Microarray on osteoblasts made from bone marrow stromal cells from Bmp2 flox/flox and Bmp2; Prx1-Cre mice. Mus musculus

Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, supporting pre-Osx1+ osteoprogenitors as a critical source and target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) is to date an unidentified transcription factor in the osteoblast gene regulatory network that is induced during bone development and bone repair, and acts upstream of Osx in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives critical regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3. Overall design: Gene level differential expression analysis was performed with Affymetrix Mouse ST 2.1 microarrays on bone marrow stromal cells from Bmp2F/F or Bmp2Prx1Δ/Δ mice that were differentiated for 7 days in osteogenic medium alone, plus BMP2 (100 ng/ml), plus Wnt3a (40 ng/ml), or BMP2 plus Wnt3a. Cultures were performed in biological triplicates using pooled cells from n≥3 mice/genotype. Traditional cDNA, generated from 1 ug total RNA using EcoDry Premix (Clonetech), was used for pre-microarray and post-microarray validation by QPCR. Eight cDNA libraries were made from 100 ng total RNA using WT Expression Kit (Ambion): 1 pooled sample from 3 independent cultures of Bmp2F/F cells grown in osteogenic medium (33.33 ng each for a total of 100 ng); 1 pooled sample from 3 independent cultures of Bmp2Prx1Δ/Δ cells + BMP2 + Wnt3a (33.33 ng each for a total of 100 ng); 3 samples from 3 independent cultures of Bmp2F/F cells + Wnt3a (100 ng each); and 3 samples from 3 independent cultures of Bmp2Prx1Δ/Δ cells + Wnt3a (100 ng each). Data was analyzed with Expression Console and Transcriptome Analysis Console v3.0 (free from Affymetrix). Gene expression was reported as linear fold change relative to Bmp2Prx1Δ/Δ cells + Wnt3a, and p-value was calculated by one-way between-subject ANOVA for unpaired samples.

增强型骨形态发生蛋白（Bone Morphogenetic Protein, BMP）或经典Wnt（canonical Wnt, cWnt）信号通路是目前用于促进骨形成与骨折修复的治疗策略，但这两条通路分别调控成骨细胞（osteoblast）细胞命运的具体机制仍未明确。在骨骼表达的所有骨形态发生蛋白中，我们发现仅特异性缺失Bmp2即可阻断经典Wnt信号通路从肢芽或骨髓祖细胞定向分化为成骨细胞的过程。当暴露于经典Wnt配体刺激时，Bmp2缺陷细胞无法通过Runx2/Osx1检查点，因此无法上调成骨细胞中多个调控矿物质代谢的基因。在Osx1诱导后缺失Bmp2的细胞，在经典Wnt刺激下可正常分化，这表明Osx1阳性前体成骨祖细胞是BMP2发挥作用的关键来源与作用靶点。本研究进一步发现，成骨细胞基因调控网络中迄今尚未被鉴定的转录因子Grainyhead样蛋白3（Grainyhead-like 3, Grhl3）会在骨发育与骨修复过程中被诱导表达，并以BMP2依赖的方式作用于Osx的上游。因此，Runx2向Osx1的转变过程依赖于BMP2提供的关键调控信号，这一信号无法由经典Wnt信号通路代偿，且该调控至少部分通过Grhl3的诱导与激活介导。

实验整体设计：采用Affymetrix Mouse ST 2.1基因芯片（Affymetrix Mouse ST 2.1 microarrays）对两组骨髓基质细胞进行基因层面的差异表达分析，两组细胞分别来自Bmp2F/F小鼠及Bmp2Prx1Δ/Δ小鼠，均在成骨诱导培养基中分别单独培养7天，或添加100 ng/ml BMP2、40 ng/ml Wnt3a，或同时添加BMP2与Wnt3a。所有培养实验均设置3次生物学重复，每个基因型的细胞均取自至少3只小鼠的混合细胞。采用EcoDry Premix试剂盒（Clonetech）从1 μg总RNA中反转录得到的常规cDNA，可用于芯片实验前后的实时定量PCR（Quantitative Real-time PCR, QPCR）验证。采用WT Expression Kit（Ambion）从100 ng总RNA中构建了8个cDNA文库，具体分组如下：1份混合样本，取自3份独立培养的Bmp2F/F成骨诱导培养基细胞（每份33.33 ng，总RNA总量100 ng）；1份混合样本，取自3份独立培养的Bmp2Prx1Δ/Δ细胞+ BMP2 + Wnt3a（每份33.33 ng，总RNA总量100 ng）；3份独立样本，分别取自3份独立培养的Bmp2F/F细胞+ Wnt3a（每份总RNA 100 ng）；以及3份独立样本，分别取自3份独立培养的Bmp2Prx1Δ/Δ细胞+ Wnt3a（每份总RNA 100 ng）。芯片数据采用Expression Console与Transcriptome Analysis Console v3.0软件（由Affymetrix官方免费提供）进行分析。基因表达量以相对于Bmp2Prx1Δ/Δ细胞+ Wnt3a组的线性倍数变化表示，P值通过针对非配对样本的单因素组间方差分析（one-way between-subject ANOVA）计算得到。