Claudin 1 Mediates TNFα-Induced Gene Expression and Cell Migration in Human Lung Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies

Biochemical Investigation of the Bacteriophage Protein HK97 gp74

Bacteriophages are viruses that infect and propagate within bacteria by making use of the host’s biosynthetic machinery. With a global population of 1031, phages pose a significant influence on microbial populations. Studies of bacteriophage proteins can elucidate the influence that bacteriophages play on the evolution of bacteria, as well as, providing the basis for the use of phage proteins as possible therapeutics and bioengineering solutions. This study aims to investigate the structural and functional role of the HK97 phage protein gp74. Sequence alignments indicate that gp74 is related to homing HNH endonucleases. Homing endonucleases are predominantly double-stranded DNases, suggesting that gp74 mediates integration of phage genes into the host genome or may target foreign phage DNA. DNA digestion experiments with gp74 reveals that gp74 mediates non-specific double-stranded cleavage of lambda phage DNA and single strand cleavage of plasmid DNA. Our initial work demonstrates that HK97 gp74 is an HNH endonuclease.MAS

Physiological Roles of the XB130/Tks5 Scaffold Protein Interaction for the Regulation of Cell Homeostasis

XB130 is a scaffold protein that regulates cell growth, survival and migration by forming dynamic complexes with other proteins that mediate signal transduction cascades for the regulation of cell function. Yeast two-hybrid screening identified another scaffold protein, tyrosine kinase substrate 5 (Tks5), as XB130’s top putative binding partner. We hypothesized that as scaffold proteins, XB130 and Tks5 interact to facilitate the formation of distinct molecular complexes for the mediation of signal transduction, which is required for the regulation of cellular processes, like growth, survival and migration for cell homeostasis. In the present study, we characterized the structural interaction between XB130 and Tks5 by demonstrating that XB130 N-terminal polyproline rich motifs bind to a WWY ligand binding motif in the 5th SH3 domain of TKS5. Tks5 is a well studied protein in cell migration processes. This study demonstrated that Tks5 plays a role in cell proliferation and survival of airway epithelial cells. Additionally, the XB130/Tks5 interaction was shown to regulate cell proliferation, growth and survival via EGFR/Src/PI3K signaling. Surprisingly, we discovered that XB130 and TKS5 play contrasting roles in cell migration; XB130 promotes lamellipodia formation and lateral cell migration, whereas TKS5 promotes podosome formation and extracellular matrix degradation for cell invasion. Consequently, XB130 was shown to dissociate from TKS5 to interact with Rac1 for the assembly and activation of the Rac1/PAK/WAVE2 lamellipodia pathway, whereas, Tks5 mediates the Cdc42/PAK/N-WASP podosome pathway for regulation of stimulus-dependent cytoskeletal rearrangement and cell morphology changes. In conclusion, the dynamic relationship between XB130 and TKS5 facilitates distinct protein-protein interactions and complex formation for the mediation of signal transduction and regulation of cell proliferation, survival and motility. Our data suggests that these dynamic scaffold protein interactions may be a common mechanism for proteins to form unique signaling complexes that facilitate the regulation or enhancement of different physiological or pathophysiological conditions.Ph.D

XB130—A Novel Adaptor Protein: Gene, Function, and Roles in Tumorigenesis

Several adaptor proteins have previously been shown to play an important role in the promotion of tumourigenesis. XB130 (AFAP1L2) is an adaptor protein involved in many cellular functions, such as cell survival, cell proliferation, migration, and gene and miRNA expression. XB130’s functional domains and motifs enable its interaction with a multitude of proteins involved in several different signaling pathways. As a tyrosine kinase substrate, tyrosine phosphorylated XB130 associates with the p85α regulatory subunit of phosphoinositol-3-kinase (PI3K) and subsequently affects Akt activity and its downstream signalling. Tumourigenesis studies show that downregulation of XB130 expression by RNAi inhibits tumor growth in mouse xenograft models. Furthermore, XB130 affects tumor oncogenicity by regulating the expression of specific tumour suppressing miRNAs. The expression level and pattern of XB130 has been studied in various human tumors, such as thyroid, esophageal, and gastric cancers, as well as, soft tissue tumors. Studies show the significant effects of XB130 in tumourigenesis and suggest its potential as a diagnostic biomarker and therapeutic target for cancer treatments.Peer Reviewe

Reducing Claudin 1 protein levels enhanced serum and/or EGF induced A549 cell migration, and Claudin 1 siRNA reduced TNFα-induced morphological changes of A549 cells.

