Adenovirus-mediated expression of a mutant IκB kinase 2 inhibits the response of endothelial cells to inflammatory stimuli

Abstract

In a variety of cell types, the transcription factor nuclear factor B (NF-B) functions as a mediator of stress and immune responses. In endothelial cells (ECs), it controls the expression of genes encoding, eg, cytokines, cell adhesion molecules, and procoagulatory proteins. This study investigates the effect of NF-B suppression on several pathophysiologic functions of ECs, including inflammation, coagulation, and angiogenesis. A recombinant adenovirus was generated for expression of a dominant negative (dn) mutant of IB kinase 2 (IKK2), a kinase that acts as an upstream activator of NF-B. dnIKK2 inhibited NF-B, resulting in strongly reduced nuclear translocation and DNA binding activity of the transcription factor and lack of expression of several proinflammatory markers, including E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and interleukin-8. Concomitantly, inhibition of leukocyte binding to dnIKK2-expressing ECs could be demonstrated in a cell adhesion assay. Furthermore, expression of tissue factor as well as the ability to form capillary tubes in a matrigel assay was impaired in dnIKK2-expressing ECs. These data demonstrate that NF-B is of central importance not only for the inflammatory response but also for a number of other EC functions. Therefore, this transcription factor as well as its upstream regulatory signaling molecules may represent favorable targets for therapeutic interference. IntroductionNuclear factor B (NF-B)/Rel transcription factors represent an ubiquitously expressed family that modulate the expression of genes involved in diverse cellular functions such as stress response, innate and adaptive immune reactions, and apoptosis. Examples include ␤-interferon, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, immunoglobulin light chain, interleukin-2 (IL-2), major histocompatibility complex I, T-cell receptor, A20, inhibitor of apoptosis protein (IAP), and acute phase genes (for recent reviews, see Baeuerle, 1 de Martin et al, 2 and Hatada et al 3 ). Viruses, such as human immunodeficiency virus 1 or human T-cell leukemia virus 1, use NF-B to control the transcription of their own genes and prevent their host cells from undergoing apoptosis. 4 In endothelial cells (ECs), genes dependent on NF-B include, among others, IL-1, IL-6, IL-8, E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), inducible nitric oxide synthase, cyclooxygenase 2, and tissue factor. In cells of the vasculature, NF-B can be activated by inflammatory cytokines, bacterial lipopolysaccharides (LPS), oxidized low-density lipoprotein (LDL), advanced glycation end products, platelet-derived growth factor, and hypoxia/reoxygenation. Several disorders that involve endothelial and smooth muscle cells, such as acute and chronic inflammation and atherogenesis, have been associated with elevated levels of NF-B.The NF-B family includes the mammalian proteins p65 (RelA), RelB, c-Rel, the viral oncogene v-Rel, ...

Heterogeneous requirement of IκB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: Implications for therapy

“…However, in dermal fibroblasts, RA synovial fibroblasts, and HUVECs, IKK-2 is essential for TNF␣-, IL-1-, and LPS-induced NF-B activation and IL-6 or IL-8 production. This is due to a direct effect of IKK-2 on I B␣ degradation and NF-B activation, and is consistent with other studies that have examined some of these aspects (29,39,58).…”

“…Ad0, Ad␤-gal, AdGFP, and AdIKK1dn were kind gifts of Dr. A. Byrnes (Oxford University, Oxford, UK), Quantum Biotech (Carlsbad, CA), and Dr. M. Karin (University of California, San Diego), respectively. AdIKK2dn and I B␣ were produced by us (39). All viruses were E1/E3-deleted, belonged to the Ad5 serotype, and had been previously used in other studies (39)(40)(41)(42)(43).…”

“…AdIKK2dn and I B␣ were produced by us (39). All viruses were E1/E3-deleted, belonged to the Ad5 serotype, and had been previously used in other studies (39)(40)(41)(42)(43). Viruses were propagated in HEK 293 cells (American Type Culture Collection, Manasas, VA), purified by ultracentifugation through 2 CsCl gradients, and viral titers were determined by plaque assay as previously described (44).…”

NF-?B in epiretinal membranes after human diabetic retinopathy

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Hyaluronan Production in Synoviocytes as a Consequence of Viral Infections

“…To this effect two adenovirus constructs were used expressing either IB or a mutated versions of IKK-2. As demonstrated before, overexpression of a mutated IKK-2 or IB␣ protein is well suited to test the involvement of NF-B (25,26). Fig.…”