Expression and role of retinoblastoma RB1-inducible
coiled-coil 1 in the subchondral bone of osteoarthritis

doi:10.3969/j.issn.2095-4344.2673

Abstract

Abstract:

BACKGROUND: Osteoarthritis is mainly characterized by degeneration of the articular cartilage and reconstruction of the subchondral bone. The specific pathogenesis of osteoarthritis is still unclear. Most studies have used cartilage and subchondral bone as the main entry point to explore the molecular mechanism and signal pathway changes in the disease progression, providing new biological targets and research direction for the diagnosis and treatment of osteoarthritis.

OBJECTIVE: To investigate the expression of RB1-inducible coiled-coil 1 (RB1CC1) in the subchondral bone during the development of osteoarthritis.

METHODS: Eight-week-old C57 mice were randomly divided into experimental group and sham operation group. Experimental group was then randomly divided into two subgroups of 4 weeks and 8 weeks. In the experimental group, the tibia ligament of the right knee was cut off to dissociate the medial meniscus to induce osteoarthritis. In the sham operation group, only the joint capsule was cut without medial ligament resection and meniscus dissociation. The study was implemented with an experimental animal ethic approval from the Third Affiliated Hospital of Southern Medical University, China (approval No. 44007200038731) on December 13, 2017.

RESULTS AND CONCLUSION: Compared with the sham operation group, the Osteoarthritis Research Society International scores were increased significantly in the experimental group. Compared with the sham operation group, the expression of collagen II was decreased, and RB1CC1 in the subchondral bone was gradually increased in the experimental group, which was consistent with the expression trend of BSP2. To conclude, with the development of osteoarthritis, the expression of RB1CC1 in the subchondral bone is gradually increased, which may be related to the increase of hyperplasia in the subchondral bone and remodeling.

Key words: osteoarthritis, subchondral bone, RB1CC1, osteogenesis, bone metabolism, BSP2, sclerosis, angiogenesis

CLC Number:

Li Junyan, Fang Hang, Feng Xiaofeng, Cai Daozhang. Expression and role of retinoblastoma RB1-inducible coiled-coil 1 in the subchondral bone of osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(17): 2642-2647.

