The Effects of Inorganic Phosphorus Levels on Phosphorus Utilization, Local Bone-Derived Regulators, and BMP/MAPK Pathway in Primary Cultured Osteoblasts of Broiler Chicks.

Understanding the underlying mechanisms that regulate the bone phosphorus (P) utilization would be helpful for developing feasible strategies to improve utilization efficiency of P in poultry. We aimed to investigate the effects of inorganic P levels on P utilization, local bone-derived regulators and bone morphogenetic protein/mitogen-activated protein kinase (BMP/MAPK) pathway in primary cultured osteoblasts of broiler chicks in order to address whether local bone-derived regulators or BMP/MAPK pathway was involved in regulating the bone P utilization of broilers using an in vitro model. The primary cultured tibial osteoblasts of broiler chicks were randomly divided into one of five treatments with six replicates for each treatment. Then, cells were respectively incubated with 0.0, 0.5, 1.0, 1.5, or 2.0 mmol/L of added P as NaH2PO4 for 24 days. The results showed that as added P levels increased, tibial osteoblastic P retention rate, number and area of mineralized nodules, the mRNA expressions of endopeptidases on the X chromosome (PHEX), dentin matrix protein 1 (DMP1), bone morphogenetic protein 2 (BMP2), and the mRNA and protein expressions of matrix extracellular phosphoglycoprotein (MEPE) increased linearly (p < 0.001) or quadratically (p < 0.04), while extracellular signal-regulated kinase 1 (ERK1) mRNA expression and c-Jun N-terminal kinase 1 (JNK1) phosphorylated level decreased linearly (p < 0.02) or quadratically (p < 0.01). Correlation analyses showed that tibial osteoblastic P retention rate was positively correlated (r = 0.452-0.564, p < 0.03) with MEPE and BMP2 mRNA expressions. Furthermore, both number and area of mineralized nodules were positively correlated (r = 0.414-0.612, p < 0.03) with PHEX, DMP1, MEPE, and BMP2 mRNA expressions but negatively correlated (r = -0.566 to -0.414, p < 0.04) with the ERK1 mRNA expression and JNK1 phosphorylated level. These results suggested that P utilization in primary cultured tibial osteoblasts of broiler chicks might be partly regulated by PHEX, DMP1, MEPE, BMP2, ERK1, and JNK1.

Dentin Matrix Protein 1 Silencing Inhibits Phosphorus Utilization in Primary Cultured Tibial Osteoblasts of Broiler Chicks.

Three experiments were carried out in the present study to investigate whether dentin matrix protein 1 (DMP1) was involved in regulating phosphorus (P) metabolic utilization in primary cultured tibial osteoblasts of broiler chicks. Experiment 1 was conducted to select the optimal osteogenic inductive culture medium and the optimal induction time in primary cultured tibial osteoblasts of broiler chicks. In experiment 2, the siRNAs against DMP1 were designed, synthesized and transfected into primary cultured tibial osteoblasts of broiler chicks, and then the inhibitory efficiencies of siRNAs against DMP1 were determined, and the most efficacious siRNA was selected to be used for the DMP1 silencing. In experiment 3, with or without siRNA against DMP1, primary cultured tibial osteoblasts of broiler chicks were treated with the medium supplemented with 0.0, 1.0 or 2.0 mmol/L of P as NaH2PO4 for 12 days. The P metabolic utilization-related parameters were measured. The results showed that the osteogenic induced medium 2 and 12 days of the optimal induction time were selected; Among the designed siRNAs, the si340 was the most effective (P < 0.05) in inhibiting the DMP1 expression; DMP1 silencing decreased (P < 0.05) the expressions of DMP1 mRNA and protein, P retention rate, mineralization formation, alkaline phosphatase activity and bone gla-protein content in tibial osteoblasts at all of added P levels. It is concluded that DMP1 silencing inhibited P utilization, and thus DMP1 was involved in regulating P metabolic utilization in primary cultured tibial osteoblasts of broiler chicks, which provides a novel insight into the regulation of the P utilization in the bone of broilers, and will contribute to develop feasible strategies to improve the bone P utilization efficiency of broilers so as to decrease its excretion.

Regulation of bone phosphorus retention and bone development possibly by related hormones and local bone-derived regulators in broiler chicks.

BACKGROUND: Phosphorus is essential for bone mineralization in broilers, however, the underlying mechanisms remain unclear. We aimed to investigate whether bone phosphorus retention and bone development might be regulated by related hormones and local bone-derived regulators in broilers. METHODS: Broilers were fed diets containing different levels of non-phytate phosphorus (NPP) 0.15%, 0.25%, 0.35%, 0.45% and 0.55% or 0.15%, 0.22%, 0.29%, 0.36% and 0.43% from 1 to 21 or 22 to 42 days of age. Serum and tibia samples were collected for determinations of bone phosphorus retention and bone development parameters, related hormones and local bone-derived regulators of broiler chickens on d 14, 28 and 42, respectively. RESULTS: Tibia ash phosphorus, total phosphorus accumulation in tibia ash (TPTA), bone mineral concentration (BMC), bone mineral density (BMD), bone breaking strength (BBS), and ash on d 14, 28 or 42, serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on d 28 and 42, mRNA expressions of tibia fibroblast growth factor 23 (FGF23) and dentin matrix protein 1 (DMP1) on d 14 and 28 increased linearly or quadratically (P < 0.05), while serum parathyroid hormone (PTH) on d 28, tibia alkaline phosphatase (ALP) on d 14, 28 and 42, bone gal protein (BGP) on d 14, and mRNA expression of tibia phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) on d 14 and 28 decreased linearly or quadratically (P < 0.04) as dietary NPP level increased. TPTA, BMC, BMD, and ash on d 28 and 42, BBS on d 28, and ash phosphorus on d 42 were positively correlated (r = 0.389 to 0.486, P < 0.03) with serum 1,25(OH)2D3. All of the above parameters were positively correlated (r = 0.380 to 0.689, P < 0.05) with tibia DMP1 mRNA expression on d 14, 28 and 42, but negatively correlated (r = - 0.609 to - 0.538, P < 0.02) with serum PTH on d 28, tibia ALP on d 14, 28 and 42, and BGP on d 14. TPTA, BMC and ash on d 14 and BMD on d 28 were negatively correlated (r = - 0.397 to - 0.362, P < 0.03) with tibia PHEX mRNA expression, and BMD on d 28 was positively correlated (r = 0.384, P = 0.04) with tibia FGF23 mRNA expression. CONCLUSIONS: These results suggested that bone phosphorus retention and bone development parameters had moderate to strong correlations with serum PTH and 1,25(OH)2D3 and tibia DMP1, PHEX, FGF23, ALP and BGP in broilers during the whole growth period, and thus they might be partly regulated by these related hormones and local bone-derived regulators.