The Effects Of Oxidized Phospholipids On Osteoblast Differentiation And The Underlying Mechanism

Background and ObjectivesPeriodontitis is one of the most common diseases of oral cavity and the major reason of tooth loss. Periodontitis is characterized by the destruction of periodontal tissues including alveolar bone, cementum and periodontal ligament. Similar to other bones, alveolar bone is remodeling through the lifetime, depending on the balance of bone formation by osteoblasts and bone resorption by osteoclasts. During the pathogenesis of periodontitis, the bone resorption by osteoclasts is promoted and bone formation by osteoblasts is repressed. Plenty of studies have identified that periodontitis is tightly associated with cardiovascular diseases (CVD). CVD are a category of chronic diseases in the cardiovascular system and are the leading cause of death worldwide. Bone mineral density (BMD) loss and fracture risk are increased in patients with CVD. Defined as elevated lipids in the blood, hyperlipidemia is one of the best-established risk factors of atherosclerotic CVD. Patients with atherogenic lipid profiles are reported to have decreased bone mineral density and an increased risk of osteopenia than those with normal lipid profiles. Hyperlipidemia induced by atherosclerotic high-fat diet in low density lipoprotein receptor knockout (Ldlr-/-) mice significantly impaires cranial bone regeneration and mechanical strength of femoral bone. Additionally, the anabolic effect of parathyroid hormone (PTH) on bone formation is also blunted in hyperlipidemic mice.Lipids are transported via blood circulation by lipoproteins, the polymolecular assemblies comprising cholesterol, triglycerides, phospholipids and apolipoproteins which facilitate the systemic distribution of lipids. Low density lipoprotein (LDL) functions to transport cholesterol and cholesteryl ester to peripheral tissues. High density lipoprotein (HDL) mediates the reverse transport of cholesterol from peripheral tissues back to the liver, facilitating its elimination from gall bladder. While raised level of LDL cholesterol (LDL-c) increases the risk of CVD, a high level of HDL cholesterol (HDL-c) possesses cardiovascular protective effects. The increased LDL-c and decreased HDL-c are acknowledged as atherogenic lipid profiles.Oxidative modification of LDL is believed to carry out principally in the arterial wall, and a variety of biologically active lipid derivatives generate in this process, including lipid hydroperoxides and aldehydes. Produced in the early stage of atherosclerosis, oxidized-LDL (ox-LDL) plays pro-atherosclerotic roles in almost all of the steps of atherosclerosis, including endothelial cell dysfunction, foam cell formation as well as fibrous cap disruption. Among the various ox-LDLs, minimally modified LDL (MM-LDL), which is not completely oxidized, possesses high pro-inflammatory and pro-adhesive capacities in the development of atherosclerosis. Compared to heavily oxidized LDL, MM-LDL has a higher content of oxidized phospholipids. The oxidized product of phospholipids, oxidized 1-palmitoyl2-arachidonoyl-sn-glycero-3-phosphatidylcholine (ox-PAPC), is considered one of the major bioactive components of MM-LDL. Similarly to MM-LDL, ox-PAPC is reported to promote adipogenic differentiation and inhibit osteogenic differentiation of mesenchymal stem cells. And in osteoblasts, ox-PAPC also shows inhibitory effects on alkaline phosphatase (ALP) activity and calcium uptake. But the effects of ox-PAPC on osteogenic gene expression and the underlying molecular mechanism have not been fully elucidated.Wnt/?-catenin is a critical signaling pathway regulating bone development and regeneration. Activation of canonical wnt pathway is initiated by the binding of wnt ligands (Wnt1, Wnt3a, Wnt5b, etc) to frizzled (FZD) receptors and low density lipoprotein receptor-related protein 5 (LRP5) or LRP6 co-receptors. It results in inactivation of ?-catenin degradation complex, hence facilitating its translocation to the nucleus. In nucleus, ?-catenin binds to members of the T cell factor (TCF) family transcription factors, and consequently activates the transcription of a variety of osteogenic genes. Whether the canonical Wnt signaling is affected by ox-PAPC, and whether it is involved in ox-PAPC-induced effects in pre-osteoblasts have not been investigated.Above evidences confirm that lipid oxidation products inhibit bone formation, and as the major active component of MM-LDL, ox-PAPC possibly influences the osteoblast functions and osteoblast differentiation. In this study, we focused on the effects of ox-PAPC on osteogenic gene expression in pre-osteoblasts and osteoblasts. Whether the critical canonical Wnt signaling participates in the regulation of osteoblast differentiation in response to ox-PAPC and the underlying molecular mechanism were also investigated.Materials and MethodsPart 1 Effects of ox-PAPC on osteoblast viability and osteogenic gene expressionMurine primary osteoblasts were isolated from cranial bone, and identified by cell immunochemistry staining of BSP. P2 cells were cultured with 0,15,30 ?g/ml ox-PAPC for 0,24,48,72 or 96 hours, and detected with CCK-8 assay kit to determine the cell viability. P2 cells were cultured with 0,15,30 ?g/ml ox-PAPC in medium containing 5% fetal bovine serum (FBS) for 48 hours, and cell apoptosis was tested using caspase-3 activity assay.MC3T3-E1 cells and murine primary osteoblasts were cultured respectively with osteogenic inducing medium, and 15 ?g/ml ox-PAPC or PAPC for 3h, Id,3d and 5d. Real time PCR was used to analyze the mRNA levels of osterix, Runx2, ALP and OC.Part 2 Effects of ox-PAPC on canonical Wnt signaling pathway and it induced osteoblast differentiation1. To investigate the effect of ox-PAPC on the canonical Wnt signaling pathway, MC3T3-E1 cells were treated with the regular culture medium supplemented with Wnt-3a and/or ox-PAPC for 1 day. To determine the activation level of canonical Wnt signaling, western blot and immunofluorescent staining were performed to analyze the distribution of ?-catenin in nucleus and cytoplasm.2. To investigate the effects of ox-PAPC on canonical Wnt signaling-induced osteoblast differentiation, MC3T3-E1 cells were treated with the regular culture medium supplemented with Wnt-3a and/or ox-PAPC. ALP activity after 2d and protein levels of Runx2 after 2d and BSP after 3d were then evaluated.3. To investigate whether ERK and p38 MAPK signaling pathways were activated by ox-PAPC, MC3T3-E1 cells were incubated with the regular culture medium supplemented with ox-PAPC. The phosphorylation levels of ERK and p38 MAPK were then determined using western blot analysis at different time points.4. To investigate the roles of activated ERK and p38 MAPK pathways in ox-PAPC-treated cells, MC3T3-E1 cells were pretreated with regular culture medium supplemented with ERK inhibitor PD98059 or p38 MAPK inhibitor SB203580 for 1 hour, and then treated with Wnt-3a, ox-PAPC and/or MAPK inhibitors. ALP activity assay and western blot analysis were used to assess the activity and expression levels of bone-related proteins, and immunofluorescent staining and western blot were used to determine the nuclear translocation of ?-catenin.ResultsPart1Primary osteoblasts were successfully isolated from cranial bone of new born mice and stained positive for BSP expressed. Ox-PAPC at 15 and 30?g/ml promotes proliferation of murine primary osteoblasts, and promotes cell apoptosis in 5% FBS. No differentiation was detected between 15 and 30 ?g/ml ox-PAPC.MC3T3-E1 treated with ox-PAPC showed significant decreased osterix, Runx2, ALP and OC mRNA expression, while those treated with PAPC did not exhibit any changes in osteogenic gene expression. Primary osteoblasts showed similar changes in osterix, Runx2, ALP and OC expression.Part 21. Wnt-3a-treated cells showed a significant increase in the nuclear translocation of P-catenin when compared with the control group. And Wnt-3a+ox-PAPC group showed significant attenuation of Wnt-3a-induced nuclear translocation of ?-catenin. These results were further confirmed by immunofluorescent staining of ?-catenin. The increased numbers of cell nuclei with positive ?-catenin fluorescence signal in the Wnt-3a group was decreased by addition of ox-PAPC2. Two days after the treatment, the ALP activity in the Wnt-3a group was enhanced when compared with the control group. However, the addition of ox-PAPC in the Wnt-3a+ox-PAPC group significantly down-regulated the Wnt-3a-activated ALP activity. No statistically significant difference was detected between the ox-PAPC group and the control group during the study period. Changes in the protein levels of Runx2 and BSP showed similar change patterns.3. ox-PAPC treatment immediately increased the phosphorylation levels of both ERK and p38 MAPK, and these effects continued after 3 hours of ox-PAPC treatment. The phosphorylation levels of ERK and p38 MAPK after 1 hour were both up-regulated by ox-PAPC but not by Wnt-3a.4. ERK inhibitor PD98059 successfully reversed the inhibitory effects of ox-PAPC on Wnt-3a-enhanced expressions of ALP, Runx2 and BSP. In contrast, the addition of p38 inhibitor SB203580 slightly enhanced the negative effects of ox-PAPC on ALP activity and expression of Runx2. ERK inhibitor PD98059 completely restored the inhibitory effect of ox-PAPC on Wnt-3a-triggered nuclear translocation of ?-catenin. In contrast, SB203580 showed no effects on nuclear translocation of ?-catenin, but further enhanced the inhibition of ALP activity and Runx2 protein expression.Conclusions1. Ox-PAPC promotes osteoblast proliferation, and induces apoptosis in medium containing 5% FBS. Ox-PAPC inhibits osterix, Runx2, ALP and OC expression in MC3T3-E1 cells and primary osteoblasts, while PAPC shows no similar effects. Oxidized phospholipids instead of unoxidized ones repress osteoblast differentiation and function.2. Ox-PAPC depresses Wnt-3a-enhanced ?-catenin nuclear translocation, and thus inhibits activation of canonical Wnt signaling. Ox-PAPC inhibits Wnt-3a-induced osteoblast differentiation.3. Ox-PAPC activates ERK and p38 MAPK pathway. Ox-PAPC inhibits canonical Wnt signaling and canonical Wnt-induced osteoblast differentiation through activation of ERK pathway.