The retinoid-related orphan receptor RORα promotes keratinocyte differentiation via FOXN1.

RORα is a retinoid-related orphan nuclear receptor that regulates inflammation, lipid metabolism, and cellular differentiation of several non-epithelial tissues. In spite of its high expression in skin epithelium, its functions in this tissue remain unclear. Using gain- and loss-of-function approaches to alter RORα gene expression in human keratinocytes (HKCs), we have found that this transcription factor functions as a regulator of epidermal differentiation. Among the 4 RORα isoforms, RORα4 is prominently expressed by keratinocytes in a manner that increases with differentiation. In contrast, RORα levels are significantly lower in skin squamous cell carcinoma tumors (SCCs) and cell lines. Increasing the levels of RORα4 in HKCs enhanced the expression of structural proteins associated with early and late differentiation, as well as genes involved in lipid barrier formation. Gene silencing of RORα impaired the ability of keratinocytes to differentiate in an in vivo epidermal cyst model. The pro-differentiation function of RORα is mediated at least in part by FOXN1, a well-known pro-differentiation transcription factor that we establish as a novel direct target of RORα in keratinocytes. Our results point to RORα as a novel node in the keratinocyte differentiation network and further suggest that the identification of RORα ligands may prove useful for treating skin disorders that are associated with abnormal keratinocyte differentiation, including cancer

The PIKfyve Inhibitor YM201636 Blocks the Continuous Recycling of the Tight Junction Proteins Claudin-1 and Claudin-2 in MDCK cells

Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation

Post-Transcriptional Regulation of Cadherin-11 Expression by GSK-3 and β-Catenin in Prostate and Breast Cancer Cells

The cell-cell adhesion molecule cadherin-11 is important in embryogenesis and bone morphogenesis, invasion of cancer cells, lymphangiogenesis, homing of cancer cells to bone, and rheumatoid arthritis. However, very little is known about the regulation of cadherin-11 expression.Here we show that cell density and GSK-3beta regulate cadherin-11 levels in cancer cells. Inactivation of GSK3beta with lithium chloride or the GSK3 inhibitor BIO and GSK3beta knockdown with siRNA repressed cadherin-11 mRNA and protein levels. RNA Polymerase II chromatin immunoprecipitation experiments showed that inhibition of GSK3 does not affect cadherin-11 gene transcription. Although the cadherin-11 3'UTR contains putative microRNA target sites and is regulated by Dicer, its stability is not regulated by GSK3 inhibition or density. Our data show that GSK3beta regulates cadherin-11 expression in two ways: first a beta-catenin-independent regulation of cadherin-11 steady state mRNA levels, and second a beta-catenin-dependent effect on cadherin-11 3'UTR stability and protein translation.Cadherin-11 mRNA and protein levels are regulated by the activity of GSK3beta and a significant degree of this regulation is exerted by the GSK3 target, beta-catenin, at the level of the cadherin-11 3'UTR

Yokukansan Inhibits Neuronal Death during ER Stress by Regulating the Unfolded Protein Response

Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death.We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein.Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs.Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD

RhoE Is Regulated by Cyclic AMP and Promotes Fusion of Human BeWo Choriocarcinoma Cells

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N6-phenyl-cAMP, and a specific inhibitor of PKA (14–22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways

Regulated Expression of ADAMTS-12 in Human Trophoblastic Cells: A Role for ADAMTS-12 in Epithelial Cell Invasion?

Metastatic carcinoma cells exploit the same molecular machinery that allows human placental cytotrophoblasts to develop an invasive phenotype. As altered expression levels of ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) subtypes have been associated with cancer progression, we have examined the function and regulation of members of this gene family in epithelial cell invasion using cultures of highly invasive extravillous cytotrophoblasts and the poorly invasive JEG-3 cytotrophoblast cell line as model systems. Of the multiple ADAMTS subtypes identified in first trimester human placenta and these two trophoblastic cell types, only ADAMTS-12 was preferentially expressed by extravillous cytotrophoblasts. Transforming growth factor-β1 and interleukin-1β, two cytokines that promote and restrain cytotrophoblast invasion in vitro, were also found to differentially regulate trophoblastic ADAMTS-12 mRNA levels. Loss- or gain-of-function studies confirmed that ADAMTS-12, independent of its proteolytic activity, plays a specific, non-redundant role in trophoblast invasion. Furthermore, we demonstrated that ADAMTS-12 regulated cell-extracellular matrix adhesion and invasion through a mechanism involving the αvβ3 integrin heterodimer. This study identifies a novel biological role for ADAMTS-12, and highlights the importance and complexity of its non-proteolytic domain(s) pertaining to its function

Plakophilin3 Loss Leads to an Increase in PRL3 Levels Promoting K8 Dephosphorylation, Which Is Required for Transformation and Metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis

Quality of Life as an outcome in Alzheimer's disease and other dementias- obstacles and goals

<p>Abstract</p> <p>Background</p> <p>The number of individuals at risk for dementia will probably increase in ageing societies as will the array of preventive and therapeutic options, both however within limited economic resources. For economic and medical purposes valid instruments are required to assess disease processes and the efficacy of therapeutic interventions for different forms and stages of illness. In principal, the impact of illness and success of an intervention can be assessed with biomedical variables, e.g. severity of symptoms or frequency of complications of a disease. However, this does not allow clear judgement on clinical relevance or comparison across different diseases.</p> <p>Discussion</p> <p>Outcome model variables such as quality of life (QoL) or health care resource utilization require the patient to appraise their own well-being or third parties to set preferences. In Alzheimer's disease and other dementias the evaluation process performed by the patient is subject to the disease process itself because over progress of the disease neuroanatomical structures are affected that mediate evaluation processes.</p> <p>Summary</p> <p>Published research and methodological considerations thus lead to the conclusion that current QoL-instruments, which have been useful in other contexts, are ill-suited and insufficiently validated to play a major role in dementia research, decision making and resource allocation. New models integrating biomedical and outcome variables need to be developed in order to meet the upcoming medical and economic challenges.</p

Epigenetic silencing of DSC3 is a common event in human breast cancer

INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure

Desmoglein 3, via an Interaction with E-cadherin, Is Associated with Activation of Src

Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion.To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation.Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation