Chi-Yuan Hong

The role of adsorbed endotoxin in particle-induced stimulation of cytokine release.

Numerous in vitro models have demonstraed the capacity of wear particles to stimulate the release of soluble pro‐inflammatory products with the ability to induce local bone resorption. Recent observations have demonstrated that binding of lipopolysaccharide (LPS) to particulate wear deris can significantly modulate the pattern of cell response in the in vitro models. These findings raise concerns over the possible role of LPS in the athogenesis of aseptic looseing after total joint replacements, and also indicates the importance of controlling for possible confounding effects of LPS contamination in the in vitro models used to study the reactive nature of wear debris. Our studies were undertaken to rigorously analyze the effects of particle‐associated LPS on cell responses and to assess the efficacy ofdifferent treatment protocols to inactivate LPS associated with different particulate materials. Particles of cobalt‐chrome alloy, titanium‐6‐aluminum‐4‐vanadium, titanium nitride and silica were pretreated with LPS and exposed to multiple treatment protocols. When cells were treated with „as‐received”︁ particles prepared by washing in ethanol, small amounts of TNF‐α, IL‐1β, and IL‐1α were detected. In contrast, all particle species pretreated with LPS produced marked increases in TNF‐α, IL‐1α, and IL‐1β release, as well as upregulation of corresponding mRNa levels even after ethanol washing. Boiling the LPS‐pretreated particles in 1% acetic acid or autoclaving and baking the particles also markedly reduced and in some instances abolished the effect of the LPS‐pretreatment. This indicates that LPS binds to the surface of particles of diverse composition and that the bound LPS is biologically active. Treatment protocols to inactivate particle‐associated LPS demonstrated significant differences in effcacy. When the most rigorous treatments were utilized, essentially all LPS activity could be eliminated. Particles treated with these methods retained some capacity to stimulated cytokine release, but activities were markedly reduced. These results provide further evidnce indicating that LPS contamination of particulate materials can markedly enhance their biological activity. This potential confounding effect needs to be carefully monitored and controlled in the in vitro model systems systems used to evaluate wear particles. Furthermore, the presence of particle‐associated endotoxin at the bone‐implant interface in vivo could markedly enhance the adverse biological activity of particulate wear debris.

Induction of Dental Pulp Fibroblast Matrix Metalloproteinase–1 and Tissue Inhibitor of Metalloproteinase–1 Gene Expression by Interleukin–1α and Tumor Necrosis Factor–α Through a Prostaglandin–Dependent Pathway

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the degradation of extracellular matrix in many inflammatory diseases. Little is known regarding the expression of these mediators in dental pulp fibroblasts. The effects of proinflammatory cytokines (interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha)) and prostaglandin E2 (PGE2) on pulp fibroblast MMP-1 and TIMP-1 gene expression were investigated. Northern hybridization showed that IL-1alpha and TNF-alpha induced significant MMP-1 gene expression, with only little effect on TIMP-1 gene. Exogenous PGE2, however, upregulated TIMP-1 mRNA synthesis but not MMP-1. Concomitant addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 suppressed MMP-1 mRNA production, compared with the groups treated with IL-1alpha or TNF-alpha alone. In contrast, PGE2 enhanced the upregulatory effects of TIMP-1 mRNA by IL-1alpha or TNF-alpha. Furthermore, cytokine stimulation of MMP-1 and TIMP-1 gene expressions can be enhanced or blocked by indomethacin, respectively, and reversed by exogenous PGE2. These results suggested that cytokine-stimulated MMP-1 and TIMP-1 gene expression in dental pulp fibroblasts was mediated, at least in part, by a prostaglandin-dependent pathway. The differential regulation of IL-1alpha or TNF-alpha-induced MMP-1 and TIMP-1 mRNA synthesis, as well as the direct upregulation of TIMP-1 gene expression by PGE2, also implied that prostaglandin may serve as a protective mechanism from excessive tissue breakdown during pulpitis.

Epigallocatechin‐3‐gallate diminishes CCL2 expression in human osteoblastic cells via up‐regulation of phosphatidylinositol 3‐Kinase/Akt/Raf‐1 interaction: A potential therapeutic benefit for arthritis

OBJECTIVE To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)-induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied. METHODS CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically. RESULTS EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1)-CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts. CONCLUSION By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis.

