Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin^ΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.Tight junctions seal the paracellular space in simple epithelia, such as those lining the lungs, intestines, and kidneys (Anderson et al., 2004 ; Fanning and Anderson, 2009 ; Shen et al., 2011 ). In the intestine, reduced paracellular barrier function is associated with disorders in which increased paracellular flux of ions and molecules contributes to symptoms such as diarrhea, malabsorption, and intestinal protein loss. Recombinant tumor necrosis factor (TNF) can be used to model this barrier loss in vitro or in vivo (Taylor et al., 1998 ; Clayburgh et al., 2006 ), and TNF neutralization is associated with restoration of intestinal barrier function in Crohn''s disease (Suenaert et al., 2002 ). Further, in vivo and in vitro studies of intestinal epithelia show that TNF-induced barrier loss requires myosin light chain kinase (MLCK) activation (Zolotarevsky et al., 2002 ; Clayburgh et al., 2005 , 2006 ; Ma et al., 2005 ; Wang et al., 2005 ). The resulting myosin II regulatory light chain (MLC) phosphorylation drives occludin internalization, which is required for cytokine-induced intestinal epithelial barrier loss (Clayburgh et al., 2005 , 2006 ; Schwarz et al., 2007 ; Marchiando et al., 2010 ). In addition, transgenic EGFP-occludin expression in vivo limits TNF-induced depletion of tight junction–associated occludin, barrier loss, and diarrhea (Marchiando et al., 2010 ). Conversely, in vitro studies show that occludin knockdown limits TNF-induced barrier regulation (Van Itallie et al., 2010 ). The basis for this discrepancy is not understood.One challenge is that, despite being identified 20 yr ago (Furuse et al., 1993 ), the contribution of occludin to tight junction regulation remains incompletely defined. The observation that occludin-knockout mice are able to form paracellular barriers and do not have obvious defects in epidermal, respiratory, or bladder tight junction function (Saitou et al., 2000 ; Schulzke et al., 2005 ) led many to conclude that occludin is not essential for tight junction barrier function. It is important to note, however, that barrier regulation in response to stress has not been studied in occludin-deficient animals.We recently showed that dephosphorylation of occludin serine-408 promotes assembly of a complex composed of occludin, ZO-1, and claudin-2 that inhibits flux across size- and charge-selective channels termed the pore pathway (Anderson and Van Itallie, 2009 ; Turner, 2009 ; Raleigh et al., 2011 ; Shen et al., 2011 ). Although this demonstrates that occludin can serve a regulatory role, it does not explain the role of occludin in TNF-induced barrier loss, which increases flux across the size- and charge-nonselective leak pathway (Wang et al., 2005 ; Weber et al., 2010 ). In vitro studies, however, do suggest that occludin contributes to leak pathway regulation, as occludin knockdown in either Madin–Darby canine kidney (MDCK) or human intestinal (Caco-2) epithelial monolayers enhances leak pathway permeability (Yu et al., 2005 ; Al-Sadi et al., 2011 ; Ye et al., 2011 ). Taken as a whole, these data suggest that occludin organizes the tight junction to limit leak pathway flux, whereas occludin removal, either by knockdown or endocytosis, enhances leak pathway flux.To define the mechanisms by which occludin regulates the leak pathway, we analyzed the contributions of occludin, as well as specific occludin domains, to basal and TNF-induced barrier regulation. The data indicate that TNF destabilizes tight junction–associated occludin via interactions mediated by the C-terminal coiled-coil occludin/ELL domain (OCEL). Further, these OCEL-mediated events are required for TNF-induced barrier regulation. Thus these data provide new insight into the structural elements and mechanisms by which occludin regulates leak pathway paracellular flux.