The G protein-coupled serotonin 5-hydroxytryptamine (5-HT) 2A receptor is primarily recognized for its role in brain neurotransmission, where it mediates a wide variety of functions, including certain aspects of cognition. However, there is significant expression of this receptor in peripheral tissues, where its importance is largely unknown. We have now discovered that activation of 5-HT 2A receptors in primary aortic smooth muscle cells provides a previously unknown and extremely potent inhibition of tumor necrosis factor (TNF)-␣-mediated inflammation. 5-HT 2A receptor stimulation with the agonist (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(R)-DOI] rapidly inhibits a variety of TNF-␣-mediated proinflammatory markers, including intracellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and interleukin (IL)-6 gene expression, nitric-oxide synthase activity, and nuclear translocation of nuclear factor B, with IC 50 values of only 10 to 20 pM.It is significant that proinflammatory markers can also be inhibited by (R)-DOI hours after treatment with TNF-␣. With the exception of a few natural toxins, no current drugs or small molecule therapeutics demonstrate a comparable potency for any physiological effect. TNF-␣-mediated inflammatory pathways have been strongly implicated in a number of diseases, including atherosclerosis, rheumatoid arthritis, psoriasis, type II diabetes, depression, schizophrenia, and Alzheimer's disease. Our results indicate that activation of 5-HT 2A receptors represents a novel, and extraordinarily potent, potential therapeutic avenue for the treatment of disorders involving TNF-␣-mediated inflammation. Note that because (R)-DOI can significantly inhibit the effects of TNF-␣ many hours after the administration of TNF-␣, potential therapies could be aimed not only at preventing inflammation but also treating inflammatory injury that has already occurred or is ongoing.Serotonin, 5-hydroxytryptamine (5-HT), is a small monoamine molecule primarily known for its role as a neurotransmitter. Within the brain, it modulates a variety of behaviors including cognition, mood, aggression, mating, feeding, and sleep . These behaviors are mediated through interactions at seven different receptor families (5-HT 1-7 ) comprised of 14 distinct subtypes . Each of these are G protein-coupled receptors, with the exception of the 5-HT 3 receptor, which is a ligandgated ion channel. Of all the serotonin receptors, the 5-HT 2A receptor, which is known to primarily couple to the G␣q effector pathway (Roth et al., 1986), has been the one most closely linked to complex behaviors. There is a high level of expression within the frontal cortex, with significant localization to the apical dendrites of cortical pyramidal cells (Willins et al., 1997), and further expression at lower levels PKC, protein kinase C; Gö 6976, 5,6,7,pyrrolo [3,4-c]carbazole-12-propanenitrile; PMA, phorbol 12-myristate 13-acetate; F-22, fragment 6 -22; LA-SS-Az, (2ЈS,4ЈS)-(ϩ)-9,10-didehydro-6-methylergoli...
Material Supplementary 6.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.100064
Although nuclear translocation of NF-kappaB and subsequent binding to promoters of ICAM-1 and VCAM-1 have been shown to be decisive for their expression, a number of discrepancies in the expression patterns of these adhesion molecules have been reported in both cell culture systems and disease settings, including atherosclerosis, asthma, and autoimmune diseases. Here we show that while p65 NF-kappaB nuclear translocation in TNF-treated smooth muscle cells (SMCs) was sufficient for the expression of VCAM-1, expression of ICAM-1 showed a critical requirement for PARP-1. I-kappaBalpha phosphorylation and subsequent degradation were virtually identical in both TNF-treated wild-type and PARP-1-/- SMCs. VCAM-1 expression in TNF-treated PARP-1-/- SMCs was completely inhibited by the NF-kappaB inhibitor, pyrrolidine dithiocarbamate, confirming that VCAM-1 expression was indeed NF-kappaB-dependent. The expression of both VCAM-1 and ICAM-1 was associated with a transient interaction between PARP-1 and p65 NF-kappaB when examined in the fibroblastic cell line, COS-7, and in the airway epithelial cell line, A549. Such interactions were confirmed using florescence resonance energy transfer analysis. Protein acetylation activity, mediated by p300/CBP, was required for both VCAM-1 and ICAM-1 expression in TNF-treated SMCs; however, the interaction of PARP-1 with p300/CBP was dispensable for VCAM-1 expression. These findings indicate that p65 NF-kappaB nuclear translocation may be sufficient for certain genes (e.g., VCAM-1) while insufficient for others (e.g., ICAM-1), thus providing a novel insight into the role of NF-kappaB in driving target gene expression. Furthermore, the data suggest a differential requirement for PARP-1 expression in inflammatory processes.
Background We recently showed that PARP-1 may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. We also reported that while IL-5 replenishment promotes reversal of eosinophilia in lungs of PARP-1−/− mice, IL-4 or IgE replenishment do not, suggesting a potentially significant regulatory relationship between PARP-1 and IL-5. Objective To explore the mechanism by which PARP-1 regulates IL-5 production and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia. Methods This study was conducted using a murine model of allergic airway inflammation and primary splenocytes. Results PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. STAT-6 protein was severely downregulated in spleens of PARP-1−/− mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1−/− mice required allergen stimulation. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6 integrity. The dowregulation of STAT-6 coincided with mRNA and protein reduction of GATA-3 and occupancy of its binding site on the IL-5 gene-promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1−/− mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes. Conclusion These results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.
The role of inducible NO synthase (iNOS) in allergic airway inflammation remains elusive. We tested the hypothesis that iNOS plays different roles during acute versus chronic airway inflammation. Acute and chronic mouse models of OVA-induced airway inflammation were used to conduct the study. We showed that iNOS deletion was associated with a reduction in eosinophilia, mucus hypersecretion, and IL-5 and IL-13 production upon the acute protocol. Such protection was completely abolished upon the chronic protocol. Interestingly, pulmonary fibrosis observed in wild-type mice under the chronic protocol was completely absent in iNOS−/− mice despite persistent IL-5 and IL-13 production, suggesting that these cytokines were insufficient for pulmonary fibrosis. Such protection was associated with reduced collagen synthesis and indirect but severe TGF-β modulation as confirmed using primary lung smooth muscle cells. Although activation of matrix metalloproteinase-2/-9 exhibited little change, the large tissue inhibitor of metalloproteinase-2 (TIMP-2) increase detected in wild-type mice was absent in the iNOS−/− counterparts. The regulatory effect of iNOS on TIMP-2 may be mediated by peroxynitrite, as the latter reversed TIMP-2 expression in iNOS−/− lung smooth muscle cells and fibroblasts, suggesting that the iNOS–TIMP-2 link may explain the protective effect of iNOS-knockout against pulmonary fibrosis. Analysis of lung sections from chronically OVA-exposed iNOS−/− mice revealed evidence of residual but significant protein nitration, prevalent oxidative DNA damage, and poly(ADP-ribose) polymerase-1 activation. Such tissue damage, inflammatory cell recruitment, and mucus hypersecretion may be associated with substantial arginase expression and activity. The results in this study exemplify the complexity of the role of iNOS in asthma and the preservation of its potential as a therapeutic a target.
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