Effect And Mechanism Of GSK-3β/β-catenin Signal Pathway On Wear Particle-induced Peri-prosthetic Osteolysis

Part ⅠPharmaceutical modulation of GSK-3β/β-catenin signal pathway suppresses wear debris-induced osteolysisObjective: To determine whether lithium chloride(Li Cl), a selective inhibitor of glycogen synthetase kinase 3 beta(GSK-3β), mitigates wear debris-induced osteolysis in a murine calvarial model of osteolysis.Methods: Eight-four female 6- to 7-week-old C57BL/6 mice were randomly divided among four experimental groups: a control group receiving no Ti particles(control), a Ti particle group(Ti), and two groups administered Ti particles together with Li Cl at either a low(L-Li Cl) or high(H-Li Cl) level. The mice in the L-Li Cl and H-Li Cl groups were gavage fed the GSK-3β selective inhibitor lithium chloride(50 or 200 mg kg-1d-1) or 300 μL of distilled water(control and Ti groups) 6 days per week for 2 weeks. Blood samples were collected from the mice just prior to sacrifice at 2 weeks after the operation, and the calvariae were dissected for molecular, micro-computed tomography(μCT), and histological analyses.Results: As assessed in the three-dimensional μCT reconstructions, the presence of Ti particles induced obvious bone resorption near the midline suture of the calvaria compared with that found in the control group. Treatment with Li Cl significantly mitigated Ti particle-induced osteolysis compared with the untreated groups. The Li Cl treatment induced a marked decrease in the number of pores(37.5% and 59.8% reductions in the L-Li Cl and H-Li Cl groups compared with the group stimulated with particles alone, p<0.05). Moreover, the Li Cl treatment significantly increased the bone mineral density(23.8% and 51.7%), bone volume(18.1% and 45.8%) and bone volume fraction(21.7% and 43.9%) parameters in the region of interest compared with those of the Ti group. The H&E staining demonstrated diminished inflammation after treatment with Li Cl while intensive macrophage infiltration into the calvariae in Ti group. Histomorphometric analysis demonstrated the area of bone eroded surface(BES) decreased to(0.07±0.01)mm2 and(0.04±0.008)mm2, while the bone thickness increased to(0.16±0.03)mm and(0.22±0.11)mm respectively in L-Li Cl and H-Li Cl group compared to(0.13±0.03)mm2 and(0.12±0.04)mm in Ti groups(p<0.05). TRAP staining revealed that multiple TRAP-positive osteoclasts located along the eroded bone surface in sections from the Ti group. However, the number of osteoclasts and the percentage of osteoclast surface relative to bone surface were clearly decreased after Li Cl treatment. Immunohistological examination showed Li Cl up-regulated the expression of phospho-Ser9-GSK-3β, β-catenin, alkaline phosphatase, Osterix and osteoprotegerin(OPG), while down-regulated the expression of receptor activator of nuclear factor kappa B ligand(RANKL), tumor necrosis factor-α(TNF-α), interleukin(IL)-1β and IL-6, even in the presence of Ti particles. However, the Li Cl treatment had little effect on the level of GSK-3β staining with or without Ti particle stimulation. RT-PCR showed that titanium particle stimulation could enhance m RNA expression of cathepsin K, calcitonin receptor, TRAP, osteoclast associated receptor compared with their levels in the control group. However, these Ti particle-induced increases were clearly mitigated by the Li Cl treatment. In addition, the RT-PCR data showed that the Ti particle stimulation significantly altered the expression of β-catenin and axin-2. The Li Cl treatment significantly increased the level of those gene transcripts compared with that in untreated mice. ELISA results showed that the circulating level of OPG was increased in Ti-stimulated mice compared with that in the control group(2730 ± 502 versus 2125 ± 305 pg/ml), whereas the RANKL level was not changed(161 ± 22 versus 156 ± 39 pg/ml). In the Li Cl-treated osteolytic mice, a significant increase in the OPG level in both the low(3410 ± 395 pg/ml) and high(3583 ± 353 pg/ml) dose groups and a clear decrease in the RANKL level(low: 134 ± 19 pg/ml; high: 103 ± 13 pg/ml) were observed. Consequently, the mean ratio of RANKL to OPG decreased from 0.072 ± 0.015 to 0.058 ± 0.009 in Ti particle-stimulated mice and was reduced in animals treated with Li Cl for 2 weeks to 0.042 ± 0.008(50 mg/kg) and 0.030 ± 0.007(200 mg/kg). We also determined the serum level of CTX, a fragment of type I collagen released from bone during bone resorption. As expected, the Ti particle stimulation increased this bone resorption marker by 37.1%, while the levels of CTX remained unchanged in Ti particle-stimulated mice after Li Cl treatment. The serum of P1 NP and OCN in Ti groups were 42.63±6.31ng/ml and 41.40±7.76ng/ml, and Li Cl treatment significantly increased the expression of P1 NP and OCN [L-Li Cl group: 52.53±9.07ng/ml, 49.92±5.54ng/ml; H-Li Cl group: 62.70±9.43ng/ml, 58.97±9.62ng/ml, p<0.05]. In addition, Ti particle stimulation increased the serum levels of TNF-α, IL-6 and IL-1β, which were significantly decreased by Li Cl treatment(p<0.05). No differences in the white blood cell count, hemoglobin, or platelet count were found between the control and Li Cl-treated mice. No evidence of liver or kidney toxicities were observed in the mice treated with either 50 or 200 mg kg-1d-1 of Li Cl.Conclusion: In summary, the results of this study clearly demonstrated that the GSK-3β/β-catenin signaling pathway is involved in the development of Ti particle-induced osteolysis. This finding is supported by evidence showing that the inhibition of GSK-3β using lithium significantly attenuated inflammation, increased bone formation and decreased bone resorption in the mouse calvarial model. Overall, the results indicate that GSK-3β inhibitors such as lithium may be therapeutic candidates for the prevention and treatment of wear debris-induced osteolysis in the clinic.Part ⅡTitanium particle inhibits osteogenic differentiation via the GSK-3β/β-catenin signaling pathwayObjective: To determine the role of GSK-3β/β-catenin signaling pathway on osteoblast differentiation, inflammatory cytokines section, and receptor activator of nuclear factor kappa B ligand(RANKL) and osteoprotegerin(OPG) expression in MC3T3-E1 cells stimulated with titanium(Ti) particles.Methods: The effect of Ti particles on the viability of MC3T3-E1 cells was determined using a cell counting kit-8(CCK-8) assay. Alkaline phosphatase(ALP) activity was measured using using a p-nitrophenyl phosphate assay. The mineralization of osteoblast, which was differentiation-induced for 21 days, was determined by Alizarin red S(ARS) staining. The m RNA levels of Runx2, Osterix, OCN, RANKL, OPG, TNF-α, IL-1β, IL-6, COX-2, β-catenin and axin-2 were detected by using quantitative reverse transcription-polymerase chain reaction(RT-PCR). The protein levels of GSK-3β, p Ser9-GSK-3β and β-catenin were determined by using Western blotting. Intracellular distribution of β-catenin in osteoblasts was determined by immunofluorescence using an anti-β-catenin antibody at a dilution of 1:250. The luciferase activity of β-catenin/TCF target genes was determined using a Dual-Luciferase Assay. Enzyme-linked immunosorbent assay(ELISA) was performed to determine the section of RANKL, OPG, IL-6 and PGE2.Results: Ti particles(0.1mg/ml) did not affect MC3T3-E1 cell viability. Ti particles significantly inhibited ALP activity, an early differentiation marker. In addition, RT-PCR results demonstrated that Ti particles significantly reduced the m RNA levels of Runx2 and Osterix, two master osteoblast-specific transcription factors. Consistent with this, Ti particles also reduced OCN expression. At the end of this study, osteoblasts were stained using ARS. Ti particles significantly decreased the staining density of ARS in visible observation quantification by spectrophotometry when compared with the control group. Interestingly, we found that Ti-particle stimulation reduced the expression of phospho-Ser9-GSK-3β and subsequently decreased the cytosolic and nuclear levels of β-catenin in MC3T3-E1 cells. Meanwhile, the m RNA levels of β-catenin and axin-2 were also significantly decreased in MC3T3-E1 cells stimulated with Ti particles. In addition, we found that Ti particles impaired β-catenin-dependent transcription induced by Wnt-3a as determined by the Topflash reporter assay. To determine whether Ti-particle-induced inhibition of osteoblast differentiation could be reversed by lithium chloride(Li Cl), a selective inhibitor of glycogen synthetase kinase 3 beta(GSK-3β), the murine MC3T3-E1 cells were pretreated with 10 m M Li Cl followed by the application of Ti particles. Western blot analysis revealed that Li Cl increased the serine-9 phosphorylation of GSK-3β and inhibited β-catenin degradation induced by Ti particles. RT-PCR results showed that the m RNA levels of β-catenin and the β-catenin target gene axin-2 were significantly increased in the Li Cl-treated group when compared with that in the untreated group. The luciferase assay demonstrated that inhibition of GSK-3β by Li Cl promotes the activation of Wnt/β-catenin signal pathway. In addition, Li Cl also inhibited osteogenic differentiation of MC3T3-E1 cells. Li Cl treatment attenuated Ti-particle inhibition of ALP activity and Runx2, Osterix, and OCN gene expression. Meanwhile, quercetin, a selective inhibitor of β-catenin, attenuated Li Cl-mediated protective effects on β-catenin activity. Moreover, we found that deletion of β-catenin activity also attenuated Li Cl-induced osteogenic mineralization mediated by MC3T3-E1 cells. We also demonstrated that Li Cl treatment increased the m RNA levels of OPG and reduced the m RNA levels of RANKL even in the presence of Ti particles. The protein levels of OPG and RANKL in MC3T3-E1 cell cultures were measured with ELISA. The results showed that levels of RANKL and OPG were 17.36±2.28pg/ml and 0.67±0.17ng/ml in Ti groups, but secretion of RANKL(11.24±2.16pg/ml) was reduced together with an increase in OPG(2.45±0.32ng/ml) by Li Cl treatment. Consequently, the mean ratio of RANKL to OPG decreased from 25.75±3.21/103(Ti group) to 4.76±0.89/103(Ti+Li Cl group). We also determined the expression of TNF-α, IL-β, IL-6 and COX-2 in MC3T3-E1 cells stimulated with Ti particles. Interestingly, IL-β and TNF-α m RNA levels were not affected by Ti treatment. The IL-6 and COX-2 m RNA levels, however, significantly increased after particle stimulation in MC3T3-E1 cells. As expected, blocking GSK-3β by Li Cl significantly decreased the gene expression of IL-6 and COX-2 even in the presence of Ti particles. Next, the levels of IL-6 and PGE2 in MC3T3-E1 cell cultures were measured by ELISA. After culture for 24 h, the protein levels of IL-6(694.86±81.59pg/ml) and PGE2(17.68±1.63ng/ml) in Ti group were significantly reduced to 262.82±41.91pg/ml and 7.32±1.18ng/ml in Ti+Li Cl group.Conclusion: These results demonstrated that Ti particle inhibits osteoblast differentiation, promotes inflammatory cytokines expression and up-regulates RANKL to OPG ratio in MC3T3-E1 cells via modulation of the GSK-3β/β-catenin signal pathway. Inactivation of GSK-3β by Li Cl significantly enhanced Wnt/β-catenin signaling activity and cell differentiation in the presence of Ti particles. Meanwhile, Li Cl also decreased the expression of IL-6 and COX-2/PGE2 as well as the ratio of RANKL to OPG. These results indicate that GSK-3β/β-catenin signal pathway play a critical role in wear particle-induced inhibition on osteoblast function.Part Ⅲ Lithium chloride inhibits wear particle-induced osteoclastogenesis via the regulation on osteoblast cellsObjective: To explore the effects of culture medium obtained from MC3T3-E1 cells stimulated with lithium chloride and Ti particle on the regulation of osteoclast differentiation from a murine macrophage cell line(RAW264.7) stimulated with Ti particles and the receptor activator of nuclear factor kappa B(NF-κB) ligand(RANKL).Methods: Osteoclast formation was measured by tartrate resistant acid phosphatase(TRAP) staining. Osteoclatic bone resorption was determined using a 24-well Osteo Assay Plate. The m RNA levels of Cath-K, CTR, TRAP, OSCAR, NFATc1, MMP-9, TNF-α and IL-1β were detected by using reverse transcription polymerase chain reaction(RT-PCR). The protein levels of NFATc1, MMP-9, IκBα and p-IκBα were determined by using Western blotting. The luciferase activity of NF-κB signal pathway was determined using a Dual-Luciferase Assay. Enzyme-linked immunosorbent assay(ELISA) was performed to determine the section of TNF-α and IL-1β.Results: Mature osteoclasts were obtained from RAW264.7 cells stimulated with RANKL and Ti particles for 5 days, and numerous TRAP-positive cells were observed in the group without Li-CM(control group). In contrast, Li-CM treatment significantly decreased the number of osteoclast-like cells in a dose-dependent manner. The m RNA levels of TRAP, CTR, OSCAR, NFATc1, Cath-K and MMP-9 were elevated after stimulation with RANKL and Ti particles for 5 days, whereas simultaneous treatment with Li-CM significantly inhibited this increase in the Cath-K, CTR, TRAP, OSCAR, NFATc1 and MMP-9 m RNA levels. In addition, the resorption area was decreased to 41.7±5.4% when 50% Li-CM was included. Although inflammation-related expression of TNF-α and IL-1β were induced in the control group, the gene copies of those genes were clearly decreased by Li-CM(50%) treatments. ELISA was used to determine the TNF-α and IL-1β protein levels in the culture medium of RAW264.7. The presence of Ti particles induced a significant increase in the protein levels of TNF-α(637.9±82.3pg/ml) and IL-1β(315.7±37.5pg/ml). Li-CM treatment significantly suppressed TNF-α and IL-1β expression in a dose-dependent manner. Western blot results showed that pretreatment with Li-CM(50%) for 4h significantly inhibited RANKL-induced phosphorylation and degradation of Ik Ba in RAW264.7 cells. In accordance with IκBα degradation and phosphorylation, RANKL and Ti particles were able to induce more than 9.1 fold of luciferase activity in RAW264.7 cells transfected with NF-κB-luc after 8h of treatment. Interestingly, the luciferase activity was significant reduced by Li-CM even in response to RANKL and Ti particles. To further determined whether Li-CM inhibits NF-κB dependent transcription in RAW264.7 cells, m RNA and protein levels of NF-κB two target genes, NFATc1 and MMP-9 which play significant role in osteoclast differentiation, were measured by RT-PCR and Western blot after RANKL and Ti particle stimulation for various time.The results demonstrated that pretreatment of Li-CM for 4h significantly reduced the expression of NFATc1 and MMP-9.Conclusion: Li-CM could significantly inhibit Ti particle-induced inflammatory osteoclatogenesis.