SGK1 negatively regulates inflammatory immune responses and protects against alveolar bone loss through modulation of TRAF3 activity.

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1ß, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection-induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun-NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.

New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains.

In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state. Generalized Polarization measurements and spectral phasor analysis revealed that truncated-TRAF2 affects the membrane fluidity, especially when vesicles are heated up at physiological temperature. The addition of nanomolar concentration of TRAF2 in GUVs also seems to exert a mechanical action, as demonstrated by the formation of intraluminal vesicles, a process in which ganglioside GM1 plays a crucial role.

Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction.

OBJECTIVE: In this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth. METHODS: Human gingival fibroblasts were cultured to semiconfluence and treated with PHT (0.025, 0.1, 0.25, and 1.0 µM) for 48 h, and then, the apoptotic cell numbers were relatively determined by absorptiometry. After 24 h of 0.25 µM PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by Western blot analysis. After 48 h of 0.25 µM PHT treatment, appearance of apoptotic cells was detected by TUNEL assay. RESULTS: PHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity. CONCLUSION: PHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.

Enhanced cortisol increase upon awakening is associated with greater pain ratings but not salivary cortisol or soluble tumor necrosis factor-α receptor II responses to acute pain.

OBJECTIVES: The cortisol awakening response (CAR) is related to psychosocial factors and health in potentially significant ways, suggesting that it may be a distinctive marker of hypothalamic-pituitary-adrenal axis function and dysfunction. This study sought to expand upon previous work that examined the association between CAR and ratings of laboratory-evoked acute pain stimulation. In addition to evoked pain ratings, this study also tested whether CAR was prospectively related with salivary cortisol and soluble tumor necrosis factor-α receptor II responses to acute pain stimulation. METHODS: This study included 36 healthy, pain-free volunteers of both sexes recruited through posted study flyers. Prior to completion of laboratory pain testing, salivary cortisol samples were obtained at home over the course of a single morning according to the following time frame: upon awakening, and 15, 30, and 60 minute after awakening. After collection of saliva, study participants brought their home saliva samples to the laboratory for assay and subsequently completed acute experimental pain testing procedures. RESULTS: Cluster analysis of CAR revealed two distinct groups with similar patterns of cortisol response to awakening; increased and flattened. Relative to flattened CAR, increased CAR was associated with greater ratings of pain intensity and unpleasantness. Salivary cortisol was significantly increased and soluble tumor necrosis factor-α receptor II significantly decreased after pain testing, but neither of these responses differed as a function of increased versus flattened CAR. DISCUSSION: CAR may be a marker for stress sensitivity and/or the anticipation of impending stress, which could explain why the increased CAR cohort reported greater acute pain ratings.

TNF-like weak inducer of apoptosis inhibits proinflammatory TNF receptor-1 signaling.

Soluble TNF-like weak inducer of apoptosis (TWEAK) trimers induce, in a variety of cell lines, translocation of cytosolic tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) to a triton X-100-insoluble compartment without changes in the total cellular TRAF2 content. TWEAK-induced TRAF2 translocation is paralleled by a strong increase in nuclear factor kappaB 2 (NFkappaB2)/p100 processing to p52, indicating that TRAF2 redistribution is sufficient for activation of the alternative NFkappaB pathway. In accordance with the crucial role of TRAF2 in proinflammatory, anti-apoptotic TNF receptor-1 (TNFR1) signaling, we observed that TWEAK-primed cells have a reduced capacity to activate the classical NFkappaB pathway or JNK (cJun N-terminal kinase) in response to TNF. Furthermore, TWEAK-primed cells are sensitized for the TNFR1-mediated induction of apoptotic and necrotic cell death. Notably, the expression of the NFkappaB-regulated, TRAF2-interacting TRAF1 protein can attenuate TWEAK-induced depletion of the triton X-100-soluble TRAF2 fraction and improve TNFR1-induced NFkappaB signaling in TWEAK-primed cells. Taken together, we demonstrate that soluble TWEAK desensitizes cells for proinflammatory TNFR1 signaling and thus identify TWEAK as a modifier of TNF signaling.

Cross-linking of 4-1BB activates TCR-signaling pathways in CD8+ T lymphocytes.

Cross-linking of 4-1BB, a member of the TNFR family, increased tyrosine phosphorylation of TCR-signaling molecules such as CD3epsilon, CD3zeta, Lck, the linker for activation of T cells, and SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76). In addition, incubation of activated CD8+ T cells with p815 cells expressing 4-1BBL led to redistribution of the lipid raft domains and Lck, protein kinase C-theta;, SLP-76, and phospholipase C-gamma1 (PLC-gamma1) on the T cell membranes to the areas of contact with the p815 cells and recruitment of 4-1BB, TNFR-associated factor 2, and phospho-tyrosine proteins to the raft domains. 4-1BB ligation also caused translocation of TNFR-associated factor 2, protein kinase C-theta;, PLC-gamma1, and SLP-76 to detergent-insoluble compartments in the CD8+ T cells, and cross-linking of 4-1BB increased intracellular Ca2+ levels apparently by activating PLC-gamma1. The redistribution of lipid rafts and Lck, as well as translocation of PLC-gamma1, and degradation of IkappaB-alpha in response to 4-1BB were inhibited by disrupting the formation of lipid rafts with methyl-beta-cyclodextrin. These findings demonstrate that 4-1BB is a T cell costimulatory receptor that activates TCR-signaling pathways in CD8+ T cells.