Induced NF-κB Activation Research Articles

Enterovirus 71 2C Protein Inhibits TNF-α–Mediated Activation of NF-κB by Suppressing IκB Kinase β Phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.

The inhibition of RANKL/RANK signaling by osteoprotegerin suppresses bone invasion by oral squamous cell carcinoma cells

Oral squamous cell carcinomas (OSCCs) are malignant tumors that frequently invade the maxilla and mandibular bone. However, the molecular mechanisms underlying bone invasion by OSCC are unclear. Recent studies showed that receptor activator of nuclear factor κB (RANK) was expressed not only in osteoclast precursors but also in tumor cells. Therefore, we examined whether RANK ligand (RANKL)/RANK signaling regulates bone invasion by OSCC cells in vivo and in vitro. We first injected human OSCC B88 cells into the masseter region of nude mice. Mice were treated for 3 weeks with osteoprotegerin (OPG), the decoy receptor for RANKL. Treatment with OPG decreased bone invasion by B88 cells, reduced the number of osteoclasts and increased B88 cell apoptosis. However, OPG did not affect apoptosis and proliferation in B88 cells in vitro, suggesting that the effects of OPG on apoptosis in B88 cells are restricted in a bone environment. RANK was expressed in the B88 cells and in OSCC cells from patients. RANKL induced NF-κB activation and extracellular signal-regulated kinase phosphorylation in B88 cells and enhanced B88 cell migration in a modified chemotaxis chamber equipped with a gelatin-coated filter. OPG inhibited RANKL-induced NF-κB activation, extracellular signal-regulated kinase phosphorylation and cell migration. Our data clearly indicate that RANKL/RANK inhibition suppresses bone invasion by inhibiting osteoclastogenesis and cancer cell migration and by inducing apoptosis of cancer cells via indirect anticancer action in vivo.

Discovery of the DIGIRR Gene from Teleost Fish: A Novel Toll–IL-1 Receptor Family Member Serving as a Negative Regulator of IL-1 Signaling

Toll-IL-1R (TIR) family members play crucial roles in a variety of defense, inflammatory, injury, and stress responses. Although they have been widely investigated in mammals, little is known about TIRs in ancient vertebrates. In this study, we report a novel double Ig IL-1R related molecule (DIGIRR) from three model fish (Tetraodon nigroviridis, Gasterosteus aculeatus, and Takifugu rubripes), adding a previously unknown homolog to the TIR family. This DIGIRR molecule contains two Ig-like domains in the extracellular region, one Arg-Tyr-mutated TIR domain in the intracellular region, and a unique subcellular distribution within the Golgi apparatus. These characteristics distinguish DIGIRR from other known family members. In vitro injection of DIGIRR into zebrafish embryos dramatically inhibited LPS-induced and IL-1β-induced NF-κB activation. Moreover, in vivo knockdown of DIGIRR by small interfering RNA significantly promoted the expression of IL-1β-stimulated proinflammatory cytokines (IL-6 and IL-1β) in DIGIRR-silenced liver and kidney tissues and in leukocytes. These results strongly suggest that DIGIRR is an important negative regulator of LPS-mediated and IL-1β-mediated signaling pathways and inflammatory responses. The Arg-Tyr-mutated site disrupted the signal transduction ability of DIGIRR TIR. Evolutionally, we propose a hypothesis that DIGIRR and single Ig IL-1R related molecule (SIGIRR) might originate from a common ancient IL-1R-like molecule that lost one (in DIGIRR) or two (in SIGIRR) extracellular Ig-like domains and intracellular Ser and Arg-Tyr amino acids. DIGIRR might be an evolutionary "transitional molecule" between IL-1R and SIGIRR, representing a shift from a potent receptor to a negative regulator. These results help define the evolutionary history of TIR family members and their associated signaling pathways and mechanisms.

