Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Observational Study
. 2021 Apr 4;13(8):11296-11314.
doi: 10.18632/aging.202817. Epub 2021 Apr 4.

Hepcidin-induced reduction in iron content and PGC-1β expression negatively regulates osteoclast differentiation to play a protective role in postmenopausal osteoporosis

Affiliations
Observational Study

Hepcidin-induced reduction in iron content and PGC-1β expression negatively regulates osteoclast differentiation to play a protective role in postmenopausal osteoporosis

Hui Zhang et al. Aging (Albany NY). .

Abstract

As a necessary trace element, iron is involved in many physiological processes. Clinical and basic studies have found that disturbances in iron metabolism, especially iron overload, might lead to bone loss and even be involved in postmenopausal osteoporosis. Hepcidin is a key regulator of iron homeostasis. However, the exact role of hepcidin in bone metabolism and the underlying mechanism remain unknown. In this study, we found that in postmenopausal osteoporosis cohort, the concentration of hepcidin in the serum was significantly reduced and positively correlated with bone mineral density. Ovariectomized (OVX) mice were then used to construct an osteoporosis model. Hepcidin overexpression in these mice significantly improved bone mass and rescued the phenotype of bone loss. Additionally, overexpression of hepcidin in OVX mice greatly reduced the number and differentiation of osteoclasts in vivo and in vitro. This study found that overexpression of hepcidin significantly inhibited ROS production, mitochondrial biogenesis, and PGC-1β expression. These data showed that hepcidin protected osteoporosis by reducing iron levels in bone tissue, and in conjunction with PGC-1β, reduced ROS production and the number of mitochondria, thus inhibiting osteoclast differentiation and bone absorption. Hepcidin could provide new targets for the clinical treatment of postmenopausal osteoporosis.

Keywords: PGC-1β; ROS; hepcidin; iron; postmenopausal osteoporosis.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
The hepcidin content is decreased in the postmenopausal women with osteoporosis. (A) Hepcidin concentration in the serum of peripheral blood in the postmenopausal women with osteoporosis (T score ≤ 2.5); (B) Lumbar BMD and serum hepcidin concentration showed a positive linear correlationship in the postmenopausal women with osteoporosis; r = 0.4560; statistical significance was considered at P = 0.0059, (n = 35; age = 62.11 ± 5.9).
Figure 2
Figure 2
Hepcidin overexpression has little effect on osteoblasts in the OVX mouse. (A, B) Hepcidin overexpression decrease the irons contents in the mouse bone. (C, D) ALP stain shows that overexpression Hepcidin does not significantly inhibit the number of osteoblasts in femoral bone in the OVX mouse; (E, F) Osteoblasts, which cultured by mouse serum for 14 or 21 days, stained with ALP or alizarin red to assess its differentiation and mineralization; (G, H) Quantitative polymerase chain reaction (q-PCR) analysis of the expression of bone formation markers including Alp and Bmp2. Scale bar, 200 μm. The asterisks (*) indicate significant differences at P < 0.05.
Figure 3
Figure 3
Hepcidin overexpression (HAMP) of hepcidin rescues bone loss induced by ovariectomy in mice. (A) The micro-CT showed that the loss of bone was rescued by overexpression of hepcidin in the OVX mouse. Micro-CT showed that (B) the distal femur bone mineral density and (C) the relevant parameters percent bone volume (BV/TV), (D) trabecular number (Tb.N), (E) trabecular thickness (Tb.Th) and (F) trabecular separation (Tb.Sp) were rescued by overexpression of hepcidin in the OVX mouse. Student's t-test was performed to determine statistic difference. The asterisks (*, **) indicate significant differences at P < 0.05, 0.01.
Figure 4
Figure 4
Hepcidin overexpression inhibits osteoclast number and differentiate in the OVX mouse. (A, B) TRAP staining shows that overexpression Hepcidin significantly inhibits the number of osteoclasts in femoral bone in the OVX mouse; (C) Overexpression hepcidin decrease CTX concentration in the OVX mouse serum. Bone marrow macrophage, which extracted from femur and cultured with M-CSF and RANKL for 8 days, stained with TRAP to assess its differentiation. (D) TRAP staining revealed that differentiation of osteoclasts in vivo M-SCF induced in the OVX mouse; (E) The osteoclasts were counted in the overexpression hepcidin and non overexpression hepcidin OVX mice; (F) The bone pits in vivo M-SCF induced osteoclasts, which was separate from OVX mouse; (G) The bone pits in vivo M-SCF induced osteoclasts, which was separate from overexpression hepcidin OVX mouse; Quantitative polymerase chain reaction (q-PCR) analysis of the expression of bone resorption markers, including (H) Mmp9, (I) Trap, (J) Ctsk and (K) Ptk2β. Scale bar, 200 μm. The asterisks (*, **) indicate significant differences at P < 0.05, 0.01.
Figure 5
Figure 5
Hepcidin overexpression inhibits ROS production, mitochondrial biogenesis, and PGC-1β expression in osteoclasts. Bone marrow macrophages were extracted from femur and cultured with M-CSF and RANKL for 8 days. (A, C) The smaller cells are undifferentiated bone marrow macrophages and the larger cells in the middle are completely differentiated mature osteoclasts; (A) DCFH-DA and (C) mitochondrion-selective probes were used for assessing ROS and mitochondrial number in un-or-differentiated osteoclasts; (B, D) Mean fluorescence density of intermediate mature osteoclasts was measured to represent ROS and mitochondrial number respectively; (E) The smaller cells are undifferentiated bone marrow macrophages and the larger cells in the middle are completely differentiated mature osteoclasts; Immunocytofluorescence was used for assessing PGC-1β (E) in the osteoclasts, which were extracted from femur and cultured with M-CSF and RANKL for 8 days; (F) Mean fluorescence density of intermediate mature osteoclasts was measured to represent PGC-1β protein level; (G) the PGC-1β expression level was evaluated using qRT-PCR in osteoclasts; (H) PGC-1β protein levels were analyzed by western blotting in osteoclasts. Scale bar, 50 μm. The asterisks (*, **) indicate significant differences at P < 0.05, 0.01.
Figure 6
Figure 6
Overexpression hepcidin decreases PGC-1β expression in OVX mouse bone. The expression of PGC-1β in bone was evaluated by immunohistofluorescence assays in overexpression hepcidin and non-overexpression hepcidin OVX mouse bone. Scale bar, 50 μm.
Figure 7
Figure 7
Mechanism pattern diagram of hepcidin overexpression to improve bone metabolism. Hepcidin overexpression in the model mice liver (1) reduces estrogen deficiency-induced bone loss (2), through reducing iron content (3), decreasing mitochondrial respiration (4) and ROS production (5), suppressing PGC-1β expression (6), inhibiting mitochondrial biogenesis (7), and depressing the function of osteoclasts (8).

Similar articles

Cited by

References

    1. Eastell R, O’Neill TW, Hofbauer LC, Langdahl B, Reid IR, Gold DT, Cummings SR. Postmenopausal osteoporosis. Nat Rev Dis Primers. 2016; 2:16069. 10.1038/nrdp.2016.69 - DOI - PubMed
    1. Miyamoto T. Mechanism underlying post-menopausal osteoporosis: HIF1α is required for osteoclast activation by estrogen deficiency. Keio J Med. 2015; 64:44–47. 10.2302/kjm.2015-0003-RE - DOI - PubMed
    1. Hughes DE, Dai A, Tiffee JC, Li HH, Mundy GR, Boyce BF. Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta. Nat Med. 1996; 2:1132–36. 10.1038/nm1096-1132 - DOI - PubMed
    1. Hofbauer LC, Schoppet M. Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases. JAMA. 2004; 292:490–95. 10.1001/jama.292.4.490 - DOI - PubMed
    1. Zhao J, Huang M, Zhang X, Xu J, Hu G, Zhao X, Cui P, Zhang X. MiR-146a deletion protects from bone loss in OVX mice by suppressing RANKL/OPG and M-CSF in bone microenvironment. J Bone Miner Res. 2019; 34:2149–61. 10.1002/jbmr.3832 - DOI - PubMed

Publication types

MeSH terms