Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid
Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid
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DOI：
10.1016/j.bbrc.2011.01.071
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发表时间：
2011-02-25
影响因子：
3.1
通讯作者：
Nishihara, Tatsuji
中科院分区：
文献类型：
--
作者：
Kawano, Michinao;Ariyoshi, Wataru;Nishihara, Tatsuji
目的：骨形态发生蛋白-2(BMP-2)有望用于修复骨缺损，促进骨折愈合。然而，它不能产生足够的临床反应用于骨再生。近年来，研究发现肝素、硫酸肝素、硫酸角质形成蛋白、硫酸皮肤素、4-硫酸软骨素、6-硫酸软骨素和透明质酸(HA)等糖胺聚糖可调节BMP-2的活性，但其调控成骨活性的机制尚未完全阐明。本研究旨在探讨透明质酸(HA)对骨形态发生蛋白-2(BMP-2)诱导成骨细胞分化的影响。材料和方法：单层培养成骨细胞系MG63细胞，分别与BMP-2和HA孵育不同时间。为了确定成骨细胞的分化，对细胞裂解产物中的碱性磷酸酶(ALP)活性进行了定量。Western印迹分析检测Smad1/5/8、p38和ERK蛋白的磷酸化。为阐明磷酸化Smad1/5/8的核转位，对刺激细胞进行免疫荧光显微镜观察。为了进一步阐明HA在BMP-2诱导的Smad信号转导中的作用，用实时荧光定量RT-PCR法检测了BMP-2受体拮抗剂noggin和Folistatin的mRNA表达。结果：BMP-2和HA共同培养MG63细胞后，BMP-2诱导的碱性磷酸酶(ALP)活化、Smad1/5/8磷酸化和核转位均上调。Western印迹分析显示，透明质酸可使ERK蛋白的磷酸化程度降低。结论：HA增强的BMP-2通过下调BMP-2拮抗剂和ERK的磷酸化，诱导MG63细胞向成骨细胞分化。皇冠版权所有(C)2011由爱思唯尔公司出版。保留所有权利。
Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2.Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR.Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA.Conclusions: These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.