Honokiol stimulates osteoblastogenesis by suppressing NF-κB activation

Magnolia officinalis, a component of Asian herbal teas, has long been employed in traditional Japanese and Chinese medicine to treat numerous maladies. Honokiol, a biphenolic compound, is now considered to be one of the major active ingredients of Magnolia extract, and is under intense investigation for its anti-angiogenic, anti-inflammatory, anti-tumor and neuroprotective properties. Biochemically, honokiol has been recognized to modulate the nuclear factor κ B (NF-κB) signal transduction pathway suggesting that it possesses anti-inflammatory properties. Inflammation is intimately associated with bone turnover and skeletal deterioration and consequently, anti-inflammatory drugs may hold significant promise as bone protective agents to stem bone loss in osteoporotic conditions. We and others have demonstrated that suppression of NF-κB blunts osteoclastic bone resorption, but promotes osteoblastic bone formation. Indeed previous studies have demonstrated the anti-osteoclastogenic effects of honokiol, however, activities on osteoblast differentiation and activity have yet to be investigated. In this study, we show that honokiol is a potent inducer of in vitro osteoblast differentiation by virtue of its capacity to suppress basal and tumor necrosis factor alpha (TNFα)-induced NF-κB activation and to alleviate the suppressive action of TNFα on bone morphogenetic protein (BMP)-2-induced Smad activation. Our data confirm that honokiol may have considerable promise as a dual anabolic/anti-catabolic agent for the amelioration of multiple osteoporotic diseases.

IgG Immune Complex Induces the Recruitment of Inflammatory Cells into the Airway and TNF-Mediated Late Airway Hyperresponsiveness via NF-κB Activation in Mice

Background. Many of the inflammatory proteins that are expressed in asthmatic airways are regulated, at least partially, by nuclear factor (NF)-κB. Blockade of NF-κB activity has resulted in attenuation of the cardinal features of asthma. Thus, delineating the mechanisms involved in NF-κB activation in asthma might provide an interesting approach to improving the management of asthma. However, despite its importance, the mechanism for NF-κB activation in asthma has not yet been determined. Objective. To examine the role of IgE and IgG antibodies (Abs) in the activation of NF-κB in mouse lungs. Methods. To examine the effect of IgE, mice underwent intratracheal (i.t.) instillation of an IgE immune complex (IgE-IC) (anti-2,4-dinitrophenyl hapten (DNP) IgE + DNP-BSA or DNP-OVA) and anaphylactogenic anti-IgE (LO-ME-2). For IgG, mice underwent i.t. instillation with a complex of anti-chicken gamma globulin (CGG) IgG1 mAb + CGG. NF-κB activation was determined by gel shift assay. Small interfering RNA was used for blockade of p50 expression. The effect of tumor necrosis factor (TNF) blockade was determined using anti-TNF Ab. A previously established murine model of asthma was used to assess airway hyperresponsiveness (AHR). Results. A single i.t. instillation of either IgE-IC or LO-ME-2 failed to induce activation of NF-κB in the lungs. In contrast, single i.t. instillation of IgG-IC was capable of inducing NF-κB activation, as well as NF-κB-dependent proinflammatory molecules, such as TNF and CXC chemokines. Pretreatment of p50 small interfering RNA decreased bronchoalveolar lavage fluid levels of TNF and macrophage inflammatory protein-2 induced by IgG-IC instillation. Single i.t. instillation of IgG-IC caused the recruitment of neutrophils and macrophages into the airway and TNF-mediated late AHR, but failed to induce Th2 cell-mediated asthmatic phenotypes. Conclusion. IgG, but not IgE, is the major Ab that induces not only NF-κB activation and NF-κB-dependent proinflammatory molecules in the lungs but also subsequent recruitment of inflammatory cells into the airway and TNF-mediated late AHR.

Platelet-Rich Plasma Releasate Inhibits Inflammatory Processes in Osteoarthritic Chondrocytes

