Claudin-1 Is a p63 Target Gene with a Crucial Role in Epithelial Development

The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of ΔNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (−80%). ΔNp63α binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a –1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon–Ectodermal dysplasia–Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias

Enhancement of metastatic ability by ectopic expression of ST6GalNAcI on a gastric cancer cell line in a mouse model

ST6GalNAcI is a sialyltransferase responsible for the synthesis of sialyl Tn (sTn) antigen which is expressed in a variety of adenocarcinomas including gastric cancer especially in advanced cases, but the roles of ST6GalNAcI and sTn in cancer progression are largely unknown. We generated sTn-expressing human gastric cancer cells by ectopic expression of ST6GalNAcI to evaluate metastatic ability of these cells and prognostic effect of ST6GalNAcI and sTn in a mouse model, and identified sTn carrier proteins to gain insight into the function of ST6GalNAcI and sTn in gastric cancer progression. A green fluorescent protein-tagged human gastric cancer cell line was transfected with ST6GalNAcI to produce sTn-expressing cells, which were transplanted into nude mice. STn-positive gastric cancer cells showed higher intraperitoneal metastatic ability in comparison with sTn-negative control, resulting in shortened survival time of the mice, which was mitigated by anti-sTn antibody administration. Then, sTn-carrying proteins were immunoprecipitated from culture supernatants and lysates of these cells, and identified MUC1 and CD44 as major sTn carriers. It was confirmed that MUC1 carries sTn also in human advanced gastric cancer tissues. Identification of sTn carrier proteins will help understand mechanisms of metastatic phenotype acquisition of gastric cancer cells by ST6GalNAcI and sTn

Transcriptome Analysis of the Octopus vulgaris Central Nervous System

Background: Cephalopoda are a class of Mollusca species found in all the world's oceans. They are an important model organism in neurobiology. Unfortunately, the lack of neuronal molecular sequences, such as ESTs, transcriptomic or genomic information, has limited the development of molecular neurobiology research in this unique model organism. Results: With high-throughput Illumina Solexa sequencing technology, we have generated 59,859 high quality sequences from 12,918,391 paired-end reads. Using BLASTx/BLASTn, 12,227 contigs have blast hits in the Swissprot, NR protein database and NT nucleotide database with E-value cutoff 1e(-5). The comparison between the Octopus vulgaris central nervous system (CNS) library and the Aplysia californica/Lymnaea stagnalis CNS ESTs library yielded 5.93%/13.45% of O. vulgaris sequences with significant matches (1e(-5)) using BLASTn/tBLASTx. Meanwhile the hit percentage of the recently published Schistocerca gregaria, Tilapia or Hirudo medicinalis CNS library to the O. vulgaris CNS library is 21.03%-46.19%. We constructed the Phylogenetic tree using two genes related to CNS function, Synaptotagmin-7 and Synaptophysin. Lastly, we demonstrated that O. vulgaris may have a vertebrate-like Blood-Brain Barrier based on bioinformatic analysis. Conclusion: This study provides a mass of molecular information that will contribute to further molecular biology research on O. vulgaris. In our presentation of the first CNS transcriptome analysis of O. vulgaris, we hope to accelerate the study of functional molecular neurobiology and comparative evolutionary biology.National fund for oceanography research in Public Interest [201005013]; National Key Technology RD Program [2011BAD13

GEF-H1 Mediated Control of NOD1 Dependent NF-κB Activation by Shigella Effectors

Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1) mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-κB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-κB activation by H-Ala-D-γGlu-DAP (γTriDAP). GEF-H1 is essential for NF-κB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK) dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens

A Membrane Fusion Protein αSNAP Is a Novel Regulator of Epithelial Apical Junctions

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins

Protective effects of N-acetylcysteine on acetic acid-induced colitis in a porcine model

BACKGROUND: Ulcerative colitis is a chronic inflammatory disease and involves multiple etiological factors. Acetic acid (AA)-induced colitis is a reproducible and simple model, sharing many characteristics with human colitis. N-acetylcysteine (NAC) has been widely used as an antioxidant in vivo and in vitro. NAC can affect several signaling pathways involving in apoptosis, angiogenesis, cell growth and arrest, redox-regulated gene expression, and inflammatory response. Therefore, NAC may not only protect against the direct injurious effects of oxidants, but also beneficially alter inflammatory events in colitis. This study was conducted to investigate whether NAC could alleviate the AA-induced colitis in a porcine model. METHODS: Weaned piglets were used to investigate the effects of NAC on AA-induced colitis. Severity of colitis was evaluated by colon histomorphology measurements, histopathology scores, tissue myeloperoxidase activity, as well as concentrations of malondialdehyde and pro-inflammatory mediators in the plasma and colon. The protective role of NAC was assessed by measurements of antioxidant status, growth modulator, cell apoptosis, and tight junction proteins. Abundances of caspase-3 and claudin-1 proteins in colonic mucosae were determined by the Western blot method. Epidermal growth factor receptor, amphiregulin, tumor necrosis factor-alpha (TNF-α), and toll-like receptor 4 (TLR4) mRNA levels in colonic mucosae were quantified using the real-time fluorescent quantitative PCR. RESULTS: Compared with the control group, AA treatment increased (P < 0.05) the histopathology scores, intraepithelial lymphocyte (IEL) numbers and density in the colon, myeloperoxidase activity, the concentrations of malondialdehyde and pro-inflammatory mediators in the plasma and colon, while reducing (P < 0.05) goblet cell numbers and the protein/DNA ratio in the colonic mucosa. These adverse effects of AA were partially ameliorated (P < 0.05) by dietary supplementation with NAC. In addition, NAC prevented the AA-induced increase in caspase-3 protein, while stimulating claudin-1 protein expression in the colonic mucosa. Moreover, NAC enhanced mRNA levels for epidermal growth factor and amphiregulin in the colonic mucosa. CONCLUSION: Dietary supplementation with NAC can alleviate AA-induced colitis in a porcine model through regulating anti-oxidative responses, cell apoptosis, and EGF gene expression