Heme oxygenase-1 (HO-1; encoded by the Hmox1 gene) catalyzes the degradation of free heme into biliverdin, via a reaction that releases iron (Fe) and carbon monoxide. We report that HO-1 down-regulates the proinflammatory phenotype associated with endothelial cell (EC) activation by reducing intracellular nonprotein-bound Fe (labile Fe). EC isolated from Hmox1−/− mice have higher levels of intracellular labile Fe and reactive oxygen species (ROS) as compared with EC isolated from Hmox1+/+ mice. Basal and TNF-induced expression of VCAM-1, ICAM-1, and E-selectin were increased in Hmox1−/− vs Hmox1+/+ EC, an effect reversed by Fe chelation using deferoxamine mesylate (DFO). Fe chelation inhibits TNF-driven transcription of Vcam-1, Icam-1, and E-selectin, as assessed using luciferase reporter assays. This effect is associated with inhibition of the transcription factor NF-κB via a mechanism that is not associated with the inhibition of IκBα phosphorylation/degradation or NF-κB (i.e., RelA) nuclear translocation, although it affects very modestly NF-κB binding to DNA κB consensus sequences in the Vcam-1 and E-selectin promoters. HO-1 inhibits NF-κB (i.e., RelA) phosphorylation at Ser276, a phosphoacceptor that is critical to sustain TNF-driven NF-κB activity in EC. This effect was mimicked by Fe chelation as well as by antioxidants (N-acetylcysteine). In conclusion, we demonstrate a novel mechanism via which HO-1 down-modulates the proinflammatory phenotype of activated EC, i.e., the inhibition of RelA phosphorylation at Ser276.

Nuclear factor (NF)-κB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-κB occurs predominantly in the cytoplasm, where Tax1 binds NF-κB Essential Modulator (NEMO/IKKγ) and triggers the activation of IκB kinases. Several independent studies have shown that Tax1-mediated NF-κB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-κB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-κB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-κB activation.

Host colonisation by lymphotropic gammaherpesviruses depends critically on the expansion of viral genomes in germinal centre (GC) B cells. Yet, host and virus molecular mechanisms involved in driving such proliferation remain largely unknown. Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-jB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-jB family member p65/RelA. The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73. Functional deletion of this SOCS-box motif ablated NF-jB inhibitory effect of ORF73, suppressed MuHV-4 expansion in GC B cells and prevented MuHV-4 persistent infection in mice. These findings demonstrate that viral inhibition of NF-jB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-jB as a regulatory mechanism of this signalling pathway.