Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Nov 1;367(Pt 3):873–879. doi: 10.1042/BJ20020746

Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure.

Marco Wachtel 1, Karl Frei 1, Elisabeth Ehler 1, Christian Bauer 1, Max Gassmann 1, Sergio M Gloor 1
PMCID: PMC1222922  PMID: 12137564

Abstract

During an ischaemic insult, oedema formation occurs as a consequence of increased vascular permeability. To study mechanisms leading to vascular barrier failure, endothelial cells were exposed to ischaemia (1% O(2) in serum- and glucose-free medium) for 5 h. In in vitro conditions, ischaemia increased paracellular permeability, disassembled actin stress fibres, displaced focal adhesion kinase (FAK) from focal adhesions and enhanced cytoskeletal association of occludin. Reoxygenation restored paracellular barrier function, actin organization and FAK distribution. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) was rapidly activated after 30 min, strongly inhibited after 5 h of continuous ischaemia and reactivated 3 times more than control during reoxygenation. Inhibition of ERK activation during reoxygenation with U0126, an inhibitor of the ERK activator, MAPK/ERK kinase 1/2, prevented both barrier restoration and stress-fibre formation, but did not prevent recruitment of FAK to focal contacts. Under normoxic conditions, ERK inhibition led to barrier failure and disassembly of stress fibres only in the absence of serum. These results demonstrate that ERK activity is essential to rebuild a disrupted endothelial barrier after ischaemia and to maintain barrier function in cells exposed to non-ischaemic stress.

