Duan Miao, Zhou Bowen, Zhou Xinrong, Yuan Gang
From the *Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology; †Department of Endocrinology, Wuhan No. 1 Hospital; and ‡Department of Endocrinology, Wuhan No. 6 Hospital, Wuhan, China.
J Investig Med. 2015 Jun;63(5):758-64. doi: 10.1097/JIM.0000000000000191.
Peroxisome proliferator-activated receptor γ (PPARγ) and Wnt play different roles in bone homeostasis. Thiazolidinediones are PPARγ agonists that cause bone mineral density loss. This study investigated the relationship between PPARγ and Wnt/β-catenin signaling in mouse osteoblastic MC3T3-E1 cells.
MC3T3-E1 cells were treated with either pioglitazone (Pio) or rosiglitazone (Rosi), thiazolidinediones, for 24 hours at 10 to 40-μM concentrations. Recombinant mouse Wnt3a protein (50 ng/mL) for 6 hours was also used to treat the 20-μM Pio and Rosi pretreated cells. Cell proliferation was measured by MTT, and apoptosis with flow cytometry using annexin V/propidium iodide staining; reverse transcriptase-polymerase chain reaction measured mRNA expression levels of LRP5/6 (low-density lipoprotein-related protein 5/6), glycogen synthase kinase 3β (GSK3β), TCF7L2 (transcription factor 7-like 2), PPARγ, and cyclin D1, and Western blots detected β-catenin and p-GSK3β proteins.
Pioglitazone and Rosi decreased MC3T3-E1 cell viability by 28.07% and 18.14% at 20 μM, respectively (P < 0.05). Apoptosis increased compared with controls (7.21%), after 20-μM treatment with Pio or Rosi, to 10.45% and 12.10%, respectively (P < 0.05). Both Pio and Rosi decreased β-catenin protein levels and increased p-GSK3β, but the LRP5/6, GSK3β, and TCF7L2 mRNA levels were constant. Upon activation of the Wnt pathway by mouse Wnt3a protein, β-catenin and p-GSK3β protein levels were reversed, accompanied with increased proliferation, but apoptosis remained high.
Activation of PPARγ in osteoblasts accompanied Wnt signaling suppression. Activation of Wnt signaling alleviated the PPARγ proliferation decreases but not the apoptosis increases. The thiazolidinedione PPARγ agonists act in part through inhibition of the Wnt signaling pathway, showing there is a relationship between PPARγ and Wnt signaling.
过氧化物酶体增殖物激活受体γ（PPARγ）和Wnt在骨稳态中发挥不同作用。噻唑烷二酮类药物是PPARγ激动剂，可导致骨密度降低。本研究调查了小鼠成骨细胞MC3T3-E1细胞中PPARγ与Wnt/β-连环蛋白信号通路之间的关系。
用10至40μM浓度的吡格列酮（Pio）或罗格列酮（Rosi）这两种噻唑烷二酮类药物处理MC3T3-E1细胞24小时。还使用重组小鼠Wnt3a蛋白（50 ng/mL）处理经20μM Pio和Rosi预处理的细胞6小时。通过MTT法测量细胞增殖，使用膜联蛋白V/碘化丙啶染色通过流式细胞术检测细胞凋亡；逆转录聚合酶链反应测量低密度脂蛋白相关蛋白5/6（LRP5/6）、糖原合酶激酶3β（GSK3β）、转录因子7样2（TCF7L2）、PPARγ和细胞周期蛋白D1的mRNA表达水平，蛋白质印迹法检测β-连环蛋白和磷酸化GSK3β蛋白。
20μM时，吡格列酮和罗格列酮分别使MC3T3-E1细胞活力降低28.07%和18.14%（P<0.05）。与对照组（7.21%）相比，用20μM Pio或Rosi处理后，细胞凋亡增加，分别达到10.45%和12.10%（P<0.05）。Pio和Rosi均降低β-连环蛋白蛋白水平并增加磷酸化GSK3β，但LRP5/6、GSK3β和TCF7L2的mRNA水平保持不变。用小鼠Wnt3a蛋白激活Wnt信号通路后，β-连环蛋白和磷酸化GSK3β蛋白水平逆转，同时细胞增殖增加，但细胞凋亡仍保持较高水平。
成骨细胞中PPARγ的激活伴随着Wnt信号通路的抑制。Wnt信号通路的激活减轻了PPARγ引起的增殖降低，但并未减轻凋亡增加。噻唑烷二酮类PPARγ激动剂部分通过抑制Wnt信号通路起作用，表明PPARγ与Wnt信号之间存在关联。
