Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that breaks down superoxide anion into oxygen and hydrogen peroxide in extracellular spaces and plays key roles in controlling pulmonary and vascular diseases in response to oxidative stresses. We aimed to investigate the role of EC-SOD in angiogenesis and inflammation in chronic inflammatory skin disorders such as psoriasis. Overexpressed EC-SOD reduced expression of angiogenic factors and proinflammatory mediators in hypoxia-induced keratinocytes and in ultraviolet B-irradiated mice, whereas the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase-1 and anti-inflammatory cytokine interleukin-10 were increased. EC-SOD decreased new vessel formation, epidermal edema, and inflammatory cell infiltration in UVB-irradiated transgenic mice. Moreover, cells treated with recombinant human EC-SOD showed inhibited endothelial tube formation and cell proliferation. Overall, the antiangiogenic and anti-inflammatory effects of EC-SOD might be due to suppression of hypoxia-inducible factor-1α, protein kinase C, and nuclear factor-κB expression. Furthermore, EC-SOD expression in tissue from psoriasis patients was markedly decreased in psoriatic lesional and nonlesional skins from psoriasis patients in comparison to normal skin from healthy volunteers. Together, these results suggest that EC-SOD may provide a novel therapeutic approach to treating angiogenic and inflammatory skin diseases such as psoriasis.     

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.     

Docosahexaenoic acid inhibition of inflammation is partially via cross-talk between Nrf2/heme oxygenase 1 and IKK/NF-κB pathways

We examined the underlying mechanisms involved in n-3 docosahexaenoic acid (DHA) inhibition of inflammation in EA.hy926 cells. The present results demonstrated that pretreatment with DHA (50 and 100 μM) inhibited tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule 1 (ICAM-1) protein, mRNA expression and promoter activity. In addition, TNF-α-stimulated inhibitory kappa B (IκB) kinase (IKK) phosphorylation, IκB phosphorylation and degradation, p65 nuclear translocation, and nuclear factor-κB (NF-κB) and DNA binding activity were attenuated by pretreatment with DHA. DHA triggered early-stage and transient reactive oxygen species (ROS) generation and significantly increased the protein expression of heme oxygenase 1 (HO-1), induced nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, DHA inhibited Nrf2 ubiquitination and proteasome activity. DHA activated Akt, p38 and ERK1/2 phosphorylation, and specific inhibitors of respective pathways attenuated DHA-induced Nrf2 nuclear translocation and HO-1 expression. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of TNF-α-induced p65 nuclear translocation and ICAM-1 expression. Importantly, we show for the first time that HO-1 plays a down-regulatory role in NF-κB nuclear translocation, and inhibition of Nrf2 ubiquitination and proteasome activity are involved in increased cellular Nrf2 level by DHA. In this study, we show that HO-1 plays a down-regulatory role in NF-κB nuclear translocation and that the protective effect of DHA against inflammation is partially via up-regulation of Nrf2-mediated HO-1 expression and inhibition of IKK/NF-κB signaling pathway.     

Insulin/IGF-1 paradox of aging: Regulation via AKT/IKK/NF-κB signaling

GH/insulin/IGF-1 signaling is a vital pathway e.g. in the regulation of protein synthesis and glucose metabolism. However, mouse dwarf strains which exhibit reduced GH secretion and subsequently a decline in IGF-1 signaling can live longer than their wild type counterparts. There is striking evidence indicating that the IGF-1/PI-3K/AKT signaling enhances growth of animals during development but later in life can potentiate the aging process. This conserved pleiotropy has been called the insulin/IGF-1 paradox. In Caenorhabditis elegans, the decline in this pathway activates the DAF-16 gene, an ortholog of mammalian FoxO genes, which regulate stress resistance and longevity. The mammalian PI-3K/AKT pathway also activates the NF-κB signaling that inhibits apoptosis and triggers inflammatory responses. Many longevity genes, e.g. FoxOs and SIRT1, are inhibitors of NF-κB signaling. We will discuss the evidence that insulin/IGF-1 signaling can enhance the NF-κB signaling and subsequently potentiate the aging process and aggravate age-related degenerative diseases.     