Figures/Tables 5

1.4.2 小鼠骨关节炎模型建立实验组小鼠行右侧膝关节内侧半月板胫骨韧带切除，游离内侧半月板。术前小鼠自由摄取食水，每周更换垫料，造模时间在小鼠10周龄、骨骼肌肉发育成熟后进行。体积分数1%戊巴比妥钠(8 μL/g)进行腹腔麻醉，麻醉起效后将小鼠右膝关节周围的鼠毛剔除，并用棉球蘸取碘伏进行消毒。随后在解剖显微镜下将小鼠右膝关节处皮肤切开，沿髌骨韧带内侧纵行切开肌肉，将其外翻后钝性分离肌肉等组织，暴露胫骨平台与内侧半月板胫骨韧带。将内侧半月板胫骨韧带切断，游离内侧半月板，造成膝关节不稳定以诱导骨关节炎[14]，最后生理盐水冲洗，并将外翻的髌骨韧带复位，缝合切口[14-15]。麻醉苏醒后，将小鼠放回原笼中继续饲养，勤换垫料与观察伤口情况，小鼠自由摄取食水、可自由活动。假手术组仅切开关节囊。 1.4.3 组织取材实验组分别在术后4周与8周进行取材，假手术组在第8周取材，将小鼠断颈处死后取其右侧膝关节，将皮肤肌肉大致剔除，用40 g/L多聚甲醛固定组织标本，放于4 ℃冰箱。第2天用去离子水冲洗后置于摇床上进行脱钙，持续4周左右，脱钙液隔天更换一次。脱钙完成后将标本放入包埋盒并标记分组，用流水冲洗过夜，洗净脱钙液。第2天进行脱水，脱水完成后取出标本，将关节的内侧面朝金属模具底下进行包埋，期间注意保持关节面的平整，包埋完成后将蜡块放入冰箱冻存。 1.4.4 组织切片与脱蜡水化蜡块切片厚度均为4 μm，切片之后将其置于37 ℃恒温箱干燥过夜。脱蜡水化前，先将组织切片置于60 ℃风箱中烤片1.0-2.0 h，随后在二甲苯(2次)和梯度乙醇(体积分数100%，100%，90%，80%，70%，50%)中按顺序进行脱蜡水化，2次二甲苯和2次无水乙醇每次10 min，其余每次5 min。 1.4.5 番红O快速绿染色与骨关节炎OARSI评分切片脱蜡水化后铁苏木精染色15 s，水洗后吹干。体积分数1%固绿溶液染色1 min，冰醋酸浸泡数秒后吹干，体积分数0.5%番红O染液染色3 min，吹去多余染料，于显微镜下观察颜色深浅是否合适。随后二甲苯透明，过夜风干，隔天用中性树胶封固，显微镜下观察。切片关节组织的结构改变情况参照国际骨关节炎研究协会(Osteoarthritis Research Society International，OARSI)评分标准，其可反映骨关节炎严重程度，评分越高，骨关节炎软骨损伤越严重。 1.4.6 苏木精-伊红染色切片脱蜡水化后铁苏木精染色15 s，水洗后吹干切片。伊红染液染色30 s，流水洗去多余染液，二甲苯透明后风干过夜，次日用中性树胶封固定，显微镜观察。 1.4.7 免疫荧光组织切片脱蜡水化后浸泡于枸橼酸，在60 ℃水浴箱中过夜修复，次日PBS洗3次，每次5 min。用山羊血清在室温下封闭1 h后加入RB1CC1(1∶100)与BSP2(1∶100)混合—抗工作液，在4 ℃条件下过夜孵育14-16 h，次日PBS洗3次，每次5 min。避光滴加兔抗羊(594)(1∶500)与鼠抗羊(488)(1∶500)混合荧光二抗，孵育1 h后PBS洗3次，每次5 min，注意避光。最后用DAPI染核封固。在荧光显微镜下，观察切片组织中RB1CC1及BSP2的表达情况。 1.4.8 免疫组化脱蜡水化与枸橼酸修复步骤同上。加体积分数3%过氧化物酶抑制剂，室温孵育10 min，PBS洗3次，每次5 min。用山羊血清在室温条件下封闭1 h。加入Ⅱ型胶原(1∶100)—抗液在4 ℃条件下过夜孵育14-16 h，次日PBS洗3次，每次5 min。室温下滴加兔抗羊(1∶200)二抗，孵育1 h后PBS洗3次，每次5 min。DAB避光显色，苏木素染核，脱水透明后中性树胶封固，显微镜下观察拍照。 1.5 主要观察指标 ①切片组织学改变与骨关节炎OARSI 评分；②组织中Ⅱ型胶原、RB1CC1及BSP2的表达水平。 1.6 统计学分析用EXCEL2013(美国微软公司)整理汇总资料，Photoshop CS6软件(美国Adobe Systems公司)处理图片，Graph Pad Prism8软件做图分析(美国Graph Pad公司)，SPSS 23.0软件(美国IBM公司)进行统计分析。进行单因素方差分析和多重比较检测评估假手术组与4周及8周实验组的差异，P < 0.05为差异有显著性意义。 "

