Marco Tripodi - Academia.edu (original) (raw)

Papers by Marco Tripodi

Cell Death & Differentiation, Dec 2, 2011

Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal ... more Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular minicircuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4a, directly represses the expression of the epithelial microRNAs (miRs)-200c and-34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4a, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and-200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4a and miRs-200a, b, c and-34a as epistatic elements controlling hepatic stem cell maintenance/differentiation.

Journal of Medicinal Chemistry, Jul 13, 2023

The human genome contains three ~l-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for sli... more The human genome contains three ~l-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for slightly different forms of the protein. The major component of human od-acid glyeoprotein found in plasma is coded by AGP-A, which is expressed in liver and in hepatoma cell lines and is induced by inflammatory stimuli. We have studied the regulation of the cloned AGP-A gene by transfeetion into cell lines of hepatic and nonhepatie origin. Unlike any other liver-specific gene investigated so far, every AGP construct tested was expressed with comparable efficiency in hepatoma and HeLa cells. In contrast, identical constructs in transgenie mice are expressed in a tissue-specific manner and are regulated by acute-phase stimuli. Transgenic mice carrying the cluster of three AGP genes secrete the human protein in the serum, and the corresponding mRNA is mainly derived from the AGP-A gene. The mRNA is liver specific, and its concentration increases several fold following experimentally induced inflammation. Additional transgenie lines carrying only the AGP-A gene showed that sufficient information for tissue-specific and regulated expression is contained within a 6.6-kb segment comprising the whole coding region plus 1.2-kb 5'-flanking and 2-kb 3'-flanking DNA.

RNA Biology, Jul 5, 2021

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in dou... more Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex. Overall, these data support the role of ADAR2 as regulator of L1 life cycle.

BACKGROUND & AIMS: In each hepatocyte, the specific repertoire of gene expression is influenc... more BACKGROUND & AIMS: In each hepatocyte, the specific repertoire of gene expression is influenced by its exact location along the portocentrovenular axis of the hepatic lobule and provides a reason for the liver functions compartmentalization defined “metabolic zonation.” So far, few molecular players controlling genetic programs of periportal (PP) and perivenular (PV) hepatocytes have been identified; the elucidation of zonation mechanisms remains a challenge for experimental hepatology. Recently, a key role in induction and maintenance of the hepatocyte heterogeneity has been ascribed to Wnt/-catenin pathway. We sought to clarify how this wide-ranging stimulus integrates with hepatocyte specificity. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) allowed the transcriptional profiling of hepatocytes derived from in vitro differentiation of liver stem cells. The GSK3 inhibitor 6-bromoindirubin-3=-oxime (BIO) was used for -catenin stabilization. Co-immunoprecipitations were used to study biochemical protein interactions while ChIP assays allowed the in vivo inspection of PV and PP genes regulatory regions. RESULTS: We found that spontaneous differentiation of liver stem cells gives rise to PP hepatocytes that, after Wnt pathway activation, switch into PV hepatocytes. Next, we showed that the Wnt downstream player LEF1 interacts with the liver-enriched transcriptional factor HNF4. Finally, we unveiled that the BIO induced activation of PV genes correlates with LEF1 binding to both its own and HNF4 consensus, and the repression of PP genes correlates with HNF4 displacement from its own consensus. CONCLUSION: Our data show a direct and hitherto unknown convergence of the canonical Wnt signaling on the HNF4-driven transcription providing evidences of a mechanism controlling liver zonated gene expression.

Journal of Hepatology, 2013

Background & Aims: The tumor fate derives from cell autonomous properties and niche microenvironm... more Background & Aims: The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor b (TGFb) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4a is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFb on the anti-EMT and tumor suppressor HNF4a activity. Methods: Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4a DNA binding activity. HNF4a post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3b activity was modulated by chemical inhibition and constitutive active mutant expression. Results: We demonstrated that the presence of TGFb impairs the efficiency of HNF4a as tumor suppressor. We found that TGFb induces HNF4a PTMs that correlate with the early loss of HNF4a DNA binding activity on target gene promoters. Furthermore, we identified the GSK3b kinase as one of the TGFb targets mediating HNF4a functional inactivation: GSK3b chemical inhibition results in HNF4a DNA binding impairment while a constitutively active GSK3b mutant impairs the TGFb-induced inhibitory effect on HNF4a tumor suppressor activity. Conclusions: Our data identify in the dominance of TGFb a limit for the HNF4a-mediated gene therapy of HCC.

