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Chiara VECCHI

Personale tecnico amministrativo
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

2023 - CREB-H is a stress-regulator of hepcidin gene expression during early postnatal development [Articolo su rivista]
Vecchi, C.; Montosi, G.; Garuti, C.; Canali, S.; Sabelli, M.; Bergamini, E.; Ricci, A.; Buzzetti, E.; Corradini, E.; Pietrangelo, A.
abstract

Hepcidin, the hepatic iron hormone, is the central regulator of iron homeostasis. Cyclic AMP-Responsive Element-Binding protein 3-like 3 (CREB3L3/CREB-H) is a liver homeostatic regulator of essential nutrients (i.e. glucose and lipids) and has been previously involved in hepcidin response to pathologic stress signals. Here, we asked whether CREB-H has also a physiologic role in iron homeostasis through hepcidin. To this end, we analyzed hepcidin gene expression and regulation in the liver of wild type and Creb3l3 knockout mice during early postnatal development, as a model of "physiologic" stressful condition. The effect of iron challenge in vivo and BMP6 stimulation in vitro have been also addressed. In addition, we investigated the BMP signaling pathway and hepcidin promoter activity following CREB3L3 silencing and hepcidin promoter mutation in HepG2 cells. Creb3l3 knockout suckling and young-adult mice showed a prominent serum and hepatic iron accumulation, respectively, due to impaired hepcidin mRNA expression which progressively returned to normal level in adult mice. Interestingly, upon iron challenge, while the upstream BMP/SMAD signaling pathway controlling hepcidin was equally responsive in both strains, hepcidin gene expression was impaired in knockout mice and more iron accumulated in the liver. Accordingly, hepcidin gene response to BMP6 was blunted in primary CREB-H knockout hepatocytes and in HepG2 cells transfected with CREB-H siRNA or carrying a hepcidin promoter mutated in the CREB-H binding site. In conclusion, CREB-H has a role in maintaining the homeostatic balance of iron traffic through hepcidin during the critical postnatal period and in response to iron challenge.Key messagesCREB-H KO mice develop liver iron overload shortly after weaning that normalizes in adulthood.CHEB-H is involved in hepcidin gene response to oral iron in vivo.CREB-H loss hampers hepcidin promoter response to BMP6.CREB-H is a key stress-sensor controlling hepcidin gene transcription in physiologic and pathophysiologic states.

2016 - The SMAD pathway is required for hepcidin response during endoplasmic reticulum stress [Articolo su rivista]
Canali, Susanna; Vecchi, Chiara; Garuti, Cinzia; Montosi, Giuliana; Babitt, Jodie L; Pietrangelo, Antonello
abstract

Hepcidin, the iron hormone, is regulated by a number of stimulatory and inhibitory signals. The cAMP responsive element binding protein 3-like 3, CREB3L3, mediates hepcidin response to endoplasmic reticulum (ER) stress. In this study we asked whether hepcidin response to ER stress also requires the SMAD1/5/8 pathway that has a major role in hepcidin regulation in response to iron and other stimuli. We analyzed hepcidin mRNA expression and promoter activity in response to ER stressors in HepG2 cells in the presence of the BMP type I receptor inhibitor LDN-193189, mutated hepcidin promoter or siRNA against different SMAD proteins. We then used a similar approach in vivo in wild-type, Smad1/5 or Creb3l3 -/- animals undergoing ER stress. In vitro, LDN-193189 prevented hepcidin mRNA induction by different ER stressors. Seemingly, mutation of a BMP-responsive element in the hepcidin promoter prevented ER stress-mediated upregulation. Moreover, in vitro silencing of SMAD proteins by siRNA, in particular SMAD5, blunted hepcidin response to ER stress. On the contrary, hepcidin induction by ER stress was maintained when using antibodies against canonical BMP receptor ligands. In vivo, hepcidin was induced by ER stress and prevented by LDN-193189. In addition, in Smad1/5 knock-out mice, ER stress was unable to induce hepcidin expression. Finally, in Creb3l3 knock-out mice, in response to ER stress, SMAD1/5 were correctly phosphorylated and hepcidin induction was still appreciable, although to a lesser extent as compared to control mice. In conclusion, our study indicates that hepcidin induction by ER stress involves the central regulatory SMAD1/5 pathway.

