Interactions of the ectodomain of HFE with the transferrin receptor are critical for iron homeostasis in cells.

Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y(2) HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.

The hereditary hemochromatosis gene (HFE): a MHC class I-like gene that functions in the regulation of iron homeostasis.

The iron overload disorder, hereditary hemochromatosis, is one of the most common genetic diseases of individuals of Northern European descent. The disorder is characterized by the progressive accumulation of dietary iron in the major organs of the body, which if not diagnosed, leads to numerous medical maladies and eventually death. The locus for this disorder was mapped by genetic linkage to the short arm of chromosome over twenty years ago, but it was not until 1996 that the gene for this disorder was cloned by an identity-by-descent positional cloning approach. The gene, called HFE, encodes a major histocompatibility complex (MHC) class I-like protein that is mutated in approx 85% of all individuals known to have hereditary hemochromatosis (HH). Since the cloning of the HFE gene, considerable work has been carried out which has furthered our understanding of the genetics of this prevalent disorder. In addition, with the identification of the transferrin receptor as a protein capable of interacting with HFE we are now beginning to understand how a protein with the structural characteristics of an MHC class I molecule can influence cellular iron homeostasis.

The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells.

HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C., Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E., Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B., Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana, L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna, D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996) Nat. Genet. 13, 399-408). At the cell surface, HFE complexes with transferrin receptor (TfR), increasing the dissociation constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998) J. Biol. Chem. 273, 22068-22074; Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N. , Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A 95, 1472-1477). HFE does not remain at the cell surface, but traffics with TfR to Tf-positive internal compartments (Gross et al., 1998). Using a HeLa cell line in which the expression of HFE is controlled by tetracycline, we show that the expression of HFE reduces 55Fe uptake from Tf by 33% but does not affect the endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to reduce cellular acquisition of iron from Tf within endocytic compartments. HFE specifically reduces iron uptake from Tf, as non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not altered. These results explain the decreased ferritin levels seen in our HeLa cell system and demonstrate the specific control of HFE over the Tf-mediated pathway of iron uptake. These results also have implications for the understanding of cellular iron homeostasis in organs such as the liver, pancreas, heart, and spleen that are iron loaded in hereditary hemochromatotic individuals lacking functional HFE.

Transferrin receptor mutation analysis in hereditary hemochromatosis patients.

The Cys282-->Tyr mutation in the HFE gene is carried by the majority of hereditary hemochromatosis patient chromosomes, yet some patients do not seem to harbor any mutation in this gene. This suggests a possibility that these patients may have a mutation in other genes in the same pathway as HFE. We analyzed the cDNA sequences of transferrin receptor (TFR), which was recently shown to interact with HFE, in twenty-one hereditary hemochromatosis patients including sixteen individuals who did not carry a Cys282-->Tyr mutation. A nucleotide substitution (424A-->G), which resulted in the Ser142-->Gly amino acid substitution, was the only amino acid polymorphism detected in the open reading frame of the TFR gene in these patients. This amino acid substitution was a rather common polymorphism in the general population (49%) and its frequency did not significantly differ in the hereditary hemochromatosis (HH) patients regardless of the HFE genotype. Thus, amino acid changes in the TFR gene do not appear to play a role in HH even when the patients do not have a HFE mutation. However, this study does not rule out the possibility of the involvement of mutations in non-coding regions.

Co-trafficking of HFE, a nonclassical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation.

