Asymmetric dimethylarginine metabolism and its involvement in the pathogenesis of pulmonary arterial hypertension

Protein arginine methylation represents a posttranslational modification undertaken by protein arginine methyltransferases (PRMT) that results in production of protein-incorporated omega-NG-monomethylarginine (L-NMMA), asymmetric omega-NG, NG-dimethylarginine (ADMA), or omega-NG, N G-dimethylarginine (SDMA). Free cellular L-NMMA, ADMA and SDMA can be generated via the proteolytic cleavage of intracellular proteins, thereby also affecting methylarginine content in the plasma. Free methylarginines can be cleared from the body by renal excretion. L-NMMA and ADMA, but not SDMA, can be degraded via enzymes called NG, NG–dimmethylarginine dimethylaminohydrolase (DDAH). ADMA is an endogenous inhibitor of nitric oxide synthases (NOS) and a marker of endothelial dysfunction. Increased plasma ADMA levels have been reported in patients with cardiovascular disorders including pulmonary arterial hypertension (PAH), a fatal disease characterized by elevated blood pressure in the pulmonary circulation, due to increased resistance of pulmonary arterioles. The major pathophysiologic hallmark of PAH is pulmonary arterial smooth muscle cell (PASMC) hypertrophy and proliferation, leading to the occlusion of pulmonary arterioles. The interplay between methylarginine synthesis and degradation in vivo, as well as specific alterations to intrapulmonary ADMA levels or distorted generation of ADMA in PAH, however, remains to be elucidated. In the current study, we hypothesized that methylarginine production and degradation is tissue-specific and that the lung has a significant impact on serum/plasma ADMA levels, possibly leading to endothelial dysfunction observed in PAH. To this end, we sought to address the following specific aims: 1) to develop a novel, HPLC-based method to assess protein-incorporated and free cellular methylarginine content in biological samples, 2) to analyze the tissue-specific methylarginine metabolism in normal subjects, and 3) to analyze the methylarginine content in the lungs of patients with PAH compared with healthy donors. First, to analyze tissue-specific methylarginine metabolism in the normal physiological state, we performed high performance liquid chromatography (HPLC)-driven assessment of protein-incorporated and free cellular methylarginine levels, together with Western blot analyses of PRMT and DDAH expression, in organs of the cardiovascular system. Our results revealed that pulmonary expression of type I PRMT was correlated with enhanced protein arginine methylation in the lung. Moreover, our studies also revealed that the kidney and the liver provide complementary routes for clearance and metabolic conversion of circulating ADMA. To address the impact of intrapulmonary ADMA metabolism in pathogenic conditions, we next analyzed lung homogenates of PAH patients. HPLC analysis revealed significantly lower levels of protein-incorporated ADMA in the lungs of PAH patients (n=12), compared with controls (n=10, transplant donors). Western Blot analyses confirmed a significantly decreased content of asymmetrically dimethylated proteins in PAH lungs. The expression of PRMT, in particular PRMT1, was decreased in PAH. Immunohistochemical staining of IPAH and control lungs localized PRMT1 to pulmonary arterial vascular smooth muscle cells (PASMC). Moreover, PRMT1 knockdown in primary PASMC by siRNA technology significantly increased PASMC proliferation. Our results demonstrate that, in the normal physiological state, methylarginine metabolism by the pulmonary system significantly contributes to circulating methylarginine levels. In pathogenic conditions, protein-incorporated ADMA concentrations do not reflect free cellular levels of ADMA in the lung. This may be explained by the alterations of DDAH activity in the lung, which, consequently, regulate ADMA content in the serum of IPAH patients. In addition, our studies demonstrated a novel regulatory role of PRMT1 in progression of PAH, by the alteration of PASMC proliferation, a major characteristic of PAH. This led to conclusions that protein arginine methylation plays a pivotal role in the pathogenesis of PAH.Posttranslationale Protein-Arginin Methylierung erfolgt durch eine Gruppe spezifischer Protein-Arginin Methyltransferasen (PRMTs), die neben der Bildung von asymmetrischem Dimethylarginin (ADMA) auch für die Synthese von Monomethylarginin (L-NMMA) und symmetrischem Dimethylarginin (SDMA) verantwortlich sind. Die