A novel nuclear function for the centrosomal serine/threonine kinase Nek2

Numerose chinasi che regolano il ciclo centrosomale sono frequentemente regolate in maniera aberrante nelle cellule neoplastiche. Cambiamenti nella loro espressione possono portare ad alterazioni nella duplicazione del centrosoma quindi aneuploide. I tumori delle cellule germinali testicolari sono caratterizzati da amplificazione dei centrosomi la cui causa è ancora largamente sconosciuta. Abbiamo individuato una nuova chinasi centrosomale, Nek2, che risulta up-regolata nei seminomi testicolari in maniera specifica e ne abbiamo caratterizzato la sua funzione nelle cellule germinali neoplastiche. Una scoperta inaspettata del nostro studio è stata la presenza di Nek2 nel nucleo delle cellule germinali di seminoma testicolare, localizzazione ritrovata anche nelle cellule di linea derivanti dai seminomi: le Tcam-2. Inoltre tale localizzazione è stata ritrovata nelle cellule germinali da cui i seminomi derivano: le cellule germinali primordiali e gli spermatogoni più indifferenziati. Tali risultati suggeriscono un ruolo di Nek2 come marcatore di staminalità delle cellule germinali maschili, mantenuto anche nella trasformazione neoplastica. Abbiamo inoltre dimostrato che la localizzazione nucleare di Nek2 non è una caratteristica peculiare dei seminomi testicolari ma è principalmente localizzata nel nucleo in diversi tipi di tumore. Nelle cellule tumorali inoltre Nek2 è presente in splicing speckles dove associa con numerosi fattori di splicing come la proteina SR (ASF/SF2), hnRNP (A1, F and H), e la proteina STAR Sam68. Il nostro studio dimostra che Nek2 è in grado di fosforilare Sam68 e di modularne l’attività di splicing. Questi risultati identificano un nuovo ruolo nucleare per Nek2Protein kinases that regulate the centrosome cycle are often aberrantly regulated in neoplastic cells. Changes in their expression or activity can lead to perturbations in centrosome duplication and aneuploidy. In addition, many centrosomal protein kinases participate to other aspects of cell cycle progression. Testicular germ cell tumors (TGCTs) are characterized by amplification of centrosomes through unknown mechanisms. We have discovered that the centrosomal kinase Nek2 is overexpressed in testicular seminomas and we have characterized its function in neoplastic germ cells. One unexpected finding of our study was the nuclear localization of Nek2 in germ cells of patients. The same nuclear localization was observed in the seminoma cell line Tcam-2. We found that Nek2 was localized in the nucleus also in undifferentiated embryonal male primordial germ cells (PGCs) and in spermatogonial stem cells from post-natal testis. These results suggest that nuclear Nek2 is a novel marker of the undifferentiated stage of male germ cells that is maintained in testicular seminomas, but not in other TGCTs. The nuclear localization of Nek2 is not a unique feature of testicular seminomas, we show that Nek2 is mainly distributed in the nucleus of cancer cells from other tissues, including breast, prostate and colon cancer cells. The subnuclear distribution of Nek2 in speckles closely resembled that of many regulators of pre-mRNA splicing. We found that Nek2 physically associates with several splicing factors, such as SR proteins (ASF/SF2), hnRNPs (A1, F and H), and the STAR protein Sam68. We focused our study on Sam68 because this splicing regulator is also up-regulated in breast and prostate carcinomas like Nek2. Our study shows that Sam68 is also overexpressed in testicular seminomas but not in other TGCTs, like Nek2. Moreover, Nek2 phosphorylates Sam68 and affects Sam68-dependent splicing of CD44v5 pre-mRNA, an alternatively spliced form of the receptor, frequently altered in cancer cells, that promotes cell proliferation and invasiveness. These results identify a novel nuclear function of Nek2 and suggest that modulation of alternative splicing events by this kinase can contribute to neoplastic transformatio