<p>Cells were transfected with Claudin 1 or control siRNA. The confluence monolayers were serum starved, mechanically wounded and then stimulated with serum (10% FBS) and/or EGF (50 ng/ml). The wounded areas at 12 h were quantified. N = 4, Mean ± SEM. *P<0.05 vs. control siRNA treated group. (B). Claudin 1 siRNA clearly reduced Claudin 1 protein levels as shown by Western blotting. (C). Claudin 1 siRNA reduced TNFα- and TNFα plus TGFβ-induced morphological changes as shown by F-actin staining at 48 h. Similar results were also found after 24 h or 72 h of TNFα treatment.</p

Down-regulation of Claudin 1 with siRNA significantly blocked TNFα-induced gene expression.

<p>(A) Claudin 1 siRNA effectively reduced both basal and TNFα-induced gene expression of Claudin 1 (reading from microarray). (B) Principle Component Analysis (PCA) showed that the overall gene expression profiles are separated based on the Claudin 1 siRNA transfection and TNFα treatment. (C) Hierarchical clustering analysis demonstrates that gene expression patterns are highly dependent upon Claudin 1 siRNA transfection and TNFα treatments. A two-way ANOVA showed that 2,490 genes were significantly different. Red: up-regulated; blue: down-regulated. (D) Claudin 1 siRNA blocked 75% of the TNFα-induced gene expression changes. The gene expressions that were significantly changed by TNFα were defined with FDR q value less than 5.0% and fold of change greater than 1.3 by SAM analysis. Venn diagram shows that 75% of TNFα-induced expression changes were not shown in Claudin 1 siRNA transfected cells.</p

TNFα-induced Claudin 1 protein is mainly in the cytosolic fraction.

<p>(A) At 24, 48 h after treating the cells with TNFα, expression of Claudin 1 protein was increased mainly in cytoplasm. A549 cells were immunostained with an anti-Claudin 1 antibody and counterstained F-actin and nuclei with rhodamine phalloidin and Hoechst 33342, respectively. (B) Confocal microscopy further confirmed the cytosolic distribution of Claudin 1 in both control and TNFα groups. (C) Claudin 1 and GAPDH were mainly found in the Triton X-100 soluble (S) cytoplasm fraction, whereas ZO-1 and Occludin were mainly in the Triton X-100 insoluble cytoskeletal pellets (P). A549 cells treated with or without TNFα (20 ng/ml for 24 h) were lyzed and separated into Triton X-100 soluble and insoluble fractions for immunoblotting.</p

TNFα and TGFβ1 induce EMT in A549 cells.

<p>(A) The combined treatment of TNFα (20 ng/ml) and TGFβ1 (10 ng/ml) for 72 h induced morphological alterations characterized as fibroblast-like cells. (B) TNFα and TGFβ1 treatment reduced expression of E-Cadherin at cell-to-cell contacts, and increased formation of F-actin stress fibers. Cells were immunostained with an anti-E-Cadherin antibody and counterstained F-actin and nuclei with rhodamine phalloidin and Hoechst 33342, respectively. (C) The redistribution of E-cadherin after TGFβ and TNFα treatment from the cell-to-cell contacts to cytosol was further demonstrated with confocal microscopy at higher maginification. (D) The 72 h treatment with TNFα and TGFβ1 decreased expressions of E-Cadherin and Occludin, epithelial markers, and increased expressions of Vimentin, a mesenchymal marker, in A549 cells. Surprisingly, the expression of Claudin 1, an epithelial marker, was increased as analyzed by western blotting.</p

Top 10 down-regulated genes induced by TNFα in Control-siRNA or Claudin 1-siRNA transfected A549 cells.

<p>Top 10 down-regulated genes induced by TNFα in Control-siRNA or Claudin 1-siRNA transfected A549 cells.</p

TNFα alone induces fibroblast-like morphology and Claudin 1 expression.

<p>(A) TNFα alone induced fibroblast-like morphological alteration, reduced cell-to-cell contacts, and increased F-actin stress fibers. A549 cells treated with TNFα, TGFβ1 or TNFα and TGFβ1 together for 72 h were examined with light microscopy and stained with rhodamine phalloidin to visualize F-actin structures. TNFα-induced changes were similar as that of the TNFα and TGFβ1 treatment, whereas TGFβ1 alone did not induce these changes. (B) Protein levels of E-Cadherin were more effectively reduced by TGFβ1 than TNFα. TNFα alone increased Claudin 1 expression, whereas TGFβ1 had little effects on Claudin 1 after 24 h or 48 h treatment, and even increased it after 72 h treatment, as determined by western blotting. (C) In MDCK cells, TGFβ1 reduced Claudin 1 after 24, 48 or 72 h treatment. (D) Expression levels of Claudin 1 mRNA were significantly increased by TNFα in a time-dependent manner as measured by real-time quantitative RT-PCR. Mean ± SEM. <i>n</i> = 3 experiments. *<i>p</i><0.05 (compared with control at 24 h). #<i>p</i><0.05 (compared with control at 48 h).</p