The HIV-1 Tat protein, essential for HIV-1 gene expression and viral replication, is known to be secreted by infected cells and has pleiotropic effects on various cell functions. It seems that extracellular Tat may exert its functions on cellular targets by at least two different mechanisms, namely, by adsorptive endocytosis, and by a possible interaction with cell surface receptor(s). Here we report that extracellular Tat activates AIM/CD69 gene transcription through an NF-kappaB-dependent pathway in the erythroleukemia cell line K562. Tat induces NF-kappaB binding to DNA as a result of IkappaBalpha phosphorylation and degradation, which depend on the intracellular redox state. We found that the second Tat-coding exon is required for CD69 gene trans-activation, but not for HIV LTR gene transcription. Fluorescein-labeled Tat proteins were used to study cell surface binding sites and cellular uptake of the proteins. Full-length Tat protein has specific binding sites on the surface of K562 cells, whereas truncated Tat1-48, which is efficiently internalized by the cells, does not bind to the cell surface. Our results suggest that extracellular Tat may activate a cell surface-mediated pathway that induces intracellular signal transduction in K562 cells, leading to the activation of NF-kappaB and the transcription of NF-kappaB-dependent genes, such as CD69.
In this study, we aimed to investigate the association between single nucleotide polymorphisms (SNPs) within two genes involved in the NF-κB cascade (GPR177 and MAP3K14) and bone mineral density (BMD) assessed at different skeletal sites, radial geometric parameters and bone turnover.
Ten GPR177 SNPs previously associated with BMD with genome-wide significance and twelve tag SNPs (r(2)≥0.8) within MAP3K14 (±10 kb) were genotyped in 2359 men aged 40-79 years recruited from 8 centres for participation in the European Male Aging Study (EMAS). Measurement of bone turnover markers (PINP and CTX-I) in the serum and quantitative ultrasound (QUS) at the calcaneus were performed in all centres. Dual energy X-ray absorptiometry (DXA), at the lumbar spine and hip, and peripheral quantitative computed tomography (pQCT), at the distal and midshaft radius, were performed in a subsample (2 centres). Linear regression was used to test for association between the SNPs and bone measures under an additive genetic model adjusting for study centre.
We validated the associations between SNPs in GPR177 and BMD(a) previously reported and also observed evidence of pleiotrophic effects on density and geometry. Rs2772300 in GPR177 was associated with increased total hip and LS BMD(a), increased total and cortical vBMD at the radius and increased cortical area, thickness and stress strain index. We also found evidence of association with BMD(a), vBMD, geometric parameters and CTX-I for SNPs in MAP3K14. None of the GPR177 and MAP3K14 SNPs were associated with calcaneal estimated BMD measured by QUS.
Our findings suggest that SNPs in GPR177 and MAP3K14 involved in the NF-κB signalling pathway influence bone mineral density, geometry and turnover in a population-based cohort of middle aged and elderly men. This adds to the understanding of the role of genetic variation in this pathway in determining bone health.
Endothelial lipase (EL) is a new member of triacylglycerol lipase family that has been shown to decrease high-density lipoprotein (HDL) cholesterol levels leading to increased risk of atherosclerosis. Its expression is increased during inflammation and by inflammatory cytokines. Sulforaphane (SFN) is a naturally occurring isothiocyanate present in cruciferous vegetables that has antioxidant and anti-inflammatory effects. Nuclear factor (NF)-κB is one of the molecular targets for SFN-mediated protective effects. Our aim was therefore to assess whether SFN could impact on EL expression via modulation of NF-κB pathway.
Quantitative PCR and Western blot results demonstrated that SFN inhibited tumor necrosis factor (TNF)-α-mediated induction of EL in human umbilical vein endothelial cells (HUVEC). Lentiviral transduction of HUVEC with mutated form of IκB-α (IκBM) as well as silencing of NF-κB subunit p65 using RNA interference revealed that TNF-α-mediated induction of EL is mediated through NF-κB pathway. In addition, a total of five NF-κB binding sites were found in LIPG gene, which encodes EL. SFN inhibited binding of NF-κB to these sites analyzed by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). SFN also inhibited TNF-α mediated phosphorylation of IκB kinase (IKK) 1/2 and IκB-α.
