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.

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/R(1692-1731) 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/R(1692-1731) 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.

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

Notch3 and pTalpha signaling events are essential for T-cell leukemogenesis and characterize murine and human T-cell acute lymphoblastic leukemia. Genetic ablation of pTalpha expression in Notch3 transgenic mice abrogates tumor development, indicating that pTalpha signaling is crucial to the Notch3-mediated leukemogenesis. Here we report a novel direct interaction between Notch3 and pTalpha. 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 pTalpha 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 theta (PKCtheta), 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/pTalpha(-/-) double-mutant thymocytes, which also display a decreased PKCtheta activity. Our data indicate that pTalpha/pre-T-cell receptor is able to regulate the different subcellular localization of c-Cbl and, by regulating PKCtheta activity, is also able to influence its ubiquitin ligase activity upon Notch3 protein.

[Italian translation and validation of the Nordic IRSST standardized questionnaire for the analysis of musculoskeletal symptoms]. / Traduzione in lingua italiana e validazione del questionario standardizzato Nordic IRSST per la rilevazione di disturbi muscoloscheletrici.

BACKGROUND: Data on self-reported symptoms and/or functional impairments are important in research on work-related musculoskeletal disorders (WRMSDs). In such cases the availability of international standardized questionnaires is extremely important since they permit comparison of studies performed in different Countries. OBJECTIVES: Translation into Italian and validation of the Nordic Musculoskeletal Questionnaire (NMQ), a tool which is widely used in studies on WRMSDs in the international scientific literature. METHODS: The extended Canadian version of the NMQ was translated into Italian. As per the current guidelines, the cross-cultural adaptation was performed by translation of the items from French, back-translation by independent mother-tongue translators and committee review. The resulting version of the questionnaire underwent pre-testing in 3 independent groups of subjects. The comprehensibility, reliability (internal consistency and reproducibility) and sensitivity were evaluated. RESULTS: After translation/back-translation and review of the items the comprehensibility of the Italian version of the questionnaire was judged good in a group of 40 workers. The internal consistency was evaluated using the Cronbach's Alpha test in the same group and in another 98 engineering workers: the results were on the whole acceptable. The reproducibility, which was tested with Cohen's Kappa test in the 40 workers, was good/excellent. In a preliminary evaluation, performed in 30 outpatients of a of Rehabilitation Service, sensitivity was very good. CONCLUSIONS: The results show that the Italian version of the Nordic Musculoskeletal Questionnaire is valid for self-administration and can be applied in 'field" studies on self-reported musculoskeletal symptoms and functional impairments in group of workers.

[The Italian version of Nordic Musculoskeletal Standardized Questionnaire]. / Versione in lingua Italiana del questionario standardizzato autocompilabile Nordic IRSST per la rile vazione di disturbi muscoloscheletrici .

We translated into Italian the Nordic musculoskelethal questionnaire, as completed by Canadian IRSST with Authors' agreement in 2001, according to OMS recommendations. This translation involved the following items: aches and troubles of neck, dorsal region, low back, shoulders, elbows, hands and wrists, hips and thighs, ankles and feet in the last 12 months. The questionnaire was then submitted to reliability and stability tests. The Italian version of the questionnaire, already used in different languages, proved to be suitable and reliable also for self administration.

[Assessment of the risk of occupational biomechanical overload: comparison of methods currently used]. / La valutazione del rischio da sovraccarico biomeccanico occupazionale: confronto tra metodi di corrente utilizzo.

Some of the most common methods for the evaluation of the ergonomic risk of Work-Related Musculo Skeletal Disorders were applied to different workplaces. The results show that an evaluation of the single components of the synthetic risk-indices given by the methods is needed to evidence the specific critical aspects.