Differentially Expressed Proteins in Malignant and Benign Adrenocortical Tumors

<div><p>We have compared the microsomal protein composition of eight malignant and six benign adrenocortical tumors with proteomic methods. IGF2 had increased level in the malignant tumors, confirming previous microarray studies on the same material. Aldolase A, a glycolytic enzyme, also showed increased levels in the malignant tissue compared to the benign. Additionally, several proteins belonging to complex I in the mitochondrial respiration chain showed decreased levels in the malignant tissue. Taken together, this may indicate a shift in energy metabolism where glycolysis may be favored over tight coupling of glycolysis and mitochondrial respiration, a phenomenon known as the Warburg effect. One of the complex I proteins that showed decreased levels in the malignant tissue was GRIM-19. This protein has been suggested as a tumor suppressive protein by being a negative regulator of STAT3. In summary, an analysis of the microsomal proteome in adrenocortical tumors identifies groups of proteins as well as specific proteins differentially expressed in the benign and malignant forms. These proteins shed light on the biology behind malignancy and could delineate future drug targets.</p></div

Western blot and immunohistochemical analyses.

<p>A–D) Expression levels of aldolase A and GRIM-19. To the left are the expression levels from the MS data and to the right are the western blot analyses. Ku70 and actin are loading controls. Western blot analyses were performed on tissue samples that showed the most significant differences in the iTRAQ experiments (two ACAs and two ACCs). E–F) Immunohistochemical analyses with anti-GRIM-19 (panels E and F). GRIM-19 staining in ACAs had a grain-like pattern, suggesting mitochondrial localization (panel E). In ACCs there was a more cytoplasmic staining (panel F).</p

Visualization of the data analysis workflow.

<p>A) Both univariate (Student's t-test) and multivariate (OPLS) analyses were performed. B) The top canonical pathway identified by Ingenuity Pathway Analysis was mitochondrial dysfunction. Many proteins in this pathway were found to be downregulated in the malignant samples. C) Hierarchical clustering of the overlapping proteins (t-test and OPLS). Class 1: ACA, class 2: ACC.</p

Correlation between protein expression levels and tumor size.

<p>Protein expression levels of the 26 proteins that overlapped in the t-test and OPLS analyses correlate with the size of the tumors. Two proteins have increased expression levels (light grey dots/lines), the rest have decreased expression levels (black dots/lines). Corresponding protein names can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087951#pone.0087951.s005" target="_blank">Table S4</a>.</p

Enrichment analysis.

<p>Panel A shows a pie chart depicting the number of transmembrane segments the 1902 identified and quantified proteins are predicted to contain (from ProteinCenter). Panel B shows the GOrilla results performed on the 1902 proteins, ranked by t-test p-value. Shown are enriched GO terms in “cellular component”.</p

Clinical data of analyzed cases.

a<p><i>Time between surgery and follow-up.</i></p>b<p><i>Dead of disease.</i></p

Mass Spectrometry-Based Proteomics Identifies UPF1 as a Critical Gene Expression Regulator in MonoMac 6 Cells

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Recently, we have demonstrated that 5-LO mRNA expression is regulated by alternative splicing and nonsense-mediated mRNA decay (NMD). In addition to this, 5-LO protein expression was reduced on translational level in UPF1 knockdown cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry-based proteomics study was performed to identify compartment-specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis revealed that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (∼21%) but not in the cytosolic fraction (<1%). The results suggest that UPF1 is a critical gene expression regulator in a compartment-specific way. During differentiation by TGFβ and calcitriol, the majority of UPF1 regulated proteins were adjusted to normal level. This indicates that the translational regulation by UPF1 can potentially be cell differentiation-dependent

Microsomal prostaglandin E synthase-1 gene deletion impairs neuro-immune circuitry of the cholinergic anti-inflammatory pathway in endotoxaemic mouse spleen

<div><p>The cholinergic anti-inflammatory pathway (CAP) is an innate neural reflex where parasympathetic and sympathetic nerves work jointly to control inflammation. Activation of CAP by vagus nerve stimulation (VNS) has paved way for novel therapeutic strategies in treating inflammatory diseases. Recently, we discovered that VNS mediated splenic acetylcholine (ACh) release and subsequent immunosuppression in response to LPS associated inflammation is impaired in mice lacking microsomal prostaglandin E synthase-1 (mPGES-1) expression, a key enzyme responsible for prostaglandin E2 synthesis. Here, we have further investigated the consequences of mPGES-1 deficiency on various molecular/cellular events in the spleen which is critical for the optimal functioning of VNS in endotoxaemic mice. First, VNS induced splenic norepinephrine (NE) release in both mPGES-1 (+/+) and (-/-) mice. Compared to mPGES-1 (+/+), immunomodulatory effects of NE on cytokines were strongly compromised in mPGES-1 (-/-) splenocytes. Interestingly, while LPS increased choline acetyltransferase (ChAT) protein level in mPGES-1 (+/+) splenocytes, it failed to exert similar effects in mPGES-1 (-/-) splenocytes despite unaltered β₂ AR protein expression. In addition, nicotine inhibited TNFα release by LPS activated mPGES-1 (+/+) splenocytes <i>in vitro</i>. However, such immunosuppressive effects of nicotine were reversed both in mPGES-1 (-/-) mouse splenocytes and human PBMC treated with mPGES-1 inhibitor. In summary, our data implicate PGE2 as an important mediator of ACh synthesis and noradrenergic/cholinergic molecular events in the spleen that constitute a crucial part of the CAP immune regulation. Our results suggest a possible link between cholinergic and PG system of CAP that may be of clinical significance in VNS treatment.</p></div

Lipopolysaccharide activated splenocytes lacking mPGES-1 gene expression display an altered response to NE stimulation.

<p>Primary splenocyte cultures established from mPGES-1 (+/+) and (-/-) mice were pretreated with norepinephrine (NE) at 1, 10 and 100 μM concentration for 30 mins and then activated with the endotoxin, LPS (100ng/ml). Cell supernatants were analyzed for cytokine production following 3 hours of treatment. ^a,b p<0.0001; LPS versus LPS+NE within WT or KO; One-way ANOVA. ^# p<0.05; mPGES-1(+/+) versus mPGES-1 (-/-) within LPS+NE treatment; student’s T-test. Each sample was run as duplicates during the assay and values are represented as mean ±SEM from 3 independent experiments.</p

beta 2 Adrenergic receptor expression analysis by flow cytometry on CD4+ T cells isolated from mPGES-1 KO or WT mouse splenocytes

Flow cytometric data from five experiments analyzing beta-2-adrenergic receptor expression on CD4+ T cells (MACS positive selection from spleen) from mPGES-1 ko (-/-) or WT (+/+) mice subjected to vagus nerve stimulation (VNS) and intraperitoneal LPS injection 30 min prior to sample collection.<br><br><u>Panel</u> (<i>more detailed info can be found in the "FCS file identification list" document</i>):<br>FITC-Viability<br>PE-b2AR<br>PCPCy5.5-CD69<br>PECy7-CD62L<br>APC-CD44<br>APCCy7-CD3<br>BV421-CD25<br>BV510-CD4<br><br>The protocol for staining, cell extraction VNS etc. is described in the publication "<u>Microsomal prostaglandin E synthase-1 gene deletion impairs neuro-immune circuitry of the cholinergic anti-inflammatory pathway in endotoxaemic mpuse spleen</u>" published in <b>PLOS ONE 2017</b> by the authors: Priya Revathikumar, Johanna Estelius, Utsa Karmakar, Erwan LeMaître, Marina Korotkova, Per-Johan Jakobsson and Jon Lampa. <br><br><br