Collectively, these results indicate that SFN inhibits EL expression via inhibition of NF-κB which may have a beneficial effect on HDL cholesterol levels.
The transforming growth factor-beta 1 (TGFβ1) and NFκB pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGFβ1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFβ1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFβ1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFβ1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFβ1 induction of NFκB-luciferase. TGFβ1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFβ1 and this was blocked in NFκB+/- and -/- keratinocytes and by the IκB super-repressor. To test the effects of the TGFβ1 pathway on a biologically relevant activator of NFκB, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGFβ1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/- mice. These studies demonstrate that intact TGFβ1 signaling is required for NFκB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFβ1.
The fibroblast growth factor (FGF23) plasma level is high in cardiac and renal failure and is associated with poor clinical prognosis of these disorders. Both diseases are paralleled by hyperaldosteronism. Excessive FGF23 levels and hyperaldosteronism are further observed in Klotho-deficient mice. The present study explored a putative aldosterone sensitivity of Fgf23 transcription and secretion the putative involvement of the aldosterone sensitive serum & glucocorticoid inducible kinase SGK1, SGK1 sensitive transcription factor NFκB and store operated Ca(2+) entry (SOCE). Serum FGF23 levels were determined by ELISA in mice following sham treatment or exposure to deoxycorticosterone acetate (DOCA) or salt depletion. In osteoblastic UMR106 cells transcript levels were quantified by qRT-PCR, cytosolic Ca(2+) concentration utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, DOCA treatment and salt depletion of mice elevated the serum C-terminal FGF23 concentration. In UMR106 cells aldosterone enhanced and spironolactone decreased SOCE. Aldosterone further increased Fgf23 transcript levels in UMR106 cells, an effect reversed by mineralocorticoid receptor blockers spironolactone and eplerenone, SGK1 inhibitor EMD638683, NFκB-inhibitor withaferin A, and Ca(2+) channel blocker YM58483. In conclusion, Fgf23 expression is up-regulated by aldosterone, an effect sensitive to SGK1, NFκB and store-operated Ca(2+) entry.
The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-alpha or IKK-beta and modulates NF-kappaB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-kappaB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-kappaB signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD.
Curcuma xanthorrhiza Roxb., commonly known as Javanese turmeric, has been reported to possess a variety of biological activities, including anti-inflammatory effects, anticarcinogenic effects, wound healing effects, and serum cholesterol-lowering effects. CPE, crude polysaccharide extract isolated from the rhizome of C. xanthorrhiza using 0.1 N NaOH, consisted of arabinose (18.69%), galactose (14.0%), glucose (50.67%), mannose (12.97%), rhamnose (2.73%), and xylose (0.94%), with an average molecular weight of 33,000 Da. In the present study, we investigated the effect of CPE on nitric oxide (NO), hydrogen peroxide (H2O2), tumor necrosis factor-alpha (TNF-alpha), and prostaglandin E2 (PGE2) production in RAW 264.7 cells. The uptake of fluorescein-labeled Escherichia coli was measured to determine whether CPE stimulates the phagocytic activity of RAW 264.7 cells. CPE significantly increased the phagocytosis of macrophages and the release of NO, H2O2, TNF-alpha, and PGE2 in a dose-dependent manner, and showed a similar activity to lipopolysaccharide (LPS). To study the mechanisms of CPE, we examined induction of iNOS and COX-2. NO and PGE2 were produced as a result of stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) respectively. Both modulations of iNOS and COX-2 expression by CPE were evaluated by Western immunoblotting and RT-PCR. Since transcription of these enzymes is under the control of nuclear factor-kappa B (NF-kappaB), we assessed the phosphorylation of inhibitor kappaBalpha (IkappaBalpha) through Western immunoblotting. CPE clearly induced phosphorylation of IkappaBalpha, suggesting a role as an NF-kappaB activator. Taking all this together, we conclude that CPE isolated from Curcuma xanthorrhiza stimulates the immune functions of macrophages, which is mediated in part by specific activation of NF-kappaB.
Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (≤10μM) and decreased at higher (>100μM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-κB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-κB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway.
The HTLV-1 oncoprotein Tax is a potent activator of classical and alternative NF-κB pathways and is thought to promote cell proliferation and transformation via NF-κB activation. We showed recently that hyperactivation of NF-κB by Tax triggers a cellular senescence response (H. Zhi et al., PLoS Pathog. 7:e1002025, 2011). Inhibition of NF-κB activation by expression of I-κBα superrepressor or by small hairpin RNA (shRNA)-mediated knockdown of p65/RelA rescues cells from Tax-induced rapid senescence (Tax-IRS). Here we demonstrate that Tax-IRS is driven by the transcriptional activity of NF-κB. Knockdown of IKKγ, the primary Tax target, by shRNAs abrogated Tax-mediated activation of both classical and alternative NF-κB pathways and rendered knockdown cells resistant to Tax-IRS. Consistent with a critical role of IKKα in the transcriptional activity of NF-κB, IKKα deficiency drastically decreased NF-κB trans-activation by Tax, although it only modestly reduced Tax-mediated I-κBα degradation and NF-κB nuclear localization. In contrast, although IKKβ knockdown attenuated Tax-induced NF-κB transcriptional activation, the residual NF-κB activation in IKKβ-deficient cells was sufficient to trigger Tax-IRS. Importantly, the phenotypes of NIK and TAK1 knockdown were similar to those of IKKα and IKKβ knockdown, respectively. Finally, double knockdown of RelB and p100 had a minor effect on senescence induction by Tax. These data suggest that Tax, through its interaction with IKKγ, helps recruit NIK and TAK1 for IKKα and IKKβ activation, respectively. In the presence of Tax, the delineation between the classical and alternative NF-κB pathways becomes obscured. The senescence checkpoint triggered by Tax is driven by the transcriptional activity of NF-κB, which depends on activated IKKα and p65/RelA.
Colorectal cancer (CRC) is one of the major causes of cancer deaths worldwide. Wild-type p53-induced protein 1 (WIP1) is overexpressed in multiple human cancers and acted as an oncogene. This study was aimed to investigate the effect of WIP1 in colorectal cancer growth and analyzed underlying mechanisms. Herein, we determined WIP1 expression in CRC tissues and cell lines, as well as evaluated its detailed function in CRC cell proliferation. Several factors have been reported to mediate WIP1 effects; herein, we examined the involvement of mTOR and p21 in WIP1 regulation of CRC cell proliferation. Moreover, NF-κB has been regarded as a positive transcriptional regulator of WIP1 to activate its expression. NF-κB knockdown suppressed CRC cell proliferation, which could be reversed by WIP1 overexpression, through p21 and mTOR. Further, we examined the binding of NF-κB to the promoter region of WIP1. In CRC tissues, NF-κB expression was significantly up-regulated, and positively correlated with WIP1 expression, suggesting that inhibiting NF-κB expression to attenuate its activating effect on WIP1 expression presented a promising strategy of controlling excess proliferation of CRC cell. In summary, WIP1 promotes CRC proliferation through p21 and mTOR, both downstream targets of p53; NF-κB served as a positive transcriptional regulator of WIP1 to activate its expression and affect its function in CRC cells. Our finding provided a novel strategy for treatment for CRC.
Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.
