1,25-dihydroxyvitamin D3 stimulates activator protein-1-dependent CaCo-2 cell differentiation

1,25-Dihydroxyvitamin D-3 (1,25(OH)(2)D-3) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)(2)D-3 specifically activated protein kinase C-alpha (PKC-alpha) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates 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 mechanisms? 1,25(OH)(2)D-3 caused a time-dependent increase in AP-I DNA binding activity 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 activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)(2)D3 also increased AP-1 gene-transactivating activity. This AP-I activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that 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, and CaCo-2 cells stably transfected with antisense PKC-alpha cDNA, demonstrated that PKC-alpha mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 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 in CaCo-2 cells by a PKC-alpha- and JNK-dependent mechanism leading to increases in cellular differentiation.