Identification of a 25-hydroxyvitamin D-3 1 alpha-hydroxylase gene transcription product in cultures of human syncytiotrophoblast cells

Although accumulating data show that placenta is able to synthesize 1,25-di hydroxyvitamin D-3, the presence of cytochrome P-450 enzyme capable of conv erting 25-hydroxyvitamin D-3 (250HD(3)) to the biologically active form of vitamin D in this tissue, has not been yet clearly established. In this stu dy, we have investigated the presence of 25-hydroxyvitamin D-3 1 alpha-hydr oxylase (1 alpha-(OH)ase) gene expression products in cultured human syncyt iotrophoblast. Total RNA was isolated from cultured placental cells and sub jected to Northern blots or RT-PCR by using 1 alpha-(OH)ase-specific primer s. The amplified complementary DNA fragments were analyzed by gel electroph oresis and nucleotide sequencing. Total RNA from kidney HEK 293 cells was s ubjected to reverse transcriptase reaction, and a 298-bp complementary DNA 1 alpha-(OH)ase probe was generated by PCR. Primary cultures of human syncy tiotrophoblasts exhibited 1 alpha-(OH)ase activity, and a transcript for th is gene could be demonstrated in these cells. Northern blot analysis reveal ed the presence of a 2.5-kb product, similar in size to that previously rep orted in kidney. RT-PCR analysis demonstrated the presence of a single tran script with nucleotide sequence identical to that previously reported for h uman 1 alpha-(OH)ase complementary DNA clones. In addition, data are presen ted 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 stu dy provide evidence for the presence of 1 alpha-(OH)ase in the human placen ta, suggesting that conversion of 250HD(3), to 1,25-dihydroxyvitamin D-3 in the trophoblast is most probably attributed to an enzymatic 1 alpha-hydrox ylation reaction.