References

[1] CROSS M, SMITH E, HOY D, et al. The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study. Ann Rheum Dis. 2014;73(7):1323-1330.
[2] HUNTER DJ, SCHOFIELD D, CALLANDER E. The individual and socioeconomic impact of osteoarthritis. Nat Rev Rheumatol. 2014;10(7): 437-441.
[3] LAMBOVA SN, MÜLLER-LADNER U. Osteoarthritis - Current Insights in Pathogenesis, Diagnosis and Treatment. Curr Rheumatol Rev. 2018; 14(2): 91-97.
[4] NELSON AE, ALLEN KD, GOLIGHTLY YM, et al. A systematic review of recommendations and guidelines for the management of osteoarthritis: The chronic osteoarthritis management initiative of the U.S. bone and joint initiative. Semin Arthritis Rheum. 2014;43(6):701-712.
[5] MURPHY L, HELMICK CG. The impact of osteoarthritis in the United States: a population-health perspective: A population-based review of the fourth most common cause of hospitalization in U.S. adults. Orthop Nurs. 2012;31(2):85-91.
[6] MURAOKA T, HAGINO H, OKANO T, et al. Role of subchondral bone in osteoarthritis development: a comparative study of two strains of guinea pigs with and without spontaneously occurring osteoarthritis. Arthritis Rheum. 2007;56(10):3366-3374.
[7] DORE DA. The role of subchondral bone in osteoarthritis. Hobart, Australia: University of Tasmania. 2011.
[8] MARUOTTI N, CORRADO A, CANTATORE FP. Osteoblast role in osteoarthritis pathogenesis. J Cell Physiol. 2017;232(11):2957-2963.
[9] OCHI Y, CHANO T, IKEBUCHI K, et al. RB1CC1 activates the p16 promoter through the interaction with hSNF5. Oncol Rep. 2011;26(4):805-812.
[10] LI L, WANG G, HU JS, et al. RB1CC1-enhanced autophagy facilitates PSCs activation and pancreatic fibrogenesis in chronic pancreatitis. Cell Death Dis. 2018;9(10):952.
[11] LI S, QIANG Q, SHAN H, et al. MiR-20a and miR-20b negatively regulate autophagy by targeting RB1CC1/FIP200 in breast cancer cells. Life Sci. 2016;147:143-152.
[12] CARAMÉS B, HASEGAWA A, TANIGUCHI N, et al. Autophagy activation by rapamycin reduces severity of experimental osteoarthritis. Ann Rheum Dis. 2012;71(4):575-581.
[13] ALMONTE-BECERRIL M, NAVARRO-GARCIA F, GONZALEZ-ROBLES A, et al. Cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of Osteoarthritis within an experimental model. Apoptosis. 2010;15(5):631-638.
[14] FANG H, BEIER F. Mouse models of osteoarthritis: modelling risk factors and assessing outcomes. Nat Rev Rheumatol. 2014;10(7):413-421.
[15] GLASSON SS, ASKEW R, SHEPPARD B, et al. Characterization of and osteoarthritis susceptibility in ADAMTS-4-knockout mice. Arthritis Rheum. 2004;50(8):2547-2558.
[16] VON DER MARK K, KIRSCH T, NERLICH A, et al. Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy. Arthritis Rheum. 1992;35(7):806-811.
[17] KOBAYASHI M, SQUIRES GR, MOUSA A, et al. Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum. 2005;52(1):128-135.
[18] CHOI MC, MARUYAMA T, CHUN CH, et al. Alleviation of Murine Osteoarthritis by Cartilage-Specific Deletion of IκBζ. Arthritis Rheumatol. 2018;70(9):1440-1449.
[19] BURR DB. Increased biological activity of subchondral mineralized tissues underlies the progressive deterioration of articular cartilage in osteoarthritis. J Rheumatol. 2005;32(6):1156-1159.
[20] LI G, YIN J, GAO J, et al. Subchondral bone in osteoarthritis: insight into risk factors and microstructural changes. Arthritis Res Ther. 2013;15(6): 223.
[21] FUNCK-BRENTANO T, COHEN-SOLAL M. Subchondral bone and osteoarthritis. Curr Opin Rheumatol. 2015;27(4):420-426.
[22] RADIN EL, PAUL IL, ROSE RM. Role of mechanical factors in pathogenesis of primary osteoarthritis. Lancet. 1972;1(7749):519-522.
[23] CHAN PMB, WEN C, YANG WC, et al. Is subchondral bone cyst formation in non-load-bearing region of osteoarthritic knee a vascular problem?. Med Hypotheses. 2017;109:80-83.
[24] LIN C, SHAO Y, ZENG C, et al. Blocking PI3K/AKT signaling inhibits bone sclerosis in subchondral bone and attenuates post-traumatic osteoarthritis [published correction appears in J Cell Physiol. 2019 Jun;234(6):9873]. J Cell Physiol. 2018;233(8):6135-6147.
[25] FANG H, HUANG L, WELCH I, et al. Early Changes of Articular Cartilage and Subchondral Bone in The DMM Mouse Model of Osteoarthritis. Sci Rep. 2018;8(1):2855.
[26] XUE J, XUE J, ZHANG J, et al. miR-130b-3p/301b-3p negatively regulated Rb1cc1 expression on myogenic differentiation of chicken primary myoblasts. Biotechnol Lett. 2017;39(11):1611-1619.
