Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells

Español, Alejandro; Goren, Nora; Ribeiro, Maria L.C.; Sales, Marı́a Elena

doi:10.1007/s00011-009-0097-4

Cited by 12 publications

(7 citation statements)

References 39 publications

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“…Both IL-1β and the cell-associated form IL-1α stimulate elongation. In other tissues, fibroblasts are thought to be intermediaries in the immune reaction during an inflammatory progression that includes chemokine signaling via prostaglandins and nitric oxide release [41]. We show that this phenomenon of cilia elongation, in response to the inflammatory cytokine IL-1, is not just active in chondrocytes but is also present in fibroblasts and therefore may have implications for all inflamed tissues.…”

Section: Discussionmentioning

confidence: 74%

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Wann

Knight

2012

Cell. Mol. Life Sci.

119

151

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Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50 % increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.

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Section: Discussionmentioning

confidence: 74%

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Wann

Knight

2012

Cell. Mol. Life Sci.

119

151

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“…It is well known that different inflammatory stimuli can activate the NF‐κB signalling pathway and that this activation can lead to an increase in the expression of several proteins (Bonizzi and Karin, ). Although we demonstrated that NF‐κB is constitutively active in NIH3T3 cells (Español et al ., ), our results indicate that this transcription factor is not involved in the de novo expression of M 3 and M 5 receptors in these cells.…”

Section: Discussionmentioning

confidence: 97%

“…In the present study, we showed for the first time that muscarinic acetylcholine receptor protein expression is induced under inflammatory conditions. In addition, we demonstrated that this effect is time‐dependent, because short‐term stimulation with the same concentrations of LPS and IFN‐γ failed to induce the expression of M 3 and M 5 receptors (Español et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Treatment with LPS plus INF‐γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX

Español

Maddaleno

Lombardi

et al. 2014

British J Pharmacology

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BACKGROUND AND PURPOSELPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. EXPERIMENTAL APPROACHNIH3T3 cells were treated with LPS (10 ng·mL ) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. KEY RESULTSThe cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. CONCLUSIONS AND IMPLICATIONSInflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation.

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“…Although, we have not established the expression of thromboxane synthase that generates TXA2, several studies have established that NIH3T3 cells express all the necessary components for activating the TPαR. This includes arachidonic acid [44] and COX-1/2 [45], [46] that are responsible for generating prostaglandin H2 (that in itself can work as an agonist on the TP receptor) [47], [48]. 2) Co-expression of AT1R with a mutant TPαR R130V, deficient in G protein coupling, enhanced the potency of AngII signaling to a much lesser degree than did the wild-type TPαR (Fig.…”

Section: Discussionmentioning

confidence: 93%

Functional Enhancement of AT1R Potency in the Presence of the TPαR Is Revealed by a Comprehensive 7TM Receptor Co-Expression Screen

et al. 2013

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BackgroundFunctional cross-talk between seven transmembrane (7TM) receptors can dramatically alter their pharmacological properties, both in vitro and in vivo. This represents an opportunity for the development of novel therapeutics that potentially target more specific biological effects while causing fewer adverse events. Although several studies convincingly have established the existence of 7TM receptor cross-talk, little is known about the frequencey and biological significance of this phenomenon.Methodology/Principal FindingsTo evaluate the extent of synergism in 7TM receptor signaling, we took a comprehensive approach and co-expressed 123 different 7TM receptors together with the angiotensin II type 1 receptor (AT1R) and analyzed how each receptor affected the angiotensin II (AngII) response. To monitor the effect we used integrative receptor activation/signaling assay called Receptor Selection and Amplification Technology (R-SAT). In this screen the thromboxane A2α receptor (TPαR) was the only receptor which significantly enhanced the AngII-mediated response. The TPαR-mediated enhancement of AngII signaling was significantly reduced when a signaling deficient receptor mutant (TPαR R130V) was co-expressed instead of the wild-type TPαR, and was completely blocked both by TPαR antagonists and COX inhibitors inhibiting formation of thromboxane A2 (TXA2).Conclusions/SignificanceWe found a functional enhancement of AT1R only when co-expressed with TPαR, but not with 122 other 7TM receptors. In addition, the TPαR must be functionally active, indicating the AT1R enhancement is mediated by a paracrine mechanism. Since we only found one receptor enhancing AT1R potency, our results suggest that functional augmentation through 7TM receptor cross-talk is a rare event that may require specific conditions to occur.

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Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells

Abstract: Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.

Cited by 12 publications

References 39 publications

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Treatment with LPS plus INF‐γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX

Functional Enhancement of AT1R Potency in the Presence of the TPαR Is Revealed by a Comprehensive 7TM Receptor Co-Expression Screen

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