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

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 hypophosphatemia͞hypercalcemia 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. (2). Rachitic growth plates also are associated with inherited mutations that impair the actions of the Phex gene (X-linked hypophosphatemic rickets) (3, 4) and the metabolism of FGF-23 (autosomal dominant hypophosphatemic rickets) (5). Both of these disorders are associated with reduced 25-hydroxyvitamin D 1-␣-hydroxylase activity, leading to a reduction in 1,25(OH) 2 D synthesis. It was, therefore, unclear whether the pathophysiologic abnormality leading to rachitic growth plates in the hereditary hypophosphatemias is similar to that underlying rickets due to VDR deficiency and whether these rachitic changes are a direct consequence of impaired 1,25(OH) 2 D action.Targeted ablation of the VDR in mice is a phenocopy of the human disease hereditary vitamin D resistant rickets (6). These mice are born metabolically normal, but as a consequence of impaired 1,25(OH) 2 D-dependent intestinal calcium absorption, they develop hypocalcemia and secondary hyperparathyroidism, the latter of which leads to an increase in urinary phosphate excretion resulting in hypophosphatemia. The VDR-null mice develop rickets and osteomalacia by the fourth week of life. 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 ...

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“…Nevertheless, the growth plate remained somewhat enlarged, and the cellular architecture, particularly in the hypertrophic zone, was disorganized. However, in the Casr À/À 1a(OH)ase À/À animals on a rescue diet, vitamin D deficiency still was present, and hypophosphatemia, which has been reported to impair caspase-mediated apoptosis of hypertrophic chondrocytes, (25) also persisted. Therefore, the fact that complete normalization of the growth plate did not occur may indicate that persistent hypophosphatemia and absent active vitamin D precluded complete recovery.…”

Parathyroid hormone/parathyroid hormone-related protein receptor signaling is required for maintenance of the growth plate in postnatal life

“…3 A-C). Normally, apoptosis is observed only in highly differentiated hypertrophic chondrocytes at the border of the primary spongiosa (11). We observed an increase in the number of TUNEL-positive cells at the expected location at the end of the hypertrophic layer of the Coll2-CreER T :PPR fl/fl mice at 4 d after administration of Tam [TUNEL-positive cells in the hypertrophic region (%): 4.11 ± 0.82 vs. 3.51 ± 0.78, n = 5 in each group], and we also observed TUNEL-positive cells in the region of columnar chondrocytes not seen in those cells in PPR fl/fl control mice (1.66 ± 0.53 vs. 0.04 ± 0.04; P < 0.05; n = 5 in each group) (Fig.…”

“…To examine the relationship between apoptosis and growth plate disappearance, we used an in vivo model known to suppress apoptosis of late hypertrophic chondrocytes. Sabbagh et al (11) showed that low levels of inorganic phosphate in the blood lead to suppression of apoptosis of late hypertrophic chondrocytes and expansion of the hypertrophic chondrocyte layer (rickets). To determine whether hypophosphatemia could suppress apoptosis in the absence of PPR signaling and, if so, whether this suppression mitigated the phenotype of the PPR KO mice, we subjected Coll2-CreER T : PPR fl/fl and PPR fl/fl control mice to a low-phosphate diet.…”

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“…The development of rachitic changes in this model paralleled the development of impaired chondrocyte apoptosis and hypophosphatemia, which lends further credence to the hypothesis that hypophosphatemia leads to rickets by impairing apoptosis of late hypertrophic chondrocytes. 10 It is interesting to note that in the models examined, impaired apoptosis was observed at a time when there was still considerable mineralized matrix surrounding the late hypertrophic chondrocytes, which suggests that circulating, rather than locally deposited, phosphate is the critical determinant of apoptosis. Supporting this hypothesis is the observation that tissue-nonspecific alkaline phosphatase-knockout mice (a model for the human disease hypophosphatasia) have normal circulating phosphate levels, have markedly impaired matrix mineralization, and do not develop rickets.…”

“…To address this issue, studies were performed to characterize the growth-plate phenotype in 2 additional murine models: diet-induced hypophosphatemia/hypercalcemia and the hyp mouse (which has a mutation in the PHEX gene and is the murine model for the human disease X-linked hypophosphatemia). 10 The mice with hypophosphatemia in the presence of hypercalcemia (and suppressed PTH levels) and the hyp mice (normal calcium and PTH levels) both demonstrated expansion of the late hypertrophic chondrocyte layer associated with impaired apoptosis of these cells, which points to hypophosphatemia as the common etiologic factor. …”