Maria Agnese Della Fazia

Aryl hydrocarbon receptor control of a disease tolerance defence pathway

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

Oxidative stress activates a specific p53 transcriptional response that regulates cellular senescence and aging.

Oxidative stress is a determining factor of cellular senescence and aging and a potent inducer of the tumour‐suppressor p53. Resistance to oxidative stress correlates with delayed aging in mammals, in the absence of accelerated tumorigenesis, suggesting inactivation of selected p53‐downstream pathways. We investigated p53 regulation in mice carrying deletion of p66, a mutation that retards aging and confers cellular resistance and systemic resistance to oxidative stress. We identified a transcriptional network of ~200 genes that are repressed by p53 and encode for determinants of progression through mitosis or suppression of senescence. They are selectively down‐regulated in cultured fibroblasts after oxidative stress, and, in vivo, in proliferating tissues and during physiological aging. Selectivity is imposed by p66 expression and activation of p44/p53 (also named Delta40p53), a p53 isoform that accelerates aging and prevents mitosis after protein damage. p66 deletion retards aging and increases longevity of p44/p53 transgenic mice. Thus, oxidative stress activates a specific p53 transcriptional response, mediated by p44/p53 and p66, which regulates cellular senescence and aging.

Cyclic AMP signalling pathway and cellular proliferation: induction of CREM during liver regeneration

The CREM gene encodes both activators and repressors of cAMP-induced gene expression. An isoform of CREM encodes the powerful transcriptional repressor ICER (Inducible cAMP Early Repressor), which has been shown to be inducible by virtue of an alternative, intronic promoter. The CREM gene belongs to the early response class and displays a characteristic neuroendocrine cell- and tissue-specific expression. To date ICER inducibility has been described in non-replicating, terminally differentiated tissues. In this paper we document a robust induction of CREM expression in the regenerating rat liver after partial hepatectomy. This represents the first link of inducible CREM expression to the phenomenon of cellular proliferation. Furthermore, it represents the first example of transcriptional activation of a cAMP-responsive factor in the regenerating liver. This has significant physiological relevance since the adenylate cyclase signalling pathway is strongly implicated in liver regeneration. Finally, we show that the repressor ICER is inducible in the hepatoma cell line H35 upon activation of the adenylate cyclase and phosphorylation of the activator CREB.

Disruption of the gene encoding 3β‐hydroxysterol Δ14‐reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

Tm7sf2 gene encodes 3β‐hydroxysterol Δ14‐reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Δ14‐unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. The C‐terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Δ14‐reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3β‐hydroxysterol Δ14‐reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3β‐hydroxysterol Δ14‐reductase activity were detectable in liver microsomes of Tm7sf2(−/−) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2(−/−) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3β‐hydroxysterol Δ14‐reductase activity determined in liver nuclei showed comparable values in wild‐type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2(−/−) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell‐cycle progression are downregulated in the liver of Tm7sf2(−/−) mice, whereas genes involved in xenobiotic metabolism are upregulated.

Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box

TM7SF2 gene encodes 3beta-hydroxysterol Delta(14)-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoter-luciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in co-transfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Role of Innate Immune Receptors in Paradoxical Caspofungin Activity In Vivo in Preclinical Aspergillosis

ABSTRACT This study investigated the possible mechanisms underlying the paradoxical caspofungin activity in vivo in preclinical aspergillosis. We evaluated the activity of escalating doses of caspofungin in vivo in different preclinical models of invasive aspergillosis, including mice deficient for selected innate immune receptors. The therapeutic efficacy of caspofungin in experimental invasive aspergillosis was strictly dose dependent, being observed at doses of 0.1 and 1 mg/kg of body weight depending on the experimental models. Paradoxical increase in pulmonary fungal burden as well as inflammatory pathology was observed at the highest dose of caspofungin (5 mg/kg), occurred independently of the so-called Eagle effect and susceptibility to caspofungin in vitro, and was contingent upon the presence of TLR2, Dectin-1, and TLR9. Increased expression of Dectin-1 and TLR9 were observed upon exposure to caspofungin in vitro and in vivo. Together, these findings suggest that the net activity of caspofungin in vivo is orchestrated by the activation, directly or indirectly, of multiple innate immune receptors.

HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation

The liver has the ability to autonomously regulate growth and mass. Following partial hepatectomy, hormones, growth factors, cytokines and their coupled signal transduction pathways have been implicated in hepatocyte proliferation. To understand the mechanisms responsible for the proliferative response, we studied liver regeneration by characterization of novel genes that are activated in residual hepatocytes. A regenerating liver cDNA library screening was performed with cDNA-subtracted probes derived from regenerating and normal liver. Here, we describe the biology of Hops (for hepatocyte odd protein shuttling). HOPS is a novel shuttling protein that contains an ubiquitin-like domain, a putative NES and a proline-rich region. HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. Evidence shows that cAMP governs HOPS export in hepatocytes of normal and regenerating liver and is mediated via CRM-1. We demonstrate that HOPS binds to elongation factor eEF-1A and interferes in protein synthesis. HOPS overexpression in H-35-hepatoma and 3T3-NIH cells strongly reduces proliferation.

Differential expression of CD44 isoforms during liver regeneration in rats.

BACKGROUND CD44 is a transmembrane glycoprotein known to bind hyaluronic acid (HA). This molecule is a multifunctional cell surface glycoprotein involved in lymphocyte homing and activation, tumor growth and metastasis. We have investigated the qualitative modification of CD44 in the regenerating liver as a model for studying cellular proliferation in vivo. Molecules involved in cell adhesion and the extracellular matrix (ECM), which influence differentiation, growth, cell-cell interactions and cellular polarity, play an important role in the liver regeneration. We studied the modulation of CD44 gene expression and its post-transcriptional modifications, analyzing the expression of different isoforms containing exon v6 in the regenerating liver, in sham operated liver and in the hepatoma cells H-35. METHODS The expression of CD44 and CD44v6 were analyzed in RNA extracted from regenerating liver at different times after partial hepatectomy (PH), and H-35 hepatoma cells by Northern blot, RT-PCR and Southern blot, and in protein extracts from regenerating liver by Western blot. H-35 hepatoma cells were assayed with the antibody cross-linked technique with CD44 antibodies. RESULTS The standard CD44 form is expressed in regenerating liver and its levels were not modified following PH. However, our analysis revealed CD44 isoforms containing v6 in the first hours after PH as well as in the H-35 hepatoma cell line. H-35 cells treated with cross-linked anti-CD44 antibodies or HA show an increased rate of incorporation of [3H]thymidine (30 and 25%, respectively) with respect to the control. CONCLUSION These findings suggest that CD44 may play a role in the proliferation of residual hepatocytes following PH.

HOPS is an essential constituent of centrosome assembly

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.

Different expression of tyrosine aminotransferase and serine deydratase in rat livers after partial hepatectomy

Tyrosine aminotransferase activity in rat liver increases during the first 24 hrs after partial hepatectomy with two peaks, one at 10 hrs and another at 18 hrs. This behaviour is due to an increase in TATmRNA synthesis. Expression of serine deydratase is also enhanced during the first 5 hrs after hepatectomy. It is suggested that the enhanced expression of the two genes is due to an increase in hormone incretion particularly glucagon and glucocorticoids.