Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

Interactions of Na+/taurocholate cotransporting polypeptide with host cellular proteins upon hepatitis B and D virus infection: novel potential targets for antiviral therapy.

Na+/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein-protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.

Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes.

Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3-112.4 µM and Vmax values of 6683-7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes.

Multitasking Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a Drug Target for HBV Infection: From Protein Engineering to Drug Discovery.

Hepatitis B virus (HBV) infections are among the major public health concerns worldwide with more than 250 million of chronically ill individuals. Many of them are additionally infected with the Hepatitis D virus, a satellite virus to HBV. Chronic infection frequently leads to serious liver diseases including cirrhosis and hepatocellular carcinoma, the most common type of liver cancer. Although current antiviral therapies can control HBV replication and slow down disease progress, there is an unmet medical need to identify therapies to cure this chronic infectious disease. Lately, a noteworthy progress in fighting against HBV has been made by identification of the high-affinity hepatic host receptor for HBV and HDV, namely Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1). Next to its primary function as hepatic uptake transporter for bile acids, NTCP is essential for the cellular entry of HBV and HDV into hepatocytes. Due to this high-ranking discovery, NTCP has become a valuable target for drug development strategies for HBV/HDV-infected patients. In this review, we will focus on a newly predicted three-dimensional NTCP model that was generated using computational approaches and discuss its value in understanding the NTCP's membrane topology, substrate and virus binding taking place in plasma membranes. We will review existing data on structural, functional, and biological consequences of amino acid residue changes and mutations that lead to loss of NTCP's transport and virus receptor functions. Finally, we will discuss new directions for future investigations aiming at development of new NTCP-based HBV entry blockers that inhibit HBV tropism in human hepatocytes.

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

Interleukin-1ß (IL-1ß) 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-1ß can cause life-threatening systemic inflammation. Hence, mechanisms controlling IL-1ß 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-1ß maturation and release. We demonstrated that SLPI dose-dependently inhibits the ATP-mediated inflammasome activation and IL-1ß release in human monocytic cells, without affecting the induction of pro-IL-1ß mRNA by LPS. In contrast, the ATP-independent IL-1ß 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 A2ß (iPLA2ß) and leads to the release of a low molecular mass factor that mediates the inhibition of IL-1ß 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-1ß. This novel signaling pathway might lead to development of therapies that are urgently needed for the prevention and treatment of systemic inflammation.

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

OBJECTIVES: Enolase-1-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 (R50me) within Eno-1. The Eno-1R50me 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, Eno-1R50me was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-1-triggered cell invasion. CONCLUSIONS: LPS-triggered Eno-1R50me enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R50me may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients.

Chemokines (CCL3, CCL4, and CCL5) Inhibit ATP-Induced Release of IL-1ß by Monocytic Cells.

Chemokines and ATP are among the mediators of inflammatory sites that can enter the circulation via damaged blood vessels. The main function of chemokines is leukocyte mobilization, and ATP typically triggers inflammasome assembly. IL-1ß, a potent inflammasome-dependent cytokine of innate immunity, is essential for pathogen defense. However, excessive IL-1ß may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1ß. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with the P2X7 agonist BzATP for 30 min to induce IL-1ß release. CCL3, CCL4, and CCL5 dose dependently inhibited BzATP-stimulated release of IL-1ß, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. It was blunted after silencing CCR1 or calcium-independent phospholipase A2 (iPLA2) by siRNA and was sensitive to antagonists of nicotinic acetylcholine receptors containing subunits α7 and α9. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1ß release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1ß from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nicotinic acetylcholine receptor subunits α7 and α9. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1ß is minimized.

Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors.

Pirfenidone is an antifibrotic drug, recently approved for the treatment of patients with idiopathic pulmonary fibrosis (IPF). Although pirfenidone exhibits anti-inflammatory, antioxidant, and antifibrotic properties, the molecular mechanism underlying its protective effects remains unknown. Here, we link pirfenidone action with the regulation of the profibrotic hedgehog (Hh) signaling pathway. We demonstrate that pirfenidone selectively destabilizes the glioma-associated oncogene homolog (GLI)2 protein, the primary activator of Hh-mediated gene transcription. Consequently, pirfenidone decreases overall Hh pathway activity in patients with IPF and in patient-derived primary lung fibroblasts and leads to diminished levels of Hh target genes, such as GLI1, Hh receptor Patched-1, α-smooth muscle actin, and fibronectin, and to reduced cell migration and proliferation. Interestingly, Hh-triggered TGF-ß1 expression potentiated Hh responsiveness of primary lung fibroblasts by elevating the available pool of glioma-associated oncogene homolog (GLI)1/GLI2, thus creating a vicious cycle of amplifying fibrotic processes. Because GLI transcription factors are not only crucial for Hh-mediated changes but are also required as mediators of TGF-ß signaling, our findings suggest that pirfenidone exerts its clinically beneficial effects through dual Hh/TGF-ß inhibition by targeting the GLI2 protein.-Didiasova, M., Singh, R., Wilhelm, J., Kwapiszewska, G., Wujak, L., Zakrzewicz, D., Schaefer, L., Markart, P., Seeger, W., Lauth, M., Wygrecka, M. Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors.

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury.

RATIONALE: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. OBJECTIVES: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. METHODS: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. CONCLUSIONS: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.

Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial cells.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.

STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization.

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca(2+) chelator BAPTA or an inhibitor of endoplasmic reticulum Ca(2+)-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca(2+) entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca(2+) channel-mediated Ca(2+) influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.

From plasminogen to plasmin: role of plasminogen receptors in human cancer.

Cell surface-associated proteolysis mediated by plasmin (PLA) is an essential feature of wound healing, angiogenesis and cell invasion, processes that are dysregulated in cancer development, progression and systemic spread. The generation of PLA, initiated by the binding of its precursor plasminogen (PLG) to the cell surface, is regulated by an array of activators, inhibitors and receptors. In this review, we will highlight the importance of the best-characterized components of the PLG/PLA cascade in the pathogenesis of cancer focusing on the role of the cell surface-PLG receptors (PLG-R). PLG-R overexpression has been associated with poor prognosis of cancer patients and resistance to chemotherapy. We will also discuss recent findings on the molecular mechanisms regulating cell surface expression and distribution of PLG-R.

The interaction of enolase-1 with caveolae-associated proteins regulates its subcellular localization.

Cell-surface-associated proteolysis plays a crucial role in embryonic development, monocyte/macrophage recruitment and tumour cell invasion. The glycolytic enzyme ENO-1 (enolase-1) is translocated from the cytoplasm to the cell surface, where it binds PLG (plasminogen) to enhance pericellular plasmin production and cell motility. In the present study, ENO-1 was found to localize to a specialized subset of lipid rafts called caveolae as demonstrated by fluorescence confocal microscopy and sucrose gradient ultracentrifugation. Co-immunoprecipitation studies revealed that ENO-1 interacts with Cav-1 (caveolin-1), but not with Cav-2, via the CSD (Cav-scaffolding domain). Moreover, an evolutionarily conserved CBM (Cav-binding motif) F296DQDDWGAW304 was identified within ENO-1. The point mutation W301A within the ENO-1 CBM was, however, not sufficient to disrupt ENO-1-Cav-1 interaction, whereas the mutations F296A and W304A markedly affected ENO-1 protein expression. Furthermore, ENO-1 was found associated with Annx2 (annexin 2), representing another caveolar protein, and this interaction was dependent on Cav-1 expression. Knockdown of Cav-1 and Annx2 markedly decreased cell surface expression of ENO-1. ENO-1 overexpression increased cell migration and invasion in a Cav-1-dependent manner. Thus the differential association of ENO-1 with caveolar proteins regulates ENO-1 subcellular localization and, consequently, ENO-1-dependent cell migration and invasion.

Protease-activated receptors (PAR)-1 and -3 drive epithelial-mesenchymal transition of alveolar epithelial cells - potential role in lung fibrosis.

Extravascular activation of the coagulation cascade in the lung is commonly observed in pulmonary fibrosis. Coagulation proteases may exert profibrotic cellular effects via protease-activated receptors (PARs)-1 and -2. Here, we investigated the potential role of two other members of the PAR family, namely PAR-3 and PAR-4, in the pathobiology of lung fibrosis. Elevated expression of PAR-3, but not PAR-4, was detected in the lungs of idiopathic pulmonary fibrosis (IPF) patients and in bleomycin-induced lung fibrosis in mice. Increased PAR-3 expression in fibrotic lungs was mainly attributable to alveolar type II (ATII) cells. Stimulation of primary mouse ATII, MLE15 and A549 cells with thrombin (FIIa) - that may activate PAR-1, PAR-3 and PAR-4 - induced epithelial-mesenchymal transition (EMT), a process that has been suggested to be a possible mechanism underlying the expanded (myo)fibroblast pool in lung fibrosis. EMT was evidenced by morphological alterations, expression changes of epithelial and mesenchymal phenotype markers, and functional changes. Single knockdown of FIIa receptors, PAR-1, PAR-3, or PAR-4, had no major impact on FIIa-induced EMT. Simultaneous depletion of PAR-1 and PAR-3, however, almost completely inhibited this process, whereas only a partial effect on FIIa-mediated EMT was observed when PAR-1 and PAR-4, or PAR-3 and PAR-4 were knocked down. PAR-1 and PAR-3 co-localise within ATII cells with both being predominantely plasma membrane associated. In conclusion, our study indicates that PARs synergise to mediate FIIa-induced EMT and provides first evidence that PAR-3 via its ability to potentiate FIIa-triggered EMT could potentially contribute to the pathogenesis of pulmonary fibrosis.

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.

TGF-ß1 induces tissue factor expression in human lung fibroblasts in a PI3K/JNK/Akt-dependent and AP-1-dependent manner.

The disturbance of hemostatic balance, associated with increased tissue factor (TF) expression and activity, occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF). However, the molecular mechanisms responsible for the regulation of TF expression under profibrotic conditions have not been assessed. We found that transforming growth factor-ß1 (TGF-ß1) markedly enhanced TF expression in primary human lung fibroblasts (HLFs), whereas platelet-derived growth factor (PDGF)-BB and IGF (insulin-like growth factor)-1 showed only a moderate effect, and PDGB-CC exerted no effect. TGF-ß1-induced TF expression correlated with its elevated cell-surface activity, it required de novo gene transcription and protein synthesis, and it was dependent on JNK and Akt activity, because pharmacological inhibition or the knockdown of the previously mentioned kinases prevented TF synthesis. Exposure of HLFs to TGF-ß1 activated JNK in a PI3K-dependent manner and induced Akt phosphorylation at threonine 308 and serine 473, but did not change the phosphorylation status of threonine 450. Akt phosphorylation at serine 473 correlated with JNK activity, and co-immunoprecipitation studies revealed a direct interaction between JNK and Akt. Furthermore, TGF-ß1-induced TF expression required the recruitment of c-Fos and JunD into a heterodimeric activator protein (AP)-1 complex. Moreover, strong immunoreactivity for phosphorylated Akt and JNK as well as c-Fos and JunD was observed in fibroblasts and myofibroblasts in IPF lungs. In conclusion, PI3K/JNK/Akt and AP-1 synergize to induce TF expression in HLFs after TGF-ß1 challenge. Our findings provide new insights into the molecular mechanisms responsible for the regulation of TF expression, and open new perspectives on the treatment of pulmonary fibrosis and other diseases characterized by the inappropriate expression of this cell-surface receptor.