2014 - GLUCONEOGENIC SIGNALS DIRECTLY CONTROL IRON HOMEOSTASIS THROUGH HEPCIDIN [Abstract in Atti di Convegno]
Vecchi, Chiara; Montosi, Giuliana; Garuti, Cinzia; Corradini, Elena; Sabelli, Manuela; Qian, J; Liu, C; Canali, S; Pietrangelo, Antonello
abstract

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2014 - Gluconeogenic Signals Regulate Iron Homeostasis via Hepcidin in Mice. [Articolo su rivista]
Vecchi, Chiara; Montosi, Giuliana; Garuti, Cinzia; Corradini, Elena; Sabelli, Manuela; Canali, Susanna; Pietrangelo, Antonello
abstract

Hepatic gluconeogenesis provides fuel during starvation, and is abnormally induced in obese individuals or those with diabetes. Common metabolic disorders associated with active gluconeogenesis and insulin resistance (obesity, metabolic syndrome, diabetes, and nonalcoholic fatty liver disease) have been associated with alterations in iron homeostasis that disrupt insulin sensitivity and promote disease progression. We investigated whether gluconeogenic signals directly control Hepcidin, an important regulator of iron homeostasis, in starving mice (a model of persistently activated gluconeogenesis and insulin resistance).|We investigated hepatic regulation of Hepcidin expression in C57BL/6Crl, 129S2/SvPas, BALB/c, and wild-type and Creb3l3-/- null mice. Mice were fed a standard, iron-balanced chow diet or an iron-deficient diet for 9 days before death, or for 7 days before a 24- to 48-hour starvation period; liver and spleen tissues then were collected and analyzed by quantitative reverse-transcription polymerase chain reaction and immunoblot analyses. Serum levels of iron, hemoglobin, Hepcidin, and glucose also were measured. We analyzed human hepatoma (HepG2) cells and mouse primary hepatocytes to study transcriptional control of Hamp (the gene that encodes Hepcidin) in response to gluconeogenic stimuli using small interfering RNA, luciferase promoter, and chromatin immunoprecipitation analyses.|Starvation led to increased transcription of encodes phosphoenolpyruvate carboxykinase 1 (a protein involved in gluconeogenesis) in livers of mice, increased levels of Hepcidin, and degradation of Ferroportin, compared with nonstarved mice. These changes resulted in hypoferremia and iron retention in liver tissue. Livers of starved mice also had increased levels of Ppargc1a messenger RNA and Creb3l3 messenger RNA, which encode a transcriptional co-activator involved in energy metabolism and a liverspecific transcription factor, respectively. Glucagon and a cyclic adenosine monophosphate analog increased promoter activity and transcription of Hamp in cultured liver cells; levels of Hamp were reduced after administration of small interfering RNAs against Ppargc1a and Creb3l3. PPARGC1A and CREB3L3 bound the Hamp promoter to activate its transcription in response to a cyclic adenosine monophosphate analog. Creb3l3-/- mice did not up-regulate Hamp or become hypoferremic during starvation.|We identified a link between glucose and iron homeostasis, showing that Hepcidin is a gluconeogenic sensor in mice during starvation. This response is involved in hepatic metabolic adaptation to increased energy demands; it preserves tissue iron for vital activities during food withdrawal, but can cause excessive iron retention and hypoferremia in disorders with persistently activated gluconeogenesis and insulin resistance.

2014 - Hypoxia induced downregulation of hepcidin is mediated by platelet derived growth factor BB [Articolo su rivista]
T., Sonnweber; D., Nachbaur; A., Schroll; M., Nairz; M., Seifert; E., Demetz; D., Haschka; A. M., Mitterstiller; A., Kleinsasser; M., Burtscher; S., Trubsbach; A. T., Murphy; V., Wroblewski; D. R., Witcher; K., Mleczko Sanecka; Vecchi, Chiara; M. U., Muckenthaler; Pietrangelo, Antonello; I., Theurl; G., Weiss
abstract

OBJECTIVE: Hypoxia affects body iron homeostasis; however, the underlying mechanisms are incompletely understood. DESIGN: Using a standardised hypoxia chamber, 23 healthy volunteers were subjected to hypoxic conditions, equivalent to an altitude of 5600 m, for 6 h. Subsequent experiments were performed in C57BL/6 mice, CREB-H knockout mice, primary hepatocytes and HepG2 cells. RESULTS: Exposure of subjects to hypoxia resulted in a significant decrease of serum levels of the master regulator of iron homeostasis hepcidin and elevated concentrations of platelet derived growth factor (PDGF)-BB. Using correlation analysis, we identified PDGF-BB to be associated with hypoxia mediated hepcidin repression in humans. We then exposed mice to hypoxia using a standardised chamber and observed downregulation of hepatic hepcidin mRNA expression that was paralleled by elevated serum PDGF-BB protein concentrations and higher serum iron levels as compared with mice housed under normoxic conditions. PDGF-BB treatment in vitro and in vivo resulted in suppression of both steady state and BMP6 inducible hepcidin expression. Mechanistically, PDGF-BB inhibits hepcidin transcription by downregulating the protein expression of the transcription factors CREB and CREB-H, and pharmacological blockade or genetic ablation of these pathways abrogated the effects of PDGF-BB toward hepcidin expression. CONCLUSIONS: Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis.

