Rickets in VDR Null Mice Is Secondary to Decreased Apoptosis of Hypertrophic Chondrocytes

Abstract

Both vitamin D deficiency and the absence of a functional vitamin D receptor (VDR) lead to a growth plate abnormality known as rickets. Prevention of abnormal mineral ion homeostasis by early institution of dietary therapy in VDR null mice prevents rickets, demonstrating that the VDR is not required for normal growth plate maturation. We, therefore, hypothesized that rickets, in the absence of a functional VDR, is due to impaired mineral ion homeostasis. Analyses of growth plate morphology in VDR null mice demonstrated normal resting and proliferating chondrocyte layers; however an expansion of the hypertrophic chondrocyte layer was present by 24 days of age. Because extracellular calcium has been shown to play a role in chondrocyte maturation, we addressed the hypothesis that hypocalcemia led to impaired chondrocyte differentiation. However, in situ hybridization analyses revealed normal expression of hypertrophic chondrocyte markers in the tibial growth plate of 24 day old VDR null mice, suggesting that the increase in the hypertrophic chondrocyte layer was not secondary to impaired differentiation. We then addressed whether expansion of the hypertrophic chondrocyte layer was secondary to increased proliferation or decreased apoptosis. BrdU labeling failed to demonstrate an increase in chondrocyte proliferation in the VDR null mice; however, apoptosis was markedly diminished in the late hypertrophic chondrocytes of the VDR null mice, suggesting that impairment in programmed cell death of these cells leads to the characteristic findings of rickets.

Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes

“…As reported in ref. 7 and shown in Fig. 1B (regular diet), VDR-null mice with abnormal mineral ion homeostasis have a marked reduction in hypertrophic chondrocyte apoptosis; however, rescue of the growth plate phenotype by normalizing mineral ion homeostasis restores apoptosis (Fig.…”

“…Investigations addressing the cellular basis for these rachitic changes demonstrate that the growth-plate abnormality in the VDR-null mice is due to an expansion of the late hypertrophic chondrocyte layer, a consequence of impaired apoptosis of these cells. Chondrocyte proliferation and acquisition of markers of chondrocyte differentiation as proliferative chondrocytes mature are unaffected by VDR ablation, as is signaling for vascular invasion assessed by VEGF mRNA expression (7). Of note, prevention of abnormal mineral ion homeostasis by institution of a diet high in calcium, phosphorus, and lactose by 18 days of age prevents the development of hyperparathyroidism, rickets, and osteomalacia in the VDR-null mice (8).…”

Phosphate-induced Apoptosis of Hypertrophic Chondrocytes Is Associated with a Decrease in Mitochondrial Membrane Potential and Is Dependent upon Erk1/2 Phosphorylation

“…Apoptosis was evaluated using the TUNEL-based in situ cell death detection kit (Roche Diagnostics) and an antibody to cleaved caspase-3 (Cell Signaling) (2). In situ hybridization was performed on fixed frozen or paraffin sections using 35 S-UTPlabeled antisense RNA probes as described previously (4). The presence of endothelial cells was evaluated using an anti-CD31 antibody (BD Biosciences).…”

“…Rickets is a growth plate anomaly observed in growing animals and humans with abnormalities of vitamin D action and renal phosphate reabsorption (3)(4)(5)(6). In vivo investigations in genetically modified and dietary-manipulated mouse models demonstrate that hypophosphatemia is the underlying metabolic abnormality that impairs growth plate maturation in these disorders: low circulating phosphate levels result in impaired apoptosis of terminally differentiated hypertrophic chondrocytes in the growth plate, leading to rickets (2).…”

Inactivation of the 25-Hydroxyvitamin D 1α-Hydroxylase and Vitamin D Receptor Demonstrates Independent and Interdependent Effects of Calcium and Vitamin D on Skeletal and Mineral Homeostasis

“…This constellation of abnormalities is observed in humans with VDR mutations in the inherited disorder vitamin D-dependent rickets type II (also called hereditary vitamin D-resistant rickets) (23). Rescue of this phenotype has been successfully accomplished with a high calcium, high phosphorus, high lactose diet administered for at least 1 month after weaning (22,24). Consequently, it has been postulated that the major action of the VDR in skeletal growth, maturation, and remodeling is its role in intestinal calcium absorption (25).…”