Comparative analysis of transcriptome of fracture healing under normal and hypophosphatemic conditions
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Abstract
Phosphate deficiency delays bone healing by disrupting various cellular and molecular processes involved in bone regeneration. This transcriptomic analysis investigates the intricate relationship between environmental effects of phosphate deficiency on the time course of fracture healing within the C57BL/6J (B6) mouse strain. To manipulate the healing environment, a low phosphate diet was used to induce a hypophosphatemic/rachitic condition. Previous research suggested that temporal patterns of chondrogenic and osteogenic lineage development varied among mouse strains, with the B6 strain exhibiting unique characteristics. This strain was selected due to its significant cartilage development, which is highly affected by phosphate restriction. Total RNA analysis was conducted in triplicate for each condition, utilizing the Affymetrix mouse 2.0A chip for microarray for transcriptomic profiling. Differential gene expression was assessed using standard Co-ANOVA methods, with a false discovery rate of 0.05 or less indicating significant differences. Temporal Clustering of gene expression was carried out using specialized methods employing an entropy-penalized expectation maximization (EPEM) algorithm. Functional gene ontologies, pathways, and key regulators associated with different dietary conditions were identified using Ingenuity Pathway Analysis (IPA) from Qiagen, Inc. The study aimed to provide transcriptomic insights into the time course of fracture healing under normal and phosphate-restricted conditions, as well as comparing temporal expression patterns between control and phosphate-restricted callus tissues.Canonical pathways associated with fracture callus were examined, revealing that the most notable downregulation in response to hypophosphatemia was in oxidative energy metabolism pathways, indicating metabolic adaptation in response to phosphate restriction. The study also evaluated top upstream regulators, revealing both activation and inhibition of mRNA expression among regulators associated with bone repair processes. Downstream effect networks shed light on the molecular pathways influenced by the activation status of these regulators.
The analysis compared the quantitative transcriptomic data with temporally grouped data to understand gene expression dynamics during fracture healing under phosphate restriction conditions. Three distinct temporal groups were identified, each exhibiting unique expression patterns. Group 1 which shifted expression of genes from earlier to later times showed Sonic Hedgehog (SHH) and Cryptochrome 2 (CRY2) emerging as potential regulators, indicating involvement in osteoblast behavior, differentiation, and circadian rhythm regulation. Temporal Group 2 showed the genes that shifted from later to earlier time showing activation of the WNT/SHH axonal guidance signaling suggesting potential interplay in cellular migration and differentiation. Group 3 showed a marked shift in return from a downregulated state during the first two weeks when the mice were on their restricted diets to higher levels of expression in the last two weeks after phosphate reintroduction. Interestingly this group of genes was predominated by those associated with oxidative metabolism, suggesting that after the return to their normal diet was restored oxidative metabolism to normal levels of activity. Selective upstream regulators associated with these pathways included RICTOR and TEAD1.
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2024