Functional analysis of RACK1 as a novel interaction partner of BMPRII in pulmonary arterial hypertension

Die Pulmonalarterielle Hypertonie (PAH) ist charakterisiert durch eine selektive Erhöhung des pulmonalarteriellen Blutdrucks. Das pathologische Korrelat der PAH ist ein Verschluss der pulmonalen Arteriolen durch eine Proliferation/Fehlfunktion der glatten Gefässmuskelzellen und des Endothels. Heterozygote Keimbahnmutationen im Bone Morphogenetic Receptor Type II (BMPRII) kodierenden Genlokus zeigen eine Assoziation mit PAH, was für einen Einfluss des BMPRII auf die Pathogenese der PAH spricht. Um die Funktion von BMPRII zu charakterisieren, war es das Ziel unserer Arbeit, neue potentielle Interaktionspartner dieses Rezeptors mittels Yeast Two-Hybrid Analyse zu identifizieren. Unter den vielen bisher unbekannten Interaktionspartnern von BMPRII wurde RACK (receptor for activated protein kinase C)-1 für weitergehende Untersuchungen ausgewählt. Glutathion S-Transferase (GST)-pulldown Experimente sowie Ko-Immunopräzipitationen bestätigten die Interaktion von RACK1 und der BMPRII Kinasedomäne. Immunohistochemische Analysen von Lungenschnitten und Immunofluoreszenzanalysen isolierter glatter Muskelzellen aus der Pulmonalarterie zeigten eine Ko-Lokalisation von BMPRII und RACK1 in vitro und in vivo. Für weitere funktionelle Analysen wurde das RACK1 Gen kloniert und in einem BMP-Reportergenassay überexprimiert. RACK1 Expression führte zu einer zweifach erhöhten Reportergenaktivität nach BMP-2 Stimulation, was einen synergistischen Einfluss der BMPRII-RACK1 Interaktion auf die BMP Signalkaskade zeigt. Dieser Befund wird durch die Tatsache unterstüzt, dass die Depletion von RACK1 mittels small interfering RNA (siRNA) Technologie zu einer verstärkten Proliferation von glatten Gefässmuskelzellen der A. Pulmonalis führt, was für eine regulatorische Rolle von RACK1 auf das Zellwachstum spricht. Mehrere BMPRII Varianten, welche aus dem internationalen PAH Patientenpool stammen, zeigten eine reduzierte Affinität für RACK1. Im Tiermodell der durch Monokrotalin induzierten pulmonalen Hypertonie wurde eine signifikant erniedrigte Expression von RACK1 auf RNA und Proteinebene gefunden. Die vorliegende Arbeit beschreibt daher einen funktionell bedeutenden Einfluss der neu identifizierten Interaktion zwischen BMPRII und RACK1 auf die BMP Signaltransduktion. Die reduzierte Affinität von RACK1 für BMPRII Varianten und die erniedrigte RACK1 Expression im Tiermodell der pulmonalen Hypertonie sprechen für einen bedeutenden Einfluss von RACK1 und der RACK1-BMPRII Interaktion auf die Pathogenese der PAH.Pulmonary arterial hypertension (PAH) is characterised by selective elevation of pulmonary arterial pressure. The pathological hallmark of PAH is the occlusion of pulmonary arterioles due to proliferation and dysfunction of smooth muscle and endothelial cells. Heterozygous mutations in BMPR2, encoding the type II BMP receptor (BMPRII), were identified in PAH suggesting that alterations to BMPRII function are involved in disease onset and/or progression. To further elucidate the function of BMPRII, we sought to identify novel interaction partners of BMPRII by yeast two-hybrid analysis using the kinase domain of BMPRII as a bait. Using this technology, several novel interaction partners of BMPRII were identified. Among these, the receptor for activated protein kinase C (RACK)-1 was selected for further investigation. The interaction between RACK1 and the BMPRII kinase domain was confirmed by Glutathione S-transferase (GST)-pull-down and co-immunoprecipitation. Immunofluorescent staining of human pulmonary artery smooth muscle cells (paSMC), as well immunohistochemistry of human lungs from healthy donors and PAH patients, demonstrated the co-localisation of BMPRII and RACK1 in vitro and in vivo. Overexpression of RACK1 in paSMC led to a two-fold increase in induction of a BMP-responsive promoter in a luciferase-based promoter reporter assay, indicating that the BMPRII-RACK1 interaction may potentiate BMP signalling. RACK1 depletion using small interfering RNA (siRNA) technology resulted in enhanced proliferation of paSMC, thus implicating a role for RACK1 and the RACK1-BMPRII interaction in paSMC growth regulation. In contrast, overexpression of RACK1 led to enhanced proliferation of paSMC. Several BMPRII variants that contained amino acid substitutions present in PAH patients exhibited a reduced affinity for RACK1. Furthermore, in the monocrotaline-induced rat model of PAH, the expression of RACK1 was significantly down-regulated at the RNA and protein level, two and four weeks after monocrotaline administration. Thus, the novel interaction of RACK1 with BMPRII is functionally significant in BMP signal transduction. The reduced affinity of RACK1 for BMPRII variants that are peculiar to PAH patients, and the reduced levels of RACK1 evident in the pulmonary vasculature in an animal model of PAH, suggest a potential role for RACK1, and the RACK1-BMPRII interaction, in the pathogenesis of PAH

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

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

Dimethylarginine metabolism during acute and chronic rejection of rat renal allografts

Background. Dimethylarginines are inhibitors of NO synthesis and are involved in the pathogenesis of vascular diseases. In this study, we ask the question if asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) levels change during fatal and reversible acute rejection, and contribute to the pathogenesis of chronic vasculopathy

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

Protein Arginine Methyltransferases (PRMTs): Promising Targets for the Treatment of Pulmonary Disorders

Protein arginine methylation is a novel posttranslational modification that plays a pivotal role in a variety of intracellular events, such as signal transduction, protein-protein interaction and transcriptional regulation, either by the direct regulation of protein function or by metabolic products originating from protein arginine methylation that influence nitric oxide (NO)-dependent processes. A growing body of evidence suggests that both mechanisms are implicated in cardiovascular and pulmonary diseases. This review will present and discuss recent research on PRMTs and the methylation of non-histone proteins and its consequences for the pathogenesis of various lung disorders, including lung cancer, pulmonary fibrosis, pulmonary hypertension, chronic obstructive pulmonary disease and asthma. This article will also highlight novel directions for possible future investigations to evaluate the functional contribution of arginine methylation in lung homeostasis and disease

Monocytic Tissue Transglutaminase in a Rat Model for Reversible Acute Rejection and Chronic Renal Allograft Injury

Acute rejection is a major risk factor for chronic allograft injury (CAI). Blood leukocytes interacting with allograft endothelial cells during acute rejection were suggested to contribute to the still enigmatic pathogenesis of CAI. We hypothesize that tissue transglutaminase (Tgm2), a multifunctional protein and established marker of M2 macrophages, is involved in acute and chronic graft rejection. We focus on leukocytes accumulating in blood vessels of rat renal allografts (Fischer-344 to Lewis), an established model for reversible acute rejection and CAI. Monocytes in graft blood vessels overexpress Tgm2 when acute rejection peaks on day 9 after transplantation. Concomitantly, caspase-3 is activated, suggesting that Tgm2 expression is linked to apoptosis. After resolution of acute rejection on day 42, leukocytic Tgm2 levels are lower and activated caspase-3 does not differ among isografts and allografts. Cystamine was applied for 4 weeks after transplantation to inhibit extracellular transglutaminase activity, which did, however, not reduce CAI in the long run. In conclusion, this is the first report on Tgm2 expression by monocytes in vivo. Tgm2 may be involved in leukocytic apoptosis and thus in reversion of acute rejection. However, our data do not support a role of extracellular transglutaminase activity as a factor triggering CAI during self-limiting acute rejection

Knockout of the CMP–Sialic Acid Transporter SLC35A1 in Human Cell Lines Increases Transduction Efficiency of Adeno-Associated Virus 9: Implications for Gene Therapy Potency Assays

Recombinant adeno-associated viruses (AAV) have emerged as an important tool for gene therapy for human diseases. A prerequisite for clinical approval is an in vitro potency assay that can measure the transduction efficiency of each virus lot produced. The AAV serotypes are typical for gene therapy bind to different cell surface structures. The binding of AAV9 on the surface is mediated by terminal galactose residues present in the asparagine-linked carbohydrates in glycoproteins. However, such terminal galactose residues are rare in cultured cells. They are masked by sialic acid residues, which is an obstacle for the infection of many cell lines with AAV9 and the respective potency assays. The sialic acid residues can be removed by enzymatic digestion or chemical treatment. Still, such treatments are not practical for AAV9 potency assays since they may be difficult to standardize. In this study, we generated human cell lines (HEK293T and HeLa) that become permissive for AAV9 transduction after a knockout of the CMP–sialic acid transporter SLC35A1. Using the human aspartylglucosaminidase (AGA) gene, we show that these cell lines can be used as a model system for establishing potency assays for AAV9-based gene therapy approaches for human diseases

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