A PDGFRβ-PI3K signaling axis mediates periosteal cell activation during fracture healing

Insufficient and delayed fracture healing remain significant public health problems with limited therapeutic options. Phosphoinositide 3-kinase (PI3K) signaling, a major pathway involved in regulation of fracture healing, promotes proliferation, migration, and differentiation of osteoprogenitors. We have recently reported that knock-in mice with a global increase in PI3K signaling (gCblYF) show enhanced femoral fracture healing characterized by an extraordinary periosteal response to injury. Interestingly, of all growth factor receptors involved in fracture healing, PI3K directly binds only to PDGFR. Given these findings, we hypothesized a PDGFR-PI3K interaction is necessary for mediating robust periosteal cell activation following fracture. In this study, we isolated primary periosteal cells from gCblYF mice to analyze cross-talk between the PDGFRβ and PI3K signaling pathways. We found PDGFRβ signaling contributes to robust Akt phosphorylation in periosteal cells in comparison with other growth factor signaling pathways. Additionally, we performed femoral fractures on gCblYF mice with a conditional removal of PDGFRβ in mesenchymal progenitors using inducible alpha smooth muscle actin (αSMA) CreERT2 mice. Our studies showed that depletion of PDGFRβ signaling within these progenitors in the early phase of fracture healing significantly abrogates PI3K-mediated periosteal activation and proliferation three days after fracture. Combined, these results suggest that PDGFRβ signaling through PI3K is necessary for robust periosteal activation in the earliest phases of fracture healing.骨折愈合不充分与延迟仍是亟待解决的重大公共卫生难题，当前可供选择的治疗方案十分有限。磷酸肌醇3-激酶（Phosphoinositide 3-kinase，PI3K）信号通路是调控骨折愈合的核心通路之一，可促进骨祖细胞（osteoprogenitors）的增殖、迁移与分化。我们近期的研究报道，全局PI3K信号通路活性上调的gCblYF敲入小鼠表现出更优异的股骨骨折愈合能力，其特征为损伤后骨膜反应显著增强。有趣的是，在参与骨折愈合的各类生长因子受体中，PI3K仅能直接结合血小板衍生生长因子受体（PDGFR）。基于上述发现，我们提出假说：PDGFR与PI3K的相互作用，是介导骨折后骨膜细胞充分激活的必要条件。本研究中，我们从gCblYF小鼠体内分离原代骨膜细胞（primary periosteal cells），以分析血小板衍生生长因子受体β（PDGFRβ）与PI3K信号通路间的交叉调控机制。我们发现，相较于其他生长因子信号通路，PDGFRβ信号可显著提升骨膜细胞内丝氨酸/苏氨酸蛋白激酶B（Akt）的磷酸化水平。此外，我们利用诱导型α平滑肌肌动蛋白（α smooth muscle actin，αSMA）CreERT2小鼠，在间充质祖细胞（mesenchymal progenitors）中条件性敲除PDGFRβ，并对gCblYF小鼠实施股骨骨折造模。研究结果显示，在骨折愈合早期，敲除这些祖细胞内的PDGFRβ信号，可显著阻断PI3K介导的骨膜细胞激活与增殖，该效应在骨折后3天尤为显著。综合以上结果，我们认为在骨折愈合的初始阶段，通过PI3K传导的PDGFRβ信号通路，是实现骨膜细胞充分激活的必要条件。