丝裂原活化蛋白激酶信号通路在骨桥蛋白介导黄韧带骨化中的作用

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

背景:丝裂原活化蛋白激酶(MAPK)信号通路是生物体内重要的信号转导系统之一。课题组前期动物实验提示骨桥蛋白可介导黄韧带骨化的发生。目的:观察MAPK信号通路在骨桥蛋白介导黄韧带骨化中的作用。方法:将16例患者后路椎板切除手术中取得黄韧带标本分为黄韧带骨化以及非骨化两组,每组8例。通过免疫组织化学染色观察骨桥蛋白及其受体CD44、整合素的表达;利用骨桥蛋白对黄韧带细胞进行诱导,并利用蛋白印记方法检测丝裂素活化蛋白激酶信号通路分子的磷酸化激活情况,随后再利用SB203580和U0126阻滞剂分别阻断丝裂素活化蛋白激酶相应信号通路观察骨桥蛋白的诱导情况。结果与结论:(1)骨桥蛋白及其受体CD44及整合素的表达:骨化组的标本中均检测到骨桥蛋白及其受体CD44的表达,但未检测到整合素的表达,非骨化组中则未见到骨桥蛋白及其受体的表达;(2)MAPK信号通路变化:在骨桥蛋白的诱导作用下黄韧带细胞中骨化指标碱性磷酸酶及骨钙素表达增加(P<0.05),同时骨桥蛋白能激活丝裂素活化蛋白激酶信号通路中的p38及ERK1/2信号分子的磷酸化(P<0.05),但JNK氨基末端激酶的磷酸化激活并不明显。利用SB203580阻滞剂阻断p38的磷酸化可明显阻断骨桥蛋白诱导的黄韧带细胞的骨向分化(P<0.05),而U0126阻滞剂阻断效果不明显;(3)结果证实,MAPK信号通路在骨桥蛋白介导黄韧带骨化中其重要作用,p38是诱导黄韧带细胞骨向分化的关键因子。

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 from16cases undergoing thoracic/lumbar posterior decompression surgery were divided into ossification and non-ossification groups(n=8per group).The expression of osteopontin and its receptors CD44and 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 SB203580or U0126blocker alone to observe the induction of osteopontin.RESULTS AND CONCLUSION:Osteopontin and its receptor CD44were 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 P38and ERK1/2in the MAPK signaling pathway(P<0.05),but the phosphorylation of JNK was not obvious.p38phosphorylation blocked with SB203580blocker could significantly inhibit the osteopontin-induced osteoblast differentiation of ligament flavum cells(P<0.05),while U0126blocker had no obvious effect.These results indicate that p38in MAPK signaling pathway is a key molecule in osteopontin-mediated ossification of the ligamentum flavum.