Nitric oxide promotes infectious bone resorption by enhancing cytokine-stimulated interstitial collagenase synthesis in osteoblasts

This experiment was undertaken to determine the role of macrophage‐derived nitric oxide (NO) in mediating lipopolysaccharide (LPS)‐induced bone resorption by using an in vitro co‐culture system and an in vivo model of infectious bone resorption. Our results demonstrated that LPS stimulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)‐α mRNAs and nitrite synthesis in the J774 mouse macrophage cell line but not in the UMR‐106 (rat) and MC3T3‐E1 (mouse) osteoblast cell lines. Conditioned media (CM) from LPS‐stimulated J774 triggered only low to moderate levels of iNOS mRNAs in MC3T3‐E1 and a trivial effect in UMR‐106. On the other hand, CM induced matrix metalloproteinase‐1 (MMP‐1) gene expression in both osteoblast cell lines. The NOS inhibitor NG‐monomethyl‐L‐arginine (L‐NMMA) did not alter this effect in MC3T3‐E1 and UMR‐106, whereas TNF‐α antibody diminished the CM‐induced MMP‐1 gene expression in both cell lines. Interestingly, SNAP, a NO donor, although by itself is not a MMP‐1 stimulator for UMR‐106, augmented the TNF‐α‐stimulated MMP‐1 mRNA production in UMR‐106. In a J774/UMR‐106 co‐culture system, LPS stimulated significant MMP‐1 gene expression in UMR‐106, and this upregulation was abolished by L‐NMMA and TNF‐α antibodies. Immunohistochemical analysis in a rat model of infectious bone resorption (periapical lesion) showed co‐distributions of iNOS+ macrophages and MMP‐1+ osteoblasts around the osteolytic areas. Administration of L‐NMMA markedly reduced the extent of bone loss and the percentage of MMP‐1‐synthesizing osteoblasts. These data suggest that NO derived from macrophages after LPS stimulation may enhance bone loss by augmenting the cytokine‐induced MMP‐1 production in osteoblasts.

Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes

Norcantharidin (NCTD) is the demethylated analogue of cantharidin. In this study, multi-parameter assessments of morphological alterations, clonogenic efficiency, cell growth curves, DNA synthesis, and DNA strand break were employed to determine and compare the cytotoxic effects of NCTD on oral cancer KB cell line and normal buccal keratinocytes. The results showed NCTD induced significant cytotoxicity in KB cells after 24 h of exposure. Normal buccal keratinocytes were more resistant to NCTD induced cytotoxicity. The IC(50) of 24 h NCTD treatment for KB and keratinocytes were 15.06 and 216.29 microg/ml, respectively with a keratinocyte/KB selective index of 14.36. Anoikis and membrane blebbing, morphological characterization of apoptosis, were observed in about 90% of KB cells after exposure to 100 microg/ml of NCTD for 24 h compared to about 30% in keratinocytes. In addition, inhibition of colony formation was noted in KB cells even when exposed to low concentration of drug (5 microg/ml) for a short period of time (6 h). NCTD inhibited subsequent cell proliferation in KB but growth of normal keratinocytes was retarded only temporarily. NCTD inhibited DNA synthesis in both KB and normal keratinocytes. However, keratinocytes were more sensitive to DNA synthesis inhibition by low dose of NCTD. Significant DNA strand break was noted in KB cells only after cell viability was reduced to less than 60% of the control. In comparison, normal keratinocytes were resistant to NCTD induced DNA strand break. These results indicated KB cells were more sensitive to NCTD induced cytotoxicity compared to normal keratinocytes. NCTD may be of value in treating oral cancers. The underlying mechanisms of the differential actions of NCTD on these two cell types are worthy of further investigations.

