c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR(2)), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR(2) is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR(2) was mono-ubiquitinated. Immunofluorescence indicated that activated PAR(2) translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR(2) lacking intracellular lysine residues (PAR(2)Delta14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR(2) Delta14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR(2) stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR(2) at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR(2) ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR(2) was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR(2) to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR(2) to irrevocably terminate signaling by this and perhaps other GPCRs.

Protease-activated receptor 2: activation, signalling and function

PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR(2), which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR(2). Proteases cleave PAR(2) to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR(2) signalling is attenuated by beta-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR(2). beta-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR(2)-deficient mice support a role for PAR(2) in inflammation, and many of the effects of PAR(2) activators promote inflammation. Inflammation is mediated in part by activation of PAR(2) in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.

Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from approximately 42 degrees C to approximately 34 degrees C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

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.

Deletion of Tumor Necrosis Factor-alpha Receptor 1 (TNFR1) Protects against Diet-induced Obesity by Means of Increased Thermogenesis

In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-alpha) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-alpha inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O(2) consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O(2) consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-alpha signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.

PAR(2) and Temporomandibular Joint Inflammation in the Rat

The proteinase-activated receptor 2 (PAR(2)) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR(2) in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR(2) immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR(2) agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK(1)) receptors abolished PAR(2)-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR(2) activation is proinflammatory in the TMJ, through a neurogenic mechanism involving NK(1) receptors. This suggests that PAR(2) is an important component of innate neuro-immune response in the rat TMJ.

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and alpha-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling. Kidney International (2011) 79, 1217-1227; doi:10.1038/ki.2011.14; published online 16 March 2011

PAR(2) and Temporomandibular Joint Inflammation in the Rat

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

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)

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

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

Graves ophthalmopathy: low-dose glucocorticoid increases peroxisome proliferator-activated receptor-gamma gene expression

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

The orexin receptor 1 in the locus coeruleus modulates the hypercapnic ventilatory response in unanesthetized rats

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

Association of apolipoprotein B and adiponectin receptor 1 genes with carcass, bone integrity and performance traits in a paternal broiler line

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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.)