René St-Arnaud¹, G. Antonio Candeliere¹, and Shoukat Dedhar²

¹Genetics Unit, Shriners Hospital, and Departments of Surgery and Human Genetics, McGill University, Montréal (Québec) Canada H3G1A6

²Division of Cancer Research and Department of Medical Biophysics, University of Toronto, Reichmann Research Building, Sunnybrook Health Science Centre, Toronto (Ontario) Canada M4N 3M5

Received 07/05/96; Accepted 07/12/96; On-line 08/01/96

2.1. Vitamin D and bone cells

The hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D₃ (1,25 (OH)₂D₃, also named calcitriol), is a known modulator of osteoblastic growth and function. A number of effects have been described when primary cultures of bone cells or established osteoblastic cell lines are treated with 1,25 (OH)₂D₃ in vitro. Vitamin D treatment inhibits type I collagen expression by osteoblasts (1-5) but enhances the expression of genes that are specific markers of osteoblastic differentiation. These include alkaline phosphatase (6-9), osteopontin (10, 11), osteocalcin (12, 13), and matrix-Gla protein (14-16). Overall, the direct effects of 1,25 (OH)₂D₃ on bone cells are consistent with the induction of differentiation of osteoblasts in vitro. Accordingly, the metabolite has been shown to stimulate mineralization in cultures of clonal osteoblast-like cells (17).

2.2. VDREs

Vitamin D mainly exerts its pleiotropic effects following binding to a specific receptor which is a member of the steroid hormone receptor superfamily (reviewed in ref. 18). The ligand-bound vitamin D receptor (VDR) then interacts with its cognate binding site, termed vitamin D-response element (VDRE), to affect the transcription of target genes (19).

A number of natural and synthetic VDREs have been identified. These response elements consist of direct repeats (DR) of two hexameric core binding sites spaced by varying numbers of nucleotide residues. Binding studies using the VDR DNA binding domain have revealed the order of relative binding affinities for various hexameric core binding sites, with GGTTCA > AGGTCA > AGGACA >GGGTGA (20). The DR3-type element, exampled by the mouse osteopontin VDRE (21), consists of two GGTTCA hexameric core sites spaced by 3 residues. DR3-type elements have been described in the promoters of the rat osteocalcin gene (22), the rat calbindin D-9k gene (23), the avian integrin 3 subunit gene (24) and the rat calcidiol 24-hydroxylase gene (25) (Table 1).

GGTTCAcgaGGTTCA

GGGTGAatgAGGACA

GGGTCAacgGGGGCA

GGGTGTcggAAGCCC

GAGGCAgaaGGGAGA

CGCACCcgcTGAACC

Several DNA sequences that diverge from the canonical DR3-type elements have also been shown to bind the VDR and to mediate vitamin D-dependent transcriptional activation. These non-classical VDREs will be described in section 3 below.

2.3. VDR dimerization

In addition to the binding of the VDR to certain VDREs as a homodimer (26), it has been known for some time that the in vitro binding affinity of the VDR is enhanced by the addition of nuclear extracts, suggesting the existence of nuclear accessory factors (27). Some of these accessory factors have now been identified. The VDR forms heterodimers with retinoid X receptors (RXRs) (28-30). The RXRs are a family of nuclear receptors binding the retinoid 9-cis retinoic acid (31-33). The molecular cross-talk involving the VDR is further evidenced by results showing that it can heterodimerize with retinoic acid receptors (RARs) (34) and thyroid hormone receptors (T₃Rs) (35). The VDR dimerizing partner seems to affect the binding affinity to and the transcriptional response of particular VDREs (35, 36).