Konstantin Adamsky

Suppressors of Cytokine Signaling 4 and 5 Regulate Epidermal Growth Factor Receptor Signaling

Suppressors of cytokine signaling (SOCS) are Src homology-2-containing proteins originally identified as negative regulators of cytokine signaling. Accumulating evidence indicates a role for SOCS proteins in the regulation of additional signaling pathways including receptor tyrosine kinases. Notably, SOCS36E, the Drosophila ortholog of mammalian SOCS5, was recently implicated as a negative regulator of the Drosophila ortholog of EGFR. In this study, we aimed at characterizing the role of SOCS5 in the negative regulation of EGFR. Here we show that the expression of SOCS5 and its closest homolog SOCS4 is elevated in cells following treatment with EGF, similar to several negative feedback regulators of EGFR whose expression is up-regulated upon receptor activation. The expression of SOCS5 led to a marked reduction in EGFR expression levels by promoting EGFR degradation. The reduction in EGFR levels and EGF-induced signaling in SOCS5-expressing cells requires both the Src homology-2 and SOCS box domains of SOCS5. Interestingly, EGFR is degraded by SOCS5 prior to EGF treatment in a ligand- and c-Cbl-independent manner. SOCS5 can associate with EGFR and can also bind the ElonginBC protein complex via its SOCS box, which may recruit an E3 ubiquitin ligase to promote EGFR degradation. Thus, we have characterized a novel function for SOCS5 in regulating EGFR and discuss its potential role in controlling EGFR homeostasis.

Decreased hepcidin mRNA expression in thalassemic mice

Beta thalassemia major is a congenital haemolytic anaemia resulting from the lack of synthesis of the b-globin chain, a major component of haemoglobin A. In the absence of lifelong transfusions the disease is lethal. When adequate transfusion support is provided the patients suffer from iron overload that can lead to endocrine deficiencies, cirrhosis and cardiac failure that frequently lead to their demise. The iron overload results from ‘transfusional’ iron as well as from an inadequate high intestinal absorption despite high levels of body iron. The understanding of the deranged regulation of intestinal iron absorption is of utmost importance and may lead to rational therapy aimed at decreasing iron overload. Hepcidin, a recently identified anti-microbial peptide expressed in the liver was shown to play a role in conditions associated with both iron overload and iron deficiency. Hepcidin inhibits iron absorption in the proximal small bowel and the release of iron from macrophages. In healthy individuals as well as in murine models hepcidin expression is increased when iron stores are elevated and decreased as a result of iron deficiency anaemia and hypoxia (Nicolas et al, 2001; Park et al, 2001; Pigeon et al, 2001; Ganz, 2003). Thalassemia major represents a unique situation where anaemia, which is expected to decrease hepcidin expression, coexists with iron overload, which ought to increase it. Therefore, it was of interest to study which of these factors has an upper hand in hepcidin expression regulation. We used the recently described severe anaemia model of C57Bl/6 Hbb mice (May et al, 2002) that exhibit anaemia, abnormal red cell morphology, splenomegaly and develop spontaneous hepatic iron deposition (demonstrated by Gomori iron stain of liver tissue), and C57Bl/6 control mice in an attempt to understand the inadequately enhanced absorption of iron in thalassemia. RNA extracted from the liver of adult mice was analysed by quantifiable real time reverse transcription polymerase chain reaction (RT-PCR) with specific primers for the following iron metabolism-related genes: hepcidin, iron-regulated gene 1 (IREG1), neutral gelatinase-associated lipocalin (NGAL), haemochromatosis (HFE), transferrin receptors 1 and 2 (TfR1 and TfR2). The expression levels were normalized to b-actin. Interestingly, despite iron overload the expression levels of both hepcidin and TfR1 were significantly lower (0Æ38-fold) in the thalassemic mice. HFE was moderately decreased (0Æ7-fold), while NGAL was significantly increased (2Æ45-fold). TfR2 and IREG1 did not change significantly (Fig 1). This would suggest that iron overload is less dominant than anaemia in regulating hepcidin expression in the setting of the thalassemia major mouse model. The decreased expression of hepcidin may explain the increased absorption of iron in thalassemia. Recently, decreased expression of hepcidin was found in hereditary haemochromatosis in association with elevated levels of nontransferrin bound iron. The elevated expression of NGAL, an alternative iron delivery vehicle, supports the role of nontranferrin bound iron in the abnormal iron regulation in thalassemia. The decreased HFE expression level is similar to the finding in hereditary haemochromatosis (Bridle et al, 2003). The exact mechanism remains to be elucidated. These preliminary findings call for confirmation in human thalassemia patients. If supported, a new therapeutic approach to iron overload states such as thalassemia can be devised based on hepcidin administration or other interventions aimed at overcoming the inadequate response to iron overload associated with elevated levels of non-transferrin bound iron. Fig 1. Expression of iron regulatory genes in the liver of a mouse model of b-thalassemia. cDNA was prepared from the total RNA extracted from the liver of three C57B1/6 Hbb mice and three normal C57B1/6 wild-type mice. Gene expression of six iron regulatory genes was measured using specific primers for quantifiable reverse transcription polymerase chain reaction. Bars represent the average fold change in mRNA expression of each gene in C57B1/6 Hbb mice (black bars), when compared with control mRNA levels in C57B1/6 wild-type mice (grey bars). Each bar represents the average of 2–4 independent experiments, normalized to corresponding b-actin expression. correspondence

Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera

β‐Thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β‐thalassaemia, we observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in β‐thalassaemia might suppress liver hepcidin expression. Sera from β‐thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β‐thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by β‐thalassaemia major sera (r = 0·852, P < 0·0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from β‐thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0·765, P < 0·0099). Our results suggest that, in β‐thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption in these patients.

Role of Iron in Inducing Oxidative Stress in Thalassemia: Can It Be Prevented by Inhibition of Absorption and by Antioxidants?

Abstract: The pathophysiology of thalassemia is, to a certain extent, associated with the generation of labile iron in the pathological red blood cell (RBC). The appearance of such forms of iron at the inner and outer cell surfaces exposes the cell to conditions whereby the labile metal promotes the formation of reactive oxygen species (ROS) leading to cumulative cell damage. Another source of iron accumulation results from increased absorption due to decreased expression of hepcidin. The presence of labile plasma iron (LPI) was carried out using fluorescent probes in the FACS. RNA expression of hepcidin was measured in two models of thalassemic mice. Hepcidin expression was also measured in human hapatoma HepG2 cells following incubation with thalassemic sera. LPI was identified and could be quantitatively measured and correlated with other parameters of iron overload. Hepcidin expression was downregulated in the livers of thalassemic mice, in major more than in intermedia. Thalassemic sera down regulated hepcidin expression in HepG2 liver cells. A possible way to decrease iron absorption could be by modulating hepcidin expression pharmacologically, by gene therapy or by its administration. Treatment with combination of antioxidants such as N‐acetylcysteine for proteins and vitamin E for lipids in addition to iron chelators could neutralize the deleterious effects of ROS and monitored by quantitation of LPI.

Hepcidin, a key regulator of iron metabolism, is transcriptionally activated by p53.

Hepcidin is an iron‐regulatory protein that is upregulated in response to increased iron or inflammatory stimuli. Hepcidin reduces serum iron and induces iron sequestration in the reticuloendothelial macrophages – the hallmark of anaemia of inflammation. Iron deprivation is used as a defense mechanism against infection, and it also has a beneficial effect on the control of cancer. The tumour‐suppressor p53 transcriptionally regulates genes involved in growth arrest, apoptosis and DNA repair, and perturbation of p53 pathways is a hallmark of the majority of human cancers. This study inspected a role of p53 in the transcriptional regulation of hepcidin. Based on preliminary bioinformatics analysis, we identified a putative p53 response‐element (p53RE) contained in the hepcidin gene (HAMP) promoter. Chromatin immunoprecipitation (ChIP), reporter assays and a temperature sensitive p53 cell‐line system were used to demonstrate p53 binding and activation of the hepcidin promoter. p53 bound to hepcidin p53RE in vivo, andthis p53RE could confer p53‐dependent transcriptional activation. Activation of p53 increased hepcidin expression, while silencing of p53 resulted in decreased hepcidin expression in human hepatoma cells. Taken together, these results define HAMP as a novel transcriptional target of p53. We hypothesise that hepcidin upregulation by p53 is part of a defence mechanism against cancer, through iron deprivation. Hepcidin induction by p53 might be involved in the pathogenesis of anaemia accompanying cancer.

