School of Pharmacy, Sungkyunkwan University, Suwon, Gyunggi-do 16419, Republic of Korea.
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia.
Biochim Biophys Acta Gen Subj. 2017 Nov;1861(11 Pt A):2663-2673. doi: 10.1016/j.bbagen.2017.07.008. Epub 2017 Jul 14.
The 53kDa protein pneumolysin (PLY) is the main virulence factor of Streptococcus pneumoniae, a leading cause of invasive pneumococcal diseases. PLY forms pores in cholesterol-containing membranes, thereby interfering with the function of cells. Bone destruction is a serious matter in chronic inflammatory diseases such as septic arthritis and osteomyelitis. S. pneumoniae is increasingly being recognized as a common cause of septic arthritis, but its pathogenesis is poorly defined.
We examined the effect of PLY on osteoblast differentiation and its mechanisms of action. The effect of PLY on osteoblast differentiation was evaluated by qRT-PCR, ALP activity assay, flow cytometric analysis, and Western blotting. We also examined the role of PLY-induced autophagy in osteoblast differentiation using RNA interference analysis.
PLY inhibited osteoblast differentiation by decreasing the expression of osteoblast marker genes such as Runx2 and OCN, along with ALP activity. ROS production was increased by PLY during osteoblast differentiation. PLY induced autophagy through ROS-mediated regulation of AMPK and mTOR, which downregulated the expression of Sp1 and subsequent inhibition of differentiation. Treatment with autophagy inhibitors or Atg5 siRNA alleviated the PLY-induced inhibition of differentiation.
The results suggest that PLY inhibits osteoblast differentiation by downregulation of Sp1 accompanied by induction of autophagy through ROS-mediated regulation of the AMPK/mTOR pathway.
This study proposes a molecular mechanism for inhibition of osteoblast differentiation in response to PLY.
53kDa 蛋白肺炎球菌溶血素（PLY）是肺炎链球菌的主要毒力因子，是侵袭性肺炎球菌病的主要原因。PLY 在含有胆固醇的膜中形成孔，从而干扰细胞的功能。骨破坏是关节炎和骨髓炎等慢性炎症性疾病的严重问题。越来越多的证据表明肺炎链球菌是关节炎的常见原因，但发病机制尚未明确。
我们研究了 PLY 对成骨细胞分化的影响及其作用机制。通过 qRT-PCR、ALP 活性测定、流式细胞术分析和 Western blot 分析评估 PLY 对成骨细胞分化的影响。我们还通过 RNA 干扰分析研究了 PLY 诱导的自噬在成骨细胞分化中的作用。
PLY 通过降低成骨细胞标记基因如 Runx2 和 OCN 的表达以及 ALP 活性来抑制成骨细胞分化。在成骨细胞分化过程中，PLY 会增加 ROS 的产生。PLY 通过 ROS 介导的 AMPK 和 mTOR 的调节诱导自噬，下调 Sp1 的表达，从而抑制分化。自噬抑制剂或 Atg5 siRNA 的处理缓解了 PLY 诱导的分化抑制。
这些结果表明，PLY 通过下调 Sp1 抑制成骨细胞分化，同时通过 ROS 介导的 AMPK/mTOR 通路调节诱导自噬。
本研究提出了PLY 抑制成骨细胞分化的分子机制。
