Tumor Suppressor Scribble Regulates Assembly of Tight Junctions in the Intestinal Epithelium

Formation of the epithelial barrier and apico-basal cell polarity represent two characteristics and mutually dependent features of differentiated epithelial monolayers. They are controlled by special adhesive structures, tight junctions (TJs), and polarity protein complexes that define the apical and the basolateral plasma membrane. The functional interplay between TJs and polarity complexes remains poorly understood. We investigated the role of Scribble, a basolateral polarity protein and known tumor suppressor, in regulating TJs in human intestinal epithelium. Scribble was enriched at TJs in T84 and SK-CO15 intestinal epithelial cell monolayers and sections of normal human colonic mucosa. siRNA-mediated knockdown of Scribble in SK-CO15 cells attenuated development of epithelial barrier and inhibited TJ reassembly independently of other basolateral polarity proteins Lgl-1 and Dlg-1. Scribble selectively co-imunoprecipitated with TJ protein ZO-1, and ZO-1 was important for Scribble recruitment to intercellular junctions and TJ reassembly. Lastly, Scribble was mislocalized from TJs and its expression down-regulated in interferon-γ-treated T84 cell monolayers and inflamed human intestinal mucosa in vivo. We conclude that Scribble is an important regulator of TJ functions and plasticity in the intestinal epithelium. Down-regulation of Scribble may mediate mucosal barrier breakdown during intestinal inflammation.The epithelial cell layer in the gut plays two crucial physiological functions. One function involves the formation of the physical barrier that separates body compartments from the gut lumen and protects underlying tissues from pathogen invasion and other harmful external stimuli.^1,2 Another function involves the regulation of bidirectional passages of solutes and macromolecules, which is essential for nutrients supply and removal of body waste.^3–5 Both barrier integrity and vectorial transport in the intestinal epithelium are regulated by specialized cellular structures known as tight junctions (TJs). TJs represent a complex network of protein fibrils within the plasma membrane, which encircle the apical region of the epithelial cell perimeter in close proximity to the gut lumen.⁶ TJ fibrils are composed of adhesive transmembrane proteins, which associate with ensembles of scaffolding proteins at the cytosolic face of the membrane. The paracellular barrier is created by homotypical interactions between transmembrane TJ components of contacting epithelial cells such as occludin, members of the claudin family and junctional adhesion molecule-A (JAM-A).^6–8 These cell-cell adhesions are enhanced and regulated by cytosolic scaffolds such as members of zonula occludens (ZO) family and AF-6/afadin, which cluster and stabilize transmembrane TJ components at the plasma membrane.^6–8 Although other junctional complexes at the plasma membrane, viz., adherens junctions (AJs) and desmosomes also mediate cell-cell adhesions, TJs play a unique role in sealing the paracellular space and creating the epithelial barrier.^1,6,7The mature TJs not only mediate barrier function of the intestinal epithelium, but also contribute to the formation and maintenance of the apico-basal cell polarity.^9–11 Such cell polarity implies that the apical and basolateral domains of the plasma membrane differ in the composition of transporters, channels and receptors; therefore ensuring directionality of secretion and adsorption processes in epithelial cells.^12,13 TJs regulate the epithelial cell polarity by creating a fence that prevents intermixing of protein and lipid constituents of the apical and basolateral plasma membrane domains.^9–11 However, TJs alone are not sufficient for the apico-basal polarization of epithelial cells. In this process apical junctions cooperate with specialized protein polarity complexes that control the “identity” of distinct plasma membrane domains.Epithelial cells have three major evolutionarily conserved polarity complexes that were initially identified and named in model invertebrates. They are known as Crumbs (composed by Crumbs, PALS, and PATJ proteins), Par (Par3, Par6, and atypical protein kinase C) and Scribble (including Scribble, Disks Large (Dlg) and Lethal Giant Larvae (Lgl)) complexes.^11,14–16 Crumbs and Par cooperate to define the apical plasma membrane, whereas Scribble is critical for establishment of the basolateral membrane domain.^11,14–16 A number of studies have demonstrated a functional interplay between epithelial junctions and the polarity complexes, where these entities mutually affect each other. Thus, several members of the Crumbs and Par complexes were shown to regulate TJ assembly via either direct binding to TJ components (ZO-1 and claudins) or indirect mechanisms, involving modulation of vesicle trafficking and actin cytoskeleton remodeling.^11,14–16 In contrast, the role of the Scribble polarity complex in the regulation of epithelial TJs is not well understood,^17,18 although such a role is supported by several lines of evidence. For example, mutations in any member of this complex in Drosophila resulted in dramatic disorganization of epithelial architecture that included loss of columnar cell shape and cell-cell adhesions.^19–21 Furthermore, several reports have linked decreased protein levels of mammalian Scribble and Lgl with progression and invasiveness of epithelial tumors,^22–24 which is also accompanied by down-regulation of TJs.²⁵ Two recent studies have addressed the role of Scribble in the regulation cell-cell adhesions in mammalian epithelia; however their results appear to be inconsistent. Indeed, siRNA-mediated depletion of this protein in Madin-Darby canine kidney (MDCK) epithelial cells resulted in altered cell morphology and disorganized E-cadherin-based AJs.²⁶ However, no changes in cell morphology or AJ structure were observed following the silencing of Scribble expression in MCF10A human mammary epithelial cells.²⁷ Such inconsistent results may reflect tissue- specific effects of Scribble depletion, and they indicate that more work is needed to establish functional links between Scribble and TJs in human epithelia under normal physiological conditions and in disease states.In this study, we examined the role of Scribble in the regulation of the intestinal epithelial barrier and reorganization of TJs. Our results demonstrate that Scribble is important for TJ barrier function and assembly, and that it may regulate junctions by interacting with the TJ scaffold, ZO-1. We also report that Scribble is mislocalized and its expression down-regulated in the intestinal epithelium by inflammatory conditions in vitro and in vivo.