Acute Phosphate Restriction Impairs Bone Formation and Increases Marrow Adipose Tissue in Growing Mice.

Phosphate plays a critical role in chondrocyte maturation and skeletal mineralization. Studies examining the consequences of dietary phosphate restriction in growing mice demonstrated not only the development of rickets, but also a dramatic decrease in bone accompanied by increased marrow adipose tissue (MAT). Thus studies were undertaken to determine the effects of dietary phosphate restriction on bone formation and bone marrow stromal cell (BMSC) differentiation. Acute phosphate restriction of 28-day-old mice profoundly inhibited bone formation within 48 hours. It also resulted in increased mRNA expression of the early osteolineage markers Sox9 and Runt-related transcription factor 2 (Runx2), accompanied by decreased expression of the late osteolineage markers Osterix and Osteocalcin in BMSCs and osteoblasts, suggesting that phosphate restriction arrests osteoblast differentiation between Runx2 and Osterix. Increased expression of PPARγ and CEBPα, key regulators of adipogenic differentiation, was observed within 1 week of dietary phosphate restriction and was followed by a 13-fold increase in MAT at 3 weeks of phosphate restriction. In vitro phosphate restriction did not alter BMSC osteogenic or adipogenic colony formation, implicating aberrant paracrine or endocrine signaling in the in vivo phenotype. Because BMP signaling regulates the transition between Runx2 and Osterix, this pathway was interrogated. A dramatic decrease in pSmad1/5/9 immunoreactivity was observed in the osteoblasts of phosphate-restricted mice on day 31 (d31) and d35. This was accompanied by attenuated expression of the BMP target genes Id1, KLF10, and Foxc2, the latter of which promotes osteogenic and angiogenic differentiation while impairing adipogenesis. A decrease in expression of the Notch target gene Hey1, a BMP-regulated gene that governs angiogenesis, was also observed in phosphate-restricted mice, in association with decreased metaphyseal marrow vasculature. Whereas circulating phosphate levels are known to control growth plate maturation and skeletal mineralization, these studies reveal novel consequences of phosphate restriction in the regulation of bone formation and osteoblast differentiation.