Molecular basis of lysophosphatidic acid-induced NF-κB activation

PKC, β-arrestin 2, CARMA3, BCL10, MALT1, TRAF6 and MEKK3 are signaling proteins that have a key role in G protein-coupled receptor (GPCR)-mediated activation of nuclear factor-κB (NF-κB) pathway in nonhematopoietic cells in response to lysophosphatidic acid (LPA) stimulation. The PKC, β-arrestin 2, CARMA3-BCL10-MALT1-TRAF6 signalosome, and MEKK3 functions as a link between GPCR signaling and IKK-NF-κB activation. Here we briefly summarize recent progress in the understanding of the molecular and biological functions of these proteins in GPCR-mediated NF-κB activation in nonhematopoietic cells.     

Role of NF-κB activation in LPS-induced endothelial barrier breakdown

Endothelial barrier breakdown contributes to organ failure in sepsis. The key mechanism by which the potent sepsis inductor lipopolysaccharide (LPS) disrupts the endothelial barrier is controversial. Here, we tested the hypothesis that NF-κB activation is critically involved in endothelial barrier breakdown. Application of LPS to monolayers of porcine pulmonary artery endothelial cells (PAEC) and human dermal microvascular endothelial cells (HDMEC) induced a rapid and sustained activation of NF-κB as revealed by translocation of its subunit p65 into the nuclei in nuclear extraction assays and by immunostaining. Measurements of transendothelial electrical resistance (TER) and intercellular gap formation demonstrated significant breakdown of endothelial barrier properties following LPS treatment for 3?h. Interestingly, monolayers recovered spontaneously beginning after 10?h. Increased cAMP prevented LPS-induced loss of endothelial barrier properties, but did not block NF-κB activation. Application of the cell-permeable NEMO-binding domain (NBD) synthetic peptide was effective to prevent NF-κB activation, but did neither block LPS-induced loss of TER nor intercellular gap formation. NBD peptide alone did not alter endothelial barrier properties, but enhanced the barrier-compromising effects when applied in combination with LPS. Similarly, siRNA-mediated knock-down of p65 in HDMECs did not prevent LPS-induced barrier breakdown. Known targets of NF-κB-derived protein expression of caveolin or vasodilator-stimulated phosphoprotein (VASP) remained unaltered by LPS treatment of endothelial cells. In summary, our data indicate that NF-κB activation by LPS is not critically involved in disruption of endothelial barrier properties. Rather, our data suggest that NF-κB activation acts as a part of a rescue mechanism.     

The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation

Background

The nuclear factor κB (NF-κB) family members regulate several biological processes as cell proliferation and differentiation, inflammation, immunity and tumor progression. Ubiquitination plays a key role in NF-κB activation and the ubiquitylated transmitters of the NF-κB signaling cascade accumulate in close proximity to endomembranes.

Findings

We performed an unbiased siRNA library screen targeting the 46 E3 ubiquitin ligases bearing transmembrane domains to uncover new modulators of NF-κB activation, using tumor necrosis factor–α (TNF-α) receptor (TNFR) stimulation as a model. We report here the identification of a new Golgi Apparatus-resident protein, RNF121, as an enhancer of NF-κB promoter activity through the catalytic function of its RING domain. From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process. However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex. Finally, we discovered that RNF121 acts as a broad regulator of NF-κB signaling since its silencing also dampens NF-κB activation following stimulation of Toll-Like Receptors (TLRs), Nod-Like Receptors (NLRs), RIG-I-Like Receptors (RLRs) or after DNA damages.

Conclusions

These results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

     

TRAF6 is functional in inhibition of TLR4-mediated NF-κB activation by resveratrol

