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

Abstract

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.

The Effect of Bone Morphogenetic Protein 2 or Extracellular Signal-Regulated Kinase 1 Silencing on Phosphorus Utilization and Related Parameters in Primary Broiler Osteoblasts

“…Osteoblasts play a major role in the bone formation and development. In broilers, bone P retention and bone mineralization-related parameters are sensitive indictors that reflect P utilization (12,13,15). The ALP and BGP have been demonstrated to participate in the process of hydroxyapatite formation and mineralization in osteoblasts (19).…”

“…The primary osteoblasts were isolated by tissue explant method, and their culture and treatments were done as previously described ( 13 ). Briefly, the tibiae were obtained from one-day-old chicks (male Arbor Acres broilers, purchased from Jinghai Poultry Group Co., Ltd., Jiangsu, China), scraped cleanly, and then cut into 1 mm 2 pieces.…”

“…At 24 h post-transfection, all cells were washed twice with the P-free DMEM medium to remove the residual P. Then, the normal cells and BMP2 or ERK1 silencing cells were incubated in the osteogenic induction medium (OIM) supplemented with 0.0 or 2.0 mmol/L of P for 12 days. Selecting the supplemental P dose of 2 mmol/L in the present study was based on our previous studies ( 11 , 13 ). The OIM was the P-free DMEM supplemented with 15% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 100 nmol/L Dex and 50 μg/mL ascorbic acid.…”

“…The total P contents in the fresh or old medium were determined as previously described ( 13 ). Briefly, 1 mL of fresh or old medium was digested with HNO 3 -HClO 4 (5:1) of mixed acid at 200 °C.…”

“…The total P contents in the fresh or old medium were determined as previously described (13). Briefly, 1 mL of fresh or old medium was digested with HNO 3 -HClO 4 (5:1) of mixed acid at 200 • C. The digested residues were dissolved in 10 mL of 2% HNO 3 and then used for the total P determination.…”