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

Abstract

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.

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

“…Furthermore, ERK1 inhibitor was shown to suppress the ALP activity, bone mineralization and downregulate the expression of BMP2 mRNA in mice bone mesenchymal stem cells ( 10 ). The results from our studies in vivo and in vitro have demonstrated that P utilization in the bone and osteoblasts of broilers might be partially regulated by BMP2 and ERK1 ( 11 , 12 ). However, it is unknown if BMP2 or ERK1 is directly involved in regulating P utilization in primary broiler osteoblasts.…”

“…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 mRNA expressions of BMP2 and ERK1 in the osteoblasts were determined by the RT-qPCR according to the procedure as previously described ( 11 ). The 2 −ΔΔCt method was used to calculate the relative mRNA expressions of target genes to reference genes including β- actin and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ).…”

“…Total and phosphorylated protein expression levels of target genes were analyzed by Western blotting as previously described (11,15,16). The primary antibodies used in the experiment included anti-BMP2 (catalog no.…”

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

“…The DMP1 has been found to be involved in regulating P homeostasis and bone mineralization in mammals ( 24 , 25 ). Latest studies in vivo and in vitro suggested that bone P retention or osteoblastic P utilization might be regulated by DMP1 ( 1 , 26 ), implying a possible contribution of DMP1 to the bone P utilization of broilers. However, whether DMP1 is involved in regulating P metabolic utilization in primary cultured tibial osteoblasts of broiler chicks has not been reported before.…”