[27] CHEN J, YAN L, WANG H, et al. ZBTB38, a novel regulator of autophagy initiation targeted by RB1CC1/FIP200 in spinal cord injury. Gene. 2018; 678:8-16.
[28] CORONA VELAZQUEZ AF, JACKSON WT. So Many Roads: the Multifaceted Regulation of Autophagy Induction. Mol Cell Biol. 2018;38(21): e00303-18.
[29] NISHIMURA I, CHANO T, KITA H, et al. RB1CC1 protein suppresses type II collagen synthesis in chondrocytes and causes dwarfism. J Biol Chem. 2011;286(51):43925-43932.
[30] BOULEFTOUR W, GRANITO RN, VANDEN-BOSSCHE A, et al. Bone Shaft Revascularization After Marrow Ablation Is Dramatically Accelerated in BSP-/- Mice, Along With Faster Hematopoietic Recolonization. J Cell Physiol. 2017;232(9):2528-2537.
[31] BOULEFTOUR W, JUIGNET L, VERDIÈRE L, et al. Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover. Bone. 2019;120:411-422.
[32] LI Z, WANG L, WEI J, et al. Bone-strengthening pill (BSP) promotes bone cell and chondrocyte repair, and the clinical and experimental study of BSP in the treatment of osteonecrosis of the femoral head. Oncotarget. 2017;8(57):97079-97089.
[33] BOLAMPERTI S, VILLA I, SPINELLO A, et al. Evidence for Altered Canonical Wnt Signaling in the Trabecular Bone of Elderly Postmenopausal Women with Fragility Femoral Fracture. Biomed Res Int. 2016;2016:8169614.
[34] SHAH M, GBURCIK V, REILLY P, et al. Local origins impart conserved bone type-related differences in human osteoblast behaviour. Eur Cell Mater. 2015;29:155-176.
[35] PESESSE L, SANCHEZ C, WALSH DA, et al. Bone sialoprotein as a potential key factor implicated in the pathophysiology of osteoarthritis. Osteoarthritis Cartilage. 2014;22(4):547-556.
[36] CUI Z, CRANE J, XIE H, et al. Halofuginone attenuates osteoarthritis by inhibition of TGF-β activity and H-type vessel formation in subchondral bone. Ann Rheum Dis. 2016;75(9):1714-1721.
[37] IKEBUCHI K, CHANO T, OCHI Y, et al. RB1CC1 activates the promoter and expression of RB1 in human cancer. Int J Cancer. 2009;125(4):861-867.
[38] SURANENI MV, MOORE JR, ZHANG D, et al. Tumor-suppressive functions of 15-Lipoxygenase-2 and RB1CC1 in prostate cancer. Cell Cycle. 2014;13(11):1798-1810.
[39] LI X, WAN X, CHEN H, et al. Identification of miR-133b and RB1CC1 as independent predictors for biochemical recurrence and potential therapeutic targets for prostate cancer. Clin Cancer Res. 2014;20(9): 2312-2325.
[40] LI J, FU Z, JIANG H, et al. IGF2-derived miR-483-3p contributes to macrosomia through regulating trophoblast proliferation by targeting RB1CC1. Mol Hum Reprod. 2018;24(9):444-452.
[41] CORONA VELAZQUEZ A, CORONA AK, KLEIN KA, et al. Poliovirus induces autophagic signaling independent of the ULK1 complex. Autophagy. 2018;14(7):1201-1213.
[42] GRUNWALD DS, OTTO NM, PARK JM, et al. GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction. Autophagy. 2019. doi:10.1080/15548627.2019.1632620.
[43] MORSELLI E, SHEN S, RUCKENSTUHL C, et al. p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200. Cell Cycle. 2011;10(16):2763-2769.
[44] GUO M, MU Y, YU D, et al. Comparison of the expression of TGF-β1, E-cadherin, N-cadherin, TP53, RB1CC1 and HIF-1α in oral squamous cell carcinoma and lymph node metastases of humans and mice. Oncol Lett. 2018;15(2):1639-1645.
[45] YAO J, JIA L, KHAN N, et al. Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium. Autophagy. 2015;11(6):939-953.
[46] ZHANG Y, VASHEGHANI F, LI YH, et al. Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis. Ann Rheum Dis. 2015;74(7):1432-1440.
[47] CARAMÉS B, TANIGUCHI N, OTSUKI S, et al. Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis. Arthritis Rheum. 2010;62(3): 791-801.
[48] ZHU X, YANG S, LIN W, et al. Roles of Cell Cyle Regulators Cyclin D1, CDK4, and p53 in Knee Osteoarthritis. Genet Test Mol Biomarkers. 2016;20(9):529-534.
[49] ZHONG G, LONG H, MA S, et al. miRNA-335-5p relieves chondrocyte inflammation by activating autophagy in osteoarthritis. Life Sci. 2019; 226:164-172.
[50] CHENG NT, MENG H, MA LF, et al. Role of autophagy in the progression of osteoarthritis: The autophagy inhibitor, 3-methyladenine, aggravates the severity of experimental osteoarthritis. Int J Mol Med. 2017;39(5): 1224-1232.
[51] XUE JF, SHI ZM, ZOU J, et al. Inhibition of PI3K/AKT/mTOR signaling pathway promotes autophagy of articular chondrocytes and attenuates inflammatory response in rats with osteoarthritis. Biomed Pharmacother. 2017;89:1252-1261.