Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1alpha-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites.
The regulation of the gene for renal 25-hydroxyvitamin D 1alpha- hydroxylase (1alpha(OH)ase; CYP27B1) by parathyroid hormone (PTH) under hypocalcemic conditions is fundamentally important for the maintenance of calcium and phosphate homeostasis. The molecular mechanism that underlies this hormonal response is of current interest and has been investigated in the present study by transfection analysis of the human 1alpha(OH)ase promoter in kidney AOK-B50 cells. We have shown that the first 305 bp of promoter can be induced by hormone in transient transfection assays and also within a chromatin environment when stably integrated. Mutagenesis of possible transcription factor binding sites within this promoter length has shown that three sites clustered within the region from -66 to -135 contribute to basal expression. A likely Sp1 and a CCAAT box site are particularly important for basal expression although these sites are not likely to functionally cooperate in a major way. Mutagenesis of the CCAAT box site consistently reduced PTH induction although mutagenesis of the Sp1, Ets and other possible binding sites in the 305 bp of promoter has no significant effect on the level of PTH induction. Other experiments showed that PTH induction but not basal expression was sensitive to the protein kinase inhibitor H89. We have therefore identified for the first time the sites in the 1alpha(OH)ase promoter responsible for basal expression and provide evidence for the role of a CCAAT box binding protein in a PTH mechanism of induction that involves an H89 sensitive step.
Gao
Xiu Hui
XH
Department of Molecular Biosciences, University of Adelaide, SA, Adelaide, Australia.
Dwivedi
Prem P
PP
Choe
Sophie
S
Alba
Frances
F
Morris
Howard A
HA
Omdahl
John L
JL
May
Brian K
BK
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics*; D024506:5' Flanking Region; D001665:Binding Sites; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase