Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation.

Activation of glycoprotein VI (GPVI) and C-type lectin-like receptor-2 (CLEC-2) underlies platelet activation by diesel exhaust particles and other charged/hydrophobic ligands

Platelets are activated by a range of stimuli that share little or no resemblance in structure to each other or to recognized ligands, including diesel exhaust particles (DEP), small peptides [4N1-1, Champs (computed helical anti-membrane proteins), LSARLAF (Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides (fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation of human and mouse platelets through the glycoprotein VI (GPVI)-FcR γ-chain complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using platelets from mice deficient in either or both of these receptors. In addition, all of these ligands stimulate tyrosine phosphorylation in GPVI/CLEC-2-double-deficient platelets, indicating that they bind to additional surface receptors, although only in the case of dextran sulfate does this lead to activation. DEP, fucoidan and dextran sulfate, but not the other agonists, activate GPVI and CLEC-2 in transfected cell lines as shown using a sensitive reporter assay confirming a direct interaction with the two receptors. We conclude that this miscellaneous group of ligands bind to multiple proteins on the cell surface including GPVI and/or CLEC-2, inducing activation. These results have pathophysiological significance in a variety of conditions that involve exposure to activating charged/hydrophobic agents.

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved

Endurance training in humans leads to fiber type-specific increases in levels of peroxisome proliferator-activated receptor-[gamma] coactivator-1 and peroxisome proliferator-activated receptor-[alpha] in skeletal muscle

The peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 (PGC-1) can induce mitochondria biogenesis and has been implicated in the development of oxidative type I muscle fibers. The PPAR isoforms α, β/δ, and γ control the transcription of genes involved in fatty acid and glucose metabolism. As endurance training increases skeletal muscle mitochondria and type I fiber content and fatty acid oxidative capacity, our aim was to determine whether these increases could be mediated by possible effects on PGC-1 or PPAR-α, -β/δ, and -γ. Seven healthy men performed 6 weeks of endurance training and the expression levels of PGC-1 and PPAR-α, -β/δ, and -γ mRNA as well as the fiber type distribution of the PGC-1 and PPAR-α proteins were measured in biopsies from their vastus lateralis muscle. PGC-1 and PPAR-α mRNA expression increased by 2.7- and 2.2-fold (P < 0.01), respectively, after endurance training. PGC-1 expression was 2.2- and 6-fold greater in the type IIa than in the type I and IIx fibers, respectively. It increased by 2.8-fold in the type IIa fibers and by 1.5-fold in both the type I and IIx fibers after endurance training (P < 0.015). PPAR-α was 1.9-fold greater in type I than in the II fibers and increased by 3.0-fold and 1.5-fold in these respective fibers after endurance training (P < 0.001). The increases in PGC-1 and PPAR-α levels reported in this study may play an important role in the changes in muscle mitochondria content, oxidative phenotype, and sensitivity to insulin known to be induced by endurance training.

Upregulation of peroxisome proliferator-activated receptor gamma coactivator gene (PGC1A) during weight loss is related to insulin sensitivity but not to energy expenditure

Aims/hypothesis We investigated whether skeletal muscle peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1A; also known as PPARGC1A) and its target mitofusin-2 (MFN2), as well as carnitine palmitoyltransferase-1 (CPT1; also known as carnitine palmitoyltransferase 1A [liver] [CPT1A]) and uncoupling protein (UCP)3, are involved in the improvement of insulin resistance and/or in the modification of energy expenditure during surgically induced massive weight loss.
Materials and methods Seventeen morbidly obese women (mean BMI: 45.9 ± 4 kg/m2) were investigated before, and 3 and 12 months after, Roux-en-Y gastric bypass (RYGB). We evaluated insulin sensitivity by the euglycaemic–hyperinsulinaemic clamp, energy expenditure and substrate oxidation by indirect calorimetry, and muscle mRNA expression by PCR.
Results Post-operatively, PGC1A was enhanced at 3 (p = 0.02) and 12 months (p = 0.03) as was MFN2 (p = 0.008 and p = 0.03 at 3 and 12 months respectively), whereas UCP3 was reduced (p = 0.03) at 12 months. CPT1 did not change. The expression of PGC1A and MFN2 were strongly (p < 0.0001) related. Insulin sensitivity, which increased after surgery (p = 0.002 at 3, p = 0.003 at 12 months), was significantly related to PGC1A and MFN2, but only MFN2 showed an independent influence in a multiple regression analysis. Energy expenditure was reduced at 3 months post-operatively (p = 0.001 vs before RYGB), remaining unchanged thereafter until 12 months. CPT1 and UCP3 were not significantly related to the modifications of energy expenditure or of lipid oxidation rate.
Conclusions/interpretation Weight loss upregulates PGC1A, which in turn stimulates MFN2 expression. MFN2 expression significantly and independently contributes to the improvement of insulin sensitivity. UCP3 and CPT1 do not seem to influence energy expenditure after RYGB.

Peroxisome proliferator-activated receptor-γ coactivator-1 and insulin resistance: acute effect of fatty acids

Aims/hypothesis Peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 (PPARGC1), a coactivator regulating the transcription of genes involved in oxidative metabolism, is downregulated in patients with type 2 diabetes and in their first-degree relatives. Whether this downregulation is a cause or effect of early aberrations in the development of insulin resistance, such as disturbances in fat metabolism, is unknown. We examined whether lipid-induced insulin resistance was associated with downregulation of expression of skeletal muscle genes involved in oxidative metabolism and mitochondrial biogenesis in humans.
Materials and methods Nine healthy lean male subjects underwent a 6-h hyperinsulinaemic–euglycaemic clamp with simultaneous infusion of either a lipid emulsion or glycerol as a control. Blood was sampled at regular time points and muscle biopsies were taken before and after every test. Intramuscular triacylglycerol (IMTG) content was determined by Oil Red O staining and gene expression was measured by quantitative PCR.
Results Lipid infusion resulted in a ∼2.7-fold increase in plasma NEFA levels and a 31±6% decrease in insulin sensitivity (p=0.001). The infusion of lipids resulted in a ∼1.6-fold increase in IMTG (p=0.02), whereas during the clamp with glycerol infusion IMTG tended to decrease to ∼53% of preinfusion levels (p=0.065). Lipid infusion decreased PPARGC1A, PPARGC1B and PPARA expression to ∼61, 77 and ∼52% of basal values respectively, whereas expression of uncoupling protein 3 was upregulated 1.8-fold (all p<0.05).
Conclusions/interpretation Acute elevation of plasma NEFA levels, leading to muscular fat accumulation and insulin resistance, downregulates PPARGC1A, PPARGC1B and PPARA expression, suggesting that the decrease in PPARGC1 expression observed in the (pre)diabetic state may be the result, rather than the cause of lipid-induced insulin resistance.

Differential tumor necrosis factor receptor 2-mediated editing of virus-specific CD8+ effector T cells

Much of the CD8+ T cell response in H2b mice with influenza pneumonia is directed at the nucleoprotein366-374 (NP366) and acid polymerase224-233 (PA224) peptides presented by the H2Db MHC class I glycoprotein. These DbNP366- and DbPA224-specific T cell populations are readily analyzed by staining with tetrameric complexes of MHC+ peptide (tetramers) or by cytokine production subsequent to in vitro stimulation with the cognate peptides. The DbPA224-specific CD8+ effector T cells make more tumor necrosis factor (TNF) α than the comparable CD8+DbNP366+ set, a difference reflected in the greater sensitivity of the CD8+DbPA224+ population to TNF receptor (TNFR) 2-mediated apoptosis under conditions of in vitro culture. Freshly isolated CD8+DbNP366+ and CD8+DbPA224+ T cells from influenza-infected TNFR2-/- mice produce higher levels of IFN-γ and TNF-α after in vitro stimulation with peptide, although the avidity of the T cell receptor-epitope interaction does not change. Increased numbers of both CD8+DbPA224+ and CD8+DbNP366+ T cells were recovered from the lungs (but not the spleens) of secondarily challenged TNFR2-/- mice, a pattern that correlates with the profiles of TNFR expression in the TNFR2+/+ controls. Thus, it seems that TNFR2-mediated editing of influenza-specific CD8+ T cells functions to limit the numbers of effectors that have localized to the site of pathology in the lung but does not modify the size of the less activated responder T cell populations in the spleen. Therefore, the massive difference in magnitude for the secondary, although not the primary, response to these DbNP366 and DbPA224 epitopes cannot be considered to reflect differential TNFR2-mediated T cell editing.

Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain

A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPARαLBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPARαLBD (aa196–468) was synthesized and ligated into the pDEST17 E. coli expression vector downstream of a MBP solubility fusion tag and an intermittent TEV protease cleavage site. Following auto-induction at 28 °C, PPARαLBD protein was purified to electrophoretic homogeneity by a nickel affinity chromatographic step, on-column TEV protease cleavage followed by Sephacryl S200 size exclusion chromatography. The recombinant protein displayed cross-reactivity with goat anti-(human PPARα) polyclonal antibody and was identified as human PPARα by trypic peptide mass finger-printing. The addition of a PPARα specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPARαLBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPARαLBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPARαLBD construct make it amenable to high through-put screening assays in drug discovery programs.

Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) gamma Activators and Pan-PPAR Partial Agonists

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Thrombin-Receptor Antagonist Vorapaxar in Acute Coronary Syndromes

BACKGROUND Vorapaxar is a new oral protease-activated receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation. METHODS In this multinational, double-blind, randomized trial, we compared vorapaxar with placebo in 12,944 patients who had acute coronary syndromes without ST-segment elevation. The primary end point was a composite of death from cardiovascular causes, myocardial infarction, stroke, recurrent ischemia with rehospitalization, or urgent coronary revascularization. RESULTS Follow-up in the trial was terminated early after a safety review. After a median follow-up of 502 days (interquartile range, 349 to 667), the primary end point occurred in 1031 of 6473 patients receiving vorapaxar versus 1102 of 6471 patients receiving placebo (Kaplan-Meier 2-year rate, 18.5010 vs. 19.9%; hazard ratio, 0.92; 95% confidence interval [CI], 0.85 to 1.01; P=0.07). A composite of death from cardiovascular causes, myocardial infarction, or stroke occurred in 822 patients in the vorapaxar group versus 910 in the placebo group (14.7% and 16.4%, respectively; hazard ratio, 0.89; 95% CI, 0.81 to 0.98; P=0.02). Rates of moderate and severe bleeding were 7.2% in the vorapaxar group and 5.2% in the placebo group (hazard ratio, 1.35; 95% CI, 1.16 to 1.58; P<0.001). Intracranial hemorrhage rates were 1.1% and 0.2%, respectively (hazard ratio, 3.39; 95% CI, 1.78 to 6.45; P<0.001). Rates of nonhemorrhagic adverse events were similar in the two groups. CONCLUSIONS In patients with acute coronary syndromes, the addition of vorapaxar to standard therapy did not significantly reduce the primary composite end point but significantly increased the risk of major bleeding, including intracranial hemorrhage. (Funded by Merck; TRACER ClinicalTrials.gov number, NCT00527943.)

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) gamma Activators and Pan-PPAR Partial Agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) gamma to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPAR gamma ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPAR gamma LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPAR gamma LBD, stronger partial agonists with full length PPAR gamma and exhibit full blockade of PPAR gamma phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPAR gamma also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/beta-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPAR gamma modulators with useful clinical profiles among natural products.

GQ-16, a Novel Peroxisome Proliferator-activated Receptor gamma (PPAR gamma) Ligand, Promotes Insulin Sensitization without Weight Gain

The recent discovery that peroxisome proliferator-activated receptor gamma (PPAR gamma) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPAR gamma activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPAR gamma. The structure of GQ-16 bound to PPAR gamma demonstrates that the compound utilizes a binding mode distinct from other reported PPAR gamma ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the beta-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPAR gamma-based therapeutics stabilize the beta-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPAR gamma modulators that retain antidiabetic actions while minimizing untoward effects.