Dual function of peroxiredoxin I in lipopolysaccharide-induced osteoblast apoptosis via reactive oxygen species and the apoptosis signal-regulating kinase 1 signaling pathway

Lipopolysaccharide (LPS)-induced osteoblast apoptosis is a prominent factor to the defect in periodontal tissue repair in periodontal disease. LPS challenge contributes to the production of

reactive oxygen species (ROS) in periodontitis, and peroxiredoxin 1 (Prx1) is an antioxidant protein that protect cells against oxidative damage from ROS. Without LPS stimulation, apoptotic

rates were higher in both Prx1 knockout (Prx1KO) and Prx1 overexpression (Prx1OE) cells compared with wild type. After LPS stimulation, intracellular ROS in Prx1KO cells showed the highest

level and Prx1OE cells showed the least. Treatment with LPS significantly elevated the expression of Bax, Cyto-c, and caspase 3 in Prx1KO cells compared with wild type, although this could

be completely abolished by NAC. In Prx1OE cells, the expression and activation of ASK1 were significantly increased, and this was slightly reduced by LPS stimulation. NQDI-1 completely

abolished the increased phosphorylation of JNK and p38 and the expression of caspase 3 in LPS-stimulated cells. These results indicate that Prx1 eliminates intracellular ROS and exhibits a

cytoprotective role in LPS-induced apoptosis. However, under physiological conditions, Prx1 overexpression acts as a H2O2 messenger, triggering the expression of ASK1 and its downstream

cascades.

Bone remodeling involves the restructuring of existing bone, which undergoes constant resorption and formation. Excessive bone resorption is the cause of bone loss observed in periodontitis,

which is a chronic microbial infectious disease resulting in destruction of the periodontium. It is characterized by tissue inflammation, alveolar bone resorption, and eventually tooth

loss1,2. The increased apoptosis of osteoblasts and osteocytes is one of the mechanisms that underlie the reduced bone formation and fragility that characterize periodontitis-induced bone

destruction3. Thus, understanding the mechanisms of apoptosis in osteoblasts is imperative to developing therapeutic strategies.

Lipopolysaccharides (LPS), an important component of the outer membrane of Gram-negative bacteria, are involved in the pathogenesis of periodontal diseases and play an important role in

alveolar bone resorption4,5,6. Accumulated data have shown that LPS directly induce apoptosis in many cell types, including macrophages, vascular endothelial cells, and hepatocytes7,8,9.

LPS-induced apoptosis in osteoblasts and periodontal ligament fibroblasts is seen as an important contributing factor leading to defects in periodontal tissue repair in periodontal and

periapical diseases10, but the mechanism of LPS-induced apoptosis in these cells remains unclear.

Following LPS recognition, Toll-like receptor undergoes oligomerization and recruits its downstream adapters to activate other molecules within the cell, leading to induction of the

inflammatory response, and the subsequent production of reactive oxygen species (ROS)11. ROS acts as a second messenger in signal transduction and gene regulation in a variety of cell types

under several biological conditions such as cell growth, differentiation, progression, and death12,13. It can directly trigger apoptosis by causing excessive protein, lipid, and nucleic acid

oxidation, but also has the potential to regulate pathways involving Bcl-2 family members and caspases14. Oxidation of the anionic phospholipid cardiolipin in the inner mitochondrial

membrane is also proposed to play an important role in mitochondrial disruption and cytochrome c (Cyto-c) expression, which occurs during apoptosis15. Recent experimental evidence has shown

that LPS-induced proinflammation and cell death are highly dependent on ROS and related signaling pathways16,17,18. Thus, ROS has a critical role in LPS-induced osteoblast apoptosis.

Peroxiredoxins (Prxs) belong to an important superfamily of small non-seleno peroxidases that scavenge hydrogen peroxide (H2O2) and organic hydroperoxide, and are essential for maintaining

intracellular ROS homeostasis19. On the basis of the numbers of conserved cysteine (Cys) residues participating in the redox reaction, Prxs are divided into typical 2-Cys Prxs (including

Prx1−6), atypical 2-Cys Prx (Prx5), and 1-Cys (Prx6)20. Prx1 is one of the typical 2-Cys Prxs, and is involved in multiple physiological and pathological processes, including proliferation,

apoptosis, inflammation, and cancer21,22,23. In particular, it has been shown that Prx1 suppresses oxidative stress-induced cell apoptosis through direct or indirect interactions with a

variety of apoptosis-regulating kinases and enzymes such as apoptosis signal-regulating kinase 1 (ASK1) and p38 in a cell type- and stimulus-dependent manner24. Moreover, Prx1 overexpression

was shown to inhibit the activation of ASK1, resulting in the inhibition of downstream signaling cascades such as c-Jun N-terminal kinase (JNK) and the p38 pathway25. In Prx1 knockdown

cells, the sensitivity of ROS was strongly increased, and ASK1, P38, and JNK were rapidly activated, leading to apoptosis in response to H2O226,27. Thus, Prx1 functions as a protective

factor in cell death, but its role in LPS-induced osteoblast apoptosis remains unclear.

Because Prxs have recently been identified as essential negative regulators of LPS-induced inflammatory and apoptosis28,29, we investigated whether Prx1 regulates LPS-induced osteoblast

apoptosis. In this study, we demonstrated that Prx1 acts as either an anti-apoptotic or a pro-apoptotic factor depending on its intracellular level, through modulating ROS or ASK1 signaling,

respectively.

To determine the effect of LPS on cell viability in osteoblasts, MC3T3-E1 cells were treated with different concentrations of LPS for 24 h or with 100 ng/ml LPS for various times. CCK8

assays showed that cell viability in osteoblasts exposed to 100, 200, or 1000 ng/ml LPS was significantly reduced compared with non-treated cultures in a dose-dependent manner (Fig. 1a).

After treatment with 100 ng/ml LPS, a significant reduction in cell viability was observed at 12 h, 24 h, or 48 h compared with the control group (Fig. 1b). Flow cytometry analysis of the

apoptotic rate of osteoblasts following treatment with different concentrations of LPS for 24 h showed dose-dependent increases in apoptosis compared with the control group (Fig. 1c, d).

a MC3T3-E1 cells were treated with LPS at different concentrations for 24 h. Cell viability was evaluated using CCK8 assays. b MC3T3-E1 cells were treated with 100 ng/ml LPS for the

indicated time. c, d MC3T3-E1 cells were treated with LPS at different concentrations for 24 h and then stained with PI and Annexin V, and the percentage of apoptotic cells was analyzed by

flow cytometry assays. *P