Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes - PubMed (original) (raw)

Comparative Study

. 2005 Jul 5;102(27):9637-42.

doi: 10.1073/pnas.0502249102. Epub 2005 Jun 23.

Affiliations

Comparative Study

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

Yves Sabbagh et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Rickets is seen in association with vitamin D deficiency and in several genetic disorders associated with abnormal mineral ion homeostasis. Studies in vitamin D receptor (VDR)-null mice have demonstrated that expansion of the late hypertrophic chondrocyte layer, characteristic of rickets, is secondary to impaired apoptosis of these cells. The observation that normalization of mineral ion homeostasis in the VDR-null mice prevents rachitic changes suggests that rickets is secondary to hypocalcemia, hypophosphatemia, or hyperparathyroidism, rather than impaired VDR action. To determine which of these abnormalities is responsible for impaired chondrocyte apoptosis and subsequent rachitic changes, two additional models were examined: diet-induced hypophosphatemia/hypercalcemia and hypophosphatemia secondary to mutations in the Phex gene. The former model is associated with suppressed parathyroid hormone levels as a consequence of hypercalcemia. The latter model demonstrates normal calcium and parathyroid hormone levels, but 1,25-dihydroxyvitamin D levels that are inappropriately low for the degree of hypophosphatemia. Our studies demonstrate that normal phosphorus levels are required for growth plate maturation and implicate a critical role for phosphate-regulated apoptosis of hypertrophic chondrocytes via activation of the caspase-9-mediated mitochondrial pathway.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Histological analysis and evaluation of hypertrophic chondrocyte apoptosis in 24-day-old mice. Shown are hematoxylin/eosin-stained sections (A and C) and TUNEL assay (B and D). (A and B) Wild-type (WT) and VDR knockout (KO) mice fed a regular diet (Left) or a rescue diet (Right) that normalizes mineral ion homeostasis. (C and D) Wild-type mice fed a regular diet, Hyp mice fed a regular diet, and wild-type mice fed a high-calcium/low-phosphate diet from 18 to 24 days of age. Brackets indicate the hypertrophic chondrocyte layer. Data are representative of experiments performed on sections from three or more mice for each condition. EV, Evans blue; T, TUNEL.

Fig. 2.

Fig. 2.

Biochemical parameters. Serum phosphate (A), serum calcium (B), and immunoreactive PTH (C) were measured in 24-day-old wild-type mice fed a regular diet (black bars), Hyp mice fed a regular diet (white bars), and wild-type mice fed a high-calcium/low-phosphate diet (gray bars). (D) Serum phosphate levels at 18.5 days postcoitum and at 0.5, 10, and 24 days of age in wild-type (♦) and Hyp (○) mice fed a regular diet. Data represent the mean ± SD of values from five mice for each assay. *, P ≤ 0.05.

Fig. 3.

Fig. 3.

Histological analysis and evaluation of hypertrophic chondrocyte apoptosis in Hyp mice. Hematoxylin/eosin staining (A and C) and TUNEL assays (B and D) were performed on sections from wild-type (WT) and Hyp mice at 0.5 day (A and B) and 10 days of age (C and D). Brackets indicate the hypertrophic chondrocyte layer. Data are representative of experiments performed on sections from three or more mice for each condition. EV, Evans blue; T, TUNEL.

Fig. 4.

Fig. 4.

Growth-plate mineralization in 24-day-old mice. von Kossa staining was performed on sections from 24-day-old wild-type mice fed a regular diet, Hyp mice fed a regular diet, and wild-type mice fed a high-calcium/low-phosphate diet from 18 to 24 days of age. Insets show a magnification of the hypertrophic chondrocytes. Brackets indicate the hypertrophic chondrocyte layer. Data are representative of sections from five mice for each condition.

Fig. 5.

Fig. 5.

Cleaved caspase-3 immunohistochemistry. Sections from 24-day-old wild-type (WT) mice fed a regular diet, Hyp mice fed a regular diet, and mice fed a high-calcium/low-phosphate diet from 18 to 24 days of age were immunostained with antibody to cleaved caspase-3. Brackets indicate the hypertrophic chondrocyte layer. Data are representative of experiments performed on sections from three or more mice for each condition.

Fig. 6.

Fig. 6.

Phosphate induces hypertrophic chondrocyte apoptosis in vitro. Hypertrophic chondrocytes were treated with 7 mM NaCl or 7 mM NaH2PO4 for 18 h in the presence or absence of 1 μM cyclosporin A (CsA). Cell lysates were subjected to Western blot analysis using anti-caspase-9. Lysates of 3T3 fibroblasts and proliferating chondrocytes (PC) treated with 7 mM NaH2PO4 were similarly analyzed. Data are representative of those obtained from three independent cell preparations.

Fig. 7.

Fig. 7.

In vivo inhibition of caspase-3 or caspase-9 leads to expansion of the hypertrophic chondrocyte layer. Shown are hematoxylin/eosin-stained sections from 24-day-old mice injected at days 18–23 with DMSO, caspase-3 inhibitor (Z-DEVD-FMK), or caspase-9 inhibitor (Z-LEHD-FMK). Brackets indicate the hypertrophic chondrocyte layer.

Similar articles

Cited by

References

    1. St.-Arnaud, R., Messerlian, S., Moir, J. M., Omdahl, J. L. & Glorieux, F. H. (1997) J. Bone Miner. Res. 12, 1552-1559. - PubMed
    1. Hughes, M. R., Malloy, P. J., Kieback, D. G., Kesterson, R. A., Pike, J. W., Feldman, D. & O'Malley, B. W. (1988) Science 242, 1702-1705. - PubMed
    1. Eicher, E. M., Southard, J. L., Scriver, C. R. & Glorieux, F. H. (1976) Proc. Natl. Acad. Sci. USA 73, 4667-4671. - PMC - PubMed
    1. Sabbagh, Y., Jones, A. O. & Tenenhouse, H. S. (2000) Hum. Mutat. 16, 1-6. - PubMed
    1. The Autosomal Dominant Hypophosphataemic Rickets Consortium (2000) Nat. Genet. 26, 345-348. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources