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.
Highlights
Nuclear receptors, such as the vitamin D3 receptor (VDR) and retinoid X receptor, interact with the transcription factor activator protein-1 (AP-1) in a complex manner [12,13,14]
We asked whether AP-1 and its upstream kinases were activated by 1,25(OH)2D3 in CaCo-2 cells and, if so, by what mechanisms? The present studies demonstrated that 1,25(OH)2D3 rapidly increased c-jun gene expression at both transcriptional and translational levels and induced rapid protein kinase C (PKC)-dependent activation of ERK2 and JNK1
1,25(OH)2D3 in CaCo-2 Cells—Since Ca2ϩ-dependent PKC isoforms were implicated in the 1,25(OH)2D3-induced AP-1 activation, and this secosteroid activated PKC-␣ in CaCo-2 cells, we examined the potential role of PKC-␣ in 1,25(OH)2D3-induced activation of AP-1
Summary
1,25(OH)2D3, 1,25-dihydroxyvitamin D3; RPA, RNase protection assay; EMSA, electrophoretic mobility shift assay; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular-signal regulated kinase; JNK, Jun N-terminal kinase; dn-Jun, dombiological metabolite of vitamin D3, has been suggested to be a potential chemopreventive agent of human colon cancer (reviewed in Ref. 1). The VDR-secosteroid complex heterodimerizes with the retinoid X receptor to bind to unique promoter sequences within the genome, i.e. vitamin D response elements, which have been demonstrated to alter the expression of genes involved in the regulation of cell growth, differentiation, and apoptosis [2,3,4]. AP-1 DNA binding and transcriptional activities generally correlate with an increase in the abundance of the AP-1 complex, as well as with changes in the phosphorylation of the regulatory sites of its subunits [15, 22] Based on these observations, we asked whether AP-1 and its upstream kinases were activated by 1,25(OH)2D3 in CaCo-2 cells and, if so, by what mechanisms? Inhibition of JNK activation or suppression of c-Jun expression demonstrated that AP-1 activation by 1,25(OH)2D3 played an important role in stimulating cell differentiation
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