转录调节因子ATF4对破骨细胞分化的调节

发布时间：2018-08-04 19:22

【摘要】：骨骼是一个不断更新的具有多种功能的器官,包括调节钙平衡,支撑软组织提供造血场所等。这些功能是通过骨组织的不管更新,即重塑而完成的。破骨细胞是唯一具有骨吸收能力的多核细胞。破骨细胞来源于定向的髓系前体细胞。多种细胞及其分泌的因子可以调节破骨细胞的分化及功能,尤其是骨髓基质细胞及其分泌的M-CSF和RANKL。过度的骨溶解是多种病理性损伤中的一个重要的临床问题,如癌症骨转移,类风湿关节炎,骨质疏松以及Paget's骨病等。因此临床上需要有效的能抑制骨过度溶解或促进骨生成的治疗方法,以预防或减轻骨质疏松的发生,提高病人的生活质量。过去的十年里,在解析破骨细胞生成方面有许多重要的突破,破骨细胞的分化过程及功能越来越清晰。许多因子在这过程中发挥重要的作用,如M-CSF, RANKL, OPG, PU.1以及MITF等。激活转录因子4(ATF4)是一种重要的转录因子,通过定点突变的方法,ATF4最早被发现对于eye lens纤维的形成至关重要。随后越来越多的研究证明其在成骨细胞的分化及骨形成的过程中发挥着重要的作用。有研究证明ATF4可以通过影响成骨细胞产生RANKL的量来间接调节破骨细胞的分化和骨吸收。但是目前尚未见有关ATF4可以直接调节破骨细胞的分化的研究报道。我们认为ATF4可以直接影响破骨细胞的分化。首先,我们通过蛋白印迹的方法以及免疫组织化学染色的方法确立了ATF4在破骨细胞系中的表达,并且通过磷酸酶处理的方法,发现了其磷酸化形式的存在。然后我们利用了功能丧失和获得的方法确立了此因子在破骨细胞形成中的直接作用。在Atf4-/-小鼠的骨中,Trap阳性区域所占的比例明显减小,信号强度也减弱；在体外的分化实验中,由Atf4-/-BMM细胞所形成的多核破骨细胞MNCs(大于等于三个核)的数量也显著减少。而且通过骨片溶解吸收实验(Pitassay)可知,在体外由Atf4-/-BMM所形成破骨细胞的骨吸收凹陷数量也明显减少,但是骨吸收凹陷数目与MNCs数目的比值没有明显的变化,说明ATF4敲除后,破骨细胞的骨吸收能力没有明显改变。在由破骨细胞特异的Trap启动子驱动下,使ATF4在破骨细胞中转基因过表达时,转基因小鼠呈现出明显的骨减少症；血清CTX的水平大幅度升高；破骨细胞的形成在体内和体外都有显著增加,并且与破骨细胞分化相关的基因的表达水平,不论是蛋白水平还是mRNA水平都显著上调。进一步研究发现,由Atf4-/-小鼠的骨髓细胞所形成的GM-CFU的集落数量也明显少于野生型对照组,GM-CFU是具有向破骨细胞分化能力的最原始的造血前体细胞。将Atf4-/-BMM细胞与野生型的成骨细胞共培养或用高浓度的RANKL来刺激其分化,都不能弥补其向破骨细胞分化的缺陷。RANKL的信号是由其受体RANK来传递的。我们通过免疫组织化学染色的方法以及免疫印迹方法发现,在Atf4-/-BMM细胞中,RANK的表达显著降低,并且其mRNA水平也不能被M-CSF所上调。此外RANKL对多条MAPK信号通路的诱导激活也受ATF4的调节。在缺乏ATF4的状态下,RANKL对于三条MAPK的通路的激活能力明显下降,而NF-kB途径、PI3K/Akt途径却不受影响。但是,ATF4对于M-CSF的信号却没有明显的调节作用。到目前为止,NFATcl是破骨细胞分化的最关键基因。无论是在体内还是在体外,ATF4的缺乏都导致NFATcl的表达水平明显下降。用逆转录病毒为载体,使NFATcl在ATF4WT和KO BMM中过表达,可以以剂量依赖性的方式使Trap阳性的MNCs增多。我们利用腺病毒作为载体,在BMM细胞中过表达ATF4,发现ATF4可以以剂量依赖性的方式上调NFATcl蛋白的表达水平。在体外,ATF4可以与NFATcl的启动子结合并能剂量依赖性的激活NFATcl (?)勺启动子,另外,通过ChIP assay的方法可以发现ATF4可以与NFATcl近启动子端的片段相作用并且该作用可以在RANKL的诱导下增强。在BMM细胞中,ATF4的蛋白水平受M-CSF以及PI3K/AKT途径的调节。在缺乏M-CSF的情况下,ATF4的蛋白水平以时间依赖性的方式明显减少,M-CSF可以阻断这一进程。M-CSF的这一作用可以被PI3K/Akt途径的抑制剂LY294002阻断,并且随着LY2094002的浓度的增高,BMM细胞向破骨细胞的分化也相应的受抑制。ATF4的缺乏可以使BMM的分化由破骨细胞系向巨噬细胞系迁移,导致巨噬细胞数量的增多。我们的研究结果证明ATF4在调节破骨细胞分化方面有着重要的内在作用,这也许可以作为治疗与破骨细胞相关的骨疾病的治疗靶点。
[Abstract]:Bone is a constantly updated organ with multiple functions, including regulating calcium balance, supporting the soft tissue to provide hematopoiesis, and so on. These functions are accomplished through the regeneration of bone tissue, that is, remolding. Osteoclasts are the only multinucleated cells with bone absorptive capacity. Osteoclasts are derived from directional myeloid precursor cells. Cells and their secreted factors can regulate the differentiation and function of osteoclasts, especially bone marrow stromal cells and their secreted M-CSF and RANKL. osteolysis, which are important clinical problems in a variety of pathological injuries, such as cancer bone metastases, rheumatoid arthritis, osteoporosis and Paget's bone disease. Effective methods of inhibiting the excessive dissolving of bone or promoting bone formation in order to prevent or reduce the occurrence of osteoporosis and improve the quality of life of the patient. In the past ten years, there are many important breakthroughs in the analysis of osteoclast formation, the differentiation process and function of osteoclast are becoming more and more clear. Many factors play a heavy role in this process. The role of M-CSF, RANKL, OPG, PU.1 and MITF. Activation of transcription factor 4 (ATF4) is an important transcription factor. Through site directed mutagenesis, the earliest discovery of ATF4 is essential to the formation of eye lens fibers. More and more studies have shown that it plays an important role in the process of osteoblast differentiation and bone formation. Some studies have shown that ATF4 can indirectly regulate the differentiation and bone resorption of osteoclasts by affecting the amount of RANKL produced by osteoblasts. However, there has been no study on the differentiation of osteoclasts directly regulated by ATF4. We think that ATF4 can directly affect the differentiation of osteoclast.