Full Text

The Full Text of this article is available as a PDF (267.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abedi H., Zachary I. Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. J Biol Chem. 1997 Jun 13;272(24):15442–15451. doi: 10.1074/jbc.272.24.15442. [DOI] [PubMed] [Google Scholar]
  2. Alessandrini A., Namura S., Moskowitz M. A., Bonventre J. V. MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12866–12869. doi: 10.1073/pnas.96.22.12866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Antonetti D. A., Barber A. J., Hollinger L. A., Wolpert E. B., Gardner T. W. Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors. J Biol Chem. 1999 Aug 13;274(33):23463–23467. doi: 10.1074/jbc.274.33.23463. [DOI] [PubMed] [Google Scholar]
  4. Bacallao R., Garfinkel A., Monke S., Zampighi G., Mandel L. J. ATP depletion: a novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton. J Cell Sci. 1994 Dec;107(Pt 12):3301–3313. doi: 10.1242/jcs.107.12.3301. [DOI] [PubMed] [Google Scholar]
  5. Chen Q., Lin T. H., Der C. J., Juliano R. L. Integrin-mediated activation of MEK and mitogen-activated protein kinase is independent of Ras [corrected]. J Biol Chem. 1996 Jul 26;271(30):18122–18127. doi: 10.1074/jbc.271.30.18122. [DOI] [PubMed] [Google Scholar]
  6. Chu C. L., Reenstra W. R., Orlow D. L., Svoboda K. K. Erk and PI-3 kinase are necessary for collagen binding and actin reorganization in corneal epithelia. Invest Ophthalmol Vis Sci. 2000 Oct;41(11):3374–3382. [PMC free article] [PubMed] [Google Scholar]
  7. Clarke H., Soler A. P., Mullin J. M. Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets. J Cell Sci. 2000 Sep;113(Pt 18):3187–3196. doi: 10.1242/jcs.113.18.3187. [DOI] [PubMed] [Google Scholar]
  8. Favata M. F., Horiuchi K. Y., Manos E. J., Daulerio A. J., Stradley D. A., Feeser W. S., Van Dyk D. E., Pitts W. J., Earl R. A., Hobbs F. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998 Jul 17;273(29):18623–18632. doi: 10.1074/jbc.273.29.18623. [DOI] [PubMed] [Google Scholar]
  9. Ferrero E., Villa A., Ferrero M. E., Toninelli E., Bender J. R., Pardi R., Zocchi M. R. Tumor necrosis factor alpha-induced vascular leakage involves PECAM1 phosphorylation. Cancer Res. 1996 Jul 15;56(14):3211–3215. [PubMed] [Google Scholar]
  10. Gamble J. R., Drew J., Trezise L., Underwood A., Parsons M., Kasminkas L., Rudge J., Yancopoulos G., Vadas M. A. Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions. Circ Res. 2000 Sep 29;87(7):603–607. doi: 10.1161/01.res.87.7.603. [DOI] [PubMed] [Google Scholar]
  11. Gloor S. M., Wachtel M., Bolliger M. F., Ishihara H., Landmann R., Frei K. Molecular and cellular permeability control at the blood-brain barrier. Brain Res Brain Res Rev. 2001 Oct;36(2-3):258–264. doi: 10.1016/s0165-0173(01)00102-3. [DOI] [PubMed] [Google Scholar]
  12. Gloor S. M., Weber A., Adachi N., Frei K. Interleukin-1 modulates protein tyrosine phosphatase activity and permeability of brain endothelial cells. Biochem Biophys Res Commun. 1997 Oct 29;239(3):804–809. doi: 10.1006/bbrc.1997.7557. [DOI] [PubMed] [Google Scholar]
  13. Han B. H., Holtzman D. M. BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci. 2000 Aug 1;20(15):5775–5781. doi: 10.1523/JNEUROSCI.20-15-05775.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kevil C. G., Oshima T., Alexander B., Coe L. L., Alexander J. S. H(2)O(2)-mediated permeability: role of MAPK and occludin. Am J Physiol Cell Physiol. 2000 Jul;279(1):C21–C30. doi: 10.1152/ajpcell.2000.279.1.C21. [DOI] [PubMed] [Google Scholar]
  15. Kinugasa T., Sakaguchi T., Gu X., Reinecker H. C. Claudins regulate the intestinal barrier in response to immune mediators. Gastroenterology. 2000 Jun;118(6):1001–1011. doi: 10.1016/s0016-5085(00)70351-9. [DOI] [PubMed] [Google Scholar]
  16. Kvietikova I., Wenger R. H., Marti H. H., Gassmann M. The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site. Nucleic Acids Res. 1995 Nov 25;23(22):4542–4550. doi: 10.1093/nar/23.22.4542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laderoute K. R., Mendonca H. L., Calaoagan J. M., Knapp A. M., Giaccia A. J., Stork P. J. Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments. A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity. J Biol Chem. 1999 Apr 30;274(18):12890–12897. doi: 10.1074/jbc.274.18.12890. [DOI] [PubMed] [Google Scholar]
  18. Lewis T. S., Shapiro P. S., Ahn N. G. Signal transduction through MAP kinase cascades. Adv Cancer Res. 1998;74:49–139. doi: 10.1016/s0065-230x(08)60765-4. [DOI] [PubMed] [Google Scholar]
  19. Li S., Kim M., Hu Y. L., Jalali S., Schlaepfer D. D., Hunter T., Chien S., Shyy J. Y. Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases. J Biol Chem. 1997 Nov 28;272(48):30455–30462. doi: 10.1074/jbc.272.48.30455. [DOI] [PubMed] [Google Scholar]
  20. Liu F., Verin A. D., Borbiev T., Garcia J. G. Role of cAMP-dependent protein kinase A activity in endothelial cell cytoskeleton rearrangement. Am J Physiol Lung Cell Mol Physiol. 2001 Jun;280(6):L1309–L1317. doi: 10.1152/ajplung.2001.280.6.L1309. [DOI] [PubMed] [Google Scholar]
