- The uncoordinated-5 homolog B receptor affects hepatic ischemia reperfusion injury (2012)
- Recent evidence has demonstrated additional roles for the neuronal guidance protein receptor UNC5B outside the nervous system. Given the fact that ischemia reperfusion injury (IRI) of the liver is a common source of liver dysfunction and the role of UNC5B during an acute inflammatory response we investigated the role of UNC5B on acute hepatic IRI. We report here that UNC5B+/− mice display reduced hepatic IRI and neutrophil (PMN) infiltration compared to WT controls. This correlated with serum levels of lactate dehydrogenase (LDH), aspartate- (AST) and alanine- (ALT) aminotransferase, the presence of PMN within ischemic hepatic tissue, and serum levels of inflammatory cytokines. Moreover, injection of an anti-UNC5B antibody resulted in a significant reduction of hepatic IR injury. This was associated with reduced parameters of liver injury (LDH, ALT, AST) and accumulation of PMN within the injured hepatic tissue. In conclusion our studies demonstrate a significant role for UNC5B in the development of hepatic IRI and identified UNC5B as a potential drug target to prevent liver dysfunction in the future.
- Ninjurin 1 contributes to TLR-induced inflammation in endothelial cells (2012)
- Background: Nerve injury induced protein 1 (Ninjurin 1 (Ninj1)) was first identified in Schwann cells and neurons contributing to cell adhesion and nerve regeneration. Recently, the role of Ninj1 has been linked to inflammatory processes in the central nervous system where functional repression reduced leukocyte infiltration and clinical disease activity during experimental autoimmune encephalomyelitis in mice . But Ninj1 is also expressed outside the nervous system in various organs such as the liver and kidney as well as on leukocytes [2,3]. Therefore, we hypothesized that Ninj1 contributes to inflammation in general; that is, also outside the nervous system, with special interest in the pathogenesis of sepsis. Methods: Ninj1 was repressed by transfecting HMEC-1 cells, a human dermal microvascular endothelial cell line with siRNA targeting Ninj1 (siNinj1) or a negative control (siC). Subsequently, cells were stimulated with 100 ng/ml LPS (TLR4 agonist), 3 μg/ml LTA (TLR2 agonist) or 100 n/ml poly(I:C) (TLR3 agonist) for 3 hours. The inflammatory response was analyzed by real-time PCR. In addition, transmigration of neutrophils across a HMEC-1 monolayer was measured using transwell plates (pore size 3 μm). Results: Repression of Ninj1 by siRNA reduced Ninj1 mRNA expression in HMEC about 90% (Figure 1A). Reduced Ninj1 expression decreased neutrophil migration to 62.5% (Figure 1B) and TLR signaling. In detail, knockdown of Ninj1 significantly reduced TLR-2 and TLR-4 triggered expression of ICAM-1 and IL-6 (Figure 1C,D) while poly(I:C)-induced expression was only slightly reduced. To analyze a more specific TLR-3 target, we measured IP-10 mRNA expression, which was also significantly reduced in siNinj1-transfected cells (Figure 1E). Conclusion: Our in vitro data strongly indicated that Ninj1 is involved in regulation of TLR signaling and therewith contributes to inflammation. In vivo experiments will clarify its impact on systemic inflammation.