Alzheimer's disease is the most common form of dementia, causing progressive cognitive dysfunction, particularly memory loss. Recently, modulation of beta-amyloid (Abeta) toxicity, one of the major potential causes of Alzheimer's disease, has emerged as a possible therapeutic approach to control the onset of Alzheimer's disease. In this study, we investigated the neuroprotective effects and possible mechanisms by which 19-hydroxy-1alpha,6-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide (named as CBNU06), a new diterpene isolated from Isodon japonicus, acts against Abeta-induced toxicity in PC12 cells. Pretreatment with CBNU06 (20 microg/ml) prior to Abeta(25)(-35) (25 microM) significantly increased the viability of PC12 cells in a dose-dependent manner when examined by Hoechst staining, MTT assay and Trypan blue exclusion assay. This protective effect was accompanied by the decrease in translocation of NF-kappaB p50 and p65 from the cytoplasm to the nucleus, and followed by the decrease in cyclooxygenase-2 (COX-2) levels. In addition, pretreatment with CBNU06 significantly reversed the effect of Abeta on Bax and Bcl-2. Taken together, these results suggest that CBNU06 protected PC12 cells against Abeta-induced neurotoxicity through the inhibition of the NF-kappaB signaling pathways. Therefore, CBNU06 has the possible beneficial effects in Alzheimer's disease by attenuating Abeta-induced toxicity.
To examine the efficacy of muscovite on non-steroidal anti-inflammatory drug (NSAID) associated intestinal injury in rats, and its influences on the expressions of inflammatory factors, tumor necrosis factor-alpha (TNF-alpha) and nuclear factor-kappaB (NF-kappaB), for researching its possible mechanism of intestinal mucosal protection.
NSAID associated intestinal injury in rat was induced by intra-gastric infusion of diclofenac. Twenty-four male Sprague-Dawley rats were randomly and equally assigned to three groups: normal control group, model control group and Muscovite group, 8 in each group. The normal control group received physiological saline 1 mL/100 g and the other two groups received diclofenac 7.8 mg/kg respectively every day for 5 days; while to the Muscovite group, Muscovite 120 mg/kg was gastric infused once on the day before modeling, followed with 120 mg/kg per day, given an hour before diclofenac infusion in the modeling days. All rats were killed on the 6th day, their gross changes and histological injury of intestinal mucosa were observed and scored, serum level of TNF-alpha was assayed in radioimmunoassay and NF-kappaB activity was determined by immunohistochemistry.
The small dosage diclofenac administration can cause intestinal damage, revealing obviously erythema, erosion, multiple ulcer, intestinal stricture, even perforation, etc. Intestinal injury in the Muscovite group was obviously milder than that in the model control group, only showed changes of local congestion, edema and erosion. The scores of gross and histological intestinal features in the model control group were 4.38 +/- 1.41 and 4.00 +/- 1.85, while in the Muscovite group were 1.25 +/- 1.58 and 1.75 +/- 0.71, respectively, all higher than those in the normal control group (0.00 +/- 0.00 and 0.00 +/- 0.00, P < 0.01 and P < 0.05), respectively, but the elevation in the model control group were more significant (P < 0.05). Similar results were shown in comparisons of TNF-alpha and NF-kappaB levels between groups, the values were 6.19 +/- 2.76 and 1.38 +/- 1.19 in normal control; 22.20 +/- 5.42 and 5.75 +/- 0.46 in model control; 9.61 +/- 4.02 and 0.13 +/- 0.35 in the Muscovite group, respectively (all P < 0.01).
Muscovite could effectively reduce the NSAID associated intestinal mucosal injury by inhibiting the activity of NF-kappaB in intestinal mucosa, and down-regulating the expression of TNF-alpha in blood plasma, so muscovite is proved to have protective function for intestine.
