Elena Gammella

Role of HIF-1 and NF-κB Transcription Factors in the Modulation of Transferrin Receptor by Inflammatory and Anti-inflammatory Signals

Inflammation generates various changes in body iron homeostasis, including iron sequestration in the reticuloendothelial system with ensuing hypoferremia and anemia of chronic disease. Increased iron accumulation is caused by hepcidin-mediated down-regulation of the iron export protein ferroportin and higher iron uptake. However, enhanced iron acquisition by macrophages cannot be accounted for by the previously reported transferrin receptor (TfR1) down-regulation in macrophages exposed to lipopolysaccharide (LPS)/interferon γ (IFNγ) because it impairs a major iron uptake mechanism. Because TfR1 is up-regulated by the hypoxia-inducible factor (HIF-1), we investigated the effect of inflammatory and anti-inflammatory signals on HIF-1-mediated TfR1 gene expression. Exposure of mouse macrophages (RAW 264.7 and J774A.1 cells or peritoneal macrophages) to LPS/IFNγ up-regulated NF-κB, which in turn rapidly and transiently activated HIF-1-dependent TfR1 expression and iron uptake. Activation of an anti-inflammatory pathway by pre-exposure to the adenosine A2A receptor agonist CGS21680 prevented the inducing effect of LPS/IFNγ on HIF-1 and TfR1 expression by inhibiting NF-κB activity, whereas treatment with CGS21680 alone increased HIF-1-mediated TfR1 expression by means of an NF-κB-independent signaling pathway. In conclusion, an interplay of the HIF-1 and NF-κB pathways controls TfR1 transcription in inflammation. The consequent changes in TfR1 expression may be involved in modulating iron retention in inflammatory macrophages, thus possibly contributing to the development of hypoferremia in the early phases preceding the down-regulation of macrophage ferroportin by hepcidin.

Iron levels in polarized macrophages: Regulation of immunity and autoimmunity

Although the hallmark of autoimmune diseases remains the generation of autoantigen-specific lynfocytic cell response, growing evidence is showing a key role for macrophages in a number of autoimmune diseases. Macrophages are characterized by phenotypical and functional heterogeneity. Different immunological signals, coming from systemic blood circulation or from microenvironment, polarize macrophages to classical (M1) or alternative (M2) phenotypes. Iron accumulation in M1 macrophages is a well known bacteriostatic mechanism and one of the mechanisms at the basis of anemia associated to chronic inflammation. Moreover, some recent data suggest that iron accumulation in macrophages can directly activate macrophages to pro-inflammatory M1 phenotype, highlighting a putative role of macrophage iron retention in the pathogenesis of chronic inflammatory and autoimmune diseases. Conversely, iron content is low in M2 macrophages, principally due to increased iron release, and increased availability of iron in the extracellular milieu supported by M2 macrophages could influence the growth rate of adjacent cell and thus play an important role in tumor growth and tissue remodeling. In this review we summarize the molecular mechanisms sustaining differential iron metabolism in polarized macrophages, discuss the relevance of this metabolic signature in chronic inflammatory and autoimmune diseases, and finally focus on potential therapeutic implications rising from a better understanding of underlying molecular mechanisms.

Adenosine-dependent activation of hypoxia-inducible factor-1 induces late preconditioning in liver cells†

The cellular mechanisms by which ischemic preconditioning increases liver tolerance to ischemia/reperfusion injury are still poorly understood. This study investigated the role of the hypoxia‐inducible factor‐1 (HIF‐1) in the protection associated with the late phase of liver preconditioning. Late preconditioning was induced in primary cultured rat hepatocytes by a transient (10 minute) hypoxic stress or by 15 minutes incubation with the adenosine A2A receptors agonist CGS21680 24 hours before exposure to 90 minutes of hypoxia in a serum‐free medium. Late preconditioning induced the nuclear translocation of HIF‐1 and the expression of carbonic anhydrase IX (CAIX), a HIF‐1–regulated transmembrane enzyme that catalyzes bicarbonate production. Such effects were associated with prevention of hepatocyte killing by hypoxia and the amelioration of intracellular acidosis and Na+ accumulation. The inhibition of PKC‐mediated and PI3‐kinase–mediated signals with, respectively, chelerythrine and wortmannin abolished HIF‐1 activation and blocked both CAIX expression and the protective action of late preconditioning. CAIX expression was also prevented by interfering with the transcriptional activity of HIF‐1 using a dominant negative HIF‐1β subunit. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium‐4‐acetamido‐4′‐isothiocyanato‐stilben‐2,2′‐disulfonate also reverted the protective effects of late preconditioning on intracellular acidosis and Na+ accumulation. Conclusion: The stimulation of adenosine A2A receptors induced late preconditioning in liver cells through the activation of HIF‐1. HIF‐1–induced expression of CAIX increases hepatocyte tolerance to ischemia by maintaining intracellular Na+ homeostasis. These observations along with the importance of HIF‐1 in regulating cell survival indicates HIF‐1 activation as a possible key event in liver protection by late preconditioning. (HEPATOLOGY 2008.)

