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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-07-2192.
RED CELLS
From the Department of Internal Medicine IV, University
Hospital Heidelberg, Heidelberg, Germany.
SFT, a stimulator of iron (Fe) transport, has been described
as a transmembrane protein that facilitates the uptake of ferrous and
ferric iron in mammalian cells. This study was initiated to investigate
the 5' regulatory region of SFT and its role in the etiology of
hereditary hemochromatosis. Sequence analyses of the putative 5'
regulatory region revealed that the SFT cDNA sequence corresponds to
intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the
hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor
suppressor (pVHL) E3 ligase complex. Further mRNA expression studies
using a sequence-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that UbcH5A is
significantly up-regulated in the liver of iron-overloaded patients
with hereditary hemochromatosis, as previously published for SFT.
However, in vitro studies on HepG2 cells failed to demonstrate any
significant UbcH5A regulation in response to iron loading or iron
chelation. In conclusion, in vivo mRNA expression data previously
obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary
hemochromatosis remains to be elucidated further.
(Blood. 2003;101:3288-3293) In mammalian cells, the major route of iron uptake
is the receptor-mediated endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Following internalization and acidification of the endosome, iron is released from Tf and transported across the endosomal membrane. This step, as well as transport of iron
across the intestinal epithelial layer, requires a Tf-independent pathway.1-4 Recently, several putative transporters
involved in the Tf-independent uptake of iron have been identified.
DMT1 (formerly called Nramp2, DCT1) is a transmembrane divalent cation transporter with a high affinity for ferrous Fe(II) iron.5 Whereas DMT1 is thought to mediate the rapid uptake of dietary iron
from the intestinal lumen,5-8 IREG1 (also known as
ferroportin, MTP1) is localized to the basolateral membrane of the
enterocyte and functions as an Fe(II) exporter.9-11
Another iron transport protein, SFT (stimulator of Fe transport), has
been found to stimulate transport of both ferrous Fe(II) and ferric
Fe(III) iron and shows high specifity for iron in Tf-independent
uptake.12-14 Accordingly, Northern analysis indicated that
SFT expression is up-regulated in iron-deficient HeLa cells exposed to
the iron chelator deferoxamine and down-regulated in iron-supplemented
HeLa and HepG2 cells.15
Remarkably, hepatic SFT expression was found to be significantly
increased in patients with hereditary hemochromatosis (HH) despite
tissue iron overload.15 In most cases, this frequent autosomal recessive disorder is associated with a missense mutation (Cys282Tyr) in the hemochromatosis gene
HFE, which codes for a protein that is homologous to
class I major histocompatibility complex (MHC) molecules and
requires The present work sheds some light on the rather controversial SFT
protein. Herewith, we demonstrate that the previously published nucleotide sequence of SFT indeed corresponds to a genomic fragment of
the ubiquitin-conjugating enzyme UbcH5A. In addition, we evaluate whether previously published data on SFT regulation in response to iron
loading or iron chelation might be attributed to UbcH5A mRNA expression.
Cells
Liver biopsies
Chemicals RPMI 1640 medium, penicillin, and streptomycin were from Life Technologies (Paisley, United Kingdom). Ferric nitrate nonahydrate and nitrilotriacetic acid disodium salt (NTA) were obtained from Sigma-Aldrich (Steinheim, Germany). Deferoxamine was from Novartis (Nürnberg, Germany).Identification of SFT/UbcH5A genomic DNA sequence In order to identify 5' sequence information of SFT, the Human Genome Walker Kit (Clontech, Palo Alto, CA) was used as described in the manufacturer's manual. The oligonucleotide sequences for the gene-specific primer GSP 1 (5'-CAA TTA CTG GGT CAA TGT GAC TTA GGT TC) and the nested GSP 2 (5'-GCC AAT CCT GAA GGA GCA TAA TCT AC) were derived from SFT cDNA (GenBank accession no. AF020761.1). The polymerase chain reaction (PCR) product generated from the EcoRV library (approximately 1 kb) was reamplified using adaptor primer 2 (provided in the kit) and nested GSP 3 (5'-CCT ACA GTA CAA GCA AGA TGA AGC AGC), subcloned in pUC18 (Sure Clone Ligation Kit; Amersham Pharmacia Biotech, Freiburg, Germany), and sequenced (ABI 310 Genetic Analyzer; Applied Biosystems, Weiterstadt, Germany). From the derived sequence information, a primer pair (SFTPro501 5'-GCA AAA GAG TGT ATG AGC ACA GTA TTC, SFTPro301 5'-CTG GTA CTA AGG GGT CAT CTG G) was chosen in order to obtain a P1 genomic clone by PCR-based screening (GenomeSystems, St Louis, MO) followed by sequencing analysis. The complete exon-intron structure of SFT/UbcH5A was identified by sequence alignment (ClustAl) with the cDNA sequence of an EST clone (IMAGE:1939023; GenomeSystems) containing full-length UbcH5A cDNA.RNA expression analysis A multiple-tissue Northern blot was obtained from Clontech. Probes specific for the UbcH5A coding sequence were generated by PCR using the sense primer 5'-ATG GGG CCT CCT GAT AGC, the antisense primer 5-'CTG GTA CTA AGG GGT CAT CTG G, and IMAGE clone 1939023 (UbcH5A cDNA) as a template. The probes were 32P-labeled by random priming; hybridization was performed at 68°C, according to the manufacturer's recommendations.Quantitative RT-PCR Total RNA was isolated from liver biopsies and from cell culture using the RNAeasy Mini Kit (Qiagen, Hilden, Germany) including DNAse digestion according to the manufacturer's instructions. Real-time quantification of mRNA transcripts was performed with a 2-step reverse transcriptase (RT) PCR using the LightCycler system and Relative Quantification Software Version 1.0 (Roche Molecular Biochemicals, Mannheim, Germany). In a first step, cDNA synthesis was performed with the First Strand cDNA Synthesis Kit for RT-PCR (Roche Molecular Biochemicals) according to the manufacturer's instructions. In a second step, UbcH5A transcripts were amplified in duplicates with sense primer UbcH5A-501 (5'-GTG ATC CTA ATC CAG ATG ACC C) and antisense primer UbcH5A-301 (5'-TCA ATG CAT ACA AAA TGA TAA ATG C) and detected by sequence-specific hybridization probes UbcH5A-LCA (5'-CAT TGC ATA TTT CTG AGT CCA TTC TCT TGC-fluorescein) and UbcH5A-LCM (5'-LightCycler-Red640-TGT CTG TTG TAT TTT TCT TTG TCT GAT TTA TAG-p). This approach prevented detection of genomic DNA as well as mRNAs homologous to UbcH5A including "SFT mRNA." To provide a positive control in all experiments, transferrin receptor (TfR) expression was determined quantitatively, as follows. TfR transcripts were amplified in duplicate with sense primer TFR1-502 (5'-TAT AGA AGG TTT GGG GGC TGT G) and antisense primer TFR1-302 (5'-GAG ACC CTA TGA ACT TTT CCC TAG) and detected using SYBR Green I (Roche Molecular Biochemicals) according to the manufacturer's instructions. Both UbcH5A and TfR transcripts were normalized to actin ( -actin) as an internal
control. Actin transcripts were amplified in duplicate with sense
primer ACTB-502 (5'-AGG ATG CAG AAG GAG ATC ACT G) and antisense primer
ACTB-302 (5'-GGG TGT AAC GCA ACT AAG TCA TAG) and detected using SYBR
Green I. UbcH5A/actin and TfR/actin ratios were calculated using
LightCycler Relative Quantification Software Version 1.0 (Roche
Molecular Biochemicals), which provides a fully automated,
efficiency-corrected, relative quantification normalized to
calibrators. According to the manufacturer's instructions, calibrators
for UbcH5A, TfR, and actin were generated from EST clones
(IMAGE:1939023, GenomeSystems, for amplification of UbcH5A;
IMAGE:841703 from RZPD, Berlin, Germany, for amplification of TfR;
IMAGE:2518720, RZPD, for amplification of actin). In addition, standard
curves were prepared based on accurately determined dilutions of the
plasmids containing cDNA of UbcH5A (IMAGE:1939023), TfR (IMAGE:841703),
and actin (IMAGE:2518720) as templates. Plasmid dilutions covered a
dynamic range of 5 logarithmic orders. Statistical analysis of
quantitative variables was performed by means of the nonparametric
Mann-Whitney test. P < .05 was considered significant.
