School of Basic Medicine, Shanxi Medical University, 030001, Taiyuan, Shanxi, China.
Mol Cell Biochem. 2019 Jul;457(1-2):21-29. doi: 10.1007/s11010-019-03508-9. Epub 2019 Feb 23.
Spermatogenesis is a complicated process that is tightly regulated by the well-coordinated expression of a series of genes in the testes. Ankyrin repeat domain-containing protein 49 (ANKRD49), an evolutionarily conserved protein highly expressed in the testes, is mainly found in spermatogonia, spermatocytes, and round spermatids. However, the exact function of ANKRD49 in spermatogenesis has remained elusive. In this study, we sought to investigate the role of ANKRD49 in apoptosis and determine the mechanism underlying this process in male germ cell-derived GC-1 cells. Nuclear staining with Hoechst 33258 and annexin V-FITC/PI, as well as analysis of caspase 3 activity, mitochondrial membrane potential, and apoptotic protein expression, showed that etoposide-induced apoptosis was attenuated by ANKRD49 overexpression but promoted by RNA interference-induced ANKRD49 knockdown. Furthermore, assessment of the levels of caspase 9, caspase 8, and proteins of the Bcl-2 family revealed ANKRD49 to be involved in an intrinsic apoptosis pathway. Examination of the subcellular distribution of the NF-κB p65 subunit after treatment with an NF-κB signaling inhibitor or p65 small interfering RNA demonstrated that ANKRD49 modulated etoposide-induced GC-1 cell apoptosis via the NF-κB pathway. Taken together, these results suggest that ANKRD49 plays an important role in reducing intrinsic apoptosis of GC-1 cells by modulating the NF-κB signaling pathway.
精子发生是一个复杂的过程，在睾丸中一系列基因的协调表达下受到严格调控。锚蛋白重复域蛋白 49（ANKRD49）是一种在睾丸中高度表达的进化上保守的蛋白质，主要存在于精原细胞、精母细胞和圆形精子细胞中。然而，ANKRD49 在精子发生中的确切功能仍不清楚。在这项研究中，我们试图研究 ANKRD49 在细胞凋亡中的作用，并确定该过程在雄性生殖细胞衍生的 GC-1 细胞中的机制。用 Hoechst 33258 和 annexin V-FITC/PI 进行核染色，以及分析 caspase 3 活性、线粒体膜电位和凋亡蛋白表达表明，ANKRD49 过表达可减轻依托泊苷诱导的细胞凋亡，但 RNA 干扰诱导的 ANKRD49 敲低则促进细胞凋亡。此外，评估 caspase 9、caspase 8 和 Bcl-2 家族蛋白的水平表明，ANKRD49 参与内在凋亡途径。在用 NF-κB 信号抑制剂或 p65 小干扰 RNA 处理后，检查 NF-κB p65 亚基的亚细胞分布表明，ANKRD49 通过 NF-κB 途径调节依托泊苷诱导的 GC-1 细胞凋亡。综上所述，这些结果表明，ANKRD49 通过调节 NF-κB 信号通路，在减少 GC-1 细胞的内在凋亡中发挥重要作用。
