Paola Grazioli

Notch3 and the Notch3-upregulated RNA-binding protein HuD regulate Ikaros alternative splicing

Constitutive activation of the transmembrane receptor, Notch3, and loss of function of the hematopoietic transcription repressor, Ikaros (IK), play direct roles in T‐cell differentiation and leukemogenesis that are dependent on pre‐T‐cell receptor (pre‐TCR) signaling. We demonstrate the occurrence of crosstalk between Notch3 and IK that results in transcriptional regulation of the gene encoding the pTα chain of the pre‐TCR. We also show that, in the presence of the pre‐TCR, constitutive activation of Notch3 in thymocytes causes increased expression of dominantnegative non‐DNA‐binding IK isoforms, which are able to restrain the IK inhibition of Notch3s transcriptional activation of pTα. This effect appears to be mediated by Notch3s pre‐TCR‐dependent upregulation of the RNA‐binding protein, HuD. Notch3 signaling thus appears to play a critical role in the diminished IK activity described in several lymphoid leukemias. By exerting transcription‐activating and transcription‐repressing effects on the pTα promoter, Notch3 and IK cooperate in the fine‐tuning of pre‐TCR expression and function, which has important implications for the regulation of thymocyte differentiation and proliferation.

The chemokine Bv8/prokineticin 2 is up-regulated in inflammatory granulocytes and modulates inflammatory pain

Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freunds adjuvant-induced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced up-regulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain.

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.

Scleroderma Mesenchymal Stem Cells display a different phenotype from healthy controls; implications for regenerative medicine

IntroductionVascular involvement is a key feature of Systemic sclerosis (SSc). Although the pericytes/endothelial cells (ECs) cross-talk regulates vessels formation, no evidences about the pericytes contribution to ineffective angiogenesis in SSc are available. Recent findings showed similarities between pericytes and Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Due to difficulties in pericytes isolation, this work explores the possibility to use BM-MSCs as pericytes surrogate, clarifying their role in supporting neo-angiogenesis during SSc.MethodsTo demonstrate their potential to normally differentiate into pericytes, both SSc and healthy controls (HC) BM-MSCs were treated with TGF-β and PDGF-BB. The expression of pericytes specific markers (α-SMA, NG2, RGS5 and desmin) was assessed by qPCR, western blot, and immunofluorescence; chemioinvasion and capillary morphogenesis were also performed. Cell-sorting of BM-MSCs co-cultured with HC-ECs was used to identify a possible change in contractile proteins genes expression.ResultsWe showed that BM-MSCs isolated from SSc patients displayed an up-regulation of α-SMA and SM22α genes and a reduced proliferative activity. Moreover during SSc, both TGF-β and PDGF-BB can specifically modulate BM-MSCs toward pericytes. TGF-β was found interfering with the PDGF-BB effects. Using BM-MSCs/MVECs co-culture system we observed that SSc BM-MSCs improve ECs tube formation in stressed condition, and BM-MSCs, sorted after co-culture, showed a reduced α-SMA and SM22α gene expression.ConclusionsBM-MSCs from SSc patients behave as pericytes. They display a more mature and myofibroblast-like phenotype, probably related to microenvironmental cues operating during the disease. After their co-culture with HC-MVECs, SSc BM-MSCs underwent to a phenotypic modulation which re-programs these cells toward a pro-angiogenic behaviour.

Acetylation controls Notch3 stability and function in T-cell leukemia

Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R1692−1731 mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R1692−1731 mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.

Mouse Sertoli Cells Sustain De Novo Generation of Regulatory T Cells by Triggering the Notch Pathway Through Soluble JAGGED1

ABSTRACT FOXP3+ regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4+CD25−EGFP− T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4+FOXP3− T cells into functional CD4+FOXP3+ Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.