Immunolocalization of Macrophages and Transforming Growth Factor-β1 in Induced Rat Periapical Lesions

Apical periodontitis was induced in Wistar rats by exposing the pulp chamber of right mandibular first molars to the oral environment. Animals were killed 0, 5, 10, 15, 20, 30, 60, and 80 days after lesion induction. Microradiographic and automated image analysis showed that the lesions expanded significantly in a time-dependent manner from day 0 to day 20 (0.039 mm2/day, p < 0.05, active phase) and stabilized thereafter (chronic phase). A linear regression test revealed a positive correlation between the numbers of ED-1 positive macrophage per microscopic high power field and the periapical lesion size during the active phase (r = 0.98, p < 0.01). Immunohistochemical studies showed that transforming growth factor-beta 1 positive macrophages distributed around the root apex and areas showing bone resorption during active lesion phase, whereas TGF-beta 1-positive osteoblasts were detected during the chronic stage (days 30, 60, and 80 after pulp exposure). Histologically TGF-beta 1 positive osteoblasts possessed a large, round nucleus as well as an abundant cytoplasm and located in close vicinity to areas exhibiting reparative bone formation. These results suggest that macrophages may play important role(s) in the initiation and development of periapical lesions and TGF-beta 1 may play dual roles in both bone resorption and deposition in induced rat periapical lesions.

Simvastatin alleviates the progression of periapical lesions by modulating autophagy and apoptosis in osteoblasts.

INTRODUCTION Autophagy is a process for recycling intracellular organelles as a survival mechanism. Apoptosis has important biological roles in the pathogenesis of many diseases. This study elucidated the effect of simvastatin on autophagy/apoptosis in MC3T3E1 murine osteoblastic cells and also the significance of this action on the progression of induced rat apical periodontitis. METHODS We examined the H2O2-stimulated expression of LC3-II (an autophagy marker) and poly (adenosine phosphate ribose) polymerase (PARP) fragmentation (an apoptosis marker) in MC3T3E1 by Western analysis. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Beclin-1 (an autophagy marker) and terminal deoxyuridine triphosphate nick end-labeling (an apoptosis marker) was studied by radiographic and immunohistochemistry analyses. RESULTS Western blot showed elevated levels of LC3-II and PARP cleavage after H2O2 treatment. An autophagy inhibitor 3-methyladenine promoted whereas rapamycin (an autophagy enhancer) diminished H2O2-induced PARP cleavage. Simvastatin enhanced H2O2-induced LC3-II formation and simultaneously decreased PARP fragmentation. Radiography and immunohistopathology demonstrated that simvastatin reduced the number of apoptotic osteoblasts and the extension of periapical lesions in rats. The number of Beclin-1-synthesizing osteoblasts also increased markedly after simvastatin treatment. CONCLUSIONS We found a negative relation between autophagy and apoptosis in osteoblastic cells. In addition, simvastatin suppressed apoptosis and enhanced autophagy both in vitro and in vivo. Our data implied that simvastain might alleviate the progression of apical periodontitis by promoting autophagy to protect osteoblasts from turning apoptotic.

Simvastatin as a Novel Strategy To Alleviate Periapical Lesions

Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used cholesterol-lowering agents that also possess anti-inflammatory activities. Cysteine-rich 61 (Cyr61) and CCL2 are potential osteolytic mediators in inflammatory bone diseases. The study assessed the effect of simvastatin on tumor necrosis factor alpha (TNF- alpha)-induced synthesis of Cyr61 and CCL2 in MG-63 human osteoblastic cells. The therapeutic effect of simvastatin on rat apical periodontitis was also examined. The synthesis of Cyr61 in MG-63 was assessed by Western analysis. Expression of CCL2 was examined by an enzyme-linked immunosorbent assay. The effect of simvastatin on induced rat periapical lesion was examined radiographically and immunohistochemically. Western blot showed that TNF-alpha stimulated Cyr61 synthesis in MG-63, whereas simvastatin attenuated this effect in a dose-dependent manner. Simvastatin also reduced the levels of TNF-alpha-induced CCL2, and exogenous Cyr61 restored the inhibitory effects. Radiography and histopathology revealed that the administration of simvastatin markedly diminished the severity of induced rat periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and CD-68-positive macrophages were also decreased. Simvastatin suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.