Background: Platelet-rich plasma (PRP) has recently been postulated as a treatment for osteoarthritis (OA). Although anabolic effects of PRP on chondrocytes are well documented, no reports are known addressing effects on cartilage degeneration. Since OA is characterized by a catabolic and inflammatory joint environment, the authors investigated whether PRP was able to counteract the effects of such an environment on human osteoarthritic chondrocytes. Hypothesis: Platelet-rich plasma inhibits inflammatory effects of interleukin-1 (IL-1) beta on human osteoarthritic chondrocytes. Study Design: Controlled laboratory study. Methods: Human osteoarthritic chondrocytes were cultured in the presence of IL-1 beta to mimic an osteoarthritic environment. Medium was supplemented with 0%, 1%, or 10% PRP releasate (PRPr, the active releasate of PRP). After 48 hours, gene expression of collagen type II alpha 1 (COL2A1), aggrecan (ACAN), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, matrix metalloproteinase (MMP)13, and prostaglandin-endoperoxide synthase (PTGS)2 was analyzed. Additionally, glycosaminoglycan (GAG) content, nitric oxide (NO) production, and nuclear factor kappa B (NFκB) activation were studied. Results: Platelet-rich plasma releasate diminished IL-1 beta–induced inhibition of COL2A1 and ACAN gene expression. The PRPr also reduced IL-1 beta–induced increase of ADAMTS4 and PTGS2 gene expression. ADAMTS5 gene expression and GAG content were not influenced by IL-1 beta or additional PRPr. Matrix metalloproteinase 13 gene expression and NO production were upregulated by IL-1 beta but not affected by added PRPr. Finally, PRPr reduced IL-1 beta–induced NFκB activation to control levels containing no IL-1 beta. Conclusion: Platelet-rich plasma releasate diminished multiple inflammatory IL-1 beta–mediated effects on human osteoarthritic chondrocytes, including inhibition of NFκB activation. Clinical Relevance: Platelet-rich plasma releasate counteracts effects of an inflammatory environment on genes regulating matrix degradation and formation in human chondrocytes. Platelet-rich plasma releasate decreases NFκB activation, a major pathway involved in the pathogenesis of OA. These results encourage further study of PRP as a treatment for OA.

Induction of senescence in melanoma: thinking outside the cell

Induction of senescence in melanoma: thinking outside the cell

Exogenous regucalcin stimulates osteoclastogenesis and suppresses osteoblastogenesis through NF-κB activation

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss, and bone marrow from these animals exhibits significantly elevated osteoclast formation. Consistent with these effects exogenous regucalcin promotes osteoclastogenesis in mouse bone marrow cultures, but interestingly regucalcin suppresses the differentiation and mineralization of MC3T3 osteoblast precursors. However, the molecular mechanisms involved are presently unclear. As the nuclear factor-kappa B (NF-κB) signal transduction pathway is critical to osteoclastogenesis but inhibitory of osteoblastogenesis, we hypothesized that regucalcin may promote osteoclastogenesis and suppress osteoblastogenesis upregulating NF-κB signal transduction. In this study, we examined the effect of regucalcin on receptor activator of NF-κB (RANK) ligand (RANKL) -induced osteoclast formation using the RAW264.7 monocytic cell line and osteoblast formation using the pre-osteoblastic cell line MC3T3. As expected, culture with exogenous regucalcin was found to enhance RANKL-induced osteoclastogenesis. Consistent with this effect regucalcin increased basal and RANKL-induced NF-κB activation as assessed by NF-κB luciferase assay. The capacity of regucalcin to augment RANKL-induced NF-κB activity was inhibited by menaquinone-7, a potent NF-κB antagonist, while the Erk inhibitor PD98059 and staurosporine had no effect, demonstrating a specific effect on NF-κB signaling. By contrast, regucalcin inhibited mineralization of MC3T3 cells and enhanced tumor necrosis factor-α (TNFα)-induced NF-κB activation. As with NF-κB induction in osteoclasts, NF-κB activation was abolished by addition of the NF-κB antagonist menaquinone-7, but not by PD98059 and staurosporine. Transforming growth factor-β (TGFβ) and bone morphogenic protein-2 (BMP2) are potent early commitment and late osteoblast differentiation factors, respectively, and both mediate their actions through the Smad-signal transduction pathway, a system that is extremely sensitive to and inhibited by TNFα-induced NF-κB. We consequently examined the effect of regucalcin on TGFβ and BMP2-induced Smad activation in the presence and absence of TNFα. While regucalcin had no effect on basal Smad activation by TGFβ and BMP2, it enhanced the suppressive effect of TNFα on both TGFβ- and BMP2-induced Smad activations. Taken together, present data suggest that regucalcin may induce bone loss in vivo by promoting osteoclasts and simultaneously suppressing osteoblasts through amplification of basal and/or cytokine-induced NF-κB activation. Regucalcin may have a role as a modulator in NF-κB activation.

