TNF-α对骨形成蛋白-2诱导的小鼠C2C12细胞向成骨细胞分化的影响及可能机制
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摘要
目的:研究肿瘤坏死因子(TNF-α)对骨形成蛋白-2(BMP-2)诱导的小鼠成肌C2C12细胞向成骨细胞分化的影响并探讨相关的作用机制。方法:分别以TNF-α(5 ng/mL)和BMP-2(100 ng/mL)单独或联合处理小鼠成肌C2C12细胞,检测细胞中碱性磷酸酶(AKP)的活性;采用BMP-2 100 ng/mL联合不同浓度TNF-α(0、2、5、8、10 ng/mL)或TNF-α5 ng/mL联合不同浓度BMP-2(0、100、200、300 ng/mL)处理细胞,检测细胞中AKP的活性。蛋白质印迹法(Western Blot)检测细胞中Smad1和NF-κB磷酸化的水平。结果:与对照组相比,BMP-2(100 ng/mL)能显著增加C2C12细胞中AKP活性(P<0.05),但TNF-α(5 ng/mL)可显著抑制C2C12细胞中AKP活性(P<0.05)。C2C12细胞中AKP活性随着TNF-α浓度的增加显著降低(P<0.05),但随着BMP-2浓度的增加而显著升高(P<0.05)。与对照组相比,TNF-α能显著降低C2C12细胞中磷酸化Smad1的水平,且显著升高炎症相关因子NF-κB磷酸化水平,但加入BMP-2对NF-κB无显著影响。结论:TNF-α能抑制BMP-2诱导的C2C12细胞向成骨细胞分化,且具有一定的剂量效应,其作用机制可能与调控炎症相关因子NF-κB活性干预BMP2-Smad1信号通路有关。
Objective: To investigate the effects of tumor necrosis factor-α(TNF-α) on osteoblast differentiation of mouse myoblasts C2C12 induced by bone morphogenetic protein-2(BMP-2) and its possible mechanism. Methods: The myogenic C2C12 cells were treated with TNF-α(5 ng/mL) and BMP-2(100 ng/mL) alone or in combination, and the activity of alkaline phosphatase(AKP)in the cells was detected; BMP-2 was maintained at 100 ng/mL combined with different concentrations of TNF-α(0 ng/mL,2ng/mL, 5 ng/mL,8 ng/m L, 10 ng/mL), or TNF-α to keep 5 ng/mL combined with different concentrations of BMP-2(0 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL)were used to treated cells, to analysis of the activity of AKP. The levels of Smad1 and NF-κB phosphorylation in cells were detected by Western Blot. Results: Compared with the control group, BMP-2(100 ng/mL) significantly increased AKP activity(P<0.05) in C2C12 cells, but TNF-α(5 ng/mL) significantly inhibited the activity of AKP in C2C12 cells(P<0.05). The activity of AKP in C2C12 cells decreased significantly with the increase of TNF-α concentration(P<0.05), but increased significantly with the increase of BMP-2 concentration(P<0.05). Compared with the control group, TNF-α were significantly reduced the level of phosphorylated Smad1 in C2C12 cells and significantly increased the phosphorylation level of inflammatory related factor NF-κB. However, BMP-2 had no significant effect on NF-κB. Conclusion: TNF-α can inhibit the differentiation of C2C12 cells into osteoblasts induced by BMP-2, and has a certain dose effect. The mechanism may be related to the regulation of the inflammation-related factor NF-κB activity in BMP2-Smad1 signaling pathway.
