Nicotinamide‐induced silencing of SIRT1 by miR‐22‐3p increases periodontal ligament stem cell proliferation and differentiation

Dental stem cell proliferation and osteoblast differentiation are key cellular processes involved in periodontitis diseases. Researchers have found that SIRT1 (sirtuin 1, silent mating type information regulation 2 homolog 1) and microRNAs play a pivotal role in the process, but a clear underlying mechanism has not been determined. In this study, the has‐miR‐22‐3p that target SIRT1 was predicted by TargetScan. Luciferase reporter assay was used to confirm that SIRT1 is the direct target of miR‐22‐3p. Importantly, miR‐22‐3p was revealed to control SIRT1 in periodontal ligament stem cell (PDLSC) and to regulate the proliferation and differentiation of PDLSC by SIRT1 silencing. Furthermore, we detected the induction of miR‐22‐3p expression by nicotinamide treatment on PDLSC. Induction of PDLSC proliferation and differentiation by nicotinamide treatment was blocked by miR‐22‐3p knockdown. These results suggested that the effect of nicotinamide on PDLSC is through miR‐22‐3p. In addition, miR‐22‐3p also upregulated the expression levels of the inflammatory cytokines tumor necrosis factor‐α, interleukin‐1β (IL‐1β), and IL‐8 in PDLSC through SIRT1 pathway and downregulated the expression of TLR‐2 and TLR‐4. miR‐22‐3p is a new target either for the treatment of periodontitis or the improvement of inflammation caused by orthodontics.     

The increased expression of receptor activator of nuclear‐κB ligand (RANKL) of multiple myeloma bone marrow stromal cells is inhibited by the bisphosphonate ibandronate

The receptor activator of nuclear factor‐kappaB ligand (RANKL) and interleukin‐1beta are osteoclast activating factors which are abnormally expressed in bone marrow stromal cells and plasma cells of multiple myeloma patients. In this work we analyzed RANKL expression in human bone marrow mesenchymal stromal cells and the effect of the bisphosphonate ibandronate on RANKL expression after IL‐1beta activation of ERK pathway. Mesenchymal stromal cells were obtained from bone marrow iliac aspirates from multiple myeloma patients at stages II/III and non‐osteoporotics control donors; these cells were maintained under long‐term culture conditions. Cells were cultured in the presence or the absence of 5 ng/ml IL‐1beta and/or 5 µM ibandronate, during selected periods. mRNA for RANKL and protein levels were assayed by RT‐PCR and Western blot, respectively. Human bone marrow stromal cell line HS‐5 was used for assessing IL 1beta‐ and ibandronate‐ERK phosphorylation responses. Multiple myeloma mesenchymal stromal cells differentiate from control cells by increased basal RANKL expression. IL‐1beta up regulated RANKL expression showed dependent on activated MEK/ERK pathway. Finally, the bisphosphonate ibandronate, that hindered activation of the MEK/ERK pathway significantly inhibited both basal and IL‐1beta dependent RANKL expression by cells. Results indicate that RANKL expression involves the MEK/ERK pathway in multiple myeloma mesenchymal stromal cells, and that early obstruction of this path, such as that achieved with ibandronate, significantly deters RANKL protein expression. J. Cell. Biochem. 111: 130–137, 2010. © 2010 Wiley‐Liss, Inc.     

Porphyromonas gingivalis lipopolysaccharide regulates interleukin (IL)-17 and IL-23 expression via SIRT1 modulation in human periodontal ligament cells

Increased interleukin (IL)-17 and IL-23 levels exist in the gingival tissue of periodontitis patients, but the precise molecular mechanisms that regulate IL-17 and IL-23 production remain unknown. The aim of this study was to explore the role of SIRT1 signaling on Porphyromonas gingivalis lipopolysaccharide (LPS)-induced IL-17 and IL-23 production in human periodontal ligament cells (hPDLCs). IL-17 and IL-23 production was significantly increased in LPS-treated cells. LPS treatment also led to the upregulation of SIRT1 mRNA and protein expression. LPS-induced IL-17 and IL-23 upregulation was attenuated by pretreatment with inhibitors of phosphoinositide 3-kinase (PI3K), p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK), and NF-κB, as well as neutralizing antibodies against Toll-like receptors (TLRs) 2 and 4. Sirtinol treatment (a known SIRT1 inhibitor) or SIRT1 knockdown by small interfering RNA blocked LPS-stimulated IL-17 and IL-23 expression. Further investigation showed that LPS decreased osteoblast markers (i.e., ALP, OPN, and BSP) and concomitantly increased osteoclast markers (i.e., RANKL and M-CSF). This response was attenuated by inhibitors of the PI3K, p38, ERK, JNK, NF-κB, and SIRT1 pathways. These findings, for the first time, suggest that human periodontopathogen P. gingivalis LPS is implicated in periodontal disease bone destruction and may mediate IL-17 and IL-23 release from hPDLCs. This process is dependent, at least in part, on SIRT1-Akt/PI3K-MAPK-NF-κB signaling.     

