Disrupted phosphate metabolism and SIBLING/ASARM peptide accumulation underlie impaired bone mineralization in klotho-deficient (kl/kl) mice.

Abstract

Klotho-deficient (kl/kl) mice exhibit severely impaired bone matrix mineralization despite marked hyperphosphatemia, suggesting that local mechanisms, rather than systemic mineral availability, regulate skeletal mineralization. Therefore, to clarify the underlying mechanisms, this study aimed to examine phosphate metabolism, pyrophosphate (PPi) homeostasis, and SIBLING/ASARM peptide accumulation in the femora of kl/kl mice maintained on either normal- or low-phosphate (low-Pi) diets. Histochemical and ultrastructural analyses revealed extensive unmineralized bone matrix, impaired mineralized nodule formation, and abnormal accumulation of organic materials around osteoblasts and osteocytes in kl/kl mice. These abnormalities were associated with reduced expression of the phosphate-supplying enzymes tissue-nonspecific alkaline phosphatase (ALP) and PHOSPHO1, together with increased expression of the PPi-generating factors ENPP1 and ANK. Consistent with these findings, bone PPi levels were significantly elevated in kl/kl mice. Dentin matrix protein 1 (DMP1), osteopontin, and phosphorylated acidic serine- and aspartate-rich motif (pASARM) peptides also accumulated in osteocytes and the surrounding bone matrix. Dietary phosphate restriction reduced serum phosphate and bone PPi levels, partially restored ALP and PHOSPHO1 expression, attenuated ENPP1, ANK, DMP1, and pASARM accumulation, and improved bone mineralization. Phosphate exposure induced phosphate- and mineralization-related genes in vitro in osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cells, whereas phosphate normalization partially reversed these changes. Collectively, these findings support the concept that hyperphosphatemia contributes to defective bone mineralization in klotho deficiency by disrupting phosphate/PPi homeostasis and enhancing the accumulation of SIBLING-derived mineralization inhibitors. These findings suggest that PPi dysregulation and the SIBLING/ASARM axis are important contributors to impaired bone mineralization in kl/kl mice.