Co-stimulatory CD28 and transcription factor NFKB1 genes are considered as a crucial player in the determination of inflammatory responses; genetic variability in these may modulate the risk for idiopathic recurrent miscarriages (IRM). We investigated the association of functional variants of CD28 (rs3116496 T/C) and NFKB1 (rs28362491 ins/del and rs696 A/G) with IRM cases. We recruited 200 IRM women with a history of at least three consecutive pregnancy losses before 20th week of pregnancy and 300 fertile control women. Determination of CD28 (rs3116496 T/C) and NFKB1 (rs28362491 ins/del and rs696 A/G) gene variants were based on the polymerase chain reaction pursued by restriction fragment length polymorphism analysis and validated with Sanger sequencing. Single marker analysis and multifactor dimensionality reduction (MDR) model used to predict the IRM risk. We observed nearly three- to twofold increased risk in single marker analysis for minor homozygous genotypes of rs3116496 T/C, rs28362491 ins/del and rs696 A/G tag-SNPs in IRM cases, suggesting the risk association. In MDR analysis, we observed 10.5-fold augmented risk among IRM women in three-SNP model (rs3116496 T/C, rs28362491 ins/del and rs696 A/G). The eQTL mapping analyses was performed to strengthen the results of our study. The eQTL mapping analysis revealed that the variations in CD28 and NFKB1 gene content might affect the abundance of transcripts of CD28 and Family with sequence similarity 177 member A1 (FAM177A1) genes, respectively. These results suggest that CD28 and NFKB1 gene variants may be associated with increased risks to IRM.
To determine whether signaling through TNF and/or nuclear factor-kappaB contributes to bacterial lipopolysaccharide (LPS)-induced activation of type 2 iodothyronine deiodinase (D2) in tanycytes lining the floor and infralateral walls of the third ventricle, the effect of a TNF antagonist on D2 gene expression and LPS-induced Ikappa-Balpha expression in tanycytes were studied. Animals treated with soluble, rat, polyethylene glycol-conjugated TNF receptor type 1 (4 mg/kg body weight) before a single ip injection of LPS showed a significant reduction in circulating IL-6 levels but no effect on LPS-induced D2 mRNA in the majority of tanycytes with the exception of a subpopulation of alpha tanycytes in the wall of the third ventricle. LPS induced a rapid increase in Ikappa-Balpha mRNA in the pars tuberalis and a delayed response in alpha tanycytes but absent in all other tanycyte subsets. The LPS-induced increase in Ikappa-Balpha in the pars tuberalis was associated with increased TSHbeta gene expression in this tissue, but cAMP response element-binding protein (CREB) phosphorylation was observed only in a subset of alpha tanycytes. These data suggest that TNF and nuclear factor-kappaB signaling are not the primary, initiating mechanisms mediating the LPS-induced D2 response in tanycytes, but may contribute in part to sustaining the LPS-induced D2 response in a subset of alpha tanycytes. We hypothesize that in addition to TSH, other factors derived from the pars tuberalis may contribute to LPS-induced D2 activation in tanycytes.
