ICMR-National Institute for Research in Reproductive Health, Mumbai 400012, India.
Institute of Bioinformatics, Bengaluru 560066, India; Syngene International Ltd, Bengaluru 560099, India.
Biochim Biophys Acta Mol Cell Res. 2019 Sep;1866(9):1498-1507. doi: 10.1016/j.bbamcr.2019.06.014. Epub 2019 Jun 28.
Estrogen increases bone formation by promoting mineralization and prolonging the lifespan of osteoblasts. To understand the underlying molecular mechanism/s, we identified estrogen-regulated proteins at different stages of human osteoblast differentiation using differential proteomics approach. Among the identified proteins, we observed that estrogen upregulated RAB3GAP1 on day 1 and 5 of differentiation. RAB3GAP1 is critically involved in the process of autophagy, a eukaryotic degradative pathway essential for cell survival. We, therefore, investigated the effect of estrogen on autophagy in differentiating human osteoblasts and their precursors, the mesenchymal stem cells (MSCs). MSCs exhibited high autophagic flux which declined during osteoblast differentiation, resulting in high basal apoptosis in osteoblasts. Estrogen reduced apoptosis in differentiating osteoblasts by promoting autophagy, thus contributing towards their longer lifespan. Further, MSCs were resistant against starvation-induced apoptosis, whereas, differentiating osteoblasts showed significant susceptibility towards it. Estrogen, in addition to promoting mineralization, protected differentiating osteoblasts from starvation-induced apoptosis by increasing autophagic flux. Autophagic flux in RAB3GAP1 knockdown osteoblasts appeared diminished, and showed increased apoptosis even in nutrient-rich conditions, and exhibited significantly impaired mineralization. However, irrespective of the presence of estrogen, starvation further enhanced apoptosis in these cells. Furthermore, estrogen failed to promote mineralization in these osteoblasts. Our study illustrates that autophagy is essential for human osteoblast survival and mineralization, and osteoblasts are susceptible to apoptosis due to reduced autophagy during differentiation. Estrogen, via upregulation of RAB3GAP1, promotes autophagy in osteoblasts during differentiation thereby increasing their survival and mineralization capacity. Our study demonstrates the positive role of autophagy in bone homeostasis.
雌激素通过促进矿化和延长成骨细胞的寿命来增加骨形成。为了了解潜在的分子机制，我们使用差异蛋白质组学方法在人类成骨细胞分化的不同阶段鉴定了雌激素调节的蛋白质。在鉴定的蛋白质中，我们观察到雌激素在分化的第 1 天和第 5 天上调了 RAB3GAP1。RAB3GAP1 在内体自噬过程中起关键作用，内体自噬是细胞存活所必需的真核降解途径。因此，我们研究了雌激素对分化中的人成骨细胞及其前体细胞间充质干细胞 (MSC) 中自噬的影响。MSC 表现出高自噬通量，该通量在成骨细胞分化过程中下降，导致成骨细胞中高基础凋亡。雌激素通过促进自噬减少分化中的成骨细胞凋亡，从而延长其寿命。此外，MSC 对饥饿诱导的凋亡具有抗性，而分化中的成骨细胞对此表现出明显的敏感性。除了促进矿化外，雌激素还通过增加自噬通量来保护分化中的成骨细胞免受饥饿诱导的凋亡。RAB3GAP1 敲低的成骨细胞中的自噬通量似乎减少，即使在营养丰富的条件下也显示出增加的凋亡，并表现出明显受损的矿化。然而，无论是否存在雌激素，饥饿都会进一步增强这些细胞中的凋亡。此外，雌激素未能促进这些成骨细胞的矿化。我们的研究表明，自噬对于人成骨细胞的存活和矿化是必不可少的，并且由于分化过程中自噬减少，成骨细胞容易发生凋亡。雌激素通过上调 RAB3GAP1，在成骨细胞分化过程中促进自噬，从而提高其存活率和矿化能力。我们的研究表明自噬在骨稳态中起积极作用。
