Knockdown of DEAD-box RNA helicase DDX5 selectively attenuates serine 311 phosphorylation of NF-κB p65 subunit and expression level of anti-apoptotic factor Bcl-2.

NF-κB plays a key role in the transcriptional regulation of genes involved in immunity, inflammation, cell proliferation, and oncogenesis. The NF-κB activation process includes nuclear translocation, followed by association with basal transcription machinery. These steps are tightly regulated by posttranslational modification of the proteins involved in this pathway. We recently reported that NF-κB transactivation activity is enhanced by knockdown of diacylglycerol kinase ζ (DGKζ), which belongs to an enzyme family that phosphorylates lipidic second messenger diacylglycerol to phosphatidic acid. To investigate details of the regulatory mechanism exerted by DGKζ, we identified DEAD-box RNA helicase DDX5 as a novel DGKζ-interacting protein and examined functional role of DDX5 in NF-κB transactivation activity. Here we show that DDX5 knockdown exerts no significant effect on nuclear translocation, but specifically attenuates Ser311 phosphorylation of p65 subunit. Luciferase reporter assay reveals that the NF-κB transcriptional activity is repressed in DDX5-knockdown cells. Furthermore, we found that DDX5 knockdown selectively downregulates the expression level of Bcl-2 of the NF-κB-inducible anti-apoptotic factors upon TNF-α stimulation. Considering the evidence collectively, we can infer that DGKζ-interacting multi-protein complex modulates the NF-κB transactivation activity in a negative and positive manner under conditions in which the expression level of a component of the complex is altered.

DGKζ Downregulation Enhances Osteoclast Differentiation and Bone Resorption Activity Under Inflammatory Conditions.

Bone homeostasis is maintained by a balance between resorption of the bone matrix and its replacement by new bone. Osteoclasts play a crucially important role in bone metabolism. They are responsible for bone resorption under pathophysiological conditions. Differentiation of these cells, which are derived from bone marrow cells, depends on receptor activator of NF-κB ligand (RANKL). RANKL-induced osteoclastogenesis is regulated by the phosphoinositide (PI) signaling pathway, in which diacylglycerol (DG) serves as a second messenger in signal transduction. In this study, we examined the functional implications of DG kinase (DGK), an enzyme family responsible for DG metabolism, for osteoclast differentiation and activity. Of DGKs, DGKζ is most abundantly expressed in osteoclast precursors such as bone marrow-derived monocytes/macrophages. During osteoclast differentiation from precursor cells, DGKζ is downregulated at the protein level. In this regard, we found that DGKζ deletion enhances osteoclast differentiation and bone resorption activity under inflammatory conditions in an animal model of osteolysis. Furthermore, DGKζ deficiency upregulates RANKL expression in response to TNFα stimulation. Collectively, results suggest that DGKζ is silent under normal conditions, but it serves as a negative regulator in osteoclast function under inflammatory conditions. Downregulation of DGKζ might be one factor predisposing a person to osteolytic bone destruction in pathological conditions. J. Cell. Physiol. 232: 617-624, 2017. © 2016 Wiley Periodicals, Inc.

Inflammatory immune cell responses and Toll-like receptor expression in synovial tissues in rheumatoid arthritis patients treated with biologics or DMARDs.

Biologic antirheumatic drugs (BIO) have been reported to be potent therapeutic agents in the prevention of inflammatory joint destruction in rheumatoid arthritis (RA). The aim of this study was to investigate the immune-inflammatory cells, including Toll-like receptor (TLR)-equipped cells, in synovial tissue samples from RA patients on BIO compared to patients, who are only on conventional disease-modifying antirheumatic drug (DMARD). We analyzed immune-inflammatory cells in RA synovitis in patients of BIO group (n = 20) or DMARD group (n = 20). The grading scores of synovitis was 1.7 and 1.8 in each BIO and DMARD group and correlated best with the CD3(+) T (r = 0.71/0.70, p < 0.05) and CD20(+) B (r = 0.80/0.84, p < 0.05) cells in the both groups, but less well with the CD68(+) macrophages and S-100(+) dendritic cells (DCs). Interestingly, both T (116 vs. 242, p < 0.05) and B (80 vs. 142, p < 0.05) cell counts were lower in the BIO than in the DMARD group, whereas macrophage and DC counts did not differ. In contrast, the C-reactive protein (CRP) and disease activity score DAS28-CRP did not show clear-cut correlations with the inflammatory grade of the synovitis (r range, 0-0.35). Similar numbers of cells immunoreactive for TLR-1 to TLR-6 and TLR-9 were found in synovitis in both groups. Patients clinically responding to biologics might still have the potential of moderate/severe local joint inflammation, composed in particular of and possibly driven by the autoinflammatory TLR(+) cells.

DGKζ is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway.

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

Toll-like receptors and their adaptors are regulated in macrophages after phagocytosis of lipopolysaccharide-coated titanium particles.

Macrophages phagocytose metallic wear particles and produce mediators, which can induce cellular host response and aseptic implant loosening. Lipopolysaccharide (LPS) on the wear debris can stimulate macrophages via Toll-like receptor 4 (TLR4) and enhance the response. However, the precise functional role and interaction of TLRs and their adaptor molecules is still unclear. Rat bone marrow macrophages were stimulated with titanium particle (Ti) coated by LPS (Ti/LPS+) and LPS-free Ti (Ti/LPS-). mRNA levels of cytokines, TLRs and their adaptor molecules were measured using real time PCR. mRNA levels of TNF-α, IL-1ß, and IL-6 increased in Ti/LPS+ than Ti/LPS-. In contrast, mRNA levels of TLR4, TLR5, and TLR9 decreased in Ti/LPS+ compared to Ti/LPS-. mRNA levels of MyD88, IRAK1, IRAK4 decreased gradually, and TRAF6 underwent an initial transient increase, followed by suppression in Ti/LPS+. However, mRNA levels of TLR2 and IRAK2 increased after phagocytosis of Ti/LPS+ than Ti/LPS-. The increased expressions of proinflammatory cytokines found in Ti/LPS+ indicated that their productions cytokines could be enhanced by phagocytosis of LPS-coated particles. Subsequent down-regulation of TLR4, TLR5, TLR9, MyD88, IRAK1, and IRAK4 suggests that self-protective mechanisms to regulate excessive host responses are activated in macrophages. Increase of TLR2 and IRAK2 and a transient increase of TRAF6 in Ti/LPS+ suggest that another possible pathway to modulate TLR-mediated cellular response to prolong inflammatory response in foreign body reaction of aseptic loosening. This down- and/or up-regulation of the potential TLR-mediated responses to LPS-coated particles reflects the proactive behavior of effector cells.