Targeting estrogen receptor β as preventive therapeutic strategy for Leber's hereditary optic neuropathy

Lebers hereditary optic neuropathy (LHON) is a maternally inherited blinding disease characterized by degeneration of retinal ganglion cells (RGCs) and consequent optic nerve atrophy. Peculiar features of LHON are incomplete penetrance and gender bias, with a marked male prevalence. Based on the different hormonal metabolism between genders, we proposed that estrogens play a protective role in females and showed that these hormones ameliorate mitochondrial dysfunction in LHON through the estrogen receptors (ERs). We also showed that ERβ localize to the mitochondria of RGCs. Thus, targeting ERβ may become a therapeutic strategy for LHON specifically aimed at avoiding or delaying the onset of disease in mutation carriers. Here, we tested the effects of ERβ targeting on LHON mitochondrial defective metabolism by treating LHON cybrid cells carrying the m.11778G>A mutation with a combination of natural estrogen-like compounds that bind ERβ with high selectivity. We demonstrated that these molecules improve cell viability by reducing apoptosis, inducing mitochondrial biogenesis and strongly reducing the levels of reactive oxygen species in LHON cells. These effects were abolished in cells with ERβ knockdown by silencing receptor expression or by using specific receptor antagonists. Our observations support the hypothesis that estrogen-like molecules may be useful in LHON prophylactic therapy. This is particularly important for lifelong disease prevention in unaffected LHON mutation carriers. Current strategies attempting to combat degeneration of RGCs during the acute phase of LHON have not been very effective. Implementing a different and preemptive approach with a low risk profile may be very helpful.

Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL.

Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

Notch3 and pTα signaling events are essential for T-cell leukemogenesis and characterize murine and human T-cell acute lymphoblastic leukemia. Genetic ablation of pTα expression in Notch3 transgenic mice abrogates tumor development, indicating that pTα signaling is crucial to the Notch3-mediated leukemogenesis. Here we report a novel direct interaction between Notch3 and pTα. This interaction leads to the recruitment and persistence of the E3 ligase protein c-Cbl to the lipid rafts in Notch3-IC transgenic thymocytes. Conversely, deletion of pTα in Notch3 transgenic mice leads to cytoplasmic retention of c-Cbl that targets Notch3 protein to the proteasomal-degradative pathway. It appears that protein kinase C θ (PKCθ), by regulating tyrosine and serine phosphorylation of Cbl, is able to control its function. We report here that the increased Notch3-IC degradation correlates with higher levels of c-Cbl tyrosine phosphorylation in Notch3-IC/pTα−/− double-mutant thymocytes, which also display a decreased PKCθ activity. Our data indicate that pTα/pre-T-cell receptor is able to regulate the different subcellular localization of c-Cbl and, by regulating PKCθ activity, is also able to influence its ubiquitin ligase activity upon Notch3 protein.

Notch and Ikaros: Not Only Converging Players in T Cell Leukemia

Notch3 overexpression has been observed in virtually 100% of T cell acute lymphoblastic leukemia (T-ALL). A high percentage of infant B- and T-ALLs also display an increased expression of non DNA-binding Ikaros isoforms. It has been suggested that increased expression of non DNA-binding Ikaros isoforms and constitutively activated Notch play cooperative role in leukemogenesis, converging on the transcriptional regulation of one or more key genes. Thus far however no demonstration of a direct link between aberrant Notch signalling and altered Ikaros isoform expression has been reported. We recently suggested that pre-TCR is the missing link between Notch and Ikaros in T cell leukemogenesis. Our studies demonstrate that the presence of pre-TCR is required to sustain a Notch3-induced altered expression of spliced Ikaros isoforms. Moreover, we identified HuD, an RNA-binding protein able to regulate both mRNA stability and alternative splicing, as the potential pre-TCR-dependent mediator of Notch3 activity. HuD is able to dysregulate the expression pattern of Ikaros isoforms, thus favouring the shift towards non DNA-binding Ikaros isoforms. We finally showed that the increased expression of non DNA-binding Ikaros isoforms is able to restrain the inhibition exerted by Ikaros on Notch3-dependent transcriptional activation of pTalphapromoter, thus resulting in its significant up-regulation.Our findings may help in clarifying the regulatory mechanism of Ikaros alternative splicing and suggest a crosstalk among Notch3, pre-TCR signalling and spliced Ikaros variants in T cell leukemogenesis, mediated by HuD.