2013 - THE BMP'S PATHWAY IN HEPCIDIN REGULATION DURING ENDOPLASMIC RETICULUM STRESS RESPONSE. [Abstract in Rivista]
Canali, Susanna; Vecchi, Chiara; Garuti, Cinzia; Pietrangelo, Antonello
abstract

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2010 - Huh-7: a human hemochromatotic cell line. [Articolo su rivista]
Vecchi, Chiara; Montosi, Giuliana; Pietrangelo, Antonello
abstract

Hereditary hemochromatosis (HC) is commonly associated with homozygosity for the cysteine-to-tyrosine substitution at position 282 (C282Y) of the HFE protein. This mutation prevents HFE from binding beta(2)-microglobulin (beta(2)M) and reaching the cell surface. We have discovered that a widely used hepatoma cell line, Huh-7, carries a HFE mutation similar to that associated with human HC. By HFE gene sequencing of Huh-7 genomic DNA, we found a TAC nucleotide deletion (c. 691_693del) responsible for loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This mutation affects a conserved hydrophobic region in a loop connecting two beta strands that make up the alpha3 domain of HFE, not far from the 282 site. HIE was detected by western blot in HepG2 but not in Huh-7 cell membrane fractions. In WRL-68 cells expressing wild-type HIE, the HIE protein was largely found at the plasma membrane where it colocalizes with beta(2)M. On the contrary, the HFE-Y231del mutant, similarly to an exogenously expressed HFE-C282Y mutant, failed to reach the plasma membrane and did not colocalize with membrane-expressed beta(2)M. C282Y mutant HFE in HC is associated with inadequate hepcidin expression. We found that Huh-7 cells display lower hepcidin messenger RNA levels as compared to HepG2 cells, which carry a wild-type HFE. Interestingly, hepcidin messenger RNA levels increased significantly in Huh-7 cells stably expressing exogenous wild-type HFE at the plasma membrane. Conclusion: Huh-7 cells may represent a novel and valuable tool to investigate the role of altered HFE traffic in iron metabolism and pathogenesis of human HIE HC. (HEPATOLOGY 2010;51:654-659.)

2009 - ER stress controls iron metabolism through induction of hepcidin. [Articolo su rivista]
Vecchi, Chiara; Montosi, Giuliana; K., Zhang; Lamberti, Igor; S. A., Duncan; R. J., Kaufman; Pietrangelo, Antonello
abstract

Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin overproduction causes anemia of inflammation, whereas its deficiency leads to hemochromatosis. Inflammation and iron are known extracellular stimuli for hepcidin expression. We found that endoplasmic reticulum (ER) stress also induces hepcidin expression and causes hypoferremia and spleen iron sequestration in mice. CREBH (cyclic AMP response element-binding protein H), an ER stress-activated transcription factor, binds to and transactivates the hepcidin promoter. Hepcidin induction in response to exogenously administered toxins or accumulation of unfolded protein in the ER is defective in CREBH knockout mice, indicating a role for CREBH in ER stress-regulated hepcidin expression. The regulation of hepcidin by ER stress links the intracellular response involved in protein quality control to innate immunity and iron homeostasis.

2008 - Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription [Articolo su rivista]
Nishina, S.; Hino, K.; Korenaga, M.; Vecchi, Chiara; Pietrangelo, Antonello; Mizukami, Y.; Furutani, T.; Sakai, A.; Okuda, M.; Hidaka, I.; Okita, K.; Sakaida, I.
abstract

