Phospholipase Cγ1 suppresses foreign body giant cell formation by maintaining RUNX1 expression in macrophages.

Foreign body giant cell (FBGC) formation is associated with the inflammatory response following material implantation. However, the intracellular signaling events that regulate the process remain unclear. Here, we investigated the potential role of phospholipase C (PLC)γ1, a crucial enzyme required for growth factor-induced signaling, on FBGC formation. Knock-down of PLCγ1 using shRNA induced FBGC formation accompanied by increased expression of cathepsin K, DC-STAMP and CD36. Re-addition of PLCγ1 decreased FBGC formation. PLCγ1-deficiency caused a decrease in RUNX1 and subsequent PU.1 upregulation while subsequent rescue of RUNX1 in sh-PLCγ1-transfected cells strongly inhibited FBGC formation. FBGC generated by knock-down of PLCγ1 using shRNA resulted in strongly increased TNF-α production, with augmented activation of ERK, p38 MAPK and JNK, and subsequently NF-κB. Taken together, we suggest that PLCγ1 plays a role in the foreign body response by regulating the RUNX1/PU.1/DC-STAMP axis in macrophages.

Role of CXCR2 on the immune modulating activity of α-iso-cubebenol a natural compound isolated from the Schisandra chinensis fruit.

Previously, we demonstrated that α-iso-cubebenol, a natural compound isolated from the fruits of Schisandra chinensis, strongly enhances therapeutic efficacy against cecal ligation and puncture challenge-induced sepsis. In this study, we found that α-iso-cubebenol stimulated calcium increase and degranulation in human neutrophils. α-Iso-cubebenol also strongly induced neutrophil chemotaxis, which was completely blocked by a CXCR2 antagonist, SB225002. The increased survival rate by α-iso-cubebenol was also significantly attenuated by SB225002. Taken together, the results indicate that α-iso-cubebenol-induced anti-septic activity was mediated by CXCR2, suggesting CXCR2 as an important target for the regulation of sepsis and inflammation.

Involvement of the visfatin/toll-like receptor 4 signaling axis in human dental pulp cell senescence: Protection via toll-like receptor 4 blockade.

Background: /purpose: Dental pulp plays an important role in the maintenance of tooth homeostasis and repair. The aging of dental pulp affects the functional life of the tooth owing to the senescence of dental pulp cells. Toll-like receptor 4 (TLR4) is involved in regulating cellular senescence in dental pulp. We have recently demonstrated that visfatin induces the senescence of human dental pulp cells (hDPCs). Here, we explored the association of TLR4 with visfatin signaling in cellular senescence in hDPCs. Materials and methods: mRNA levels were determined using reverse transcription polymerase chain reaction (PCR) and quantitative real time-PCR. Protein levels were determined using immunofluorescence staining and Western blot analysis. Gene silencing was performed using small interfering RNA. The degree of cellular senescence was measured by senescence-associated-ß-galactosidase (SA-ß-gal) staining. Oxidative stress was determined by measurement of NADP/NADPH levels and intracellular reactive oxygen species (ROS) levels. Results: Neutralizing anti-TLR4 antibodies or TLR4 inhibitor markedly blocked visfatin-induced hDPCs senescence, as revealed by an increase in the number of SA-ß-gal-positive hDPCs and upregulation of p21 and p53 proteins. Moreover, visfatin-induced senescence was associated with excessive ROS production; NADPH consumption; telomere DNA damage induction; interleukin (IL)-1ß, IL-6, IL-8, cyclooxygenase-2, and tumor necrosis factor-α upregulation; and nuclear factor-κB and mitogen-activated protein kinase activation. All of these alterations were attenuated by TLR4 blockade. Conclusion: Our findings indicate that TLR4 plays an important role in visfatin-induced senescence of hDPCs and suggest that the visfatin/TLR4 signaling axis can be a novel therapeutic target for the treatment of inflammaging-related diseases, including pulpitis.

FK866 Protects Human Dental Pulp Cells against Oxidative Stress-Induced Cellular Senescence.

