Accelerated CCl4-Induced Liver Fibrosis in Hjv-/- Mice, Associated with an Oxidative Burst and Precocious Profibrogenic Gene Expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl4. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis

Tumorigenic Properties of Iron Regulatory Protein 2 (IRP2) Mediated by Its Specific 73-Amino Acids Insert

Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2Δ73 (lacking a specific insert of 73 amino acids), were injected subcutaneously into nude mice. The induction of IRP2 profoundly stimulated the growth of tumor xenografts, and this response was blunted by addition of tetracycline in the drinking water of the animals, to turnoff the IRP2 transgene. Interestingly, IRP2Δ73 failed to promote tumor growth above control levels. As expected, xenografts expressing the IRP2 transgene exhibited high levels of transferrin receptor 1 (TfR1); however, the expression of other known IRP targets was not affected. Moreover, these xenografts manifested increased c-MYC levels and ERK1/2 phosphorylation. A microarray analysis identified distinct gene expression patterns between control and tumors containing IRP2 or IRP1 transgenes. By contrast, gene expression profiles of control and IRP2Δ73-related tumors were more similar, consistently with their growth phenotype. Collectively, these data demonstrate an apparent pro-oncogenic activity of IRP2 that depends on its specific 73 amino acids insert, and provide further evidence for a link between IRPs and cancer biology

Divergent modulation of iron regulatory proteins and ferritin biosynthesis by hypoxia/reoxygenation in neurons and glial cells

Il ferro è il metallo di transizione più abbondante nel sistema nervoso centrale in quanto cofattore di enzimi eminici e non-eminici coinvolti nel metabolismo energetico cellulare. Questo metallo è, inoltre, coinvolto nella sintesi di numerosi neurotrasmettitori, nella produzione della mielina e nello sviluppo delle ramificazioni dendritiche. Tuttavia, quando presente in eccesso, il ferro può catalizzare la generazione di specie radicaliche reattive dell’ossigeno (ROS) capaci di provocare danni irreversibili al tessuto cerebrale. La ferritina, la principale proteina di deposito del ferro, svolge un’importante funzione citoprotettiva limitando la produzione dei ROS indotta dal ferro. L’espressione di questa proteina è regolata principalmente a livello post-trascrizionale dall’azione di due proteine citosoliche, IRP1 ed IRP2 (Iron Regulatory Proteins), capaci di legare specifiche sequenze nucleotidiche, note come IREs (Iron responsiva Elements), localizzate nella regione 5’-UTR dei messaggeri della ferritina. Considerando la notevole vulnerabilità delle cellule cerebrali allo stress ossidativo indotto dal ferro, importante causa di diverse patologie neurodegenerative, scopo del lavoro della tesi di Dottorato è stato quello di analizzare, in cellule di origine cerebrale, i meccanismi molecolari che controllano la biosintesi della ferritina e l’eventuale effetto protettivo di questa proteina in risposta ai danni indotti dall’ipossia e dalla successiva riossigenazione. I risultati ottenuti mostrano che l’ipossia riduce sensibilmente l’attività di RNA-binding di IRP1 nelle cellule gliali (astrociti di tipo-1 e cellule di glioma), laddove tale attività risulta incrementata nelle cellule neuronali (neuroni corticali). In tutte le colture cellulari analizzate questi effetti risultano revertiti dalla successiva fase di riossigenazione (3 e 24 h). Inoltre, nelle cellule gliali si osserva un rapido incremento della sintesi di ferritina già durante la fase di ipossia, mentre nei neuroni la sintesi di questa proteina incrementa tardivamente, solo nell’ultima fase di riossigenazione (24 h). L’analisi dei livelli degli mRNA codificanti per la ferritina suggerisce che la sintesi di questa proteina è regolata nei neuroni corticali, principalmente a livello trascrizionale, mentre nelle cellule gliali è controllata sia a livello trascrizionale che post-trascrizionale. La diversa regolazione dell’espressione della ferritina potrebbe spiegare la maggiore vulnerabilità dei neuroni corticali al danno ipossico rispetto alle cellule gliali. Infatti, l’esposizione dei neuroni corticali all’ipossia/riossigenazione determina una marcata compromissione dell’attività ossido-reduttiva mitocondriale, una notevole perossidazione lipidica ed l’attivazione dei principali marcatori dell’apoptosi. Nelle stesse condizioni sperimentali, le cellule gliali non mostrano alcuna apprezzabile variazione della vitalità cellulare, nè perossidazione lipidica né attivazione dell’apoptosi. La notevole vulnerabilità delle cellule neuronali al danno indotto dall’ipossia risulta significativamente attenuata dal pre-trattamento delle colture neuronali con apoferritina esogena. Questo risultato conferma il ruolo citoprotettivo esercitato dalla ferritina nella risposta cellulare alle condizioni di ridotta disponibilità di ossigeno

