Hepcidin promotes osteogenic differentiation through the bone morphogenetic protein 2/small mothers against decapentaplegic and mitogen-activated protein kinase/P38 signaling pathways in mesenchymal stem cells

Abstract

The ability of mesenchymal stem cells (MSCs) to differentiate into osteogenic lineages requires management for their future use in treating bone destruction and osteoporosis. Hepcidin is closely associated with bone metabolism, however, it remains to be elucidated whether hepcidin affects osteogenic differentiation in MSCs. The present study demonstrated that hepcidin enhanced osteoblastic differentiation and mineralization, which was manifested by an upregulation in the differentiation markers alkaline phosphatase and osteogenic genes. Furthermore, the expression levels of bone morphogenetic proteins and small mothers against decapentaplegic homologs were concomitantly increased following hepcidin treatment. In addition, the p38 mitogen-activated protein kinase may be an upstream kinase for osteoblastic differentiation. Thus, hepcidin may be important in the osteogenic differentiation of MSCs and may be considered as a target in the development of therapies for pathological bone loss.

Figure 1

Effect of hepcidin on the osteogenic differentiation and protein expression of ALP and OCN and mineralization in MSCs. (A) MSCs were cultured in osteogenic medium with or without 0.2 mmol/l hepcidin for 0, 14 and 21 days to induce osteogenic differentiation. Intracellular calcium deposition was assessed using alizarin red staining (magnification, ×100). (B) MSCs were cultured in osteogenic medium with or without hepcidin for 0, 5 and 7 days and the levels of ALP were detected using an ALP assay (^**P<0.01, vs.0 days). (C) MSCs were cultured in osteogenic medium with or without hepcidin for 0, 3 and 5 days and the levels of ALP were detected by western blotting (^**P<0.01, vs. 0 days; ^#P<0.01 hepcidin group, vs. control group). (D) MSCs were cultured in osteogenic medium with or without hepcidin for 0, 3 and 5 days and the levels of OCN were detected by western blotting (^**P<0.01, vs. 0 days; ^#P<0.01 hepcidin group, vs. control group). MSC, mesenchymal stem cell; ALP, alkaline phosphatase; OCN, osteocalcin.

Figure 2

Effect of hepcidin on the BMP2/Smads pathway in MSCs. (A) MSCs were cultured in osteogenic medium with or without 0.2 mmol/l hepcidin for 24, 48 and 72 h and the expression level of BMP2 mRNA was detected using RT-qPCR. The data are expressed as the means ± SEM (n=3). GAPDH was used as a loading control (^**P<0.01, vs. control; ^#P<0.01, vs. control). (B) Cells were cultured, as described above, and the expression level of Smad1 mRNA was detected using RT-qPCR. The data are expressed as the means ± SEM (n=3). GAPDH was used as a loading control. (^**P<0.01, vs. control; ^#P<0.01, vs. control). (C) Cells were cultured, as described above, and the expression level of Smad5 mRNA was detected using RT-qPCR. The data are expressed as the means ± SEM (n=3). GAPDH was used as a loading control (^**P<0.01, vs. control 24 h; ^#P<0.01, vs. control). (D) Cells were cultured, as described above, and the expression level of Smad8 mRNA was detected using RT-qPCR. The data are expressed as the means ± SEM (n=3). GAPDH was used as a loading control (^**P<0.01, vs. control; ^#P<0.01, vs. control). MSC, mesenchymal stem cell; BMP2, bone morphogenetic protein 2; Smads; small mothers against decapentaplegic; RT-qPCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean.

Figure 3

BMP2/Smad signaling pathways contribute to hepcidin-induced osteogenic differentiation by hepcidin. (A) RT-qPCR analysis was performed using total RNA isolated from BMP2 mRNA following BMP2-siRNA transfection at different times in the MSCs. Each value is expressed as the mean ± SEM (n=3). GAPDH was used as a loading control (^*P<0.05, vs. control; ^**P<0.01, vs. control). (B) Cells in the control, siRNA control and BMP2-siRNA groups were cultured in osteogenic medium with or without 0.2 mmol/l hepcidin for 48 h and the expression level of p-Smad1, p-Smad5 and p-Smad8 were detected by western blotting. Each value is expressed as the mean ± SEM (n=3). β-actin was used as a loading control. (C) Cells were cultured, and the expression levels of ALP were detected using an ALP assay. Each value is expressed as the mean ± SEM (n=3; ^*P<0.05, vs. control; ^**P<0.01, vs. control; ^#P<0.01, vs. hepcidin + BMP2-siRNA group). (D) Cells were cultured, as described above, and the expression levels of OCN were detected by western blotting. Each value is expressed as the mean ± SEM (n=3). β-actin was used as a loading control. (^*P<0.01, vs. control; ^#P<0.01, vs. hepcidin + BMP2-siRNA group). MSC, mesenchymal stem cell; BMP2, bone morphogenetic protein 2; Smad; small mothers against decapentaplegic; p-Smad, phosphorylated Smad; RT-qPRC, reverse transcription polymerase chain reaction; SEM, standard error of the mean.

Figure 4

MAPK/P38 signaling pathways contribute to osteogenic differentiation induced by hepcidin. (A) Cells in the control, siRNA control group and BMP2-siRNA groups were cultured in osteogenic medium with or without 0.2 mmol/l hepcidin for 48 h and the expression levels of p-ERK, p-P38 and p-JNK were detected using wetern blot analysis. (B) Each value is expressed as the mean ± SEM (n=3) and β-actin was used as a loading control (^*P<0.05, vs. control; ^**P<0.01, vs. control; ^#P<0.01, vs. hepcidin. (C) Cells were cultured in osteogenic medium and exposed to 0.2 mmol/l hepcidin with or without 10 μM SB203580 for 48 h and the expression levels of p-P38 were detected by western blotting. (D) Each value is expressed as the mean ± SEM (n=3) and β-actin was used as a loading control (^*P<0.05, vs. control; ^**P<0.01, vs. control; ^#P<0.01 hepcidin, vs. hepcidin + SB203580). (E) Cells were cultured, as described above, and the expression levels of ALP were detected using an alkaline phosphatase assay kit. Each value is expressed as the mean ± SEM (n=3; ^**P<0.01, vs. control; ^#P<0.01 hepcidin, vs. hepcidin + SB203580). (F) Cells were cultured, as described above, and the expression levels of OCN were detected by western blotting. Each value is expressed as the mean ± SEM (n=3) and β-actin was used as a loading control (^**P<0.01, vs. control; ^#P<0.01 hepcidin, vs. hepcidin + SB203580). MSC, mesenchymal stem cell; MAPK, mitogen-activated protein kinase; RT-qPRC, reverse transcription polymerase chain reaction; SEM, standard error of the mean.