  21. Lu Q., Paredes M., Zhang J., Kosik K. S. Basal extracellular signal-regulated kinase activity modulates cell-cell and cell-matrix interactions. Mol Cell Biol. 1998 Jun;18(6):3257–3265. doi: 10.1128/mcb.18.6.3257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lum H., Malik A. B. Regulation of vascular endothelial barrier function. Am J Physiol. 1994 Sep;267(3 Pt 1):L223–L241. doi: 10.1152/ajplung.1994.267.3.L223. [DOI] [PubMed] [Google Scholar]
  23. Madara J. L., Barenberg D., Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol. 1986 Jun;102(6):2125–2136. doi: 10.1083/jcb.102.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mandel L. J., Bacallao R., Zampighi G. Uncoupling of the molecular 'fence' and paracellular 'gate' functions in epithelial tight junctions. Nature. 1993 Feb 11;361(6412):552–555. doi: 10.1038/361552a0. [DOI] [PubMed] [Google Scholar]
  25. Mitic L. L., Anderson J. M. Molecular architecture of tight junctions. Annu Rev Physiol. 1998;60:121–142. doi: 10.1146/annurev.physiol.60.1.121. [DOI] [PubMed] [Google Scholar]
  26. Miyamoto S., Teramoto H., Coso O. A., Gutkind J. S., Burbelo P. D., Akiyama S. K., Yamada K. M. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol. 1995 Nov;131(3):791–805. doi: 10.1083/jcb.131.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Namura S., Iihara K., Takami S., Nagata I., Kikuchi H., Matsushita K., Moskowitz M. A., Bonventre J. V., Alessandrini A. Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia. Proc Natl Acad Sci U S A. 2001 Aug 14;98(20):11569–11574. doi: 10.1073/pnas.181213498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nusrat A., Parkos C. A., Verkade P., Foley C. S., Liang T. W., Innis-Whitehouse W., Eastburn K. K., Madara J. L. Tight junctions are membrane microdomains. J Cell Sci. 2000 May;113(Pt 10):1771–1781. doi: 10.1242/jcs.113.10.1771. [DOI] [PubMed] [Google Scholar]
  29. Oshima T., Flores S. C., Vaitaitis G., Coe L. L., Joh T., Park J. H., Zhu Y., Alexander B., Alexander J. S. HIV-1 Tat increases endothelial solute permeability through tyrosine kinase and mitogen-activated protein kinase-dependent pathways. AIDS. 2000 Mar 31;14(5):475–482. doi: 10.1097/00002030-200003310-00002. [DOI] [PubMed] [Google Scholar]
  30. Sakakibara A., Furuse M., Saitou M., Ando-Akatsuka Y., Tsukita S. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol. 1997 Jun 16;137(6):1393–1401. doi: 10.1083/jcb.137.6.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sanders M. A., Basson M. D. Collagen IV-dependent ERK activation in human Caco-2 intestinal epithelial cells requires focal adhesion kinase. J Biol Chem. 2000 Dec 1;275(48):38040–38047. doi: 10.1074/jbc.M003871200. [DOI] [PubMed] [Google Scholar]
  32. Schaeffer H. J., Weber M. J. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol. 1999 Apr;19(4):2435–2444. doi: 10.1128/mcb.19.4.2435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shin D. Y., Ishibashi T., Choi T. S., Chung E., Chung I. Y., Aaronson S. A., Bottaro D. P. A novel human ERK phosphatase regulates H-ras and v-raf signal transduction. Oncogene. 1997 Jun 5;14(22):2633–2639. doi: 10.1038/sj.onc.1201106. [DOI] [PubMed] [Google Scholar]
  34. Staddon J. M., Herrenknecht K., Smales C., Rubin L. L. Evidence that tyrosine phosphorylation may increase tight junction permeability. J Cell Sci. 1995 Feb;108(Pt 2):609–619. doi: 10.1242/jcs.108.2.609. [DOI] [PubMed] [Google Scholar]
  35. Stevenson B. R., Keon B. H. The tight junction: morphology to molecules. Annu Rev Cell Dev Biol. 1998;14:89–109. doi: 10.1146/annurev.cellbio.14.1.89. [DOI] [PubMed] [Google Scholar]
  36. Todd J. L., Tanner K. G., Denu J. M. Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway. J Biol Chem. 1999 May 7;274(19):13271–13280. doi: 10.1074/jbc.274.19.13271. [DOI] [PubMed] [Google Scholar]
  37. Tsukamoto T., Nigam S. K. Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins. Am J Physiol. 1999 May;276(5 Pt 2):F737–F750. doi: 10.1152/ajprenal.1999.276.5.F737. [DOI] [PubMed] [Google Scholar]
  38. Tsukamoto T., Nigam S. K. Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly. J Biol Chem. 1997 Jun 27;272(26):16133–16139. doi: 10.1074/jbc.272.26.16133. [DOI] [PubMed] [Google Scholar]
  39. Verin A. D., Liu F., Bogatcheva N., Borbiev T., Hershenson M. B., Wang P., Garcia J. G. Role of ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction. Am J Physiol Lung Cell Mol Physiol. 2000 Aug;279(2):L360–L370. doi: 10.1152/ajplung.2000.279.2.L360. [DOI] [PubMed] [Google Scholar]
  40. Wachtel M., Frei K., Ehler E., Fontana A., Winterhalter K., Gloor S. M. Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition. J Cell Sci. 1999 Dec;112(Pt 23):4347–4356. doi: 10.1242/jcs.112.23.4347. [DOI] [PubMed] [Google Scholar]
  41. Wong V., Gumbiner B. M. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier. J Cell Biol. 1997 Jan 27;136(2):399–409. doi: 10.1083/jcb.136.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yap A. S., Mullin J. M., Stevenson B. R. Molecular analyses of tight junction physiology: insights and paradoxes. J Membr Biol. 1998 Jun 1;163(3):159–167. doi: 10.1007/s002329900380. [DOI] [PubMed] [Google Scholar]
  43. Yue T. L., Wang C., Gu J. L., Ma X. L., Kumar S., Lee J. C., Feuerstein G. Z., Thomas H., Maleeff B., Ohlstein E. H. Inhibition of extracellular signal-regulated kinase enhances Ischemia/Reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res. 2000 Mar 31;86(6):692–699. doi: 10.1161/01.res.86.6.692. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES