Phosphocholine – an agonist of metabotropic but not of ionotropic functions of alpha9-containing nicotinic acetylcholine receptors

We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1beta from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1beta is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1beta release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing alpha9 and alpha10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of alpha9 subunits or heteromeric receptors containing alpha9 and alpha10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-kappa B and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4

Relation of nNOS isoforms to mitochondrial density and PGC-1alpha expression in striated muscles of mice

The expression of neuronal NO synthase (nNOS) alpha- and beta-isoforms in skeletal muscle is well documented but only little information is available about their regulation/functions. Using different mouse models, we now assessed whether the expression of nNOS-isoforms in muscle fibers is related to mitochondria content/activity and regulated by peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Catalytic histochemistry revealed highest nNOS-concentrations to be present in type-2 oxidative muscle fibers. Differences in mitochondrial density between nNOS-KO-mice and WT-littermates established by morphometry after transmission electron microscopy were significant in the oxidative portion of the tibialis anterior muscle (TA) but not in rectus femoris muscle (RF) indicating an nNOS-dependent mitochondrial pool in TA. Quantitative immunoblotting displayed the nNOS alpha-isoform to preponderate in those striated muscles of C57BL/6-mice that comprise of many type-2 oxidative fibers, e.g. TA, while roughly even levels of the two nNOS-isoforms were expressed in those muscles that mainly consist of type-2 glycolytic fibers, e.g. RF. Differences in citrate synthase-activity in muscle homogenates between nNOS-KO-mice and WT-littermates were positively related to nNOS alpha-isoform levels. In transgenic-mice over-expressing muscular PGC-1alpha compared to WT-littermates, immunoblotting revealed a significant shift in nNOS-expression in favor of the alpha-isoform in six out of eight striated muscles (exceptions: soleus muscle and tongue) without consistent relationship to changes in the expression of mitochondrial markers. In summary, our study demonstrated the nNOS alpha-isoform expression to be related to mitochondrial content/activity and to be up-regulated by up-stream PGC-1alpha in striated muscles, particularly in those enriched with type-2 oxidative fibers implying a functional convergence of the two signaling systems in these fibers

SLPI Inhibits ATP-Mediated Maturation of IL-1β in Human Monocytic Leukocytes: A Novel Function of an Old Player

Interleukin-1β (IL-1β) is a potent, pro-inflammatory cytokine of the innate immune system that plays an essential role in host defense against infection. However, elevated circulating levels of IL-1β can cause life-threatening systemic inflammation. Hence, mechanisms controlling IL-1β maturation and release are of outstanding clinical interest. Secretory leukocyte protease inhibitor (SLPI), in addition to its well-described anti-protease function, controls the expression of several pro-inflammatory cytokines on the transcriptional level. In the present study, we tested the potential involvement of SLPI in the control of ATP-induced, inflammasome-dependent IL-1β maturation and release. We demonstrated that SLPI dose-dependently inhibits the ATP-mediated inflammasome activation and IL-1β release in human monocytic cells, without affecting the induction of pro-IL-1β mRNA by LPS. In contrast, the ATP-independent IL-1β release induced by the pore forming bacterial toxin nigericin is not impaired, and SLPI does not directly modulate the ion channel function of the human P2X7 receptor heterologously expressed in Xenopus laevis oocytes. In human monocytic U937 cells, however, SLPI efficiently inhibits ATP-induced ion-currents. Using specific inhibitors and siRNA, we demonstrate that SLPI activates the calcium-independent phospholipase A2β (iPLA2β) and leads to the release of a low molecular mass factor that mediates the inhibition of IL-1β release. Signaling involves nicotinic acetylcholine receptor subunits α7, α9, α10, and Src kinase activation and results in an inhibition of ATP-induced caspase-1 activation. In conclusion, we propose a novel anti-inflammatory mechanism induced by SLPI, which inhibits the ATP-dependent maturation and secretion of IL-1β. This novel signaling pathway might lead to development of therapies that are urgently needed for the prevention and treatment of systemic inflammation

High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes

Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxidative stress and syndecan-4 (SDC-4) in human podocytes. Human podocytes were exposed to control conditions (5.6mmol/L d-glucose), high glucose (30mmol/L d-glucose or l-glucose), 100mumol/L peroxynitrite, or high glucose and the superoxide dismutase mimetic tempol (100mumol/L). TRPC6 and SDC-4 transcripts and protein expression were measured using RT-PCR and in-cell Western assay. Intracellular reactive oxygen species (ROS) and cytosolic calcium were measured using fluorescent dye techniques. High d-glucose increased TRPC6 transcripts to 8.66+/-4.08 (p<0.05) and TRPC6 protein expression to 1.44+/-0.07 (p<0.05) without altering SDC-4 transcripts or protein expression. The d-glucose induced increase of TRPC6 expression was blocked by tempol. Increased oxidative stress using peroxynitrite significantly increased TRPC6 transcripts to 4.29+/-1.26 (p<0.05) and TRPC6 protein expression to 1.28+/-0.05 (p<0.05) without altering SDC-4 transcripts or protein expression. In human podocytes transfected with scrambled siRNA, high d-glucose increased ROS after 90min to 3.55+/-0.08 arbitrary units while 5.6mmol/L d-glucose increased ROS to 2.49+/-0.09 (p<0.001) only. The increase in ROS was inhibited by tempol and by SDC-4 knockdown. High glucose modifies TRPC6 channels and ROS production via SDC-4 in human podocytes

