The X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism but also by factor(s) locally released by osteoblast cells (ObCs). The identity of these ObC-derived pathogenic factors remains unclear. In our present study, we report our finding of a prominent protein in the culture media derived from ObC of the hypophosphatemic (Hyp) mice, a murine homolog of human XLH, which was identified as the murine procathepsin D (Cat D). By metabolic labeling studies, we further confirmed that Hyp mouse ObCs released greater amount of Cat D into culture media. This increased Cat D release by Hyp mouse ObCs was unlikely to be due to nonspecific cell damage or heterogeneous cell population and was found to be associated with an increased Cat D expression at the protein level, possibly due to a reduced Cat D degradation. However, we were not able to detect a direct effect of PHEX protein on Cat D cleavage. In support of the involvement of Cat D in mediating the inhibitory effect of Hyp mouse ObC-conditioned media on ObC calcification, we found that exposure to Cat D inhibited ObC 45 Ca incorporation and that inhibition of Cat D abolished the inhibitory effect of Hyp mouse-conditioned media on ObC calcification. In conclusion, results from our present study showed that Hyp mouse ObCs release a greater amount of Cat D, which may contribute to the inhibitory effect of Hyp mouse ObCconditioned media on ObC mineralization. hypophosphatemia; rickets; bone; pepstatin X-LINKED HYPOPHOSPHATEMIA (XLH) is an X-linked dominant disorder characterized by rachitic bone disease and hypophosphatemia with renal phosphate wasting (38). Since its first description by Albright et al. in 1937 (1), significant progress has been made in our understanding of this disorder, aided particularly by the discovery of a murine homolog, the hypophosphatemic (Hyp) mouse in 1976 (15), and the identification of the XLH candidate gene PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) in 1995 (39). Prevailing evidence today suggests that inactivating mutations of PHEX in bones and possibly other tissues lead to the release of factors that cause renal phosphate wasting and a bone mineralization defect (30).However, despite extensive studies performed in Hyp mice and XLH patients, the etiology of the bone mineralization defect in XLH remains poorly understood. Although factors extrinsic to the bone, such as hypophosphatemia and deranged vitamin D metabolism, can contribute to the bone defect, available evidence indicates that the bone mineralization defect is also caused by intrinsic abnormalities in bones. Thus bone cross-transplant studies showed that the Hyp mouse bone defect could not be complete...

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