1,25-dihydroxyvitamin D-3 stimulates activator protein-1-dependent CaCo-2 cell differentiation

1,25-Dihydroxyvitamin D-3 (1,25(OH)(2)D-3) is a potential chemopreventive a gent for human colon cancer. We have reported that 1,25(OH)(2)D-3 specifica lly activated protein kinase C-alpha (PKC-alpha) and also caused a reductio n in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by wh ich this secosteroid influences these important cellular processes, however , remain unclear. The transcription factor, activator protein-1 (AP-1), reg ulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)(2)D-3 activated AP-1 in CaCo-2 cells and, if so, by what mechanism s? 1,25(OH)(2)D-3 caused a time-dependent increase in AP-I DNA binding acti vity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1,25(OH)(2)D-3 also induced a rapid and transient activa tion of ERK2 (where ERK is extracellular signal-regulated kinase) and a mor e persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfec tion experiments revealed that 1,25(OH)(2)D3 also increased AP-1 gene-trans activating activity. This AP-I activation was completely blocked by PD 0980 59, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as we ll as by a dominant negative JNK or a dominant negative Jun, indicating tha t the AP-1 activation induced by 1,25(OH)(2)D-3 was mediated by ERK and JNK . Using a specific inhibitor of the Ca2+-dependent PKC isoforms, Go6976, an d CaCo-2 cells stably transfected with antisense PKC-alpha cDNA, demonstrat ed that PKC-alpha mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly red uced 1,25(OH)(2)D-3-induced alkaline phosphatase activity, a marker of CaCo -2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)(2)D-3 stimulated AP-1 activation i n CaCo-2 cells by a PKC-alpha- and JNK-dependent mechanism leading to incre ases in cellular differentiation.