Calcitriol regulates the expression of the genes encoding the three key vitamin D3 hydroxylases and the drug-metabolizing enzyme CYP3A4 in the human fetal intestine.
OBJECTIVE
The human fetal jejunum has been shown to harbour the vitamin D3 (D3) nuclear receptor (VDRn) and to be responsive to calcitriol/1,25-dihydroxyvitamin D3[1,25(OH)2D3] through modulation of proliferation and differentiation processes. The aim of the study was to evaluate the presence as well as the effect of 1,25(OH)2D3 exposure on the expression levels of the three key D3-hydroxylase gene transcripts (25-hydroxylase, CYP27A; 24-hydroxylase, CYP24; 1alpha-hydroxylase, CYP27B1) as well as that of the 1,25(OH)2D3-responsive endobiotic/xenobiotic metabolizing enzyme CYP3A4 (which is also considered a major detoxifiying enzyme) in the human proximal and distal intestine.methods Specimens from normal fetuses ranging from 15 to 20 weeks of gestation were obtained following elective termination of normal pregnancies. Intestinal explants were cultured for a period of 24 h or 48 h with 10-7 m 1,25(OH)2D3. All data were compared to paired-control cultures without 1,25(OH)2D3. Total RNA was extracted and cDNA synthesized by RT-PCR. The cDNA obtained was amplified by radioactive PCR, the signal intensity evaluated by densitometric analyses and expressed in relation to the levels of GAPDH.


RESULTS
Data indicate that VDRn, the three D3-hydroxylases as well as CYP3A4 are expressed in all segments of the human fetal small intestine and in the colon. Basal expression levels of VDRn, CYP27A, CYP24 and CYP3A4 were found to be similar in the proximal, median and distal jejunum as well an in the proximal and distal colon. In contrast, basal 1alpha-hydroxylase CYP27B1 expression levels were found to be 65% higher in the colon than in the small intestine (P < 0.02). The 1alpha-hydroxylase was also found to be sensitive to 1,25(OH)2D3 with a 31% decrease in its expression levels within 24 h of 1,25(OH)2D3 exposure to reach a 55% decrease after 48 h of incubation in the presence of the hormone (P < 0.05). Furthermore, the levels of the 25-hydroxylase gene transcript were also decreased by 10% within the first 24 h and by 29% after 48 h of incubation in the presence of 1,25(OH)2D3 (P < 0.003). VDRn expression levels were also found to be reduced following incubation in the presence of 1,25(OH)2D3. In contrast, exposure to 1,25(OH)2D3 contributed to a 4.8 fold increase in the expression of the 24-hydroxylase gene transcript within the first 24 h of exposure (P < 0.03), and to a highly significant induction (24, 22 and 1.5 fold over basal values) of the CYP3A4 gene transcript in 3 of the 4 specimens studies.


CONCLUSIONS
Collectively, the data illustrate that at mid-gestation 1,25(OH)2D3 is fully active in the modulation of all D3-hydroxylases in the human developing intestine. They also show that the detoxifying enzyme CYP3A4 is not only present along the intestinal tract but is also sensitive to 1,25(OH)2D3, indicating that the hormone may be a key element in intestinal development and in the maintenance of the intestinal mucosa integrity in the basal state and in response to damage-inducing agents.
Theodoropoulos
Catherine
C
Département de Pharmacologie, Faculté de médecine, Centre de recherche, Centre Hospitalier de l'Université de Montréal, Université de Montréal, Québec, Canada.
Demers
Christian
C
Delvin
Edgard
E
Ménard
Daniel
D
Gascon-Barré
Marielle
M
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics; D002117:Calcitriol / Q000494:pharmacology*; D002762:Cholecalciferol / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase; D003106:Colon / Q000196:embryology / Q000201:enzymology; D046508:Culture Techniques; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D005865:Gestational Age; D006801:Humans; D007422:Intestines / Q000196:embryology / Q000201:enzymology*; D007583:Jejunum / Q000196:embryology / Q000201:enzymology; D007668:Kidney / Q000196:embryology / Q000201:enzymology; D008099:Liver / Q000196:embryology / Q000201:enzymology; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000235:genetics; D065668:Vitamin D3 24-Hydroxylase
D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol