P65 Subunit Of Nuclear factor-kappaB Research Articles

The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages.

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

MiR-217 Regulates SIRT1 Expression and Promotes Inflammatory and Apoptotic Responses in Osteoarthritis.

Previous studies have reported miR-217 uregulation in age-related pathologies. We investigated the impact of miR-217-5p on sirtuin 1 (SIRT1) regulation in human osteoarthritic (OA) chondrocytes. MiR-217 target enrichment analyses were performed using three public databases, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. MiR-217-5p expression levels were quantified in normal and OA chondrocytes. SIRT1 expression levels, nuclear factor kappa-B p65 subunit (NF-κBp65) and p53 acetylation levels, and expression levels of OA-related pro-inflammatory markers [tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), IL-6], pro-apoptotic markers [Bax, pro-caspase 3, cleaved caspase 3] and matrix regulators [matrix metalloproteinase (MMP)-1, MMP-13, MMP-9, Collagen 2 (COL2A1), Aggrecan (ACAN)] were evaluated in miR-217 mimic-treated and/or miR-217 inhibitor-treated OA chondrocytes, with/without subsequent treatment with siRNA against SIRT1 (siSIRT1). MiR-217-5p was upregulated in OA chondrocytes, while target prediction/enrichment analyses revealed SIRT1 as miR-217 target-gene. Deacetylation of NF-κBp65 and p53 in miR-217 inhibitor-treated OA chondrocytes was reversed by siSIRT1 treatment. MiR-217 inhibitor-treated OA chondrocytes showed increased COL2A1, ACAN and decreased IL-1β, IL-6, TNFα, Bax, cleaved caspase 3 and MMPs expression levels, which were reversed following miR-217 inhibitor/siSIRT1 treatment. Our findings highlight the impact of miR-217-5p on SIRT1 downregulation contributing to OA pathogenesis.

DNA methylation regulates Sirtuin 1 expression in osteoarthritic chondrocytes

PurposeSirtuin 1 (SIRT1) comprises a major anti-aging longevity factor with multiple protective effects on chondrocyte homeostasis. Previous studies have reported that downregulation of SIRT1 is linked to osteoarthritis (OA) progression. In this study, we aimed to investigate the role of DNA methylation on SIRT1 expression regulation and deacetylase activity in human OA chondrocytes. Materials and methodsMethylation status of SIRT1 promoter was analyzed in normal and OA chondrocytes using bisulfite sequencing analysis. CCAAT/enhancer binding protein alpha (C/EBPα) binding to SIRT1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Subsequently, C/EBPα′s interaction with SIRT1 promoter and SIRT1 expression levels were evaluated after treatment of OA chondrocytes with 5-Aza-2′-Deoxycytidine (5-AzadC). Acetylation and nuclear levels of nuclear factor kappa-B p65 subunit (NF-κΒp65) and expression levels of selected OA-related inflammatory mediators, interleukin 1β (IL-1β) and IL-6 and catabolic genes (metalloproteinase-1 (MMP-1) and MMP-9) were evaluated in 5-AzadC-treated OA chondrocytes with or without subsequent transfection with siRNA against SIRT1. ResultsHypermethylation of specific CpG dinucleotides on SIRT1 promoter was associated with downregulation of SIRT1 expression in OA chondrocytes. Moreover, we found decreased binding affinity of C/EBPα on the hypermethylated SIRT1 promoter. 5-AzadC treatment restored C/EBPα′s transcriptional activity inducing SIRT1 upregulation in OA chondrocytes. Deacetylation of NF-κΒp65 in 5-AzadC-treated OA chondrocytes was prevented by siSIRT1 transfection. Similarly, 5-AzadC-treated OA chondrocytes exhibited decreased expression of IL-1β, IL-6, MMP-1 and MMP-9 which was reversed following 5-AzadC/siSIRT1 treatment. ConclusionsOur results suggest the impact of DNA methylation on SIRT1 suppression in OA chondrocytes contributing to OA pathogenesis.

Long-term exercise preserves pancreatic islet structure and β-cell mass through attenuation of islet inflammation and fibrosis.

Islet fibrosis is associated with the disruption of islet structure and contributes to β-cell dysfunction, playing an essential role in the pathogenesis of type 2 diabetes. Physical exercise has been shown to attenuate fibrosis in various organs; however, the effect of exercise on islet fibrosis has not been defined. Male Sprague-Dawley rats were divided into four groups: normal diet sedentary [N-Sed], normal diet + exercise [N-Ex], high-fat diet sedentary [H-Sed], and high-fat diet + exercise [H-Ex]. After 60 weeks of exercise, 4452 islets from Masson-stained slides were analyzed. Exercise led to a 68% and 45% reduction in islet fibrosis in the normal and high-fat diet groups and was correlated with a lower serum blood glucose. Fibrotic islets were characterized by irregular shapes and substantial loss of β-cell mass, which were significantly reduced in the exercise groups. Remarkably, the islets from exercised rats at week 60 were morphologically comparable to those of sedentary rats at 26 weeks. In addition, the protein and RNA levels of collagen and fibronectin, and the protein levels of hydroxyproline in the islets were also attenuated by exercise. This was accompanied by a significant reduction in inflammatory markers in the circulation Interleukin-1 beta (IL-1β)] and pancreas [IL-1β, Tumor Necrosis Factor-alpha, Transforming Growth Factor-β, and Phosphorylated Nuclear Factor Kappa-B p65subunit], lower macrophage infiltration, and stellate cell activation in the islets of exercised rats. In conclusion, we have demonstrated that long-term exercise preserves pancreatic islet structure and β-cell mass through anti-inflammatory and anti-fibrotic actions, suggesting additional rationales for the success of exercise training in the prevention and treatment of type 2 diabetes that should be further explored.

Effects of Mixed Nuts on Colonic Cell Proliferation and Ptgs2 and Rela Gene Expression.

Nut consumption is associated with lower risk of colorectal cancer (CRC). Previously, single nut varieties have been investigated but there is limited research on the consumption of a nut mixture and the underlying mechanisms. This study examined mixed nut consumption's effect on colonic cell proliferation, apoptosis, and gene expression involved in CRC. Thirty 21-day old Sprague Dawley rats were divided into three groups: control (no nuts), pistachio or mixed nut for 8 weeks. Ki-67 quantitative immunostaining was used to mark proliferative cells and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptotic cells. Real-time quantitative polymerase chain reaction analysis was used to determine colonic gene expression of prostaglandin endoperoxide synthase 2 (Ptgs2), nuclear factor kappa-B p65 subunit (Rela), cyclin D1 (Ccnd1), peroxisome proliferator-activated receptor gamma (Pparg), O6-methylguanine-DNA-methyltransferase (Mgmt), 8-oxoguanine glycosylase (Ogg1), superoxide dismutase (Sod), and catalase (Cat). DNA damage, determined using 8-oxo-deoxyguanosin, was found to be lower in the mixed nut group only (p<0.05). Differences in proliferation and apoptosis among all three groups were not significant. Lower levels of the inflammatory marker, Ptgs2, were observed between the pistachio group and the control (p=0.035). The pistachio and mixed nut groups had lower levels of Rela compared to the control (p=0.029). Differences among diets for Ccnd1, Pparg, Mgmt, Ogg1, Sod, and Cat were not significant. Mixed nut consumption reduced DNA damage possibly via down-regulation of Rela inflammation gene expression without changes to colonic cell proliferation and apoptosis.

Chromatin accessibility analysis identifies GSTM1 as a prognostic marker in human glioblastoma patients

BackgroundGlioblastoma (GBM) is a malignant human brain tumor that has an extremely poor prognosis. Classic mutations such as IDH (isocitrate dehydrogenase) mutations, EGFR (epidermal growth factor receptor) alternations, and MGMT (O6-methylguanine-methyltransferase) promoter hypermethylation have been used to stratify patients and provide prognostic significance. Epigenetic perturbations have been demonstrated in glioblastoma tumorigenesis. Despite the genetic markers used in the management of glioblastoma patients, new biomarkers that could predict patient survival independent of known biomarkers remain to be identified.MethodsATAC-seq (assay for transposase accessible chromatin followed by sequencing) and RNA-seq have been used to profile chromatin accessible regions using glioblastoma patient samples with short-survival versus long-survival. Cell viability, cell cycle, and Western blot analysis were used to characterize the cellular phenotypes and identify signaling pathways.ResultsAnalysis of chromatin accessibility by ATAC-seq coupled with RNA-seq methods identified the GSTM1 (glutathione S-transferase mu-1) gene, which featured higher chromatin accessibility in GBM tumors with short survival. GSTM1 was confirmed to be a significant prognostic marker to predict survival using a different GBM patient cohort. Knockdown of GSTM1 decreased cell viability, caused cell cycle arrest, and decreased the phosphorylation levels of the NF-kB (nuclear factor kappa B) p65 subunit and STAT3 (signal transducer and activator of transcription 3) (pSer727).ConclusionsThis report demonstrates the use of ATAC-seq coupled with RNA-seq to identify GSTM1 as a prognostic marker of GBM patient survival. Activation of phosphorylation levels of NF-kB p65 and STAT3 (pSer727) by GSTM1 is shown. Analysis of chromatin accessibility in patient samples could generate an independent biomarker that can be used to predict patient survival.

Saccharomyces boulardii attenuates inflammatory response induced by Clostridium perfringens via TLR4/TLR15-MyD8 pathway in HD11 avian macrophages

Macrophages are professional phagocytic cells that play a critical role in initiating immune responses by presenting antigen and phagocytic clearance. The macrophages can be targeted for immunomodulation by beneficial microbes, such as probiotics. The aim of this study is to investigate the protective effect of Saccharomyces boulardii against Clostridium perfringens infection in avian macrophage cell line HD11. In this study, HD11 macrophages were prestimulated with S. boulardii for 6 h and then infected with C. perfringens for 3 h. Results showed that S. boulardii enhanced phagocytosis and bactericidal capacity against C. perfringens by HD11 cells. The S. boulardii effectively promoted the mRNA expression of CD80, CD83, and CD197 cell-surface molecules in C. perfringens-infected HD11 cells. Moreover, we found that prestimulation with S. boulardii reduced the mRNA expression of CD40, toll-like receptor [TLR] 4, and TLR15 induced by C. perfringens and thereby downregulated the mRNA expression of myeloid differentiation primary response 88, TNF receptor associated factor 6, nuclear factor kappa-B p65 subunit, and c-Jun N-terminal kinase genes in HD11 cells. The upregulation of cytokines (interleukin [IL]-6, tumor necrosis factor alpha, and IL-10) and inducible nitric oxide synthase mRNA expression in C. perfringens-infected HD11 cells were noticeably inhibited by S. boulardii pretreatment. Conclusively, these results might provide a new insight into the role of S. boulardii in regulating avian immune defense against C. perfringens invasion and immune escape.

Effects of Nut Consumption on Colon Cell Proliferation, Apoptosis, and Inflammation Gene Expression

Abstract Objectives A disruption to the colon cell kinetic balance can increase one's risk for colorectal cancer (CRC). Studies using single nut consumption has been found to decrease CRC risk. This study aimed to determine if mixed nut consumption would be as effective as or even better than single nut consumption at preventing CRC through modulation of colon cell proliferation and apoptosis cell numbers in a healthy rat model. It was hypothesized that rats consuming a mixed nut diet would exhibit fewer proliferative cells and greater apoptotic cells compared to a single nut diet and a control. Methods Thirty 21-day-old Sprague Dawley male rats were assigned to one of three isocaloric diets: control diet (no nuts), pistachio diet, or mixed nut diet for eight weeks. The mixed nut diet contained almonds, Brazil nuts, cashews, macadamia nuts, peanuts, pecans, pistachios, and walnuts. Proliferative cells were marked using Ki-67 quantitative immunostaining and apoptotic cells with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Colonic gene expression of prostaglandin endoperoxide synthase 2 (Ptgs2), nuclear factor kappa-B p65 subunit (Rela), Cyclin D1 (Ccnd1), peroxisome proliferator-activated receptor gamma (Pparγ), superoxide dismutase (SOD), and catalase (Cat) were determined by real-time quantitative polymerase chain reaction analysis. Results No significant differences were found in proliferation and apoptosis cell numbers between all three groups. Significantly lower levels of the inflammatory marker, Ptgs2, were only observed between the pistachio group and the control (p = 0.045). While both the pistachio and mixed nut groups had significantly lower levels of Rela compared to the control (p = 0.041). No significant differences were found among diets for Cyclind1, Pparγ, Sod, and Cat. Conclusions The results of this study show that nut groups were able to downregulate inflammation gene expression without alteration to colon cell proliferation and apoptosis. A longer study duration is needed to investigate if the lower gene expression involved in inflammation from nut consumption may change cell kinetics and further reduce colon cancer. Funding Sources American Heart Association (16GRNT31360007).

