NF-κB p100 limits TNF-induced bone resorption in mice by a TRAF3-dependent mechanism

TNF and RANKL mediate bone destruction in common bone diseases, including osteoarthritis and RA. They activate NF-κB canonical signaling directly in osteoclast precursors (OCPs) to induce osteoclast formation in vitro. However, unlike RANKL, TNF does not activate the alternative NF-κB pathway efficiently to process the IκB protein NF-κB p100 to NF-κB p52, nor does it appear to induce osteoclast formation in vivo in the absence of RANKL. Here, we show that TNF limits RANKL- and TNF-induced osteoclast formation in vitro and in vivo by increasing NF-κB p100 protein accumulation in OCPs. In contrast, TNF induced robust osteoclast formation in vivo in mice lacking RANKL or RANK when the mice also lacked NF-κB p100, and TNF-Tg mice lacking NF-κB p100 had more severe joint erosion and inflammation than did TNF-Tg littermates. TNF, but not RANKL, increased OCP expression of TNF receptor–associated factor 3 (TRAF3), an adapter protein that regulates NF-κB p100 levels in B cells. TRAF3 siRNA prevented TNF-induced NF-κB p100 accumulation and inhibition of osteoclastogenesis. These findings suggest that upregulation of TRAF3 or NF-κB p100 expression or inhibition of NF-κB p100 degradation in OCPs could limit bone destruction and inflammation-induced bone loss in common bone diseases.     

Topical hypochlorite ameliorates NF-κB–mediated skin diseases in mice

Nuclear factor-κB (NF-κB) regulates cellular responses to inflammation and aging, and alterations in NF-κB signaling underlie the pathogenesis of multiple human diseases. Effective clinical therapeutics targeting this pathway remain unavailable. In primary human keratinocytes, we found that hypochlorite (HOCl) reversibly inhibited the expression of CCL2 and SOD2, two NF-κB–dependent genes. In cultured cells, HOCl inhibited the activity of inhibitor of NF-κB kinase (IKK), a key regulator of NF-κB activation, by oxidizing cysteine residues Cys114 and Cys115. In NF-κB reporter mice, topical HOCl reduced LPS-induced NF-κB signaling in skin. We further evaluated topical HOCl use in two mouse models of NF-κB–driven epidermal disease. For mice with acute radiation dermatitis, topical HOCl inhibited the expression of NF-κB–dependent genes, decreased disease severity, and prevented skin ulceration. In aged mice, topical HOCl attenuated age-dependent production of p16^INK4a and expression of the DNA repair gene Rad50. Additionally, skin of aged HOCl-treated mice acquired enhanced epidermal thickness and proliferation, comparable to skin in juvenile animals. These data suggest that topical HOCl reduces NF-κB–mediated epidermal pathology in radiation dermatitis and skin aging through IKK modulation and motivate the exploration of HOCl use for clinical aims.     

NF-κB Mediates Tumor Necrosis Factor α-Induced Expression of Optineurin, a Negative Regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB.     

DEAD-box helicase DP103 defines metastatic potential of human breast cancers

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β–activated kinase-1 (TAK1) phosphorylation of NF-κB–activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB–mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.     

Positive feedback between NF-κB and TNF-α promotes leukemia-initiating cell capacity

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from leukemia-initiating cells (LICs). The identification of common mechanisms underlying LIC development will be important in establishing broadly effective therapeutics for AML. Constitutive NF-κB pathway activation has been reported in different types of AML; however, the mechanism of NF-κB activation and its importance in leukemia progression are poorly understood. Here, we analyzed myeloid leukemia mouse models to assess NF-κB activity in AML LICs. We found that LICs, but not normal hematopoietic stem cells or non-LIC fractions within leukemia cells, exhibited constitutive NF-κB activity. This activity was maintained through autocrine TNF-α secretion, which formed an NF-κB/TNF-α positive feedback loop. LICs had increased levels of active proteasome machinery, which promoted the degradation of IκBα and further supported NF-κB activity. Pharmacological inhibition of the proteasome complex markedly suppressed leukemia progression in vivo. Conversely, enhanced activation of NF-κB signaling expanded LIC frequency within leukemia cell populations. We also demonstrated a strong correlation between NF-κB activity and TNF-α secretion in human AML samples. Our findings indicate that NF-κB/TNF-α signaling in LICs contributes to leukemia progression and provide a widely applicable approach for targeting LICs.     

The Human Toll Signaling Pathway: Divergence of Nuclear Factor κB and JNK/SAPK Activation Upstream of Tumor Necrosis Factor Receptor–associated Factor 6 (TRAF6)

The human homologue of Drosophila Toll (hToll) is a recently cloned receptor of the interleukin 1 receptor (IL-1R) superfamily, and has been implicated in the activation of adaptive immunity. Signaling by hToll is shown to occur through sequential recruitment of the adapter molecule MyD88 and the IL-1R–associated kinase. Tumor necrosis factor receptor–activated factor 6 (TRAF6) and the nuclear factor κB (NF-κB)–inducing kinase (NIK) are both involved in subsequent steps of NF-κB activation. Conversely, a dominant negative version of TRAF6 failed to block hToll-induced activation of stress-activated protein kinase/c-Jun NH₂-terminal kinases, thus suggesting an early divergence of the two pathways.     

