Mechanisms of nuclear vitamin D receptor resistance to transcriptional activity in ras-transfected cells
The active form of vitamin D, 1,25dihydroxyvitamin D 3 (1,25(OH)2D3) has been shown to regulate cell growth and apoptosis. Gene transcription is regulated by 1,25(OH)2D 3 when it binds to the nuclear vitamin D receptor (nVDR), which heterodimerizes with the retinoid X receptor (RXR), and this complex enters the nucleus and associates with vitamin D responsive elements in the promoter of target genes. The mitochondrial enzyme, cytochrome P45024 (CYP24, or 24-hydroxylase), converts 1,25(OH)2D3 to an inactive metabolite. The gene for this enzyme is a target of 1,25(OH)2D3. Previous results have shown a significantly greater increase in the transcriptional activity of the nVDR, as assessed by a CYP24 luciferase reporter gene, in 1,25(OH)2D3-treated murine fibroblast (C3H10T1/2) cells compared with 1,25(OH)2D3-treated C3HIOT1/2 cells stably transfected with the Harvey ras oncogene. The purpose of these studies was to determine the mechanisms for the differential activation of the nVDR, in this cellular model of multistage carcinogenesis. Previous studies showed that the difference in activation was not due to a difference in the expression of the nVDR or RXR protein, or phosphorylation of RXR by extracellular signal-regulated protein kinase (ERK). The current studies show that the nVDR does not bind DNA in 1,25(OH)2D3-treated ras- transfected cells. There was a significant increase in the nVDR activity when phosphatidylinositol 3-kinase (PI3K) was inhibited with LY294002 in 1,25(OH)2D 3-treated ras-transfected fibroblasts compared to their vehicle control cells. These results suggest that PI3K may be mediating the ras oncogene suppression of the nVDR transcriptional activity. Additional studies in our laboratory show that 1,25(OH)2D3 protects C3H10T1/2 cells but not ras-transfected cells from apoptosis and that mitogen activated protein kinase phosphatase-1 (MKP-1) plays a role in 1,25(OH)2D3-mediated inhibition of apoptosis in C3H10T1/2 cells. MKPs are a family of dual-specificity phosphatases that de-phosphorylate and thus inactivate mitogen activated protein kinases. The other purpose of the current studies was to determine if 1,25(OH)2D3 was regulating the expression of MKP-1 in this cell model. However, 1,25(OH) 2D3 did not change the expression of MKP-1 in C3H10T1/2 or ras-transfected cells. Thus, the PI3K pathway is implicated in 1,25(OH)2D3-mediated differential activity of the nVDR.
Teegarden, Purdue University.
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