Emma E. Ramsay

Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1

The mechanisms by which corticosteroids reduce airway inflammation are not completely understood. Traditionally, corticosteroids were thought to inhibit cytokines exclusively at the transcriptional level. Our recent evidence, obtained in airway smooth muscle (ASM), no longer supports this view. We have found that corticosteroids do not act at the transcriptional level to reduce TNF-alpha-induced IL-6 gene expression. Rather, corticosteroids inhibit TNF-alpha-induced IL-6 secretion by reducing the stability of the IL-6 mRNA transcript. TNF-alpha-induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (t(1/2) = 2.4 h), compared to vehicle (t(1/2) = 9.0 h; P < 0.05) (results are expressed as decay constants [k] [mean +/- SEM] and half-life [h]). Interestingly, the underlying mechanism of inhibition by corticosteroids is via the up-regulation of an endogenous mitogen-activated protein kinase (MAPK) inhibitor, MAPK phosphatase-1 (MKP-1). Corticosteroids rapidly up-regulate MKP-1 in a time-dependent manner (44.6 +/- 10.5-fold increase after 24 h treatment with dexamethasone; P < 0.05), and MKP-1 up-regulation was temporally related to the inhibition of TNF-alpha-induced p38 MAPK phosphorylation. Moreover, TNF-alpha acts via a p38 MAPK-dependent pathway to stabilize the IL-6 mRNA transcript (TNF-alpha, t(1/2) = 9.6 h; SB203580 + TNF-alpha, t(1/2) = 1.5 h), exogenous expression of MKP-1 significantly inhibits TNF-alpha-induced IL-6 secretion and MKP-1 siRNA reverses the inhibition of TNF-alpha-induced IL-6 secretion by dexamethasone. Taken together, these results suggest that corticosteroid-induced MKP-1 contributes to the repression of IL-6 secretion in ASM cells.

Dual ERK and phosphatidylinositol 3-kinase pathways control airway smooth muscle proliferation: differences in asthma

Hyperplasia of airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely due to increased muscle proliferation. We have shown that ASM cells obtained from asthmatic patients proliferate faster than those obtained from non‐asthmatic patients. In ASM from non‐asthmatics, mitogens act via dual signaling pathways (both ERK‐ and PI 3‐kinase‐dependent) to control growth. In this study we are the first to examine whether dual pathways control the enhanced proliferation of ASM from asthmatics. When cells were incubated with 0.1% or 1% FBS, ERK activation was significantly greater in cells from asthmatic subjects (P < 0.05). In contrast, when cells were stimulated with 10% FBS, ERK activity was significantly greater in the non‐asthmatic cells. However, cell proliferation in asthmatic cells was still significantly higher in cells stimulated by both 1% and 10% FBS. Pharmacological inhibition revealed that although dual proliferative pathways control ASM growth in cells from non‐asthmatics stimulated with 10% FBS to an equal extent ([3H]‐thymidine incorporation reduced to 57.2 ± 6.9% by the PI 3‐kinase inhibitor LY294002 and 57.8 ± 1.1% by the ERK‐pathway inhibitor U0126); in asthmatics, the presence of a strong proliferative stimulus (10% FBS) reduces ERK activation resulting in a shift to the PI 3‐kinase pathway. The underlying mechanism appears to be upregulation of an endogenous MAPK inhibitor—MKP‐1—that constrains ERK signaling in asthmatic cells under strong mitogenic stimulation. This study suggests that the PI 3‐kinase pathway may be an attractive target for reversing hyperplasia in asthma. J. Cell. Physiol. 216: 673–679, 2008,

Corticosteroids and β2-agonists upregulate mitogen-activated protein kinase phosphatase 1: In vitro mechanisms

Airway remodelling is a consequence of long‐term inflammation and MAPKs are key signalling molecules that drive pro‐inflammatory pathways. The endogenous MAPK deactivator – MAPK phosphatase 1 (MKP‐1) – is a critical negative regulator of the myriad pro‐inflammatory pathways activated by MAPKs in the airway.

Long-Acting β2-Agonists Increase Fluticasone Propionate-Induced Mitogen-Activated Protein Kinase Phosphatase 1 (MKP-1) in Airway Smooth Muscle Cells

Mitogen-activated protein kinase phosphatase 1 (MKP-1) represses MAPK-driven signalling and plays an important anti-inflammatory role in asthma and airway remodelling. Although MKP-1 is corticosteroid-responsive and increased by cAMP-mediated signalling, the upregulation of this critical anti-inflammatory protein by long-acting β2-agonists and clinically-used corticosteroids has been incompletely examined to date. To address this, we investigated MKP-1 gene expression and protein upregulation induced by two long-acting β2-agonists (salmeterol and formoterol), alone or in combination with the corticosteroid fluticasone propionate (abbreviated as fluticasone) in primary human airway smooth muscle (ASM) cells in vitro. β2-agonists increased MKP-1 protein in a rapid but transient manner, while fluticasone induced sustained upregulation. Together, long-acting β2-agonists increased fluticasone-induced MKP-1 and modulated ASM synthetic function (measured by interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion). As IL-6 expression (like MKP-1) is cAMP/adenylate cyclase-mediated, the long-acting β2-agonist formoterol increased IL-6 mRNA expression and secretion. Nevertheless, when added in combination with fluticasone, β2-agonists significantly repressed IL-6 secretion induced by tumour necrosis factor α (TNFα). Conversely, as IL-8 is not cAMP-responsive, β2-agonists significantly inhibited TNFα-induced IL-8 in combination with fluticasone, where fluticasone alone was without repressive effect. In summary, long-acting β2-agonists increase fluticasone-induced MKP-1 in ASM cells and repress synthetic function of this immunomodulatory airway cell type.

Novel p38 MAPK inhibitor ML3403 has potent anti-inflammatory activity in airway smooth muscle

SB203580 is the prototypical p38 MAPK inhibitor; however it cannot be used clinically due to liver toxicity. We developed a structural analogue of SB203580 - ML3403 - with equal in vitro and ex vivo p38alpha MAPK inhibition as SB203580, but with reduced activity towards liver cytochrome P450 enzymes. In addition, we developed a selective p38alpha MAPK inhibitor - CP41. The aim of this study is to compare the anti-inflammatory activity of ML3403 and CP41, with SB203580. We compare and contrast the ability of the p38 MAPK inhibitors to repress tumour necrosis factor alpha (TNFalpha)-induced interleukin 6 (IL-6) and interleukin 8 (IL-8) mRNA expression and protein secretion from airway smooth muscle cells. We also examined and compared the binding affinities of ML3403 and SB203580 to the active and inactive p38alpha MAPK. We demonstrate that ML3403 binds to both active and inactive p38 MAPK with high affinity and that it inhibits p38 MAPK-mediated airway smooth muscle synthetic function to an equivalent degree with SB203580. CP41 was not able to reduce IL-6 and IL-8 secretion in airway smooth muscle cells; a function of its higher IC(50) against p38alpha MAPK when compared to SB203580 and ML3403. We show that p38 MAPK-mediated pro-inflammatory pathways in airway smooth muscle cells can be inhibited by ML3403. The anti-inflammatory activity is equivalent to the prototypical p38 MAPK inhibitor SB203580. Our results implicate a future pharmacotherapeutic strategy towards reducing inflammation in asthma and airway remodelling.

Proteasomal inhibition upregulates the endogenous MAPK deactivator MKP-1 in human airway smooth muscle: mechanism of action and effect on cytokine secretion.

