Functional Analysis of Membrane-Associated Scaffolding Tight Junction (TJ) Proteins in Tumorigenic Characteristics of B16-F10 Mouse Melanoma Cells.

Tight junction (TJ) proteins (Tjps), Tjp1 and Tjp2, are tight junction-associated scaffold proteins that bind to the transmembrane proteins of tight junctions and the underlying cytoskeleton. In this study, we first analyzed the tumorigenic characteristics of B16-F10 melanoma cells, including cell proliferation, migration, invasion, metastatic potential, and the expression patterns of related proteins, after the CRISPR-Cas9-mediated knockout (KO) of Tjp genes. The proliferation of Tjp1 and Tjp2 KO cells significantly increased in vitro. Other tumorigenic characteristics, including migration and invasion, were significantly enhanced in Tjp1 and Tjp2 KO cells. Zonula occludens (ZO)-associated protein Claudin-1 (CLDN-1), which is a major component of tight junctions and functions in controlling cell-to-cell adhesion, was decreased in Tjp KO cells. Additionally, Tjp KO significantly stimulated tumor growth and metastasis in an in vivo mouse model. We performed a transcriptome analysis using next-generation sequencing (NGS) to elucidate the key genes involved in the mechanisms of action of Tjp1 and Tjp2. Among the various genes affected by Tjp KO-, cell cycle-, cell migration-, angiogenesis-, and cell-cell adhesion-related genes were significantly altered. In particular, we found that the Ninjurin-1 (Ninj1) and Catenin alpha-1 (Ctnna1) genes, which are known to play fundamental roles in Tjps, were significantly downregulated in Tjp KO cells. In summary, tumorigenic characteristics, including cell proliferation, migration, invasion, tumor growth, and metastatic potential, were significantly increased in Tjp1 and Tjp2 KO cells, and the knockout of Tjp genes significantly affected the expression of related proteins.

Tea Tree Oil Terpinen-4-ol Protects Gut Barrier Integrity by Upregulation of Tight Junction Proteins via the ERK1/2-Signaling Pathway.

Tea tree oil (TTO) exhibits a potent antioxidant, antibacterial, and anti-inflammatory activity and is commonly used in skincare products. However, it is not clear whether TTO can protect gut barrier damage in inflammatory bowel disease (IBD) patients. Herein, we report the impact of terpinen-4-ol (TER, the primary constituent of TTO), on lipopolysaccharide (LPS)-induced intestinal epithelial cell barrier function impairment in intestinal porcine epithelial cell lines (IPEC-J2) and dextran sulfate sodium (DSS)-induced IBD in mice. TER protected against LPS-induced damage in IPEC-J2 cells in vitro and attenuated DSS-induced colitis in vivo. Added TER promoted the tight junction (TJ) proteins expressing in vitro and in vivo and attenuated the LPS-induced upregulation of ERK phosphorylation in IPEC-J2 cells. However, when an inhibitor of ERK phosphorylation was added, TER did not promote the expression of TJ protein, denoting that the ERK signaling pathway mediates the upregulation of TJ proteins. Our data may propose the potential application of TER in treating IBD.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma.

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.