Journal of Chromatography B, Mar 1, 2008

The interest in therapeutic drug monitoring (TDM) of antiretroviral drugs has grown significantly... more The interest in therapeutic drug monitoring (TDM) of antiretroviral drugs has grown significantly since highly active antiretroviral therapy (HAART) became a standard of care in clinical practice. TDM is useful to determine the best dosage regimen adapted to each patient. Here, we apply MALDI-TOF/TOF technology to quantify abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine in the plasma of HIVinfected patients, by standard additions analysis. Regression of standard additions was linear over the whole anti-HIV concentration range explored (1.00 × 10 −2-1.00 pmol/L). The absolute recovery ranged between 80% and 110%. Values of the drug concentration determined by MALDI-TOF/TOF were in the range of 1.00 × 10 −2-1.00 pmol/L. The limit of quantification value was 1.00 × 10 −2 pmol/L for abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine.

Journal of Hepatology, Apr 1, 2010

POSTERS quiescent cell (fat storing) phenotype evaluated by Oil-RedO staining. TGFb-1 levels were... more POSTERS quiescent cell (fat storing) phenotype evaluated by Oil-RedO staining. TGFb-1 levels were measure in the supernatant using an enzyme-linked immunosorbent assay kit. Gene expression of TGFb-1, MMP-13, MMP-2, collagen a1(I), TIMP-1, a-smooth muscle actin (a-SMA) and urokinase-type plasminogen activator (uPA) were evaluated by quantitative real time PCR (qPCR). Results: There was no cytotoxicity on murine HSCs treated with SNAC or NAC. SNAC-induced conversion of myofibroblast into quiescent cell (fat storing) phenotype evaluated by Oil-RedO staining of fat droplets. Also, SNAC decreased TGFb-1 levels in the supernatant compared to NAC or control groups. Besides, there was a reduction of profibrogenic molecules TGFb-1, collagen a1(I), MMP-2, TIMP-1 and uPA expression. SNAC promoted up-regulation the expession of interstitial collagenase MMP-13 (8-fold higher) in cells treated in comparison to NAC or control groups. Conclusions: 1. SNAC is able to induce involution of myofibroblast into lipocyte phenotype on GRX cell lines, with concomitant reduction in TGF-b1 levels; 2. SNAC is able to down-regulate profibrogenic molecules and upregulate MMP-13, that plays a key role in the degradation of interstitial collagen in liver fibrosis. These findings demonstrate that SNAC can modulate efficiently the function of activated HSCs and could be used as a antifibrogenic drug. These results are currently being investigated in an in vivo model of hepatic fibrogenesis.

Nucleic Acids Research, 1990

We have developed a technique of homologous recombination in bacteria which allows the mutagenesi... more We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.

Nucleic Acids Research, 1987

al-antitrypsin (al AT) present in large amounts in human serum and synthesized predominantly in h... more al-antitrypsin (al AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and al AT mutant proteins are associated with heriditary disorders. To investigate the regulation of the normal human aiAT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alAT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.

Cellular Signalling, Nov 1, 2008

Extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase, spe... more Extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase, specifically activated by MEK5, and involved in the regulation of many cellular functions including proliferation, survival, differentiation and apoptosis. MEK5/ERK5 module is an important element of different signal transduction pathways. The aim of this study was to investigate whether ERK5 participates to the signalling of the multifunctional cytokine TGFβ, known to play an important role in the regulation of hepatic growth. Here, we reported that ERK5 is phosphorylated and activated by TGFβ in hepatocytes, with a rapid and sustained kinetic, through a Src-dependent pathway. Moreover, we demonstrated that ERK5 participates to the TGFβinduced Snail protein regulation being required for its stabilization. We also found that the functional inactivation of ERK5 impedes the TGFβ-mediated glycogen synthase kinase-3β inactivation suggesting this as mechanism responsible for ERK5-mediated Snail stabilization. Thus, results presented in this study uncovered for the first time a role for ERK5 in the TGFβ-induced cellular responses.

Cell differentiation, Jun 1, 1984

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation ... more In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.

Research Square (Research Square), Jul 11, 2022

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of s... more YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several physio-pathological cellular processes, by integrating multiple cell autonomous and microenvironmental cues. YAP is the main downstream effector of the Hippo pathway, a tumor-suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signal-regulated kinase 5 (ERK5)/mitogen-activated protein kinase in the regulation of YAP activity. By means of ERK5 inhibition/silencing and overexpression experiments, and by using as model liver stem cells, hepatocytes, and hepatocellular carcinoma (HCC) cell lines, we provided evidence that ERK5 is required for YAP-dependent gene expression. Mechanistically, ERK5 controls the recruitment of YAP on promoters of target genes and its physical interaction with the transcriptional partner TEAD; moreover, it mediates the YAP activation occurring in cell adhesion, migration, and TGFβ-induced EMT of liver cells. Furthermore, we demonstrated that ERK5 signaling modulates YAP activity in a LATS1/2-independent manner. Therefore, our observations identify ERK5 as a novel upstream Hippo-independent regulator of YAP activity, thus unveiling a new target for therapeutic approaches aimed at interfering with its function.

Hepatology, Dec 1, 1998

It was recently reported that transgenic expression in the liver of truncated human Met renders h... more It was recently reported that transgenic expression in the liver of truncated human Met renders hepatocytes constitutively resistant to apoptosis and reproducibly permits their immortalization. The derived stable cell lines (MMH from Met murine hepatocyte) are highly differentiated and nontransformed. In this report, the capacity of MMHs to support in vitro hematopoiesis is characterized. By reversetranscription polymerase chain reaction, the expression by MMHs of cytokines involved in the survival and selfrenewal of early progenitor cells (stem cell factor and FLT3 ligand) as well as those acting at different stages of progenitor differentiation (interleukin [IL] 1␤, IL-3, leukemia inhibitory factor, IL-6, granulocyte-macrophage colonystimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and thrombopoietin) was shown. A ribonuclease protection assay further substantiated the presence of at least six cytokine transcripts in MMH lines. Cocultures between MMH layers and progenitor-enriched fetal liver hematopoietic cells resulted in a 40-fold to 80-fold expansion of total hematopoietic cells and in a 2.5-fold expansion of clonogenic progenitors after 1 to 2 weeks. Hematopoiesis was maintained for up to 6 weeks with formation of typical cobblestone cell areas and continuous differentiation of precursor into cells at various degrees of maturation. At 5 weeks of coculture, clonogenic progenitors were maintained at 20% of the input level in coculture with embryonic-derived hepatocytes, showing the ability of hepatocyte feeder layer to support survival and possibly self-renewal of clonogenic progenitors. Therefore, the data emphasize a direct role of the hepatocyte in sustaining hematopoietic cell proliferation and differentiation.

Cell & Bioscience

In recent years, progress in nanotechnology provided new tools to treat cancer more effectively. ... more In recent years, progress in nanotechnology provided new tools to treat cancer more effectively. Advances in biomaterials tailored for drug delivery have the potential to overcome the limited selectivity and side effects frequently associated with traditional therapeutic agents. While autophagy is pivotal in determining cell fate and adaptation to different challenges, and despite the fact that it is frequently dysregulated in cancer, antitumor therapeutic strategies leveraging on or targeting this process are scarce. This is due to many reasons, including the very contextual effects of autophagy in cancer, low bioavailability and non-targeted delivery of existing autophagy modulatory compounds. Conjugating the versatile characteristics of nanoparticles with autophagy modulators may render these drugs safer and more effective for cancer treatment. Here, we review current standing questions on the biology of autophagy in tumor progression, and precursory studies and the state-of-the-...

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of s... more YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several cellular processes, including proliferation, differentiation and epithelial-to-mesenchymal transition (EMT), by integrating multiple cell autonomous and microenvironmental cues. YAP overexpression was found in different types of human cancer where it is associated with the acquisition of stemness properties, chemoresistance, increased cell proliferation and survival, metastasis and, ultimately, poor prognosis. YAP is the main downstream effector of the Hippo pathway, a tumor suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signa...

Frontiers in Molecular Biosciences, 2021

Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely u... more Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely understood. In particular, no direct evidence of pleura implication in COVID-19-related fibrotic damage has been reported so far. In this study, the expression of epithelial cytokeratins and Wilms tumor 1 (WT1), specific markers of mesothelial cells (MCs), was analyzed in COVID-19 and unrelated pleura autoptic samples. SARS-CoV-2 replication was analyzed by RT-PCR and confocal microscopy in MeT5A, a pleura MC line. SARS-CoV-2 receptors were analyzed by RT-PCR and western blot. Inflammatory cytokines from the supernatants of SARS-CoV-2-infected MeT5A cells were analysed by Luminex and ELLA assays. Immunohistochemistry of COVID-19 pleura patients highlighted disruption of pleura monolayer and fibrosis of the sub-mesothelial stroma, with the presence of MCs with fibroblastoid morphology in the sub-mesothelial stroma, but no evidence of direct infection in vivo. Interestingly, we found eviden...

Acta biologica et medica Germanica, 1981

Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two ... more Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two are alpha-like and two are beta-like. Pure globin messenger RNAs from these cells were used as templates for two successive polymerizing reactions and a mixture of double stranded cDNAs coding for the four globins was obtained. These molecules were blunt-end ligated to an ECoR1 digested pBR322 plasmid and the recombinant plasmids were used to transform E. coli Hb101. Bacterial clones which proved positive upon hybridization with 32P-labelled embryonic globin cDNA were amplified and their plasmid DNA was isolated. Three different plasmids were studied, namely no. 2, 16 and 54. The restriction map of these plasmids showed that: 1) plasmid no. 2 and 54 had lost extensive DNA sequences comprising the genes responsible for tetracycline resistance; 2) the size of inserted sequences ranges from 427 base pairs of plasmid no. 16 to about 280 base pairs of plasmid no. 54; 3) plasmid no. 2 does not...

Cell Death & Differentiation, Dec 2, 2011

Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal ... more Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular minicircuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4a, directly represses the expression of the epithelial microRNAs (miRs)-200c and-34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4a, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and-200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4a and miRs-200a, b, c and-34a as epistatic elements controlling hepatic stem cell maintenance/differentiation.

Journal of Medicinal Chemistry, Jul 13, 2023

The human genome contains three ~l-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for sli... more The human genome contains three ~l-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for slightly different forms of the protein. The major component of human od-acid glyeoprotein found in plasma is coded by AGP-A, which is expressed in liver and in hepatoma cell lines and is induced by inflammatory stimuli. We have studied the regulation of the cloned AGP-A gene by transfeetion into cell lines of hepatic and nonhepatie origin. Unlike any other liver-specific gene investigated so far, every AGP construct tested was expressed with comparable efficiency in hepatoma and HeLa cells. In contrast, identical constructs in transgenie mice are expressed in a tissue-specific manner and are regulated by acute-phase stimuli. Transgenic mice carrying the cluster of three AGP genes secrete the human protein in the serum, and the corresponding mRNA is mainly derived from the AGP-A gene. The mRNA is liver specific, and its concentration increases several fold following experimentally induced inflammation. Additional transgenie lines carrying only the AGP-A gene showed that sufficient information for tissue-specific and regulated expression is contained within a 6.6-kb segment comprising the whole coding region plus 1.2-kb 5'-flanking and 2-kb 3'-flanking DNA.

RNA Biology, Jul 5, 2021

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in dou... more Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex. Overall, these data support the role of ADAR2 as regulator of L1 life cycle.

