1,25-dihydroxycholecalciferol inhibits apoptosis in C3H10T1/2 murine fibroblast cells through activation of nuclear factor kappaB.

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.