Resveratrol was suggested to inhibit Toll-like receptor (TLR)4-mediated activation of nuclear factor-κB (NF-κB) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF)–(TANK)-binding kinase 1, but the myeloid differentiation primary response gene 88–tumor necrosis factor receptor-associated factor 6 (TRAF6) pathway is not involved in this effect. However, involvement of TRAF6 in this process is still elusive since cross talk between TRIF and TRAF6 has been reported in lipopolysaccharide (LPS)-induced signaling. Using RAW 264.7 macrophages, we determined the effect of resveratrol on LPS-induced TRAF6 expression, ubiquitination as well as activation of mitogen-activated protein (MAP) kinases and Akt in order to elucidate its involvement in TLR4 signaling. LPS-induced transient elevation in TRAF6 mRNA and protein expressions is suppressed by resveratrol. LPS induces the ubiquitination of TRAF6, which has been reported to be essential for Akt activation and for transforming growth factor-β activated kinase-1–NAP kinase kinase 6 (MKK6)-mediated p38 and c-Jun N-terminal kinase (JNK) activation. We found that resveratrol diminishes the effect of LPS on TRAF6 ubiquitination and activation of JNK and p38 MAP kinases, while it has no effect on the activation of extracellular-signal-regulated kinase (ERK)1/2. The effect of resveratrol on MAP kinase inhibition is significant since TRAF6 activation was reported to induce activation of JNK and p38 MAP kinase while not affecting ERK1/2. Moreover, Akt was identified previously as a direct target of TRAF6, and we found that, similarly to MAPKs, phosphorylation pattern of Akt followed the activation of TRAF6, and it was inhibited by resveratrol at all time points. Here, we provide the first evidence that resveratrol, by suppressing LPS-induced TRAF6 expression and ubiquitination, attenuates the LPS-induced TLR4–TRAF6, MAP kinase and Akt pathways that can be significant in its anti-inflammatory effects.     

TRADD is critical for resistance to TRAIL-induced cell death through NF-κB activation

One major obstacle in the clinical application of TRAIL as a cancer therapeutic agent is the acquisition of TRAIL resistance. We found that deficiency of TRADD sensitizes cells to TRAIL-induced apoptosis. Enhanced cell death in TRADD(-/-) MEFs is associated with defective NF-κB activation, indicating that the pro-survival function of TRADD in TRAIL signaling is mediated at least in part via NF-κB activation. Moreover, siRNA knock-down of TRADD in cancer cells sensitizes them to TRAIL-induced apoptosis. Thus, TRADD has a survival role in TRAIL signaling and may be one potential target for overcoming TRAIL resistance in cancer therapy.     

Role of post-translational modifications of HTLV-1 Tax in NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered, is the etiological agent of adult-T-cell leukemia/lymphoma. The HTLV-1 encoded Tax protein is a potent oncoprotein that deregulates gene expression by constitutively activating nuclear factor-κB (NF-κB). Tax activation of NF-κB is critical for the immortalization and survival of HTLV-1-infected T cells. In this review, we summarize the present knowledge on mechanisms underlying Tax-mediated NF-κB activation, with an emphasis on post-translational modifications of Tax.     

Cysteine 38 holds the key to NF-κB activation

The importance of parallel signaling pathways controlling NF-κB subunit posttranslational modifications is demonstrated by Sen et al. (2012), who reveal that RelA (p65) sulfhydration, at its highly conserved cysteine 38 residue, regulates association with the coactivator RPS3, DNA binding, and antiapoptotic gene expression.     

Optineurin is required for CYLD-dependent inhibition of TNFα-induced NF-κB activation

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.     

Apoptosis of Dedifferentiated Hepatoma Cells is Independent of NF-κB Activation in Response to LPS

Dedifferentiated hepatoma cells, in contrast to most other cell types including hepatoma cells, undergo apoptosis when treated with lipopolysaccharide (LPS) plus the protein synthesis inhibitor cycloheximide (CHx). We recently reported that the dedifferentiated hepatoma cells also exhibit a strong and prolonged NF-κB induction phenotype upon exposure to LPS, suggesting that NF-κB signaling may play a pro-survival role, as reported in several other cell systems. To test the role of NF-κB in preventing LPS-mediated apoptosis, we examined the dedifferentiated cell line M38. Results show that antioxidants strongly inhibited LPS + CHx-mediated cell death in the M38 cells, yet only modestly inhibited NF-κB induction. In addition, inhibition of NF-κB translocation by infection of the M38 cells with an adenoviral vector expressing an IκBα super-repressor did not result in LPS-mediated cell death. These results suggest that unlike TNFα induction, the cell survival pathway activated in response to LPS is independent of NF-κB translocation in the dedifferentiated cells. Addition of inhibitors of JNK, p38 and ERK pathways also failed to elicit LPS-mediated apoptosis similar to that observed when protein synthesis is prevented. Thus, cell survival pathways other than those involving NF-κB inducible gene expression or other well-known pathways appear to be involved in protecting the dedifferentiated hepatoma variant cells from LPS-mediated apoptosis. Importantly, this pro-apoptotic function of LPS appears to be a function of loss of hepatic gene expression, as the parental hepatoma cells resist LPS-mediated apoptosis in the presence of protein synthesis inhibitors.