Tight Junction Proteins Form Large Complexes and Associate with the Cytoskeleton in an ATP Depletion Model for Reversible Junction Assembly

Abstract

A key feature of the ischemic epithelial cell phenotype is the disruption of tight junctions (TJ). In a ManinDarby canine kidney cell model for ischemia-reperfusion/hypoxia-reoxygenation injury which employs inhibitors of glycolysis (2-deoxy-D-glucose) and oxidative phosphorylation (antimycin A), transepithelial electrical resistance, a measure of TJ integrity, dropped rapidly, correlating well with declining ATP levels. Although immunocytochemical studies revealed only subtle changes in the distribution of the TJ proteins, zonula occludens (ZO)-1, ZO-2, and cingulin, examination of the Triton X-100 solubilities of these proteins, an indicator of cytoskeletal association, revealed a striking shift of all three TJ proteins into the insoluble pool, consistent with increased cytoskeletal interaction during ATP depletion. In addition, rate-zonal centrifugation analysis of a detergent-soluble fraction showed an increase in the amount of ZO-1 and ZO-2 in high density fractions following ATP depletion, providing further evidence for association of TJ proteins into a large complex possibly involving the cytoskeleton. Analysis of immunoprecipitation data from [ 35 S]methionine-labeled cells revealed that ATP depletion led to the association of a 240-kDa protein with the ZO-1-containing complex. Western blots of this protein immunoprecipitated with anti-ZO-1 antibodies confirmed its identity as fodrin, a protein believed to link membrane and other proteins to the actin-based cytoskeleton. Together, our data suggest that in the absence of major immunocytochemical changes, ATP depletion leads TJ proteins to form large insoluble complexes and associate with the cytoskeleton. We propose a model in which a key, potentially regulated, step in the generation of the ischemic epithelial cell phenotype is the interaction between TJ proteins and fodrin and/or other cytoskeletal proteins.The epithelial intercellular permeability barrier is maintained largely by the tight junction (TJ) 1 (1). The TJ, the most apical of intercellular junctions, consists of a number of proteins, including ZO-1, ZO-2, occludin, cingulin, 7H6, p130, and potentially other proteins (2-9). Considerable indirect evidence suggests that proteins of the TJ are intimately associated with the actin-based cytoskeleton (10 -12).Ischemia and subsequent reperfusion/reoxygenation causes a number of lesions in epithelial cells including mispolarization of at least some membrane proteins, perturbation of the actin cytoskeleton, and disruption of the permeability barrier (13, 14). These lesions have been reproduced in cell culture models for hypoxia-reoxygenation injury using agents that deplete cellular ATP, which has allowed for the analysis of molecular mechanisms underlying ischemic injury (15, 16). Although mechanistic insights into the disruption of the actin-based cytoskeleton are beginning to emerge, little is known about the biochemical basis of the disruption of the TJ after ischemic insult or how the TJ reassembles during recovery of epithelial cells from ische...

Reassembly of the Tight Junction after Oxidative Stress Depends on Tyrosine Kinase Activity

“…Consistent with this finding, in dGlc-treated cells, no increase in trypan blue uptake was recorded (n ϭ 10). The finding that TER is unchanged in cells ATP depleted to 20% of control levels is consistent with previous work that indicates that the threshold for ATP depletioninduced declines in TER occurs at around 5-10% of normal ATP levels (4,5,7,17,33 induces a reversible, partial breakdown of ZO-1 in the TJ after 30 min (Fig. 6A, b, f, and j).…”

“…The two most widely used reagents for ATP depletion, 2-deoxy-D-glucose and antimycin A, consistently lower intracellular ATP levels in MDCK cells to 2-5% of the initial value within minutes (16,17,43). This "maximal" ATP depletion induces the disassembly of the tight junction, and increases the association of TJ proteins with the cytoskeleton (17). The reversibility of the effects of ATP depletion on the TJ of MDCK cells has been established in recent years (16,17,33,44).…”

“…Interestingly, this ATP level is higher than ATP levels obtained with the established ATP depletion model of TJ disassembly in which both glycolysis and mitochondrial oxidative phosphorylation are inhibited. The two most widely used reagents for ATP depletion, 2-deoxy-D-glucose and antimycin A, consistently lower intracellular ATP levels in MDCK cells to 2-5% of the initial value within minutes (16,17,43). This "maximal" ATP depletion induces the disassembly of the tight junction, and increases the association of TJ proteins with the cytoskeleton (17).…”

“…Tyrosine Kinase or PKC Inhibition Prevents Recovery of TER-TJ assembly is known to be modulated by a variety of signaling mechanisms (9,16,17,(23)(24)(25)(26)(27)(28). In a broad screen of many inhibitors of signaling events known to modulate the TJ, the most consistent results were obtained with the two tyrosine kinase inhibitors PP-2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)-pyrazolo [3,4-d]pyrimidine, 1 or 10 M) and genistein (4Ј,5,7-trihydroxyisoflavone, 50 or 100 M), both of which inhibit Src family tyrosine kinases, although genistein is also a broad spectrum tyrosine kinase inhibitor.…”

Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

“…Confluent monolayers, uninfected or infected for the indicated times, were extracted as described previously (Tsukamoto and Nigam, 1997). After specified times of infection, bacteria were rinsed twice with buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 1 mM PMSF.…”