Regulation of the 25-hydroxyvitamin D-1alpha-hydroxylase gene and its splice variant

Recent Results Cancer Res. 2003;164:157-67. doi: 10.1007/978-3-642-55580-0_12.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase / genetics*
  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase / metabolism
  • Alternative Splicing*
  • Animals
  • Blotting, Western
  • Cells, Cultured
  • DNA Primers / chemistry
  • Gene Expression Regulation, Enzymologic*
  • Keratinocytes / enzymology*
  • Keratinocytes / metabolism
  • Kidney / enzymology*
  • Kidney / metabolism
  • Luciferases / metabolism
  • Mice
  • Mutagenesis, Site-Directed
  • Promoter Regions, Genetic*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Deletion

Substances

  • DNA Primers
  • Luciferases
  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase