The authors have declared that no competing interests exist.
Conceived and designed the experiments: YX. Performed the experiments: XW LMY WHC. Analyzed the data: XW LMY WHC YX. Wrote the paper: YX XW PBY JLB HYL.
Hepcidin is an antimicrobial peptide, which also negatively regulates iron in circulation by controlling iron absorption from dietary sources and iron release from macrophages. Hepcidin is synthesized mainly in the liver, where hepcidin is regulated by iron loading, inflammation and hypoxia. Recently, we have demonstrated that bone morphogenetic protein (BMP)-hemojuvelin (HJV)-SMAD signaling is central for hepcidin regulation in hepatocytes. Hepcidin is also expressed by macrophages. Studies have shown that hepcidin expression by macrophages increases following bacterial infection, and that hepcidin decreases iron release from macrophages in an autocrine and/or paracrine manner. Although previous studies have shown that lipopolysaccharide (LPS) can induce hepcidin expression in macrophages, whether hepcidin is also regulated by BMPs in macrophages is still unknown. Therefore, we examined the effects of BMP signaling on hepcidin expression in RAW 264.7 and J774 macrophage cell lines, and in primary peritoneal macrophages. We found that BMP4 or BMP6 alone did not have any effect on hepcidin expression in macrophages although they stimulated Smad1/5/8 phosphorylation and Id1 expression. In the presence of LPS, however, BMP4 and BMP6 were able to stimulate hepcidin expression in macrophages, and this stimulation was abolished by the NF-κB inhibitor Ro1069920. These results suggest that hepcidin expression is regulated differently in macrophages than in hepatocytes, and that BMPs regulate hepcidin expression in macrophages in a LPS-NF-κB dependent manner.
Hepcidin, a small cationic peptide, was first identified based on its antimicrobial and antifungal properties
Hepcidin is mainly synthesized by the liver. The expression of hepcidin in hepatocytes increases in response to infection/inflammation and elevated systemic iron. In isolated primary hepatocytes, hepcidin expression is stimulated by IL-6, IL-1 and lipopolysaccharide (LPS), but not by TNF-α
Hepcidin is also expressed by myeloid cells including monocytes, macrophages and neutrophils
Based on the pivotal role of BMP signaling in hepcidin regulation in hepatocytes, we investigated whether or not BMP signaling regulates hepcidin expression in macrophages. Surprisingly, we found that macrophages did not increase hepcidin expression in response to BMP stimulation, unless BMP signaling was accompanied by co-stimulation with LPS.
BMP4 and BMP6 were purchased from R & D Systems (Minneapolis, MN). Lipopolysaccharides (LPS) from Escherichia coli 0127:B8 was purchased from Sigma-Aldrich (Saint Louis, MO). Phospho-Smad1/5/8, Smad1, phospho-NF-κB p65 and NF-κB p65 antibodies were purchased from Cell Signaling Technology (Beverly, MA). β-actin antibodies were purchased from Sigma-Aldrich. NF-κB inhibitor Ro1069920 was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
RAW264.7 and J774 macrophage cell lines (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 2 mM L-glutamine, and antibiotics. Cells were starved with serum-free DMEM supplemented with 0.1% bovine serum albumin before they were incubated with BMP4, BMP6, LPS or a combination of BMP ligands and LPS as indicated. Cells were lysed for real-time PCR quantification of mRNA transcripts or for Western blotting analyses for phosporylated Smad1/5/8 and phosphorylated NF-κB p65 levels.
Mature resident peritoneal macrophage were harvested and cultured as previously described
To examine the effect of HJV overexpression on hepcidin expression in RAW264.7 macrophages, cells were transfected with Flag-HJV cDNA in 12-well plates using Lipofectamine 2000 (Invitrogen). Approximately 24 h after transfection, the media was replaced with serum-free DMEM medium supplemented with 0.1% BSA. 16 h later, the cells were harvested for measurement of hepcidin mRNA expression.
Real-time quantification of mRNA transcripts was performed using an AB 7600 real-time system (Applied Biosystems). First-strand cDNA was amplified with previously described mouse hepcidin and Id1 primers (11), and detected using SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions. In parallel, Rpl19 (ribosomal protein-like 19) transcripts were amplified and detected in a similar manner to serve as an internal control. Standard curves were generated from accurately determined dilutions of plasmid cDNAs or purified PCR fragments as templates.
