1,25-dihydroxyvitamin D3 regulation of vascular endothelial growth factor in the C3H10T1/2 cell model of multi-stage carcinogenesis

Evidence suggests 1,25(OH)2D3 regulates production of the pro-angiogenic vascular endothelial growth factor (VEGF). Since angiogenesis is important to both cancer progression and bone formation, these studies investigated 1,25(OH)2D3 regulation of VEGF from multi-potent C3H10T½ mouse fibroblasts and C3H10T½ cells transfected with oncogenic Harvey-ras (ras, rasneo11a) cells, representing a model of multi-stage carcinogenesis. 1,25(OH)2D 3 caused C3H10T½ and rasneo11a cells to increase the release of an endothelial cell (EC) mitogen as treatment with conditioned media from 1,25(OH)2D3-treated C3H10T½ or rasneo11a cells significantly induced EC prolieration. 1,25(OH)2D3 induced 2–3 fold increases in VEGF release from C3H10T½ and rasneo11a cells. Contrary to published evidence of the pro-angiogenic effects of ras, rasneo11a cells secreted significantly less VEGF than C3H10T½ cells. The suppressive effect of ras was confirmed in MCF10A and MCF10Aras human breast epithelial cells. In C3H10T½ cells, 1,25(OH)2D3 significantly induced mRNA expression (2-fold, 2 hrs) and VEGF release (8 hrs) and 1,25(OH) 2D3 also induced VEGF promoter activation. Further, confluent cultures were more responsive to 1,25(OH)2D3 than subconfluent cultures even though basal levels of VEGF decreased with confluence. The apparent absence of vitamin D response elements within the VEGF promoter suggested 1,25(OH)2D3-induction of VEGF was due to activation of signal transduction pathways. Subsequently, the molecular mechanism by which 1,25(OH)2D3 stimulates VEGF mRNA expression and protein secretion from C3H10T½ cells was explored. VEGF mRNA expression and release required transcription, however VEGF release was not mediated through p38 or extracellular regulated kinase (ERK) mitogen activated protein kinase (MAPK) pathways. Instead, the primary mediator of VEGF promoter activity, mRNA expression, and VEGF release was phosphatidylinositol-3 kinase (PI3K). The VEGF promoter contains binding sites for nuclear factor κB (NFκB) and hypoxia inducible factor-1 (HIF-1) transcription factors which known to be downstream of PI3K. HIF-1 is a well defined regulator of VEGF. Inhibition of NFκB transcriptional activity did not block 1,25D-induced VEGF release. Alternatively, a role for HIF-1 is suggested because protein expression of HIF-1α, the regulated subunit of HIF-1, was induced by 1,25(OH) 2D3 in a PI3K-dependent manner. Therefore, these results are the first to suggest 1,25(OH)2D3 regulation of angiogenesis may be through induction of VEGF via a PI3K → HIF-1-dependent pathway