Transmission of ER stress response by ATF6 promotes endochondral bone growth.
Transmission of ER stress response by ATF6 promotes endochondral bone growth.
复制标题
ATF6 传递内质网应激反应促进软骨内骨生长
DOI：
10.1186/s13018-015-0284-7
复制
发表时间：
2015-09-15
影响因子：
2.6
通讯作者：
Guo FJ
中科院分区：
文献类型：
--
作者：
Xiong Z;Jiang R;Zhang P;Han X;Guo FJ
我们之前报道过X-box结合蛋白1剪接（XBP1S）是未折叠蛋白反应（UPR）的关键调节因子，作为骨形态发生蛋白2 （BMP2）诱导的转录因子，通过激活颗粒-上皮前体（GEP）软骨生长因子，积极调节软骨内骨形成。在内质网（ER）错误折叠或未折叠蛋白的胁迫下，哺乳动物的细胞可通过UPR受到保护。然而，活化转录因子6 (ATF6)， UPR的另一个转录臂，在bmp2诱导的软骨细胞分化中的影响尚未被阐明。在目前的研究中，我们调查和探索ATF6在软骨内骨形成中的作用，重点关注增生性软骨细胞分化的相关分子，以及这一过程背后的分子事件。方法采用高密度微块培养法诱导ATDC5和C3H10T1/2细胞向软骨细胞分化。通过实时荧光定量PCR、免疫印迹分析和免疫组织化学检测(1)ATF6、ATF6α、II型胶原、X型胶原和基质金属蛋白酶-13 （MMP13）的表达；(2)ATF6是否刺激软骨形成，ATF6是否增强Runx2介导的软骨细胞肥大。培养胎鼠骨外植体，检测ATF6是否刺激软骨细胞肥大、矿化和软骨内骨生长。采用共免疫沉淀法测定ATF6是否与Runx2在软骨细胞分化中有关联。结果satf6在bmp2诱导的软骨细胞分化过程中存在差异表达。ATF6的过表达加速了软骨细胞的分化，离体研究表明ATF6是软骨细胞肥大、矿化和软骨内骨生长的有效刺激物。通过siRNA途径敲低ATF6可抑制软骨形成。此外，ATF6与Runx2结合，增强Runx2诱导的软骨细胞肥大。并且通过siRNA途径抑制Runx2时，ATF6的刺激作用降低，提示Runx2需要ATF6的促进作用。结论ATF6通过激活runx2介导的增生性软骨细胞分化，积极调节软骨细胞肥大和软骨内骨形成。
BackgroundWe reported earlier that X-box binding protein1 spliced (XBP1S), a key regulator of the unfolded protein response (UPR), as a bone morphogenetic protein 2 (BMP2)-inducible transcription factor, positively regulates endochondral bone formation by activating granulin-epithelin precursor (GEP) chondrogenic growth factor. Under the stress of misfolded or unfolded proteins in the endoplasmic reticulum (ER), the cells can be protected by the mammalian UPR. However, the influence of activating transcription factor 6 (ATF6), another transcriptional arm of UPR, in BMP2-induced chondrocyte differentiation has not yet been elucidated. In the current study, we investigate and explore the role of ATF6 in endochondral bone formation, focus on associated molecules of hypertrophic chondrocyte differentiation, as well as the molecular events underlying this process.MethodsHigh-cell-density micromass cultures were used to induce ATDC5 and C3H10T1/2 cell differentiation into chondrocytes. Quantitative real-time PCR, immunoblotting analysis, and immunohistochemistry were performed to examine (1) the expression of ATF6, ATF6α, collagen II, collagen X, and matrix metalloproteinase-13 (MMP13) and (2) whether ATF6 stimulates chondrogenesis and whether ATF6 enhances runt-related transcription factor 2 (Runx2)-mediated chondrocyte hypertrophy. Culture of fetal mouse bone explants was to detect whether ATF6 stimulates chondrocyte hypertrophy, mineralization, and endochondral bone growth. Coimmunoprecipitation was employed to determine whether ATF6 associates with Runx2 in chondrocyte differentiation.ResultsATF6 is differentially expressed in the course of BMP2-triggered chondrocyte differentiation. Overexpression of ATF6 accelerates chondrocyte differentiation, and the ex vivo studies reveal that ATF6 is a potent stimulator of chondrocyte hypertrophy, mineralization, and endochondral bone growth. Knockdown of ATF6 via a siRNA approach inhibits chondrogenesis. Furthermore, ATF6 associates with Runx2 and enhances Runx2-induced chondrocyte hypertrophy. And, the stimulation effect of ATF6 is reduced during inhibition of Runx2 via a siRNA approach, suggesting that the promoting effect is required for Runx2.ConclusionsOur observations demonstrate that ATF6 positively regulates chondrocyte hypertrophy and endochondral bone formation through activating Runx2-mediated hypertrophic chondrocyte differentiation.