- Regulation of peroxisome proliferator-activated receptor gamma in macrophages during inflammatory processes (2009)
- The peroxisome proliferator activated receptor gamma (PPARgamma) plays an eminent role during alternative activation of macrophages and resolution of inflammation. As an antiinflammatory signaling molecule, it seems likely that it is tightly regulated dependent on the state of the immune response. There is growing evidence that PPARgamma expression is reduced during inflammation, whereas molecular mechanisms are illdefined. Even though, its role in immunosuppression is getting more definite. Apoptotic cells (AC) provoke an active repression of pro-inflammatory responses inter alia by the inhibition of pro-inflammatory cytokine expression or attenuated generation of reactive oxygen species (ROS). The reduced formation of ROS was attributed to PPARgamma activation, while mechanisms behind the reduced cytokine expression remained unclear. Therefore, my Ph.D. thesis addressed the role of PPARgamma during inhibited cytokine synthesis in response to AC and the regulation of PPARgamma expression during an inflammatory response, which was initiated by lipopolysaccharide (LPS) exposure. In the first part of the thesis, I investigated the role of PPARgamma in coordinating the attenuation of pro-inflammatory cytokine expression in response to AC. Exposing murine RAW264.7 macrophages to AC prior to LPS-stimulation, reduced NFKB transactivation and lowered target gene expression of e.g. TNFalpha and IL-6 compared to controls. In macrophages over-expressing a dominant negative (d/n) mutant of PPARgamma, NFKB transactivation in response to LPS was restored, while using macrophages from myeloid lineage-specific conditional PPARgamma knock-out mice proved that PPARgamma transmitted the anti-inflammatory response delivered by AC. Domain analysis revealed that amino acids 32-250 are essential for inhibition of NFKB. Mutation of a SUMOylation (SUMO: small-ubiquitin related modifier) site in this region (K77R) and interfering SUMOylation by silencing the SUMO E3 ligase PIAS1 (protein inhibitor of activated Stat1) eliminated AC-provoked NFKB inhibition and concomitant TNFalpha expression. Chromatin-immunoprecipitation assays demonstrated that AC prevented the LPS-induced removal of nuclear receptor co-repressor (NCoR) from the KB response element within the TNFalpha promoter. I concluded that AC induce PPARgamma SUMOylation to attenuate the removal of NCoR, thereby blocking transactivation of NFKB. This contributes to an anti-inflammatory phenotype shift in macrophages in response to AC, by lowering pro-inflammatory cytokine production. The second part addressed molecular mechanisms responsible for reduced PPARgamma expression upon LPS exposure. PPARgamma gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer’s disease is associated with impaired PPARgamma expression. Initiation of an inflammatory response by exposing primary human macrophages to LPS revealed a rapid decline of PPARgamma1 expression. PPARgamma1 mRNA decrease was prevented by inhibition of NFKB and also after pre-treatment with the PPARgamma agonist rosiglitazone, suggesting a NFKB-dependent pathway, because activated PPARgamma is known to inhibit NFKB transactivation. Since promoter activities were not affected by LPS, I focused on mRNA stability and noticed a decreased PPARgamma1 mRNA half-life. RNA stability is often regulated via 3’ untranslated regions (UTRs). Therefore, I analyzed the impact of the PPARgamma-3’UTR by luciferase assays. LPS significantly reduced luciferase activity of pGL3-PPARgamma-3’UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion of a potential miR-27a/b binding site within the 3’UTR completely restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS-exposure, partially reversed PPARgamma1 mRNA decay, whereas the mature miR-27 mimicked the effect of LPS. MiR-27b was at least partially induced by NFKB, thus correlating with NFKB-dependent PPARgamma1 mRNA decrease. Since deletion of the miR-27 site also containing an AU-rich element (ARE) completely abrogated LPS-induced reduction but inhibition of miR-27b only partially restored PPARgamma1 mRNA expression, I suggested an additional implication of an ARE-binding protein. I provide evidence that LPS induces miR-27b, which in turn destabilizes PPARgamma1 mRNA. Understanding the molecular mechanism of PPARgamma mRNA destabilization, might help to rationalize inflammatory diseases associated with impaired PPARgamma expression. Even though, further experiments are needed to clarify the potential involvement of ARE-binding proteins.
- Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma cells (2013)
- As inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the second mitochondrial activator of caspases (Smac) mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical nuclear factor-κB (NF-κB) signaling and a tumor necrosis factor-α (TNFα)/TNF receptor 1 (TNFR1) autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cellular IAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. Electrophoretic mobility shift assay supershift shows that the NF-κB DNA-binding subunits consist of p50, p52 and RelB further confirming the activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e., interleukin 8, monocyte chemoattractant protein 1, CXC chemokine receptor 4) and invasion (i.e., matrix metalloproteinase-9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics.