Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-κB pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-κB) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-κB DNA binding activity (NF-κBp50 and NF-κBp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-κB pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-κB (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-κB (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-κB signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals.     

NF-κB- and AP-1-mediated DNA looping regulates osteopontin transcription in endotoxin-stimulated murine macrophages

Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression.     

Chronic inhibition of nuclear factor kappa B attenuates aldosterone/salt-induced renal injury

AimsRecent studies suggested that nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of renal injury. This study investigated whether NF-κB inhibition attenuates progressive renal damage in aldosterone/salt-induced renal injury and its mechanisms.Main methodsAdult male rats were uninephrectomized and treated with one of the following for 4 weeks: vehicle (0.5% ethanol, subcutaneously); vehicle/1% NaCl (1% NaCl in drinking solution); aldosterone/1% NaCl (1% NaCl in drinking solution and aldosterone, 0.75 μg/h, subcutaneously); or aldosterone/1%NaCl + pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB (100 mg/kg/day, by gavage). The activity of NF-κB was measured by EMSA and immunohistochemistry, CTGF and ICAM-1 were measured by Western blot and real-time PCR, and TGF-β and CTGF were measured by immunohistochemistry.Key findingsRats that received aldosterone/1% NaCl exhibited hypertension and severe renal injury. Renal cortical mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, protein expression of CTGF and ICAM-1, and NF-κB–DNA binding activity were significantly upregulated in rats that received aldosterone/1% NaCl. Treatment with PDTC significantly decreased the percentage of cells positive for CTGF and TGF-β; mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, and protein levels of CTGF and ICAM-1 were also inhibited by PDTC.SignificanceThese data suggest that the NF-κB signal pathway plays a role in the progression of aldosterone/salt-induced renal injury.     

Vibrio vulnificus AphB is involved in interleukin-8 production via an NF-κB-dependent pathway in human intestinal epithelial cells

We previously reported that the aphB gene mutant of Vibrio vulnificus had significantly impaired motility and adherence to host cells. In this study, we investigated the role of V. vulnificus AphB on the production of interleukin-8 (IL-8), a proinflammatory cytokine, as well as its underlying mechanism in human intestinal epithelial INT-407 cells. The aphB gene mutation significantly reduced the ability of V. vulnificus to stimulate IL-8 production and IL-8 gene promoter activation in INT-407 cells. The V. vulnificusaphB mutant also induced lower levels of NF-κB DNA binding activity and NF-κB minimal promoter activation than did the wild-type of V. vulnificus. Importantly, the observed reductions in IL-8 production, IL-8 gene promoter activation and NF-κB DNA binding activity were significantly restored by complementing the aphB gene into the V. vulnificusaphB mutant. These results indicate that V. vulnificus AphB is involved in the IL-8 production via an NF-κB dependent pathway in human intestinal epithelial cells.     

Hyperglycemia Exacerbates Burn-Induced Liver Inflammation via Noncanonical Nuclear Factor-κB Pathway Activation

Hyperglycemia and inflammation are hallmarks of burn injury. In this study, we used a rat model of hyperglycemia and burn injury to investigate the effects of hyperglycemia on inflammatory responses in the liver. Hyperglycemia was induced in male Sprague-Dawley rats with streptozotocin (STZ) (35-40 mg/kg), followed by a 60% third-degree scald burn injury. Cytokine levels (by multiplex, in cytosolic liver extracts), hormones (by enzyme-linked immunosorbent assay [ELISA], in serum), nuclear factor (NF)-κB protein deoxyribonucleic acid (DNA) binding (by ELISA, in nuclear liver extracts) and liver functional panel (using VetScan, in serum) were measured at different time points up to 7 d after burn injury. Blood glucose significantly increased after burn injury in both groups with different temporal patterns. Hyperglycemic rats were capable of endogenous insulin secretion, which was enhanced significantly versus controls 12 h after burn injury. DNA binding data of liver nuclear extracts showed a robust and significant activation of the noncanonical NF-κB pathway in the hyperglycemic versus control burn animals, including increased NF-κB-inducing kinase expression (p < 0.05). Liver acute-phase proteins and cytokine expression were increased, whereas secretion of constitutive proteins was decreased after burn injury in hyperglycemic versus control animals (p < 0.05). These results indicate that burn injury to the skin rapidly activated canonical and noncanonical NF-κB pathways in the liver. Robust activation of the NF-κB noncanonical pathway was associated with increased expression of inflammatory markers and acute-phase proteins, and impaired glucose metabolism. Hyperglycemia is detrimental to burn outcome by augmenting inflammation mediated by hepatic noncanonical NF-κB pathway activation.     

Thiophenacetamide as a potential modulator to NF-κB: structure and dynamics study using in silico and molecular biology assays

Abstract

Nuclear factor kappa B (NF-κB) plays critical roles in the regulation of many pathophysiological processes, including inflammation and immune responses, cell growth and apoptosis. This DNA-binding protein receptor is considered an important molecular target to treat many diseases through host-directed therapy. In this line, several drugs containing thiophene cores have been extensively evaluated due to their ability to interfere on NF-κB translocation to the nucleus. In this work, assays using drug affinity responsive target stability (DARTS) revealed that the parent compound N-(Aryl)-2-thiophen-2-ylacetamide referred to as thiophenacetamide (TAA) specifically binds to the p65 subunit of the NF-κB. Since no experimental binding mode of TAA with p65 is available, we explored TAA within putative sites in silico to gain insights into its possible binding mode and behavior. The binding mode of TAA found in Site 1 formed hydrogen bonds with Lys37 and Asp125 on p65, important residues near DNA-binding region. Molecular dynamics simulations showed the stability of this mode of binding in contrast to the other also tested modes. Our results suggest that TAA binding could occur in regions close to residues responsible for DNA binding, increasing NF-κB protein rigidity and affecting the association between DNA and NF-κB.

Communicated by Ramaswamy H. Sarma