Maria Pia Felli

Constitutive activation of NF‐κB and T‐cell leukemia/lymphoma in Notch3 transgenic mice

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T‐cell development. Notch3 is expressed in CD4−8− thymocytes and is down‐regulated across the CD4−8− to CD4+8+ transition, controlled by pre‐T‐cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter‐driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4−8− cells, a failure to down‐regulate CD25 in post‐CD4−8− subsets and sustained activity of NF‐κB. Subsequently, aggressive multicentric T‐cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pTα and activated NF‐κB via IKKα‐dependent degradation of IκBα and enhancement of NF‐κB‐dependent anti‐apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF‐κB activation and T‐cell tumorigenesis. The phenotypes of pre‐malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co‐ordinating growth and differentiation of thymocytes, across the pre‐T/T cell transition, consistent with the normal expression pattern of Notch3.

Notch3 and pre-TCR interaction unveils distinct NF-κB pathways in T-cell development and leukemia

Notch signaling plays a critical role in T‐cell differentiation and leukemogenesis. We previously demonstrated that, while pre‐TCR is required for thymocytes proliferation and leukemogenesis, it is dispensable for thymocyte differentiation in Notch3‐transgenic mice. Notch3‐transgenic premalignant thymocytes and T lymphoma cells overexpress pTα/pre‐TCR and display constitutive activation of NF‐κB, providing survival signals for immature thymocytes. We provide genetic and biochemical evidence that Notch3 triggers multiple NF‐κB activation pathways. A pre‐TCR‐dependent pathway preferentially activates NF‐κB via IKKβ/IKKα/NIK complex, resulting in p50/p65 heterodimer nuclear entry and recruitment onto promoters of Cyclin D1, Bcl2‐A1 and IL7‐receptor‐α genes. In contrast, upon pTα deletion, Notch3 binds IKKα and maintains NF‐κB activation through an alternative pathway, depending on an NIK‐independent IKKα homodimer activity. The consequent NF‐κB2/p100 processing allows nuclear translocation of p52/RelB heterodimers, which only trigger transcription from Bcl2‐A1 and IL7‐receptor‐α genes. Our data suggest that a finely tuned interplay between Notch3 and pre‐TCR pathways converges on regulation of NF‐κB activity, leading to differential NF‐κB subunit dimerization that regulates distinct gene clusters involved in either cell differentiation or proliferation/leukemogenesis.

Notch signaling and diseases: An evolutionary journey from a simple beginning to complex outcomes

Notch signaling pathway regulates a wide variety of cellular processes during development and it also plays a crucial role in human diseases. This important link is firmly established in cancer, since a rare T-ALL-associated genetic lesion has been initially reported to result in deletion of Notch1 ectodomain and constitutive activation of its intracellular region. Interestingly, the cellular response to Notch signaling can be extremely variable depending on the cell type and activation context. Notch signaling triggers signals implicated in promoting carcinogenesis and autoimmune diseases, whereas it can also sustain responses that are critical to suppress carcinogenesis and to negatively regulate immune response. However, Notch signaling induces all these effects via an apparently simple signal transduction pathway, diversified into a complex network along evolution from Drosophila to mammals. Indeed, an explanation of this paradox comes from a number of evidences accumulated during the last few years, which dissected the intrinsic canonical and non-canonical components of the Notch pathway as well as several modulatory extrinsic signaling events. The identification of these signals has shed light onto the mechanisms whereby Notch and other pathways collaborate to induce a particular cellular phenotype. In this article, we review the role of Notch signaling in cells as diverse as T lymphocytes and epithelial cells of the epidermis, with the main focus on understanding the mechanisms of Notch versatility.

