Mtss1 promotes cell-cell junction assembly and stability through the small GTPase Rac1

Cell-cell junctions are an integral part of epithelia and are often disrupted in cancer cells during epithelial-to-mesenchymal transition (EMT), which is a main driver of metastatic spread. We show here that Metastasis suppressor-1 (Mtss1; Missing in Metastasis, MIM), a member of the IMD-family of proteins, inhibits cell-cell junction disassembly in wound healing or HGF-induced scatter assays by enhancing cell-cell junction strength. Mtss1 not only makes cells more resistant to cell-cell junction disassembly, but also accelerates the kinetics of adherens junction assembly. Mtss1 drives enhanced junction formation specifically by elevating Rac-GTP. Lastly, we show that Mtss1 depletion reduces recruitment of F-actin at cell-cell junctions. We thus propose that Mtss1 promotes Rac1 activation and actin recruitment driving junction maintenance. We suggest that the observed loss of Mtss1 in cancers may compromise junction stability and thus promote EMT and metastasis

Adherens junctions remain dynamic

One of the four principal categories of cell-cell junctions that hold together and shape distinct tissues and organs in vertebrates, adherens junctions (AJs) form cell-cell contacts that connect transmembrane proteins with cytoskeletal actin filaments to provide architectural strength, aid in morphogenesis, and help to maintain proper tissue homeostasis. The classical organization of AJs, consisting of transmembrane cadherins and cytoplasmically attached β-catenins and α-catenins assembled together into a multiprotein complex, was once thought obligatory to craft a robust and stable connection to actin-based cytoskeletal elements, but this architecture has since been challenged and questioned to exist. In a stimulating paper published in a recent issue of BMC Biology, Millán et al. provide convincing evidence that in confluent vascular endothelial cells a novel dynamic vascular endothelial (VE)-cadherin-based AJ type exists that interacts with and physically connects prominent bundles of tension-mediating actin filaments, stress fibers, between neighboring cells. Stress fibers were known previously to link to integrin-based focal adhesion complexes but not to cell-cell adhesion mediating AJs. These new findings, together with previous results support the concept that different AJ subtypes, sharing the same transmembrane cadherin types, can assemble in various configurations to either increase barrier function and promote physical cell-cell adhesion, or to lessen cell-cell adhesion and promote cell separation and migration

Rapid Suppression of Activated Rac1 by Cadherins and Nectins during De Novo Cell-Cell Adhesion

Cell-cell adhesion in simple epithelia involves the engagement of E-cadherin and nectins, and the reorganization of the actin cytoskeleton and membrane dynamics by Rho GTPases, particularly Rac1. However, it remains unclear whether E-cadherin and nectins up-regulate, maintain or suppress Rac1 activity during cell-cell adhesion. Roles for Rho GTPases are complicated by cell spreading and integrin-based adhesions to the extracellular matrix that occur concurrently with cell-cell adhesion, and which also require Rho GTPases. Here, we designed a simple approach to examine Rac1 activity upon cell-cell adhesion by MDCK epithelial cells, without cell spreading or integrin-based adhesion. Upon initiation of cell-cell contact in 3-D cell aggregates, we observed an initial peak of Rac1 activity that rapidly decreased by ∼66% within 5 minutes, and further decreased to a low baseline level after 30 minutes. Inhibition of E-cadherin engagement with DECMA-1 Fab fragments or competitive binding of soluble E-cadherin, or nectin2alpha extracellular domain completely inhibited Rac1 activity. These results indicate that cadherins and nectins cooperate to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion, which may be important in inhibiting the migratory cell phenotype and allowing the establishment of initially weak cell-cell adhesions

Anomalous Features of EMT during Keratinocyte Transformation

During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike “conventional EMT”, these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated

Sfrp Controls Apicobasal Polarity and Oriented Cell Division in Developing Gut Epithelium

Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut

Stabilisation of β-Catenin Downstream of T Cell Receptor Signalling

The role of TCF/β-catenin signalling in T cell development is well established, but important roles in mature T cells have only recently come to light.Here we have investigated the signalling pathways that are involved in the regulation of β-catenin in primary human T cells. We demonstrate that β-catenin expression is upregulated rapidly after T cell receptor (TCR) stimulation and that this involves protein stabilisation rather than an increase in mRNA levels. Similar to events in Wnt signalling, the increase in β-catenin coincides with an inhibition of GSK3, the kinase that is required for β-catenin degradation. β-catenin stabilisation in T cells can also be induced by the activation of PKC with phorbol esters and is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PKC). Upon TCR signalling, β-catenin accumulates in the nucleus and, parallel to this, the ratio of TCF1 isoforms is shifted in favour of the longer β-catenin binding isoforms. However, phosphorylated β-catenin, which is believed to be inactive, can also be detected and the expression of Wnt target genes Axin2 and dickkopf is down regulated.These data show that in mature human T cells, TCR signalling via PI3K and PKC can result in the stabilisation of β-catenin, allowing β-catenin to migrate to the nucleus. They further highlight important differences between β-catenin activities in TCR and Wnt signalling

CA125/MUC16 Is Dispensable for Mouse Development and Reproduction

Cancer antigen 125 (CA125) is a blood biomarker that is routinely used to monitor the progression of human epithelial ovarian cancer (EOC) and is encoded by MUC16, a member of the mucin gene family. The biological function of CA125/MUC16 and its potential role in EOC are poorly understood. Here we report the targeted disruption of the of the Muc16 gene in the mouse. To generate Muc16 knockout mice, 6.0 kb was deleted that included the majority of exon 3 and a portion of intron 3 and replaced with a lacZ reporter cassette. Loss of Muc16 protein expression suggests that Muc16 homozygous mutant mice are null mutants. Muc16 homozygous mutant mice are viable, fertile, and develop normally. Histological analysis shows that Muc16 homozygous mutant tissues are normal. By the age of 1 year, Muc16 homozygous mutant mice appear normal. Downregulation of transcripts from another mucin gene (Muc1) was detected in the Muc16 homozygous mutant uterus. Lack of any prominent abnormal phenotype in these Muc16 knockout mice suggests that CA125/MUC16 is not required for normal development or reproduction. These knockout mice provide a unique platform for future studies to identify the role of CA125/MUC16 in organ homeostasis and ovarian cancer