Tumor suppressor scribble regulates assembly of tight junctions in the intestinal epithelium

Abstract

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-gamma-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.

Figure 1

Scribble localizes at TJs in polarized human intestinal epithelial cell monolayers. T84 and SK-CO15 cells were grown on membrane filters until they formed confluent high-resistance monolayers. T84 cells (A) were double-immunolabeled for Scribble (green) and occludin (red), whereas SK-CO15 cells (B) were labeled for Scribble (red) and ZO-1 (green). Note significant colocalization of Scribble and TJ proteins (yellow) in both epithelial cell lines according to x-y (arrows) and reconstructed x-z (arrowheads) confocal images. Scale bar = 10 μm.

Figure 2

Down-regulation of Scribble attenuates formation of the paracellular barrier in model intestinal epithelium. SK-CO15 cells were transfected with either Scribble-specific or control (cyclophilin B-specific) siRNAs. Development of the paracellular barrier was examined by measuring TEER and fluoresceinated dextran flux. A: Immunoblotting analysis shows siRNA-mediated decrease of Scribble protein level on day 4 post-transfection. Permeability assays show significant attenuation of TEER development (B) and increase in dextran flux (C) in Scribble-depleted cell monolayers on days 2–4 and day 4 post-transfection, respectively. Data are presented as mean ± SE (n = 3); *P < 0.05 compared to control siRNA-transfected cells.

Figure 3

Down-regulation of Scribble attenuates TJ assembly. SK-CO15 cells were transfected with either control or Scribble-specific siRNAs and on day 3 post-transfection were subjected to overnight calcium depletion to disrupt cell-cell adhesion. Reassembly of TJs in control and Scribble-deficient cell monolayers was analyzed after 5 hours of calcium repletion by monitoring the formation of characteristic chicken wire labeling pattern of the TJ proteins occludin and ZO-1 (red). Control SK-CO15 cell monolayers show almost complete restoration of normal TJ localization of occludin and ZO-1 (arrows). By contrast, Scribble-deficient cell monolayers display abnormal discontinuous occludin and ZO-1 labeling at intercellular contacts (arrowheads). Scale bar = 10 μm.

Figure 4

ZO-1 physically associates with Scribble and regulates development of the paracellular barrier. A: Scribble was immunoprecipitated from SK-CO15 cell monolayers after 5 hours of calcium repletion and was analyzed for association with TJ proteins. Immunoblotting analysis shows presence of ZO-1 but not occludin in Scribble immunoprecipitates. B: ZO-1 was immunoprecipitated from confluent SK-CO15 cell monolayers and analyzed for association with Scribble. Immunoblot shows pulldown of Scribble with ZO-1 antibody but not with control IgG. C and D: Effect of silencing of ZO-1 expression on development of the paracellular barrier was analyzed in SK-CO15 cell monolayers. Immunoblotting analysis shows significant down-regulation of ZO-1 expression on day 4 post-siRNA transfection (C). Development of TEER was significantly attenuated in ZO-1-depleted cell monolayers as compared with control siRNA-transfected cells (D). Data are presented as mean ± SE (n = 3); *P < 0.05 compared to control siRNA-transfected cells.

Figure 5

Down-regulation of ZO-1 attenuates assembly of TJs and recruitment of Scribble to apical junctions. SK-CO15 cells transfected with either control or ZO-1-specific siRNAs were subjected to calcium switch on day 3 post-transfection. Cells were fixed 5 hours after calcium repletion and double-immunolabeled for ZO-1 (green) and either occludin or Scribble (red). Control cell monolayers show complete restoration of occludin and Scribble labeling at TJs (arrows). By contrast, TJ reassembly and Scribble recruitment to apical junctions is attenuated in ZO-1-depleted cell monolayers (arrowheads). Scale bar = 10 μm.

Figure 6

Lgl-1 localizes at TJs in the model intestinal epithelium. Polarized and calcium-depleted for 1 hour T84 cell monolayers were double-immunolabeled for occludin (green) and Lgl-1 (red). Note significant colocalization of occludin and Lgl-1 (yellow) at intact (arrows) and internalized (arrowheads) TJs. Scale bar = 10 μm.

Figure 7

Lgl-1 and Dlg-1 differently associate with Scribble and differently regulate the epithelial barrier. A: Scribble was immunoprecipitated from SK-CO15 cell monolayers after 4 hours of calcium repletion and was analyzed for association with Lgl-1 and Dlg-1. Immunoblotting analysis shows presence of Lgl-1 but not Dlg-1 in Scribble immunoprecipitates. B and C: Effects of Lgl-1 and Dlg-1 knockdown on development of paracellular barrier were analyzed in SK-CO15 cell monolayers. Immunoblotting analysis demonstrates selective down-regulation of Lgl-1 and Dlg-1 expression on day 4 post-siRNA transfection with no effects on each other or Scribble expression (B). TEER measurement shows significant attenuation in development of the paracellular barrier in Dlg-1-depleted but not in Lgl-1-deficient SK-CO15 cell monolayers (C). Data are presented as mean ± SE (n = 3); *P < 0.05 compared to control siRNA-transfected cells.

Figure 8

Interferon- γ induces mislocalization and down-regulation of epithelial Scribble in vitro, which parallel TJ disassembly. Polarized T84 cell monolayers were treated with either vehicle or IFN-γ (100 U/ml). A: Cells were fixed after 48 hours of the cytokine exposure and double-immunolabeled for Scribble (green) and occludin (red). Note profound disruption of TJs and disappearance of junctional Scribble in IFN-γ-treated cells (arrows). Scale bar = 10 μm. B: Total cell lysates of vehicle- and IFN-γ-treated T84 cells were analyzed for Scribble expression by immunoblotting. Note that cytokine treatment resulted in significant decrease in Scribble protein expression.

Figure 9

Mucosal inflammation in vivo down-regulates Scribble expression and induces TJ disassembly. Tissue sections of colonic mucosa isolated from a normal human subject and a patient with active Crohn's disease were double-immunolabeled for Scribble (green) and occludin (red). Note significant colocalization (yellow) of Scribble and occludin at intact TJs in normal intestinal mucosa (arrows) and disappearance of junctional Scribble and occludin in inflamed intestinal epithelium of Crohn's disease patient. Scale bar = 20 μm.