Gprc5a-deficiency confers susceptibility to endotoxin-induced acute lung injury via NF-κB pathway

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1β and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.     

Astragalus polysaccharides alleviates LPS-induced inflammation via the NF-κB/MAPK signaling pathway

Early weaning usually causes intestinal disorders, enteritis, and diarrhea in young animals and human infants. Astragalus polysaccharides (APS) possesses anti-inflammatory activity. To study the anti-inflammatory mechanisms of APS and its potential effects on intestinal health, we performed an RNA sequencing (RNA-seq) study in lipopolysaccharide (LPS)-stimulated porcine intestinal epithelial cells (IPEC-J2) in vitro. In addition, LPS-stimulated BALB/c mice were used to study the effects of APS on intestinal inflammation in vivo. The results from the RNA-seq analysis show that there were 107, 756, and 5 differentially expressed genes in the control versus LPS, LPS versus LPS+APS, and control versus LPS+APS comparison groups, respectively. The results of Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways play significant roles in the regulation of inflammatory factors and chemokine expression by APS. Further verification of the above two pathways by using western blot and immunofluorescence analysis revealed that the gene expression levels of the phosphorylated p38 MAPK, ERK1/2, and NF-κB p65 were inhibited by APS, while the expression of IκB-α protein was significantly increased (p < .05), indicating that APS inhibits the production of inflammatory factors and chemokines by the inhibition of activation of the MAPK and NF-κB inflammatory pathways induced by LPS stimulation. Animal experiments further demonstrated that prefeeding APS in BALB/c mice can alleviate the expression of the jejunal inflammatory factors interleukin 6 (IL-6), IL-Iβ, and tumor necrosis factor-α induced by LPS stimulation and improve jejunal villus morphology.     

Microtubule-mediated NF-κB activation in the TNF-α signaling pathway

The microtubule cytoskeleton is known to play a role in cell structure and serve as a scaffold for a variety of active molecules in processes as diverse as motility and cell division. The literature on the role of microtubules in signal transduction, however, is marked by inconsistencies. We have investigated a well-studied signaling pathway, TNF-α-induced NF-κB activation, and found a connection between the stability of microtubules and the regulation of NF-κB signaling in C2C12 myotubes. When microtubules are stabilized by paclitaxel (taxol), there is a strong induction of NF-κB even in the absence of TNF-α . Although there was no additive effect of taxol and TNF-α on NF-κB activity suggesting a shared mechanism of activation, taxol strongly induced the NF-κB reporter in the presence of a TNF receptor (TNFR) blocking antibody while TNF-α did not. Both TNF-α and taxol induce the degradation of endogenous IκBα and either taxol or TNF-α induction of NF-κB activity was blocked by inhibitors of NF-κB acting at different sites in the signaling pathway. Both TNF-α and taxol strongly induce known NF-κB chemokine target genes. On the other hand, if microtubules are destabilized by colchicine, then the induction of NF-κB by TNF-α or taxol is greatly reduced. Taken together, we surmise that the activity of microtubules is at the level of the TNFR intracellular domain. This phenomenon may indicate a new level of signaling organization in cell biology, actively created by the state of the cytoskeleton, and has ramifications for therapies where microtubule regulating drugs are used.     

Galangin suppresses RANKL-induced osteoclastogenesis via inhibiting MAPK and NF-κB signalling pathways

Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti-inflammation and anti-oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose-dependently decreased osteoclastogenesis in bone marrow–derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V-ATPase d2) and dendritic cell–specific transmembrane protein (DC-STAMP), were down-regulated by galangin treatment. Furthermore, the results of the pit formation assay and F-actin ring staining revealed impaired osteoclastic bone resorption in the galangin-treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C-Jun and C-Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)-induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF-κB signalling pathways.