A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A

Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/CCdh1. Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/CCdh1-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis

Nitroxyl (HNO) Stimulates Soluble Guanylyl Cyclase to Suppress Cardiomyocyte Hypertrophy and Superoxide Generation

Background: New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NON attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated. Methods: Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli’s salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 mmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. Results: We now demonstrate that Angeli’s salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and b-myosin heavy chain expression. Angeli’s salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli’s salt were mimicked by BNP. We also demonstrate that the effects of Angeli’s salt are specifically mediated by HNO (with no role for NON or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependen

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged <it>in vivo </it>with <it>S. aureus</it>, <it>E. coli</it>, and <it>S. uberis</it>, samples from goats challenged <it>in vivo </it>with <it>S. aureus</it>, as well as cattle macrophages and ovine dendritic cells infected <it>in vitro </it>with <it>S. aureus</it>. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific.</p> <p>Results</p> <p>Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including <it>XBP1 </it>and <it>SREBF1</it>.</p> <p>The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of <it>E. coli </it>and <it>S. aureus </it>infections in cattle <it>in vivo </it>revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that <it>E. coli </it>caused a stronger host response.</p> <p>Conclusions</p> <p>This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.</p

BNP mimics the antihypertrophic and cGMP-dependent cardiomyocyte effects of Angeli's salt.

<p>BNP (1 µmol/L, over 48 h) prevents Ang II (1 µmol/L)-stimulated cardiomyocyte hypertrophy. This is evident on both <b>A</b> cell size (n = 11 myocyte preparations); and <b>B </b><i>de novo</i> protein synthesis (n = 9 myocyte preparations, P&lt;0.001); in addition to <b>C</b> cardiomyocyte superoxide generation (n = 11 myocyte preparations). <b>D</b> These antihypertrophic actions of BNP are blocked by the cGK-I inhibitor KT5823 (KT, 250 nmol/L, n = 6 myocyte preparations). <b>E</b> BNP acutely stimulates cardiomyocyte cGMP accumulation, over 5 min and 15 min (both n = 5 myocyte preparations). <b>F</b> Furthermore, 8BrcGMP (1 mmol/L, n = 4) also mimics the ROS-suppressing actions of both AS and BNP. *P&lt;0.05, **P&lt;0.01 and ***P&lt;0.001 vs control; ^#P&lt;0.05, ^##P&lt;0.01 and ^###P&lt;0.001 vs Ang II alone, ^†P&lt;0.05 vs Ang II+BNP.</p

NaOH (0.01 mol/L), the vehicle used for Angeli's salt, does not affect neonatal rat cardiomyocyte responses, alone or in the presence of Ang II (1 µmol/L).

*<p>P&lt;0.05 and</p>**<p>P&lt;0.01 vs control.</p

Angeli's salt also blunts endothelin-1 (ET₁)-induced cardiomyocyte responses.

<p>AS (1 µmol/L, added 4×/day over 48 h) inhibits ET₁ (60 nmol/L)-stimulated actions in neonatal cardiomyocytes. This is evident on <b>A</b> cardiomyocyte area (n = 3 myocyte preparations); <b>B</b> cardiomyocyte NADPH oxidase activity, on ET₁-induced superoxide generation (lucigenin chemiluminescence, n = 7 myocyte preparations); and <b>C</b> cardiomyocyte Nox2 NADPH oxidase gene expression, induced by ET₁ (n = 4 myocyte preparations). *P&lt;0.05 vs control; ^#P&lt;0.05 vs ET₁ alone.</p

Antihypertrophic actions of Angeli's salt.

<p>Ang II (1 µmol/L, 48 h)-stimulated cardiomyocyte hypertrophy is abolished by Angeli's salt (AS, 1 µmol/L, added 4×/day over 48 h). This is evident on <b>A</b> cardiomyocyte area (n = 10 myocyte preparations); <b>B </b><i>de novo</i> protein synthesis (on [³H]phenylalanine incorporation, n = 9 myocyte preparations); and <b>C</b> hypertrophic gene expression (using the fetal isoform of the contractile protein, β-myosin heavy chain, n = 6 myocyte preparations). *P&lt;0.05 and ***P&lt;0.001 vs control; ^#P&lt;0.05 and ^###P&lt;0.001 vs Ang II alone.</p

Mechanism of antihypertrophic action of HNO in cardiomyocytes.

<p>Angeli's salt utilizes HNO/sGC/cGMP/cGK-I signaling to suppress key triggers of the hypertrophic response, including expression and activity of NADPH oxidase (Nox2 subunit, a major source of reactive oxygen species, ROS) and activity of p38MAPK (the latter possibly as a result of enhanced activity of MAPK phosphatase-1, MKP-1). Activity of the cell survival kinase Akt (and its downstream target GSK-3β) remain intact in the presence of Angeli's salt. Cardiomyocyte hypertrophic responses across cell size, <i>de novo</i> protein synthesis and upregulated expression of β-myosin heavy chain are all ameliorated by HNO in the face of preserved cardiomyocyte ERK1/2 activation. Both the antihypertrophic and antioxidant actions of HNO are mediated via serial activation of sGC, cGMP production and cGK-I stimulation. Dashed lines indicate sites of inhibition. See text for references.</p

Impact of Angeli's salt on cardiomyocyte pro-hypertrophic signaling.

<p>AS (1 µmol/L, added 4×/day over 48 h) selectively inhibits Ang II (1 µmol/L, final 10 min)-stimulated p38MAPK phosphorylation of pro-hypertrophic signaling. Ang II-stimulated phosphorylation of ERK1/2 and Akt (and its downstream target GSK-3β) are preserved. Phosphorylation of <b>A</b> ERK1/2 (n = 8 myocyte preparations); <b>B</b> p38MAPK (n = 7 myocyte preparations); <b>C</b> Akt (n = 5 myocyte preparations, P&lt;0.05); and <b>D</b> GSK-3β (n = 9 myocyte preparations, P&lt;0.01), all as a ratio of total kinase. Representative images for phospho- and total kinases (from the same blot) are shown in the inset of each panel. *P&lt;0.05 and ***P&lt;0.001 vs control; ^#P&lt;0.05 and ^###P&lt;0.001 vs Ang II alone.</p