Hypophosphatemia Regulates Molecular Mechanisms of Circadian Rhythm

Abstract

Transcriptomic analysis showed that the central circadian pathway genes had significantly altered expression in fracture calluses from mice fed a low phosphate diet. This led us to hypothesize that phosphate deficiency altered the circadian cycle in peripheral tissues. Analysis of the expression of the central clock genes over a 24–36 hour period in multiple peripheral tissues including fracture callus, proximal tibia growth plate and cardiac tissues after 12 days on a low phosphate diet showed higher levels of gene expression in the hypophosphatemia groups (p < 0.001) and a 3 to 6 hour elongation of the circadian cycle. A comparative analysis of the callus tissue transcriptome genes that were differentially regulated by hypophosphatemia with published data for the genes in bone that are diurnally regulated identified 1879 genes with overlapping differential regulation, which were shown by ontology assessment to be associated with oxidative metabolism and apoptosis. Network analysis of the central circadian pathway genes linked their expression to the up regulated expression of the histone methyltransferase gene EZH2, a gene that when mutated in both humans and mice controls overall skeletal growth. These data suggest that phosphate is an essential metabolite that controls circadian function in both skeletal and non skeletal peripheral tissues and associates its levels with the overall oxidative metabolism and skeletal growth of animals.

Advances and Promises of Nutritional Influences on Natural Bone Repair

“…Calcium deficiency does not appear to significantly impact fracture healing, which is likely a result of stored calcium that is liberated from the intact skeleton following fracture. Along with calcium, the hydroxyapatite inorganic component of bone also requires phosphorus, and consumption of phosphate‐restricted diets negatively impacts secondary fracture healing . Severe phosphate deficiency impairs the proliferation and differentiation of mesenchymal progenitor cells, leading to an overall decrease in callus volume and an early, yet sustained increase in callus cartilage in mice with femoral fractures .…”

“…[56][57][58] Severe phosphate deficiency impairs the proliferation and differentiation of mesenchymal progenitor cells, leading to an overall decrease in callus volume and an early, yet sustained increase in callus cartilage in mice with femoral fractures. 56,57 These adverse effects were attributed to decreased bone morphogenetic protein signaling-2 (BMP-2) in fracture associated mesenchymal stem cells as well as disruption of the circadian cycle that slows the progression of chondrogenic differentiation of mesenchymal stem cells. Although pre-clinical animal studies have shed light on the negative impact of severe phosphate deficiency on fracture healing, it is important to recognize that deficiency would be rarely observed clinically, as most Americans consume two to three times the recommended dietary allowances (RDA) due to the widespread availability and high bioavailability of phosphorus.…”

“…Along with calcium, the hydroxyapatite inorganic component of bone also requires phosphorus, and consumption of phosphate‐restricted diets negatively impacts secondary fracture healing . Severe phosphate deficiency impairs the proliferation and differentiation of mesenchymal progenitor cells, leading to an overall decrease in callus volume and an early, yet sustained increase in callus cartilage in mice with femoral fractures . These adverse effects were attributed to decreased bone morphogenetic protein signaling‐2 (BMP‐2) in fracture associated mesenchymal stem cells as well as disruption of the circadian cycle that slows the progression of chondrogenic differentiation of mesenchymal stem cells.…”

Timing metabolism in cartilage and bone: links between circadian clocks and tissue homeostasis

“…Hypophosphatemia during skeletal development impairs the mitochondria-mediated apoptosis of hypertrophic chondrocytes, leading to abnormal growth plate maturation and bone mineralisation (Sabbagh et al 2005, Miedlich et al 2010. Expression of clock genes in fracture calluses from hypophosphataemic mice was higher than that in control littermates, suggesting that phosphate metabolism is involved in the regulation of the circadian clock in bone (Noguchi et al 2018).…”

“…Remarkably, network analysis identified Ezh2 (enhancer of zeste homolog 2), a key epigenetic regulator of skeletal longitudinal growth, as one of the main molecules associated with hypophosphatemia (Dudakovic et al 2015, Lui et al 2016, Noguchi et al 2018. Previous work revealed that EZH2 binds to the promoter region of Per1 and Per2; and targeted silencing of this histone methyltransferase disrupted circadian rhythms (Etchegaray et al 2006).…”