1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation

Abstract

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-1 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)D(3) also increased AP-1 gene-transactivating activity. This AP-1 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 Ca(2+)-dependent PKC isoforms, Gö6976, 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.