Wang Yongmei, Menendez Alicia, Fong Chak, ElAlieh Hashem Z, Chang Wenhan, Bikle Daniel D
Endocrine Unit, University of California, Veterans Affairs Medical Center, San Francisco, CA, USA.
J Bone Miner Res. 2014 Aug;29(8):1900-13. doi: 10.1002/jbmr.2196.
Ephrin B2/EphB4 mediates interactions among osteoblasts (OBs), osteoclasts (OCLs), and chondrocytes to regulate their differentiation. We investigated the role of ephrin B2/EphB4 signaling in mediating the anabolic effects of insulin-like growth factor-I (IGF-I) and parathyroid hormone (PTH) on those cells and overall endochondral bone formation. Immunohistochemistry demonstrated that the expression of ephrin B2 in OBs, OCLs, and osteocytes, and the expression of EphB4 in OBs and osteocytes was dramatically decreased in global IGF-I knockout mice. Inactivation of EphB4 by EphB4 small, interfering RNA (siRNA) in cultured bone marrow stromal cells significantly decreased the mRNA levels of OB differentiation markers and abolished the stimulatory effects of IGF-I on these markers. Blocking the interaction of EphB4 and ephrin B2 in the OB-OCL cocultures with the EphB4 specific peptide TNYL-RAW or deletion of ephrin B2 in OCL prior to coculture led to fewer and smaller tartrate-resistant acid phosphatase (TRAP)-positive cells, decreased expression of OB differentiation markers, and blunted response to IGF-I for both OCL and OB differentiation. In the growth plate, both ephrin B2 and EphB4 are expressed in late stage proliferating and prehypertrophic chondrocytes, and their expression was decreased in mice lacking the IGF-I receptor specifically in chondrocytes. In vitro, blocking the interaction of EphB4 and ephrin B2 in chondrogenic ATDC5 cells with TNYL-RAW significantly decreased both basal and IGF1-induced expression of type II and type X collagen. In the cocultures of ATDC5 cells and spleen cells (osteoclast precursors), TNYL-RAW decreased the numbers of TRAP-positive cells and the expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and receptor activator of NF-κB (RANK), and blocked their stimulation by IGF-I. Our data indicate that IGF-I/IGF-IR signaling promotes OB, OCL, and chondrocyte differentiation via ephrin B2/EphB4 mediated cell-cell communication.
Ephrin B2/EphB4介导成骨细胞（OBs）、破骨细胞（OCLs）和软骨细胞之间的相互作用，以调节它们的分化。我们研究了ephrin B2/EphB4信号在介导胰岛素样生长因子-I（IGF-I）和甲状旁腺激素（PTH）对这些细胞的合成代谢作用以及整体软骨内骨形成中的作用。免疫组织化学显示，在全身性IGF-I基因敲除小鼠中，OBs、OCLs和骨细胞中ephrin B2的表达以及OBs和骨细胞中EphB4的表达显著降低。在培养的骨髓基质细胞中，用EphB4小干扰RNA（siRNA）使EphB4失活，显著降低了OB分化标志物的mRNA水平，并消除了IGF-I对这些标志物的刺激作用。在OB-OCL共培养物中，用EphB4特异性肽TNYL-RAW阻断EphB4与ephrin B2的相互作用，或在共培养前使OCL中的ephrin B2缺失，导致抗酒石酸酸性磷酸酶（TRAP）阳性细胞数量减少且体积变小，OB分化标志物的表达降低，并且OCL和OB分化对IGF-I的反应减弱。在生长板中，ephrin B2和EphB4在晚期增殖和前肥大软骨细胞中均有表达，并且在软骨细胞中特异性缺乏IGF-I受体的小鼠中它们的表达降低。在体外，用TNYL-RAW阻断软骨生成性ATDC5细胞中EphB4与ephrin B2的相互作用，显著降低了II型和X型胶原的基础表达以及IGF1诱导的表达。在ATDC5细胞和脾细胞（破骨细胞前体）的共培养物中，TNYL-RAW减少了TRAP阳性细胞的数量以及活化T细胞核因子细胞质1（NFATc1）和NF-κB受体激活剂（RANK）的表达，并阻断了IGF-I对它们的刺激。我们的数据表明，IGF-I/IGF-IR信号通过ephrin B2/EphB4介导的细胞间通讯促进OB、OCL和软骨细胞的分化。
