Transcriptomic analysis of dietary hypophosphatemia shows downregulated oxidative metabolism in fracture callus tissues
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Phosphate deficiency mimics Vitamin D deficiency and generates a rachitic state that impairs the ability of bone to mineralize and delays fracture healing. Previous studies have shown that hypophosphatemia alters many biological functions, and oxidative phosphorylation was the top biological process that was predicted affected by this perturbation. The goals of this study were to characterize the temporal mRNA expression for genes associated with oxidative phosphorylation and determine if phosphate deficiency more broadly altered the expression of genes associated with intermediate metabolism during fracture healing. Three in bred strains of skeletally mature male mice had stabilized fractures made in the right femur. The experimental group (Pi) was fed a low phosphate diet starting two days before fracture and continued for 16 days after which, a normal diet was re-introduced. The control group (Ctrl) was fed a normal diet throughout the study. RNA was extracted from fracture calluses and the RNA was quantified by microarray analysis. Analysis of covariance (ANCOVA) identified genes that were differentially expressed between the Pi and Ctrl groups (q-value ≤ 0.005), and analysis was performed on a subset of 577 genes related to the central elements of intermediate metabolism that were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG). To categorize genes that have similar temporal responses to the phosphate perturbation, the ratio of Pi/Ctrl expressions for each strain was independently clustered using normal mixtures method. These clusters were compared across the three strains to ascertain which groups of genes responded together in all three strains, and which biological functions bifurcated due to genetic variation between the strains. Oxidative phosphorylation and the tricarboxylic acid pathways showed the tightest temporal control in response to dietary phosphate restriction across all strains of mice. Analysis of the temporal profile showed the response to phosphate was strain dependent with the B6 strain showing the strongest response to Pi restriction. The re-introduction of phosphate also restored and/or abolished the delay in oxidative phosphorylation expression in a strain specific manner. AJ and C3 strains showed more similar rebounding profiles compared to the B6 stain. Additionally, it was found that Complex IV was affected by the phosphate deficient diet in manner different from the other components of ETC. Some intermediate metabolic pathways that also appeared to be affected in similar manners across multiples strains included glycolysis, and arginine and proline metabolism. Overlaying these various functions, temporal coordination was observed and suggests the functions may be mediated by a common upstream regulator. Further investigation of this potential upstream regulator can help to better describe the mechanisms that cause a delayed fracture healing in a phosphate deficient state.