Biomedical subjects

Barbara Marinari

Publications and source records attributed to Barbara Marinari.

11 recordsLinked to original sources

PubMed2006

Cross-talks in the p53 family: deltaNp63 is an anti-apoptotic target for deltaNp73alpha and p53 gain-of-function mutants.

The p53 family of transcription factors plays a pivotal role in the control of the cellular response to DNA damaging agents. In addition to pro-apoptotic molecules such as p53, TAp73 and TAp63, this gene family also encodes for the anti-apoptotic molecules deltaNp73, deltaNp63, deltaNp53, and p53 mutants are often found in tumor cells, that have the role to limit and to modulate the pro-apoptotic side of the family. The ratio between the different members of the family is critical to make the life or death decision following DNA damage and is tightly regulated by post-translational and transcriptional mechanisms. In this study we have uncovered a novel positive feedback that involves the transcriptional activation of the anti-apoptotic molecule deltaNp63 by the anti-apoptotic molecules deltaNp73 and mutant p53, and that is put into motion upon treatment with a subset of DNA damaging agents such as Doxorubicin and 5-FU. DeltaNp73 and mutant p53 associate with the deltaNp63 promoter inducing its transcription and this is enhanced by doxorubicin treatment. Furthermore we have observed that deltaNp73- and mutp53-mediated activation of the deltaNp63 promoter requires the functionality of the proximal CCAAT boxes of this promoter, being hampered by mutation of CCAAT boxes or by dominant negative NFYA expression. This mechanism may serve as an additional control of the response of a normal cell to DNA damage or as an anti-apoptotic barrier of cancer cells subjected to DNA damage.

Animals↗

PubMed2006

Herpes simplex virus disrupts NF-kappaB regulation by blocking its recruitment on the IkappaBalpha promoter and directing the factor on viral genes.

Herpes simplex viruses (HSVs) are able to hijack the host-cell IkappaB kinase (IKK)/NF-kappaB pathway, which regulates critical cell functions from apoptosis to inflammatory responses; however, the molecular mechanisms involved and the outcome of the signaling dysregulation on the host-virus interaction are mostly unknown. Here we show that in human keratinocytes HSV-1 attains a sophisticated control of the IKK/NF-kappaB pathway, inducing two distinct temporally controlled waves of IKK activity and disrupting the NF-kappaB autoregulatory mechanism. Using chromatin immunoprecipitation we demonstrate that dysregulation of the NF-kappaB-response is mediated by a virus-induced block of NF-kappaB recruitment to the promoter of the IkappaBalpha gene, encoding the main NF-kappaB-inhibitor. We also show that HSV-1 redirects NF-kappaB recruitment to the promoter of ICP0, an immediate-early viral gene with a key role in promoting virus replication. The results reveal a new level of control of cellular functions by invading viruses and suggest that persistent NF-kappaB activation in HSV-1-infected cells, rather than being a host response to the virus, may play a positive role in promoting efficient viral replication.

Blotting, Western↗

PubMed2005

A p38-dependent pathway regulates DeltaNp63 DNA binding to p53-dependent promoters in UV-induced apoptosis of keratinocytes.

The p53 protein plays a pivotal role in determining the quality of the response to DNA damage through its transcriptional activity. Upon DNA damage, p53 is activated by post-translational modifications, binds its cognate sequences on the promoters of its target genes and stimulates transcription. In proliferating keratinocytes, the activity of p53 is blunted by its inhibitor DeltaNp63alpha. Here, we describe a novel mechanism through which DeltaNp63 functions in order to prevent the survival and propagation of ultraviolet (UV)-damaged keratinocytes. We found that UVB stimulation induces the rapid phosphorylation of DeltaNp63, which precedes DeltaNp63 transcriptional downregulation and protein degradation, which is mediated by the p38 MAPK. Phosphorylated DeltaNp63 has a lower affinity for p53REs and detaches from cell cycle arrest and apoptotic promoters, thus allowing the rapid activation of p53-dependent transcriptional apoptotic program.

Apoptosis↗

PubMed2004

CD28 and lipid rafts coordinate recruitment of Lck to the immunological synapse of human T lymphocytes.

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.

Amino Acid Motifs↗

PubMed2004

CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappaB subunits on IL-8 and Bcl-xL gene promoters.

