Increased Expression of Glutaminase in Osteoblasts Promotes Macrophage Recruitment in Periapical Lesions.

INTRODUCTION: Recently, we have shown that tissue hypoxia stimulates the progression of periapical lesions by up-regulating glycolysis-dependent apoptosis of osteoblasts. Other facets of hypoxia-induced metabolic reprogramming in disease pathogenesis require further investigation. In this study, we examined the connection between hypoxia-augmented glutamine catabolism in osteoblasts and the development of periapical lesions. METHODS: Primary human osteoblasts were cultured under hypoxia. The expression of glutaminase 1 (GLS1) was examined using Western blot analysis. The production of glutamate was measured by colorimetric assay. Knockdown of GLS1 was performed with small interfering RNA technology. C-C motif chemokine ligand 2 (CCL2) secretion and chemotaxis of J774 macrophages were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced periapical lesions, the relations between disease progression and osteoblastic expression of GLS1 or macrophage recruitment were studied. RESULTS: Hypoxia enhanced GLS1 expression and subsequent glutamate production in osteoblasts. Glutamate induced chemoattraction of macrophages by osteoblasts through up-regulation of CCL2 synthesis. Hypoxia promoted CCL2 secretion and macrophage recruitment through augmentation of glutaminolysis. Knockdown of GLS1 abolished hypoxia-induced effects. In rat periapical lesions, progressive bone resorption was significantly related to elevated GLS1 expression in osteoblasts and increased macrophage recruitment. CONCLUSIONS: In addition to the rise in glycolytic activity, the progression of periapical lesions is also associated with enhanced glutamine catabolism in osteoblasts. GLS1 may be a potential therapeutic target in the management of periapical lesions.

Sirtuin 6 Modulates Hypoxia-induced Apoptosis in Osteoblasts via Inhibition of Glycolysis: Implication for Pathogenesis of Periapical Lesions.

INTRODUCTION: Osteoblast apoptosis is important in the regulation of inflammatory bone resorption. Hypoxia resulting from inflammation enhances glycolysis and apoptosis. Sirtuin 6 (SIRT6) is a modulator of glucose metabolism and apoptosis. In the study we assessed the role of SIRT6 in hypoxia-induced glycolysis and apoptosis in osteoblasts, with special attention on the significance of these cellular processes in periapical lesions. METHODS: Human bone marrow-derived osteoblasts were cultured under hypoxia. Expression of lactate dehydrogenase A was examined by Western blot, and production of lactate was measured by colorimetric assay. Cleavage of poly (adenosine diphosphate ribose) polymerase was used as an apoptosis marker and assessed by Western blot. SIRT6 was overexpressed in osteoblasts by lentiviral gene transduction, and then glycolytic and apoptotic responses were studied. In a rat model of bacteria-induced periapical lesions, expressions of SIRT6 and markers of glycolysis and apoptosis in osteoblasts were examined. RESULTS: Hypoxia enhanced lactate dehydrogenase A expression and lactate production in osteoblasts. Poly (adenosine diphosphate ribose) polymerase cleavage was induced by hypoxia or lactate treatment. SIRT6 suppressed hypoxia-augmented glycolysis and inhibited apoptosis induced by hypoxia or lactate treatment. Expression of SIRT6 in osteoblasts was downregulated by hypoxia and inflammatory mediators. Development of periapical lesions in rats was associated with decreased expression of SIRT6 and increased glycolysis and apoptosis in osteoblasts. CONCLUSIONS: Our study suggested that hypoxia-induced apoptosis of osteoblasts is dependent on glycolytic activity. SIRT6 is a negative regulator of inflammation and may alleviate periapical lesions by suppressing osteoblastic glycolysis and apoptosis.

Simvastatin suppresses osteoblastic expression of Cyr61 and progression of apical periodontitis through enhancement of the transcription factor Forkhead/winged helix box protein O3a.

INTRODUCTION: In this study, the role of transcription factor Forkhead/winged helix box protein O3a (FoxO3a) in Cyr61 expression and its modulation by simvastatin were investigated in cultured murine osteoblasts and a rat model of induced apical periodontitis. We also examined the effects of simvastatin on the synthesis of chemokine CCL2 and chemotaxis of macrophages in vitro. METHODS: We assessed tumor necrosis factor (TNF)-α-stimulated expression of Cyr61 and phosphorylated inactive FoxO3a (p-FoxO3a) in MC3T3-E1 murine osteoblasts by Western analysis. Forced expression of FoxO3a by lentiviral-based gene transduction was performed, and its effect on Cyr61 expression was evaluated. The modulation of CCL2 secretion and macrophage chemotaxis by simvastatin were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Cyr61, p-FoxO3a, and CCL2 and macrophage recruitment were studied by radiographic and immunohistochemistry analyses. RESULTS: Western blot analysis showed enhanced expression of Cyr61 and p-FoxO3a after TNF-α treatment in a time-dependent manner. Simvastatin significantly counteracted the actions of TNF-α. Forced expression of FoxO3a reduced TNF-α-stimulated Cyr61 synthesis. Simvastatin and FoxO3a diminished TNF-α-induced CCL2 secretion and macrophage recruitment, whereas Cyr61 partially restored the stimulating action. In rat periapical lesions, simvastatin significantly attenuated bone resorption, reduced osteoblastic expressions of Cyr61, p-FoxO3a, and CCL2, and suppressed macrophage recruitment. CONCLUSIONS: Simvastatin may alleviate periapical lesions by enhancing FoxO3a activity to suppress the synthesis of Cyr61 in osteoblasts. Moreover, the downstream effector mechanism of Cyr61 may involve CCL2 production and macrophage recruitment.

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.

An extract of green tea, epigallocatechin-3-gallate, reduces periapical lesions by inhibiting cysteine-rich 61 expression in osteoblasts.

Recent investigations indicate that epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has anti-inflammatory properties. This study assessed the effect of EGCG on oncostatin M (OSM)-induced synthesis of cysteine-rich 61 (Cyr61), a potential osteolytic mediator, in MG-63 human osteoblastic cells. The therapeutic effect of EGCG in apical periodontitis in rats was also examined. Western blot analysis showed that OSM stimulated Cyr61 synthesis in MG-63 in a time-dependent manner, whereas EGCG readily attenuated this effect. On the other hand, Cyr61 treatment of MG-63 cells induced the release of CCL2, a chemokine responsible for macrophage chemotaxis. In a rat model of induced apical periodontitis, radiography and histopathology revealed that administration of EGCG markedly diminished the severity of periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and infiltrating macrophages were also decreased. Thus, EGCG suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.