Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity.
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.
Meyer
T N
TN
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Schwesinger
C
C
Ye
J
J
Denker
B M
BM
Nigam
S K
SK
D000255:Adenosine Triphosphate / Q000378:metabolism; D022005:Adherens Junctions / Q000187:drug effects*; D000818:Animals; D002460:Cell Line; D002614:Chelating Agents / Q000737:chemistry; D017630:Connexins / Q000378:metabolism; D004285:Dogs; D019833:Genistein / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000187:drug effects; D019216:Metals, Heavy / Q000737:chemistry; D018384:Oxidative Stress; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*
D002614:Chelating Agents; D017630:Connexins; D019216:Metals, Heavy; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D019833:Genistein; D011505:Protein-Tyrosine Kinases