CD28 is one of the most important costimulatory receptors necessary for full T lymphocyte activation. The CD28 receptor can enhance T cell antigen receptor (TCR) signals, as well as deliver independent signals. Indeed, CD28 engagement by B7 can generate TCR-independent signals leading to IkappaB kinase and NF-kappaB activation. Here we demonstrate that the TCR-independent CD28 signal leads to the selective transcription of survival (Bcl-xL) and inflammatory (IL-8 and B cell activation factor, but not proliferative (IL-2), genes, in a NF-kappaB-dependent manner. CD28-stimulated T cells actively secrete IL-8, and Bcl-xL up-regulation protects T cells from radiation-induced apoptosis. The transcription of CD28-induced genes is mediated by the specific recruitment of RelA and p52 NF-kappaB subunits to target promoters. In contrast, p50 and c-Rel, which preferentially bind NF-kappaB sites on the IL-2 gene promoter after anti-CD3 stimulation, are not involved. Thus, we identify CD28 as a key regulator of genes important for both survival and inflammation.

Base Sequence↗

PubMed2004

The p73 gene is an anti-tumoral target of the RARbeta/gamma-selective retinoid tazarotene.

Tazarotene, a member of the new class of acetylenic retinoids, has been shown to be effective in the treatment of several hyperproliferative skin diseases, including non-melanoma skin cancer. Its effectiveness is thought to rely on the ability to activate retinoic acid receptors beta and gamma and to induce a number of downstream anti-proliferative genes. Here, we show that the p53-related gene p73 is a target of tazarotene. Indeed, tazarotene modulates the expression of the p73 gene in immortalized keratinocyte cell lines by inducing the pro-apoptotic and anti-proliferative TAp73 isoforms and by repressing the anti-apoptotic and pro-proliferative DeltaNp73 isoforms. This occurs at the transcriptional level through a coordinated action on P1p73 and P2p73 promoters that control the expression of TA and DeltaN isoforms, respectively. The selective downregulation of DeltaNp73 expression by small interfering RNA led to an enhancement of tazarotene-induced bax activation and apoptosis, whereas the downregulation of both TA and DeltaN isoforms impairs tazarotene-mediated apoptosis. These results indicate the relevance of p73 gene products in tazarotene-induced growth inhibition and effectiveness in the treatment of skin tumors.

Animals↗

PubMed2003

Vav-1 and the IKK alpha subunit of I kappa B kinase functionally associate to induce NF-kappa B activation in response to CD28 engagement.

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of I kappa B kinase (IKK) complex and, consequently, to NF-kappa B activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKK alpha in both Jurkat and primary CD4(+) T cells. Vav-1/IKK alpha association is mediated by their helix-loop-helix domains, does not involve IKK beta, and is functionally relevant in that Vav-1-associated IKK alpha kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-kappa B activation is augmented by both IKK alpha and Vav-1, but not IKK beta. Confocal microscopy showed that endogenous Vav-1 and IKK alpha, but not IKK beta, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-kappa B pathway by targeting IKK alpha in the T cell membrane and favoring its activation in response to CD28 stimulation.

Animals↗

PubMed2003

Vav exchange factor counteracts the HIV-1 Nef-mediated decrease of plasma membrane GM1 and NF-AT activity in T cells.

Several findings support the importance of GM1-enriched lipid microdomains of plasma membrane and of Vav, an essential regulator of actin cytoskeletal rearrangement, in the regulation of T cell activation. Moreover, a functional link among lipid microdomains, Vav and the HIV product Nef has been described. These observations suggest that Nef can modify plasma membrane GM1, affecting the behavior of HIV-infected cells towards antigen recognition and Vav towards counteracting such an effect. We observed that Nef expression, either following viral infection or ectopic expression, significantly decreased the level of plasma membrane GM1 in unstimulated T cells. This down-regulation was associated with the inhibition of NF-AT activation, but not with NF-kappaB activation induced by TCR engagement. Dissecting the signaling pathway that regulates NF-AT activation, we found that Nef inhibited exclusively the Ca(2+)/calcineurin cascade, whereas the JNK cascade and AP-1 transcriptional activity were not affected. Our evidence that Vav overexpression counteracted both the Nef-induced decrease of GM1 expression and the inhibition of NF-AT activity, suggests a novel mechanism by which Nef may interfere with TCR-mediated activation through the modulation of intracellular trafficking and clustering of GM1-enriched microdomains at the cell surface.

