Identification of a 25-hydroxyvitamin D3 1alpha-hydroxylase gene transcription product in cultures of human syncytiotrophoblast cells.
Although accumulating data show that placenta is able to synthesize 1,25-dihydroxyvitamin D3, the presence of cytochrome P(450) enzyme capable of converting 25-hydroxyvitamin D3 (250HD(3)) to the biologically active form of vitamin D in this tissue, has not been yet clearly established. In this study, we have investigated the presence of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-(OH)ase) gene expression products in cultured human syncytiotrophoblast. Total RNA was isolated from cultured placental cells and subjected to Northern blots or RT-PCR by using 1alpha-(OH)ase-specific primers. The amplified complementary DNA fragments were analyzed by gel electrophoresis and nucleotide sequencing. Total RNA from kidney HEK 293 cells was subjected to reverse transcriptase reaction, and a 298-bp complementary DNA 1alpha-(OH)ase probe was generated by PCR. Primary cultures of human syncytiotrophoblasts exhibited 1alpha-(OH)ase activity, and a transcript for this gene could be demonstrated in these cells. Northern blot analysis revealed the presence of a 2.5-kb product, similar in size to that previously reported in kidney. RT-PCR analysis demonstrated the presence of a single transcript with nucleotide sequence identical to that previously reported for human 1alpha-(OH)ase complementary DNA clones. In addition, data are presented which suggest that differentiation of cytotrophoblast to the syncytial state was not necessary for this gene to be expressed, which may indicate a role of this enzyme all through pregnancy. The overall results of this study provide evidence for the presence of 1alpha-(OH)ase in the human placenta, suggesting that conversion of 25OHD(3) to 1,25-dihydroxyvitamin D3 in the trophoblast is most probably attributed to an enzymatic 1alpha-hydroxylation reaction.
Díaz
L
L
Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, México City, México.
Sánchez
I
I
Avila
E
E
Halhali
A
A
Vilchis
F
F
Larrea
F
F
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics; D000328:Adult; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015726:Giant Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014327:Trophoblasts / Q000201:enzymology*
D018076:DNA, Complementary; D012333:RNA, Messenger; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase