XIAP regulates bi-phasic NF-kappaB induction involving physical interaction and ubiquitination of MEKK2.

The transcription factor NF-kappaB is transiently activated by a wide variety of stress signals, including pro-inflammatory mediators, and regulates the expression of genes with e.g., immune, inflammatory, and anti-apoptotic functions. The strength and kinetics of its induction, as well as its ultimate down-regulation is subject to multiple levels of regulation. One such regulatory protein is X chromosome-linked inhibitor of apoptosis (XIAP) that, besides its anti-apoptotic properties, has been shown to enhance NF-kappaB activity, however, the underlying molecular mechanism has remained elusive. We show here that following TNFalpha stimulation XIAP regulates a second wave of NF-kappaB activation. XIAP interacts with and ubiquitinates MEKK2, a kinase that has previously been associated with bi-phasic NF-kappaB activation. We conclude that, through interaction with MEKK2, XIAP functions in an ubiquitin ligase dependent manner to evoke a second wave of NF-kappaB activation, resulting in the modulation of NF-kappaB target gene expression.

Resolution of inflammation: intracellular feedback loops in the endothelium.

Timely termination of the inflammatory reaction is equally important as its elicitation, since a persistent or exaggerated response may lead to detrimental effects in the affected tissues and organs. Therefore, and in accordance with the complex and highly coordinated activation phase, negative regulatory mechanisms have evolved which function on multiple levels to ensure the appropriate termination of the inflammatory response. This review will focus on the mechanisms that are operative in endothelial cells to shut down the activity of specific signaling pathways and transcription factors that have been activated in response to pro-inflammatory mediators, and provide evidence that the stage for resolution is set already early in the activation phase of the inflammatory response. The elucidation of these feedback mechanisms is of importance for the understanding of acute versus chronic inflammation, and for novel strategies for therapeutic intervention.

X-linked inhibitor of apoptosis protein regulates human interleukin-6 in umbilical vein endothelial cells via stimulation of the nuclear factor-kappaB and MAP kinase signaling pathways.

X-linked inhibitior of apoptosis (XIAP) is known as a potent inhibitor of apoptosis, but more recently has been shown to also act as a modulator of the nuclear factor kB (NF-kappaB) signaling pathway. To investigate whether XIAP also affects other signalling pathways, we studied the transcriptional regulation of interleukin 6 (IL-6), a gene that is strongly affected by XIAP, in more detail. The human IL-6 gene contains transcription factor binding sites for activator protein 1 (AP1), enhancer binding protein beta (C/EBP-beta) and NF-kappaB. In reporter gene assays, mutation of these binding sites revealed the necessity of functional NF-kappaB and AP1-sites for its ability to respond to XIAP. In contrast, IL-6 promoter activity was slightly increased in the C/EBP deletion mutant. Pharmacologic inhibition of extracellular signal regulated kinase (ERK) kinases (MEK1/2) as well as inhibition of the p38 signaling pathway both reduced XIAP-induced IL-6 promoter activity. In conclusion, these results suggest that XIAP regulates IL-6 transcription via NF-kappaB in cooperation with AP1 and C/EBP-beta.

Peptide-based anti-PCSK9 vaccines - an approach for long-term LDLc management.

BACKGROUND: Low Density Lipoprotein (LDL) hypercholesterolemia, and its associated cardiovascular diseases, are some of the leading causes of death worldwide. The ability of proprotein convertase subtilisin/kexin 9 (PCSK9) to modulate circulating LDL cholesterol (LDLc) concentrations made it a very attractive target for LDLc management. To date, the most advanced approaches for PCSK9 inhibition are monoclonal antibody (mAb) therapies. Although shown to lower LDLc significantly, mAbs face functional limitations because of their relatively short in vivo half-lives necessitating frequent administration. Here, we evaluated the long-term efficacy and safety of PCSK9-specific active vaccines in different preclinical models. METHODS AND FINDING: PCSK9 peptide-based vaccines were successfully selected by our proprietary technology. To test their efficacy, wild-type (wt) mice, Ldlr+/- mice, and rats were immunized with highly immunogenic vaccine candidates. Vaccines induced generation of high-affine PCSK9-specific antibodies in all species. Group mean total cholesterol (TC) concentration was reduced by up to 30%, and LDLc up to 50% in treated animals. Moreover, the PCSK9 vaccine-induced humoral immune response persisted for up to one year in mice, and reduced cholesterol levels significantly throughout the study. Finally, the vaccines were well tolerated in all species tested. CONCLUSIONS: Peptide-based anti-PCSK9 vaccines induce the generation of antibodies that are persistent, high-affine, and functional for up to one year. They are powerful and safe tools for long-term LDLc management, and thus may represent a novel therapeutic approach for the prevention and/or treatment of LDL hypercholesterolemia-related cardiovascular diseases in humans.

Siva1 is a XIAP-interacting protein that balances NFkappaB and JNK signalling to promote apoptosis.

XIAP is known as a potent inhibitor of apoptosis, but in addition is involved in cellular signalling, including the NFkappaB, JNK and TGFbeta pathways. Our search for XIAP-interacting partners led us to Siva1, a proapoptotic protein that is known to play a role in T-cell apoptosis through a caspase-dependent mitochondrial pathway. The interaction sites between XIAP and Siva1 were mapped to the RING domain of XIAP and the N-terminal, SAH-containing and death-homology-region-containing domains of Siva1. Co-immunoprecipitation experiments showed that XIAP, Siva1 and TAK1 form a ternary complex in Jurkat T cells. Reporter-gene analysis revealed that Siva1 inhibits XIAP- and TAK1-TAB1-mediated NFkappaB activation. By contrast, Siva1 increased XIAP- and TNFalpha-mediated AP1 activity and prolonged TNFalpha-induced JNK activation, whereas knock down of Siva1 resulted in reduced JNK activation. This suggests that Siva1 differentially modulates signalling by JNK and NFkappaB and shifts the balance between these pathways towards enhanced JNK activation, a situation that promotes apoptosis. Ectopically expressed Siva1 increased caspase-3 activity, which was inhibited by XIAP in a ubiquitin-ligase-dependent manner. In line with this, Siva1 was lysine-48-linked polyubiquitylated by XIAP. Our findings suggest that, via physical interaction with XIAP and TAK1, Siva1 diminishes NFkappaB and enhances JNK activity to favour apoptosis.