1,25-Dihydroxycholecalciferol inhibits apoptosis in C3H10T1/2 murine fibroblast cells through activation of nuclear factor κB

Abstract

1,25-dihydroxycholecalciferol [1,25(OH)2D3] is important in the regulation of cell growth, differentiation, and apoptosis. Previous results from our laboratory demonstrate that 1,25(OH)2D 3 inhibits vitamin E succinate (VES) mediated apoptosis in untransformed C3H10T1/2 mouse fibroblast cells. The current work investigated cell survival signaling pathways that may be activated by 1,25(OH) 2D3, leading to protection from apoptosis. Results showed that nuclear factor κB (NF κB) transcriptional activity was significantly increased 1.8-fold over vehicle controls by 1,25(OH) 2D3 after 4 h of treatment. Protein kinase B/AKT, a downstream effector of phosphoinositide 3-kinase (PI3K), was activated 4-fold and 8-fold at 2 and 4 h, respectively, after treatment with 1,25(OH) 2D3. Pretreatment with two PI3K inhibitors, LY294002 and wortmannin, abolished the activation of NF κB by 1,25(OH) 2D3, suggesting that this pathway is essential for NF κB transcriptional activation. Additionally, the use of a p-21 activated kinase (PAK1) inhibitory construct (PAKR299) demonstrated that PAK1 was also required for NF κB transcriptional activation by 1,25(OH) 2D3. Inhibition of NF κB activity with transfection of the NF κB inhibitory construct (IκBAta32) abolished the protective effect of 1,25(OH)2D3 on VES-mediated apoptosis. In summary, NF κB transcriptional activation was essential to 1,25(OH)2D3 protection from VES-mediated apoptosis and 1,25(OH)2D3 regulated NF κB activity through PI3K and PAK pathways.