Identification of RNA editing sites in the SNP database

The relationship between human inherited genomic variations and phenotypic differences has been the focus of much research effort in recent years. These studies benefit from millions of single-nucleotide polymorphism (SNP) records available in public databases, such as dbSNP. The importance of identifying false dbSNP records increases with the growing role played by SNPs in linkage analysis for disease traits. In particular, the emerging understanding of the abundance of DNA and RNA editing calls for a careful distinction between inherited SNPs and somatic DNA and RNA modifications. In order to demonstrate that some of the SNP database records are actually somatic modification, we focus on one type of these modifications, namely A-to-I RNA editing, and present evidence for hundreds of dbSNP records that are actually editing sites. We provide a list of 102 RNA editing sites previously annotated in dbSNP database as SNPs, and experimentally validate seven of these. Interestingly, we show how dbSNP can serve as a starting point to look for new editing sites. Our results, for this particular type of RNA editing, demonstrate the need for a careful analysis of SNP databases in light of the increasing recognition of the significance of somatic sequence modifications.

P53 in blind subterranean mole rats--loss-of-function versus gain-of-function activities on newly cloned Spalax target genes.

A tumor suppressor gene, p53, controls cellular responses to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Recently, we demonstrated that in blind subterranean mole rats, Spalax, a model organism for hypoxia tolerance, the p53 DNA-binding domain contains a specific Arg174Lys amino acid substitution. This substitution reduces the p53 effect on the transcription of apoptosis genes (apaf1, puma, pten and noxa) and enhances it on human cell cycle arrest and p53 stabilization/homeostasis genes (mdm2, pten, p21 and cycG). In the current study, we cloned Spalax apaf1 promoter and mdm2 intronic regions containing consensus p53-responsive elements. We compared the Spalax-responsive elements to those of human, mouse and rat and investigated the transcriptional activity of Spalax and human Arg174Lys-mutated p53 on target genes of both species. Spalax and human-mutated p53 lost induction of apaf1 transcription, and increased induction of mdm2 transcription. We conclude that Spalax evolved hypoxia-adaptive mechanisms, analogous to the alterations acquired by cancer cells during tumor development, with a bias against apoptosis while favoring cell arrest and DNA repair.

Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in β-thalassemia

Abstract: To develop new treatments for β‐thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp‐knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of β‐thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.

Apaf1 in chronic myelogenous leukemia (CML) progression: reduced Apaf1 expression is correlated with a H179R p53 mutation during clinical blast crisis.

Chronic myelogenous leukemia (CML) is a stem cell disorder that eventually progresses to ablast crisis phase (BC) characterized by distorted apoptotic pathways. The exact mechanismleading to failure in apoptotic pathways during CML progression is unclear. In view of thecentral role of p53 and apaf1 in the apoptotic machinery we examined six human pairedchronic and BC phases samples for their expression. Real-time PCR (RQ-PCR) experiments showed an elevation of p53 mRNA in all patientsduring transition to BC. However, elevation of apaf1 during BC was observed in five patientsonly. In contrast, one patient displayed a significant 11.5-fold reduction of apaf1 expressionduring the transition to BC. No apaf1 promoter methylation was observed. The reduced apaf1expression was accompanied by a trans-dominant point mutation (H179R) in one p53 alleleand the loss of the other. This mutant p53, when tested using functionality assays, was unableto activate apaf1, consequently explaining the reduced expression observed in this patient.Furthermore, the same mutant failed to activate either genes involved in apoptotic or cellcycle arrest pathways, and can be considered as a complete loss of function mutation. Thisspecific mutation was reported in several types of cancer, but was not implicated in CML. Toconclude, in this study we have demonstrated mRNA elevation of p53 and apaf1 during CMLblast crisis, indicating that genes and proteins involved in cellular apoptosis might be involvedin disease progression/response to therapy. Moreover, the mutated p53 discovered in thepatient exhibiting lowered apaf1 expression provides, in a clinical case, the first correlationbetween p53 and apaf1 transcription regulation in humans.