IL‐1α and IL‐1β have different effects on formation and activity of large osteoclasts

Interleukin 1 (IL‐1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL‐1α and IL‐1β, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2–5 nuclei). Moreover, large OCs express higher levels of the IL‐1 activating receptor IL‐1RI, integrins αv and β3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL‐1RII. We hypothesized that IL‐1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL‐1α or IL‐1β (1 and 10 ng/ml). In the presence of sRANKL, both IL‐1α and IL‐1β increased total OC number and resorptive activity of large OCs. IL‐1α stimulated formation of large OCs and increased the number of resorption pits, while IL‐1β changed the morphology of large OCs and integrin‐β3 phosphorylation. No effects were seen in small OCs in response to either IL‐1 isoform. These results demonstrate that IL‐1 predominantly affects large OCs. The dissimilarity of responses to IL‐1α and IL‐1β suggests that these isoforms activate different signaling pathways within the two OC populations. J. Cell. Biochem. 109: 975–982, 2010. © 2010 Wiley‐Liss, Inc.     

Vibration activates the actin/NF‐κB axis and upregulates IL‐6 and IL‐8 expression in human periodontal ligament cells

We previously reported that mechanical vibration‐induced proinflammatory cytokines, interleukin‐6 (IL‐6) and IL‐8, expression in human periodontal ligament (hPDL) cells, however, the underlying mechanism remained unclear. Mechanical stimuli are able to activate cellular responses by inducing the activation of several signaling pathways including cytoskeletal changes and inflammation. The actin cytoskeleton is a highly dynamic network and plays many important roles in intracellular events. Here, we aimed to investigate the involvement of a pivotal mediator of inflammatory responses, nuclear factor‐κB (NF‐κB), and actin polymerization in vibration‐induced upregulation of IL‐6 and IL‐8 expression in hPDL cells. hPDL cells were pretreated with the NF‐κB inhibitor BAY 11‐7082 or cytochalasin D, respectively, before exposure to vibration. IL‐6 and IL‐8 messenger RNA (mRNA) and protein expression were quantified by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assays, respectively. Subcellular localization of the NF‐κB p65 subunit was visualized by immunofluorescent staining. We found an increase in NF‐κB nuclear translocation in vibrated cells compared with control cells. Pretreatment with BAY 11‐7082 significantly inhibited vibration‐induced IL‐6 and IL‐8 mRNA and protein expression in hPDL cells. Moreover, pretreatment with cytochalasin D inhibited NF‐κB nuclear translocation and attenuated upregulation of IL‐6 and IL‐8 mRNA and protein in vibrated cells. Therefore, modulation of actin cytoskeletal polymerization in response to vibration may activate the NF‐κB signaling pathway and subsequently upregulate IL‐6 and IL‐8 expression in hPDL cells.     

Sodium hydrogen sulfide inhibits nicotine and lipopolysaccharide‐induced osteoclastic differentiation and reversed osteoblastic differentiation in human periodontal ligament cells

Although previous studies have demonstrated that hydrogen sulfide (H₂S) stimulated or inhibited osteoclastic differentiation, little is known about the effects of H₂S on the differentiation of osteoblasts and osteoclasts. To determine the possible bioactivities of H₂S on bone metabolism, we investigated the in vitro effects of H₂S on cytotoxicity, osteoblastic, and osteoclastic differentiation as well as the underlying mechanism in lipopolysaccharide (LPS) and nicotine‐stimulated human periodontal ligament cells (hPDLCs). The H₂S donor, NaHS, protected hPDLCs from nicotine and LPS‐induced cytotoxicity and recovered nicotine‐ and LPS‐downregulated osteoblastic differentiation, such as alkaline phosphatase (ALP) activity, mRNA expression of osteoblasts, including ALP, osteopontin (OPN), and osteocalcin (OCN), and mineralized nodule formation. Concomitantly, NaHS inhibited the differentiation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in mouse bone marrow cells and blocked nicotine‐ and LPS‐induced osteoclastogenesis regulatory molecules, such as RANKL, OPG, M‐CSF, MMP‐9, TRAP, and cathepsin K mRNA. NaHS blocked nicotine and LPS‐induced activation of p38, ERK, MKP‐1, PI3K, PKC, and PKC isoenzymes, and NF‐κB. The effects of H₂S on nicotine‐ and LPS‐induced osteoblastic and osteoclastic differentiation were remarkably reversed by MKP‐1 enzyme inhibitor (vanadate) and expression inhibitor (triptolide). Taken together, we report for the first time that H₂S inhibited cytotoxicity and osteoclastic differentiation and recovered osteoblastic differentiation in a nicotine‐ and periodontopathogen‐stimulated hPDLCs model, which has potential therapeutic value for treatment of periodontal and inflammatory bone diseases. J. Cell. Biochem. 114: 1183–1193, 2013. © 2012 Wiley Periodicals, Inc.     