Ferroptosis involves in intestinal epithelial cell death in ulcerative colitis

Ferroptosis has recently emerged as an iron-dependent form of nonapoptotic cell death, which is also a regulated necrosis process and a response to tumor suppression. However, whether ferroptosis is involved in ulcerative colitis (UC) is unknown. The aims of this study were to investigate whether the ferroptosis is involved in UC, particularly intestinal epithelial cell (IEC) death, and to analyze the effect of the nuclear factor kappa Bp65 subunit (NF-κBp65) on ferroptosis. The gene expression of ferroptosis-related proteins was assessed in intestinal mucosal samples from human UC. The experimental model of UC was induced with dextran sulfate sodium (DSS). Ferroptosis of IECs was evaluated, the effect of NF-κBp65 on ferroptosis was analyzed by using IEC-specific NF-κBp65-deleted mice (p65IEC-KO), and the ferroptosis signaling pathway was investigated in vitro and in vivo. The results showed that ferroptosis was significantly induced in the IECs from UC patients and mice with colitis, and the ferroptosis was mediated by endoplasmic reticulum (ER) stress signaling. The specific deletion of IEC NF-κBp65 clearly upregulated ferroptosis and exacerbated colitis, and the result showed that phosphorylated-NF-κBp65 significantly inhibited ER stress signaling by directly binding eukaryotic initiation factor 2α. These data indicate that ferroptosis contributes to UC via ER stress-mediated IEC cell death, and that NF-κBp65 phosphorylation suppresses ER stress-mediated IEC ferroptosis to alleviate UC. The results suggest that ferroptosis involves in IEC death in UC, NF-κBp65 play a critical role in the ferroptotic inhibition, and ferroptosis is a potential therapeutic target for UC.

Quercetin improves immune function in Arbor Acre broilers through activation of NF-κB signaling pathway

Quercetin, the main component of flavonoids, has a wide range of biological actions. Quercetin can be made into a variety of additives for practice, because of the stable chemical structure and water-soluble derivatives. This study was intended to explore the effects of quercetin on immune function and its regulatory mechanism in Arbor Acre broiler to provide a practical basis for improving poultry immune function and figure out the optimum supplementation as functional feed additives. A total of 240 one-day-old healthy Arbor Acre broilers, similar in body weight, were randomly allotted to 4 treatments with 6 replicates, 10 broilers in each replicate and fed with diets containing quercetin at 0, 0.02, 0.04, and 0.06% for 6 wk. Blood and immune organs (spleen, thymus, and bursa) were collected from chickens at the end of the experiment. Growth performance, immune organs indexes, contents of serum immune molecules, splenic T lymphocyte proliferative responses, and expression of immune related genes were evaluated. The results showed that dietary quercetin had no significant effect (P > 0.05) on growth performance of broilers. Compared with control, 0.06% quercetin supplementation in diet significantly increased spleen index and thymus index (P < 0.05). It also increased the secretion of immune molecules including immunoglobulin A (IgA), interleukin-4 (IL-4) (P < 0.001), immunoglobulin M (IgM) (P = 0.007), complement component 4 (C4) (P = 0.001), and tumor necrosis factor-α (TNF-α) (P < 0.05). On the other hand, 0.02% quercetin supplementation significantly increased complement component 3 (C3) (P < 0.05). Additionally, both 0.04 and 0.06% quercetin supplementation significantly increased expression of TNF-α, TNF receptor associated factor-2 (TRAF-2), TNF receptor superfamily member 1B (TNFRSF1B), nuclear factor kappa-B p65 subunit (NF-κBp65), and interferon-γ (IFN-γ) mRNA (P < 0.05), and expression of NF-κB inhibitor-alpha (IκB-α) mRNA were significantly decreased (P < 0.05). Thus, quercetin improved immune function via NF-κB signaling pathway triggered by TNF-α.

Renoprotective effect of celecoxib against gentamicin-induced nephrotoxicity through suppressing NFκB and caspase-3 signaling pathways in rats

Gentamicin-induced nephrotoxicity has been well documented, although the causing mechanisms and preventative measures need further investigation. The current study aimed to explore the potential protective impacts of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on gentamicin-induced nephrotoxicity and the potential mechanisms in rats. Rats were randomly divided into four groups as follows: group1: normal control, group 2: received gentamicin only (100 mg/kg intraperitoneally), group 3: concurrently received gentamicin and celecoxib (30 mg/kg, orally) and group 4: received celecoxib.Celecoxib administration decreased gentamicin-induced rise in kidney weight, renal somatic index (RSI), blood urea nitrogen (BUN), serum creatinine (Cr), protein in urine, lactate dehydrogenase (LDH), nitric oxide (NOx), meanwhile, it increased serum albumin, urine Cr level and creatinine clearance (CCr), increased the renal endogenous antioxidant status, revealed by decreased malondialdehyde (MDA) and increased superoxide dismutase (SOD) and reduced glutathione (GSH). Gentamicin-induced elevated nuclear factor kappa B-P65 subunit (NFκB-p65), tumor necrosis factor-alpha (TNF-α) and apoptotic markers (tumor suppressor protein (p53) and caspase-3) protein levels were significantly decreased upon celecoxib treatment. Moreover, celecoxib suppressed renal myeloperoxidase (MPO) activity and posed improvement of histological features. In immunohistochemistry, celecoxib-treated rats showed decreased immunoreactivity against COX-2 in tubular cells and a mild positive immunoreactivity against heat shock protein 70 (HSP70) in renal interstitial cells. These findings propose that celecoxib treatment mitigates renal dysfunction via decreasing renal inflammation, oxidative/nitrosative stress, and apoptosis.

Anti-inflammatory action of YHQ by regulating 5-LOX/COX-2/NF-κB/MAPKs/Akt signaling pathways in RAW 264.7 macrophage cells

Yan-Hou-Qing (YHQ), a traditional Chinese medicine (TCM) formula including fifteen herbal medicines has been used for acute pharyngitis and cough treatment in Oriental medicine. However, anti-inflammatory activities of YHQ are poorly understood. The anti-inflammatory activities of YHQ were evaluated via enzyme-linked immunosorbent assay (ELISA), and the underlying mechanisms were further determined using western blot in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells in vitro. Anti-inflammation study revealed that YHQ inhibits the release of prostaglandin E2 (PGE2) potently by suppressing the protein expression of cyclooxygenase-2 (COX-2) and leukotriene B4 (LTB4) by reducing arachidonate 5-lipoxygenase (5-LOX) in LPS-stimulated RAW 264.7 macrophages. Further study demonstrated that YHQ inhibits the production of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophages by attenuating the phosphorylation p65 subunit of nuclear factor-kappaB (NF-κB). The suppressive effects of YHQ on LPS-stimulated inflammatory cytokines and mediators can be attributed to the suppression of YHQ on the phosphorylation of Akt and c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs. This study suggested that YHQ can be a preventive and potent therapeutic candidate for the management of inflammatory-mediated immune disorders.

Anti-inflammation action of xanthones from Swertia chirayita by regulating COX-2/NF-κB/MAPKs/Akt signaling pathways in RAW 264.7 macrophage cells

BackgroundSwertia chirayita, has been commonly used under the name “Zang-yin-chen” for the treatment of liver infections, inflammation, abdominal pain, and bacterial infection in traditional Tibetan medicine. However, the bioactive components with anti-inflammatory activities and underlying mechanisms remain poorly evaluated. Study design/methodsRepeated column chromatography yielded two main xanthones from petroleum ether (PE) and ethyl acetate fractions of whole plants of S. chirayita, and their structures were determined as bellidifolin (1) and swerchirin (2) on the basis of spectroscopic data and literature analysis. The anti-inflammatory activities and mechanisms of anti-inflammation of these two isolated xanthones were determined via enzyme-linked immunosorbent assay (ELISA) and western blot in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages in vitro. ResultsAnti-inflammation assay demonstrated that 1 and 2 inhibit the production of the pro-inflammatory cytokines interleukin-6 (IL-6) and TNF-α in LPS-stimulated RAW 264.7 macrophages. Xanthone 1 also potently inhibited the production of prostaglandin E2 (PGE2) by suppressing the protein expression of cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. Western blot showed that the phosphorylation of c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs were remarkably attenuated by 1 in a concentration-dependent manner. Particularly, Compound 1 suppressed the phosphorylation of the inhibitor κB kinase-β (IKK-β), Akt, and p65 subunit of nuclear factor-kappaB (NF-κB). ConclusionThe potent suppressive effects of 1 from S. chirayita on inflammatory mediators by blocking the expression of COX-2 and phosphorylation of Akt, IKK-β, MAPK and NF-κB, activation in LPS-stimulated macrophages suggest that 1 can be a preventive therapeutic candidate for the management of inflammatory-mediated immune disorders.

PAK5-mediated phosphorylation and nuclear translocation of NF-\u03baB-p65 promotes breast cancer cell proliferation in vitro and in vivo

BackgroundAbnormal proliferation is significantly associated with the promotion of malignant tumor. Growing evidence suggest that the signal pathways of p21cdc42/rac1-activated kinase 5 (PAK5) have been found in various tumor progression, however, the role of PAK5 in breast cancer remains largely unclear.MethodsWe evaluated PAK5 and p65 staining in breast cancer tissues (BCTs) and paired non-cancerous tissues (NTs) using tissue microarray (TMA) technology. The functions of PAK5 were studied in vitro and in vivo. Cell Counting Kit-8 (CCK-8) and flow cytometry were performed to determine proliferation of breast cancer cells. Phosphorylation assay and co-immunoprecipitation (co-IP) were employed to identify the regulation mechanism of p65 by PAK5. The activation of Cyclin D1 promoter was measured with luciferase reporter assay. Xenograft models in nude mice were established to explore the roles of PAK5 in breast cancer growth.ResultsIn this study, we show that PAK5 is highly expressed in breast cancer tissues and the increased PAK5 is significantly associated with breast cancer progression. Overexpression of PAK5 promotes the proliferation and cell-cycle progression by increasing the expression of Cyclin D1 in vitro and in vivo. Mechanistic studies demonstrated that PAK5 can promote the phosphorylation and the nuclear translocation of p65 subunit of nuclear factor-kappaB (NF-κB). Furthermore, p65 can directly bind to the promoter of Cyclin D1 and mediate an increase in its protein expression.ConclusionsTaken together, our findings suggest that PAK5 may serve as a potential prognosis marker and therapeutic target for human breast cancer.

GPR120 in adipocytes has differential roles in the production of pro-inflammatory adipocytokines

How nutritional excess leads to inflammatory responses in metabolic syndrome is not well characterized. Here, we evaluated the effects of ω-3 polyunsaturated fatty acid specific G-protein coupled receptor 120 (GPR120) activation on inflammatory pathways in adipocytes, and the influence of this process on macrophage migration. Using 3T3-L1 adipocytes, we found that agonizing GPR120 using its synthetic ligand, GSK137647, attenuated both basal and lipopolysaccharide-induced production of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2). Moreover, the intervention reduced the phosphorylation of nuclear factor kappa B inhibitor alpha (IκBα) and nuclear translocation of nuclear factor kappa-B p65 subunit (p65). Furthermore, the silencing of GPR120 itself reduced IL-6 and CCL2 mRNA expression. Inhibition of protein kinase C (PKC) augmented the down-regulatory effect of GSK137647 on IL-6 and CCL2 mRNA. Using a luciferase assay to measure promoter activity of the IL-6 gene in mouse embryonic fibroblasts, we demonstrated that exogenous transfection of GPR120 alone reduced the promoter activity, which was augmented by GSK137647. Inhibition of PKC further reduced the promoter activity. Nevertheless, RAW 264.7 macrophages grown in conditioned medium collected from GSK137647-treated adipocytes attenuated the expressions of matrix metalloproteinases-9 and -3, and tissue inhibitor of metalloproteinase-1. Conditioned medium also inhibited the lipopolysaccharide-induced migration of these macrophages. Taken together, these findings provide critical evidence that although GPR120 is associated with a PKC-mediated pro-inflammatory pathway, the direct inhibitory effects of GPR120 on the nuclear factor kappa B pathway are anti-inflammatory. Moreover, GPR120 activity can attenuate the adipocyte-mediated enhanced production of extracellular matrix-modulating factors in macrophages and can reduce their migration by a paracrine mechanism.

Editorial: Transcriptional Regulation of Memory.

Editorial: Transcriptional Regulation of Memory.

Increased expression of nuclear factor kappa-B p65 subunit in adenomyosis.

ObjectiveNuclear factor kappa-B (NF-κB) is a critical proinflammatory regulator that has been suggested to play a pivotal role in the pathogenesis and pathophysiology of endometriosis. In the present study, we aimed to evaluate whether the expression of NF-κB p65 subunit is increased in the eutopic endometrium and/or in the adenomyosis nodule of women with adenomyosis.MethodsThirty-three women with histologically confirmed adenomyosis after laparoscopic or transabdominal hysterectomy were recruited. Women with carcinoma in situ of uterine cervix without evidence of adenomyosis or endometriosis (n=32) served as controls. Formalin-fixed, paraffin-embedded archival tissues were sectioned and immunostained utilizing a monoclonal anti-human NF-κB p65 subunit antibody, and the immunoreactivity of NF-κB p65 subunit was compared between women with and without adenomyosis.ResultsThe immunoreactivities of both the nuclear and the cytoplasmic NF-κB p65 subunit were significantly increased in the stromal cells in the eutopic endometrium as well as in the adenomyosis nodule of women with adenomyosis compared with controls, respectively. The nuclear expression of NF-κB p65 subunit was significantly higher in the glandular cells in the eutopic endometrium as well as the adenomyosis nodule of women with adenomyosis compared with controls, respectively.ConclusionThe expression of NF-κB p65 is increased in the eutopic endometrium and adenomyosis nodule of women with adenomyosis, which strongly suggest that NF-κB plays a critical role in the pathogenesis and/or pathophysiology of adenomyosis.

A Pro-Inflammatory Role for Nuclear Factor Kappa B in Childhood Obstructive Sleep Apnea Syndrome

Childhood obstructive sleep apnea syndrome (OSAS) is associated with an elevation of inflammatory markers such as C-reactive protein (CRP) that correlates with specific morbidities and subsides following intervention. In adults, OSAS is associated with activation of the transcription factor nuclear factor kappa B (NF-kB). We explored the mechanisms underlying NF-kB activation, based on the hypothesis that specific NF-kB signaling is activated in children with OSAS. Adenoid and tonsillar tissues from children with OSAS and matched controls were immunostained against NF-kB classical (p65 and p50) and alternative (RelB and p52) pathway subunits, and NF-kB-dependent cytokines: interleukin (IL)- 1α, IL-1β, tumor necrosis factor-α, and IL-8). Serum CRP levels were measured in all subjects. NF-kB induction was evaluated by a luciferase-NF-kB reporter assay in L428 cells constitutively expressing NF-kB and in Jurkat cells with inducible NF-kB expression. p65 translocation to the nucleus, reflecting NF-kB activation, was measured in cells expressing fluorescent NF-kB-p65-GFP (green fluorescent protein). Sleep research laboratory. Twenty-five children with OSAS and 24 without OSAS. N/A. Higher expression of IL-1α and classical NF-kB subunits p65 and p50 was observed in adenoids and tonsils of children with OSAS. Patient serum induced NF-kB activity, as measured by a luciferase-NF-kB reporter assay and by induction of p65 nuclear translocation in cells permanently transfected with GFP-p65 plasmid. IL-1β showed increased epithelial expression in OSAS tissues. Nuclear factor kappa B is locally and systemically activated in children with obstructive sleep apnea syndrome. This observation may motivate the search for new anti-inflammatory strategies for controlling nuclear factor kappa B activation in obstructive sleep apnea syndrome.

Increased nuclear expression of nuclear factor kappa-B p65 subunit in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis

ObjectiveTo evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis.DesignCase-controlled laboratory study in a university hospital.Materials and MethodsWe recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β).ResultsThe immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding.ConclusionThe nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis. ObjectiveTo evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis. To evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis. DesignCase-controlled laboratory study in a university hospital. Case-controlled laboratory study in a university hospital. Materials and MethodsWe recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). We recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ResultsThe immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding. The immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding. ConclusionThe nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis. The nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis.

Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone

Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.