The mechanism by which a novel major histocompatibility complex class I protein, HFE, regulates iron uptake into the body is not known. HFE is the product of the gene that is mutated in >80% of hereditary hemochromatosis patients. It was recently found to coprecipitate with the transferrin receptor (Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1472-1477; Parkkila, S., Waheed, A., Britton, R. S., Bacon, B. R., Zhou, X. Y., Tomatsu, S., Fleming, R.E. , and Sly, W. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13198-13202) and to decrease the affinity of transferrin for the transferrin receptor (Feder et al.). In this study, HeLa cells were transfected with HFE under the control of the tetracycline-repressible promoter. We demonstrate that HFE and the transferrin receptor are capable of associating with each other within 30 min of their synthesis with pulse-chase experiments. HFE and the transferrin receptor co-immunoprecipitate throughout the biosynthetic pathway. Excess HFE is rapidly degraded, whereas the HFE-transferrin receptor complex is stable. Immunofluorescence experiments indicate that they also endocytose into transferrin-positive compartments. Combined, these results suggest a role for the transferrin receptor in HFE trafficking. Cells expressing HFE have modestly increased levels of transferrin receptor and drastically reduced levels of ferritin. These results implicate HFE further in the modulation of iron levels in the cell.

Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor.

HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.

The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with beta2-microglobulin (beta2m) and prevents cell-surface expression. A second mutation that has no effect on beta2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/beta2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4 degrees C, the added soluble complex of HFE/beta2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.

Hereditary hemochromatosis: effects of C282Y and H63D mutations on association with beta2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells.

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282-->Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with beta2-microglobulin; a second mutation, His-63-->Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with beta2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

The hemochromatosis founder mutation in HLA-H disrupts beta2-microglobulin interaction and cell surface expression.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 --> tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 --> aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds beta2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.

A 1.1-Mb transcript map of the hereditary hemochromatosis locus.

In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A. A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments. As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes. Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein. Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2. The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC. The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC. Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA.

Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression.

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

Immunohistochemistry of HLA-H, the protein defective in patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism that leads to excessive iron storage in the liver and other organs. Recently, between 83 and 100% of HH patients have been found to be homozygous for the same mutation in a novel major histocompatibility complex class I-like gene, called the HLA-H gene. The Cys-282 --> Tyr mutation in HH patients would be expected to disrupt the function of the HLA-H gene product by altering a critical disulfide bridge. As a first step in understanding the function of the HLA-H gene product, we generated an antibody to a C-terminal peptide and used it for immunolocalization of the HLA-H protein in the gastrointestinal tract of Finnish and American subjects presumed not to have HH. Although staining for the HLA-H protein was seen in some epithelial cells in every segment of the alimentary canal, its cellular and subcellular expression in the small intestine were quite distinct from those seen in other segments. In contrast to the stomach and colon, where staining was polarized and restricted to the basolateral surfaces, and in contrast to the epithelial cells of the esophagus and submucosal leukocytes, which showed nonpolarized staining around the entire plasma membrane, the staining in small intestine was mainly intracellular and perinuclear, limited to cells in deep crypts. Prior genetic evidence suggested that a defective HLA-H protein is the molecular basis of HH. Here we show that the HLA-H protein not only varies in its pattern of expression along the cranial/caudal axis of the gastrointestinal tract but that it has a unique subcellular localization in the crypts of the small intestine in proximity to the presumed sites of iron absorption.

A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

Asymmetric localization of a mammalian numb homolog during mouse cortical neurogenesis.

During Drosophila neurogenesis, differential segregation of Numb is necessary for daughter cells of asymmetric divisions to adopt distinct fates, at least partly by biasing the Notch-mediated cell-cell interaction. We have isolated a highly conserved mammalian homolog of Drosophila numb, m-numb. During mouse cortical neurogenesis, m-Numb is asymmetrically localized to the apical membrane of dividing ventricular neural progenitors. Depending upon the orientation of the cleavage plane, m-Numb may be distributed into one or both of the daughter cells. When expressed in Drosophila embryos, m-Numb is localized asymmetrically in dividing neural precursors and rescues the numb mutant phenotype. Furthermore, m-Numb can physically interact with mouse Notch1. We propose that some shared molecular mechanisms, both cell-intrinsic and cell-extrinsic, generate asymmetric cell divisions during neurogenesis of vertebrates and invertebrates.