BACKGROUND & AIMS: In each hepatocyte, the specific repertoire of gene expression is influenc... more BACKGROUND & AIMS: In each hepatocyte, the specific repertoire of gene expression is influenced by its exact location along the portocentrovenular axis of the hepatic lobule and provides a reason for the liver functions compartmentalization defined “metabolic zonation.” So far, few molecular players controlling genetic programs of periportal (PP) and perivenular (PV) hepatocytes have been identified; the elucidation of zonation mechanisms remains a challenge for experimental hepatology. Recently, a key role in induction and maintenance of the hepatocyte heterogeneity has been ascribed to Wnt/-catenin pathway. We sought to clarify how this wide-ranging stimulus integrates with hepatocyte specificity. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) allowed the transcriptional profiling of hepatocytes derived from in vitro differentiation of liver stem cells. The GSK3 inhibitor 6-bromoindirubin-3=-oxime (BIO) was used for -catenin stabilization. Co-immunoprecipitations were used to study biochemical protein interactions while ChIP assays allowed the in vivo inspection of PV and PP genes regulatory regions. RESULTS: We found that spontaneous differentiation of liver stem cells gives rise to PP hepatocytes that, after Wnt pathway activation, switch into PV hepatocytes. Next, we showed that the Wnt downstream player LEF1 interacts with the liver-enriched transcriptional factor HNF4. Finally, we unveiled that the BIO induced activation of PV genes correlates with LEF1 binding to both its own and HNF4 consensus, and the repression of PP genes correlates with HNF4 displacement from its own consensus. CONCLUSION: Our data show a direct and hitherto unknown convergence of the canonical Wnt signaling on the HNF4-driven transcription providing evidences of a mechanism controlling liver zonated gene expression.

Journal of Hepatology, 2013

Background & Aims: The tumor fate derives from cell autonomous properties and niche microenvironm... more Background & Aims: The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor b (TGFb) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4a is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFb on the anti-EMT and tumor suppressor HNF4a activity. Methods: Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4a DNA binding activity. HNF4a post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3b activity was modulated by chemical inhibition and constitutive active mutant expression. Results: We demonstrated that the presence of TGFb impairs the efficiency of HNF4a as tumor suppressor. We found that TGFb induces HNF4a PTMs that correlate with the early loss of HNF4a DNA binding activity on target gene promoters. Furthermore, we identified the GSK3b kinase as one of the TGFb targets mediating HNF4a functional inactivation: GSK3b chemical inhibition results in HNF4a DNA binding impairment while a constitutively active GSK3b mutant impairs the TGFb-induced inhibitory effect on HNF4a tumor suppressor activity. Conclusions: Our data identify in the dominance of TGFb a limit for the HNF4a-mediated gene therapy of HCC.

Journal of Chromatography B, Mar 1, 2008

The interest in therapeutic drug monitoring (TDM) of antiretroviral drugs has grown significantly... more The interest in therapeutic drug monitoring (TDM) of antiretroviral drugs has grown significantly since highly active antiretroviral therapy (HAART) became a standard of care in clinical practice. TDM is useful to determine the best dosage regimen adapted to each patient. Here, we apply MALDI-TOF/TOF technology to quantify abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine in the plasma of HIVinfected patients, by standard additions analysis. Regression of standard additions was linear over the whole anti-HIV concentration range explored (1.00 × 10 −2-1.00 pmol/L). The absolute recovery ranged between 80% and 110%. Values of the drug concentration determined by MALDI-TOF/TOF were in the range of 1.00 × 10 −2-1.00 pmol/L. The limit of quantification value was 1.00 × 10 −2 pmol/L for abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine.

Journal of Hepatology, Apr 1, 2010

POSTERS quiescent cell (fat storing) phenotype evaluated by Oil-RedO staining. TGFb-1 levels were... more POSTERS quiescent cell (fat storing) phenotype evaluated by Oil-RedO staining. TGFb-1 levels were measure in the supernatant using an enzyme-linked immunosorbent assay kit. Gene expression of TGFb-1, MMP-13, MMP-2, collagen a1(I), TIMP-1, a-smooth muscle actin (a-SMA) and urokinase-type plasminogen activator (uPA) were evaluated by quantitative real time PCR (qPCR). Results: There was no cytotoxicity on murine HSCs treated with SNAC or NAC. SNAC-induced conversion of myofibroblast into quiescent cell (fat storing) phenotype evaluated by Oil-RedO staining of fat droplets. Also, SNAC decreased TGFb-1 levels in the supernatant compared to NAC or control groups. Besides, there was a reduction of profibrogenic molecules TGFb-1, collagen a1(I), MMP-2, TIMP-1 and uPA expression. SNAC promoted up-regulation the expession of interstitial collagenase MMP-13 (8-fold higher) in cells treated in comparison to NAC or control groups. Conclusions: 1. SNAC is able to induce involution of myofibroblast into lipocyte phenotype on GRX cell lines, with concomitant reduction in TGF-b1 levels; 2. SNAC is able to down-regulate profibrogenic molecules and upregulate MMP-13, that plays a key role in the degradation of interstitial collagen in liver fibrosis. These findings demonstrate that SNAC can modulate efficiently the function of activated HSCs and could be used as a antifibrogenic drug. These results are currently being investigated in an in vivo model of hepatic fibrogenesis.

Nucleic Acids Research, 1990

We have developed a technique of homologous recombination in bacteria which allows the mutagenesi... more We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.

Nucleic Acids Research, 1987

al-antitrypsin (al AT) present in large amounts in human serum and synthesized predominantly in h... more al-antitrypsin (al AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and al AT mutant proteins are associated with heriditary disorders. To investigate the regulation of the normal human aiAT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alAT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.

Cellular Signalling, Nov 1, 2008

Extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase, spe... more Extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase, specifically activated by MEK5, and involved in the regulation of many cellular functions including proliferation, survival, differentiation and apoptosis. MEK5/ERK5 module is an important element of different signal transduction pathways. The aim of this study was to investigate whether ERK5 participates to the signalling of the multifunctional cytokine TGFβ, known to play an important role in the regulation of hepatic growth. Here, we reported that ERK5 is phosphorylated and activated by TGFβ in hepatocytes, with a rapid and sustained kinetic, through a Src-dependent pathway. Moreover, we demonstrated that ERK5 participates to the TGFβinduced Snail protein regulation being required for its stabilization. We also found that the functional inactivation of ERK5 impedes the TGFβ-mediated glycogen synthase kinase-3β inactivation suggesting this as mechanism responsible for ERK5-mediated Snail stabilization. Thus, results presented in this study uncovered for the first time a role for ERK5 in the TGFβ-induced cellular responses.

Cell differentiation, Jun 1, 1984

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation ... more In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.

Research Square (Research Square), Jul 11, 2022

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of s... more YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several physio-pathological cellular processes, by integrating multiple cell autonomous and microenvironmental cues. YAP is the main downstream effector of the Hippo pathway, a tumor-suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signal-regulated kinase 5 (ERK5)/mitogen-activated protein kinase in the regulation of YAP activity. By means of ERK5 inhibition/silencing and overexpression experiments, and by using as model liver stem cells, hepatocytes, and hepatocellular carcinoma (HCC) cell lines, we provided evidence that ERK5 is required for YAP-dependent gene expression. Mechanistically, ERK5 controls the recruitment of YAP on promoters of target genes and its physical interaction with the transcriptional partner TEAD; moreover, it mediates the YAP activation occurring in cell adhesion, migration, and TGFβ-induced EMT of liver cells. Furthermore, we demonstrated that ERK5 signaling modulates YAP activity in a LATS1/2-independent manner. Therefore, our observations identify ERK5 as a novel upstream Hippo-independent regulator of YAP activity, thus unveiling a new target for therapeutic approaches aimed at interfering with its function.

