Expression of 25-hydroxyvitamin D3-24-hydroxylase activity in Caco-2 cells. An in vitro model of intestinal vitamin D catabolism.
The C-24 oxidation pathway plays a major role in the degradation of vitamin D metabolites in kidney and other target tissues. The aim of the present study was to establish an intestinal cell culture system to study the mechanisms regulating the vitamin D catabolic pathway. 25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the catabolic sequence, was examined in Caco-2 cells, a human colon adenocarcinoma cell line which exhibits differentiated functions of absorbing intestinal epithelial cells. While untreated Caco-2 cells did not exhibit 24-hydroxylase activity, significant catabolic activity was induced by prior treatment of cell monolayers with 1,25-dihydroxyvitamin D3(1,25-(OH)2D3). Induced 24-hydroxylase D3 (25OHD3) and 1,25-(OH)2D3 was detected 6 h after treatment of cells with 10(-8)M 1,25-(OH)2D3, peaked at 16 h, and decreased thereafter. Treatment of cells with 10(-7) M 1,25-(OH)2D3 elicited a maximal 24-hydroxylase response. Comparable time courses of induction by 1,25-(OH)2D3 and 1,25-(OH)2D3-dose response curves were observed in cultured human skin fibroblasts and Caco-2 cells. 25OHD3 was not as good an inducer of the vitamin D catabolic pathway in Caco-2 cells as 1,25-(OH)2D3. Induction of 24-hydroxylase activity by 1,25-(OH)2D3 was inhibited by pretreatment of Caco-2 cells with either actinomycin D, alpha-amanitin, or cycloheximide suggesting that mRNA and protein synthesis are required for induction. The present study demonstrates that 1,25-(OH)2D3-treated Caco-2 cells express the vitamin D catabolic pathway and, therefore, constitute a useful in vitro model to study the mechanism of induction by 1,25-(OH)2D3.
Tomon
M
M
Medical Research Council Genetics Group, McGill University-Montreal Children's Hospital, Quebec, Canada.
Tenenhouse
H S
HS
Jones
G
G
D000230:Adenocarcinoma; D000546:Amanitins / Q000494:pharmacology; D002112:Calcifediol / Q000378:metabolism / Q000494:pharmacology; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology; D003110:Colonic Neoplasms; D003513:Cycloheximide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System; D003609:Dactinomycin / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006900:Hydroxylation; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D013250:Steroid Hydroxylases / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D014807:Vitamin D / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D000546:Amanitins; D014807:Vitamin D; D003609:Dactinomycin; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol; D002112:Calcifediol