Objective: To investigate the effects of tumor necrosis factor-α(TNF-α) on osteoblast differentiation of mouse myoblasts C2C12 induced by bone morphogenetic protein-2(BMP-2) and its possible mechanism. Methods: The myogenic C2C12 cells were treated with TNF-α(5 ng/mL) and BMP-2(100 ng/mL) alone or in combination, and the activity of alkaline phosphatase(AKP)in the cells was detected; BMP-2 was maintained at 100 ng/mL combined with different concentrations of TNF-α(0 ng/mL,2ng/mL, 5 ng/mL,8 ng/m L, 10 ng/mL), or TNF-α to keep 5 ng/mL combined with different concentrations of BMP-2(0 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL)were used to treated cells, to analysis of the activity of AKP. The levels of Smad1 and NF-κB phosphorylation in cells were detected by Western Blot. Results: Compared with the control group, BMP-2(100 ng/mL) significantly increased AKP activity(P<0.05) in C2C12 cells, but TNF-α(5 ng/mL) significantly inhibited the activity of AKP in C2C12 cells(P<0.05). The activity of AKP in C2C12 cells decreased significantly with the increase of TNF-α concentration(P<0.05), but increased significantly with the increase of BMP-2 concentration(P<0.05). Compared with the control group, TNF-α were significantly reduced the level of phosphorylated Smad1 in C2C12 cells and significantly increased the phosphorylation level of inflammatory related factor NF-κB. However, BMP-2 had no significant effect on NF-κB. Conclusion: TNF-α can inhibit the differentiation of C2C12 cells into osteoblasts induced by BMP-2, and has a certain dose effect. The mechanism may be related to the regulation of the inflammation-related factor NF-κB activity in BMP2-Smad1 signaling pathway.
引文
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[2]Liu J,Liang C,Guo B,et al.Increased PLEKHO1 within osteoblasts suppresses Smad-dependent BMP signaling to inhibit bone formation during aging[J].Aging cell,2017,16(2):360-376
[3]Wu M,Chen G,Li Y P.TGF-βand BMP signaling in osteoblast,skeletal development,and bone formation,homeostasis and disease[J].Bone Research,2016,4(1):16009
[4]Li A,Yang L,Geng X,et al.Rocaglamide-A Potentiates Osteoblast Differentiation by Inhibiting NF-κB Signaling[J].Molecules&Cells,2015,38(11):941
[5]Liu S P,Wang G D,Du X J,et al.Triptolide inhibits the function of TNF-αin osteoblast differentiation by inhibiting the NF-κB signaling pathway[J].Experimental&Therapeutic Medicine,2017,14(3):2235
[6]Noguchi T,Ebina K,Hirao M,et al.Progranulin plays crucial roles in preserving bone mass by inhibiting TNF-α-induced osteoclastogenesis and promoting osteoblastic differentiation in mice[J].Biochemical and biophysical research communications,2015,465(3):638-643
[7]王链链,郭晓英.破骨细胞分化过程中的信号通路及信号因子的研究进展[J].中国骨质疏松杂志,2015,27(6):742-748Wang Lian-lian,Guo Xiao-ying.Advances in Signal Pathways and Signal Factors during Osteoclast Differentiation[J].Chinese Journal of Osteoporosis,2015,27(6):742-748
[8]Chen W,Gao B,Hao L,et al.The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation[J].Journal of periodontal research,2016,51(5):647-660
[9]陈丽珍,周英,黄俊飞,等.大血藤对破骨细胞活性及成骨细胞增殖分化作用的研究[J].中国中药杂志,2015,40(22):4463-4468Chen Li-zhen,Zhou Ying,Huang Jun-fei,et al.Study on the activity of osteopontin and the proliferation and differentiation of osteoblasts in Rhodiola sachalinensis[J].Chinese Journal of Medicine,2015,40(22):4463-4468
[10]Dixon S J,Sims S M.P2 purinergic receptors on osteoblasts and osteoclasts:Potential targets for drug development[J].Drug Development Research,2015,49(3):187-200
[11]Baum R,Gravallese E M.Bone as a Target Organ in Rheumatic Disease:Impact on Osteoclasts and Osteoblasts[J].Clinical Reviews in Allergy&Immunology,2016,51(1):1
[12]Chen Q,Shou P,Zheng C,et al.Fate decision of mesenchymal stem cells:adipocytes or osteoblasts?[J].Cell Death&Differentiation,2016,23(7):1128-1139
[13]Millard S M,Pettit A R,Ellis R,et al.Intrauterine bone marrow transplantation in osteogenesis imperfecta mice yields donor osteoclasts and osteomacs but not osteoblasts[J].Stem cell reports,2015,5(5):682-689
[14]刘彩宏,王军强.转化生长因子β1在骨形态发生蛋白2诱导的人牙周膜干细胞成骨分化中的作用[J].牙体牙髓牙周病学杂志2016,26(6):332-337Liu Cai-hong,Wang Jun-qiang.Role of transforming growth factorβ1 in osteogenic differentiation of human periodontal ligament stem cells induced by bone morphogenetic protein 2[J].Journal of endodontics,periodontal diseases,2016,26(6):332-337
[15]Zhao W,Wang T,Luo Q,et al.Cartilage degeneration and excessive subchondral bone formation in spontaneous osteoarthritis involves altered TGF-βsignaling[J].Journal of Orthopaedic Research Official Publication of the Orthopaedic Research Society,2016,34(5):763
[16]Hyuck C,Byung-Chul J,Min-Suk K,et al.Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8and p38 pathways[J].Journal of Biomedical Science,2016,23(1):45
[17]李蓉,杜军辉,常远.肿瘤坏死因子-α对RF/6A细胞自噬促进细胞增殖、迁移和管腔形成的影响[J].眼科新进展,2015,35(12):1132-1136Li Rong,Du Jun-hun,Chang Yuan.Effects of TNF-alpha on autophagy promoting cell proliferation,migration and lumen formation in RF/6A cells[J].New progress in Ophthalmology,2015,35(12):1132-1136
[18]Raehtz S,Bierhalter H,Schoenherr D,et al.Estrogen Deficiency Exacerbates Type 1 Diabetes-Induced Bone TNF-αExpression and Osteoporosis in Female Mice[J].Endocrinology,2017,158(7):2086-2101
[19]Toussiroté,Aubin F.Paradoxical reactions under TNF-αblocking agents and other biological agents given for chronic immune-mediated diseases:an analytical and comprehensive overview[J].RMD open,2016,2(2):e000239
[20]Jackaman C,Yeoh T L,Acuil M L,et al.Murine mesothelioma induces locally-proliferating IL-10+TNF-α+CD206-C X3CR1+M3macrophages that can be selectively depleted by chemotherapy or immunotherapy[J].Oncoimmunology,2016,5(6):e1173299
[21]Cao Y,Jansen I D C,Sprangers S,et al.TNF-αhas both stimulatory and inhibitory effects on mouse monocyte-derived osteoclastogenesis[J].Journal of Cellular Physiology,2017,232(12):3273-3285
[22]Zhao G,Yu Y M,Kaneki M,et al.Simvastatin Reduces Burn Injuryinduced Splenic Apoptosis via Down-regulation of the TNF-α/NF-κB Pathway[J].Annals of surgery,2015,261(5):1006
[23]Fu H,Li G,Liu C,et al.Probucol Prevents Atrial Remodeling by Inhibiting Oxidative Stress and TNF-α/NF-κB/TGF-βSignal Transduction Pathway in Alloxan-Induced Diabetic Rabbits[J].Journal of cardiovascular electrophysiology,2015,26(2):211-222
[24]Kato.Nanotopography Directs Mesenchymal Stem Cells to Osteoblast Lineage Through Regulation of microRNA-SMAD-BMP-2Circuit(vol 229,pg 1690,2014)[J].Journal of Cellular Physiology,2016,231(11):2548-2548
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[27]Mao C Y,Wang Y,Zhang X,et al.Double-edged-sword effect of IL-1βon the osteogenesis of periodontal ligament stem cells via crosstalk between the NF-κB,MAPK and BMP/Smad signaling pathways[J].Cell death&disease,2016,7(7):e2296
[28]Luo K.Signaling Cross Talk between TGF-β/Smad and Other Signaling Pathways[J].Cold Spring Harbor Perspectives in Biology,2017,9(1):a022137
[29]Li A,Cong Q,Xia X,et al.Pharmacologic Calcitriol Inhibits Osteoclast Lineage Commitment via the BMP-Smad1-IκB-NF-κB Pathway[J].Journal of Bone&Mineral Research,2017,32(7):1406-1420
[30]Hirata-Tsuchiya S,Fukushima H,Kokabu S,et al.Fine-tuning between BMP and NF-κB pathways regulates osteoblastic bone formation[J].Journal of Oral Biosciences,2016,58(3):73-77
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