Freisetzung von Methylarginine in das Blutplasma erfolgt nach heutigem Wissensstand über die Proteolyse zellulärer, methylierter Proteine. Alle Methylargininformen werden über renale Exkretion aus dem Körper eliminiert. Neuere Studien heben die Metabolisierung von ADMA und L-NMMA durch das Enzym Dimethylarginin-Dimethylaminohydrolase (DDAH) als Hauptabbauweg hervor. ADMA ist ein endogener Inhibitor der NO-Synthase und ein Marker für endotheliale Fehlfunktion. Eine erhöhte ADMA Konzentration im Blut wird bei verschiedenen kardiovaskulären Erkrankungen, so auch bei pulmonal-arterieller Hypertonie (PAH), für einen Mangel an biologisch verfügbarem NO verantwortlich gemacht. Die pulmonal-arterielle Hypertonie ist durch eine pathologische Hypertrophie und Proliferation pulmonalarterieller glatter Muskelzellen (PASMC) gekennzeichnet, die eine Okklusion pulmonaler Arteriolen zur Folge hat. Ob ein Zusammenhang zwischen Arginin- und Dimethylargininstoffwechsel und den bei PAH zu beobachtenden Symptomen vorliegt, wurde bislang nicht untersucht. Deshalb sollte in der vorliegenden Studie geprüft werden, ob der Methylarginin-metabolismus der Lunge signifikant zur ADMA Konzentration im Blut beiträgt und somit an der Ausbildung endothelialer Fehlfunktionen beteiligt sein könnte. Im Konkreten sollten hierfür folgende Vorhaben realisiert werden: (1) Entwicklung einer auf Hochdruckflüssigkeitschromatographie-basierenden Methode zur Quantifizierung von protein-inkorporiertem und freiem Methylarginin in biologischen Proben, (2) Analyse des gewebespezifischen Methylargininmetabolismus und (3) Bestimmung des pulmonalen Methylarginingehaltes von PAH Patienten und gesunden Organspendern. Zur Beschreibung des Methylargininmetabolismus unter normalen physiologischen Bedingungen wurden protein-inkorporiertes und freies Methylarginin in Organen des kardiovaskulären Systems bestimmt. Zudem wurde vergleichend Proteinexpression und Aktivität der PRMTs und DDAHs ermittelt. Unsere Untersuchungen ergaben eine klare Korrelation zwischen pulmonaler Typ I PRMT Proteinexpression und erhöhter Protein-Arginin Methylierung. Zudem konnte gezeigt werden, dass Niere und Leber komplementär an der Eliminierung und Metabolisierung von ADMA und L-NMMA beteiligt sind. Zur Beurteilung der Frage, ob bei PAH ein geänderter Dimethylargininstoffwechsel zu beobachten ist, wurde Lungenhomogenat mittels HPLC untersucht. Die Analyse bei PAH Patienten (n=12) und gesunden Organspendern (n=10) ergab eine signifikante Abnahme an protein-inkorporiertem ADMA bei PAH Patienten. Zudem konnte über Western-Blot Analyse ein reduzierter Gehalt an asymmetrisch dimethylierten Proteinen nachgewiesen werden. Bei PAH Patienten zeigte sich auch eine signifikant reduzierte Expression jener Protein-Arginin-Methyltransferasen, insbesondere PRMT 1, die für eine asymmetrische Dimethylierung von Zielproteinen verantwortlich sind. Immunohistochemische Untersuchungen führten zu dem Ergebnis, dass PRMT 1 überwiegend in PASMCs lokalisiert ist. Zudem resultierte die Reduktion der PRMT 1 Expression mittels siRNA Technologie in einer Zunahme der PASMC Proliferation. Aus den vorliegenden Ergebnissen lässt sich somit schlussfolgern, dass der pulmonale Dimethylargininmetabolismus maßgeblich zum Plasma ADMA-Spiegel beiträgt. Bei PAH Patienten konnte keine Korrelation zwischen protein-inkorporiertem ADMA und freiem Methylarginin nachgewiesen werden. Dieses Ergebnis deutet auf eine Änderung der pulmonalen DDAH Aktivität und Plasma ADMA-Werte bei PAH Patienten hin. Des Weiteren konnte eindeutig demonstriert werden, dass PRMT 1 an der Regulation der PASMC Proliferation beteiligt ist. Zusammenfassend lässt sich somit feststellen, dass Protein-Arginin Methylierung an der Entwicklung und am Fortschreiten von PAH beteiligt sein könnte

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

From arginine methylation to ADMA: A novel mechanism with therapeutic potential in chronic lung diseases

Protein arginine methylation is a novel posttranslational modification regulating a diversity of cellular processes, including protein-protein interaction, signal transduction, or histone function. It has recently been shown to be dysregulated in chronic renal, vascular, and pulmonary diseases, and metabolic products originating from protein arginine methylation have been suggested to serve as biomarkers in cardiovascular and pulmonary diseases

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

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

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