Abstract
Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent.
Highlights
Endochondral ossification is critical to the development and growth of mammals
Our results suggest that cellular ATP synthesis consequent to Pi uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent
We have reported previously that osteoclast number was decreased in Hyp mice compared with WT mice and that a high-Pi diet partially restored this, showing that Pi influences osteoclastogenesis and suggesting that this Pi effect on osteoclastogenesis may be associated with the pathogenesis of abnormal skeletogenesis in Hyp mice [6]
Summary
Endochondral ossification is critical to the development and growth of mammals. The process begins with condensation of undifferentiated mesenchymal cells, and these cells differentiate into proliferating chondrocytes that express type II, IX, and XI collagen and sulfated glycosaminoglycans (GAG)2 [1]. Expression of the type III NPT Pit-1 and Pi uptake were diminished, and intracellular ATP levels were reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. Consistent with these in vivo results, chondrocytes isolated from Hyp mice (Hyp chondrocytes) in culture showed decreased apoptosis assessed by DNA fragmentation using a commercially available ELISA kit (Fig. 1C), and mineralization was determined by alizarin red staining (Fig. 1D, lower).
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