Flaxseed orbitides,linusorbs, inhibit LPS-induced THP-1 macrophage inflammation

Linusorbs (flaxseed orbitides) are a family of naturally-occurring cyclic peptides. Previously, we reported that their anticancer effects were associated with their structures. In this study, we investigated the anti-inflammatory activities of 2 different linusorbs ([1–9-NαC]-linusorb B2 and [1–9-NαC]-linusorb B3) in lipopolysaccharide (LPS)-induced THP-1 macrophage activation as well as the underlying mechanism of this inflammatory response. Both molecules suppressed pro-inflammatory mediators (TNF-α, IL-1β, IL-6, NO and COX-2) and were involved in downregulating the NF-κB signaling pathway. The suppressive effects on pro-inflammatory mediators were comparable and the concentration range of action was similar (1–4 μM). However, the concentration of compound that induced downregulation of the NF-κB pathway was different for each compound. While [1–9-NαC]-linusorb B3 could inhibit the activation of the NF-κB pathway at concentrations of 1 and 2 μM, [1–9-NαC]-linusorb B2 induced a comparable inhibitory effect at a concentration of 4 μM.

Linusorbs (flax orbitides) are a family of plant cyclic peptides. We investigate the anti-inflammatory activities of two different linusorbs ([1–9-NaC]-linusorb B2 and [1–9-NaC]-linusorb B3) and the underlying mechanism of this inflammatory response.     

Inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal H1N1 influenza infection in mice

Introduction

Factors implicated in influenza-mediated morbidity and mortality include robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction. Tumor necrosis factor-alpha (TNF-α) is an important pro-inflammatory cytokine present during influenza infection, but it is unclear whether direct inhibition of TNF-α can elicit protection against influenza infection.

Methods

In this study, the commercially available TNF-α inhibitor etanercept was used to inhibit TNF-α induced by lethal A/FM/1/47 (H1N1) influenza virus infection of mice. The effects of TNF-α inhibition on mouse survival, pathologic changes, immune cell infiltration, inflammatory cytokine secretion, Toll-like receptor expression, and activation of the NF-κB (nuclear factor kappa B) signaling pathway were evaluated.

Results

The intranasal delivery of etanercept provided significant protection against mortality (30% of mice survived up to 14 days after infection) in mice treated with etanercept. In contrast, no survivors were found beyond 6 days in mice treated with saline after lethal challenge with H1N1 influenza virus. It was observed that etanercept significantly reduced inflammatory cell infiltration (for example, macrophages and neutrophils), inflammatory cytokine secretion (for example, interleukin-6, TNF-α, and interferon gamma), and expression of Toll-like receptors (TLR-3, TLR-4, and TLR-7). Etanercept also downregulated and inhibited the cascade proteins of the NF-κB signaling pathway (for example, MyD88, TRIF, NF-κB, and p65), as well as enhanced host control of virus replication.

Conclusions

These findings indicate that etanercept, by blocking TNF-α, can significantly downregulate excessive inflammatory immune responses and provide protection against lethal influenza infection, making its use a novel strategy for controlling severe influenza-induced viral pneumonia.     

TGF-β induces miR-182 to sustain NF-κB activation in glioma subsets

The strength and duration of NF-κB signaling are tightly controlled by multiple negative feedback mechanisms. However, in cancer cells, these feedback loops are overridden through unclear mechanisms to sustain oncogenic activation of NF-κB signaling. Previously, we demonstrated that overexpression of miR-30e* directly represses IκBα expression and leads to hyperactivation of NF-κB. Here, we report that miR-182 was overexpressed in a different set of gliomas with relatively lower miR-30e* expression and that miR-182 directly suppressed cylindromatosis (CYLD), an NF-κB negative regulator. This suppression of CYLD promoted ubiquitin conjugation of NF-κB signaling pathway components and induction of an aggressive phenotype of glioma cells both in vitro and in vivo. Furthermore, we found that TGF-β induced miR-182 expression, leading to prolonged NF-κB activation. Importantly, the results of these experiments were consistent with an identified significant correlation between miR-182 levels with TGF-β hyperactivation and activated NF-κB in a cohort of human glioma specimens. These findings uncover a plausible mechanism for sustained NF-κB activation in malignant gliomas and may suggest a new target for clinical intervention in human cancer.     

Gastric Hyperplasia and Increased Proliferative Responses of Lymphocytes in Mice Lacking the COOH-terminal Ankyrin Domain of NF-κB2

The nfkb2 gene encodes the p100 precursor which produces the p52 protein after proteolytic cleavage of its COOH-terminal domain. Although the p52 product can act as an alternative subunit of NF-κB, the p100 precursor is believed to function as an inhibitor of Rel/NF-κB activity by cytoplasmic retention of Rel/NF-κB complexes, like other members of the IκB family. However, the physiological relevance of the p100 precursor as an IκB molecule has not been understood. To assess the role of the precursor in vivo, we generated, by gene targeting, mice lacking p100 but still containing a functional p52 protein. Mice with a homozygous deletion of the COOH-terminal ankyrin repeats of NF-κB2 (p100^−/−) had marked gastric hyperplasia, resulting in early postnatal death. p100^−/− animals also presented histopathological alterations of hematopoietic tissues, enlarged lymph nodes, increased lymphocyte proliferation in response to several stimuli, and enhanced cytokine production in activated T cells. Dramatic induction of nuclear κB–binding activity composed of p52-containing complexes was found in all tissues examined and also in stimulated lymphocytes. Thus, the p100 precursor is essential for the proper regulation of p52-containing Rel/NF-κB complexes in various cell types and its absence cannot be efficiently compensated for by other IκB proteins.     

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage–TNF–IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.