MAPK Signaling has Stage-dependent Osteogenic Effects on Human Adipose-derived Stem Cells in vitro

<b>PURPOSE</b>: The use of pro-osteogenic growth factors, such as BMP2, in human adipose-derived stem cell (ASC) osteogenesis is well described. Because these growth factors work via signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascade, a study of the relationship between MAPK signaling and ASC osteogenesis was conducted. <b>METHODS</b>: ERK, JNK, and p38MAPK activation were measured in ASCs osteo-induced using either dexamethasone or vitamin D3 and correlated to mineralization. Activation and mineralization were also measured without dexamethasone or using the glucocorticoid, cortisone. The expression of the MAPK phosphatase, MKP1, and its relationship to mineralization was also assessed. The effect of decreasing MAPK activation on mineralization through the use of exogenous inhibitors was examined along with siRNA-knockdown and adenoviral overexpression of ERK1/2. Finally, the effect of ERK1/2 overexpression on ASCs induced on PLGA scaffolds was assessed. <b>RESULTS</b>: ASC mineralization in dexamethasone or vitamin D3-induced ASCs correlated with both increased ERK1/2 and JNK1/2 activation. ASCs induced without dexamethasone also mineralized, with JNK1/2 signaling possibly mediating this event. No link between cortisone induction and MAPK signaling could be ascertained. ASCs treated with ERK, JNK, or p38MAPK inhibitors showed decreased osteogenic gene expression and diminished mineralization. Mineralization levels were also affected by viruses designed to inhibit or augment ERK1/2 expression and activity. Finally, ASC mineralization appeared to be a balance between MAPK kinase activity and MKP1. <b>CONCLUSIONS</b>: It is likely that MAPK signaling plays a significant role in ASC osteogenesis, affecting differentiation in kinase- and stage-specific manners.

**研究目的**：促骨生成生长因子（如骨形态发生蛋白2（Bone Morphogenetic Protein 2, BMP2））应用于人脂肪来源干细胞（Adipose-derived Stem Cell, ASC）进行成骨分化的相关研究已有充分报道。此类生长因子通过丝裂原活化蛋白激酶（Mitogen-activated Protein Kinase, MAPK）级联反应等信号转导通路发挥作用，因此本研究针对MAPK信号通路与ASC成骨分化之间的关联展开。**研究方法**：分别采用地塞米松或维生素D3诱导ASC成骨分化，检测细胞外调节蛋白激酶（Extracellular Regulated Protein Kinase, ERK）、c-Jun氨基末端激酶（c-Jun N-terminal Kinase, JNK）及p38丝裂原活化蛋白激酶（p38 Mitogen-activated Protein Kinase, p38MAPK）的激活水平，并将其与细胞矿化程度进行关联分析。同时在未添加地塞米松、或使用糖皮质激素可的松的条件下，检测上述激酶激活水平与细胞矿化程度。此外，本研究还评估了MAPK磷酸酶MKP1（MAPK Phosphatase 1, MKP1）的表达水平及其与细胞矿化的关联。通过外源性抑制剂处理、小干扰RNA（small interfering RNA, siRNA）敲低以及腺病毒介导的ERK1/2过表达，探究降低MAPK激活水平对细胞矿化的影响。最后，检测了ERK1/2过表达对在聚乳酸-羟基乙酸共聚物（Poly(lactic-co-glycolic acid), PLGA）支架上诱导的ASC成骨分化的作用。**研究结果**：在地塞米松或维生素D3诱导的ASC中，细胞矿化程度与ERK1/2及JNK1/2的激活水平均呈正相关。未添加地塞米松诱导的ASC同样可发生矿化，该过程可能由JNK1/2信号通路介导。未发现可的松诱导与MAPK信号通路激活之间存在明确关联。经ERK、JNK或p38MAPK抑制剂处理的ASC，其成骨相关基因表达水平降低，矿化程度也显著减弱。ERK1/2表达与活性的抑制或增强型病毒载体处理同样会影响细胞矿化水平。最终发现，ASC的矿化程度取决于MAPK激酶活性与MKP1之间的动态平衡。**研究结论**：MAPK信号通路在ASC成骨分化过程中发挥重要调控作用，其可通过激酶特异性及阶段特异性的方式影响细胞分化进程。