SFT mRNA shows high sequence homology with intron 6 and exon 7 of UbcH5A Using oligonucleotide primers specific for SFT, a 1-kb PCR product was generated from the EcoRV library of the Human Genome Walker Kit (Clontech). Sequence analysis of the subcloned PCR product was expected to reveal further 5' sequence information of SFT, including the promotor and regulatory elements. In contrast, we found a nucleotide sequence that had no homology to a previously published short fragment of the SFT 5' regulatory region.23 Therefore, a genomic clone was isolated by PCR-based screening with primers specific for our 1-kb PCR product. Sequence analysis of the genomic clone confirmed the nucleotide sequence of the 1-kb PCR product and demonstrated the reliability of our results. A BlastN2 search of our putative 5' regulatory region of SFT revealed a 94-nucleotide fragment showing 100% identity with nucleotides 305 through 398 of UbcH5A (ubiquitin-conjugating enzyme E2) mRNA coding sequence (GenBank accession no. NM_003338.1; Figure 1). This observation strongly suggests that the fragment represents an exon of the UbcH5A gene. To prove this hypothesis, full-length mRNA sequence of UbcH5A was obtained by sequencing an EST clone with high homology to the published UbcH5A coding sequence (GenBank accession no. NM_003338.1). Sequence analysis revealed full-length UbcH5A mRNA including the 5' and 3' untranslated regions (submitted to GenBank, accession no. AJ272367). The exon/intron organization of the UbcH5A gene was then determined with the information being submitted to GenBank (chromosome 10 genomic clone RP11-373P23; GenBank accession no. AC023170). According to this, the UbcH5A gene is located on chromosome 10, spans 35 kb, and contains 7 exons (submitted to GenBank, accession no. AJ293565). In addition, sequence alignment demonstrates 99% identity between intron 6/exon 7 of UbcH5A and the published SFT cDNA (GenBank accession no. AF020761, now designated as unknown RNA sequence).
UbcH5A mRNA tissue expression UbcH5A was ubiquitously expressed with a high level of expression in heart and skeletal muscles (Figure 2). As demonstrated for SFT,12 Northern analysis revealed 2 transcripts of approximately 1.5 kb and approximately 2.4 kb. Both transcripts were observed, although the probes used were specific for UbcH5A mRNA coding sequence and did not show any homology to the published SFT nucleotide sequence.
UbcH5A transcript levels are not regulated in response to cellular iron content in HepG2 cells In all previous studies on SFT mRNA expression, probes matching the 3' untranslated region (UTR) were used.12,15,24 Since the UbcH5A 3' UTR (GenBank accession no. AJ272367) is almost identical to the previously published SFT 3' UTR (GenBank accession no. AF020761.1), those studies might have detected UbcH5A transcripts instead of SFT transcripts. Therefore, we analyzed UbcH5A mRNA expression in response to cellular iron content, using a highly specific assay. UbcH5A transcript levels were determined quantitatively with sequence-specific hybridization probes on the LightCycler PCR system (real-time RT-PCR), and were normalized to actin transcript levels (UbcH5A/actin ratio). To provide a positive control, transferrin receptor (TfR) mRNA expression in response to cellular iron content was measured and normalized to actin (TfR/actin ratio). As demonstrated in Figure 3, HepG2 cells treated with 50 µM deferoxamine (DFO) showed an increased TfR/actin ratio and those treated with 65 µM Fe-NTA a decreased TfR/actin ratio (P < .01). In contrast, neither iron chelation (50 µM DFO) nor iron loading (65 µM Fe-NTA) induced significant changes in the UbcH5A/actin ratio in HepG2 cells.
