Aldo Bernelli-Zazzera

Transferrin Receptor Induction by Hypoxia HIF-1-MEDIATED TRANSCRIPTIONAL ACTIVATION AND CELL-SPECIFIC POST-TRANSCRIPTIONAL REGULATION

The tight relationship between oxygen and iron prompted us to investigate whether the expression of transferrin receptor (TfR), which mediates cellular iron uptake, is regulated by hypoxia. In Hep3B human hepatoma cells incubated in 1% O2 or treated with CoCl2, which mimics hypoxia, we detected a 3-fold increase of TfR mRNA despite a decrease of iron regulatory proteins activity. Increased expression resulted from a 4-fold stimulation of the nuclear transcription rate of the TfR gene by both hypoxia and CoCl2. A role for hypoxia-inducible factor (HIF-1), which activates transcription by binding to hypoxia-responsive elements in the activation of TfR, stems from the following observations. (a) Hypoxia and CoCl2-dependent expression of luciferase reporter gene in transiently transfected Hep3B cells was mediated by a fragment of the human TfR promoter containing a putative hypoxia-responsive element sequence, (b) mutation of this sequence prevented hypoxic stimulation of luciferase activity, (c) binding to this sequence of HIF-1α, identified by competition experiments and supershift assays, was induced in Hep3B cells by hypoxia and CoCl2. In erythroid K562 cells, the same treatments did not affect iron regulatory proteins activity, thus resulting in a stimulation of TfR gene expression higher than in hepatoma cells.

Multiple mechanisms of iron-induced ferritin synthesis in HeLa cells

Iron administration to HeLa cells stimulates the accumulation of H-subunit and L-subunit rich isoferritins at similar extent. The increase in both types of isoferritins is accompanied by an increase in the amount of messenger RNAs specific for H and L subunits. The increase in the amount of these messenger RNAs, which occurs in the nucleus as well as in the cytoplasm, is proportionately lower than the increase in the protein. These results, together with analysis of transcription in isolated nuclei indicate the existence of a mechanism of transcriptional control of ferritin synthesis, associated to the translational control described so far.

Adriamycin: Energy metabolism and mitochondrial oxidations in the heart of treated rabbits

Abstract Heart mitochondria isolated from rabbits subjected to intermittent treatment with adriamycin show a reduced respiratory control, resulting from increase in state 4b oxidation. Continuous daily treatment causes an impairment of respiratory control which is more severe, is due both to increase of state 4b and decrease of state 3 oxygen uptake and occurs after a total amount of drug which does not produce appreciable effects with the intermittent schedule of treatment: however, these changes disappear within 2 weeks from the interruption of the treatment. Mitochondria isolated from adriamycin-treated rabbits show constantly increased permeability to the addition of NADH. On the contrary the ADP/O ratio measured in vitro is essentially unchanged: the same happens with the tissue contents of ATP, ADP, AMP and the metabolites chosen to estimate cytoplasmic and mitochondrial redox states and measured in quick-frozen hearts in vivo . The results are discussed in relation to the possible role of mitochondrial functional defects in the onset of adriamycin cardiomyopathy.

The MAP kinase cascades are activated during post-ischemic liver reperfusion

We have investigated the involvement of MAP kinase cascades in the response of the liver to post‐ischemic reperfusion. Both JNKs and ERKs are activated but the duration and magnitude of the increase in their activities appear to be different. JNK activation is more marked but shorter than that of ERKs. The increase observed in the phosphotyrosine content of the 52 kDa Shc protein, accompanied by an increased amount of co‐immunoprecipitated Grb2, and the activation of Raf‐1 kinase provide evidence of the involvement of a Ras‐Raf‐dependent pathway, with a time course that is similar to that of ERK activation. The treatment of rats with IL‐1 receptor antagonist modified all of the described effects, suggesting that IL‐1 plays a role in the response of the liver to reperfusion.

