ANKRD49 inhibits etoposide-induced intrinsic apoptosis of GC-1 cells by modulating NF-κB signaling.

ABSTRACT

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.