Jorge P. Pinto

Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPα

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPα showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPα binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.

ER Stress-Inducible Factor CHOP Affects the Expression of Hepcidin by Modulating C/EBPalpha Activity

Endoplasmic reticulum (ER) stress induces a complex network of pathways collectively termed the unfolded protein response (UPR). The clarification of these pathways has linked the UPR to the regulation of several physiological processes. However, its crosstalk with cellular iron metabolism remains unclear, which prompted us to examine whether an UPR affects the expression of relevant iron-related genes. For that purpose, the HepG2 cell line was used as model and the UPR was activated by dithiothreitol (DTT) and homocysteine (Hcys). Here, we report that hepcidin, a liver secreted hormone that shepherds iron homeostasis, exhibits a biphasic pattern of expression following UPR activation: its levels decreased in an early stage and increased with the maintenance of the stress response. Furthermore, we show that immediately after stressing the ER, the stress-inducible transcription factor CHOP depletes C/EBPα protein pool, which may in turn impact on the activation of hepcidin transcription. In the later period of the UPR, CHOP levels decreased progressively, enhancing C/EBPα-binding to the hepcidin promoter. In addition, analysis of ferroportin and ferritin H revealed that the transcript levels of these iron-genes are increased by the UPR signaling pathways. Taken together, our findings suggest that the UPR can have a broad impact on the maintenance of cellular iron homeostasis.

Hepcidin messenger RNA expression in human lymphocytes

Hepcidin regulates intracellular iron levels by interacting with and promoting the degradation of ferroportin, a membrane protein and the only known cellular iron exporter. Studies of hepcidin expression and regulation have focused on its effects in innate immunity and as a regulator of systemic iron metabolism. In the present study we characterized the expression of hepcidin messenger RNA (mRNA) in human peripheral blood mononuclear cells (PBMCs) with a focus on peripheral blood lymphocytes (PBLs). We found that (1) all human PBMCs analyzed express basal hepcidin mRNA levels; (2) hepcidin mRNA expression increases after T‐lymphocyte activation; (3) expression by PBLs increases in response to challenge by holotransferrin (Fe‐TF) and by ferric citrate in vitro; (4) the Fe‐TF‐mediated up‐regulation of hepcidin decreases ferroportin expression at the cytoplasmic membrane of PBLs; and (5) silencing of tumour necrosis factor‐α (TNF‐α) abrogates the effect of Fe‐TF. In summary, we show that hepcidin expression determines intracellular iron levels by regulating the expression of ferroportin, as described in other cells, and that inappropriately low expression of hepcidin impairs normal lymphocyte proliferation. The results establish hepcidin as a new player in lymphocyte biology.

Non-Transferrin-Bound Iron (NTBI) Uptake by T Lymphocytes: Evidence for the Selective Acquisition of Oligomeric Ferric Citrate Species

Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI). NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.

Two novel mutations in the tmprss6 gene associated with iron‐refractory iron‐deficiency anaemia (irida) and partial expression in the heterozygous form

Arceci, R.J., Sande, J., Lange, B., Shannon, K., Franklin, J., Hutchinson, R., Vik, T.A., Flowers, D., Aplenc, R., Berger, M.S., Sherman, M. L., Smith, F.O., Bernstein, I. & Sievers, E.L. (2005) Safety and efficacy of gemtuzumab ozogamicin in pediatric patients with advanced CD33+ acute myeloid leukemia. Blood, 106, 1183–1188. Bornhauser, M., Illmer, T., Oelschlaegel, U., Schetelig, J., Ordemann, R., Schaich, M., Hanel, M., Schuler, U., Thiede, C., Kiani, A., Platzbecker, U. & Ehninger, G. (2008) Gemtuzumab ozogamicin as part of reducedintensity conditioning for allogeneic hematopoietic cell transplantation in patients with relapsed acute myeloid leukemia. Clinical Cancer Research, 14, 5585–5593. Burnett, A.K., Hills, R.K., Milligan, D., Kjeldsen, L., Kell, J., Russell, N.H., Yin, J.A., Hunter, A., Goldstone, A.H. & Wheatley, K. (2011) Identification of patients with acute myeloblastic leukemia who benefit from the addition of gemtuzumab ozogamicin: results of the MRC AML15 trial. Journal of Clinical Oncology, 29, 369–377. Cooper, T.M., Franklin, J., Gerbing, R.B., Alonzo, T.A., Hurwitz, C., Raimondi, S.C., Hirsch, B., Smith, F.O., Mathew, P., Arceci, R.J., Feusner, J., Iannone, R., Lavey, R.S., Meshinchi, S. & Gamis, A. (2012) AAML03P1, a pilot study of the safety of gemtuzumab ozogamicin in combination with chemotherapy for newly diagnosed childhood acute myeloid leukemia: a report from the Children’s Oncology Group. Cancer, 118, 761–769. de Lima, M., Champlin, R.E., Thall, P.F., Wang, X., Martin, T.G., 3rd, Cook, J.D., McCormick, G., Qazilbash, M., Kebriaei, P., Couriel, D., Shpall, E.J., Khouri, I., Anderlini, P., Hosing, C., Chan, K.W., Andersson, B.S., Patah, P.A., Caldera, Z., Jabbour, E. & Giralt, S. (2008) Phase I/ II study of gemtuzumab ozogamicin added to fludarabine, melphalan and allogeneic hematopoietic stem cell transplantation for high-risk CD33 positive myeloid leukemias and myelodysplastic syndrome. Leukemia, 22, 258–264. Petersdorf, S., Kopecky, K., Stuart, R.K., Larson, R. A., Nevill, T.J., Stenke, L., Slovak, M.L., Tallman, M.S., Willman, C.L., Erba, H. & Appelbaum, F.R. (2009) Preliminary Results of Southwest Oncology Group Study S0106: An International Intergroup Phase 3 Randomized Trial Comparing the Addition of Gemtuzumab Ozogamicin to Standard Induction Therapy Versus Standard Induction Therapy Followed by a Second Randomization to Post-Consolidation Gemtuzumab Ozogamicin Versus No Additional Therapy for Previously Untreated Acute Myeloid Leukemia. Blood (ASH Annual Meeting Abstracts), 114, 790. Sakaguchi, H., Watanabe, N., Muramatsu, H., Doisaki, S., Yoshida, N., Matsumoto, K. & Kato, K. (2010) Danaparoid as the prophylaxis for hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation in childhood hematological malignancy. Pediatric Blood & Cancer, 55, 1118–1125. Sibson, K., Steward, C., Moppett, J., Cornish, J. & Goulden, N. (2009) Dismal long-term prognosis for children with refractory acute myeloid leukaemia treated with gemtuzumab ozogamicin and stem cell transplantation: where now? British Journal of Haematology, 146, 342–344. Wadleigh, M., Richardson, P.G., Zahrieh, D., Lee, S.J., Cutler, C., Ho, V., Alyea, E.P., Antin, J.H., Stone, R.M., Soiffer, R.J. & DeAngelo, D.J. (2003) Prior gemtuzumab ozogamicin exposure significantly increases the risk of veno-occlusive disease in patients who undergo myeloablative allogeneic stem cell transplantation. Blood, 102, 1578–1582.

CAT53 and HFE alleles in Alzheimer's disease: a putative protective role of the C282Y HFE mutation.

Alzheimers disease (AD) is a complex disorder, resulting from an interaction between environmental and genetic factors. Several studies addressed the association of AD with MHC class-I polymorphisms without definite conclusions. Considering the remarkable linkage disequilibrium at the MHC region, it is not possible to assume if the reported associations result from a direct effect of the respective genes or result from associations with other closely linked genes transmitted in an extended conserved haplotype. Recent evidence pointed to CAT53, a newly described gene located at the MHC class-I region in the vicinity of HLA-C, as a candidate modifier gene in AD. CAT53 encodes a phosphatase 1 nuclear inhibitor protein and is strongly expressed in brain regions involved in memory and AD. Here we tested the potential association of CAT53 with the risk of developing AD and searched for potential haplotypic associations of CAT53 with two common mutations (H63D, C282Y) in the HFE gene, also located at chromosome 6p21.3. The allele frequencies of these mutations in AD patients were compared to the expected frequencies previously established in the normal Portuguese population. We detected only one polymorphism (G>A) in CAT53, at position 8232, in intron 17. Screening of this polymorphism in 113 AD patients and 82 controls did not show any evidence of association, therefore excluding the hypothetical role of the CAT53 polymorphism as modifier in AD. In contrast, we found a significant negative association of the C282Y HFE mutation with AD, thus supporting a putative protective role of this protein variant in neurodegeneration.