Nicotinamide phosphoribosyltransferase/sirtuin 1 pathway is involved in human immunodeficiency virus type 1 Tat‐mediated long terminal repeat transactivation

Tat is a multifunctional transactivator encoded by human immunodeficiency virus type 1 (HIV‐1). Tat transactivating activity is controlled by nicotinamide adenine nucleotide⁺ (NAD⁺)‐dependent deacetylase sirtuin 1 (SIRT1). Nicotinamide phosphoribosyltransferase (Nampt) is a rate‐limiting enzyme in the conversion of nicotinamide into NAD⁺, which is crucial for SIRT1 activation. Thus, the effect of Nampt on Tat‐regulated SIRT activity was studied in Hela‐CD4‐β‐gal (MAGI) cells. We demonstrated that Tat caused NAD⁺ depletion and inhibited Nampt mRNA and protein expression in MAGI cells. Resveratrol reversed Tat‐induced NAD⁺ depletion and inhibition of Nampt mRNA and protein expression. Further investigation revealed that Tat‐induced inhibition of SIRT1 activity was potentiated in Nampt‐knockdown by Nampt siRNA compared to treatment with Tat alone. Nampt siRNA potentiated Tat‐induced HIV‐1 transactivation in MAGI cells. Altogether, these results indicate that Nampt is critical in the regulation of Tat‐induced inhibition of SIRT1 activity and long terminal repeat (LTR) transactivation. Nampt/SIRT1 pathway could be a novel therapeutic tool for the treatment of HIV‐1 infection. J. Cell. Biochem. 110: 1464–1470, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of human bronchial epithelial cells by inflammatory cytokines IL‐27 and TNF‐α: Implications for immunopathophysiology of airway inflammation

Interleukin (IL)‐27 is a member of IL‐6/IL‐12 family cytokines produced by antigen‐presenting cells in immune responses. IL‐27 can drive the commitment of naive T cells to a T helper type 1 (Th1) phenotype and inhibit inflammation in later phases of infection. Human bronchial epithelial cells have been shown to express IL‐27 receptor complex. In this study, we investigated the in vitro effects of IL‐27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)‐α on the pro‐inflammatory activation of human primary bronchial epithelial cells and the underlying intracellular signaling mechanisms. IL‐27 was found to enhance intercellular adhesion molecule 1 (ICAM‐1) expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL‐27 and TNF‐α on the expression of ICAM‐1. Although IL‐27 did not alter the basal IL‐6 secretion from bronchial epithelial cells, it could significantly augment TNF‐α‐induced IL‐6 release. These synergistic effects on the up‐regulation of ICAM‐1 and IL‐6 were partially due to the elevated expression of TNF‐α receptor (p55TNFR) induced by IL‐27. Further investigations showed that the elevation of ICAM‐1 and IL‐6 in human bronchial epithelial cells stimulated by IL‐27 and TNF‐α was differentially regulated by phosphatidylinositol 3‐OH kinase (PI3K)‐Akt, p38 mitogen‐activated protein kinase, and nuclear factor‐κB pathways. Our results therefore provide a new insight into the molecular mechanisms involved in airway inflammation. J. Cell. Physiol. 223:788–797, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin‐1α and β

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)‐β and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF‐β‐induced expression of CTGF in fibroblasts by an interleukin (IL)‐1 α‐dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL‐1α and β. Human dermal fibroblasts and NIH 3T3 cells were treated with IL‐1α or β in presence or absence of TGF‐β1. IL‐1 suppressed basal and TGF‐β‐induced CTGF mRNA and protein expression. IL‐1α and β inhibited TGF‐β‐stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3‐binding CAGA elements. Furthermore, IL‐1α and β inhibited TGF‐β‐stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF‐β activated kinase1 (TAK1) is necessary for IL‐1 inhibition of TGF‐β‐stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226–1233, 2010. Published 2010 Wiley‐Liss, Inc.     