The role of iron in anthracycline cardiotoxicity

The clinical use of the antitumor anthracycline Doxorubicin is limited by the risk of severe cardiotoxicity. The mechanisms underlying anthracycline-dependent cardiotoxicity are multiple and remain uncompletely understood, but many observations indicate that interactions with cellular iron metabolism are important. Convincing evidence showing that iron plays a role in Doxorubicin cardiotoxicity is provided by the protecting efficacy of iron chelation in patients and experimental models, and studies showing that iron overload exacerbates the cardiotoxic effects of the drug, but the underlying molecular mechanisms remain to be completely characterized. Since anthracyclines generate reactive oxygen species, increased iron-catalyzed formation of free radicals appears an obvious explanation for the aggravating role of iron in Doxorubicin cardiotoxicity, but antioxidants did not offer protection in clinical settings. Moreover, how the interaction between reactive oxygen species and iron damages heart cells exposed to Doxorubicin is still unclear. This review discusses the pathogenic role of the disruption of iron homeostasis in Doxorubicin-mediated cardiotoxicity in the context of current and future pharmacologic approaches to cardioprotection.

Erythropoietin's inhibiting impact on hepcidin expression occurs indirectly

Under conditions of accelerated erythropoiesis, elevated erythropoietin (Epo) levels are associated with inhibition of hepcidin synthesis, a response that ultimately increases iron availability to meet the enhanced iron needs of erythropoietic cells. In the search for erythroid regulators of hepcidin, many candidates have been proposed, including Epo itself. We aimed to test whether direct interaction between Epo and the liver is required to regulate hepcidin. We found that prolonged administration of high doses of Epo in mice leads to great inhibition of liver hepcidin mRNA levels, and concomitant induction of the hepcidin inhibitor erythroferrone (ERFE). Epo treatment also resulted in liver iron mobilization, mediated by increased ferroportin activity and accompanied by reduced ferritin levels and increased TfR1 expression. The same inhibitory effect was observed in mice that do not express the homodimeric Epo receptor (EpoR) in liver cells because EpoR expression is restricted to erythroid cells. Similarly, liver signaling pathways involved in hepcidin regulation were not influenced by the presence or absence of hepatic EpoR. Moreover, Epo analogs, possibly interacting with the postulated heterodimeric β common EpoR, did not affect hepcidin expression. These findings were supported by the lack of inhibition on hepcidin found in hepatoma cells exposed to various concentrations of Epo for different periods of times. Our results demonstrate that hepcidin suppression does not require the direct binding of Epo to its liver receptors and rather suggest that the role of Epo is to stimulate the synthesis of the erythroid regulator ERFE in erythroblasts, which ultimately downregulates hepcidin.

Indirect down‐regulation of nuclear NF‐κB levels by cobalamin in the spinal cord and liver of the rat

We used electrophoretic mobility shift assays to investigate the effects of cobalamin (Cbl) deficiency on the levels of activated nuclear factor‐kappa B (NF‐κB) in the spinal cords (SCs) and livers of rats made Cbl‐deficient (Cbl‐D) by total gastrectomy or a Cbl‐D diet. We chose the SC and liver because they are severely or scarcely affected, respectively, by Cbl deficiency in terms of histological damage. We found permanently increased NF‐κB levels (particularly the p50 and p65 subunits) in the SCs and livers of both types of Cbl‐D rats, and Western blot analysis demonstrated increased p65 levels. NF‐κB and p65 protein levels normalized when the totally gastrectomized (TGX) rats were treated with Cbl replacement. As we have previously demonstrated that Cbl deficiency increases tumor necrosis factor (TNF)–α and nerve growth factor (NGF) levels in the SC (each of which is a known NF‐κB activator), we redetermined NF‐κB levels in the SCs and livers of TGX rats treated with anti‐TNF‐α or anti‐NGF antibodies and found that NF‐κB levels normalized in both tissues after either treatment. These results demonstrate that: (1) Cbl physiologically and indirectly down‐regulates NF‐κB levels in rat SC and liver, and (2) NF‐κB is an important signaling molecule after Cbl deficiency injury.

