Ha Woo-Hun, Seong Hwa-Sik, Choi Na-Rae, Park Bong-Soo, Kim Yong-Deok
1Department of Oral and Maxillofacial Surgery, Pusan National University, Beomeori, Mulgeom, Yangsan, 50612 Korea.
2Department of Oral Anatomy, Pusan National University, Yangsan, 50612 Korea.
Tissue Eng Regen Med. 2017 Mar 8;14(4):433-441. doi: 10.1007/s13770-017-0046-1. eCollection 2017 Aug.
Hypoxia suppresses osteoblastic differentiation and the bone-forming capacity. As the leading osteoinductive growth factor used clinically in bone-related regenerative medicine, recombinant human bone morphogenic protein-2 (rhBMP-2) has yielded promising results in unfavorable hypoxic clinical situations. Although many studies have examined the effects of rhBMP-2 on osteoblastic differentiation, mineralization and the related signaling pathways, those of rhBMP-2 on osteoblastic cells remain unknown, particularly under hypoxic conditions. Therefore, this study was conducted under a 1% oxygen tension to examine the differentiating effects of rhBMP-2 on osteoblastic cells under hypoxia. rhBMP-2 could also induce the differentiation and mineralization of Osteoblastic (MC3T3-E1) cells under 1% hypoxic conditions. rhBMP-2 could also induce the differentiation and mineralization of MC3T3-E1 cells under 1% hypoxic conditions. rhBMP-2 increased the alkaline phosphatase {ALP} activity in a time dependent manner, and expression of ALP, collagen type-1 (Col-1) and osteocalcin (OC) mRNA were up-regulated significantly in a time- and concentration-dependent manner. In addition, the area of the mineralized nodules increased gradually in a concentration-dependent manner. Western blot analysis, which was performed to identify the signaling pathways underlying rhBMP-2-induced osteoblastic differentiation under hypoxic conditions, showed that rhBMP-2 significantly promoted the phosphorylation of the p38 mitogen-activated protein kinase (MAPK) in a time-dependent manner. A pretreatment with SB203580, a p38 MAPK inhibitor, inhibited the rhBMP-2-mediated differentiation and mineralization. Moreover, the phosphorylation of p38 induced by rhBMP-2 was inhibited in response to a pretreatment of the cells with Go6976, a protein kinase D {PKD) inhibitor. These findings suggest that rhBMP-2 induces the differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions via activation of the PKD and p38 MAPK signaling pathways.
缺氧会抑制成骨细胞分化和骨形成能力。作为临床上在骨相关再生医学中使用的主要骨诱导生长因子，重组人骨形态发生蛋白-2（rhBMP-2）在不利的缺氧临床情况下已取得了令人鼓舞的结果。尽管许多研究已经考察了rhBMP-2对成骨细胞分化、矿化及相关信号通路的影响，但其对成骨细胞的影响仍不清楚，尤其是在缺氧条件下。因此，本研究在1%氧张力条件下进行，以考察rhBMP-2在缺氧情况下对成骨细胞的分化作用。rhBMP-2在1%缺氧条件下也能诱导成骨细胞（MC3T3-E1）的分化和矿化。rhBMP-2在1%缺氧条件下还能诱导MC3T3-E1细胞的分化和矿化。rhBMP-2以时间依赖性方式增加碱性磷酸酶（ALP）活性，并且ALP、Ⅰ型胶原（Col-1）和骨钙素（OC）mRNA的表达以时间和浓度依赖性方式显著上调。此外，矿化结节面积以浓度依赖性方式逐渐增加。为了确定缺氧条件下rhBMP-2诱导成骨细胞分化的信号通路而进行的蛋白质印迹分析表明，rhBMP-2以时间依赖性方式显著促进p38丝裂原活化蛋白激酶（MAPK）的磷酸化。用p38 MAPK抑制剂SB203580预处理可抑制rhBMP-2介导的分化和矿化。此外，用蛋白激酶D（PKD）抑制剂Go6976预处理细胞可抑制rhBMP-2诱导的p38磷酸化。这些发现提示，rhBMP-2在缺氧条件下通过激活PKD和p38 MAPK信号通路诱导MC3T3-E1细胞的分化和矿化。