RAW264.7 cells or peritoneal primary macrophages were lysed in TBS (50 mM Tris-HCl, 150 mM NaCl, and 1% Triton X-100 [pH 7.4]) containing protease inhibitor mixture (Pierce) and phosphatase inhibitor mixture (Pierce) for 30 min on ice. After centrifugation for 10 min at 4°C, the supernatant was assayed for protein concentration by colorimetric assay (BCA kit; Pierce). A total of 20–40 µg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with anti–phospho-Smad1/5/8 (1∶1000) or anti–phospho-NF-κB p65 (1∶1000). Membranes were stripped in 0.2 M glycine (pH 2.5) and 0.5% Tween 20 for 10 min, and reprobed with anti-Smad1 (1∶1000), anti-NF-κB p65 (1∶1000) or anti-β-actin (1∶10,000) antibodies.
Results from real time PCR analyses are expressed as the means ± S.D. of three replicates. Differences were assessed by Student's
It is well documented that BMPs stimulate hepcidin expression in hepatocytes. To examine whether or not BMP signaling plays a role in hepcidin expression in macrophages, we incubated RAW264.7 macrophages with BMP4. Surprisingly, hepcidin mRNA levels were not stimulated by BMP4 with concentrations of up to 200 ng/ml (
(A, B, C and D) Effects of BMP4 on hepcidin and Id-1 expression in RAW264.7 macrophages. Cells were incubated overnight with increasing amounts of BMP4 (A & B) or with 50 ng/ml of BMP4 for increasing periods of time (C &D) in serum free DMEM supplemented with 0.1% BSA. Cells were collected for measurements of hepcidin (A & C), Id1 (B & D) and RPL19 mRNA levels. (E) Effects of BMP4 on phosphorylation levels of Smad1/5/8 in RAW264.7 macrophages. Cells were incubated with BMP4 for 1 h before cells were lysed for Western blotting for phospho-Smad1/5/8 and Smad1 levels. (F) Left panel: Effects of BMP4 on hepcidin expression in primary mouse peritoneal macrophages. Cells were incubated for 8 h with increasing amounts of BMP4 before cells were collected for quantification of hepcidin and RPL19 mRNA levels. Right panel: Effects of BMP4 and BMP6 on phosphorylation levels of Smad1/5/8 in primary mouse peritoneal macrophages. Cells were incubated with BMP4 or BMP6 for 1 h before cells were lysed for Western blotting for phospho-Smad1/5/8 and Smad1 levels. **,
BMP6 appears to be a key endogenous BMP ligand for hepcidin expression in the liver
(A and B) Cells were incubated for 8 h with increasing amounts of BMP6 in serum free DMEM supplemented with 0.1% BSA. Cells were collected for real time RT-PCR analyses to qualify hepcidin (A), Id1 (B) and RPL19 mRNA levels. (C) Effects of BMP6 on phosphorylation levels of Smad1/5/8 in RAW264.7 macrophages. Cells were incubated with BMP6 for 1 h before cells were lysed for Western blotting for phospho-Smad1/5/8 and Smad1 levels. (D) Effects of BMP6 on hepcidin expression in primary mouse peritoneal macrophages. Cells were incubated for 8 h with increasing amounts of BMP6 before cells were collected for quantification of hepcidin and RPL19 mRNA levels. ***,
HJV is a co-receptor for BMP signaling that increases BMP signaling and hepcidin expression in hepatocytes. Our previous study showed that expression of endogenous HJV in RAW264.7 cells is low
Cells were tranfected with HJV cDNA (4 ng/ml). 48 hours after transfection, the cells were collected for real time RT-PCR analyses to qualify hepcidin (A) and Id1 (B). *,
These results suggest that, unlike hepatocytes, macrophages do not respond to BMP stimulation with an increase in hepcidin expression. This unresponsiveness cannot be attributed to defects in BMP signaling in macrophages because Smad1/5/8 phosphorylation and Id1 expression were induced by treatment with BMP4 or BMP6, and Id1 expression was induced by overexpression of HJV.
