Karin Hochrainer

Nuclear Factor-κB Activation and Postischemic Inflammation Are Suppressed in CD36-Null Mice after Middle Cerebral Artery Occlusion

CD36, a class-B scavenger receptor involved in multiple functions, including inflammatory signaling, may also contribute to ischemic brain injury through yet unidentified mechanisms. We investigated whether CD36 participates in the molecular events underlying the inflammatory reaction that accompanies cerebral ischemia and may contribute to the tissue damage. We found that activation of nuclear factor-κB, a transcription factor that coordinates postischemic gene expression, is attenuated in CD36-null mice subjected to middle cerebral artery occlusion. The infiltration of neutrophils and the glial reaction induced by cerebral ischemia were suppressed. Treatment with an inhibitor of inducible nitric oxide synthase, an enzyme that contributes to the tissue damage, reduced ischemic brain injury in wild-type mice, but not in CD36 nulls. In contrast to cerebral ischemia, the molecular and cellular inflammatory changes induced by intracerebroventricular injection of interleukin-1β were not attenuated in CD36-null mice. The findings unveil a novel role of CD36 in early molecular events leading to nuclear factor-κB activation and postischemic inflammation. Inhibition of CD36 signaling may be a valuable therapeutic approach to counteract the deleterious effects of postischemic inflammation.

Progranulin deficiency promotes post-ischemic blood-brain barrier disruption.

Loss-of-function mutations of progranulin (PGRN) have been linked to frontotemporal dementia, but little is known about the effects of PGRN deficiency on the brain in health and disease. PGRN has been implicated in neurovascular development, inflammation, and Wnt signaling, a pathway involved in the formation of the blood–brain barrier (BBB). Because BBB alterations and inflammation contribute to ischemic brain injury, we examined the role of PGRN in the brain damage produced by ischemia-reperfusion. PGRN+/− and PGRN−/− mice underwent middle cerebral artery occlusion (MCAO) with monitoring of cerebral blood flow. Infarct volume and motor deficits were assessed 72 h later. Post-ischemic inflammation was examined by expression of inflammatory genes and flow cytometry. BBB structure and permeability were examined by electron microscopy (EM) and Evans blue (EB) extravasation, respectively. MCAO resulted in ∼60% larger infarcts in PGRN+/− and PGRN−/− mice, an effect independent of hemodynamic factors or post-ischemic inflammation. Rather, massive hemorrhages and post-ischemic BBB disruption were observed, unrelated to degradation of tight junction (TJ) proteins or matrix metalloproteinases (MMPs). By EM, TJ were 30–52% shorter, fewer, and less interlocking, suggesting a weaker seal between endothelial cells. Intracerebral injection of platelet-derived growth factor-CC (PDGF-CC), which increases BBB permeability, resulted in a more severe BBB breakdown in PGRN+/− and PGRN−/− than wild-type mice. We describe a previously unrecognized involvement of PGRN in the expression of key ultrastructural features of the BBB. Such a novel vasoprotective role of PGRN may contribute to brain dysfunction and damage in conditions associated with reduced PGRN function.

Site-specific Phosphorylation of the p65 Protein Subunit Mediates Selective Gene Expression by Differential NF-κB and RNA Polymerase II Promoter Recruitment

Background: Phosphorylation of nuclear factor-κB (NF-κB) subunits is critical for NF-κB activity. Results: Mutation of phospho-acceptor sites within the p65 Rel homology domain influences NF-κB activity in a gene-dependent manner by altering p65 and RNA polymerase II promoter recruitment. Conclusion: Differential p65 phosphorylation serves as a code to target NF-κB transcriptional activity to distinct gene subsets. Significance: Our data provide insight into how NF-κB transcriptional specificity is achieved. Phosphorylation of NF-κB plays an important role in modulating transcriptional activity of NF-κB independently of inhibitor of κB (IκB) proteins. For the p65 subunit, multiple phosphorylation sites have been mapped in and adjacent to both the N-terminal Rel homology domain and the C-terminal transactivation domain. Their impact on NF-κB-dependent transcription, however, has never been assessed at a broader level. In this study, we evaluate the importance of differential p65 phosphorylation on four serine acceptor sites in the Rel homology domain for the expression of an array of NF-κB-dependent genes in endothelial cells. We find that inhibition of p65 phosphorylation on these serine residues targets NF-κB activity to distinctive gene subsets in a κB enhancer element-specific context. We show that the phosphorylation-dependent alterations in gene and protein expression are reflective of the amount of p65 and phosphorylated RNA polymerase II (p-RNAP II) bound to respective gene promoter regions. Depending on the gene subset, impaired gene expression was either a result of decreased p65 promoter recruitment or of a failure of bound p65 to recruit p-RNAP II. In conclusion, our findings demonstrate that site-specific p65 phosphorylation targets NF-κB activity to particular gene subsets on a global level by influencing p65 and p-RNAP II promoter recruitment.