Staphylococcus aureus inhibits nuclear factor kappa B activation mediated by prolactin in bovine mammary epithelial cells

The hormone prolactin (PRL) regulates differentiation and lactation in the bovine mammary epithelium. This tissue is especially prone to contracting mastitis, a disease characterized by an inflammatory response in the mammary gland. Staphylococcus aureus is the infectious agent primarily responsible for mastitis. In a previous study, we have shown that bovine PRL (bPRL) stimulates S. aureus internalization in bovine mammary epithelial cells (bMECs) by regulating several host innate immune elements, which are often modulated by nuclear factor kappa B (NF-κB). However, it is unknown whether the activation of the NF-κB transcription factor is regulated by bPRL during S. aureus internalization. The objective of this study was to determine the role of NF-κB in bPRL-stimulated bMECs during S. aureus internalization. Our results showed that bPRL (5 ng/ml) induced NF-κB activation in bMECs; however, it was inhibited by S. aureus in presence of the hormone. When we blocked NF-κB activation with acetylsalicylic acid, we detected an inhibition in S. aureus internalization (48%) in bPRL-stimulated bMECs. The infection-induced inhibition of NF-κB activation in the presence of bPRL correlates with the downregulation in bPRL-mediated tumor necrosis factor (TNF)-α (27%) and tracheal antimicrobial peptide (TAP, 70%) mRNA expression and nitric oxide (NO) production in bMECs. We also detected an inhibition in the expression of the bPRL target gene κ-casein (50%) under these conditions. Interestingly, these effects are not achieved through increased PRL receptor expression (PRLR), as it was inhibited (48%) compared to control cells. In conclusion, NF-κB activation in bMECs is inhibited by S. aureus in the presence of bPRL, suggesting a mechanism by which the host innate immune response may be compromised during subclinical mastitis.

Non-structural protein 1 of avian influenza A viruses differentially inhibit NF-κB promoter activation

BackgroundInfluenza virus infection activates NF-κB and is a general prerequisite for a productive influenza virus infection. On the other hand, non-structural protein 1 (NS1) suppresses this viral activated NF-κB, presumably to prevent expression of NF-κB mediated anti-viral response. NS1 proteins of influenza A viruses are divided into two groups, known as allele A and allele B. The possible functional relevance of this NS1 division to viral pathogenicity is lacking.FindingsThe ability of NS1 protein from two avian influenza subtypes, H6N8 and H4N6, to inhibit NF-κB promoter activation was assessed. Further, efforts were made to characterize the genetic basis of this inhibition. We found that allele A NS1 proteins of H6N8 and H4N6 are significantly better in preventing dsRNA induced NF-κB promoter activation compared to allele B of corresponding subtypes, in a species independent manner. Furthermore, the ability to suppress NF-κB promoter activation was mapped to the effector domain while the RNA binding domain alone was unable to suppress this activation. Chimeric NS1 proteins containing either RNA binding domain of allele A and effector domain of allele B or vice versa, were equally potent in preventing NF-κB promoter activation compared to their wt. NS1 protein of allele A and B from both subtypes expressed efficiently as detected by Western blotting and predominantly localized in the nucleus in both A549 and MiLu cells as shown by in situ PLA.ConclusionsHere, we present another aspect of NS1 protein in inhibiting dsRNA induced NF-κB activation in an allele dependent manner. This suggests a possible correlation with the virus's pathogenic potential.

Effects of impressic acid from Acanthopanax koreanum on NF-κB and PPARγ activities

Impressic acid, 3α,11α-dihydroxylup-20(29)-en-28-oic acid, is a lupane-type triterpenoid isolated from Acanthopanax koreanum, which has been used as a Korean folk medicine for rheumatism, hepatitis, diabetes, and inflammatory disorders. Recently, it was reported that impressic acid has inhibitory effects on the LPS-stimulated pro-inflammatory cytokine production in bone marrow-derived dendritic cells and on the nuclear factor of activated T-cells transcription factor activity. Thus, to investigate whether impressic acid has effects on the inhibition of nuclear factor kappa B (NF-κB) and activation of peroxisome proliferator-activated receptor gamma (PPARγ), luciferase reporter assays were used. The effects on the expression of NF-κB and PPARγ target genes were also examined by reverse transcription-polymerase chain reaction. In this study, impressic acid was found to inhibit tumor necrosis factor (TNF)-α induced NF-κB activity and to up-regulate transcriptional activity of PPARγ.

Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax

BackgroundOncoprotein Tax, encoded by the human T-cell leukemia virus type 1 (HTLV1), persistently induces NF-κB activation, which contributes to HTLV1-mediated T-cell transformation. Recent studies suggest that the signaling function of Tax requires its ubiquitination, although how the Tax ubiquitination is regulated remains unclear.ResultsWe show here that the deubiquitinase CYLD physically interacts with Tax and negatively regulates the ubiquitination of this viral protein. This function of CYLD is associated with inhibition of Tax-mediated activation of IKK although not that of Tak1. Interestingly, CYLD undergoes constitutive phosphorylation in HTLV1-transformed T cells, a mechanism known to inactivate the catalytic activity of CYLD. Consistently, a phospho-mimetic CYLD mutant fails to inhibit Tax ubiquitination.ConclusionThese findings suggest that CYLD negatively regulates the signaling function of Tax through inhibition of Tax ubiquitination. Conversely, induction of CYLD phosphorylation may serve as a mechanism by which HTLV1 overrides the inhibitory function of CYLD, leading to the persistent activation of NF-κB.

RETRACTED: Triptolide, histone acetyltransferase inhibitor, suppresses growth and chemosensitizes leukemic cells through inhibition of gene expression regulated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK pathway

Triptolide, a diterpene triepoxide, from the Chinese herb Tripterygium wilfordii Hook.f, exerts its anti-inflammatory and immunosuppressive activities by inhibiting the transcription factor nuclear factor-κB (NF-κB) pathway, through a mechanism not yet fully understood. We found that triptolide, in nanomolar concentrations, suppressed both constitutive and inducible NF-κB activation, but did not directly inhibit binding of p65 to the DNA. The diterpene did block TNF-induced ubiquitination, phosphorylation, and degradation of IκBα, the inhibitor of NF-κB and inhibited acetylation of p65 through suppression of binding of p65 to CBP/p300. Triptolide also inhibited the IκBα kinase (IKK) that activates NF-κB and phosphorylation of p65 at serine 276, 536. Furthermore, the NF-κB reporter activity induced by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKKβ was abolished by the triepoxide. Triptolide also abrogated TNF-induced expression of cell survival proteins (XIAP, Bcl-x(L), Bcl-2, survivin, cIAP-1 and cIAP-2), cell proliferative proteins (cyclin D1, c-myc and cyclooxygenase-2), and metastasis proteins (ICAM-1 and MMP-9). This led to enhancement of apoptosis induced by TNF, taxol, and thalidomide by the diterpene and to suppression of tumor invasion. Overall, our results demonstrate that triptolide can block the inflammatory pathway activated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK, sensitizes cells to apoptosis, and inhibits invasion of tumor cells.

Low-Level Laser Therapy Activates NF-kB via Generation of Reactive Oxygen Species in Mouse Embryonic Fibroblasts

BackgroundDespite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear.Methodology/Principal FindingsIn this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm2 and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration.ConclusionWe conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

4-Hydroxy-2-hexenal-induced apoptosis in human renal proximal tubular epithelial cells

The aldehyde products of lipid peroxidation such as 4-hydroxy-2-hexenal (HHE) might be responsible for the pathogenesis of kidney injury. The present study was aimed to investigate the effects of HHE on renal tubular epithelial cells and its signaling mechanisms. Human proximal tubular epithelial (HK-2) cells were treated with 10 μM of HHE. Cell viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The fluorescent probe 2',7'-dichlorofluorescein diacetate was used to measure intracellular levels of reactive oxygen species (ROS). The protein expression of NF-κB, mitogen-activated protein kinase (MAPK), pro-apoptoic Bax and anti-apoptotic protein Bcl-2 was determined by semiquantitative immunoblotting. Apoptosis was assessed by flow cytometry analysis after the cells were stained by fluorescein isothiocyanate-conjugated annexin V protein and propidium iodine. Treatment with various doses of HHE resulted in dose-dependent decreases of cell viability and increases of ROS. HHE increased the expression of p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). HHE induced NF-κB activation and IκB-α degradation. Increased nuclear NF-κB activation was blocked by inhibitors of ERK (PD98059) or JNK (SP600125), but not affected by p38 MAPK inhibitor (SB203580). Flow cytometry analysis revealed HHE-induced apoptosis. HHE decreased the expression of Bcl-2, while it increased that of Bax, which was attenuated by the treatment of NF-κB inhibitor (Bay 11-7082) or N-acetyl-L-cysteine (NAC). An inhibition of NF-κB prevented HHE-induced apoptosis. HHE-induced tubular cell apoptosis is mediated by modulation of Bax and Bcl-2 via ROS generation. HHE-mediated accumulation of ROS may induce redox-sensitive transcription factor, NF-κB, through activation of ERK and JNK, resulting in cellular apoptosis in HK-2 cells.

