Abstract:Background & Aims:TGFβ/BMP signalling in the liver plays a critical role in liver disease. Growth factors, such as BMP2, BMP6 and TGFβ1, are released from liver sinusoidal endothelial cells (LSECs) and signal in a paracrine manner to hepatocytes and hepatic stellate cells to control systemic iron homeostasis and fibrotic processes, respectively. The misregulation of the TGFβ/BMP pathway affects expression of the iron-regulated hormone hepcidin causing frequent iron overload and deficiency diseases. However, wh… Show more
“…In addition, using induced pluripotent stem cell–derived brain ECs as a human blood–brain barrier model, these ECs were shown to engage in iron transport and serve as a critical regulator on the human blood–brain barrier ( 32 ). As for LSECs, it was stated that only in murine LSEC–hepatocytes coculture but not in hepatocyte monoculture, the induction of hepcidin by an ALK5 inhibitor can be observed ( 14 ). Recently, Nrf2 has been suggested as a potential key molecule in mediating iron sensing and controlling BMP6 in LSECs ( 38 ).…”
Section: Discussionmentioning
confidence: 99%
“…The latter interacts with SMAD4 to form the SMAD complex and then translocates into the nucleus and binds to the hepcidin promoter ( 12 ). Inhibitor of DNA binding 1 (Id1), protein atonal homolog 8, and inhibitory Smad6 and Smad7 are other important modulators of the BMP–SMAD pathway ( 13 , 14 ). In addition, typical mediators of inflammation ( e.g.…”
Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin
in vitro
in contrast to
in vivo
conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using
in vitro
coculture models that mimic hepcidin signaling
in vivo
. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron
in vivo
. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.
“…In addition, using induced pluripotent stem cell–derived brain ECs as a human blood–brain barrier model, these ECs were shown to engage in iron transport and serve as a critical regulator on the human blood–brain barrier ( 32 ). As for LSECs, it was stated that only in murine LSEC–hepatocytes coculture but not in hepatocyte monoculture, the induction of hepcidin by an ALK5 inhibitor can be observed ( 14 ). Recently, Nrf2 has been suggested as a potential key molecule in mediating iron sensing and controlling BMP6 in LSECs ( 38 ).…”
Section: Discussionmentioning
confidence: 99%
“…The latter interacts with SMAD4 to form the SMAD complex and then translocates into the nucleus and binds to the hepcidin promoter ( 12 ). Inhibitor of DNA binding 1 (Id1), protein atonal homolog 8, and inhibitory Smad6 and Smad7 are other important modulators of the BMP–SMAD pathway ( 13 , 14 ). In addition, typical mediators of inflammation ( e.g.…”
Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin
in vitro
in contrast to
in vivo
conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using
in vitro
coculture models that mimic hepcidin signaling
in vivo
. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron
in vivo
. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.
“…We first explored the requirement of iron accumulation in LSECs for increased Bmp6 messenger RNA (mRNA) expression in vivo by comparing wild‐type (wt) mice maintained on a “high iron” diet’ (Fe high diet) 14 and Fpn(C326S) mice exerting iron overload due to a point mutation in Fpn that disrupts hepcidin binding 15 (http://links.lww.com/HS/A302). From these mice, we isolated HCs and LSECs and analyzed ferritin L protein and transferrin receptor ( Tfr ) 1 mRNA levels, as markers of intracellular iron content.…”
Section: Figurementioning
confidence: 99%
“…As previously shown, this cell preparation is highly pure and maintains fenestrae, denoting lack of cell trans-differentiation. 14 Exposure of LSECs to iron nitrilotriacetate (FeNTA) decreases Tfr1 mRNA expression and activates hemoxygenase1 ( Ho1 ) transcription (Figure 1G, H), indicating that intracellular iron accumulation induces oxidative stress (Figure 1G). By contrast, Bmp6 transcription was not induced (Figure 1I).…”
“…Hepa 1–6 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (Invitrogen) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma–Aldrich), 10% (v/v) FBS (Gibco). Primary hepatocytes were isolated from C57BL/6 J mice (9 weeks old, males) following a previously described protocol [ 38 ]. Primary hepatocytes were plated in a density of 5 × 10 4 cells/cm 2 and cultured in William's E medium (Life Technologies) supplemented with 4% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.…”
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