A novel nuclear function for the centrosomal serine/threonine kinase Nek2

Numerose chinasi che regolano il ciclo centrosomale sono frequentemente regolate in maniera aberrante nelle cellule neoplastiche. Cambiamenti nella loro espressione possono portare ad alterazioni nella duplicazione del centrosoma quindi aneuploide. I tumori delle cellule germinali testicolari sono caratterizzati da amplificazione dei centrosomi la cui causa è ancora largamente sconosciuta. Abbiamo individuato una nuova chinasi centrosomale, Nek2, che risulta up-regolata nei seminomi testicolari in maniera specifica e ne abbiamo caratterizzato la sua funzione nelle cellule germinali neoplastiche. Una scoperta inaspettata del nostro studio è stata la presenza di Nek2 nel nucleo delle cellule germinali di seminoma testicolare, localizzazione ritrovata anche nelle cellule di linea derivanti dai seminomi: le Tcam-2. Inoltre tale localizzazione è stata ritrovata nelle cellule germinali da cui i seminomi derivano: le cellule germinali primordiali e gli spermatogoni più indifferenziati. Tali risultati suggeriscono un ruolo di Nek2 come marcatore di staminalità delle cellule germinali maschili, mantenuto anche nella trasformazione neoplastica. Abbiamo inoltre dimostrato che la localizzazione nucleare di Nek2 non è una caratteristica peculiare dei seminomi testicolari ma è principalmente localizzata nel nucleo in diversi tipi di tumore. Nelle cellule tumorali inoltre Nek2 è presente in splicing speckles dove associa con numerosi fattori di splicing come la proteina SR (ASF/SF2), hnRNP (A1, F and H), e la proteina STAR Sam68. Il nostro studio dimostra che Nek2 è in grado di fosforilare Sam68 e di modularne l’attività di splicing. Questi risultati identificano un nuovo ruolo nucleare per Nek2Protein kinases that regulate the centrosome cycle are often aberrantly regulated in neoplastic cells. Changes in their expression or activity can lead to perturbations in centrosome duplication and aneuploidy. In addition, many centrosomal protein kinases participate to other aspects of cell cycle progression. Testicular germ cell tumors (TGCTs) are characterized by amplification of centrosomes through unknown mechanisms. We have discovered that the centrosomal kinase Nek2 is overexpressed in testicular seminomas and we have characterized its function in neoplastic germ cells. One unexpected finding of our study was the nuclear localization of Nek2 in germ cells of patients. The same nuclear localization was observed in the seminoma cell line Tcam-2. We found that Nek2 was localized in the nucleus also in undifferentiated embryonal male primordial germ cells (PGCs) and in spermatogonial stem cells from post-natal testis. These results suggest that nuclear Nek2 is a novel marker of the undifferentiated stage of male germ cells that is maintained in testicular seminomas, but not in other TGCTs. The nuclear localization of Nek2 is not a unique feature of testicular seminomas, we show that Nek2 is mainly distributed in the nucleus of cancer cells from other tissues, including breast, prostate and colon cancer cells. The subnuclear distribution of Nek2 in speckles closely resembled that of many regulators of pre-mRNA splicing. We found that Nek2 physically associates with several splicing factors, such as SR proteins (ASF/SF2), hnRNPs (A1, F and H), and the STAR protein Sam68. We focused our study on Sam68 because this splicing regulator is also up-regulated in breast and prostate carcinomas like Nek2. Our study shows that Sam68 is also overexpressed in testicular seminomas but not in other TGCTs, like Nek2. Moreover, Nek2 phosphorylates Sam68 and affects Sam68-dependent splicing of CD44v5 pre-mRNA, an alternatively spliced form of the receptor, frequently altered in cancer cells, that promotes cell proliferation and invasiveness. These results identify a novel nuclear function of Nek2 and suggest that modulation of alternative splicing events by this kinase can contribute to neoplastic transformatio

PDE5 inhibitors in type 2 diabetes cardiovascular complications

Pharmacological inhibition of Phosphodiesterase type 5 (PDE5) proved its efficacy treating several pathological conditions, such as erectile dysfunction and pulmonary hypertension. Nowadays, its benefits on cardiovascular diseases are well documented, particularly in the treatment of type 2 diabetes (T2DM)-related cardiovascular complications. In this context, treatment of T2DM with PDE5 inhibitors, such as sildenafil, tadalafil or vardenafil ameliorates endothelial dysfunction both in patients and animal models through an augmented flow mediated dilation rate and an up-regulation of endothelial markers; it also reduces the inflammatory state by down-regulating inflammatory cytokines expression and improves diabetic cardiomyopathy and ischemia-reperfusion injury mainly through the activation of NO-cGMP-PKG pathway. The present review summarizes the state of art on PDE5 inhibition in the treatment of cardiovascular complications in T2DM

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved

Carbon balance and energy fluxes of a Mediterranean crop

This paper is based on the analysis of a long-term mass (carbon dioxide, water vapour) and energy (solar radiation) balance monitoring programme carried out during years 2010 and 2012 in an irrigated orange orchard in Sicily, using the Eddy Covariance (EC) method. Orange (Citrus sinensis L.) is one of the main fruit crops worldwide and its evergreen orchard may have a great potential for carbon sequestration, but few data are currently available. In the study, the role of the orchard system in sequestering atmospheric CO2 was analyzed, thus contributing to assess the carbon balance of the specie in the specific environment.Vertical energy fluxes of net radiation, soil heat, sensible heat and latent heat fluxes were measured at orchard scale by EC. Evapotranspiration (ET) values were compared with upscaled transpiration data determined by the sap flow heat pulse technique, evidencing the degree of correspondence between instantaneous transpirational flux at tree level and the micrometeorological measurement of ET at orchard level

Diagnostic value of qualitative and strain ratio elastography in the differential diagnosis of non-palpable testicular lesions

