【Abstract】
Fibroblast growth factor 9 (Fgf9) is a well-known factor that regulates bone development; however, its function in bone homeostasis is still unknown. Previously, we identified a point mutation in the FGF9 gene (p.Ser99Asn, S99N) and generated an isogeneic knock-in mouse model, which revealed that this loss-of-function mutation impaired early joint formation and was responsible for human multiple synostosis syndrome 3 (SYNS3). Moreover, newborn and adult S99N mutant mice exhibited significantly increased bone mass, suggesting that Fgf9 also participated in bone homeostasis. Histomorphology, tomography and serological analysis of homozygous newborns and heterozygous adults showed that the Fgf9S99N mutation immensely increased bone mass and bone formation in perinatal and adult bones and decreased osteoclastogenesis in adult bone. An in vitro differentiation assay further revealed that the S99N mutation enhanced bone formation by promoting osteogenesis and mineralization of bone marrow mesenchymal stem cells (BMSCs) and attenuating osteoclastogenesis of bone marrow monocytes (BMMs). Considering the loss-of-function effect of the S99N mutation, we hypothesized that Fgf9 itself inhibits osteogenesis and promotes osteoclastogenesis. An in vitro differentiation assay revealed that Fgf9 prominently inhibited BMSC osteogenic differentiation and mineralization and showed for the first time that Fgf9 promoted osteoclastogenesis by enhancing preosteoclast aggregation and cell-cell fusion. Furthermore, specific inhibitors and in vitro differentiation assays were used and showed that Fgf9 inhibited BMSC osteogenesis mainly via the MEK/ERK pathway and partially via the PI3K/AKT pathway. Fgf9 also promoted osteoclastogenesis as a potential costimulatory factor with M-CSF and RANKL by coactivating the MAPK and PI3K/AKT signaling pathways. Taken together, our study demonstrated that Fgf9 is a negative regulator of bone homeostasis by regulating osteogenesis and osteoclastogenesis and provides a potential therapeutic target for bone degenerative diseases.
【中文摘要】
我們前期研究發現並證實人類成纖維細胞生長因子9（FGF9）基因單鹼基錯義突變（p.Ser99Asn, S99N）造成的FGF9功能缺失是人類多發性骨性連接綜合症（SYNS3）的致病原因。有意思的是，在構建的同源突變小鼠模型中，FGF9突變小鼠骨密度顯著升高，提示FGF9在骨代謝中也有重要作用，但目前其在骨穩態中的作用尚不明確。組織形態學、X光斷層掃描和血清學檢測實驗數據提示，與野生型小鼠相比，S99N突變小鼠成骨作用增強，破骨作用減弱，導致松質骨和皮質骨顯著增厚。體外分化實驗證實，S99N突變的成骨前體細胞向成骨細胞分化和礦化作用顯著減弱，而破骨前體細胞向成熟破骨細胞分化以及骨吸收能力均明顯下調。基於上述結果和前期研究基礎，我們提出假說，FGF9蛋白具有抑制成骨、促進破骨的作用。通過體外分化實驗首次證實FGF9通過誘導破骨前體細胞融合，促進破骨細胞分化和骨吸收。同時證實FGF9蛋白顯著抑制成骨細胞分化和礦化。機制研究發現，FGF9主要通過MEK/ERK和PI3K信號通路發揮抑制成骨，促進破骨的作用。綜上，我們的研究首次在體內外水平證實FGF9可抑制成骨、促進破骨，雙向調控骨穩態，為骨代謝異常疾病診治提供新的理論基礎。