Oncostatin M–induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: An implication for the pathogenesis of arthritis

OBJECTIVE To examine the roles of STATs 1 and 3 in CCL2 production in human osteoblastic cells and their influences on arthritis development. METHODS The expression of CCL2 in primary human osteoblasts and U2OS human osteoblastic cells was examined by Northern blotting and enzyme-linked immunosorbent assay. The roles of STAT-1/3 and c-Fos were assessed using short hairpin RNAs (shRNA) to silence their functions. Serine phosphorylation of STATs was assessed by Western blotting. Promoter activities of c-Fos and CCL2 were assessed by chloramphenicol acetyltransferase and luciferase assays, respectively. Interactions of STAT-1, STAT-3, and c-Fos with DNA were evaluated by electrophoretic mobility shift assay (EMSA) and immunoprecipitation. The effect of the JAK inhibitor AG-490 on collagen-induced arthritis (CIA) in rats was examined using immunohistochemistry. RESULTS Oncostatin M (OSM) stimulated CCL2 expression in primary human osteoblasts and U2OS cells. In U2OS cells, STAT-1 and STAT-3 were involved in OSM-stimulated CCL2 expression, and both the phosphatidylinositol 3-kinase/Akt and MEK/ERK pathways were implicated in the activation of these STATs. STAT-1 and STAT-3 modulated the expression of c-Fos and directly transactivated the CCL2 promoter. Moreover, EMSA showed formation of a DNA-protein complex containing STAT-1, STAT-3, and interestingly, c-Fos. Immunoprecipitation confirmed the binding between c-Fos and STAT-1/3. Reporter assay revealed synergistic attenuation of CCL2 promoter activity by shRNA targeting of STAT-1, STAT-3, and c-Fos. AG-490 suppressed OSM-stimulated activation of STAT-1/3 and synthesis of CCL2 in vitro and diminished the severity of CIA and the number of CCL2-synthesizing osteoblasts in vivo. CONCLUSION These findings show that multiple levels of STAT-1/3 signaling modulate OSM-stimulated CCL2 expression in human osteoblastic cells. Clinically, this pathway may be related to the pathogenesis of arthritis.

Simvastatin inhibits cysteine‐rich protein 61 expression in rheumatoid arthritis synovial fibroblasts through the regulation of sirtuin‐1/FoxO3a signaling

OBJECTIVE To examine the role of sirtuin-1 (SIRT-1)/FoxO3a in the expression of cysteine-rich protein 61 (CYR-61) in rheumatoid arthritis synovial fibroblasts (RASFs) and the influence of simvastatin on this pathway, and to determine the relationship between disease progression and FoxO3a/CYR-61 signaling in synovial fibroblasts in vivo using a rat model of collagen-induced arthritis (CIA). METHODS In RASFs, the expression of CYR-61 and SIRT-1, the localization of FoxO3a in the nucleus/cytoplasm, and the phosphorylation/acetylation of FoxO3a were examined by Western blotting. Secretion of CCL20 was assessed by enzyme-linked immunosorbent assay. Promoter activity of the Cyr61 gene was evaluated by luciferase assay, with or without forced expression of FoxO3a and SIRT-1 by lentiviral transduction. FoxO3a-Cyr61 promoter interaction was examined by chromatin immunoprecipitation. In rats with CIA, the expression of CYR-61 and phosphorylated FoxO3a in synovial fibroblasts was examined by immunohistochemistry. RESULTS In RASFs, simvastatin suppressed the tumor necrosis factor α (TNFα)-induced production of CYR-61 and CCL20. Nuclear levels of FoxO3a were decreased after TNFα stimulation of RASFs, and forced expression of FoxO3a reversed the inductive effects of TNFα on CYR-61. Simvastatin inhibited the nuclear export, phosphorylation, and acetylation of FoxO3a and maintained its binding to the Cyr61 promoter. Forced expression of SIRT-1 in RASFs led to decreased levels of CYR-61 and deacetylation of FoxO3a. Following treatment with simvastatin, the expression of SIRT-1 was up-regulated and SIRT-1/FoxO3a binding was enhanced in RASFs. In rats with CIA, intraarticular injection of simvastatin alleviated arthritis and suppressed CYR-61 expression and FoxO3a phosphorylation in synovial fibroblasts. CONCLUSION CYR-61 is important in the pathogenesis of RA, and SIRT-1/FoxO3a signaling is crucial to induction of CYR-61 in RASFs. Simvastatin plays a beneficial role in inflammatory arthritis through its up-regulation of SIRT-1/FoxO3a signaling in synovial fibroblasts. Continued study of the pathways linking sirtuins, FoxO proteins, and the inflammatory responses of RASFs may provide new insights into the pathophysiology of RA.