Lipopolysaccharide-Induced NF-κB Activation in Human Endothelial Cells Involves Degradation of IκBα but Not IκBβ

We studied the signal transduction pathways involved in NF-κB activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitorN-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-κB DNA-binding activity induced by LPS treatment. LPS induced IκBα degradation at 30–60 min after treatment, but did not induce IκBβ degradation up to 120 min. In contrast, TNF-α rapidly induced IκBα degradation within 5 min and IκBβ degradation within 15 min. Cycloheximide did not prevent LPS-induced IκBα degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IκBα degradation. LPS-induced IκBα degradation was inhibited by ALLN, confirming that ALLN inhibits NF-κB activation by preventing IκBα degradation. Of note, HMA also inhibited LPS-induced IκBα degradation. However, tyrosine phosphorylation of IκBα itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IκBα is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-κB-dependent genes through degradation of IκBα, not IκBβ, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IκBα.     

Role of Novel Serine 316 Phosphorylation of the p65 Subunit of NF-κB in Differential Gene Regulation

Nuclear factor κB (NF-κB) is a central coordinator in immune and inflammatory responses. Constitutive NF-κB is often found in some types of cancers, contributing to oncogenesis and tumor progression. Therefore, knowing how NF-κB is regulated is important for its therapeutic control. Post-translational modification of the p65 subunit of NF-κB is a well known approach for its regulation. Here, we reported that in response to interleukin 1β, the p65 subunit of NF-κB is phosphorylated on the novel serine 316. Overexpression of S316A (serine 316 → alanine) mutant exhibited significantly reduced ability to activate NF-κB and decreased cell growth as compared with wtp65 (wild type p65). Moreover, conditioned media from cells expressing the S316A-p65 mutant had a considerably lower ability to induce NF-κB than that of wtp65. Our data suggested that phosphorylation of p65 on Ser-316 controls the activity and function of NF-κB. Importantly, we found that phosphorylation at the novel Ser-316 site and other two known phosphorylation sites, Ser-529 and Ser-536, either individually or cooperatively, regulated distinct groups of NF-κB-dependent genes, suggesting the unique role of each individual phosphorylation site on NF-κB-dependent gene regulation. Our novel findings provide an important piece of evidence regarding differential regulation of NF-κB-dependent genes through phosphorylation of different p65 serine residues, thus shedding light on novel mechanisms for the pathway-specific control of NF-κB. This knowledge is key to develop strategies for prevention and treatment of constitutive NF-κB-driven inflammatory diseases and cancers.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

Vascular calcification is a predictor of cardiovascular mortality and is prevalent in patients with atherosclerosis and chronic renal disease. It resembles skeletal osteogenesis, and many bone cells as well as bone-related factors involved in both formation and resorption have been localized in calcified arteries. Previously, we showed that aortic medial cells undergo osteoblastic differentiation and matrix calcification both spontaneously and in response to PKA agonists. The PKA signaling pathway is also involved in regulating bone resorption in skeletal tissue by stimulating osteoblast-production of osteoclast regulating cytokines, including receptor-activator of nuclear κB ligand (RANKL) and interleukins. Therefore, we investigated whether PKA activators regulate osteoclastogenesis in aortic smooth muscle cells (SMC). Treatment of murine SMC with the PKA agonist forskolin stimulated RANKL expression at both mRNA and protein levels. Forskolin also stimulated expression of interleukin-6 but not osteoprotegerin (OPG), an inhibitor of RANKL. Consistent with these results, osteoclastic differentiation was induced when monocytic preosteoclasts (RAW264.7) were cocultured with forskolin-treated aortic SMC. Oxidized phospholipids also slightly induced RANKL expression in T lymphocytes, another potential source of RANKL in the vasculature. Because previous studies have shown that RANKL treatment alone induces matrix calcification of valvular and vascular cells, we next examined whether RANKL mediates forskolin-induced matrix calcification by aortic SMC. RANKL inhibition with OPG had little or no effect on osteoblastic differentiation and matrix calcification of aortic SMC. These findings suggest that, as in skeletal tissues, PKA activation induces bone resorptive factors in the vasculature and that aortic SMC calcification specifically induced by PKA, is not mediated by RANKL.     

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.     

Photoprotective Potential of Penta-O-Galloyl-β-DGlucose by Targeting NF-κB and MAPK Signaling in UVB Radiation-Induced Human Dermal Fibroblasts and Mouse Skin

Exposure of the skin to ultraviolet radiation can cause skin damage with various pathological changes including inflammation. In the present study, we identified the skin-protective activity of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (pentagalloyl glucose, PGG) in ultraviolet B (UVB) radiation-induced human dermal fibroblasts and mouse skin. PGG exhibited antioxidant activity with regard to intracellular reactive oxygen species (ROS) generation as well as ROS and reactive nitrogen species (RNS) scavenging. Furthermore, PGG exhibited anti-inflammatory activity, inhibiting the activation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, resulting in inhibition of the expression of pro-inflammatory mediators. Topical application of PGG followed by chronic exposure to UVB radiation in the dorsal skin of hairless mice resulted in a significant decrease in the progression of inflammatory skin damages, leading to inhibited activation of NF-κB signaling and expression of pro-inflammatory mediators. The present study demonstrated that PGG protected from skin damage induced by UVB radiation, and thus, may be a potential candidate for the prevention of environmental stimuli-induced inflammatory skin damage.     

Activation of EMT in colorectal cancer by MTDH/NF-κB p65 pathway

Molecular and Cellular Biochemistry - Epithelial-mesenchymal transition (EMT) leads to tumor dissemination and metastasis. Metadherin (MTDH) is an oncogene that plays an important role in...     

Regulation of Xylosyltransferase I Gene Expression by Interleukin 1β in Human Primary Chondrocyte Cells: MECHANISM AND IMPACT ON PROTEOGLYCAN SYNTHESIS*

Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1β regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation. Based on promoter deletions, the region up to −850 bp was defined as a major element of XT-I gene displaying both constitutive and IL-1β-regulated promoter activity. Point mutation and signaling analyses revealed that IL-1β-induced promoter activity is achieved through AP-1 response elements and mediated by SAP/JNK and p38 signaling pathways. Transactivation and chromatin immunoprecipitation assays indicated that AP-1 is a potent transactivator of XT-I promoter and that IL-1β-induced activity is mediated through increased recruitment of AP-1 to the promoter. Finally, we show that Sp3 is a repressor of XT-I promoter and bring evidence that the repressive effect of IL-1β during the late phase is mediated through Sp3 recruitment to the promoter. This suggests that modulation of Sp3 in cartilage could prevent IL-1β inhibition of PG synthesis and limit tissue degradation.