Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and Induces Osteocyte Apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.Bcl2家族蛋白（Bcl2 subfamily proteins）包括Bcl2与Bcl-XL，可抑制细胞凋亡（apoptosis）。由于成骨细胞（osteoblast）凋亡在性激素缺乏、糖皮质激素过量及衰老状态下的骨质疏松症中具有部分致病作用，因此上调Bcl2的表达或可抑制骨丢失；然而，Bcl2过表达对成骨细胞分化、骨骼发育与稳态维持的影响尚未得到充分研究。为探究上述问题，本研究构建了两株成骨细胞特异性BCL2转基因小鼠。与野生型小鼠相比，BCL2转基因小鼠在6周龄时骨量升高，但10周龄时无此差异。在10周龄高表达BCL2的转基因小鼠中，成骨细胞与骨细胞（osteocyte）数量增加，但类骨质厚度与骨形成速率降低。在2周龄与6周龄时，BrdU阳性细胞数量升高，而TUNEL阳性细胞数量无明显变化。成骨细胞分化受到抑制，具体表现为Col1a1与骨钙素（osteocalcin）表达水平降低。从BCL2转基因小鼠颅盖骨中分离的原代成骨细胞，其体外分化能力同样下降。在与骨髓细胞共培养时，原代成骨细胞中过表达BCL2对破骨细胞生成（osteoclastogenesis）无影响。出乎意料的是，成骨细胞中过表达BCL2最终会引发骨细胞凋亡。骨细胞的突起数量减少，并逐渐出现凋亡相关结构改变与凋亡相关分子表达上调，最终死亡的骨细胞在皮质骨（cortical bone）中累积。上述研究结果表明，成骨细胞中过表达BCL2可抑制成骨细胞分化、减少骨细胞突起，并诱导骨细胞凋亡。