SUMMARY Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-γ (IFN-γ) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-β-catenin and Wingless-Int (Wnt)-β-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-γ resulted in activation of β-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-γ treatment, and reduced proliferation through depletion of the Wnt co-receptor LRP6. These effects were enhanced by tumor necrosis factor-α (TNF)-α, suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased β-catenin-T-cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-γ - deficient mice. Thus, we propose that IFN-γ regulates intestinal epithelial homeostasis by sequential regulation of converging β-catenin signaling pathways.

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated downregulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation. INTRODUCTIONMucosal epithelial barriers are dependent upon the association of neighboring cells with each other through adhesive multiprotein complexes at sites of cell-cell contacts. Simple epithelial cells (such as those lining the intestine, lungs, and kidneys) associate through a series of intercellular junctions that maintain epithelial integrity, regulate paracellular movement of solutes, and restrict access of luminal antigens/pathogens from underlying tissue compartments. Intercellular junctions include tight junctions (TJs), adherens junctions (AJs), desmosomes (DMs), and in some epithelia gap junctions (Cereijido et al., 1978;Gonzalez-Mariscal et al., 2003). DMs have been visualized as punctuate "spot welds" that hold cells together and provide mechanical strength to epithelial tissues by forming stable cell-cell contacts that are anchored to the keratin intermediate filaments (Getsios et al., 2004b). Transmembrane proteins in DMs include the cadherin superfamily members desmoglein (Dsg 1-4) and desmocollin (Dsc 1-3) (Getsios et al., 2004b;Kottke et al., 2006) that mediate calcium-dependent cell-cell adhesion. Simple epithelia such as in the intestine, express Dsg2 and Dsc2, which affiliate with underlying keratin intermediate filaments via the armadillo family of proteins and desmoplakin (DSP) (Bornslaeger et al., 1996;Hatzfeld, 1999;Jonkman et al., 2005).Epithelial cells differentiate as they migrate toward the lumen along the intestinal crypt villus axis where they detach from the basement membrane and undergo apoptosis (Dufour et al., 2004). This cycle of progenitor crypt cell proliferation, migration, differentiation, and apoptosis is vital for maintaining integrity of the epithelium and therefore mucosal barrier function. The survival of most normal intestinal epithelial cells (IECs) requires cell-cell and cellmatrix adhesion, and loss of such cell adhesion induces epithelial...

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The development of luminal organs begins with the formation of spherical cysts composed of a single layer of epithelial cells. Using a model three-dimensional cell culture, this study examines the role of a cytoskeletal motor, myosin II, in cyst formation. Caco-2 and SK-CO15 intestinal epithelial cells were embedded into Matrigel, and myosin II was inhibited by blebbistatin or siRNA-mediated knockdown. Whereas control cells formed spherical cysts with a smooth surface, inhibition of myosin II induced the outgrowth of F-actin-rich surface protrusions. The development of these protrusions was abrogated after inhibition of F-actin polymerization or of phospholipase C (PLC) activity, as well as after overexpression of a dominant-negative ADF/cofilin. Surface protrusions were enriched in microtubules and their formation was prevented by microtubule depolymerization. Myosin II inhibition caused a loss of peripheral F-actin bundles and a submembranous extension of cortical microtubules. Our findings suggest that inhibition of myosin II eliminates the cortical F-actin barrier, allowing microtubules to reach and activate PLC at the plasma membrane. PLC-dependent stimulation of ADF/cofilin creates actin-filament barbed ends and promotes the outgrowth of F-actin-rich protrusions. We conclude that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface.

Vassilieva EV, Gerner-Smidt K, Ivanov AI, Nusrat A. Lipid rafts mediate internalization of ␤ 1-integrin in migrating intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 295: G965-G976, 2008. First published August 28, 2008 doi:10.1152 doi:10. /ajpgi.00082.2008 mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of ␤ 1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, ␤ 1-integrin is internalized in a dynamindependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent ␤ 1-integrin internalization. However, ␤ 1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized ␤ 1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased ␤ 1-integrin endocytosis. Our data suggest that, in migrating IEC, ␤ 1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure. mucosal restitution; endocytosis; recycling; dynamin; caveolin; flotillin EPITHELIAL EROSIONS AND ULCERATIONS are observed in active inflammatory disorders of the intestine. In response to injury, the epithelium migrates to rapidly cover denuded surfaces and reseal wounds (17,38,59). In this process, referred to as restitution, epithelial cells adjoining the wound migrate as a cohesive sheet of cells. Migrating cells transiently adhere to the matrix via an array of specialized basal structures that evolve from focal complexes to focal adhesions to fibrillar adhesions (42,34,52,69,71). Cell adhesion complexes contain a structural core of transmembrane integrins (25) that form heterodimers consisting of ␣-and ␤-subunits. Intestinal epithelial cells (IECs) are enriched in integrin dimers containing a ␤ 1 -subunit that plays an important role in cell migration (38,39). Integrins affiliate with the matrix via their extracellular domains and also interact with the cytoskeleton via cytosolic scaffolding proteins. Such dual interactions make integrinbased adhesion complexes not simply substrate anchors but critical mechanical sensors and signal transducers that orchestrate the entire process of cell movement (21, 57).Cell ...