UbcH5A expression is enhanced in the liver of patients with hereditary hemochromatosis To investigate the role of UbcH5A in hereditary hemochromatosis, levels of UbcH5A transcripts normalized to actin (UbcH5A/actin ratio) were measured in liver biopsies from iron-overloaded patients homozygous for the Cys282Tyr mutation in HFE. In the liver of these patients, the UbcH5A/actin ratio was approximately 2-fold higher compared with liver biopsies from controls with negative iron staining and serum ferritin levels within normal limits (P < .05; Figure 4). In contrast, the TfR/actin ratio used as a positive control was significantly decreased (P < .01) in patients with untreated hemochromatosis. To exclude a significant impact of inflammation on these observations, levels of UbcH5A transcripts normalized to actin (UbcH5A/actin ratio) were measured in liver biopsies from patients with inflammation due to chronic HCV infection and were compared with those in liver biopsies from patients without intrahepatic inflammation. The mean UbcH5A/actin ×103 ratios (± SD) did not differ significantly between the 2 groups (2.60 ± 0.64 vs 2.83 ± 0.89; P = .9468).
Recently, several genes involved in iron metabolism, including the hemochromatosis gene HFE, have been isolated.5,9-12,16,25-28 From these, the apical iron transporter DMT1 and the basolateral iron exporter IREG1 mediate iron absorption in the duodenal enterocyte.2,3 Interestingly, both iron transport molecules are up-regulated in the duodenum during iron deficiency as they are in the duodenum of HH patients.29 This paradoxical up-regulation of DMT1 and IREG1 might explain progressive iron accumulation in HH. However, the exact mechanism by which defective HFE leads to these phenomena is not yet clear. Most authors favor the hypothesis that the absence of functional HFE results in an erroneous sensing of body iron stores in immature duodenal crypt cells.1-4 In addition, signaling molecules such as the putative iron store regulator hepcidin,28 or proteins such as SFT, a stimulator of Fe transport,12-15 may contribute to the etiology of HH. SFT was first described in 1997 as a transmembrane protein stimulating Tf-independent and Tf-dependent iron uptake.12 Although functional data suggest that SFT stimulates the uptake of of ferrous Fe(II) and ferric Fe(III) iron in mammalian cells,13 Northern analyses indicate an up-regulation of SFT in response to iron loading and a down-regulation in response to iron chelation.15 In contrast, SFT expression studies in iron-overloaded HH patients demonstrated an up-regulation in liver by Northern analysis15 and in the duodenum by immunohistochemistry.30 This paradoxical up-regulation, despite iron overload conditions, strongly suggests an important role of SFT in the etiology of HH, although these data are not reproducible in the mouse model of HH.31,32 In order to analyze transcriptional regulation of SFT, we began to investigate 5' sequence information of SFT. In contrast to the previously published short segment of the putative SFT promotor,23 our 5' sequence data revealed a 94-nucleotide fragment sharing 100% identity with UbcH5A, a member of the E2 ubiquitin-conjugating enzymes. After obtaining an UbcH5A genomic clone and UbcH5A full-length cDNA, sequence alignment demonstrated that the SFT cDNA published in 1997 is almost identical with intron 6 and exon 7 of the UbcH5A gene. Subsequently, the authors retracted some of their original results12 due to sequencing errors and noted that their sequence matches a part of a ubiquitin-conjugating enzyme.21 As a consequence, the original SFT cDNA GenBank entry was deleted and replaced by a sequence now designated as "unknown RNA involved in iron metabolism" (GenBank accession no. AF020761). Identity between this corrected sequence and intron 6/exon 7 of the UbcH5A gene is higher than 99%. Therefore, our data strongly suggest that the putative SFT nucleotide sequence corresponds to a fragment of UbcH5A genomic DNA. Such a hypothesis is also supported by the fact that the putative SFT lost its previously reported open reading frame due to the correction of sequencing errors.21 It is unclear how the previously published data on SFT, particularly the protein and functional data,12-14,30 should be interpreted. Regarding mRNA expression analyses, all previous