Negative Regulation of HIV-1 Transcription by a Heterodimeric NF-κB1/p50 and C-Terminally Truncated STAT5 Complex

Signal transducers and activator of transcription (STAT) proteins are often constitutively activated in leukocytes of HIV-1 + individuals, which frequently show a dominant expression of a C-terminally truncated isoform of STAT5 (STAT5Δ) . STAT5Δ can act as a negative regulator of human immunodeficiency virus type 1 (HIV-1) expression in both CD8-depleted primary leukocytes and chronically infected promonocytic U1 cells stimulated with granulocyte–macrophage colony-stimulating factor (GM-CSF). Activated STAT5Δ can directly bind to two consensus sequences in the HIV-1 long terminal repeat (LTR) promoter; binding impairs recruitment of RNA polymerase II (Crotti, A., Lusic, M., Lupo, R., Lievens, P. M., Liboi, E., Della Chiara, G., et al. (2007). Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression. Blood, 109, 5380–5389). One of the STAT consensus sequences overlaps with one nuclear factor κB (NF-κB) binding site; interestingly, NF-κB1/p50 homodimers, frequently detected in monocytic cells, are negative regulators of HIV transcription. Here, we show that GM-CSF stimulation of U1 cells, while not inducing NF-κB activation, leads to STAT5Δ phosphorylation and binding to the NF-κB/STAT target sequence in the HIV LTR promoter, which already associates with p50 under unstimulated conditions. STAT5Δ was found to associate with p50, but not with RelA/p65, in both U1 cells expressing endogenous proteins and 293T cells overexpressing these factors. Furthermore, GM-CSF stimulation promoted concurrent binding of STAT5Δ and p50 at the HIV LTR promoter in U1 cells. Immunoprecipitation of chromatin from GM-CSF-stimulated U1 cells confirmed in vivo binding of p50 to the viral promoter together with STAT5Δ. Thus, cytokine-activated STAT5Δ/p50 complexes can contribute to the maintenance of HIV-1 latency in monocytic cells.

PGE2‐EP2 signalling in endothelium is activated by haemodynamic stress and induces cerebral aneurysm through an amplifying loop via NF‐κB

Cerebral aneurysm is a frequent cerebrovascular event and a major cause of fatal subarachnoid haemorrhage, but there is no medical treatment for this condition. Haemodynamic stress and, recently, chronic inflammation have been proposed as major causes of cerebral aneurysm. Nevertheless, links between haemodynamic stress and chronic inflammation remain ill-defined, and to clarify such links, we evaluated the effects of prostaglandin E(2) (PGE(2) ), a mediator of inflammation, on the formation of cerebral aneurysms. Expression of COX and prostaglandin E synthase (PGES) and PGE receptors were examined in human and rodent cerebral aneurysm. The incidence, size and inflammation of cerebral aneurysms were evaluated in rats treated with COX-2 inhibitors and mice lacking each prostaglandin receptor. Effects of shear stress and PGE receptor signalling on expression of pro-inflammatory molecules were studied in primary cultures of human endothelial cells (ECs). COX-2, microsomal PGES-1 and prostaglandin E receptor 2 (EP(2) ) were induced in ECs in the walls of cerebral aneurysms. Shear stress applied to primary ECs induced COX-2 and EP(2) . Inhibition or loss of COX-2 or EP(2) in vivo attenuated each other's expression, suppressed nuclear factor κB (NF-κB)-mediated chronic inflammation and reduced incidence of cerebral aneurysm. EP(2) stimulation in primary ECs induced NF-κB activation and expression of the chemokine (C-C motif) ligand 2, essential for cerebral aneurysm. These results suggest that shear stress activated PGE(2) -EP(2) pathway in ECs and amplified chronic inflammation via NF-κB. We propose EP(2) as a therapeutic target in cerebral aneurysm.

Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and β-cell apoptosis

Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing genes in T1D, we performed an in silico "phenome-interactome analysis" on a genome-wide linkage scan dataset. This method prioritizes candidates according to their physical interactions at the protein level with other proteins involved in diabetes. A total of 11 genes were predicted to be likely disease genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated rat and human islets. Overexpression of HIP14 was associated with a decrease in IL-1β-induced NF-κB activity and protection against IL-1β-mediated apoptosis. Our study demonstrates that the current network biology approach is a valid method to identify genes of importance for T1D and may therefore embody the basis for more rational and targeted therapeutic approaches.

IκBβ Attenuates Angiotensin II–Induced Cardiovascular Inflammation and Fibrosis in Mice

The development of cardiovascular fibrosis is associated with chronic inflammation, where activation of nuclear factor κB (NF-κB) signaling may play a critical role. NF-κB activation is tightly regulated by the cellular inhibitor of κB (IκB) family of proteins, such as IκBα and IκBβ. IκBα and IκBβ display different regulation kinetics in response to inflammatory stimulation. The present study tested the hypothesis that IκBα and IκBβ may have different roles in modulating cardiovascular inflammation and fibrosis, using a model of angiotensin II infusion-induced hypertension in wild-type mice and IκBβ knock-in mice, in which the IκBα gene is replaced by IκBβ cDNA (AKBI). In WT mice, subcutaneous angiotensin II infusion for 7 days induced increased perivascular and interstitial collagen deposition and fibrotic lesions, associated with myocardial interstitial hemosiderin accumulation and extensive macrophage infiltration. These effects of angiotensin II were dramatically limited in AKBI mice. Replacement of IκBα with IκBβ significantly attenuated angiotensin II infusion-induced expression of interleukin 1β, interleukin 6, monocyte chemotactic protein 1, collagen I and III, fibronectin, and tissue inhibitor of metalloproteinase 1 in the hearts. Furthermore, using cultured vascular smooth muscle cells, we demonstrated that interleukin 1β-induced NF-κB activation and monocyte chemotactic protein 1, vascular cell adhesion molecule 1, and tissue inhibitor of metalloproteinase 1 expressions were suppressed in the AKBI cells because of the replacement of IκBα with IκBβ. These results indicate that NF-κB has an essential role in mediating the cardiovascular inflammatory response to angiotensin II and suggest that targeting the balance of IκBα and IκBβ expression might be a novel therapeutic modality in preventing fibrosis in hypertensive cardiovascular disease.

Radiation-triggered Tumor Necrosis Factor (TNF) α-NFκB Cross-signaling Favors Survival Advantage in Human Neuroblastoma Cells

Induced radioresistance in the surviving cancer cells after radiotherapy could be associated with clonal selection leading to tumor regrowth at the treatment site. Previously we reported that post-translational modification of IκBα activates NFκB in response to ionizing radiation (IR) and plays a key role in regulating apoptotic signaling. Herein, we investigated the orchestration of NFκB after IR in human neuroblastoma. Both in vitro (SH-SY5Y, SK-N-MC, and IMR-32) and in vivo (xenograft) studies showed that IR persistently induced NFκB DNA binding activity and NFκB-dependent TNFα transactivation and secretion. Approaches including silencing NFκB transcription, blocking post-translational NFκB nuclear import, muting TNF receptor, overexpression, and physiological induction of either NFκB or TNFα precisely demonstrated the initiation and occurrence of NFκB → TNFα → NFκB positive feedback cycle after IR that leads to and sustains NFκB activation. Selective TNF-dependent NFκB regulation was confirmed with futile inhibition of AP-1 and SP-1 in TNF receptor muted cells. Moreover, IR increased both transactivation and translation of Birc1, Birc2, and Birc5 and induced metabolic activity and clonal expansion. This pathway was further defined to show that IR-induced functional p65 transcription (not NFκB1, NFκB2, or c-Rel) is necessary for activation of these survival molecules and associated survival advantage. Together, these results demonstrate for the first time the functional orchestration of NFκB in response to IR and further imply that p65-dependent survival advantage and initiation of clonal expansion may correlate with an unfavorable prognosis of human neuroblastoma.