Reperfusion Rather than Ischemia Drives the Formation of Ubiquitin Aggregates After Middle Cerebral Artery Occlusion

Background and Purpose— Cerebral ischemia leads to accumulation of ubiquitinated protein aggregates. However, the factors triggering ubiquitination and their impact on the outcome of cerebral ischemia remain poorly understood. Here we investigate the relationship between ubiquitin aggregation and duration of ischemia/reperfusion, infarct volume, and proteasomal activity in a mouse model of focal ischemia. Methods— Free ubiquitin and ubiquitin aggregate levels were examined by Western blotting in the mouse neocortex and striatum after different periods of ischemia/reperfusion and permanent ischemia induced by middle cerebral artery occlusion. Infarct volumes were measured in thionin-stained brain sections. Proteasome activity was studied by fluorometric peptidase activity assay. Results— Following transient ischemia, ubiquitin aggregates were detected in the ipsilateral neocortex and, to a lesser extent, striatum only after induction of reperfusion. In permanent ischemia, no ubiquitin aggregates were found. Shorter ischemic periods producing no or minimal tissue damage (10–15 minutes) resulted in ubiquitin aggregate levels similar to those produced by ischemia resulting in substantial infarction (30 minutes). Proteasomal impairment was greatest in ischemia without reperfusion, in which no ubiquitin aggregates were detected. Conclusions— The data demonstrate that reperfusion rather than ischemia leads to the appearance of ubiquitinated aggregates, which form in the absence of major tissue damage and are not correlated with decreased proteasomal peptidase activity. Ubiquitin aggregates may form in potentially viable brain tissue, which may be later recruited into infarction by factors independent of ubiquitination.

Highly homologous HERC proteins localize to endosomes and exhibit specific interactions with hPLIC and Nm23B

Abstract.Small HERC proteins are defined by the presence of one RCC1-like domain and a HECT domain. Having evolved out of one common ancestor, the four members of the family exhibit a high degree of homology in genomic organization and amino acid sequence, thus it seems possible that they might accomplish similar functions. Here we show that small HERC proteins interact with each other and localize to the same cellular structures, which we identify as late endosomes and lysosomes. We demonstrate interaction of HERC3 with the ubiquitin-like proteins hPLIC-1 and hPLIC-2 and we establish interaction of HERC5 with the metastasis suppressor Nm23B. While hPLIC proteins are not ubiquitinated by HERC3, HERC5 plays an important role in ubiquitination of Nm23B. In summary, although small HERC proteins are highly homologous showing the same subcellular distribution, they undergo different molecular interactions.

SUMO2/3 is Associated with Ubiquitinated Protein Aggregates in the Mouse Neocortex after Middle Cerebral Artery Occlusion

Protein modifications cooperatively act to protect the proteome from cellular stress. Focal cerebral ischemia increases protein ubiquitination, resulting in formation of ubiquitin-rich aggregates. A concurrent elevation in small ubiquitin-related modifier (SUMO)-conjugated proteins has also been reported, but a potential connection to ubiquitin remains unexplored. Here we show that SUMO2/3 conjugates are present in postischemic ubiquitin-rich aggregates, physically associated with ubiquitin. The coaggregation of SUMO2/3 and ubiquitin is induced rapidly after ischemia, depends on reperfusion, and is also observed in the absence of ischemic damage. The association between SUMO and ubiquitin suggests overlapping functional roles after ischemia/reperfusion.

Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation

Termination and resolution of inflammation are tightly linked to the inactivation of one of its strongest inducers, NF-κB. While canonical post-stimulus inactivation is achieved by upregulation of inhibitory molecules that relocate NF-κB complexes to the cytoplasm, termination of the NF-κB response can also be accomplished directly in the nucleus by posttranslational modifications, e.g., ubiquitination of the RelA subunit. Here we reveal a functional role for RelA monoubiquitination in regulating NF-κB activity. By employing serine-to-alanine mutants, we found that hypo-phosphorylated nuclear RelA is monoubiquitinated on multiple lysine residues. Ubiquitination was reversed by IκBα expression and was reduced when nuclear translocation was inhibited. RelA monoubiquitination decreased NF-κB transcriptional activity despite prolonged nuclear presence and independently of RelA degradation, possibly through decreased CREB-binding protein (CBP) co-activator binding. Polyubiquitin-triggered proteasomal degradation has been proposed as a model for RelA inactivation. However, here we show that proteasomal inhibition, similar to RelA hypo-phosphorylation, resulted in nuclear translocation and monoubiquitination of RelA. These findings indicate a degradation-independent mechanism for regulating the activity of nuclear RelA by ubiquitination.

Regulation of Nuclear Factor κB (NF-κB) Transcriptional Activity via p65 Acetylation by the Chaperonin Containing TCP1 (CCT)

The NF-κB family member p65 is central to inflammation and immunity. The purpose of this study was to identify and characterize evolutionary conserved genes modulating p65 transcriptional activity. Using an RNAi screening approach, we identified chaperonin containing TCP1 subunit η (CCTη) as a regulator of Drosophila NF-κB proteins, Dorsal and Dorsal-related immunity factor (Dif). CCTη was also found to regulate NF-κB-driven transcription in mammalian cells, acting in a promoter-specific context, downstream of IκB kinase (IKK). CCTη knockdown repressed IκBα and CXCL2/MIP2 transcription during the early phase of NF-κB activation while impairing the termination of CCL5/RANTES and CXCL10/IP10 transcription. The latter effect was associated with increased DNA binding and reduced p65 acetylation, presumably by altering the activity of histone acetyltransferase CREB-binding protein (CBP). We identified p65 lysines (K) 122 and 123 as target residues mediating the CCTη-driven termination of NF-κB-dependent transcription. We propose that CCTη regulates NF-κB activity in a manner that resolves inflammation.

Crosstalk between the NF-κB activating IKK-complex and the CSN signalosome

A great variety of signalling pathways regulating inflammation, cell development and cell survival require NF‐κB transcription factors, which are normally inactive due to binding to inhibitors, such as IκBα. The canonical activation pathway of NF‐κB is initiated by phosphorylation of the inhibitor by an IκB kinase (IKK) complex triggering ubiquitination of IκB molecules by SCF‐type E3‐ligase complexes and rapid degradation by 26S‐proteasomes. The ubiquitination machinery is regulated by the COP9 signalosome (CSN). We show that IκB kinases interact with the CSN‐complex, as well as the SCF‐ubiquitination machinery, providing an explanation for the rapid signalling‐induced ubiquitination and degradation of IκBα. Furthermore, we reveal that IKK’s phosphorylate not only IκBα, but also the CSN‐subunit Csn5/JAB1 (c‐Jun activation domain binding protein‐1) and that IKK2 influences ubiquitination of Csn5/JAB1. Our observations imply that the CSN complex acts as an inhibitor of constitutive NF‐κB activity in non‐activated cells. Knock‐down of Csn5/JAB1 clearly enhanced basal NF‐κB activity and improved cell survival under stress. The inhibitory effect of Csn5/JAB1 requires a functional MPN+ metalloprotease domain, which is responsible for cleaving ubiquitin‐like Nedd8‐modifications. Upon activation of cells with tumour necrosis factor‐α, the CSN complex dissociates from IKK’s allowing full and rapid activation of the NF‐κB pathway by the concerted action of interacting protein complexes.

Hypo-phosphorylation leads to nuclear retention of NF-κB p65 due to impaired IκBα gene synthesis

Subcellular localization guided by IκBα is crucial for regulation of nuclear factor‐κB function. Here, we show that p65 Rel homology domain phosphorylation mutants are transported into the nucleus after IκBα degradation, but as a consequence of lower IκBα levels their relocation to the cytosol is blocked. We demonstrate that phosphorylation of residues S205, S276, and S281 of p65 is not required for interaction between p65 and IκBα, but is pivotal for regulating cellular IκBα levels by positively affecting gene synthesis. Our findings indicate that reduction of phosphorylation leads to nuclear retention of p65, which might be partly responsible for altered transcriptional behavior of p65 serine mutants.