The purpose of this study was to evaluate prospectively the accuracy of qualitative and strain ratio elastography (SE) in the differential diagnosis of non-palpable testicular lesions. The local review board approved the protocol and all patients gave their consent. One hundred and six patients with non-palpable testicular lesions were consecutively enrolled. Baseline ultrasonography (US) and SE were correlated with clinical and histological features and ROC curves developed for diagnostic accuracy. The non-palpable lesions were all ≤1.5 cm; 37/106 (34.9%) were malignant, 38 (35.9%) were benign, and 31 (29.2%) were non-neoplastic. Independent risk factors for malignancy were as follows: size (OR 17.788; p = 0.002), microlithiasis (OR 17.673, p < 0.001), intralesional vascularization (OR 9.207, p = 0.006), and hypoechogenicity (OR, 11.509, p = 0.036). Baseline US had 89.2% sensitivity (95% CI 74.6-97.0) and 85.5% specificity (95% CI 75.0-92.8) in identifying malignancies, and 94.6% sensitivity (95% CI 86.9-98.5) and 87.1% specificity (95% CI 70.2-96.4) in discriminating neoplasms from non-neoplastic lesions. An elasticity score (ES) of 3 out of 3 (ES3, maximum hardness) was recorded in 30/37 (81.1%) malignant lesions (p < 0.001). An intermediate score of 2 (ES2) was recorded in 19/38 (36.8%) benign neoplastic lesions and in 22/31 (71%) non-neoplastic lesions (p = 0.005 and p = 0.001 vs. malignancies). None of the non-neoplastic lesions scored ES3. Logistic regression analysis revealed a significant association between ES3 and malignancy (χ2 = 42.212, p < 0.001). ES1 and ES2 were predictors of benignity (p < 0.01). Overall, SE was 81.8% sensitive (95% CI 64.8-92.0) and 79.1% specific (95% CI 68.3-88.4) in identifying malignancies, and 58.6% sensitive (95% CI 46.7-69.9) and 100% specific (95% CI 88.8-100) in discriminating non-neoplastic lesions. Strain ratio measurement did not improve the accuracy of qualitative elastography. Strain ratio measurement offers no improvement over elastographic qualitative assessment of testicular lesions; testicular SE may support conventional US in identifying non-neoplastic lesions when findings are controversial, but its added value in clinical practice remains to be proven

Characterization of three PDE5 isoforms in murine cardiomyocytes

Phosphodiesterase 5 (PDE5A) is responsible for hydrolysis of cGMP, a second messenger regulating many physiological functions in cardiac myocytes. PDE5A involvement in cardiac hypertrophy has been reported and the use of its inhibitor, sildenafil, has reverted the pathological increase of cardiac size in humans and in animal models (Nagendran et al., 2007). In humans, a single PDE5 gene encodes for three isoforms (PDE5A1, A2 and A3), which differ in their N-terminus being translated from alternative initiation sites (Lin et al., 2000). The isoforms exhibit specific tissue expression patterns and different sensitivities to pharmacological inhibitors. However, little is known about their specific biological roles. The existence of three murine PDE5A isoforms was predicted through human gene homology and confirmed by RT-PCR. Tissue expression pattern of each variant was uncovered by RT-PCR and western blot analysis. In adult heart, transcripts encoding for the three isoforms were detected. In cardiomyocytes primary cultures and cell lines PDE5A isoforms localization was revealed by fluorescence microscopy analysis and subcellular fractioning. Their phosphodiesterasic activities and sildenafil sensibilities were measured by radioactive assays. Finally, post-translational modifications were explored. Hypertrophic stimuli resulted in Ser 92 phosphorylation of PDE5A isoforms, possibly through by Protein Kinase A. In summary, the understanding of PDE5A isoforms localization and differential activation and activity might be an important step toward the improvement of the diagnostic, prognostic, and predictive values of PDE5A in hypertrophy treatment

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation

Molecular profiling of follicular fluid microRNAs in young women affected by Hodgkin lymphoma.

Research question Treatments for Hodgkin lymphoma have improved but one of their common effects is gonadal toxicity, which contributes to fertility damage of patients and induces temporary or irreversible loss of fertility. Could micro-RNA (miRNA) expression profiles in follicular fluid be influenced by Hodgkin lymphoma? Could their alteration affect molecular pathways involved in follicle growth and oocyte maturation? Design miRNA expression profile was investigated in follicular fluid samples from young women affected by Hodgkin lymphoma compared with healthy controls by NanoString technology. Bioinformatic analysis was used to verify miRNA involvement in follicle development and miRNA deregulation with Hodgkin lymphoma in a larger cohort of follicular fluid samples was confirmed by real-time quantitative polymerase chain reaction. Results Thirteen miRNAs are deregulated in Hodgkin lymphoma samples compared with controls and are involved in molecular pathways related to cancer, gametogenesis and embryogenesis. Among them, let-7b-5p, miR-423-5p, miR-503-5p, miR-574-5p and miR-1303 are implicated in biological processes related to follicle development and oocyte maturation. Let-7b-5p holds the central position in the regulatory network of miRNA-mRNA interactions, has the highest number of mRNA target genes shared with the other differentially expressed miRNAs and is significantly downregulated in Hodgkin lymphoma follicular fluid samples. Conclusions These data led us to question the potential influence of miRNA deregulation on oocyte quality. Further studies are needed to verify the reproductive potential of young patients with Hodgkin lymphoma before starting chemotherapy protocols and an adequate protocol of fertility preservation needs to be guaranteed