Role of phosphate restriction in regulating HIPPO and hypoxia signaling pathway gene expression during bone fracture repair
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Abstract
Bone, a hybrid tissue composed of carbonate-rich hydroxyapatite mineral and bone matrix proteins, serves various functions including mechanical support, metabolic homeostasis, and hematopoiesis. As hydroxyapatite is composed of calcium and phosphate, chronic phosphate deficiency or wasting leads to impaired skeletal mineralization. Phosphate is essential for mineralization, thus, an important regulator for the differentiation of chondrocytes. Previous studies showed hypophosphatemic conditions by food intake affect bone fracture healing process. Several recent studies implicated the involvement of a set of molecular metabolic/signaling pathways to control bone fracture healing process. However, the involvement of HIPPO and hypoxia signaling pathways during fracture healing with hypophosphatemic condition is still largely unknown. Therefore, the current study was conducted to test the hypothesis whether HIPPO and hypoxia signaling pathway is associated with the fracture healing process with dietary phosphate restriction. In this study, we examine their involvement during fracture healing under control and hypophosphatemic conditions and analyze both gene and protein expression by quantitative real-time polymerase chain reaction (qPCR) and histological analyses. RNA was isolated, purified, and used for reverse transcription to synthesize cDNA. The cDNA was then subjected to qPCR using specific primers for markers related to chondrogenesis, osteogenesis, phosphate regulation, HIPPO pathway, and hypoxia. Histological staining analyses were performed on femur samples to assess callus tissue formation and collagen matrix organization. Safranin-O staining, picro-sirius red staining, and immunohistochemical staining were conducted to evaluate cellular and matrix characteristics. The results provide insights into the effects of dietary phosphate restriction on the expression of key genes, the quality of callus extracellular matrix during bone fracture healing, oxidative metabolic collagen gene regulation, and HIPPO regulatory activity during bone fracture healing. The qPCR analysis revealed that phosphate restriction led to enhanced expression levels of chondrogenesis and early/intermediate osteogenesis markers, while late-stage osteogenesis marker expression was impaired. Additionally, phosphate restriction showed a differential effect on phosphate-regulation markers, inhibiting the expression of fibroblast growth factor 23 (FGF23) but leaving phosphate regulating endopeptidase homolog, X-linked (Phex), essentially unaffected. Furthermore, phosphate restriction markedly inhibited the expression levels of HIPPO signaling pathway markers.
Histological analysis demonstrated that phosphate restriction resulted in disorganized chondrocyte zones with more hypertrophic chondrocytes and inhibited collagen matrix organization and maturation during bone fracture healing. Immunohistochemical analysis revealed reduced immunoreactivity of type I collagen and connective tissue growth factor (CTGF) in the phosphate-restricted group compared to the control, indicating impaired bone formation and reduced cellular activity. Other key findings included alterations in gene expression profiles, callus tissue formation, collagen matrix organization, and protein expression patterns of HIPPO and hypoxia markers during bone fracture healing. The findings suggested that phosphate restriction modulates the expression of YAP/TAZ target genes, potentially influencing fracture healing outcomes. The study also explored the effects of phosphate restriction on hypoxia signaling and collagen matrix quality during fracture healing. Overall, the results supported the hypothesis that dietary phosphate restriction inhibits the HIPPO and hypoxia signaling pathways, affecting the quality of callus extracellular matrix organization and maturation during bone healing. Future directions for research include investigating the regulation of collagen-modifying enzymes, examining additional YAP/TAZ target genes, and exploring hypoxia-related gene expressions to further elucidate the mechanisms underlying the effects of dietary phosphate restriction on fracture healing.
Description
2024