The aim of these in vitro studies was to examine the involvement of transcription factor NF-κB (p50/p50, p65/p65) and FSH in control of porcine ovarian granulosa cells functions and the possible role of dimers p50/p50, p65/p65 in mediating FSH actions on these cells. Monolayer of primary granulosa cells was transfected with plasmids encoding human p50 cDNA and p65 cDNA, and cultured with or without addition of FSH (0, 1, 10 or 100 ng/ml). The accumulation of proteins p50 and p65, as well as of proliferation markers (PCNA and MAPK/ERK1,2) and marker of apoptosis (Bax) in cells was detected by using SDS-PAGE-Western immunoblotting and immunocytochemistry. DNA fragmentation was evaluated by TUNEL assay. Release of hormones insulin-like growth factor I (IGF-I), progesterone (P(4)), oxytocin (OT), prostaglandins E(2) (PGE(2)) and F(2α) (PGF(2α)) was measured by using RIA. We observed, that p50/p50 promoted the accumulation of PCNA, MAPK/ERK1,2, the release of OT, PGF(2α); inhibited the occurrence of TdT-positive cells, the release of IGF-I and P(4), and did not influence the accumulation of Bax and the release of PGE(2). p65/p65 enhanced the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, PGE(2) and PGF(2α); decreased the percentage of cell containing TdT and did not affect the release of P(4). FSH stimulated the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, P(4), PGE(2); but reduced the proportion of TdT-positive cells and the release of PGF(2α). These observations suggest (1) the involvement of NF-κB (p50/p50) in stimulation of proliferation, inhibition of apoptosis and in either stimulation (OT, PGE(2)) or inhibition (IGF-I, P(4), but not PGF(2)) of hormones release by porcine ovarian granulosa cells; (2) the involvement of NF-κB (p65/p65) in stimulation of proliferation and mitochondrial/Bax-related apoptosis, inhibition of nuclear/TdT-related apoptosis and in stimulation of ovarian hormones (IGF-I, OT, PGE(2), PGF(2α), but not P(4)) release; (3) the role of FSH in up-regulation of both ovarian cell proliferation and mitochondrial/Bax-related apoptosis, in inhibition of nuclear/TdT-related apoptosis, in promotion of IGF-I, P(4), OT, PGE(2) and suppression of PGF(2α) release by porcine ovarian cells. The majority of results demonstrates the involvement of NF-κB (p50/p50 and p65/p65) and FSH in control of basic ovarian functions (proliferation, apoptosis, and secretory activity), but not the functional interrelationships between these regulators.
Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERα)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERα66 and upregulation of the 36-kDa variant of ER (ERα36). We identified that NF-κB, HIF1α, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-κB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-κB activation, combining didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-κB activation. Finally, inhibition of RRM2 by didox reversed tamoxifen-resistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.
S6K (ribosomal S6 kinase p70, p70S6K) activation requires phosphorylation at two stages. The first phosphorylation is independent of insulin stimulation and mediated by an unknown kinase. The second phosphorylation is mediated by mTOR in insulin dependent manner. In this study, we identified JNK1 (c-Jun N-terminal kinase 1) as a kinase in the first phosphorylation. S6K protein was phosphorylated by JNK1 at S411 and S424 in the carboxyl terminal autoinhibitory domain. The phosphorylation was observed in kinase assay with purified S6K as a substrate, and in cells after JNK1 activation by TNF-α or MEKK1 expression. The phosphorylation was detected in JNK2 null cells, but not in JNK1 null cells after TNF-α treatment. When JNK1 activation was inhibited by MKK7 knockdown, the phosphorylation was blocked in cells. The phosphorylation led to S6K protein degradation in NF-κB deficient cells. The degradation was blocked by inhibition of proteasome activity with MG132. In wide type cells, the phosphorylation did not promote S6K degradation when IKK2 (IKKβ, IκB kinase beta) was activated. Instead, the phosphorylation allowed S6K activation by mTOR, which stabilizes S6K protein. In IKK2 null cells or cells treated by IKK2 inhibitor, the phosphorylation led to S6K degradation. These data suggest that S6K is phosphorylated by JNK1 and the phosphorylation makes S6K protein unstable in the absence of IKK2 activation. This study provides a mechanism for regulation of S6K protein stability.
The enteric pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli employ a type 3 secretion system (T3SS) to manipulate the host inflammatory response during infection. Previously, it has been reported that EPEC, in a T3SS-dependent manner, induces an early proinflammatory response through activation of NF-κB via extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase Cζ (PKCζ). However, the activation of NF-κB during infection has not yet been attributed to an effector. At later time points postinfection, NF-κB signaling is inhibited through the translocation of multiple effectors, including NleE and NleC. Here we report that the highly conserved non-LEE (locus of enterocyte effacement)-encoded effector F (NleF) shows both diffuse and mitochondrial localization during ectopic expression. Moreover, NleF induces the nuclear translocation of NF-κB p65 and the expression of interleukin 8 (IL-8) following ectopic expression and during EPEC infection. Furthermore, the proinflammatory activity and localization of NleF were dependent on the C-terminal amino acids LQCG. While the C-terminal domain of NleF has previously been shown to be essential for interaction with caspase-4, caspase-8, and caspase-9, the proinflammatory activity of NleF was independent of interaction with caspase-4, -8, or -9. In conclusion, EPEC, through the T3SS-dependent translocation of NleF, induces a proinflammatory response in an NF-κB-dependent manner in the early stages of infection.