Iron-Induced Damage in Cardiomyopathy: Oxidative-Dependent and Independent Mechanisms

The high incidence of cardiomyopathy in patients with hemosiderosis, particularly in transfusional iron overload, strongly indicates that iron accumulation in the heart plays a major role in the process leading to heart failure. In this context, iron-mediated generation of noxious reactive oxygen species is believed to be the most important pathogenetic mechanism determining cardiomyocyte damage, the initiating event of a pathologic progression involving apoptosis, fibrosis, and ultimately cardiac dysfunction. However, recent findings suggest that additional mechanisms involving subcellular organelles and inflammatory mediators are important factors in the development of this disease. Moreover, excess iron can amplify the cardiotoxic effect of other agents or events. Finally, subcellular misdistribution of iron within cardiomyocytes may represent an additional pathway leading to cardiac injury. Recent advances in imaging techniques and chelators development remarkably improved cardiac iron overload detection and treatment, respectively. However, increased understanding of the pathogenic mechanisms of iron overload cardiomyopathy is needed to pave the way for the development of improved therapeutic strategies.

Liver iron modulates hepcidin expression during chronically elevated erythropoiesis in mice

The liver‐derived peptide hepcidin controls the balance between iron demand and iron supply. By inhibiting the iron export activity of ferroportin, hepcidin modulates iron absorption and delivery from the bodys stores. The regulation of hepcidin, however, is not completely understood and includes a variety of different signals. We studied iron metabolism and hepcidin expression in mice constitutively overexpressing erythropoietin (Epo) (Tg6 mice), which leads to excessive erythropoiesis. We observed a very strong down‐regulation of hepcidin in Tg6 mice that was accompanied by a strong increase in duodenal expression of ferroportin and divalent metal tranporter‐1, as well as enhanced duodenal iron absorption. Despite these compensatory mechanisms, Tg6 mice displayed marked circulating iron deficiency and low levels of iron in liver, spleen, and muscle. To elucidate the primary signal affecting hepcidin expression during chronically elevated erythropoiesis, we increased iron availability by either providing iron (thus further increasing the hematocrit) or reducing erythropoiesis‐dependent iron consumption by means of splenectomy. Both treatments increased liver iron and up‐regulated hepcidin expression and the BMP6/SMAD pathway despite continuously high plasma Epo levels and sustained erythropoiesis. This suggests that hepcidin expression is not controlled by erythropoietic signals directly in this setting. Rather, these results indicate that iron consumption for erythropoiesis modulates liver iron content, and ultimately BMP6 and hepcidin. Analysis of the BMP6/SMAD pathway targets showed that inhibitor of DNA binding 1 (ID1) and SMAD7, but not transmembrane serine protease 6 (TMPRSS6), were up‐regulated by increased iron availability and thus may be involved in setting the upper limit of hepcidin. Conclusion: We provide evidence that under conditions of excessive and effective erythropoiesis, liver iron regulates hepcidin expression through the BMP6/SMAD pathway. (Hepatology 2013; 58:2122–2132)

Dual Role of ROS as Signal and Stress Agents: Iron Tips the Balance in favor of Toxic Effects.

Iron is essential for life, while also being potentially harmful. Therefore, its level is strictly monitored and complex pathways have evolved to keep iron safely bound to transport or storage proteins, thereby maintaining homeostasis at the cellular and systemic levels. These sequestration mechanisms ensure that mildly reactive oxygen species like anion superoxide and hydrogen peroxide, which are continuously generated in cells living under aerobic conditions, keep their physiologic role in cell signaling while escaping iron-catalyzed transformation in the highly toxic hydroxyl radical. In this review, we describe the multifaceted systems regulating cellular and body iron homeostasis and discuss how altered iron balance may lead to oxidative damage in some pathophysiological settings.

Cobalamin deficiency-induced down-regulation of p75-immunoreactive cell levels in rat central nervous system

We investigated immunoreactivity for p75 neurotrophin receptor (NTR) in the spinal cord white matter and septum of rats made cobalamin-deficient (Cbl-D) by means of total gastrectomy or a Cbl-D diet. Cbl deficiency down-regulates p75NTR-immunoreactive cell levels in spinal cord white matter and septum with different time courses. On the whole, the spinal cord white matter seems to be more affected in terms of p75NTR-immunoreactive cells, most of which are astrocytes. The p75NTR-immunoreactive cell levels in the spinal cord white matter and septum normalized in rats treated with Cbl (scheme b) and killed 4 months after total gastrectomy. However, Western blot analysis of p75NTR in the spinal cords of Cbl-D rats shows increased p75NTR protein levels, which are resistant to Cbl replacement. These findings demonstrate that a neurotrophic vitamin (Cbl) positively regulates the levels of a neurotrophic receptor (p75NTR) (at least in terms of immunohistochemistry) in rat central nervous system, although the underlying mechanism(s) are still unknown.