Expression of metabotropic glutamate receptors in murine thymocytes and thymic stromal cells

RT-PCR combined with immunoblotting showed the expression of group-I (mGlu1 and 5) and group-II (mGlu2 and 3) metabotropic glutamate receptors in whole mouse thymus, isolated thymocytes and TC-1S thymic stromal cell line. Cytofluorimetric analysis showed that mGlu-5 receptors were absent in CD4(-)/CD8(-) but present in more mature CD4(+) CD8(+) and CD4(+)CD8(-) thymocytes. mGlu-1a receptors showed an opposite pattern of expression with respect to mGlu5, whereas mGlu2/3 receptor expression did not differ between double negative and double positive cells. mGlu receptors expressed in both thymic cell components were functional, as indicated by measurements of polyphosphoinositide hydrolysis or cAMP formation. These data suggest a possible role for mGlu receptor signalling in the thymus.

Notch3: from subtle structural differences to functional diversity.

The Notch3 gene was identified, at the beginning of 90s, as the third mammalian Notch and was initially reported as being expressed in proliferating neuroepithelium. Since then, increasing evidence has demonstrated a number of structural and functional differences between Notch3 and both Notch1 and Notch2, which exhibit the highest structural similarity among the four mammalian Notch receptors. Possibly due to its more restricted tissue distribution, targeted deletion of murine Notch3 does not lead to embryonic lethality as is observed with targeted deletion of Notch1 and Notch2. However, genetic mutation, amplification and deregulated expression of Notch3 have been correlated with the disruption of cell differentiation in transgenic mice and to development of diseases in mice and humans. This review discusses the possible relationships between the structural differences and the nonredundant roles that Notch3 plays in the pathogenesis of the human disease cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy and in the regulation of murine T-cell differentiation and leukemogenesis.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)Cθ is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-κB activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pTα/pre-TCR and constitutive activation of NF-κB characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKCθ is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-κB activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKCθ in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-κB activation. This paper therefore suggests that PKCθ mediates the activation of NF-κB by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

Aspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4-Mediated Transport Evidence of a Reduced Drug Action in Patients After Coronary Artery Bypass Grafting

OBJECTIVES In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production). CONCLUSIONS Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

Notch3 and canonical NF-κB signaling pathways cooperatively regulate Foxp3 transcription

Notch3 overexpression has been previously shown to positively regulate the generation and function of naturally occurring regulatory T cells and the expression of Foxp3, in cooperation with the pTα/pre-TCR pathway. In this study, we show that Notch3 triggers the trans activation of Foxp3 promoter depending on the T cell developmental stage. Moreover, we discovered a novel CSL/NF-κB overlapping binding site within the Foxp3 promoter, and we demonstrate that the activation of NF-κB, mainly represented by p65-dependent canonical pathway, plays a positive role in Notch3-dependent regulation of Foxp3 transcription. Accordingly, the deletion of protein kinase Cθ, which mediates canonical NF-κB activation, markedly reduces regulatory T cell number and per cell Foxp3 expression in transgenic mice with a constitutive activation of Notch3 signaling. Collectively, our data indicate that the cooperation among Notch3, protein kinase Cθ, and p65/NF-κB subunit modulates Foxp3 expression, adding new insights in the understanding of the molecular mechanisms involved in regulatory T cell homeostasis and function.

Clinical ResearchAntiplatelet TherapyAspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4–Mediated Transport: Evidence of a Reduced Drug Action in Patients After Coronary Artery Bypass Grafting

OBJECTIVES In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production). CONCLUSIONS Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

Inheritance of ribosomal gene activity and level of DNA methylation of individual gene clusters in a three generation family

SummaryRibosomal gene activity and levels of DNA methylation were investigated by cytochemical and immunological methods in the nucleolar organizer regions (NORs) of individually recognised acrocentric chromosomes. Mendelian inheritance of ribosomal gene activity in a three generation family was demonstrated, together with consistent behaviour of individual gene clusters in different carriers, even when environmental conditions were changed. For most chromosomes, an inverse relationship between gene activity and the level of DNA methylation was observed. Exceptions were the two chromosomes 15 and chromosomes 13cp and 22p, all being strongly chromomycin-A3-positive in their short arms. These chromosomes bound to anti-5-MeC antibodies with differential frequencies in the different carriers. The possibility of involvement of repetitive GC-rich DNA in this behaviour is discussed.