Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts
Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts
复制标题
膜去极化调节非兴奋性成骨细胞的细胞内 RANKL 运输
DOI：
10.1016/j.bone.2015.07.031
复制
发表时间：
2015
期刊：
影响因子：
4.1
通讯作者：
Yao H and Noda M.
中科院分区：
文献类型：
--
作者：
Notomi T;Kuno M;Ezura Y;Honma M;Ishizuka T;Ohura K;Yao H and Noda M.
甲状旁腺激素（PTH）和1α，25-二羟维生素D3（VD 3）是维持钙稳态的重要因素，通过调节核因子κ B受体激活因子配体（RANKL）mRNA的表达促进破骨细胞的生成。然而，其对RANKL细胞内转运（RANKLiT）的贡献，包括RANKL溶酶体囊泡（RANKL-lv）融合至细胞膜的触发因素尚不清楚。在神经元中，膜电位的去极化增加细胞内Ca 2+水平（[Ca 2 +]i），并通过突触囊泡与细胞膜的融合促进神经递质释放。为了确定膜去极化是否也调节细胞过程，如MC 3 T3-E1成骨细胞（OB）中的RANKLiT，我们产生了一个光敏OB细胞系，并开发了一个系统，通过传递蓝光刺激来改变其膜电位。在RANKL过表达OB的膜组分中，PTH和VD 3在应用后10 min增加膜结合RANKL（mbRANKL）水平，而不影响mRNA表达水平，并使细胞膜去极化，同时瞬时增加[Ca 2 +]i。在我们稳定表达通道视紫红质受体的新OB细胞系中，蓝光诱导的去极化增加了mbRANKL水平，这可通过L型电压门控Ca 2+通道和内质网Ca 2+释放阻断剂的治疗逆转。在破骨细胞趋化因子样RAW264.7细胞和过度表达RANKL的光敏OB的共培养物中，光刺激诱导抗酒石酸酸性磷酸酶活性增加并促进破骨细胞分化。这些结果表明，细胞膜的去极化是RANKL-lv融合到膜的触发器，并且膜电位有助于OB的功能。此外，VD 3诱导的RANKL-lv融合的非基因组作用包括膜结合的VD 3受体（1，25 D3-MARRS受体）。阐明PTH和VD 3对RANKLiT的调控机制，将有助于开发预防骨质疏松症和其他骨疾病骨丢失的药物。
Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3(VD3) are important factors in Ca2 +homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca2 +level ([Ca2 +]i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD3increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca2 +]i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers forL-type voltage-gated Ca2 +channels and Ca2 +release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD3-induced RANKL-lv fusion included the membrane-bound VD3receptor (1,25D3-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD3will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.