First, we established the expression of ATF4 in the osteoclast cell line by the method of Western blot and immunohistochemical staining, and found the form of phosphorylation by the method of phosphatase treatment. Then we used the method of function loss and acquisition to establish the direct of this factor in the formation of osteoclast. In the bone of Atf4-/- mice, the proportion of the Trap positive region was significantly reduced and the signal intensity was weakened; in the differentiation experiment in vitro, the number of MNCs (more than three nuclei) formed by Atf4-/-BMM cells was also significantly reduced. And by the bone dissolving absorption experiment (Pitassay), it is known that Atf4 in vitro The number of bone resorption sags of osteoclasts formed in -/-BMM also decreased significantly, but the ratio of the number of bone resorption depression to the number of MNCs did not change significantly, indicating that the bone resorption capacity of osteoclasts was not significantly changed after ATF4 knockout. Under the drive of osteoclast specific Trap promoter, ATF4 was genetically modified in osteoclasts. At the time of arrival, the transgenic mice showed obvious osteopenia, the level of serum CTX increased significantly, and the formation of osteoclasts increased significantly in both in vivo and in vitro, and the level of gene expression related to osteoclast differentiation, both in protein level and in mRNA level, was significantly up-regulated.
Further studies have found that the number of colonies of GM-CFU formed by bone marrow cells from Atf4-/- mice is also significantly less than that in the wild type control group, and GM-CFU is the most primitive hematopoietic progenitor cells that have the ability to differentiate into osteoclasts.
To co culture Atf4-/-BMM cells with wild type osteoblasts or to stimulate their differentiation with high concentration of RANKL, the signal to the defect to osteoclast differentiation,.RANKL, is transmitted by its receptor RANK. We found that RANK in Atf4-/-BMM cells through immunohistochemical staining and immunological trace methods. The expression was significantly reduced and its mRNA level could not be raised by M-CSF. In addition, the activation of RANKL on multiple MAPK signaling pathways was also regulated by ATF4. In the absence of ATF4, the activation ability of RANKL to the three MAPK pathway was significantly decreased, while the NF-kB pathway was not affected by the PI3K/Akt path. There is no obvious adjustment.
So far, NFATcl is the most critical gene for osteoclast differentiation. The lack of ATF4 in both in vivo and in vitro leads to a significant decline in the expression level of NFATcl. Using retrovirus as the carrier, NFATcl is overexpressed in ATF4WT and KO BMM, and the positive MNCs of Trap can be increased in a dose-dependent manner. We use adenosis. As a carrier, ATF4 is overexpressed in BMM cells, and it is found that ATF4 can increase the expression level of NFATcl protein in a dose dependent manner. In vitro, ATF4 can be combined with NFATcl promoter and can activate the NFATcl (?) spoon promoter in a dose-dependent manner. In addition, ATF4 can be found by ChIP assay method and NFATcl near promoter. In BMM cells, the protein level of ATF4 is regulated by the M-CSF and the PI3K/AKT pathway. In the absence of M-CSF, the protein level of ATF4 is significantly reduced in a time dependent manner, and M-CSF can block this process of.M-CSF by PI3K/Akt, which can be blocked by M-CSF. The inhibitor LY294002 of the diameter is blocked, and with the increase of the concentration of LY2094002, the differentiation of BMM cells to the osteoclast is also corresponding to the lack of.ATF4, which can cause the differentiation of BMM to migrate from the osteoclast line to the macrophage system, which leads to the increase of the number of macrophages. Our research results show that ATF4 is regulating the osteoclast differentiation side. The surface has important internal effects, which may be used as a therapeutic target for osteoclast related bone diseases.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.1

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