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

1,25-dihydroxycholecalciferol [1,25(OH)(2)D-3] is important in the regulation of cell growth, differentiation, and apoptosis. Previous results from our laboratory demonstrate that 1,25(OH)(2)D-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)(2)D-3, leading to protection from apoptosis. Results showed that nuclear factor kappaB (NFkappaB) transcriptional activity was significantly increased 1.8-fold over vehicle controls by 1,25(OH)(2)D-3 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)(2)D-3. Pretreatment with two PI3K inhibitors, LY294002 and wortmannin, abolished the activation of NFkappaB by 1,25(OH)(2)D-3, suggesting that this pathway is essential for NFkappaB transcriptional activation. Additionally, the use of a p-21 activated kinase (PAK1) inhibitory construct (PAK(R299)) demonstrated that PAK1 was also required for NFkappaB transcriptional activation by 1,25(OH)(2)D-3. Inhibition of NFkappaB activity with transfection of the NFkappaB inhibitory construct (IkappaB(Ala32)) abolished the protective effect of 1,25(OH)(2)D-3 on VES-mediated apoptosis. In summary, NFkappaB transcriptional activation was essential to 1,25(OH)(2)D-3 protection from VES-mediated apoptosis and 1,25(OH)(2)D-3 regulated NFkappaB activity through PI3K and PAK pathways.