Background & Aims: Despite abundant clinical evidence, the mechanisms by which hepatic iron overload develops in patients with hepatitis C virus (HCV)-associated chronic liver disease remain unknown. The aim of this study was to investigate how hepatic iron overload develops in the presence of HCV proteins. Methods: Male transgenic mice expressing the HCV polyprotein and nontransgenic control mice (C57BL/6) were assessed for iron concentrations in the liver, spleen, and serum and iron regulatory molecules in vivo and ex vivo. Results: Transgenic mice had increased hepatic and serum iron concentrations, decreased splenic iron concentration, and lower hepcidin expression in the liver accompanied by higher expression of ferroportin in the duodenum, spleen, and liver. In response to hepatocellular iron excess, transferrin receptor 1 expression decreased and ferritin expression increased in the transgenic liver. Transgenic mice showed no inflammation in the liver but preserved the ability to induce hepcidin in response to proinflammatory cytokines induced by lipopolysaccharide. Hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBP) were down-regulated concomitant with increased expression of C/EBP homology protein, an inhibitor of C/EBP DNA binding activity, and with increased levels of reactive oxygen species in transgenic mice at the ages of 8 and 14 months. Conclusions: HCV-induced reactive oxygen species may down-regulate hepcidin transcription through inhibition of C/EBP alpha DNA binding activity by C/EBP homology protein, which in turn leads to increased duodenal iron transport and macrophage iron release, causing hepatic iron accumulation.

2005 - Kupffer cells and macrophages are not required for hepatic hepcidin activation during iron overload [Articolo su rivista]
Montosi, Giuliana; Corradini, Elena; Garuti, Cinzia; S., Barelli; S., Recalcati; G., Cairo; L., Valli; Pignatti, Elisa; Vecchi, Chiara; F., Ferrara; Pietrangelo, Antonello
abstract

Hepcidin, the iron hormone, is produced by the liver in response to iron and inflammation. Its synthesis during inflammation is triggered by cytokines, but the details of iron activation are obscure. We tested the role of Kupffer cells and macrophages by studying iron-loaded or inflamed mice with selective inactivation of Kupffer cells or the in vitro effect of conditioned human macrophages on hepcidin expression. Hepcidin messenger RNA (mRNA) expression was studied by Northern blot and reverse transcriptase polymerase chain reaction analysis in mice that were treated with 40 mg/kg gadolinium (III) chloride (GdCl3) as a Kupffer cell inactivating agent and subjected to inflammatory challenges with either lipopolysaccharide (LPS) and turpentine or iron overload by iron-dextran administration. Similar analyses were performed in human hepatoma cells (HepG2) cultured with medium from LPS- or iron-conditioned macrophages from blood donors or patients with HFE-linked hereditary hemochromatosis (HH). In vivo, LPS and particularly turpentine stimulated hepcidin mRNA expression, and this effect was prevented by the inactivation of Kupffer cells. Also, iron overload markedly upregulated hepatic hepcidin mRNA, but this activity persisted in spite of Kupffer cell blockade. In vitro, the medium of LPS-treated normal or hemocromatotic macrophages turned on hepcidin expression. On the contrary, medium of iron-manipulated macrophages, regardless of their HFE status, did not affect hepcidin mRNA steady-state levels. In conclusion, Kupffer cells are required for the activation of hepcidin synthesis during inflammation, and HH inflamed macrophages are capable of mounting a normal response, eventually leading to hepcidin stimulation. However, both Kupffer cells and human macrophages are dispensable for the regulatory activity exerted by iron on hepatic hepcidin.

2003 - Dynamic recruitment of NF-Y and histone acetyltransferases on cell-cycle promoters [Articolo su rivista]
Caretti, G; Salsi, Valentina; Vecchi, Chiara; Imbriano, Carol; Mantovani, Roberto
abstract

Regulation of transcription during the cell-cycle is under the control of E2 factors (E2Fs), often in cooperation with nuclear factor Y (NF-Y), a histone-like CCAAT-binding trimer. NF-Y is paradigmatic of a constitutive, ubiquitous factor that pre-sets the promoter architecture for other regulatory proteins to access it. We analyzed the recruitment of NF-Y, E2F1/4/6, histone acetyltransferases, and histone deacetylase (HDAC) 1/3/4 to several cell-cycle promoters by chromatin immunoprecipitation assays in serum-starved and restimulated NIH3T3 cells. NF-Y binding is not constitutive but timely regulated in all promoters tested, being displaced when promoters are repressed. p300 association correlates with activation, and it is never found in the absence of NF-Y, whereas PCAF/hGCN5 is often found before NF-Y association. E2F4 and E2F6, together with HDACs, are bound to repressed promoters, including the G(2)/M Cyclin B2. As expected, an inverse relationship between HDACs association and histones H3/H4 acetylation is observed. Blocking cells in G(1) with the cyclin-dependent kinase 2 inhibitor R-roscovitine confirms that NF-Y is bound to G(1)/S but not to G(2)/M promoters in G(1). These data indicate that following the release of E2Fs/HDACs, a hierarchy of PCAF-NF-Y-p300 interactions and H3-H4 acetylations are required for activation of cell-cycle promoters.