Cell Biology Poster Session
Before acquiring all its biological potential, vitamine D3 (D3) must first be transformed into 25-hydroxyvitamin D3 by the 25-hydroxylase situated mainly in the liver, and then into 1,25 dihydroxyvitamin D3 by the 1a-hydroxylase found principally in the kidney. 1, 25 dihydroxyvitamin D3 is the hormonal form of the vitamin . Once the 1,25 dihydroxyvitamin D3 levels reach a certain plateau, CYP24, a 24-hydroxylase situated mainly in the kidney and intestin, transforms 1,25 dihydroxyvitamin D3 into 1,24, 25 trihydroxyvitamin D3, a product with a decreased hormonal activity. The mitochondrial sterol 27-hydroxylase (CYP27) catalyses the first reaction in the oxidation of the side chain in the acidic bile acid biosynthesis pathway. It has also been shown to be active in the C-25 hydroxylation of vitamin D3 and 1a-vitamin D3 in animal models and in the human Hepatoma cell line HepG2. Moreover, studies have indicated that the hydroxylation of D3 was regulated by the D3 status and suggestions have been made that 1,25 dihydroxyvitamin D3 was an inhibitor of the enzyme. Previous studies in our laboratory have shown similar CYP27 mRNA levels in normal and diseased human livers. In addition, rat steady state mRNA levels of the rat mitochondrial D3 25-hydroxylase encoded by CYP27 was found not to be regulated by extracellular calcium, nor by the hormonal or nutritional status in vitamin D. The aim of the present study was to investigate the regulation and expression of CYP27 mRNA levels in intestin.
Materials and Methods
-2 groups of male Sprague-Dawley rats (200-250g) were used, depleted in vitamin D3 and hypocalcemic -group 1 was injected i.v.with 2.4,12,120 and 240nmol/kg of 1, 25 dihydroxyvitamin D3, 6 hours before being sacrificed (n=3, for each dose used) -group 2 was implanted i.p.with osmotic mini-pumps releasing 28pmol/day of 25OHD3 and sacrificed after 1, 3, 5 and 7 days of treatment (n=3 for each day of treatment) -the duodena were removed (first 2 cm of small intestin) -RNA was extracted by the guanidium isothyocyanate method and placed on a nylon membrane -a CYP27 404bp fragment (corresponding to the 399 and 803bp within the sequence) , and a CYP24 247bp fragment were used as hybridization probes
Intestinal CYP27 mRNA levels were found to be progressively downregulated with increasing doses (2.4 to 240nmol/kg) of 1,25(OH)2D3. The levels were decreased 50%, compared to vitamin D3 and hypocalcemic rats, in the rats treated with 120 and 240nmol/kg (p<0.001). In parallel, CYP24 steady state expression level increased with the increasing pharmacological doses (p<0,05).
figure 1: Influence of an acute dose of 1,25(OH)2D3 on intestinal CYP27 and CYP24 mRNA steady state levels
Intestinal CYP27 exhibits a progressive decrease in mRNA level steady state expression, starting at day 1 (10% decrease) and becoming statistically significant after 48 hours of 25OHD3 administration (p<0.05, 33% decrease), and decreasing down to 45% of levels observed at day 0 after one week of treatment. The decrease in CYP27 mRNA was shown to be linear over the one week period with an r2 value of 0.67 (p<0.002)
figure 2: Effect of 25OHD3 on intestinal CYP27 and CYP24 mRNA steady state levels
Discussion and Conclusion
We have been able to demonstrate for the first time that CYP27 mRNA steady state levels can by downregulated in the intestin by the physiological levels of 25OHD3 and pharmacological levels of 1,25(OH)2D3.
It has been known for some years that the hepatic calciferol 25-hydroxylase activity could be modulated by vitamin D3. However, studies with the livers of the same rats in this study were inconclusive, and suggested an upregulation of CYP27 mRNA levels, for those treated with the pharmacological levels of 1,25(OH)2D3.
Our studies in the intestine suggest a reciprocal regulation between the two cytochrome P450, CYP27 and CYP24. Indeed, acute administration of 1,25(OH)2D3 resulted in a dose dependent down regulation of CYP27 mRNA levels and an upregulation of CYP24 levels, when compared to calcium and vitamin D3 deficient controls.
The present studies, thus, suggest that the intestine is a tissue involved in both the anabolism and catabolism of vitamin D3.
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|Theodoropoulos, C; Demers, C; Gascon-Barré, M; (1998). The Intestinal Vitamin D3-25 Hydroxylase. Regulation by 25OHD3 and 1,25(OH)2D3.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/cellbio/theodoropoulos0496/index.html|
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