Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity

TY - JOUR

T1 - Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity

AU - Meyer, T N

AU - Meyer-Schwesinger, Catherine

AU - Ye, J

AU - Denker, B M

AU - Nigam, S K

PY - 2001/6/22

Y1 - 2001/6/22

N2 - Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavenger N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress.

AB - Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavenger N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress.

KW - Adenosine Triphosphate

KW - Adherens Junctions

KW - Animals

KW - Cell Line

KW - Chelating Agents

KW - Connexins

KW - Dogs

KW - Genistein

KW - Hydrogen Peroxide

KW - Immunohistochemistry

KW - Membrane Potentials

KW - Metals, Heavy

KW - Oxidative Stress

KW - Protein-Tyrosine Kinases

U2 - 10.1074/jbc.M011477200

DO - 10.1074/jbc.M011477200

M3 - SCORING: Journal article

C2 - 11294856

VL - 276

SP - 22048

EP - 22055

JO - J BIOL CHEM

JF - J BIOL CHEM

SN - 0021-9258

IS - 25

ER -