Involvement of oxidative stress in NF-kappaB activation in endothelial cells treated by photodynamic therapy.
In human endothelial cells ECV 304 and HMEC-1 photosensitized by pyropheophorbide-a methylester (PPME) in sublethal conditions transcription factor Nuclear Factor kappa B (NF-kappaB) activation takes place for several hours. Activated NF-kappaB was functional because it stimulated the transcriptional activation of either a transfected reporter gene or the endogenous gene encoding interleukin (IL)-8. Concomitant with NF-kappaB activation, inhibitor of NF-kappaB alpha (IkappaB alpha) was degraded during photosensitization and IkappaB beta, p100, p105 and IkappaB epsilon were slightly modified. Reactive oxygen species (ROS) were shown to be crucial intermediates in the activation because antioxidants strongly decreased NF-kappaB activation. Using both a fluorescent probe and isotope substitution, it was shown that ROS, and especially singlet oxygen (1O2), were important in the activation process. Because NF-kappaB activation in the presence of ROS was suspected to proceed through a pathway independent of the IkappaB kinases (IKK), we demonstrated that the IKK were indeed not activated by photosensitization but required an intact tyrosine residue at position 42 on IkappaB alpha, suggesting the involvement of a tyrosine kinase in the activation process. This was further reinforced by the demonstration that herbimycin A, a tyrosine kinase inhibitor, prevented NF-kappaB activation by photosensitization but not by TNF alpha, a cytokine known to activate NF-kappaB through an IKK-dependent mechanism.
Volanti
Cédric
C
Laboratory of Virology & Immunology, Institute of Pathology, University of Liège, Belgium.
Matroule
Jean-Yves
JY
Piette
Jacques
J
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D018384:Oxidative Stress; D018462:Photobiology; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology; D011166:Porphyrins / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000737:chemistry
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D017319:Photosensitizing Agents; D011166:Porphyrins; D017382:Reactive Oxygen Species; C452939:pyropheophorbide-a methylester; D000072000:NF-KappaB Inhibitor alpha; D014443:Tyrosine; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human