studies used hybridization probes matching the putative SFT 3' untranslated region (UTR).12,15,24 As the UbcH5A 3' UTR is almost identical to the previously published SFT 3' UTR, those studies most likely detected UbcH5A instead of SFT transcripts. This hypothesis is supported by our finding that Northern analysis for UbcH5A reveals 2 transcripts of approximately 1.5 kb and approximately 2.4 kb. Interestingly, 2 transcripts of the same length have been described for SFT.12 In our study, both transcripts were observed, although the probes used were specific for UbcH5A mRNA coding sequence and did not show any homology to the published SFT nucleotide sequence. Based on this observation, it might be conceivable that UbcH5A mRNA expression is iron-regulated as previously demonstrated for SFT.15 However, we were not able to demonstrate significant changes in UbcH5A mRNA expression in response to iron loading or chelation in HepG2 cells. On the other hand, UbcH5A transcripts are significantly up-regulated in the liver of iron-overloaded HH patients. This up-regulation of UbcH5A in HH patients is statistically significant but is only 2-fold. Therefore, the question arises whether UbcH5A plays an important role in iron metabolism or hemochromatosis as suggested for SFT.12-14 Classical iron-related genes such as DMT1 or IREG1 are also up-regulated only by a factor of 2 in the duodenum of HH patients.29,33 In addition, we observed a less than 3-fold down-regulation of TfR in the liver of HH patients. These data demonstrate that the level of UbcH5A up-regulation in HH patients is in good agreement with other iron-related genes. On the other hand, it might be conceivable that the up-regulation of UbcH5A transcripts in the liver of HH patients is unspecific. Fully expressed HH is associated with intrahepatic inflammation and fibrosis.1,19,20 As these factors might lead to an increased expression of UbcH5A, we evaluated the influence of intrahepatic inflammation or fibrosis on the expression of UbcH5A using liver biopsies from patients with chronic HCV infection and patients showing regular liver histology. These data do not suggest an up-regulation of UbcH5A in response to an inflammatory reaction or to fibrosis. Nevertheless, it is unclear whether UbcH5A plays a significant role in
iron metabolism or hemochromatosis. UbcH5A is a member of the
ubiquitin-conjugating enzymes (E2) involved in ubiquitination of
specific proteins. In general, ubiquitination requires sequential actions of activating (E1), conjugating (E2), and ligase (E3) enzymes.
First, ubiquitin is activated to form a thiol ester intermediate with
the E1. Activated ubiquitin is then transferred to an E2 from E1. In
the presence of an E3, the E2, such as UbcH5A, transfers ubiquitin to
the specific substrate.34 UbcH5A itself is known to be
involved in the E6-AP-dependent degradation of p5335 and the ubiquitination of the
Submitted July 23, 2002; accepted November 25, 2002.
Prepublished online as Blood First Edition Paper, December 12, 2002; DOI 10.1182/blood-2002-07-2192.
Supported by grants from the Deutsche Forschungsgemeinschaft (STR 216/10-1) and the Dietmar Hopp Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Wolfgang Stremmel, University Hospital Heidelberg, Department of Internal Medicine IV, Bergheimer Strasse 58-69115 Heidelberg, Germany; e-mail: stremmel{at}medizin-online.com.
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Top
Abstract
Introduction
-glutamyl
transferase elevation and normal liver histology and from 3 patients
with chronic hepatitis C and mild histological activity. All controls had serum ferritin levels below 300 µg/L, showed no significant fibrosis, were negative for stainable iron in liver biopsy, and did not
show any abnormalities in serological iron parameters. To evaluate the
influence of inflammation on gene expression, 13 iron stain-negative
liver biopsy samples from patients with chronic hepatitis C were
obtained. Histological grading of activity according to the METAVIR
score
20°C in
RNAlater solution (Ambion, Austin, TX) prior to RNA isolation. The
study was approved by the local ethics committee. Informed consent was
obtained from all patients.
subunit of the hypoxia-inducible factor (HIF-
a new member of the transferrin receptor-like family.
J Biol Chem.
1999;274:20826-20832