Regulation of glucose transporter (GLUT) expression and activity plays a vital role in the supply of glucose to embryonic stem (ES) cells.
To observe the effect of 6-phenyl cyclic monophosphate (cAMP) on glucose uptake and cell proliferation, 2-deoxyglucose (2-DG) uptake, immunohistochemistry, Western blotting, and immunoprecipitation were carried out.
Among GLUT isoforms in mouse ES cells, GLUT1 was predominantly expressed and 6-phenyl cAMP increased GLUT mRNA levels. Among cAMP agonists, 6-phenyl cAMP increased 2-DG uptake more than that of 8-p-chlorophenylthio-2'-O-methyl-cAMP. 6-Phenyl cAMP increased GLUT1 expression and translocation from the cytosol to the plasma membrane. 6-Phenyl cAMP increased 2-DG uptake in a time- and concentration-dependent manner due to an increase in V(max) but not K(m). 6-Phenyl cAMP increased phosphorylation of nuclear factor-κB (NF-κB) and cAMP response element binding (CREB) and expression of the CREB protein (CBP) and transducer of regulated CREB activity 2 (TORC2) in sequence. 6-Phenyl cAMP induced complex formation of NF-κB/CREB/CBP/TORC2, which are involved in the increase of gluconeogenic enzyme expression. 6-Phenyl cAMP also increased cell cycle regulatory protein expression levels, the proportion of S-phase cells, and proto-oncogene expression via protein kinase A (PKA)-dependent NF-κB signaling. Finally, GLUT1 siRNA blocked the 6-phenyl cAMP-induced increase in ES cell proliferation. We conclude that PKA stimulated the complex formation of CREB/CBP/TORC2 via NF-κB, which induced effective coordination of glucose uptake as well as proliferation in ES cells.
6-Phenyl cAMP-induced PKA activation modified the proliferation, which may be beneficial for expanding ES cell use to cell therapy.
Chemoresistance has been one of the major problems in anticancer therapy. In our effort to find a potential molecular target for overcoming the chemoresistance in prostate cancer, a promising anticancer drug trichostatin A (TSA) induced cell death was found to be compromised by enhanced NF-kappaB activation in 267B1/K-ras human prostate epithelial cancer cells. However, both the NF-kappaB activation and chemoresistance were reduced by pretreatment with proteasome inhibitor-I (ProI), accompanied by accumulations of both IkappaBalpha and p65/RelA (but not p50/NF-kappaB1) in the cytoplasm. Clonogenic cell survival and soft agar assays further confirmed the increased TSA chemosensitivity of 267B1/K-ras cells by ProI treatment. Moreover, dominant negative mutant of IKKbeta, IkappaBalpha and p65 enhanced the chemosensitization, too. Unexpectedly, using LY294002 and PD98059, phosphatidylinositol-3-kinase and mitogen-activated protein kinase were also implied in TSA chemoresistance through NF-kappaB activation, while these compounds had showed no effect on radiosensitization in the cells. On the other hand, together with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, activations of caspase-8 and caspase-3 by TSA and ProI were noticed, suggesting the involvement of apoptotic process in chemosensitization of 267B1/K-ras cells. Altogether, these results suggest that blocking the NF-kappaB activation pathway could be an efficient target for improving the TSA chemosensitization and applying to the development of anticancer therapeutics in Ki-Ras-overexpressing tumorigenic cells, including prostate cancer.