Induction of ferritin synthesis in ischemic-reperfused rat liver: Analysis of the molecular mechanisms

BACKGROUND & AIMS Iron may catalyze the production of reactive oxygen species (ROS) during postischemic reoxygenation. Ferritin, a cellular iron storage protein, can either represent a source of iron or perform a cytoprotective action against ROS. The aim of this study was to address the role of ferritin in postischemic reperfusion. METHODS Transcriptional and posttranscriptional mechanisms controlling ferritin gene expression were studied in reperfused rat livers. RESULTS Proteolysis reduced ferritin levels 2 hours after reperfusion, but a concomitant increase of synthesis, accompanied by enhanced transcription and accumulation of H and L ferritin subunit messenger RNAs (mRNAs), almost re-established normal ferritin content at 4 hours. Pretreatment with interleukin 1 receptor antagonist (IL-1RA) did not prevent the rise of ferritin mRNAs. RNA bandshift assays showed that the activity of the iron regulatory proteins (IRPs), which control ferritin mRNA translation, declined early after reperfusion and recovered progressively thereafter. Pretreatment with either the antioxidant N-acetyl cysteine or IL-1RA was sufficient to prevent almost completely down-regulation of IRP activity. CONCLUSIONS Postischemic reperfusion causes degradation of ferritin, possibly increasing iron levels. However, induction of ferritin gene transcription, possibly mediated by ferritin-derived iron and ROS-mediated inactivation of IRP, which allows translation of ferritin mRNAs, counteracts this effect and concurs to reestablish the amount of ferritin, which may thus act to limit reperfusion damage.

Some aspects of glycogen metabolism following reversible or irreversible liver ischemia

Abstract Some aspects of glycogen metabolism have been investigated in rat liver lobes that were subjected to pedicle ligation for different periods and then followed by reversible or irreversible cell damage according to the duration of ischemia. In fasted rats the “free” glycogen level diminished promptly after the onset of ischemia, but the depletion of liver glycogen was not significantly influenced by the duration of ischemia. The “free” glycogen content after ischemia decreased even in the livers of fed rats; but the amount of glycogen lost in fed animals was significantly influenced by the duration of ischemia. Two hours after the re-establishment of a normal blood supply, the oxygen uptake of liver slices, which was reduced immediately after ischemia, returned to normal if the period of ischemia did not exceed the limit of reversibility; but the liver glycogen level progressively decreased. The equation which describes the fall of glycogen is different according to the nutritional state of the animals. The alterations of the “residual” glycogen follow the pattern observed for the “free” fraction. The slices of ischemic livers practically lost the capacity to synthesize glycogen in vitro , even after very short periods of ischemia; in the case of fed rats, after 30 minutes ischemia or more, the concentration of glycogen in the slices at the end of incubation was less than the concentration at the beginning of the experiment. The meaning of this observation is discussed in relation to the mechanism of glycogen synthesis and degradation in liver cells. The rate of glycogen synthesis following re-establishment of normal blood supply depends both on the nutritional state of the animal and the duration of ischemia previously suffered by liver lobes. The results seem to emphasize that glycogen synthesis recovers better in the liver cells of fed animals than in the corresponding cells of fasted rats.

Differential activation of some transcription factors during rat liver ischemia, reperfusion, and heat shock

Cells respond to external stimuli by changes in gene expression that are largely dependent on transcription factors (TFs). We studied the behavior of some TFs in rat liver during ischemia, postischemic reperfusion, and heat shock. Knowledge of the conditions at the end of ischemia is essential to understand changes occurring at reperfusion. The TFs investigated are known to be typically responsive to heat shock (HSF), hypoxia (HIF‐1), pro‐ and antioxidant conditions (AP‐1), or to various environmental changes (HNF‐1 and ATF/CREB family). The most relevant new information includes the following: 1) Liver ischemia activates extremely rapidly the DNA binding capacity of HSF, soon followed by analogous activation of HIF‐1 and AP‐1. 2) After a certain lag time from the activation of HIF‐1, mRNAs accumulate for two glycolytic enzymes, in particular Aldolase A and Heme Oxygenase 1, which contain HIF‐1 sequences in their promoters. 3) Reperfusion, which is known to further increase the binding of HSF and to induce NFkB binding, abrogates or decreases the binding of HIF‐1 and AP‐1, stimulated by ischemia, and activates the binding of ATF/CREB. Later on, a second peak of AP‐1 binding is induced. 4) Heat shock activates both ischemia‐responsive and reperfusion‐responsive TFs. 5) Preliminary experiments of supergelshift reveal that the activation of AP‐1 at reperfusion or upon heat shock may result from the different involvement of the component subunits. J. Cell. Physiol. 180:255–262, 1999.