The role of dimethylarginine dimethylaminohydrolase in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, dysregulated response to alveolar injury that culminates in compromised lung function from excess extracellular matrix production. Associated with high morbidity and mortality, IPF is generally refractory to current pharmacological therapies. We examined fibrotic lungs from mice and from patients with IPF and detected increased expression of dimethylarginine dimethylaminohydrolases (DDAHs)--key enzymes that metabolize asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of nitric oxide synthase, to form l-citrulline and dimethylamine. DDAHs are up-regulated in primary alveolar epithelial type II cells from these mice and patients where they are colocalized with inducible nitric oxide synthase. In cultured alveolar epithelial type II cells from bleomycin-induced fibrotic mouse lungs, inhibition of DDAH suppressed proliferation and induced apoptosis in an ADMA-dependent manner. In addition, DDAH inhibition reduced collagen production by fibroblasts in an ADMA-independent but transforming growth factor/SMAD-dependent manner. In mice with bleomycin-induced pulmonary fibrosis, the DDAH inhibitor L-291 reduced collagen deposition and normalized lung function. In bleomycin-induced fibrosis, inducible nitric oxide synthase inhibition decreased fibrosis, but an even stronger reduction was observed after inhibition of DDAH. Thus, DDAH inhibition reduces fibroblast-induced collagen deposition in an ADMA-independent manner and reduces abnormal epithelial proliferation in an ADMA-dependent manner, offering a possible therapeutic avenue for attenuation of pulmonary fibrosis.

Role of protease-activated receptor-2 in idiopathic pulmonary fibrosis.

RATIONALE: Activation of the coagulation cascade has been demonstrated in pulmonary fibrosis. In addition to its procoagulant function, various coagulation proteases exhibit cellular effects that may also contribute to fibrotic processes in the lung. OBJECTIVE: To investigate the importance of protease-activated receptor (PAR)-2 and its activators, coagulation factor VIIa (FVIIa)/tissue factor (TF), in the development of idiopathic pulmonary fibrosis (IPF). METHODS: Expression and localization of PAR-2 and its activators were examined in IPF lung tissue. The ability of PAR-2 to mediate various cellular processes was studied in vitro. MEASUREMENTS AND MAIN RESULTS: Expression of PAR-2 was strongly elevated in IPF lungs and was attributable to alveolar type II cells and fibroblasts/myofibroblasts. Transforming growth factor-ß(1), a key profibrotic cytokine, considerably enhanced PAR-2 expression in human lung fibroblasts. FVIIa stimulated proliferation of human lung fibroblasts and extracellular matrix production in a PAR-2-dependent manner, but did not initiate differentiation of fibroblasts into myofibroblasts. PAR-2/FVIIa-driven mitogenic activities were mediated via the p44/42 mitogen-activated protein kinase pathway and were independent of factor Xa and thrombin production. Proproliferative properties of FVIIa were markedly potentiated in the presence of TF and abrogated by TF antisense oligonucleotides. Hyperplastic alveolar type II cells overlying fibroblastic foci were found to be the source of FVII in IPF lungs. Moreover, TF colocalized with PAR-2 on fibroblasts/myofibroblasts in IPF lungs. CONCLUSIONS: The PAR-2/TF/FVIIa axis may contribute to the development of pulmonary fibrosis; thus, interference with this pathway confers novel therapeutic potential for the treatment of IPF.

Quantitative assessment of arginine methylation in free versus protein-incorporated amino acids in vitro and in vivo using protein hydrolysis and high-performance liquid chromatography.

Arginine methylation constitutes a posttranslational modification dependent on the action of protein arginine methyltransferases (PRMTs). Using S-adenosylmethionine as a methyl donor, PRMTs catalyze the formation of monomethylarginine (L-NMMA), asymmetric dimethylarginine (ADMA), or symmetric dimethylarginine (SDMA). Protein arginine methylation is involved in the regulation of signal transduction, RNA export, and cell proliferation, but a quantitative view of arginine methylation of the cell and tissue proteome remains to be performed. In this study, we developed a high-performance liquid chromatography (HPLC)-based method to accurately quantify methylated arginines in free and protein-incorporated amino acid pools of cell and tissue extracts, using protein precipitation and hydrolysis, HPLC separation, and fluorescence detection for the simultaneous quantification of L-arginine (L-Arg), L-NMMA, ADMA, and SDMA. This method permits accurate assessment of the degree of protein arginine methylation in complex biological samples. Using this method, we determined dynamic changes in protein methylation in vitro in cells subjected to proteasome inhibition. We furthermore demonstrate differential methylation patterns in heart and kidney lysates in vivo. Thus, the described method will greatly facilitate our understanding of the role of arginine methylation in physiology and pathophysiology and of the effects of pharmacological interventions on arginine methylation in select cell culture models.