IL‐17 induces osteoclastogenesis from human monocytes alone in the absence of osteoblasts,which is potently inhibited by anti‐TNF‐α antibody: A novel mechanism of osteoclastogenesis by IL‐17

IL‐17 is a proinflammatory cytokine crucial for osteoclastic bone resorption in the presence of osteoblasts or synoviocytes in rheumatoid arthritis. However, the role of IL‐17 in osteoclastogenesis from human monocytes alone remains unclear. Here, we investigated the role of IL‐17 in osteoclastogenesis from human monocytes alone and the direct effect of infliximab on the osteoclastogenesis induced by IL‐17. Human peripheral blood mononuclear cells (PBMC) were cultured for 3 days with M‐CSF. After non‐adherent cells were removed, IL‐17 was added with either infliximab or osteoprotegerin (OPG). Seven days later, adherent cells were stained for vitronectin receptor. On the other hand, CD11b‐positive monocytes purified from PBMC were also cultured and stained as described above. CD11b‐positive cells were cultured with TNF‐α and receptor activator of NF‐κB ligand (RANKL). In the cultures of both adherent cells and CD11b‐positive cells, IL‐17 dose‐dependently induced osteoclastogenesis in the absence of soluble‐RANKL. OPG or infliximab inhibited IL‐17‐induced osteoclastogenesis. Interestingly, in the culture of CD11b‐positive cells, the osteoclastogenesis was more potently inhibited by infliximab than by OPG. TNF‐α and RANKL synergistically induced osteoclastogenesis. The present study clearly demonstrated the novel mechanism by which IL‐17 directly induces osteoclastogenesis from human monocytes alone. In addition, infliximab potently inhibits the osteoclastogenesis directly induced by IL‐17. J. Cell. Biochem. 108: 947–955, 2009. © 2009 Wiley‐Liss, Inc.     

Dexmedetomidine protects against high mobility group box 1‐induced cellular injury by inhibiting pyroptosis

Dexmedetomidine (DEX) is a widely used clinical anesthetic with proven anti‐inflammatory effects. Both high mobility group box 1 (HMGB1) and pyroptosis play an important role in the inflammatory response to infection and trauma. Thus far, there have been no studies published addressing the effect of DEX on HMGB1 and pyroptosis. In order to fill this gap in the literature, bone marrow‐derived macrophages (BMDMs) were exposed to HMGB1 (4 µg/mL) with or without DEX (50 μM) pretreatment. The production of pro‐inflammatory cytokines [such as tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), and IL‐18], phosphorylation of extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) and P38, and the activation of caspase‐1 were measured by enzyme immunosorbent assay, western blot analysis, confocal microscope, and flow cytometry, respectively. We found that DEX protected against HMGB1‐induced cell death of BMDMs. In addition, DEX suppressed the generation of TNF‐α, IL‐1β, and IL‐18 as well as the phosphorylation of ERK1/2 and P38. Moreover, DEX inhibited caspase‐1 activation and decreased pyroptosis. Taken together, these findings demonstrate the protective effect of DEX in mediating HMGB1‐induced cellular injury, thus indicating that DEX may be a potential therapeutic candidate for the management of infection and trauma‐derived inflammation.     

Effect of heme oxygenase‐1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase‐1 (HO‐1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO‐1 recombinant adenovirus (HO‐MSCs) for stable expression of HO‐1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor‐α (TNF‐α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad‐MSCs, Ad‐HO + MSCs or HO‐MSCs. mRNA and protein expression of Zona occludens‐1 (ZO‐1) and human HO‐1 and the release of cytokines were measured. ZO‐1 and human HO‐1 in Caco2 were significantly decreased after treatment with TNF‐α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO‐1 was not significantly affected by Caco2 treatment with TNF‐α, Ad‐HO, and MSCs. In contrast, ZO‐1 and human HO‐1 increased significantly when the damaged Caco2 was treated with HO‐MSCs. HO‐MSCs showed the strongest effect on the expression of ZO‐1 in colon epithelial cells. Coculture with HO‐MSCs showed the most significant effects on reducing the expression of IL‐2, IL‐6, IFN‐γ and increasing the expression of IL‐10. HO‐MSCs protected the intestinal epithelial barrier, in which endogenous HO‐1 was involved. HO‐MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti‐inflammatory factors. These results suggested that HO‐MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO‐1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.