Localization of iron metabolism–related mRNAs in rat liver indicate that HFE is expressed predominantly in hepatocytes

Abstract

The mRNAs of proteins involved in iron metabolism were measured in isolated hepatocytes, Kupffer cells, sinusoidal endothelial cells (SECs), and hepatic stellate cells (HSCs). Levels of type I hereditary hemochromatosis gene (HFE), transferrin, hepcidin, transferrin receptors 1 and 2 (TfR1, TfR2), ferroportin 1 (FPN1), divalent metal transporter 1 (DMT1), natural resistance-associated macrophage protein 1 (Nramp1), ceruloplasmin, hephaestin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were measured by quantitative reversetransriptase polyerase chain reaction (qRT-PCR). We show that hepatocytes express almost all the iron-related genes tested, in keeping with their central role in iron metabolism. In addition, hepatocytes had 10-fold lower TfR1 mRNA levels than TfR2 and the lowest levels of TfR1 of the 4 cell types isolated. Kupffer cells, which process senescent red blood cells and recycle the iron, had high levels of ferroportin 1, ceruloplasmin, and hephaestin mRNA. Most important, of all the cell types tested, hepatocytes had the highest level of HFE mRNA, a factor of 10 higher than Kupffer cells. In situ hybridization analysis was conducted with rat liver sections. Consistent with the qRT-PCR analysis, HFE gene expression was localized mainly in hepatocytes. Western blot analysis confirmed this finding. Unexpectedly, HSCs also had high levels of DMT1 and ferroportin, implicating them in either iron sensing or iron cycling. IntroductionThe liver plays a central role in iron homeostasis. It is the major site of the body's excess iron storage, which accounts for approximately 12.5% to 25% (0.5 to 1 g) of total body iron in a healthy adult man. 1 Iron is mainly sequestered in hepatocytes as ferritin. Under low-iron conditions, the stored iron can be actively mobilized into circulation. 2 Hepatocytes are also the source of transferrin (Tf), the iron transport protein in blood; ceruloplasmin, a serum multicopper ferric oxidase; and hepcidin, a recently discovered peptide involved in the regulation of iron absorption from the intestine. Thus, they play a major role in iron homeostasis in the body. Kupffer cells reprocess iron from senescent red blood cells. The roles that 2 other cell types in the liver, hepatic stellate cells (HSCs) and sinusoidal endothelial cells (SECs), play in iron homeostasis are not known.In the iron-overload disease hereditary hemochromatosis, excess iron accumulates in the liver as well as a number of other organs. The protein that is mutated in the most common form of hereditary hemochromatosis is HFE. By Northern analysis, the liver contains the highest levels of the mRNA for HFE, but the cell type that expresses HFE has been controversial. [3][4][5] Knowing the cell types that express HFE is important in determining the mechanism by which HFE regulates iron homeostasis in the liver.In this study, isolated cell populations of rat liver hepatocytes, Kupffer cells, SECs, and HSCs were examined for their expression of genes implicated in iron transport and regulation. A series of...

Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice

“…HFE, TfR2, and hepcidin are expressed predominantly in hepatocytes, suggesting that HFE and TfR2 regulate iron metabolism upstream of hepcidin. 33 The findings that macrophage/granulocyte-specific depletion of HFE in mice (Hfe) has no detectable effect on Hamp1 expression, whereas hepatic-specific depletion of Hfe is sufficient to decrease Hamp1 expression reinforces the importance of Hfe in regulating hepcidin. 34 TfR2 and HFE are capable of forming a complex, 35,36 suggesting that the complex is involved in hepcidin regulation.…”

“…Hamp1, Hfe, Tfr2, and Gapdh mRNA were measured with the use of the mouse primers listed in Table 1 as previously described. 33,40 Results were expressed as the level relative to the corresponding Gapdh. All primers were verified for linearity of amplification.…”

“…33 Oligo (dT) primers and Superscript II reverse transcriptase were used to synthesize cDNA according to the manufacturer's instructions. Hamp1, Hfe, Tfr2, and Gapdh mRNA were measured with the use of the mouse primers listed in Table 1 as previously described.…”

Transferrin Receptor 2: Evidence for Ligand-induced Stabilization and Redirection to a Recycling Pathway

“…TfR2 binds Tf in a pH-dependent manner, but its affinity for Fe 2 Tf (K D ϳ30 nM; Kawabata et al, 2000;West et al, 2000) is significantly lower than that of TfR1 (K D ϳ1 nM, Tsunoo and Sussman, 1983;Enns et al, 1991;Richardson and Ponka, 1997). Unlike TfR1, TfR2 expression is limited predominantly to hepatocytes (Kawabata et al, 1999;Fleming et al, 2000Fleming et al, , 2002Vogt et al, 2003;Calzolari et al, 2004;Zhang et al, 2004) and is not regulated by intracellular iron (Fleming et al, 2000;Kawabata et al, 2000Kawabata et al, , 2001. TfR2 cannot compensate for TfR1, whose knockout in mice results in embryonic lethality due to severe anemia (Levy et al, 1999).…”

Iron‐ and inflammation‐induced hepcidin gene expression in mice is not mediated by Kupffer cells in vivo†

“…[4][5][6][7] The hepcidin gene is expressed predominantly in the liver, where its expression is strictly restricted to hepatocytes. [8][9][10][11] It is synthesized as an 84 -amino acid precursor that is subsequently processed and secreted as a 25-amino acid peptide. This circulating form has been purified from human blood ultrafiltrate and from urine.…”