Fig 1: EC-derived BMP6 is rapidly secreted in response to hemin.A, hemin 100 nM or 500 nM both significantly increased heme oxygenase 1 (HO-1) mRNA expression of HUVECs and BMP6 protein secretion in the culture supernatant. B, hemin treatments (100 nM or 500 nM) also upregulated HO-1 transcription and BMP6 protein secretion of SK hep. Representative data of three independent experiments are presented. C, HUVECs conditioned medium (CM) from cells preincubated with 500 nM hemin increased hepcidin expression compared with the HUVEC-CM without any pretreatment. The ALK2/3 inhibitor (20 nM LDN) blocked all the induction of hepcidin by HUVEC-CM. Data are shown as the mean ± SD and n = 3. D, CM from SK hep preincubated with hemin (500 nM) also increased hepcidin in Huh7 cells that could be blocked by the ALK2/3 inhibitor (LDN 20 nM). Data are shown as the mean ± SD and n = 3. E, hemin treatment (100 nM or 500 nM) did not significantly affect the hepcidin mRNA expression in Huh7 monocultured in fresh endothelial cell growth medium (ECGM). Representative data are from three independent experiments. F, Huh7 cells were seeded on top of HUVECs transfected with control siRNA or BMP6 siRNA. Transfection reagents were completely removed. After attachment, the cocultured HUVEC–Huh7 were treated with 500 nM hemin. Hemin still induced hepcidin upregulation in controls, whereas it failed to do so in BMP6-knockdown cells. Representative data are from three independent experiments. G, Huh7 cells were seeded on top of siBMP6-transfected SK heps. BMP6 silencing also blocked hepcidin induction by hemin in this Huh7/SK hep coculture system. Representative data of three independent experiments are presented. Cells in panels A, C, E, and F were cultured in ECGM containing 2% FCS, and cells in panels B, D, and G were cultured in DMEM containing 2% FCS (only Huh7 cell seeding procedures required the DMEM containing 10% FCS). BMP6 concentrations in the supernatant were measured by the direct ELISA assay. HO-1 and hepcidin mRNA expression was determined by qRT-PCR, and the results were normalized to beta-2-microglobulin (ß2MG). Data are presented as dot plots with the mean ± SD, and significant differences are marked by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001). ALK, ALK receptor tyrosine kinase; BMP6, Bone morphogenetic protein 6; ECs, endothelial cells; FCS, fetal calf serum; hemin, ferric chloride heme; HUVECs, human umbilical vein endothelial cells; LDN, LDN193189 hydrochloride; n.s., not significant; qRT-PCR, real-time quantitative PCR.
Fig 2: Crosstalk between endothelial cells and hepatocytes in iron regulation. Liver sinusoidal endothelial cells (LSECs) recognize surrounding ferric iron or heme and moderately deliver iron signaling to hepatocytes by modifying BMP6 and BMPER secretion. BMP6 can bind to ALK2/3 on hepatocytes to activate the downstream SMAD–hepcidin pathway. ALK, ALK receptor tyrosine kinase; BMP6, Bone morphogenetic protein 6; BMPER, BMP-binding endothelial regulator; SMAD, small mothers against decapentaplegic homolog.
Fig 3: Silencing endothelial BMP6 blocks hepatocellular hepcidin induction.A, BMP6 siRNA (50 nM/well) and negative siRNA (50 nM/well) as control were separately transfected into HUVECs by Lipofectamine 2000 (3 µl/well). All reagents were removed and replaced by fresh endothelial cell growth medium (EGCM) after 6 h. Knockdown efficiency was validated at 48 h by Western blot and qRT-PCR. BMP6 expression was significantly suppressed by BMP6 siRNA compared with the nontargeting control siRNA on both mRNA and protein levels. Representative data of three independent experiments (n = 3) are shown. B, after transfection reagents were removed and HUVECs recovered stable in fresh ECGM for 18 h, Huh7 cells (in DMEM with 10% FCS) were directly seeded on top of these transfected HUVECs (cell ratio 1:1) with Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS. The seeding medium was removed at 48 h after Huh7 cells were well attached, and just fresh ECGM with 2% FCS was added to the coculture system. All cells were collected together at 72 h. In the HUVEC and Huh7 coculture system, the induction of hepatocellular hepcidin was drastically impaired by BMP6 siRNA interference in HUVECs. Representative data are shown for three independent experiments. C, the protocols of BMP6 siRNA transfection in SK hep and coculture with Huh7 cells were the same as those used for HUVECs except DMEM containing 2% FCS for SK heps culture. BMP6 transcription and protein levels were reduced in SK hep by BMP6 siRNA as well. Representative data are shown for three independent experiments. D, BMP6 silencing inhibited the hepatocellular hepcidin expression in a direct SK hep/Huh7 coculture system. Representative data are shown for three independent experiments. E and F, the hepcidin induction in Huh7 cells by BMP6 (50 ng/ml) was completely blocked by the ALK2/3 inhibitor (LDN 20 nM) after 24 h of treatment, whereas the LDN did not significantly affect basal hepcidin expression. This experiment on Huh7 cells was operated under normal medium for Huh7 culture (DMEM with 10% FCS). G, ALK2/3 inhibitor (LDN 20 nM) in HUVEC conditioned medium (CM) decreased hepcidin expression in Huh7 after 24 h. Representative data are from three independent experiments (n = 3). H, hepcidin transcription level was inhibited by the ALK2/3 inhibitor (LDN 20 nM) as well. Representative data of three independent experiments (n = 3 of each time) are used for statistical analysis. Target proteins were determined by Western blot and normalized to GADPH. Transcriptional changes of target genes were determined by qRT-PCR, and the results were normalized to beta-2-microglobulin (ß2MG) or hypoxanthine guanine phosphoribosyltransferase (HPRT). Data are presented as dot plots with the mean ± SD, and significant differences are marked by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001). ALK, ALK receptor tyrosine kinase; BMP6, Bone morphogenetic protein 6; HUVECs, human umbilical vein endothelial cells; FCS, fetal calf serum; LDN, LDN193189 hydrochloride; n.s., not significant; qRT-PCR, real-time quantitative PCR.
Fig 4: Endothelial cells are highly responsive to surrounding ferric iron changes.A, concentration gradient of iron (ferric ammonium citrate [FAC] 0.5 µM, 5 µM, 50 µM in medium) gradually upregulated BMP6 mRNA expression in HUVECs. B, BMP6 transcription in HUVECs was upregulated by 50 µM FAC after 6 h, and this upregulation still lasted after 12 h of treatment. C, iron (FAC 50 µM) significantly induces BMP6 concentration in the supernatants of cultured HUVECs over a time period of 24 h as measured by human BMP6 ELISA. D, treatment of HUVECs with 5 µM FAC or with 5 µM hemin for 24 h could both induce BMP6 expression and inhibited transferrin receptor 1 (TFR1) expression at the protein level. TFR1 was induced in the presence of the membrane-permeable iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) (5 µM). Representative data of three independent Western blots are used for statistical analysis. E, endothelial-released BMPER, an autocrine BMP inhibitor, was likewise induced both in vivo (left) and in vitro (right) in response to iron at the mRNA level. Mice were iron-loaded using SFG (three mice in each group), whereas HUVECs were treated with 50 µM FAC for 24 h. F, human recombinant BMPER protein (50 ng/ml in medium) had no influence on basal hepcidin expression in Huh7 cells, whereas it strongly blocked the hepcidin expression in response to recombinant BMP6 (50 ng/ml). Data are shown as the mean ± SD and n = 3. Further conditions are as follows: HUVECs in panels A–E were under endothelial cell growth medium containing 2% FCS, and Huh7 cells in panel F were cultured with Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS. The mRNA expression was determined by qRT-PCR. PCR results were normalized to hypoxanthine guanine phosphoribosyltransferase (HPRT) or beta-2-microglobulin (ß2MG). Data are presented as dot plots and the mean ± SD; significant differences are marked by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001). BMP6, Bone morphogenetic protein 6; BMPER, BMP-binding endothelial regulator; FCS, fetal calf serum; hemin, ferric chloride heme; HUVECs, human umbilical vein endothelial cells; n.s., not significant; qRT-PCR, real-time quantitative PCR; SFG, sodium ferric gluconate.
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