Genomic cloning and chromosomal localization of HRY, the human homolog to the Drosophila segmentation gene, hairy.

The Drosophila hairy gene encodes a basic helix-loop-helix protein that functions in at least two steps during Drosophila development: (1) during embryogenesis, when it partakes in the establishment of segments, and (2) during the larval stage, when it functions negatively in determining the pattern of sensory bristles on the adult fly. In the rat, a structurally homologous gene (RHL) behaves as an immediate-early gene in its response to growth factors and can, like that in Drosophila, suppress neuronal differentiation events. Here, we report the genomic cloning of the human hairy gene homolog (HRY). The coding region of the gene is contained within four exons. The predicted amino acid sequence reveals only four amino acid differences between the human and rat genes. Analysis of the DNA sequence 5' to the coding region reveals a putative untranslated exon. To increase the value of the HRY gene as a genetic marker and to assess its potential involvement in genetic disorders, we sublocalized the locus to chromosome 3q28-q29 by fluorescence in situ hybridization.

A rat gene with sequence homology to the Drosophila gene hairy is rapidly induced by growth factors known to influence neuronal differentiation.

Several genes encoding transcription factors with a helix-loop-helix (HLH) motif are involved in the early process of neural development in Drosophila spp. We report the isolation from the rat a homolog of one of these genes, called hairy. The rat-hairy-like (RHL) gene is expressed early during embryogenesis. In contrast to the restricted expression of hairy mRNA in Drosophila spp., however, the mRNA encoded by RHL is detectable in all tissues examined. Stimulation of PC12 pheochromocytoma cells by nerve growth factor, basis fibroblast growth factor, or epidermal growth factor or of Rat-1 fibroblasts by epidermal growth factor causes a rapid and transient induction of the RHL gene. Thus, RHL acts as an immediate-early gene that can potentially transduce growth factor signals during the development of the mammalian embryo.

Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts.

We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to the lipophilic antifolates trimetrexate and piritrexim. These data suggested that R50 cells may possess additional mechanism(s) of antifolate resistance, such as MTX transport alteration. Flow cytometric analysis using fluorescein methotrexate revealed comparable levels of fluorescein MTX displacement with lipophilic antifolates in viable R50 and B11 0.5 cells, but marked insensitivity of R50 cells to MTX competition, thus suggesting a poor uptake of MTX into R50 cells. Analysis of the kinetic parameters of dihydrofolate reductase from R50 cells neither showed alterations in enzyme affinities for various antifolates nor in the Michaelis constant for folic acid and NADPH nor a change in the pH activity optimum. R50 cell-free extracts contained wild-type levels of folylpoly-gamma-glutamyl synthetase activity. However, following metabolic labeling with [3H]MTX, no MTX polyglutamates could be detected in R50 cells. We conclude that the high level of MTX resistance in R50 cells is multifactorial, including overexpression of dihydrofolate reductase, reduced MTX transport, and possibly altered formation of MTX polyglutamates. The potential interactions between the different modalities of MTX resistance in R50 cells are being discussed.

Functional analysis of the tobacco mosaic virus tRNA-like structure in cytoplasmic gene regulation.

The 3'-untranslated region (UTR) of tobacco mosaic virus (TMV), which terminates in a tRNA-like structure, functionally substitutes for a poly(A) tail in both plant and animal cells. The addition of the TMV 3'-UTR to chimeric mRNA constructs increases their expression up to 100-fold, increasing both translational efficiency and mRNA stability. The domain largely responsible for the regulation maps to a 72 base region immediately upstream of the tRNA-like structure, however, the 3'-terminal, tRNA-like structure is required for full function. Its contribution is lost if separated from the upstream pseudoknot domain by as few as 5 bases or if 6 bases are removed from the 3'-terminus. Sequence addition to the 3'-terminus of the TMV 3'UTR or the upstream pseudoknot domain inhibits function in both tobacco and Chinese hamster ovary cells.

Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression.

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.