FK866 possesses various functional properties, such as anti-angiogenic, anti-cancer, and anti-inflammatory activities. We previously demonstrated that premature senescence of human dental pulp cells (hDPCs) was induced by hydrogen peroxide (H2O2). The present study aimed to investigate whether H2O2-induced premature senescence of hDPCs is affected by treatment with FK866. We found that FK866 markedly inhibited the senescent characteristics of hDPCs after exposure to H2O2, as revealed by an increase in the number of senescence-associated ß-galactosidase (SA-ß-gal)-positive hDPCs and the upregulation of the p21 and p53 proteins, which acts as molecular indicators of cellular senescence. Moreover, the stimulatory effects of H2O2 on cellular senescence are associated with oxidative stress induction, such as excessive ROS production and NADPH consumption, telomere DNA damage induction, and upregulation of senescence-associated secretory phenotype factors (IL-1ß, IL-6, IL-8, COX-2, and TNF-α) as well as NF-κB activation, which were all blocked by FK866. Thus, FK866 might antagonize H2O2-induced premature senescence of hDPCs, acting as a potential therapeutic antioxidant by attenuating oxidative stress-induced pathologies in dental pulp, including inflammation and cellular senescence.

Visfatin Induces Senescence of Human Dental Pulp Cells.

Dental pulp plays an important role in the health of teeth. The aging of teeth is strongly related to the senescence of dental pulp cells. A novel adipokine, visfatin, is closely associated with cellular senescence. However, little is known about the effect of visfatin on the senescence of human dental pulp cells (hDPCs). Here, it was found that in vivo visfatin levels in human dental pulp tissues increase with age and are upregulated in vitro in hDPCs during premature senescence activated by H2O2, suggesting a correlation between visfatin and senescence. In addition, visfatin knockdown by small interfering RNA led to the reduction in hDPC senescence; however, treatment with exogenous visfatin protein induced the senescence of hDPCs along with increased NADPH consumption, which was reversed by FK866, a chemical inhibitor of visfatin. Furthermore, visfatin-induced senescence was associated with both the induction of telomere damage and the upregulation of senescence-associated secretory phenotype (SASP) factors as well as NF-κB activation, which were all inhibited by FK866. Taken together, these results demonstrate, for the first time, that visfatin plays a pivotal role in hDPC senescence in association with telomere dysfunction and the induction of SASP factors.

Regulation of cancer cell death by a novel compound, C604, in a c-Myc-overexpressing cellular environment.

The proto-oncogene c-Myc has been implicated in a variety of cellular processes, such as proliferation, differentiation and apoptosis. Several c-Myc targets have been studied; however, selective regulation of c-Myc is not easy in cancer cells. Herein, we attempt to identify chemical compounds that induce cell death in c-Myc-overexpressing cells (STF-cMyc and STF-Control) by conducting MTS assays on approximately 4000 chemical compounds. One compound, C604, induced cell death in STF-cMyc cells but not STF-Control cells. Apoptotic proteins, including caspase-3 and poly(ADP-ribose) polymerase (PARP), were cleaved in C604-treated STF-cMyc cells. In addition, SW620, HCT116 and NCI-H23 cells, which exhibit higher basal levels of c-Myc, underwent apoptotic cell death in response to C604, suggesting a role for C604 as an inducer of apoptosis in cancer cells with c-Myc amplification. C604 induced cell cycle arrest at the G2/M phase in cells, which was not affected by apoptotic inhibitors. Interestingly, C604 induced accumulation of c-Myc and Cdc25A proteins. In summary, a chemical compound was identified that may induce cell death in cancer cells with c-Myc amplification specifically through an apoptotic pathway.

Inhibition of human neutrophil activity by an RNA aptamer bound to interleukin-8.

Interleukin-8 (IL-8) is a proinflammatory CXC chemokine that has been associated with the promotion of neutrophil chemotaxis, degranulation, and the pathogenesis of several neutrophil-infiltrating chronic inflammatory diseases. In the current study, we generated and characterized a 2'-fluoro-pyrimidine modified RNA aptamer (8A-35) against human IL-8. The 8A-35 aptamer binds to IL-8 with high specificity and affinity, yielding an estimated K(D) of 1.72 pM. NMR data revealed that the residues of Lys8, Leu10, Val63, Val66, Lys69 and Ala74 of IL-8 interact with aptamer. Moreover, the 8A-35 aptamer has a potent IL-8-neutralizing activity that can modulate multiple biological activities of IL-8 in human neutrophils, including migration, intracellular signaling, and intracellular Ca(2+) mobilization. Our results suggest that the 8A-35 aptamer has great potential to be a lead structure in the development of effective therapeutic agents against inflammatory diseases.