Physiological implications of NTBI uptake by T lymphocytes.

In iron overload disorders a significant fraction of the total iron circulates in the plasma as low molecular weight complexes not bound to transferrin, known as non-transferrin-bound iron (NTBI). By catalyzing the formation of free radicals, NTBI accumulation results in oxidative stress and cellular damage, being a major cause of organ toxicity. NTBI is rapidly and preferentially cleared from circulation by the liver and the myocardium, the main disease targets in iron overload conditions. We have recently demonstrated that human peripheral blood T lymphocytes take up NTBI in vitro, with a pattern that resembles that of hepatocytes. Since T lymphocytes constitute a numerically important component of the circulating cell pool, these findings support a putative role for this cell type in the systemic protection against iron toxicity. Here we tested the hypothesis that the circulating peripheral blood T lymphocyte pool constitutes an important storage compartment for NTBI and is thus a modifier of NTBI deposition in target organs. First we show that NTBI uptake by human T lymphocytes increases the expression of the iron-storage protein ferritin and of the iron exporter ferroportin via an IRE-dependent mechanism. NTBI retention by T lymphocytes is shown to be critically controlled by the hepcidin-mediated modulation of ferroportin both in vitro and in vivo. Finally, the protective effect of T lymphocytes was tested by analyzing the patterns of iron accumulation in the T lymphocyte-deficient mouse model Foxn1nu before and after reconstitution with T lymphocytes by adoptive transfer. The results confirmed a significant increase of liver and pancreas iron accumulation in T lymphocyte-deficient mice. NTBI accumulation in the liver and spleen was prevented by reconstitution with syngeneic T lymphocytes. Altogether, our results demonstrate that T lymphocytes are important components of a circulating “NTBI storage compartment” and show its physiological relevance as a modifier of tissue iron overload.

Efficient screening of the cystinuria-related C663T Slc3a1 nonsense mutation in Newfoundland dogs by denaturing high-performance liquid chromatography.

Cystinuria in Newfoundland dogs is a metabolic disease associated with a nonsense mutation in the exon 2 of the Slc3a1 gene. Similar to type I human cystinuria, heterozygote carriers are not affected by the disease and do not reveal differences in urinary concentration of dibasic amino acids when compared with normal dogs. However, through a recessive mode of inheritance, these dogs are able to transmit the disease to their offspring. Early detection of mutation carriers through cost-effective reliable methods is therefore essential for the implementation of breeding methods aimed at the eradication of the disease. Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid and efficient screening of nucleotide polymorphisms in polymerase chain reaction–amplified products. This technique was used for the identification of the C663T Slc3a1 mutation in Portuguese Newfoundland dogs. Polymerase chain reaction products amplified from a region containing the C663T locus were subjected to DHPLC analysis, and results were double checked by DNA sequencing. Results showed the presence of the mutation in 6 of the 22 dogs tested. Urine biochemical parameters correlated well with the number of mutated Slc3a1 copies, and homozygotes for the C663T mutation were the only dogs diagnosed with cystinuria. Sequence analysis confirmed the DHPLC results, demonstrating that the technique could be a reliable alternative to sequencing for the rapid and cost-effective identification of mutations in canine breeds.

Hepcidin: a clue for a new role for lymphocytes

Jorge P Pinto1, PhD; Vera Dias1; Heinz Zoller2, MD; Pedro N Rodrigues1, PhD; Helena Gomes3, Pedro Ramos1; Felix Carvalho3, PhD; Graca Porto1, MD, PhD; Maria de Sousa1, MD, PhD. 1Iron Genes and Immune System, IBMC Institute for Molecular and Cell Biology, Oporto, Portugal; 2Internal Medicine II, Gastroenterology & Hepatology, Medical University, Innsbruck, Austria; 3Toxicology Department, REQUIMTE (Rede de Quimica e Tecnologia), Oporto, Portugal