the actin filaments and increased the motility of these cells. Forced expression of plakoglobin in SLUG-high cells had the reverse effects on cellular motility. This study thus implicates SLUG-induced repression of plakoglobin as a motility determinant in highly disseminating breast cancer.

DeltaN TP63 reactivation, epithelial phenotype maintenance, and survival in lung squamous cell carcinoma.

Genes, active during normal development, are frequently reactivated during neoplastic transformation and may be related to progression. One of them, the transcription factor TP63, is crucial for pulmonary epithelial development and a possible target of the recurrent 3q amplifications in lung squamous cell carcinoma (SCC). Here, we explored whether TP63 reactivation could be associated to cancer progression in lung SCC through an epithelial to mesenchymal transition. We studied TP63 amplification and TP63 expression at RNA and protein levels and we analyzed the ΔNTP63/TATP63 ratio that quantifies the proportion of the isoform lacking the transactivation domain/the isoform containing the transactivation domain. We correlated TP63 status to survival and to the expression of epithelial (E-cadherin and plakoglobin) and mesenchymal (N-cadherin, vimentin, TWIST1, and SNAIL) markers. We found that high ΔN/TA TP63 ratio was related to high E-cadherin and plakoglobin mRNA levels (P < 0.05) and that E-cadherin mRNA level was the only marker related to survival. Kaplan-Meier survival curves stratified according to the expression level of E-cadherin showed, as already reported in breast cancer, that patients with low (first quartile) or high (last quartile) E-cadherin expression had a worse survival with respect to patients with intermediate E-cadherin expression. Altogether, our results indicate that a reactivation of ΔNTP63 is linked to the maintenance of epithelial markers and suggest that E-cadherin has a dual role in lung SCC.

Cytokeratin-related loss of cellular integrity is not a major driving force of human intrinsic skin aging.

The contribution of extracellular matrix components to intrinsic skin aging has been investigated thoroughly, however, there is little information as to the role of the cytoskeletal proteins in this process. Therefore, we compared the expression of the constituents of the cytoskeleton, keratins 1-23 (K1-K23) as well as junction-plakoglobin (JUP), alpha-tubulin (TUBA), and beta-actin (ACTB) in human foreskins of both young (mean 6.4 years) and aged (mean 54.3 years) individuals. By applying RNA expression analysis to intrinsically aged human skin, we demonstrated that the mRNA levels of the genes for K1, K3, K4, K9, K13, K15, K18, K19 and K20 are downregulated in aged skin, K5 and K14 are unchanged, and K2, K16 and K17 are upregulated in aged skin. The mRNA data were confirmed on the protein level. This diverse picture is in contrast to other cytoskeletal proteins including components of the desmosome (JUP), microtubuli (TUBA) and microfilaments (ACTB) - often regarded as house-keeping genes - that were all reduced in aged skin. These cytoskeletal features of intrinsic aging highlight the importance of the cellular compartment in this process and demonstrate that special attention has to be given to RNA as well as protein normalization in aging studies.

Modulation of gingival epithelial phenotypes by interactions with regionally defined populations of fibroblasts.

BACKGROUND AND OBJECTIVE: The unusual structure and functions of junctional epithelium, together with its pattern of migration in periodontal disease, raise interesting questions about the factors associated with the maintenance of its unique phenotype. To explore the effects of regionally differing fibroblast populations on the growth and patterns of differentiation of oral epithelia, this study used an organotypical in vitro model in an attempt to detect interactions occurring between populations of human oral fibroblasts and keratinocytes. MATERIAL AND METHODS: Keratinocytes and fibroblasts, isolated from the gingival region and periodontal ligament, were characterized by their patterns of growth and by their expression of known differentiation markers. Changes in cell behaviour and phenotypic marker expression were examined during in vitro passage as an indication of the maintenance of in vivo phenotypic traits. Using early passage cells, organotypical cultures were generated and patterns of epithelial growth and expression of phenotypic markers were examined. RESULTS: Phenotypically different populations of junctional and oral-gingival keratinocytes, and of oral-gingival and periodontal ligament fibroblasts, were successfully isolated, cultured and characterized. In the organotypic culture system, oral-gingival fibroblasts were found to have a markedly greater ability than periodontal ligament fibroblasts to support and maintain the growth of either type of epithelium. Shifts of epithelial phenotype were induced by different fibroblasts. CONCLUSION: Periodontal and gingival fibroblast subpopulations have differential effects on the growth and patterns of differentiation of oral and junctional epithelia. By modulating the epithelial phenotype, regionally differing fibroblasts can influence the stability and behaviour of the gingival attachment apparatus in health and disease.

Comparison of P75 NTR-positive and -negative etcomesenchymal stem cell odontogenic differentiation through epithelial-mesenchymal interaction.

OBJECTIVES: The aim of this study was to investigate differences of odonto-differentiation between P75 -neurotrophin receptor (P75 -NTR)-positive ectomesenchymal stem cells (P75+EMSCs) and P75 -NTR-negative ectomesenchymal stem cells (P75-EMSCs), and their underlying mechanisms. MATERIALS AND METHODS: Primary cranial neural crest-derived cells (CNC) were isolated from the first branchial arches, and P75+EMSCs and P75-EMSCs were sorted by fluorescence-activated cell sorting. Differentiation of P75+EMSCs or P75-EMSCs into odontoblast-like cells was induced by dental epithelial cells in vitro or in vivo. Differential gene expression profiles between P75+EMSCs and P75-EMSCs were analysed by microarray assay. Smad4-specific small interfering RNA and activator kartogenin were used to treat the cells, to evaluate effects of Smad4 in odonto-differentiation of P75+EMSCs or P75-EMSCs. RESULTS: Under induction of dental epithelium conditioned medium, P75+EMSCs had more mineralized node formation and higher expression of Dmp1 and Dspp compared to P75-EMSCs. In our in vivo study, graft of P75+EMSCs recombination with dental epithelium showed higher expression of DMP1 and DSP. Knock-down of Smad4 in P75+EMSCs significantly downregulated expression of DMP1 and DSP, while activation of Smad4 in P75-EMSCs by the activator kartogenin, significantly increased DSP and DMP1 expression. CONCLUSIONS: P75+EMSCs showed more odonto-differentiation potential than P75-EMSCs both in vivo and in vitro. Smad4 played a critical role in determination of odonto-differentiation potential of CNC-derived EMSCs.