Induction of ferritin expression by oxalomalate

Ferritin is a ubiquitous protein required for intracellular iron storage; its biosynthesis is mainly regulated by iron-regulatory proteins (IRP1 and IRP2) at post-transcriptional level. This regulation prevents iron excess from promoting the formation of reactive oxygen species (ROS). IRP1 is regulated by such factors as intracellular iron levels, the oxidants H2O2 and NO. We recently demonstrated that oxalomalate (OMA, α-hydroxy-β-oxalosuccinic acid), a competitive inhibitor of aconitase, which is an enzyme of the citric acid cycle, remarkably decreases the binding activity of IRP1. The aim of the present study was to investigate whether this molecule could affect the expression of ferritin. The RNA-binding activity of IRP1, evaluated by gel retardation assay, decreased after treatment of several cell lines with 5 mM OMA, with a maximal decrease of about 3-fold after 6 h. This effect remained almost constant up to 48 h after which it returned to basal levels. Intracellular ferritin levels, determined by Western blot analysis, increased in correlation with the OMA-induced decrease of IRP1 binding activity. Furthermore, treatment of cells with OMA caused a rise in ferritin mRNA levels. Interestingly, in cells exposed to iron challenge, OMA-induced overexpression of ferritin prevented formation of ROS and cellular lipid peroxidation. These data show that an inhibitor of aconitase, OMA, besides being involved in energetic metabolism, is able to control ferritin expression, probably through molecular mechanisms of either post-transcriptional regulation or transcriptional modulation, with advantageous consequences for the cell

Induction of H-ferritin synthesis by oxalomalate is regulated at both the transcriptional and post-transcriptional levels

Ferritin gene expression is complex and is controlled at transcriptional level in response to a variety of stimuli such as hormones, cytokines and cAMP. Iron, hemin and several compounds, chemically different, also activate the transcription of the ferritin gene. Ferritin biosynthesis is mainly regulated at post-transcriptional level by iron regulatory proteins (IRP1 and IRP2). We previously reported that oxalomalate, a competitive inhibitor of aconitase, remarkably decreases the IRP1 RNA-binding activity and induces a significant increase of ferritin expression. Here, we examined in cells cultured in presence of OMA the IRP1 intracellular content, ferritin biosynthesis and the transcriptional efficiency of H-ferritin gene promoter. Our results demonstrate a peculiar role of OMA that rapidly inactivates IRP1 without affecting IRP1 protein content and subsequently activates H-ferritin gene transcription leading to an overall increase of ferritin biosynthesis. We conclude that OMA regulates H-ferritin biosynthesis acting early at the post-transcriptional level and later on at transcriptional level

Oxygenated cembranoids of the decaryiol type from the Indonesian soft coral Lobophytum sp.

Three novel cembrane diterpenoids, decaryiols B-D (5-7), characterized by a bicyclic skeleton of the decaryiol-type, have been isolated from the Indonesian soft coral Lobophytum sp., along with three known cembranoids. The stereostructures of these metabolites have been established through extensive NMR spectroscopic analysis, application of the modified Mosher method, and chemical conversion. Cembranoids obtained from Lobophytum sp. (2-7) and six semisynthetic derivatives (9-14) prepared from decaryiol were tested for cell growth inhibitory activity against three different cell lines. O-Methyl decaryiol (10) exhibited a significant and selective activity against glioma cell lines