Role of MAPK signaling pathway in osteopontin-mediated ossification of the ligamentum flavum

doi:10.3969/j.issn.2095-4344.2017.28.007

Abstract

Abstract:

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway is an important signal transduction system. Our precious animal experiments have shown that osteopontin can mediate the ossification of the ligamentum flavum.
OBJECTIVE: To investigate the role of MAPK signaling pathway in osteopontin-induced ossification of the ligamentum flavum.
METHODS: The ligamentum flavum specimens obtained from 16 cases undergoing thoracic/lumbar posterior decompression surgery were divided into ossification and non-ossification groups (n=8 per group). The expression of osteopontin and its receptors CD44 and integrin was observed by immunohistochemical staining. The activation of phosphorylation in MAPK signaling pathway was detected by western blot assay. The MAPK signaling pathway was blocked by SB203580 or U0126 blocker alone to observe the induction of osteopontin.
RESULTS AND CONCLUSION: Osteopontin and its receptor CD44 were expressed in the ossification group, but not in the non-ossififcation group. However, the expression of integrin was not detected in the ossification group. The expression levels of alkaline phosphatase and osteocalcin in the ligamentum flavum were significnatly increased under the induction of osteopontin (P < 0.05), and osteopontin could activate the phosphorylation of P38 and ERK1/2 in the MAPK signaling pathway (P < 0.05), but the phosphorylation of JNK was not obvious. p38 phosphorylation blocked with SB203580 blocker could significantly inhibit the osteopontin-induced osteoblast differentiation of ligament flavum cells (P < 0.05), while U0126 blocker had no obvious effect. These results indicate that p38 in MAPK signaling pathway is a key molecule in osteopontin-mediated ossification of the ligamentum flavum.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tissue Engineering, Ligmentum Flavum, Alkaline Phosphatase

CLC Number:

R318

Li Xue-bin, Xu Zheng, Zhou Sheng-yuan, Liu Xiao-dong, Wang Zhi-qing, Xu Guo-feng, Chen Xiong-sheng. Role of MAPK signaling pathway in osteopontin-mediated ossification of the ligamentum flavum[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(28): 4469-4474.

Figures/Tables 2

2.3 黄韧带组织形态变化免疫组织化学染色显示，通过对临床获得的黄韧带骨化症患者黄韧带和骨化组织标本进行免疫组织化学染色可见骨桥蛋白及其受体CD44呈阳性表达，而Integrin的表达呈阴性。视野大体可以分为上下两块区域，下半区域为胸椎椎板，上半区域为黄韧带骨化区。在两区交界可见软骨层，存在大量的软骨陷窝，内有增生肥大的软骨细胞，胞核以及胞质透明，细胞周围可见成片的淡褐色染色。而在骨化区域内，可见大小不一，排列杂乱无章，形状不规则，胞核以及胞质蓝染的细胞，如图1。在非骨化的黄韧带标本中骨桥蛋白及其受体CD44，Integrin在非骨化的黄韧带细胞表达均呈阴性。 2.4 黄韧带细胞分离培养形态将临床取得的黄韧带骨化的标本通过反复无菌生理盐水冲洗发现，标本中存在骨化的黄韧带以及未骨化的黄韧带。取下未骨化的部分行组织爬片后可观察到黄韧带细胞从组织块边缘爬出，并呈辐射状分散生长，爬出的黄韧带细胞呈纺锤形，如图2。 2.5 骨桥蛋白诱导情况利用骨桥蛋白对骨化组和非骨化组体外培养的黄韧带细胞进行干预后发现只有骨化组的碱性磷酸酶以及骨钙素等骨化指标的表达增加(P < 0.05；图3)，同时发现MAPK信号通路分子p38，ERK1/2被磷酸化激活(P < 0.05)，但JNK的磷酸化激活并不明确(图4)。 2.6 MAPK信号通路通路阻断后相关蛋白的表达在质量浓度20 μg/L的ERK1/2特异的磷酸化抑制剂U0126作用下，骨桥蛋白诱导骨化组黄韧带成骨的过程并未被阻断，表现为碱性磷酸酶和骨钙素的表达与单用骨桥蛋白诱导的阳性对照组相比没有减弱的趋势(图5)。而在质量浓度20 μg/L的p38特异的磷酸化抑制剂SB203580的阻断下，可明显观察到碱性磷酸酶以及骨钙素的表达明显下调降低到空白组的表达水平(P < 0.05)。与单用骨桥蛋白的阳性对照组相比表达量明显降低(P < 0.05)，说明在p38信号分子磷酸化被阻断的情况下，骨桥蛋白诱导黄韧带骨化症-黄韧带细胞成骨的作用被抑制(图5)。"

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