Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells.
Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells.
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DOI：
10.1002/jcb.23426
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发表时间：
2012-03
影响因子：
4
通讯作者：
Xiao, Zhousheng
中科院分区：
文献类型：
--
作者：
Qiu, Ni;Zhou, Honghao;Xiao, Zhousheng
在小鼠模型中，PKD 1的突变和/或缺失导致成骨细胞功能减弱和骨形成缺陷;然而，PKD 1在人类成骨细胞和骨中的功能仍然不确定。在本研究中，我们使用慢病毒介导的shRNA技术稳定地敲低人成骨细胞MG-63细胞系中的PKD 1，并研究PKD 1对人成骨细胞功能的作用和分子机制。我们发现，在稳定转染的MG-63细胞中，PKD 1 shRNA使PKD 1减少53%，导致细胞增殖增加，成骨细胞分化受损，这反映在与对照shRNA MG-63细胞相比，BrdU掺入增加，碱性磷酸酶活性降低，钙沉积增加，RUNX 2和OSTERIX表达降低。此外，PKD 1 mRNA的敲低导致稳定的PKD 1 shRNA MG-63细胞中脂肪生成增强，如脂质蓄积增加和脂肪细胞相关标志物（如PPARγ和aP 2）表达增加所证明。稳定的PKD 1 shRNA MG-63细胞表现出较低的基础细胞内钙，这导致响应于流体流动剪切应力的细胞溶质钙信号减弱，以及响应于毛喉素（10 μM）刺激的细胞内cAMP信息增加。此外，当用PKA抑制剂H89（1 μM）处理稳定的PKD 1 shRNA MG-63细胞时，细胞增殖增加，成骨细胞分化抑制，成骨和成脂基因标记物显著逆转。这些结果表明，PKD 1在人MG-63细胞中的下调通过细胞内钙-cAMP/PKA信号通路导致成骨细胞功能缺陷。
Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARγ and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 μM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1 μM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.
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