Plakoglobin represses SATB1 expression and decreases in vitro proliferation, migration and invasion.

Plakoglobin (γ-catenin) is a homolog of ß-catenin with dual adhesive and signaling functions. Plakoglobin participates in cell-cell adhesion as a component of the adherens junction and desmosomes whereas its signaling function is mediated by its interactions with various intracellular protein partners. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in the human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We observed that the mRNA levels of SATB1, the oncogenic chromatin remodeling factor, were decreased approximately 3-fold in SCC9-PG cells compared to parental SCC9 cells. Here, we showed that plakoglobin decreased levels of SATB1 mRNA and protein in SCC9-PG cells and that plakoglobin and p53 associated with the SATB1 promoter. Plakoglobin expression also resulted in decreased SATB1 promoter activity. These results were confirmed following plakoglobin expression in the very low plakoglobin expressing and invasive mammary carcinoma cell line MDA-MB-231 cells (MDA-231-PG). In addition, knockdown of endogenous plakoglobin in the non-invasive mammary carcinoma MCF-7 cells (MCF-7-shPG) resulted in increased SATB1 mRNA and protein. Plakoglobin expression also resulted in increased mRNA and protein levels of the metastasis suppressor Nm23-H1, a SATB1 target gene. Furthermore, the levels of various SATB1 target genes involved in tumorigenesis and metastasis were altered in MCF-7-shPG cells relative to parental MCF-7 cells. Finally, plakoglobin expression resulted in decreased in vitro proliferation, migration and invasion in different carcinoma cell lines. Together with the results of our previous studies, the data suggests that plakoglobin suppresses tumorigenesis and metastasis through the regulation of genes involved in these processes.

Novel 5'TOPmRNAs regulated by ribosomal S6 kinase are important for cardiomyocyte development: S6 kinase suppression limits cardiac differentiation and promotes pluripotent cells toward a neural lineage.

Moving stem cells from bench to bedside has been a challenging task. Undermining this task is comprehending and optimizing the underlying regulatory mechanisms that drive differentiation of stem cells into desired cell and tissue types. Here we present evidence that ribosomal S6 kinase (S6K) is among the proteins upregulated as embryonic stem cells (ESCs) and human induced pluripotent stem cells differentiate into beating cardiomyocytes. We hypothesized that S6K plays a pivotal role in cardiomyogenesis, primarily because it regulates the translation of 3 cardiac-involved genes recently shown to have 5' terminal oligopyrimidine (5'TOP) sequences: connexin 43 (Cx43), desmoplakin (Dsp), and phosphatase and tensin homolog (PTEN). Along with another independent laboratory, we confirmed that S6K is indeed upregulated in beating ESC-derived cardiomyocytes compared to the surrounding nonbeating, differentiated cells. S6K short interfering RNA-transfected stem cell cultures indicate that inhibition of S6K strongly hinders development of cardiomyocyte beating and translation of Cx43, Dsp, and PTEN; these cardiac 5'TOP mRNAs were only properly translated in cells with S6K, supporting our hypothesis. An unexpected discovery took the role of S6K one step further: S6K-knockdown stem cell cultures developed significantly more neurons than seen in embryoid bodies subjected to a typical cardiac differentiation protocol. These results introduced the novel idea that in addition to its critical cardiac roles, S6K may be a significant factor that prevents stem cells from pursuing a neuronal pathway. Overall, results have indicated the necessity of S6K for normal stem cell cardiomyogenesis, as well as lowered S6K expression for stem cell neurogenesis.

Dioscorin protects tight junction protein expression in A549 human airway epithelium cells from dust mite damage.

BACKGROUND AND PURPOSE: In addition to being an allergen, the trypsin activity of dust mite extract also destroys the tight junctions of bronchial epithelium. Such damage can lead to airway leakage, which increases airway exposure to allergens, irritants, and other pathogens. Dioscorin, the storage protein of yam, demonstrates anti-trypsin activity, as well as other potential anti-inflammatory effects. This study investigated the protective role of dioscorin for tight junctions. METHODS: The immunofluorescence stains of zonula occludens (ZO-1), E-cadherin (EC) and desmoplakin (DP) proteins were compared. A cultured A549 cell line was used as a control and A549 cells were incubated with mite extract 100 mg/mL for 16 h, with or without dioscorin 100 mg/mL pretreatment for 8 h and with dioscorin 100 mg/mL alone for 16 h. Western blot was performed to detect changes in ZO-1, EC, and DP in the treated A549 cell lines. RESULTS: Loss of tight junction protein expression (ZO-1, EC, DP) was demonstrated after 16-h mite extract incubation. The defect could be restored if cells were pretreated with dioscorin for 8 h. In addition, dioscorin did not cause damage to the A549 cell lines in terms of cell survival or morphology. Western blot showed no change in the amount of tight junction protein under various conditions. CONCLUSION: Dioscorin is a potential protector of airway damage caused by mite extract.