Silvia Franceschelli

bag3 gene expression is regulated by heat shock factor 1

BAG3 protein, a member of the BAG co‐chaperones family, sustains cell survival, through its interaction with the heat shock protein (HSP) 70, in a variety of normal and neoplastic cell types. bag3 gene expression is induced by stressful stimuli. Here we report for the first time that two of the three putative heat shock‐responsive elements (HSEs) in bag3 promoter interact with the heat shock factor (HSF) 1 in vitro and in vivo. Furthermore, downmodulation of HSF1 protein levels by specific small interfering (si) RNAs results in reducing BAG3 protein levels, indicating that the transcription factor plays a major role in bag3 gene expression. Because of the anti‐apoptotic role of BAG3 protein, these results disclose a previously unrecognized pathway, through which HSF1 maintains cell survival. J. Cell. Physiol. 215: 575–577, 2008.

The identification of a novel natural activator of p300 histone acetyltranferase provides new insights into the modulation mechanism of this enzyme.

Many severe human pathologies are related to alterations of the fine balance between histone acetylation and deacetylation; because not all such diseases involve hypoacetylation, but also hyperacetylation, compounds able to enhance or repress the activities of histone acetyltransferases (HATs) could be promising therapeutic agents. We evaluated in vitro and in cell the ability of eleven natural polyisoprenylated benzophenone derivatives to modulate the HAT activity of p300/CBP, an enzyme that plays a pivotal role in a variety of cellular processes. Some of the tested compounds bound efficiently to the p300/CBP protein: in particular, guttiferone A, guttiferone E and clusianone inhibit its HAT activity, whereas nemorosone showed a surprising ability to activate the enzyme. The ability of nemorosone to penetrate cell membranes and modulate histone acetylation into the cell together with its high affinity for the p300/CBP enzyme made this compound a suitable lead for the design of optimized anticancer drugs. Besides, the studies performed at a cellular and molecular level on both the inhibitors and the activator provided new insights into the modulation mechanism of p300/CBP by small molecules.

Role of Annexin A1 in Mouse Myoblast Cell Differentiation

Annexin A1 (ANXA1) is a calcium‐ and phospholipid‐binding protein involved in a broad range of cellular events. This study used molecular and microscopy approaches to explore the role of ANXA1 in mouse myoblast C2C12 cell differentiation. We report that ANXA1 expression increases during differentiation and that the down‐regulation of ANXA1 significantly inhibits the differentiation process. ANXA1 is expressed in vivo in both quiescent and activated satellite cells and is highly localized in the cells that migrate in the lumen of regenerating fibers after an acute injury. Endogenous ANXA1 co‐localizes with actin fibers at the protruding ends of undifferentiated but not differentiated cells suggesting a role of the protein in cell migration. Furthermore, ANXA1 neutralizing antibody reduces MyHC expression, decreases myotube formation and significantly inhibits cell migration. The data reported here suggest for the first time that ANXA1 plays a role in myogenic differentiation. J. Cell. Physiol. 224: 757–765, 2010.

Endoplasmic reticulum stress reduces the export from the ER and alters the architecture of post-ER compartments

In eukaryotic cells several physiologic and pathologic conditions generate the accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. To restore normal function, some ER transmembrane proteins sense the ER stress and activate coordinated signalling pathways collectively called the Unfolded Protein Response (UPR). Little is known on how the UPR relates to post-ER compartments and to the export from the ER of newly synthesized proteins. Here, we report that the ER stress response induced by either thapsigargin or nitric oxide modifies the dynamics of the intracellular distribution of ERGIC-53 and GM130, two markers of the ER Golgi Intermediate Compartment and of the cis-Golgi, respectively. In addition, induction of ER stress alters the morphology of the ERGIC and the Golgi complex and interferes with the reformation of both compartments. Moreover, ER stress rapidly reduces the transport to the Golgi complex of the temperature sensitive mutant of the Vesicular Stomatitis Virus G Glycoprotein (VSV-G) fused with the Green Fluorescent Protein (ts045G), without apparently decreasing the amount of the protein competent for export. Interestingly, a parallel rapid reduction of the number of Sec31 labelled fluorescent puncta on the ER membranes does occur, thus suggesting that the ER stress alters the ER export and the dynamic of post-ER compartments by rapidly targeting the formation of COPII-coated transport intermediates.

In the Huh7 Hepatoma Cells Diclofenac and Indomethacin Activate Differently the Unfolded Protein Response and Induce ER Stress Apoptosis

Non-steroidal anti-inflammatory drugs (NSAIDs) are cyclooxygenases (COXs) inhibitors frequently used in the treatment of acute and chronic inflammation. Side effects of NSAIDs are often due to their ability to induce apoptosis. Located at the Endoplasmic Reticulum membranes a tripartite signalling pathway, collectively known as the Unfolded Protein Response (UPR), decides survival or death of cells exposed to cytotoxic agents. To shed light on the molecular events responsible for the cytotoxicity of NSAIDs, we analysed the ability of diclofenac and indomethacin to activate the UPR in the human hepatoma cell line Huh7. We report that both NSAIDs can induce differently the single arms of the UPR. We show that indomethacin turns on the PERK and, only in part, the ATF6 and IRE1 pathways. Instead, diclofenac reduces the expression of ATF6 and does not stimulate the IRE1 endonuclease, which drives the expression of the prosurvival factor XBP1. Diclofenac, as well as indomethacin, is able to activate efficiently only the PERK pathway of the UPR, which induces the expression of the proapoptotic GADD153/CHOP protein. Our results highlight the importance of the UPR in evaluating the potential of drugs to induce apoptosis.

Genetic Modification of the Salmonella Membrane Physical State Alters the Pattern of Heat Shock Response

It is now recognized that membranes are not simple physical barriers but represent a complex and dynamic environment that affects membrane protein structures and their functions. Recent data emphasize the role of membranes in sensing temperature changes, and it has been shown that the physical state of the plasma membrane influences the expression of a variety of genes such as heat shock genes. It has been widely shown that minor alterations in lipid membranes are critically involved in the conversion of signals from the environment to the transcriptional activation of heat shock genes. Previously, we have proposed that the composition, molecular arrangement, and physical state of lipid membranes and their organization have crucial roles in cellular responses during stress caused by physical and chemical factors as well as in pathological states. Here, we show that transformation of Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) with a heterologous Delta(12)-desaturase (or with its trans-membrane regions) causes major changes in the pathogens membrane dynamic. In addition, this pathogen is strongly impaired in the synthesis of major stress proteins (heat shock proteins) under heat shock. These data support the hypothesis that the perception of temperature in Salmonella is strictly controlled by membrane order and by a specific membrane lipid/protein ratio that ultimately causes transcriptional activation of heat shock genes. These results represent a previously unrecognized mode of sensing temperature variation used by this pathogen at the onset of infection.

Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence

So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Delta(12)-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MPhi) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Delta(9)-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MPhi along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MPhi of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogens membrane fluid state and stress response.

Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach

IntroductionMicroRNAs (miRs) regulate gene expression to support important physiological functions. Significant evidences suggest that miRs play a crucial role in many pathological events and in the cell response to various stresses.MethodsWith the aim to identify new miRs induced by perturbation of intracellular calcium homeostasis, we analysed miR expression profiles of thapsigargin (TG)-treated cells by microarray. In order to identify miR-663a-regulated genes, we evaluated proteomic changes in miR-663a-overexpressing cells by two-dimensional differential in-gel electrophoresis coupled to mass spectrometric identification of the differentially represented proteins. Microarray and proteomic analyses were supported by biochemical validation.ResultsResults of microarray revealed 24 differentially expressed miRs; among them, miR-663a turned out to be by ER stress and under the control of the PERK pathway of the unfolded protein response. Proteomic analysis revealed that PLOD3, which is the gene encoding for collagen-modifying lysyl hydroxylase 3 (LH3), is regulated by miR-663a. Luciferase reporter assays demonstrated that miR-663a indeed reduces LH3 expression by targeting to 3′-UTR of PLOD3 mRNA. Interestingly, miR-663a inhibition of LH3 expression generates reduced extracellular accumulation of type IV collagen, thus suggesting the involvement of miR-663a in modulating collagen 4 secretion in physiological conditions and in response to ER stress.ConclusionThe finding of the ER stress-induced PERK-miR-663a pathway may have important implications in the understanding of the molecular mechanisms underlying the function of this miR in normal and/or pathological conditions.

Inhibition of Wnt/ β -Catenin pathway and Histone acetyltransferase activity by Rimonabant: a therapeutic target for colon cancer

In a high percentage (≥85%) of both sporadic and familial adenomatous polyposis forms of colorectal cancer (CRC), the inactivation of the APC tumor suppressor gene initiates tumor formation and modulates the Wnt/β-Catenin transduction pathways involved in the control of cell proliferation, adhesion and metastasis. Increasing evidence showed that the endocannabinoids control tumor growth and progression, both in vitro and in vivo. We evaluated the effect of Rimonabant, a Cannabinoid Receptor 1 (CB1) inverse agonist, on the Wnt/β-Catenin pathway in HCT116 and SW48 cell lines carrying the genetic profile of metastatic CRC poorly responsive to chemotherapies. In these models, Rimonabant inhibited the Wnt/β-Catenin canonical pathway and increased β-Catenin phosphorylation; in HCT116 cells, but not in SW48, the compound also triggered the Wnt/β-Catenin non canonical pathway activation through induction of Wnt5A and activation of CaMKII. The Rimonabant-induced downregulation of Wnt/β-Catenin target genes was partially ascribable to a direct inhibition of p300/KAT3B histone acetyltransferase, a coactivator of β-Catenin dependent gene regulation. Finally, in HCT116 xenografts, Rimonabant significantly reduced tumor growth and destabilized the nuclear localization of β-Catenin. Obtained data heavily supported the rationale for the use of cannabinoids in combined therapies for metastatic CRC harbouring activating mutations of β-Catenin.

Matrine modulates HSC70 levels and rescues ΔF508-CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP‐dependent Cl− channel located in the plasma membrane, and its malfunction results in cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Most CF patients carry the deletion of Phe508 (ΔF508 mutation); this mutation prevents the delivery of the CFTR to its correct cellular location, the apical (lumen‐facing) membrane of epithelial cells. Molecular chaperones play a central role in determining the fate of ΔF508‐CFTR. In this report, we show that the Matrine, a quinolizidine alkaloid, downregulates the expression of the molecular chaperone HSC70 and increases the protein levels of ΔF508‐CFTR in human alveolar basal epithelial cells (A549 cell line), stably transfected with a ΔF508‐CFTR‐expressing construct. Moreover, Matrine induced ΔF508‐CFTR release from endoplasmic reticulum to cell cytosol and its localization on the cell membrane. Interestingly, downregulation of HSC70 resulted in increased levels of ΔF508‐CFTR complexes with the co‐chaperone BAG3 that in addition appeared to co‐localize with the mutated protein on the cell surface. These results shed new light on ΔF508‐CFTR interactions with proteins of the chaperones/co‐chaperones system and could be useful in strategies for future medical treatments for CF. J. Cell. Physiol. 227: 3317–3323, 2012.