Hepcidin in macrophages is stimulated by bacterial pathogens and LPS
Cells were incubated with LPS (10 ng/ml) in serum free DMEM supplemented with 0.1% BSA, and were collected at the indicated times for real time RT-PCR analyses to qualify hepcidin and RPL19 mRNA levels. *,
(A and B) RAW264.7 cells were incubated with increasing amounts of BMP4 (A) or BMP6 (B) in the presence of LPS (10 ng/ml) for 8 h. Cells were collected for real time RT-PCR analyses to qualify hepcidin and RPL19 mRNA levels. (C and D) Primary mouse peritoneal macrophages were incubated with BMP4 (C) or BMP6 (D) in the presence of LPS (10 ng/ml) for 8 h. Cells were collected for real time RT-PCR analyses to qualify hepcidin and RPL19 mRNA levels. (B) *,
A previous study showed that LPS-induced hepcidin expression is mediated by the NF-κB signaling pathway in human leukocytes
(A and B) Cells were incubated for 8 h with LPS alone, or LPS in combination with BMP4 in the presence or absence of Ro1069920 (5 µM) in serum free DMEM supplemented with 0.1% BSA. Cells were collected for Western blotting analyses using the indicated antibodies (A), and densitometry analysis from two replicates was performed (B). (C and D) Cells were incubated for 8 h with LPS alone, or LPS in combination with BMP4 (C) or BMP6 (D) in the presence or absence of Ro1069920 (5 µM) in serum free DMEM supplemented with 0.1% BSA. Cells were collected for real time RT-PCR analyses to qualify hepcidin and RPL19 mRNA levels. *,
Hepcidin enhances the host immune defense by its antimicrobial and antifungal activities, and by its negative regulation of serum iron levels. Hepcidin is mainly produced by the liver, but it is also expressed in myeloid cells including macrophages and monocytes. Infections stimulate hepcidin expression in both hepatocytes and myeloid cells, leading to an increase in hepcidin concentrations in circulation and in urine. Although the contribution of myeloid cells to the circulating hepcidin relative to that of hepatocytes during infections is difficult to determine, emerging evidence suggests that hepcidin produced from macrophages and monocytes under the stimulation of bacterial pathogens or LPS inhibits the growth of bacteria while inducing iron retention in these cells. Depletion of iron from monocytic lineage impairs LPS-induced expression of inflammatory cytokines including interferon β, whereas iron retention appears to potentiate these cytokine responses
A number of differences in the regulatory mechanisms of hepcidin expression have been identified for myeloid cells and hepatocytes. For example, hepcidin expression in macrophages in the spleen is not regulated by serum iron levels, while hepatocytes increase hepcidin expression in response to iron loading. In leukocytes
The mechanism underlying the failure of BMP signaling to stimulate hepcidin expression in macrophages remains unknown. Phosphorylation of Smad1/5/8 and Id1 expression was stimulated by BMP4 or BMP6 in macrophages. This suggests that the BMP signaling pathway is not defective. Whether there are any transcription factors that are important for hepcidin transcription but are not present or not activated in macrophages, and whether there are any epigenetic differences in the hepcidin promoter between hepatocypes and macrophages remain to be addressed. A recent study showed that the binding motif of HNF4α, a liver specific transcription factor, in the hepcidin promoter is critical for the HJV and BMP response of hepcidin in hepatocytes
STAT3 is a key transcription factor involved in regulation of hepcidin in hepatocytes in inflammatory conditions
The mechanisms responsible for the effects of BMPs on hepcidin expression in the presence of LPS co-stimulation were not examined in the present study. Previous studies have shown that LPS induces SOCS3 expression, and SOCS3 inhibits hepcidin expression as a negative feedback. Studies have also shown that TGF-β1 inhibits SOCS3 expression
Augmented BMP signaling via the enhanced expression of BMP ligands, Smad effectors or their downstream activation, has been frequently observed to be a hallmark of infection, inflammation and local tissue injury
The authors thank Mr. Yueshui Zhao, Dr. Xiaoling Li, Prof. Hui Zhao, Prof. Chao Wan and Prof. Ya Ke for helpful discussions.