Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion.

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.

IFN-gamma promotes generation of IL-10 secreting CD4+ T cells that suppress generation of CD8 responses in an antigen-experienced host.

Ags characterizing tumors or chronic viral infection are generally presented to the host immune system before specific immunotherapy is initiated, and consequent generation of regulatory CD4(+) T cells can inhibit induction of desired effector CD8 T cell responses. IL-10 produced in response to ongoing Ag exposure inhibits generation of CD8 T cells in an Ag-experienced host. We now show that this IL-10 is produced by Ag experienced CD4(+) glucocorticoid-induced tumor necrosis factor receptor(+) T cells that also secrete IFN-gamma upon antigenic stimulation, that IL-10 secretion by these cells is enhanced through IFN-gamma signaling, and, unexpectedly, that IFN-gamma signaling is required for inhibition of generation of Ag-specific CD8 T cell responses in an Ag-experienced host. Systemic inhibition of both IL-10 and IFN-gamma at the time of immunization may therefore facilitate induction of effective immunotherapeutic responses against tumor specific and viral Ags.

Tension-sensitive actin assembly supports contractility at the epithelial zonula adherens.

BACKGROUND: Actomyosin-based contractility acts on cadherin junctions to support tissue integrity and morphogenesis. The actomyosin apparatus of the epithelial zonula adherens (ZA) is built by coordinating junctional actin assembly with Myosin II activation. However, the physical interaction between Myosin and actin filaments that is necessary for contractility can induce actin filament turnover, potentially compromising the contractile apparatus itself. RESULTS: We now identify tension-sensitive actin assembly as one cellular solution to this design paradox. We show that junctional actin assembly is maintained by contractility in established junctions and increases when contractility is stimulated. The underlying mechanism entails the tension-sensitive recruitment of vinculin to the ZA. Vinculin, in turn, directly recruits Mena/VASP proteins to support junctional actin assembly. By combining strategies that uncouple Mena/VASP from vinculin or ectopically target Mena/VASP to junctions, we show that tension-sensitive actin assembly is necessary for junctional integrity and effective contractility at the ZA. CONCLUSIONS: We conclude that tension-sensitive regulation of actin assembly represents a mechanism for epithelial cells to resolve potential design contradictions that are inherent in the way that the junctional actomyosin system is assembled. This emphasizes that maintenance and regulation of the actin scaffolds themselves influence how cells generate contractile tension.

Vinculin, cadherin mechanotransduction and homeostasis of cell-cell junctions.

Cell adhesion junctions characteristically arise from the cooperative integration of adhesion receptors, cell signalling pathways and the cytoskeleton. This is exemplified by cell-cell interactions mediated by classical cadherin adhesion receptors. These junctions are sites where cadherin adhesion systems functionally couple to the dynamic actin cytoskeleton, a process that entails physical interactions with many actin regulators and regulation by cell signalling pathways. Such integration implies a potential role for molecules that may stand at the interface between adhesion, signalling and the cytoskeleton. One such candidate is the cortical scaffolding protein, vinculin, which is a component of both cell-cell and cell-matrix adhesions. While its contribution to integrin-based adhesions has been extensively studied, less is known about how vinculin contributes to cell-cell adhesions. A major recent advance has come with the realisation that cadherin adhesions are active mechanical structures, where cadherin serves as part of a mechanotransduction pathway by which junctions sense and elicit cellular responses to mechanical stimuli. Vinculin has emerged as an important element in cadherin mechanotransduction, a perspective that illuminates its role in cell-cell interactions. We now review its role as a cortical scaffold and its role in cadherin mechanotransduction.

Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens.

Classic cadherin receptors cooperate with regulators of the actin cytoskeleton to control tissue organization in health and disease. At the apical junctions of epithelial cells, the cadherin ring of the zonula adherens (ZA) couples with a contiguous ring of actin filaments to support morphogenetic processes such as tissue integration and cellular morphology. However, the molecular mechanisms that coordinate adhesion and cytoskeleton at these junctions are poorly understood. Previously we identified non-muscle myosin II as a target of Rho signalling that supports cadherin junctions in mammalian epithelial cells. Myosin II has various cellular functions, which are increasingly attributable to the specific biophysical properties and regulation of its different isoforms. Here we report that myosin II isoforms have distinct and necessary roles at cadherin junctions. Although two of the three mammalian myosin II isoforms are found at the ZA, their localization is regulated by different upstream signalling pathways. Junctional localization of myosin IIA required E-cadherin adhesion, Rho/ROCK and myosin light-chain kinase, whereas junctional myosin IIB depended on Rap1. Further, these myosin II isoforms support E-cadherin junction integrity by different mechanisms. Myosin IIA RNA-mediated interference (RNAi) selectively perturbed the accumulation of E-cadherin in the apical ZA, decreased cadherin homophilic adhesion and disrupted cadherin clustering. In contrast, myosin IIB RNAi decreased filament content, altered dynamics, and increased the lateral movement of the perijunctional actin ring. Myosin IIA and IIB therefore identify two distinct functional modules, with different upstream signals that control junctional localization, and distinct functional effects. We propose that these two isoform-based modules cooperate to coordinate adhesion receptor and F-actin organization to form apical cadherin junctions.

Phosphatidylinositol 3'-kinase signalling supports cell height in established epithelial monolayers.

Cell-cell interactions influence epithelial morphogenesis through an interplay between cell adhesion, trafficking and the cytoskeleton. These cellular processes are coordinated, often by cell signals found at cell-cell contacts. One such contact-based signal is the phosphatidylinositol 3'-kinase (PI3-kinase; PI3K) pathway. PI3-kinase is best understood for its role in mitogenic signalling, where it regulates cell survival, proliferation and differentiation. Its precise morphogenetic impacts in epithelia are, in contrast, less well-understood. Using phosphoinositide-specific biosensors we confirmed that E-cadherin-based cell-cell contacts are enriched in PIP(3), the principal product of PI3-kinase. We then used pharmacologic inhibitors to assess the morphogenetic impact of PI3-kinase in MDCK and MCF7 monolayers. We found that inhibiting PI3-kinase caused a reduction in epithelial cell height that was reversible upon removal of the drugs. This was not attributable to changes in E-cadherin expression or homophilic adhesion. Nor were there detectable changes in cell polarity. While Myosin II has been implicated in regulating keratinocyte height, we found no effect of PI3-kinase inhibition on apparent Myosin II activity; nor did direct inhibition of Myosin II alter epithelial height. Instead, in pursuing signalling pathways downstream of PI3-kinase we found that blocking Rac signalling, but not mTOR, reduced epithelial cell height, as did PI3-kinase inhibition. Overall, our findings suggest that PI3-kinase exerts a major morphogenetic impact in simple cultured epithelia through preservation of cell height. This is independent of potential effects on adhesion or polarity, but may occur through PI3-kinase-stimulated Rac signaling.