The proinflammatory cytokines interleukin-1 beta (IL-1β) and interferon gamma (IFN-γ) play important roles in the progressive loss of beta-cell mass and function during development of both type 1 and type 2 diabetes. We have recently showed that bone morphogenetic protein (BMP)-2 and -4 are expressed in pancreatic islets and inhibit beta-cell growth and function. In this study, we describe that IL-1β and IFN-γ induce the expression of BMP-2 suggesting a possible role for BMP-2 in mediating the effects of IL-1β and IFN-γ on beta-cell apoptosis and dysfunction. IL-1β increased BMP-2 mRNA levels 6- and 3-fold in isolated islets of Langerhans from neonatal rat and human. Downstream target genes of the BMP pathway were also increased by cytokine treatment and could be reversed by neutralization of endogenous BMP activity. Nuclear factor kappa B- (NFκB) binding sites were identified in the rat BMP-2 promoter, and reporter assays verified the role of NFκB in cytokine-induced BMP-2 expression. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed NFκB binding to BMP-2 promoter upon IL-1β stimulation in beta cells. In conclusion, we suggest that NFκB stimulates BMP-2 mRNA expression in rat and human beta cells upon cytokine exposure.
The expression of transcriptional factor nuclear factor kappaB (NF-kappaB) in untreated esophageal cancer specimens from patients who receive preoperative chemoradiation is associated with aggressive clinical biology. We hypothesized that nuclear NF-kappaB would define clinical biology even when surgery is used as primary therapy.
Consecutive patients who did not receive any preoperative therapy were selected. Surgical cancer specimens were examined for nuclear NF-kappaB and correlated with overall survival (OS) and disease-free survival (DFS).
One hundred twenty-three patients (stage I, 9%; stage II, 24%; stage III, 53%; stage IV, 15%) with adenocarcinoma who underwent surgery as primary therapy were analyzed. Most patients were men (90%) and the median age was 63 years. For all 123 patients, the median DFS was 21 months and the median OS was 28 months. Nuclear NF-kappaB was associated with shortened DFS (P = 0.001) in 123 patients but also in stage II (P = 0.03) and stage III (P = 0.04). Nuclear NF-kappaB was associated with shortened OS (P = 0.002) in 123 patients and in stage II (P = 0.04) and showed trend in stage III (P = 0.17). Numbers are too small for stages I and IV. In multivariate models, nuclear NF-kappaB was an independent predictor for both DFS and OS (P = 0.005 and P = 0.01).
Our data are the first to show that NF-kappaB status significantly correlates with DFS and OS for patients with esophageal adenocarcinoma undergoing surgery as primary therapy. NF-kappaB is an independent prognosticator of outcome, even for individual stages (e.g., stages II and III). More comprehensive molecular studies could help the design of strategies to individualize therapy of esophageal adenocarcinoma.
TCR signaling is mediated by intracellular signaling molecules and nuclear transcription factors, which are tightly regulated by interaction with regulatory proteins such as Grb2 and SLAP. We reported recently that TCR stimulation induces the expression of cytokine-induced SH2 protein (CIS). The expression of CIS promotes TCR-mediated activation. We have now found specific interactions between CIS and activated protein kinase C (PKC) alpha, beta and theta in TCR-stimulated T cells. CIS was shown by in vitro kinase assay to associate with activated PKC. In CIS-expressing T cells isolated from CIS-transgenic mice, the amount of activated PKC associated with CIS was found to increase following TCR stimulation. By immunohistochemical analysis, CIS was also found to co-localize with PKCtheta at the plasma membrane of activated T cells. In addition to the interaction and intracellular co-localization of the CIS and PKC, an increase in the activation of AP-1 and NF-kappaB was noted in CIS-expressing T cells, after stimulation by either anti-CD3/CD28 or phorbol myristate acetate + ionomycin. These results suggest that CIS regulates PKC activation, and that this may be important for the activation of both the AP-1 and NF-kappaB pathways in TCR signaling.