Evidence for Actin Cytoskeleton-dependent and -independent Pathways for RelA/p65 Nuclear Translocation in Endothelial Cells

Abstract

Activation of the transcription factor NF-B involves its release from the inhibitory protein IB␣ in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-B remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor ␣, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-B activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-B activation occurs downstream of IB␣ degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor ␣, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells. NF-B2 is an ubiquitously expressed family of transcription factors controlling varied biological effects ranging from inflammatory, immune, and stress-induced responses to cell fate decisions such as proliferation, differentiation, tumorigenesis, and apoptosis (1-3). NF-B dimers, typically a heterodimer of p50 and RelA/p65 subunits, are mostly sequestered in the cytoplasm by IB␣, the prototype of a family of inhibitory proteins IBs that mask the nuclear localization signal of RelA/ p65 (4, 5). Activation of NF-B requires serine phosphorylation (Ser 32 and Ser 36 ) of IB␣ by a macromolecular IB kinase (IKK) complex (6 -8). Phosphorylation marks IB␣ for polyubiquitination by the E3-SCF␤-TrCP ubiquitin ligase leading to its degradation by the 26 S proteasome (9 -11). The released NF-B undergoes rapid nuclear translocation and subsequent binding to NF-B responsive elements to activate transcription of target genes including intercellular adhesion molecule-1 (ICAM-1; CD54), an inducible endothelial adhesive protein that serves as a counter-receptor for ␤ 2 -integrins (CD11/CD18) present on the surface of leukocytes (12-14). Interaction of ICAM-1 with ␤ 2 -integrins enables polymorphonuclear leukocytes to adhere firmly and stably to the vascular endothelium, and to migrate across the endothelial barrier (15-18). have shown that activation of NF-B is essential for ICAM-1 expression in endothelial cells after stimulation with the proinflammatory cytokine tumor necrosis factor ...

Vasodilator-stimulated phosphoprotein protects against vascular inflammation and insulin resistance

“…Whether the specific mechanism involves an interaction of VASP directly with NF-B or with upstream signaling molecules is unknown, and several possibilities exist. For one, VASP regulates actin polymerization (17), and the actin cytoskeleton may in turn regulate NF-B translocation to the nucleus in response to thrombin (5). This model is consistent with the dynamic nature of the actin cytoskeleton and the fact that its polymerization and depolymerization are critical determinants of many cellular responses, including inflammatory activation.…”

Rho-kinase regulation of TNF-α-induced nuclear translocation of NF-κB RelA/p65 and M-CSF expression via p38 MAPK in mesangial cells

“…The possibility that transcriptional activity of NF-B may depend on arrangement of the cytoskeleton has been previously proposed by Mackenzie et al (27), who found that cytoskeletal integrity is essential for the nuclear translocation of NF-B in neuronal cells. Moreover, Fazal et al (10) reported that actin dynamics play a role in thrombin-induced nuclear uptake of RelA/p65 in endothelial cells. Although the mechanism by which the actin cytoskeleton participates in regulating NF-B signaling remains unclear, several possibilities are open to speculation.…”

Ectromelia Virus Encodes a BTB/kelch Protein, EVM150, That Inhibits NF-κB Signaling

“…That EVM150 inhibits NF-B independently of the kelch domain and cullin-3 is not surprising since proteins containing BTB domains regulate a wide variety of cellular processes, including transcriptional regulation and cytoskeletal rearrangement (66)(67)(68). Interestingly, it has recently been demonstrated that NF-B nuclear translocation relies on rearrangement of the actin cytoskeleton after degradation of IB␣ (69)(70)(71). Consequently, it is possible that EVM150 may interact with or degrade cellular BTB/kelch proteins regulate the actin rearrangement following release of NF-B from IB␣, thus inhibiting nuclear translocation.…”