Department of Endocrinology, Xiangya Hospital of Central South University, Changsha, Hunan, China.
Life Sci. 2012 Apr 9;90(13-14):509-18. doi: 10.1016/j.lfs.2012.01.005. Epub 2012 Jan 20.
This study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells.
Rat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5mM d-glucose) and HG media (25.5mM d-glucose) for up to 14days. l-Glucose (20mM plus 5.5mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot.
Measurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs.
HG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.
本研究旨在探讨高糖（HG）对骨髓基质细胞中 TGF-β1 表达的影响及其机制。
将大鼠骨髓多能成体祖细胞（MAPCs）分别在正常（5.5mM d-葡萄糖）和 HG 培养基（25.5mM d-葡萄糖）中培养，最长可达 14 天。l-葡萄糖（20mM 加 5.5mM d-葡萄糖）用作高渗透压对照。通过定量 RT-PCR、ELISA 和免疫荧光染色检测细胞和条件培养基中 TGF-β1 的 mRNA 和蛋白水平来评估 TGF-β1 的表达。用 Western blot 检测 MAPCs 在 HG 培养基中培养时 ERK1/2 和 STAT3 的表达和激活。
在正常培养基中培养的细胞中检测到可测量水平的 TGF-β1。在 HG 培养基中培养 36 小时后，MAPCs 中 TGF-β1 的表达显著增加，mRNA 增加超过 20 倍，蛋白水平增加 5 倍以上。有趣的是，HG 培养基中 MAPCs 的 ERK1/2 磷酸化显著增加，而 STAT3（Tyr705）而非 STAT3（Ser727）磷酸化显著减少。用特定的 MEK1 抑制剂 PD98059 或 U0126 处理细胞可抑制 ERK1/2 磷酸化和 TGF-β1 表达，并完全恢复 HG 培养基中 MAPCs 中 STAT3（Tyr705）磷酸化水平。用特异性 STAT3 磷酸化抑制剂 AG490 处理细胞可显著阻断 STAT3（Tyr705）磷酸化，增加 TGF-β1 表达，而对 MAPCs 中 ERK1/2 磷酸化无影响。
HG 通过增强 ERK1/2 信号抑制 MAPCs 中 STAT3（Tyr705）来增加 TGF-β1 表达。
