Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction

Abstract

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1alpha-hydroxylase [1alpha(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1alpha(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1alpha(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1alpha(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

Figure 1

Gene targeting of mouse 1α(OH)ase. (A) Schematic representation of the genomic region encoding the 1α(OH)ase and the creation of a mutant allele by homologous recombination. The neomycin resistance cassette (1.1 kb), which is introduced in the antisense orientation, is flanked by ≈1.4 and 2.8 kb of 5′ and 3′ 1α(OH)ase gene sequences and replaces exons VI and VII encoding the hormone-binding domain, exon VIII encoding the heme-binding domain, and part of exon IX. Genomic organization of the mouse 1α(OH)ase region was determined by cloning the gene from a 129sv/J library as described (17). (B) Analysis of genomic DNA isolated from pups born to two heterozygotes. For Southern blot, purified DNA was digested with BamHI. The mutated allele (3.4 kb) is distinguished from the wild-type allele (5.4 kb) using the 5′ probe indicated in A. For multiplex PCR, a 500-bp neomycin gene product and a 376-bp 1α(OH)ase exons VI and VII gene product were amplified. The positions of the primers are shown in A. (C) Reverse-transcriptase (RT)-PCR of total kidney RNA (20 μg) isolated from wild-type (+/+), heterozygous (+/−), or homozygous (−/−) 1α(OH)ase-deleted littermates with primers generating a 176-bp 1α(OH)ase product and a 508-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) product. Densitometric analysis values of 1α(OH)ase/GAPDH for (+/+) = 100 ± 4%; (+/−) = 60 ± 5%; (−/−) = 0, mean ± SEM, n = 3. (D) Analysis of target gene expression in wild-type (+/+), heterozygous (+/−), and (−/−) 1α(OH)ase mice. Northern blot analyses of 24(OH)ase, calbindin D_9K, and calbindin D_28K were performed on intestinal and/or kidney RNA of mice of each genotype at 7 weeks. Densitometric analysis values for specific mRNA/18S of (+/+) vs. (+/−) vs. (−/−): intestine Calb9k, 100 ± 5% vs. 85 ± 5% vs. 2 ± 2%; kidney 24(OH)ase, 100 ± 4% vs. 103 ± 5% vs. 4 ± 2%; kidney Calb9k, 100 ± 5% vs. 91 ± 5% vs. 36 ± 4%; kidney Calb28k, 100 ± 5% vs. 96 ± 4% vs. 61 ± 3%, mean ± SEM, n = 3.

Figure 2

Histology of bone from 1α(OH)ase null mutant mice (−/−) and wild-type littermates (+/+). (A) Proximal tibial epiphysis, (GP) growth plate, and metaphysis at 4 weeks, hematoxylin and eosin. (Upper) Bar = 500 μm; (Lower) Bar = 250 μm. (B) Von Kossa stain of undemineralized sections at 4 weeks. Counterstaining with toluidine blue. (DF) distal femur, bar = 500 μm; (GP) growth plate, bar = 100 μm; (TB) trabecular and (CB) cortical bone, bar = 50 μm, *, osteoid. (C) Tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts in the primary spongiosa. Counterstaining with methylene blue. (Upper) Bar = 250 μm; (Lower) Bar = 50 μm

Figure 3

Histology of parathyroid glands and adjacent thyroid tissue of 1α(OH)ase null mutant mice (−/−) and wild-type littermates (+/+). (Upper), Hematoxylin and eosin; (Lower), Immunostaining for calcium-sensing receptor (CaSR). Bar = 250 μm. Parathyroid glands are denoted by arrows and lymph nodes by arrowheads.

Figure 4

Comparison of uterus and ovary of wild-type mice (+/+) (Left) and 1α(OH)ase mutant littermates (−/−) (Right). Histology, hematoxylin and eosin staining. Uterus and ovary (Upper), bar = 250 μm; ovary (Lower, Insets), bar = 50 μm. In the uterus, the arrow indicates the normal endometrium. In the ovary Upper panels, the Insets (enlarged in the Lower panels) show the normal corpora lutea in (+/+) mice and that they are absent in the (−/−) mice, which have hypertrophied interstitial cells.