Association of toll-like receptor 4 signaling pathway with steroid-induced femoral head osteonecrosis in rats

Abstract

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramuscularly with 20 mg/kg methylprednisolone (MP) for 8 weeks, twice per week. The animals were sacrificed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups (n=24 each). Rats in the control group (n=12) were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase (TRAP) in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-κB p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR (RT-PCR) and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentration of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in control rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased significantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.

Keywords

osteonecrosis / Toll-like receptor 4 / corticosteroids / femoral head / rat

References

[1] CompstonJ. Glucocorticoid-induced osteoporosis. Hormone Research, 2003, 60(3): 77-79 PMID: 14671402

[2] HougardyDM, PetersonGM, BleaselMD, et al. . Is enough attention being given to the adverse effects of corticosteroid therapy?. J Clin Pharmacol Ther, 2000, 25(3): 227-234

[3] MontMA, JonesLC, HungerfordDS. Nontraumatic osteonecrosis of the femoral head: ten years later. J Bone Joint Surg Am, 2006, 88(5): 1117-1132 PMID: 16651589

[4] FisherDE, BickelWH. Corticosteroid-induced avascular necrosis. J Bone Joint Surg, 1971, 53(5): 859-873 PMID: 4326745

[5] MankinHJ. Nontraumatic necrosis of bone (osteonecrosis). N Engl J Med, 1992, 326(22): 1473-1479 PMID: 1574093

[6] ChangCC, GreenspanA, GershwinME. Osteonecrosis: current perspectives on pathogenesis and treatment. Semin Arthritis Rheum, 1993, 23(1): 47-69 PMID: 8235665

[7] DrescherW, WeigertKP, BungerMH, et al. . Femoral head blood flow reduction and hypercoagulability under 24h megadose steroid treatment in pigs. J Orthop Res, 2004, 22(3): 501-508 PMID: 15099627

[8] ZizicTM, MarcouxC, HungerfordDS, et al. . The early diagnosis of ischemic necrosis of bone. Arthritis Rheum, 1986, 29(10): 1177-1186 PMID: 3768054

[9] JonesJPJr. Intravascular coagulation and osteonecrosis. Clin Orthop Relat Res, 1992, 277: 41-53 PMID: 1532547

[10] ZalavrasC, DailianaZ, ElisafM, et al. . Potential aetiological factors concerning the development of osteonecrosis of the femoral head. Eur J Clin Invest, 2000, 30(3): 215-221 PMID: 10691998

[11] YamamotoT, HiranoK, TsutsuiH, et al. . Corticosteroid enhances the experimental induction of osteonecrosis in rabbits with Schwartzman reaction. Clin Orthop Relat Res, 1995, 316: 235-243 PMID: 7634712

[12] IchisekiT, KaneujiA, UedaY, et al. . Osteonecrosis development in a novel rat model characterized by a single application of oxidative stress. Arthritis Rheum, 2011, 63(7): 2138-2141 PMID: 21437877

[13] OkazakiS, NagoyaS, TatedaK, et al. . Weight bearing does not contribute to the development of osteonecrosis of the femoral head. Int J Exp Path, 2012, 93(6): 458-462

[14] JankeLJ, LiuCC, VogelP, et al. . Primary epiphyseal arteriopathy in a mouse model of steroid-induced osteonecrosis. Am J Path, 2013, 183(1): 19-25 PMID: 23673001 PMCID: 3702740

[15] OkazakiS, NishitaniY, NagoyaS, et al. . Femoral head osteonecrosis can be caused by disruption of the systemic immune response via the toll-like receptor 4 signalling pathway. Rheum, 2009, 48(3): 227-232

[16] LiH, SunB. Toll-like receptor 4 in atherosclerosis. J Cell Mol Med, 2007, 11(1): 88-95 PMID: 17367503

[17] XuY, TaoX, ShenB, et al. . Structural basis for signal transduction by the Toll/interleukin-l recptor domains. Nature, 2000, 408(6808): 111-115 PMID: 11081518

[18] DougallWC. Molecular pathways: osteoclast dependent and osteoclast independent roles of the RANKL/RANK/OPG pathway in tumorigenesis and metastasis. Clin Cancer Res, 2012, 18(2): 326-335 PMID: 22031096

[19] NakashimaT, TakayanagiH. The dynamic interplay between osteoclasts and the immune system. Arch Biochem Biophys, 2008, 473(2): 166-171 PMID: 18410741

[20] NakashimaT, TakayanagiH. New regulation mechanisms of osteoclast differentiation. Ann N.Y. Acad Sci, 2011, 1240(1): 13-18

[21] NakashimaT, HayashiM, TakayanagiH. New insights into osteoclastogenic signaling mechanisms. Cell, 2012, 23(11): 582-590

[22] AkiraS, TakedaK, KaishoT. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol, 2001, 2(8): 675-680 PMID: 11477402

[23] TakayanagiH. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nat Rev Immunol, 2007, 7(4): 292-304 PMID: 17380158

[24] MAXL, LIUZP, MAJX, et al. . Dynamic expression of Runx2, Osterix and AJl8 in the femoral head of steroid-induced osteonecrosis in rats. Chin J Orthop, 2010, 30(1): 67-72

[25] SuganoN, KuboT, TakaokaK, et al. . Diagnostic criteria for non-traumatic osteonecrosis of the femoral head. A multicentre study. J Bone Joint Surg Br, 1999, 81B(4): 590-595

[26] MendoncaDM, ChimelliL, MartinezAM. Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis. Braz J Med Biol Res, 2005, 38(6): 925-933 PMID: 15933787

[27] TakedaK, KaishoT, AkxraS. Toll-like receptors. Annu Rev Immunol, 2003, 21: 335-376 PMID: 12524386

[28] JanewayCA, MedzhltovJR. Innate immune recognition. Annu Rev immunol, 2002, 20(1): 197-216 PMID: 11861602

[29] GoldringSR, GravalleseEM. Pathogenesis of bone lesions in rheumatoid arthritis. Curr Reumatol Rep, 2002, 4(3): 226-231

[30] RomasE, SimsNA, HardsDK, et al. . Osteoprotegerin reduces osteoelast numbers and prevents bone erosion in collagen-induced arthritis. Am J Pathol, 2002, 161(4): 1419-1427 PMID: 12368214 PMCID: 1867274

[31] Soares-SchanoskiA, Gómez-PiñaV, del FresnoC, et al. . 6-Methylprednisolone down-regulates IRAK-M in human and murine osteoclasts and boosts bone resorbing activity: a putative mechanism for corticoid induced osteoporosis. J Leukoc Biol, 2007, 82(3): 700-709 PMID: 17576820

[32] CominaciniL, Fratta PasiniA, GarbinU, et al. . Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 in endothelial cells induces the activation of NF-κB through an increased production of intracellular reactive oxygen species. J Biol Chem, 2000, 275(17): 12633-12638 PMID: 10777555

[33] FrantzS, KobzikL, KimYD. TLR4 expression in cardiac myocytes in normal and failing myocardium. J Clin Invest, 1999, 104(3): 271-280 PMID: 10430608 PMCID: 408420

[34] DengYY, LuJ, LingEA, et al. . Monocyte chemoattractant protein-1 (MCP-1) produced Via NF-κB signaling pathway mediates migration of amoeboid microglia in the periventricular white matter in hypoxic neonatal rats. GLIA, 2009, 57(6): 604-621 PMID: 18942743

[35] ViedtC, DechendR, FeiJ, et al. . MCP-1 induces inflammatory activation of human tubular epithelial cells: Involvement of the transcription factors, nuclear factor-κB and activating protein-1. J Am Soc Nephrol, 2002, 13(6): 1534-1547 PMID: 12039983

[36] KimYY, KimSH, OhS, et al. . Increased fat due to estrogen deficiency induces bone loss by elevating monocyte chemoattractant protein-1 (MCP-1) production. Mol Cells, 2010, 29(3): 277-282 PMID: 20108169

[37] KimMS, DayCJ, MorrisonNA. MCP-1 is induced by receptor activator of nuclear factor-kB ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating factor suppression of osteoclast formation. J Biol Chem, 2005, 280(16): 16163-16169 PMID: 15722361

[38] KabataT, KuboT, MatsumotoT, et al. . Apoptotic cell death in steroid-induced osteonecrosis: an experimental study in rabbits. J Rheumatol, 2000, 27(9): 166-171

[39] IchisekiT, MatsumotoT, NishinoM, et al. . Oxidative stress and vascular permeability in steroid-induced osteonecrosis model. J Orthop Sci, 2004, 9(5): 509-515 PMID: 15449127

[40] IchisekiT, KaneujiA, KatsudaS, et al. . DNA oxidation injury in bone early after steroid administration is involved in the pathogenesis of steroid-induced osteonecrosis. Rheumatol, 2005, 44(4): 456-460

[41] IchisekiT, KaneujiA, KatsudaS, et al. . Initial MRI findings of non-traumatic osteonecrosis of the femoral head in renal allograft recipients. Magn Reson Imaging, 1997, 15(9): 1017-1023

[42] SuganoN, OhzonoK, MasuharaK, et al. . Prognostication of osteonecrosis of the femoral head in patients with systemic lupus erythematosus by magnetic resonance imaging. Clin Orthop, 1994, 305: 190-199 PMID: 8050228

[43] SuganoN, MasuharaK, NakamuraN, et al. . MRI of early osteonecrosis of the femoral head after transcervical fracture. J Bone Joint Surg Br, 1996, 78(2): 253-257 PMID: 8666637

[44] HiranoT, IwasakiK, OdaJ, et al. . Osteonecrosis of the femoral head in spontaneously hypertensive rats-relation to ossific nuclei during growth. Acta Orthop Scand, 1992, 63(1): 37-40 PMID: 1738967

[45] IchisekiT, UedaY, KatsudaS, et al. . Oxidative stress by glutathione depletion induces osteonecrosis in rats. Rheumatol, 2006, 45(3): 287-290