Hepatology, Dec 1, 1998

It was recently reported that transgenic expression in the liver of truncated human Met renders h... more It was recently reported that transgenic expression in the liver of truncated human Met renders hepatocytes constitutively resistant to apoptosis and reproducibly permits their immortalization. The derived stable cell lines (MMH from Met murine hepatocyte) are highly differentiated and nontransformed. In this report, the capacity of MMHs to support in vitro hematopoiesis is characterized. By reversetranscription polymerase chain reaction, the expression by MMHs of cytokines involved in the survival and selfrenewal of early progenitor cells (stem cell factor and FLT3 ligand) as well as those acting at different stages of progenitor differentiation (interleukin [IL] 1␤, IL-3, leukemia inhibitory factor, IL-6, granulocyte-macrophage colonystimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and thrombopoietin) was shown. A ribonuclease protection assay further substantiated the presence of at least six cytokine transcripts in MMH lines. Cocultures between MMH layers and progenitor-enriched fetal liver hematopoietic cells resulted in a 40-fold to 80-fold expansion of total hematopoietic cells and in a 2.5-fold expansion of clonogenic progenitors after 1 to 2 weeks. Hematopoiesis was maintained for up to 6 weeks with formation of typical cobblestone cell areas and continuous differentiation of precursor into cells at various degrees of maturation. At 5 weeks of coculture, clonogenic progenitors were maintained at 20% of the input level in coculture with embryonic-derived hepatocytes, showing the ability of hepatocyte feeder layer to support survival and possibly self-renewal of clonogenic progenitors. Therefore, the data emphasize a direct role of the hepatocyte in sustaining hematopoietic cell proliferation and differentiation.

Cell & Bioscience

In recent years, progress in nanotechnology provided new tools to treat cancer more effectively. ... more In recent years, progress in nanotechnology provided new tools to treat cancer more effectively. Advances in biomaterials tailored for drug delivery have the potential to overcome the limited selectivity and side effects frequently associated with traditional therapeutic agents. While autophagy is pivotal in determining cell fate and adaptation to different challenges, and despite the fact that it is frequently dysregulated in cancer, antitumor therapeutic strategies leveraging on or targeting this process are scarce. This is due to many reasons, including the very contextual effects of autophagy in cancer, low bioavailability and non-targeted delivery of existing autophagy modulatory compounds. Conjugating the versatile characteristics of nanoparticles with autophagy modulators may render these drugs safer and more effective for cancer treatment. Here, we review current standing questions on the biology of autophagy in tumor progression, and precursory studies and the state-of-the-...

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of s... more YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several cellular processes, including proliferation, differentiation and epithelial-to-mesenchymal transition (EMT), by integrating multiple cell autonomous and microenvironmental cues. YAP overexpression was found in different types of human cancer where it is associated with the acquisition of stemness properties, chemoresistance, increased cell proliferation and survival, metastasis and, ultimately, poor prognosis. YAP is the main downstream effector of the Hippo pathway, a tumor suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signa...

Frontiers in Molecular Biosciences, 2021

Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely u... more Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely understood. In particular, no direct evidence of pleura implication in COVID-19-related fibrotic damage has been reported so far. In this study, the expression of epithelial cytokeratins and Wilms tumor 1 (WT1), specific markers of mesothelial cells (MCs), was analyzed in COVID-19 and unrelated pleura autoptic samples. SARS-CoV-2 replication was analyzed by RT-PCR and confocal microscopy in MeT5A, a pleura MC line. SARS-CoV-2 receptors were analyzed by RT-PCR and western blot. Inflammatory cytokines from the supernatants of SARS-CoV-2-infected MeT5A cells were analysed by Luminex and ELLA assays. Immunohistochemistry of COVID-19 pleura patients highlighted disruption of pleura monolayer and fibrosis of the sub-mesothelial stroma, with the presence of MCs with fibroblastoid morphology in the sub-mesothelial stroma, but no evidence of direct infection in vivo. Interestingly, we found eviden...

Acta biologica et medica Germanica, 1981

Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two ... more Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two are alpha-like and two are beta-like. Pure globin messenger RNAs from these cells were used as templates for two successive polymerizing reactions and a mixture of double stranded cDNAs coding for the four globins was obtained. These molecules were blunt-end ligated to an ECoR1 digested pBR322 plasmid and the recombinant plasmids were used to transform E. coli Hb101. Bacterial clones which proved positive upon hybridization with 32P-labelled embryonic globin cDNA were amplified and their plasmid DNA was isolated. Three different plasmids were studied, namely no. 2, 16 and 54. The restriction map of these plasmids showed that: 1) plasmid no. 2 and 54 had lost extensive DNA sequences comprising the genes responsible for tetracycline resistance; 2) the size of inserted sequences ranges from 427 base pairs of plasmid no. 16 to about 280 base pairs of plasmid no. 54; 3) plasmid no. 2 does not...