转录因子NFIB通过影响Wnt/β-Catenin信号调控成骨细胞分化和骨稳态的作用及其机制研究

The nuclear factor I/B (NFIB) transcription factor is a site-specific DNA-binding protein that plays a critical role in regulating stem cell differentiation, with Nfib null mice exhibiting defects such as severely delayed brain and lung maturation. The role of NFIB in bone development and homeostasis has never been explored. Our preliminary data showed that Nfib mRNA was induced in primary cultured murine stromal cell and established murine progenitor cell lines after adipogenic treatment but remained relatively stable after osteogenic treatment. Overexpressing Nfib in murine ST2 stromal cell and MC3T3-E1 preosteoblastic cell inhibited the cells to fully differentiate into osteoblasts, evidenced by alleviated alkaline phosphatase staining and reduced expression of osteoblast-specific genes including Runx2, osterix, osteocalcin, ALP and osteopontin. Conversely, knockdown of Nfib induced osteogenic differentiation of the cells. By contrast, overexpressing Nfib promoted ST2 stromal cell and C3H10T1/2 mesenchymal cell to differentiate into mature adipocytes, along with the induction of adipocyte-specific transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα), and the marker genes adipocyte protein 2 (aP2) and adipsin. Conversely, knockdown of the endogenous Nfib suppressed adipogenic differentiation from ST2 and C3H10T1/2. Further investigation demonstrated that NFIB positively regulated Dkk1 and Sfrp4 expression and negatively affected nuclear protein levels of β-Catenin and TCF7L2 in ST2, suggesting that NFIB might reciprocally regulate osteoblast and adipocyte differentiation from marrow stromal stem cell and progenitor cell through interacting with Wnt/β-Catenin signaling. In the present study we will further investigate the regulatory role of NFIB in osteoblast and adipocyte differentiation using primary cultured marrow stromal cell. Then the promoter studies and ChIP assay will be done to see if NFIB could activate the promoter of Dkk1 and/or Sfrp4 gene(s) through binding the putative NFI binding sequences. We will develop Nfib-floxed (Nfibfx/fx) mice and then make osteoblast progenitor-specific Nfib knockout mice by crossing the Nfibfx/fx mice with Osx-Cre mice. The phenotypes of the conditional knockout (cKO) mice will be analyzed. In brief, bone mass and bone mineral density of tibias will be studied by using micro-CT, X-ray, and histomorphological approaches. The numbers of active osteoblasts and osteoclasts in the tibia and adipocytes in the marrow will be measured. The levels of osteoblast- and osteoclast-related proteins including DKK1 and SFRP4 in serum as well as in the tibia tissues will be studied. The bone formation rate and mineral apposition rate will be determined as well. Osteoblast and osteoclast formation assays, using the bone marrow cells and calvarial cells isolated from the cKO mice and the control mice, will also be carried out to demonstrate if the Nfib deletion in progenitor cells results in altered differentiation of osteoblasts and activity of Wnt/β-Catenin signaling. If the above mentioned measurements give us positive results, we will perform ovariectomy with the female adult mice and study if the Nfib deletion in progenitor cell helps to block the development of osteoporosis. Our proposed studies will provide novel insights into the mechanisms of osteoblast development and bone homeostasis, and implicate potential novel approach for bone loss recovery.

我们前期研究发现转录因子NFIB调节骨髓基质细胞系和间充质细胞系等的定向分化，抑制其成骨分化而促进成脂分化。NFIB与经典Wnt通路存在相互作用，促进其抑制因子Dkk1和Sfrp4的表达，减少胞核中β-Catenin和TCF7L2的蛋白水平。因此提出NFIB通过抑制经典Wnt通路而调控成骨细胞发育，继而影响骨稳态。本研究将进一步确证NFIB对原代骨髓基质细胞成骨/成脂分化的调节作用；探讨NFIB是否通过蛋白-DNA相互作用而促进Dkk1和Sfrp4转录，或通过蛋白-蛋白相互作用而下调经典Wnt通路活性，进而调节干细胞和前体细胞的分化方向；建立成骨前体细胞Nfib基因敲除鼠模型，从整体和组织学水平，结合体外细胞和分子生物学实验，观察NFIB缺失对成骨细胞分化和骨稳态的影响及其与Wnt/β-Catenin信号的关系，观察NFIB缺失后小鼠抗骨质疏松能力。本研究将深化对骨发育和骨稳态规律的认识。

我们前期研究发现转录因子NFIB调节骨髓基质细胞系和间充质细胞系等的定向分化，抑制其成骨分化而促进成脂分化。本项目在此基础上，进一步确证了NFIB对原代骨髓基质细胞成骨/成脂分化的调节作用，发现NFIB抑制骨髓基质干细胞成骨分化而促进其成脂分化；探讨了NFIB调节骨髓基质干细胞定向分化的分子机制，发现NFIB通过结合Sfrp4基因启动子而促进Sfrp4转录，进而抑制经典Wnt通路活性；SFRP4促进基质细胞向脂肪细胞分化而抑制其向成骨细胞分化，敲减Sfrp4水平可以有效缓解NFIB对成骨细胞分化的抑制作用而对脂肪细胞分化的促进作用。建立了小鼠体内转染siRNA模型，发现在小鼠骨髓腔转染Nfib siRNA可使成骨细胞增加而脂肪细胞减少。建立了Nfib基因敲除鼠和Nfia基因敲入鼠模型，进行了生物表型分析。与对照小鼠相比，3月龄Osx-Cre; Nfibfx/fx小鼠未表现出明显骨量变化。分离骨髓基质细胞进行成骨/成脂诱导，与对照小鼠细胞相比，Osx-Cre; Nfibfx/fx小鼠的骨髓基质细胞成骨分化明显增强而成脂分化明显减弱，与体外研究结论一致。考虑到NFIA和NFIB可能存在功能冗余，也同时进行了NFIA调节骨髓基质干细胞分化的体内外研究。体外研究表明NFIA通过抑制经典Wnt信号通路而减弱骨髓基质干细胞成骨分化并促进成脂分化。成骨细胞条件性Nfia基因敲入鼠主要表现为长骨干骺端松质骨小梁数量显著增加而破骨细胞减少，但其成骨细胞Rankl/Opg比值不下降，提示NFIA除了调节成骨细胞分化外，以不依赖RANKL方式调控破骨细胞分化而影响机体骨稳态和骨量。本研究将深化对骨发育和骨稳态规律的认识。

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