Khalid Aysha B, Pence Jacquelyn, Suthon Sarocha, Lin Jianjian, Miranda-Carboni Gustavo A, Krum Susan A
Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center, Memphis, TN, United States of America.
Department of Medicine, University of Tennessee Health Science Center, Memphis, TN, United States of America; Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN, United States of America.
Bone. 2021 Mar;144:115819. doi: 10.1016/j.bone.2020.115819. Epub 2020 Dec 16.
GATA4 is a transcription factor that regulates osteoblast differentiation. However, GATA4 is expressed at a higher level in mesenchymal stem cells (MSCs) than in osteoblasts. Therefore, the role of GATA4 in limb bud mesenchyme differentiation was investigated in mice by knocking out Gata4 using Cre-recombinase controlled by the Prx1 promoter (herein called Gata4 Prx-cKO mice). μCT analysis of the Gata4 Prx-cKO mice showed a decrease in trabecular bone properties compared with wildtype (Gata4) littermates. Gata4 Prx-cKO mice have fewer MSCs as measured by CFU-F assays, mesenchymal progenitor cells (MPC2) (flow cytometry of Sca1/CD45/CD34/CD44) and nestin immunofluorescence. Gata4 Prx-cKO bone marrow-derived MSCs have a significant reduction in WNT ligands, including WNT10B, and WNT signalosome components compared to control cells. Chromatin immunoprecipitation demonstrates that GATA4 is recruited to enhancers near Wnt3a, Wnt10b, Fzd6 and Dkk1. GATA4 also directly represses YAP in wildtype cells, and the absence of Gata4 leads to increased YAP expression. Together, we show that the decrease in MSCs is due to loss of Gata4 and a WNT10B-dependent positive autoregulatory loop. This leads to a concurrent increase of YAP and less activated β-catenin. These results explain the decreased trabecular bone in Gata4 Prx-cKO mice. We suggest that WNT signalosome activity in MSCs requires Gata4 and Wnt10b expression for lineage specification.
GATA4是一种调节成骨细胞分化的转录因子。然而，GATA4在间充质干细胞（MSC）中的表达水平高于成骨细胞。因此，通过使用由Prx1启动子控制的Cre重组酶敲除Gata4（在此称为Gata4 Prx-cKO小鼠），在小鼠中研究了GATA4在肢芽间充质分化中的作用。对Gata4 Prx-cKO小鼠的μCT分析显示，与野生型（Gata4）同窝小鼠相比，小梁骨特性有所下降。通过CFU-F分析、间充质祖细胞（MPC2）（Sca1/CD45/CD34/CD44的流式细胞术）和巢蛋白免疫荧光检测，Gata4 Prx-cKO小鼠的MSC较少。与对照细胞相比，Gata4 Prx-cKO骨髓来源的MSC中WNT配体（包括WNT10B）和WNT信号体成分显著减少。染色质免疫沉淀表明，GATA4被招募到Wnt3a、Wnt10b、Fzd6和Dkk1附近的增强子。GATA4在野生型细胞中也直接抑制YAP，Gata4的缺失导致YAP表达增加。我们共同表明，MSC的减少是由于Gata4的缺失和WNT10B依赖性正自调节环的丧失。这导致YAP同时增加，β-连环蛋白活化减少。这些结果解释了Gata4 Prx-cKO小鼠小梁骨减少的原因。我们认为，MSC中的WNT信号体活性需要Gata4和Wnt10b表达来进行谱系定向。