Activation of transcription factors by drugs inducing oxidative stress in rat liver.

Chemically induced oxidative stress of the liver associates with gene reprogramming and activation of some transcription factors (TFs), in particular nuclear factor-kappaB (NF-kappaB). We have now investigated other TFs, such as activator protein-1 (AP-1) and hypoxia inducible factor-1 (HIF-1) that we had shown to be activated in rat liver during heat shock, ischemia or post-ischemic reperfusion, and signal transducer and activator of transcription (STAT), CCAAT/enhancer binding protein (C/EBP) and hepatocyte nuclear factor-1 (HNF-1), which may be involved in the response of the liver to injury. The expression of target genes, containing consensus sequences for these TFs was assessed by Northern and Western blot analysis. The rats were treated with buthionine-sulfoximine, nitrofurantoin (NF) or phorone (Ph), which cause liver oxidative stress with different mechanisms. All these agents activated AP-1, known to depend on redox state, HIF-1, initially described as an hypoxia-responsive TF, and STAT3, generally connected to the response to cytokines. HNF-1 a constitutive TF associated with liver-specific gene expression was not affected. The composition of AP-1 was slightly different according to the drug used for treatment. The levels of the mRNAs for heme oxygenase-1 (HO-1), Aldolase A and alpha(1)-acid glycoprotein as well as the corresponding proteins increased after the treatments, thus, indicating that the activation of the TFs was functional. These observations suggest that the treatment of rats with drugs inducing oxidative stress causes a broad spectrum of changes in gene expression with features common to stresses generally considered as separate entities.

Oxidative stress induces a subset of heat shock proteins in rat hepatocytes and MH1C1 cells

Lipoperoxidative damage caused by exposure of isolated hepatocytes or cultivated hepatoma cells to ADP-iron or to 4-hydroxynonenal induces the synthesis of some proteins which are different under these two conditions but are always a subset of the proteins induced in each type of cells upon heat-shock (heat-shock proteins). For at least one of these proteins (hsp 31), induced by 4-hydroxynonenal, the increase is dose-dependent and the effect of heat and the chemical seems to be additive. Lipoperoxidation may be implicated in the induction of some of the heat shock proteins, but reproduces only incompletely the response of protein synthesis typical of heat-shock conditions.

Phosphorylation and redox states in ischemic liver

Abstract Metabolite concentrations, redox and phosphorylation states were studied in ischemic and postischemic rat livers; oxidative phosphorylation and related functional parameters also were investigated with mitochondria derived from these tissues. Time periods were chosen to assess the effects of short-duration (15 min), long-duration reversible (60 min), and necrogenic ischemia (120 min). The energy-charge of liver cells drops promptly and deeply within the first 15 min of ischemia, with a concurrent profound decrease of the [NAD + ] [NADH] ratio both in cytoplasm and mitochondria. Once affected by this initial change, redox and phosphorylation states do not vary appreciably by prolonging blood deprivation for 120 min. The functional capacity of isolated mitochondria declines steadily with increasing duration of ischemia and severe reductions—especially of the ADP O ratios—are attained only at the end of 120 min of ischemia. The reestablishment of the blood supply promotes a good recovery of the energy-charge and redox states (at a slightly different pace) only if previous ischemia did not last more than 60 min. Results with isolated mitochondria correlate well with chemical determinations carried out on postischemic tissue, but respiratory control index does not recover completely over the investigated period following reversible ischemia, thus indicating a less tight coupling of phosphorylation to oxidation during the repair of reversible cellular damage. The relationships between impaired mitochondrial functions, phosphorylation and redox states, and the relevance of their changes to the fate of ischemic cells are discussed.