Asthma is a chronic inflammatory condition. Inhibition of the ubiquitin-proteasome system offers promise as a anti-inflammatory strategy, being responsible for the degradation of key proteins involved in crucial cellular functions, including gene expression in inflammation (e.g. inhibitory IkappaB-alpha and the endogenous MAPK deactivator - MKP-1). As MKP-1 inhibits MAPK-mediated pro-remodeling functions in human airway smooth muscle (ASM; a pivotal immunomodulatory cell in asthma) in this study we investigate the effect of the proteasome inhibitor MG-132 on MKP-1 and evaluate the anti-inflammatory effect of MG-132 on cytokine secretion from ASM cells. Examining the time-course of induction of MKP-1 mRNA and protein by MG-132 (10microM) we show that MKP-1 mRNA was first detected at 30min, increased to significant levels by 4h, resulting in a 12.6+/-1.5-fold increase in MKP-1 mRNA expression by 24h (P<0.05). MKP-1 protein levels corroborate the mRNA results. Investigating the effect of MG-132 on secretion of the cytokine IL-6 we show that while short-term pretreatment with MG-132 (30min) partially reduced TNFalpha-induced IL-6 via inhibition of IkappaB-alpha degradation and the NF-kappaB pathway, longer-term proteasome inhibition (up to 24h) robustly upregulated MKP-1 and was temporally correlated with repression of p38-mediated IL-6 secretion from ASM cells. Moreover, utilizing a cytokine array we show that MG-132 represses the secretion of multiple cytokines implicated in asthma. Taken together, our results demonstrate that MG-132 upregulates MKP-1 and represses cytokine secretion from ASM and highlight the potential of the proteasome as a therapeutic target in asthma.

Corticosteroids Inhibit Sphingosine 1-Phosphate–Induced Interleukin-6 Secretion from Human Airway Smooth Muscle via Mitogen-Activated Protein Kinase Phosphatase 1–Mediated Repression of Mitogen and Stress-Activated Protein Kinase 1

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

Cyclin D1 in ASM Cells from Asthmatics Is Insensitive to Corticosteroid Inhibition

Hyperplasia of airway smooth muscle (ASM) is a feature of the remodelled airway in asthmatics. We examined the antiproliferative effectiveness of the corticosteroid dexamethasone on expression of the key regulator of G1 cell cycle progression—cyclin D1—in ASM cells from nonasthmatics and asthmatics stimulated with the mitogen platelet-derived growth factor BB. While cyclin D1 mRNA and protein expression were repressed in cells from nonasthmatics in contrast, cyclin D1 expression in asthmatics was resistant to inhibition by dexamethasone. This was independent of a repressive effect on glucocorticoid receptor translocation. Our results corroborate evidence demonstrating that corticosteroids inhibit mitogen-induced proliferation only in ASM cells from subjects without asthma and suggest that there are corticosteroid-insensitive proliferative pathways in asthmatics.

Effect of the cyclopyrrolones suriclone and RP 59037 on body temperature in mice

The effects of the cyclopyrrolones suriclone and RP 59037 on body temperature were investigated in male TO mice. The full agonist suriclone (3, 10, 30 mg/kg i.p.) produced significant hypothermia which was inhibited by concurrent administration of benzodiazepine receptor antagonists of both benzodiazepine (flumazenil; 10 mg/kg i.p.) and beta-carboline (ZK 93426; 3 mg/kg i.p.) structure. The response to suriclone (10 mg/kg i.p.) was also attenuated by benzodiazepine (Ro 17-1812; 10 mg/kg i.p.) and beta-carboline (ZK 91296; 30 mg/kg i.p.) partial agonists - which have no effect on body temperature per se. In contrast with these compounds, the cyclopyrrolone partial agonist RP 59037 (10, 30 mg/kg i.p.) produced significant hypothermia itself (although it was much less efficacious in this respect than the full agonist) and at a dose of 30 mg/kg failed to block the decrease in body temperature induced by suriclone (10 mg/kg i.p.). Thus suriclone acts as a full agonist at benzodiazepine receptors in the body temperature paradigm. RP 59037 possesses some partial agonist properties in this model, however, it appears to have greater intrinsic activity than other partial agonists tested previously.