TSC1蛋白调节破骨细胞的分化和吸收功能的研究

Tuberous sclerosis complex 1 (TSC1) is an important modulator of mammalian target of rapamycin(mTOR) activation in cell survival, apoptosis, proliferation and differentiation. Our previous data demonstrate that mice with osteoclast precursor-specific deletion of TSC1 exhibit high trabecular bone mass and lower urinary DPD/Creatinine, an effect predominantly associated with decreased osteoclast-mediated bone resorption. The knockout of TSC1 potently inhibited primary mouse osteoclast formation, measured as the number of tartrate-resistant acid phosphatase (TRAP) multinucleated cells in the bone marrow cell culture. Moreover, TSC1-deficient in mature osteoclast cells showed a high degree of cortical porosity in the long bones by microCT assay. To explore those dramatical phenotype, rapamycin, the inhibitor of mTOR, will be used to rescure the decreased bone resorption in TSC1 knockout mice. Furthermore, the deletion of mTOR in those osteoclast precursor cells and mature osteoclast cells will also be set up. Those experiments will determine if the function of TSC1 in osteoclast is dependent on mTOR signal pathway. The cell fusion is an important step for osteoclast formation, which involved in the upregulation of some essential regulators, such as DC-STAMP and ATP6V0d2. The protein level and mRNA level of them will be determined. As reported in other models of TSC1 deficiency, TSC1 deletion cells in osteoclast precursor cells were larger due likely to a role of mTORC1 in regulating cell size. These observations confirmed high anabolic level, which will raise an endoplasmic reticulum (ER) stress response. The association of ER stress marker, including Chop and BiP with osteoclast differentiation will be probed. The high cortical porosity in cortical bone implicates higher cell absorption, or higher cell survival, or lower cell apoptosis. Accordingly, the protein level and mRNA level of H+-ATPase and CTSK will be determined. NF-kappaB activity, which is the most important molecule for osteoclast survival, will be traced. In addition, the apoptosis pathway will be evaluated in those knockout mice. In conclusion, the aim of this study was to investigate the potential of setting TSC1 as the target for the treatment of osteoporosis.

TSC1蛋白是细胞内mTOR信号通路的一个关键抑制性调节蛋白，参与细胞的生存、凋亡、增殖和分化等。我们前期的工作发现，在小鼠破骨前体细胞（LysM-Cre）中敲除TSC1，小鼠股骨和腰椎骨的骨量显著增加，破骨细胞数显著减少，尿液中DPD/Creatinine显著减少；离体培养的骨髓细胞形成TRAP阳性的多核破骨细胞显著减少。而在成熟的破骨细胞（CTSK-Cre）中敲除TSC1后，小鼠股骨皮质骨中出现大量多孔的结构。本项目将通过给药rapamycin来建立针对TSC1敲除的挽救实验。随后在破骨前体细胞和成熟的破骨细胞中敲除mTOR，进行类比性实验。同时通过骨髓细胞的离体培养系统，进一步分析TSC1蛋白缺失后破骨前体细胞和成熟破骨细胞的生存、凋亡、增殖和分化，深入考察内质网应激和NF-kappaB信号通路，希望为临床防治骨质疏松提供新的靶点和思路。

TSC1蛋白是细胞内mTOR信号通路的一个关键抑制性调节蛋白，参与细胞的生存、凋亡、增殖和分化等。M-CSF/RANKL信号是破骨细胞胞膜上的触发开关，而mTOR信号通路是破骨前体细胞增殖、分化和破骨细胞生存和功能的胞内的控制开关，是研究破骨细胞分化和吸收功能的一个重要方向。.本研究发现在小鼠破骨前体细胞中，TSC1蛋白的缺失会促发mTOR信号通路中的磷酸化S6蛋白水平显著增加，导致单核细胞/巨噬细胞的细胞体积显著增大，而成熟的多核的破骨细胞数显著减少，最终小鼠尿液中DPD/Creatinine显著减少，股骨和腰椎骨的骨量显著增加。而在成熟的破骨细胞中，TSC1蛋白的缺失也会促发mTOR信号通路中的磷酸化S6蛋白水平显著增加，TRAP阳性染色的细胞体积增大，然而其吸收功能却降低，表现为actin-ring阳性染色的细胞数目显著减少，骨吸收陷窝面积显著减少，最终小鼠股骨和腰椎骨的骨小梁显著增加。腹腔连续注射rapamycin（3ug/g体重）40天(类似于促进TSC1蛋白的功能)，野生小鼠骨量（BV/TV）显著降低，而破骨细胞前体细胞中敲除小鼠（LysM-Cre; Tsc1f/f）给药后骨量也显著降低。.本研究证实了TSC1蛋白是破骨细胞增值、分化和吸收功能的重要的胞内的控制开关，失活TSC1蛋白的作用能够抑制破骨细胞的吸收功能，增加松质骨骨量，可为临床防治骨质疏松提供新的靶点和方法。