Calcium Signaling↗

PubMed2003

The activation of Csk by CD4 interferes with TCR-mediated activatory signaling.

CD4-Lck recruitment to TCR/CD3, as well as Lck activation is essential for T cell activation. Indeed, the blockage of CD4-Lck recruitment to TCR during antigen recognition exerts a drastic inhibitory effect on T cell activation by interfering with both early and late phases of T cell signaling. In the present work, we report a novel inhibitory mechanism by which CD4 can shut down proximal T cell-activating signals. Indeed, we show that upon ligation of CD4 by antibodies the inhibitory kinase, p50(csk), is strongly induced and prolonged during the time. In contrast, p50(csk) was not activated when TCR and CD4 were properly engaged by their ligands. We also demonstrate that anti-CD4 treatment stimulated Csk kinase associated to the membrane adapter, PAG/Cbp, without affecting the total amount of Csk bound to PAG/Cbp. As a consequence, early tyrosine phosphorylation events as well as downstream signaling pathways leading to IL-2 gene expression induced by TCR were inhibited in anti-CD4 pretreated cells. We suggest a new model to explain the activation of negative signals by CD4 molecule.

CD4 Antigens↗

PubMed2002

CD4-Lck through TCR and in the absence of Vav exchange factor induces Bax increase and mitochondrial damage.

In the present study, we aimed to demonstrate that CD4 may represent a critical turning point that governs the apoptotic and survival programs in T cells, without modifying the physical association with the TCR-CD3 complex. To address this issue, we have explored the possibility that the activation of CD4 may transduce apoptotic signals unless signaling effectors neutralize them. Our data show that in Jurkat T cells CD4 engagement by Leu3a mAb results in a rapid and strong increase of Lck kinase activity, subsequent alterations of mitochondrial membrane potential, and apoptosis. Critical parameters are coassociation of CD4/Lck with TCR/CD3 and up-regulation of the proapoptotic protein Bax. Indeed, Leu3a-mediated Lck activation failed to induce apoptotic features in Jurkat cells either defective for TCR/CD3 or overexpressing the antiapoptotic protein Bcl-2. Furthermore, we demonstrate that Leu3a treatment of Jurkat cells overexpressing Vav results in the inhibition of mitochondrial damage and apoptosis; this rescue effect is accompanied with a significant decrease of Bax expression observed in apoptotic cells. Our evidence that the activation of Lck activates in T cells apoptotic pathways which are counteracted by Vav, a signaling molecule that cooperates with CD28 to boost TCR signals, suggests a novel role for costimulation in protecting T cells from CD4-mediated cell death.

Antibodies, Monoclonal↗

PubMed2002

Vav cooperates with CD28 to induce NF-kappaB activation via a pathway involving Rac-1 and mitogen-activated kinase kinase 1.

CD28-delivered costimulatory signals are required to induce NF-kappaB activation in response to TCR stimulation. We have recently demonstrated that the mitogen-activated kinase kinase 1 (MEKK1), a kinase known to regulate the c-jun N-terminal kinase (JNK) pathway, is also involved in the CD28- and TCR-induced inhibitor of kappaB factor (IkappaB) kinases (IKK) and NF-kappaB activation. Searching for molecules that couple TCR and CD28 to MEKK1, we found that the guanine nucleotide exchange factor Vav synergized with CD28 stimulation in Jurkat cells to induce NF-kappaB transcriptional activity through the activation of IKKalpha and IKKbeta. Dominant negative mutants of Vav inhibited TCR- and CD28-NF-kappaB-dependent transcription by interfering with the activation of the IKK complex. Blocking Rac signaling downstream of Vav by dominant negative RacN17 exerts similar effects on IKK and NF-kappaB activation after TCR/CD28 stimulation. Finally, Vav-induced NF-kappaB activation in CD28 costimulated cells was inhibited by dominant negative MEKK(KM). These results identify Vav, Rac-1 and MEKK1 as components of a common pathway regulating both NF-kappaB and AP-1 that contributes to full activation of the CD28 response element (CD28RE).

CD28 Antigens↗