Regulation of the 25-hydroxyvitamin D-1alpha-hydroxylase gene and its splice variant.
The 25-hydroxyvitamin D-1alpha-OHase (1alpha-OHase) is responsible for producing the active form of vitamin D, 1alpha,25-dihydroxyvitamin D. The enzyme not only is expressed in kidneys, but also is expressed in many nonrenal tissues, including skin. In this study, we compared the regulation of the 1alpha-OHase expression in kidney cells and keratinocytes. Using transfected luciferase reporter gene constructs, we compared the activity and regulatory features of the human 1alpha-OHase gene promoter in C-21 human kidney cells (PTH/PTHrP receptor positive) and cultured human keratinocytes (NHKs). We found that two regions, -1,100 bp and -396 bp from the ATG, were highly sensitive to parathyroid hormone (PTH) in C-21 cells but not in NHK. Furthermore, three CRE-like sequences (CLS) were identified within this PTH-sensitive area of the 1alpha-OHase promoter and when deleted they reduced induction of PTH by 50%-95% in C-21 cells. To further investigate the differential regulation profile, we examined the protein products of 1alpha-OHase in kidney and skin. Western blot analysis of whole cell extracts from these tissues with a 1alpha-OHase-specific antibody revealed the predicted 1alpha-OHase protein product of 56 kDa in kidney and a larger protein product of 59 kDa in skin. Using RT-PCR for the 1alpha-OHase in skin and kidney, we detected an insertion between exons 2 and 3 in skin but not in kidney. These results suggest that the regulation of renal and skin 1alpha-OHase gene expression may be tissue specific and possibly produce different splice variants, and that this specificity is likely conferred by differential expression of CRE-binding proteins in different cell types. In conclusion, the differential tissue expression of 1alpha-OHase gene variants and the tissue-specific regulation profile open up a new paradigm in the understanding of the role of 25-hydroxyvitamin D3 1alpha-hydroxylase gene in the regulation of vitamin D physiology.
Flanagan
John N
JN
Department of Medicine, Endocrine Section, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
Wang
Lilin
L
Tangpricha
Vin
V
Reichrath
Jörg
J
Chen
Tai C
TC
Holick
Michael F
MF
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D015603:Keratinocytes / Q000201:enzymology* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion
D017931:DNA Primers; D008156:Luciferases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase