Targeted Disruption of the Hepatic Transferrin Receptor 2 Gene in Mice Leads to Iron Overload
2006; Elsevier BV; Volume: 132; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2006.11.028
ISSN1528-0012
AutoresDaniel F. Wallace, Lesa Summerville, V. Nathan Subramaniam,
Tópico(s)Hemoglobinopathies and Related Disorders
ResumoBackground & Aims: Transferrin receptor 2 (TfR2) plays a key role in the regulation of iron metabolism. Mutations of TfR2 in humans cause type 3 hereditary hemochromatosis. Although highly expressed in liver, several studies have reported TfR2 expression in other tissues. To determine the contribution of liver expressed TfR2 in iron homeostasis, we have generated and characterized a liver-specific TfR2-knockout (KO) mouse. Methods: Liver-specific TfR2-KO mice were generated by crossing TfR2-floxed mice with transgenic albumin-Cre mice. Tissue and serum from homozygous TfR2-floxed mice with and without albumin-Cre were analyzed. Serum transferrin saturation, hepatic, and splenic iron concentrations were determined. The expression of iron-related mRNA transcripts was analyzed by real-time PCR. Levels of the iron-related proteins TfR1, TfR2, ferritin, and prohepcidin were analyzed by immunoblotting. Results: Liver-specific TfR2-KO mice develop significant iron overload comparable to complete TfR2-KO mice. At all ages studied, transferrin saturation, hepatic iron concentration, and hepatic ferritin were significantly elevated. Hepatic TfR2 mRNA and protein were absent in the livers of liver-specific TfR2-KO mice, and TfR1 expression was reduced consistent with liver iron loading. At 5 weeks of age, hepcidin1 mRNA, and prohepcidin protein were decreased in liver-specific TfR2-KO compared to control mice. Conclusions: The significant iron loading and modulation of expression of iron-related genes in liver-specific TfR2-KO mice demonstrates that the liver is the primary site for TfR2 expression and activity and that liver-expressed TfR2 is required for the regulation of hepcidin1. Background & Aims: Transferrin receptor 2 (TfR2) plays a key role in the regulation of iron metabolism. Mutations of TfR2 in humans cause type 3 hereditary hemochromatosis. Although highly expressed in liver, several studies have reported TfR2 expression in other tissues. To determine the contribution of liver expressed TfR2 in iron homeostasis, we have generated and characterized a liver-specific TfR2-knockout (KO) mouse. Methods: Liver-specific TfR2-KO mice were generated by crossing TfR2-floxed mice with transgenic albumin-Cre mice. Tissue and serum from homozygous TfR2-floxed mice with and without albumin-Cre were analyzed. Serum transferrin saturation, hepatic, and splenic iron concentrations were determined. The expression of iron-related mRNA transcripts was analyzed by real-time PCR. Levels of the iron-related proteins TfR1, TfR2, ferritin, and prohepcidin were analyzed by immunoblotting. Results: Liver-specific TfR2-KO mice develop significant iron overload comparable to complete TfR2-KO mice. At all ages studied, transferrin saturation, hepatic iron concentration, and hepatic ferritin were significantly elevated. Hepatic TfR2 mRNA and protein were absent in the livers of liver-specific TfR2-KO mice, and TfR1 expression was reduced consistent with liver iron loading. At 5 weeks of age, hepcidin1 mRNA, and prohepcidin protein were decreased in liver-specific TfR2-KO compared to control mice. Conclusions: The significant iron loading and modulation of expression of iron-related genes in liver-specific TfR2-KO mice demonstrates that the liver is the primary site for TfR2 expression and activity and that liver-expressed TfR2 is required for the regulation of hepcidin1. Transferrin receptor 2 (TfR2) is thought to play an important role in iron metabolism.1Subramaniam V.N. Summerville L. Wallace D.F. Molecular and cellular characterization of transferrin receptor 2.Cell Biochem Biophys. 2002; 36: 235-239Crossref PubMed Scopus (16) Google Scholar, 2Trinder D. Baker E. Transferrin receptor 2: a new molecule in iron metabolism.Int J Biochem Cell Biol. 2003; 35: 292-296Crossref PubMed Scopus (79) Google Scholar, 3Deicher R. Horl W.H. New insights into the regulation of iron homeostasis.Eur J Clin Invest. 2006; 36: 301-309Crossref PubMed Scopus (65) Google Scholar, 4Camaschella C. Why do humans need two types of transferrin receptor? Lessons from a rare genetic disorder.Haematologica. 2005; 90: 296PubMed Google Scholar It is a homologue of the well-described and ubiquitously expressed transferrin receptor (TfR1), which is the main means by which cells acquire iron.5Ponka P. Lok C.N. The transferrin receptor: role in health and disease.Int J Biochem Cell Biol. 1999; 31: 1111-1137Crossref PubMed Scopus (469) Google Scholar, 6Aisen P. 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The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22.Nat Genet. 2000; 25: 14-15Crossref PubMed Scopus (748) Google Scholar A transgenic mouse with a targeted mutation of TfR29Fleming R.E. Ahmann J.R. Migas M.C. Waheed A. Koeffler H.P. Kawabata H. Britton R.S. Bacon B.R. Sly W.S. Targeted mutagenesis of the murine transferrin receptor–2 gene produces hemochromatosis.Proc Natl Acad Sci U S A. 2002; 99: 10653-10658Crossref PubMed Scopus (218) Google Scholar (Y245X, orthologous to human Y250X) develops iron overload with a similar phenotype to patients with TFR2-associated hemochromatosis. We have previously described a complete TfR2-knockout (KO) mouse that develops iron overload in a similar fashion to that observed in humans.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar The role of TfR1 in providing iron to cells through the binding and internalization of diferric transferrin and its regulation by intracellular iron levels has helped to elucidate many important aspects of cellular iron homeostasis.11Aisen P. Transferrin, the transferrin receptor, and the uptake of iron by cells.Met Ions Biol Syst. 1998; 35: 585-631PubMed Google ScholarTfR2, however, does not have any iron-responsive elements, and its mRNA does not appear to be regulated in a similar fashion to TfR1.12Fleming R.E. Migas M.C. Holden C.C. Waheed A. Britton R.S. Tomatsu S. Bacon B.R. Sly W.S. 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Diferric transferrin regulates transferrin receptor 2 protein stability.Blood. 2004; 104: 4287-4293Crossref PubMed Scopus (199) Google Scholar, 14Robb A. Wessling-Resnick M. Regulation of transferrin receptor 2 protein levels by transferrin.Blood. 2004; 104: 4294-4299Crossref PubMed Scopus (176) Google Scholar Hepcidin is synthesized and secreted by the liver in response to iron loading or inflammation and functions to lower serum iron levels by binding, internalizing, and degrading the iron exporter ferroportin.15Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3847) Google Scholar Most forms of hemochromatosis result from inappropriately low levels or absence of hepcidin. 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Hepatic iron metabolism.Semin Liver Dis. 2005; 25: 420-432Crossref PubMed Scopus (109) Google Scholar To elucidate the contribution of hepatic TfR2 to iron homeostasis, we have generated and characterized mice with a liver-specific deletion of the TfR2 gene. Using Cre-loxP technology, the TfR2 gene was modified by flanking exons 2 to 6 with loxP sites. We have previously shown that Cre-mediated deletion of exons 2 to 6 results in loss of TfR2 protein expression.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar The targeting construct contained a neomycin selection cassette flanked by FRT sites to enable FLPe recombinase-mediated excision and generation of a floxed (flanked by loxP sites) TfR2 allele for the development of conditional KOs.32Wallace D.F. Tonks I.D. Zournazi A. Kay G.F. Subramaniam V.N. Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system.Genesis. 2004; 39: 38-41Crossref PubMed Scopus (12) Google Scholar Crossing of the floxed mice with albumin-Cre mice generated mice with a hepatocyte-specific deletion of the TfR2 gene. We have used these mice to investigate the contribution of hepatic TfR2 to iron homeostasis and the expression of hepcidin and other iron-related molecules. Approval for all studies with animals was obtained from the Animal Research Ethics Committee of the Queensland Institute of Medical Research. Anti-TfR1 antibodies were purchased from Zymed Laboratories, San Francisco, CA, anti-ferritin antibodies from The Binding Site, Birmingham, England, and anti-actin antibodies from Sigma-Aldrich, Castle Hill, Australia. Anti-TfR2 and anti-prohepcidin antibodies were generated in rabbits using recombinant mouse TfR2 and prohepcidin, respectively.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar, 33Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. Prohepcidin localises to the Golgi compartment and secretory pathway in hepatocytes.J Hepatol. 2005; 43: 720-728Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Transferrin saturation was measured using an iron and iron binding capacity kit (Sigma-Aldrich, Castle Hill, Australia). Hepatic (HIC) and splenic iron concentrations (SIC) were determined using the method of Torrance and Bothwell.34Torrance J.D. Bothwell T.H. A simple technique for measuring storage iron concentrations in formalinised liver samples.S Afr J Med Sci. 1968; 33: 9-11PubMed Google Scholar Perls' Prussian blue staining was used for detecting iron in liver sections. Three to 10 mice per group were used in all the analyses. The generation and characterization of complete TfR2 null mice with deletion of exons 2 to 6 of TfR2 in all tissues has been described previously.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google ScholarTfR2-floxed mice were generated as described previously.32Wallace D.F. Tonks I.D. Zournazi A. Kay G.F. Subramaniam V.N. Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system.Genesis. 2004; 39: 38-41Crossref PubMed Scopus (12) Google Scholar The TfR2-floxed allele contains exons 2 to 6 of TfR2 flanked by loxP sites and results in normal expression levels of TfR2 protein.32Wallace D.F. Tonks I.D. Zournazi A. Kay G.F. Subramaniam V.N. Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system.Genesis. 2004; 39: 38-41Crossref PubMed Scopus (12) Google Scholar Both the TfR2-deleted and TfR2-floxed alleles were created on a 129T2/SvEms background, and mice had been backcrossed twice to C57BL/6J. The albumin-Cre mice35Yakar S. Liu J.L. Stannard B. Butler A. Accili D. Sauer B. LeRoith D. Normal growth and development in the absence of hepatic insulin-like growth factor I.Proc Natl Acad Sci U S A. 1999; 96: 7324-7329Crossref PubMed Scopus (1215) Google Scholar had been backcrossed to C57BL/6J 7 times. TfR2-floxed mice were bred with albumin-Cre transgenic mice to produce mice with hepatocyte-specific deletion of TfR2. Mice homozygous for the TfR2-floxed allele and heterozygous for the albumin-Cre transgene (TfR2F/F, Alb-Cre+/−) were considered to be liver- specific TfR2-KOs (LSKO). Mice homozygous for the TfR2-floxed allele but not carrying the albumin-Cre transgene (TfR2F/F, Alb-Cre−/−) were used as controls. For the complete TfR2 null mice, wild-type mice of the same background were used as controls.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google ScholarTfR2F/F, Alb-cre+/− mice were bred with TfR2F/F, Alb-cre−/− mice to produce equal numbers of LSKO and control offspring. Male mice were sacrificed at 5, 10, and 26 weeks of age, and tissues and blood were taken for analysis. All mice were fed on a normal chow diet. Genomic DNA was isolated from liver, duodenum, kidney, and bone marrow. A real-time polymerase chain reaction (PCR) method was used to quantitate relative amounts of the TfR2-floxed and TfR2-deleted alleles. Primers TfR2-5′LoxP-F (ggg gtc tac ttc gga gag tgg taa g) and TfR2-5′LoxP-R (ctg agg gtt agg caa gaa tgg tgt) were used to detect the TfR2-floxed allele and primers TfR2-5′LoxP-F, and TfR2-3′LoxP-R (ttc tgc caa cat tct ctc cct ctc) were used to detect the TfR2-deleted allele.32Wallace D.F. Tonks I.D. Zournazi A. Kay G.F. Subramaniam V.N. Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system.Genesis. 2004; 39: 38-41Crossref PubMed Scopus (12) Google Scholar Primers specific for mouse hepcidin1: mHepc1+2-F (aga gct gca gcc ttt gca c) and mHepc1-R (aca ctg gga att gtt aca gca ttt a) were used as a normalization control.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar Real-time reactions were performed using 200 nmol/L of each primer, and SYBR green PCR master mix (Applied Biosystems, Warrington, England). Reactions were performed on the Rotorgene RG3000 (Corbett Research, Brisbane, Australia). Relative amounts of each allele were determined after normalizing to the mouse hepcidin1 gene. Immunohistochemical staining of liver sections was performed as described.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar, 33Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. Prohepcidin localises to the Golgi compartment and secretory pathway in hepatocytes.J Hepatol. 2005; 43: 720-728Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Briefly, sections of formalin-fixed mouse livers were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked. Antigen retrieval was performed in 1 M trisodium citrate pH 6.0. Specimens were blocked in 20% goat serum/PBS for 1 hour at room temperature (RT) and then incubated in 10 μg/mL anti-TfR2 or anti-rabbit IgG overnight at 4°C. Sections were then incubated with biotinylated goat anti-rabbit antibody (DAKO A/S, Glostrup, Denmark) diluted 1:400 in PBS for 1 hour at RT, followed by the Streptavidin-HRP for 30 minutes at RT and then DAB substrate. Sections were counterstained with hematoxylin, mounted, examined by light microscopy, and photographed. Western blotting was performed as described.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar Briefly, 100 μg of mouse liver homogenate was electrophoresed on a 10% SDS-PAG or 4% to 12% Novex Bis-Tris SDS-PAG (Invitrogen, Mulgrave, Australia) and then transferred onto either Hybond-C+ membrane or polyvinylidene difluoride membrane. The blots were blocked in 10% skim milk/0.5%Tween 20 in PBS (blocking buffer) and primary antibodies, diluted in blocking buffer, and were applied to the blot for 1 hour at RT at the following concentrations: ferritin (1:5000), prohepcidin (1.5 μg/mL), TfR1 (2.5 μg/mL), TfR2 (0.1 μg/mL), and actin (1:1000). Blots were then washed and incubated with anti-mouse or anti-rabbit HRP diluted 1:10,000 in blocking buffer. Washed blots were incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Sydney, Australia) and subjected to autoradiography. Bands were quantified by densitometry using a Syngene GeneGenius (Syngene, Cambridge, England). Five to 7 mice per group were used in all the analyses. Real-time RT-PCR quantification of mRNA was performed as described previously using the same primer sets and PCR conditions.10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar Briefly, RNA isolated from liver and duodenum was reverse transcribed using Superscript III (Invitrogen, Mulgrave, Australia). Real-time reaction mixes contained 2 μL of cDNA (diluted 1 in 5), 200 nmol/L of each primer, and SYBR green PCR master mix (Applied Biosystems). Reactions were performed on the Rotorgene RG3000 (Corbett Research, Brisbane, Australia). Expression of all target genes was determined by normalizing to the respective β-actin levels. For the 10-week studies, 10 mice per group were analyzed. For the 5-, 10-, and 26-week studies, 4 to 5 mice per group were analyzed. Variables were compared between groups using both parametric (1-way ANOVA and the Student's t test) and nonparametric (Kruskal–Wallis and Mann–Whitney) statistical tests using the SPSS software package (SPSS Inc., Chicago, IL). A P value < .05 was considered statistically significant. Real-time PCR was used to detect the relative amounts of the TfR2-floxed allele and TfR2-deleted allele in genomic DNA isolated from liver, duodenum, kidney, and bone marrow of a LSKO mouse. The relative amounts were quantified by comparison with genomic DNA isolated from the livers of a complete TfR2-KO and a homozygous TfR2-floxed mouse (without albumin-Cre). Primers designed to pick up the deleted allele detected approximately 61% of the signal compared with the complete TfR2-KO in the liver of the LSKO. In the 3 other tissues, the deleted allele was not detected at all. Primers designed to pick up the TfR2-floxed allele detected approximately 25% of the signal compared with the homozygous TfR2-floxed mouse. Although the values for the deleted and floxed alleles do not add up to exactly 100%, they are consistent with TfR2 being deleted exclusively in hepatocytes.36Zhang A.S. Xiong S. Tsukamoto H. Enns C.A. Localization of iron metabolism-related mRNAs in rat liver indicate that HFE is expressed predominantly in hepatocytes.Blood. 2004; 103: 1509-1514Crossref PubMed Scopus (155) Google Scholar Importantly, in the 3 other tissues studied, duodenum, kidney and bone marrow, no deletion of the TfR2 gene was observed. Measurements of iron indices in LSKO mice showed that they developed significant iron loading at all ages studied. Transferrin saturation was significantly elevated at all ages (Figure 1A). Hepatic iron concentration was also significantly elevated at all ages (Figure 1C), being 5- to 9-fold higher in the LSKOs compared to controls. In order to discern any difference in phenotype between the LSKOs and complete TfR2-KOs, transferrin saturation, HIC, and SIC were measured in 5-, 10-, and 26-week-old complete TfR2-KO mice and compared with the LSKO mice (Figure 1B, D, F and A, C, E, respectively). All measurements were done in a single run to exclude any interexperiment variation. At 5 weeks and 26 weeks of age, the HIC was similar in the 2 KO strains (Figure 1C and D). However, the HIC was significantly reduced in the 10-week-old complete TfR2-KOs when compared to the LSKOs. The SIC significantly increased with age in all groups but was similar between the 2 KO strains at all ages studied (Figure 1E and F). Perls' staining of liver sections was performed to study the pattern of iron loading in the liver of 10-week-old control and LSKO mice. Although no iron was detected in livers of control mice (Figure 2A), a predominantly hepatocellular accumulation of iron in the liver with a gradient from the periportal to the pericentral regions was observed in livers of LSKO mice (Figure 2B). The expression of TfR2 was examined by immunohistochemical staining with a specific anti-TfR2 antibody10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar in the livers of 10-week-old mice. As seen previously,10Wallace D.F. Summerville L. Lusby P.E. Subramaniam V.N. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005; 54: 980-986Crossref PubMed Scopus (131) Google Scholar a predominantly basolateral surface staining in the hepatocytes of control animals was observed (Figure 3A). This staining was absent in the LSKO mice confirming hepatocyte-specific deletion of TfR2 (Figure 3C).Figure 3Immunohistochemical localization of TfR2 in the liver. Liver sections from 10-week-old control (A, B) and LSKO (C, D) mice were incubated with anti-TfR2 antibodies (A, C) or control rabbit IgG (B, D). Staining for TfR2 expression is observed on the basolateral surface of hepatocytes in livers from control mice. No staining is observed in LSKO mice or with rabbit IgG. Original magnification 40X.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The levels of mRNA transcripts were initially measured in the liver and duodenum of 10-week-old LSKO and control mice (Table 1) (10 mice per group). TfR2 mRNA was absent in the livers of the LSKO mice indicating that TfR2 is expressed exclusively in the hepatocyte (albumin-expressing) cell population of the liver. Liver TfR1 and Dmt1 mRNA were significantly reduced and ferroportin mRNA significantly increased in the LSKO mice consistent with hepatic iron loading. A small but significant increase in Hjv mRNA was also observed in the livers of the LSKOs compared to controls. The levels of total hepcidin and hepcidi
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