Han Xiaofeng, Zhang Peng, Jiang Rong, Xia Fei, Li Meiling, Guo Feng-Jin
Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing, 400016, China.
Histochem Cell Biol. 2014 Nov;142(5):497-509. doi: 10.1007/s00418-014-1233-y. Epub 2014 Jun 17.
We previously report that BMP2 mediates mild ER stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. The transcription factor activating transcription factor 6 (ATF6), a key regulator of the UPR, is known to be important for ER stress-mediated apoptosis and cell growth, but the molecular mechanism underlying these processes remains unexplored. In this study, we demonstrate that ATF6 is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. ATF6 can enhance the level of IRE1a-spliced XBP1S protein in chondrogenesis. IRE1a and ATF6 can synergistically regulate endogenous XBP1S gene expression in chondrogenesis. Furthermore, overexpression ATF6 inhibited, while ATF6-knockdown enhanced, the cell proliferation in chondrocyte development with G1 phase arresting, S phase reducing and G2-M phase delaying. Besides, Ad-ATF6 can activate, whereas knockdown ATF6 by an siRNA-silencing approach inhibited, ER stress-mediated apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, CHOP, p-JNK expression in the course of chondrocyte differentiation. On the other hand, FCM, TUNEL assay and immunohistochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that Ad-ATF6 activation of the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of ATF6 in regulating ER stress-mediated apoptosis in chondrocyte differentiation and the molecular mechanisms involved.
我们之前报道过，骨形态发生蛋白2（BMP2）介导轻度内质网应激激活的活化转录因子6（ATF6），并在软骨形成过程中直接调节XBP1S的剪接。哺乳动物未折叠蛋白反应（UPR）可保护细胞免受内质网（ER）中错误折叠蛋白的应激。无法适应内质网应激会导致UPR触发细胞凋亡。转录因子ATF6是UPR的关键调节因子，已知其对内质网应激介导的细胞凋亡和细胞生长很重要，但这些过程背后的分子机制仍未被探索。在本研究中，我们证明ATF6在BMP2刺激的软骨细胞分化过程中差异表达，并在生长板软骨细胞中表现出显著表达。ATF6可增强软骨形成过程中肌醇需求酶1α（IRE1α）剪接的XBP1S蛋白水平。IRE1α和ATF6可协同调节软骨形成过程中内源性XBP1S基因的表达。此外，过表达ATF6会抑制软骨细胞发育中的细胞增殖，而敲低ATF6则会增强细胞增殖，导致G1期停滞、S期减少和G2-M期延迟。此外，腺病毒载体-ATF6（Ad-ATF6）可激活软骨形成过程中BMP2诱导的内质网应激介导的细胞凋亡，而通过小干扰RNA（siRNA）沉默方法敲低ATF6则会抑制这种凋亡，这通过软骨细胞分化过程中裂解的半胱天冬酶3、C/EBP同源蛋白（CHOP）、磷酸化应激活化蛋白激酶（p-JNK）的表达来检测。另一方面，流式细胞术（FCM）、末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）检测和免疫组织化学分析也在体外和体内证实了这一结果。结果表明，Ad-ATF6激活了发育中的软骨细胞组织中的内质网应激特异性半胱天冬酶级联反应。总的来说，这些发现揭示了ATF6在调节软骨细胞分化过程中内质网应激介导的细胞凋亡中的新关键作用及其涉及的分子机制。