ZNF580 – a brake on Interleukin-6

Abstract Background Zinc finger protein 580 (ZNF580) was reported to modulate angiogenesis, endothelial homeostasis and blood pressure control. ZNF580 regulated genes include VEGF-A and IL-8. However, it is unknown if ZNF580 could play a role during inflammation. The aim of this study was to find out if ZNF580 affects the expression of IL-6, if it occurs in monocytic cells and responds to inflammatory mediators. Results Overexpression of ZNF580 reduced LPS-induced promotor activity of IL-6. Consistently, overexpression of ZNF580 reduced by half the LPS-induced expression of IL-6. ZNF580 was strongly expressed in the nucleus of MonoMac6, a human monocytic cell line. LPS-stimulated IL-6 secretion increased when ZNF580 was suppressed with siRNA. After stimulation of MonoMac6 with LPS for 24 h, ZNF580 negatively correlated with the amount of secreted IL-6. In response to LPS, ZNF580 was increased within the first 8 h, followed by a marked decrease after 16 h. This decrease coincided with sustained IL-6 production. Conclusion This study demonstrated that ZNF580 inhibits LPS-induced expression of IL-6. ZNF580 was highly expressed in monocytic cells and therefore may contribute to the modulation of its IL-6 production, at least in response to LPS. This suggests cooperation between ZNF580 and NFκB, which could play a role during sepsis

Protein arginine methyltransferase 5 mediates enolase-1 cell surface trafficking in human lung adenocarcinoma cells

Objectives: Enolase-l-dependent cell surface proteolysis plays an important role in cell invasion. Although enolase-1 (Eno-1), a glycolytic enzyme, has been found on the surface of various cells, the mechanism responsible for its exteriorization remains elusive. Here, we investigated the involvement of post-translational modifications (PTMs) of Eno-1 in its lipopolysaccharide (LPS)-triggered trafficking to the cell surface. Results: We found that stimulation of human lung adenocarcinoma cells with LPS triggered the monomethylation of arginine 50 (R5Ome) within Eno-1. The Eno-1R5Ome was confirmed by its interaction with the tudor domain (TD) from TD-containing 3 (TDRD3) protein recognizing methylarginines. Substitution of R50 with lysine (R50K) reduced Eno-1 association with epithelial caveolar domains, thereby diminishing its exteriorization. Similar effects were observed when pharmacological inhibitors of arginine methyltransferases were applied. Protein arginine methyltransferase 5 (PRMT5) was identified to be responsible for Eno-1 methylation. Overexpression of PRMT5 and caveolin-1 enhanced levels of membrane-bound extracellular Eno-1 and, conversely, pharmacological inhibition of PRMT5 attenuated Eno-1 cell-surface localization. Importantly, Eno1R5Ome was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-l-triggered cell invasion. Conclusions: LPS-triggered Eno-1R5Ome enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R5Ome may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients

Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA

Protein arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). Three forms of methylarginine have been identified in eukaryotes: monomethylarginine (l-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA), all characterized by methylation of one or both guanidine nitrogen atoms of arginine. l-NMMA and ADMA, but not SDMA, are competitive inhibitors of all nitric oxide synthase isoforms. SDMA is eliminated almost entirely by renal excretion, whereas l-NMMA and ADMA are further metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To explore the interplay between methylarginine synthesis and degradation in vivo, we determined PRMT expression and DDAH activity in mouse lung, heart, liver, and kidney homogenates. In addition, we employed HPLC-based quantification of protein-incorporated and free methylarginine, combined with immunoblotting for the assessment of tissue-specific patterns of arginine methylation. The salient findings of the present investigation can be summarized as follows: 1) pulmonary expression of type I PRMTs was correlated with enhanced protein arginine methylation; 2) pulmonary ADMA degradation was undertaken by DDAH1; 3) bronchoalveolar lavage fluid and serum exhibited almost identical ADMA/SDMA ratios, and 4) kidney and liver provide complementary routes for clearance and metabolic conversion of circulating ADMA. Together, these observations suggest that methylarginine metabolism by the pulmonary system significantly contributes to circulating ADMA and SDMA levels