Sphingosine 1-phosphate (S1P), a bioactive sphingolipid elevated in asthmatic airways, is increasingly recognized as playing an important role in respiratory disease. S1P activates receptor-mediated signaling to modulate diverse cellular functions and promote airway inflammation. Although many of the stimulatory pathways activated by S1P have been delineated, especially mitogen-activated protein kinases (MAPK), the question of whether S1P exerts negative feedback control on its own signaling cascade via upregulation of phosphatases remains unexplored. We show that S1P rapidly and robustly upregulates mRNA and protein expression of the MAPK deactivator-MAPK phosphatase 1 (MKP-1). Utilizing the pivotal airway structural cell, airway smooth muscle (ASM), we confirm that S1P activates all members of the MAPK family and, in part, S1P upregulates MKP-1 expression in a p38 MAPK-dependent manner. MKP-1 is a cAMP response element binding (CREB) protein-responsive gene and here, we reveal for the first time that an adenylate cyclase/PKA/CREB-mediated pathway also contributes to S1P-induced MKP-1. Thus, by increasing MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways, S1P temporally regulates MAPK signaling pathways by upregulating the negative feedback controller MKP-1. This limits the extent and duration of pro-inflammatory MAPK signaling and represses cytokine secretion in ASM cells. Taken together, our results demonstrate that S1P stimulates both kinases and the phosphatase MKP-1 to control inflammation in ASM cells and may provide a greater understanding of the molecular mechanisms responsible for the pro-asthmatic functions induced by the potent bioactive sphingolipid S1P in the lung.
BACKGROUND AND PURPOSEAirway 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. EXPERIMENTAL APPROACHHerein we investigated the molecular mechanisms responsible for the upregulation of MKP-1 in airway smooth muscle (ASM) by the corticosteroid dexamethasone and the b2-agonist formoterol, added alone and in combination. KEY RESULTSMKP-1 is a corticosteroid-inducible gene whose expression is enhanced by long-acting b2-agonists in an additive manner. Formoterol induced MKP-1 expression via the b2-adrenoceptor and we provide the first direct evidence (utilizing overexpression of PKIa, a highly selective PKA inhibitor) to show that PKA mediates b2-agonist-induced MKP-1 upregulation. Dexamethasone activated MKP-1 transcription in ASM cells via a cis-acting corticosteroid-responsive region located between -1380 and -1266 bp of the MKP-1 promoter. While the 3′-untranslated region of MKP-1 contains adenylate + uridylate elements responsible for regulation at the post-transcriptional level, actinomycin D chase experiments revealed that there was no increase in MKP-1 mRNA stability in the presence of dexamethasone, formoterol, alone or in combination. Rather, there was an additive effect of the asthma therapeutics on MKP-1 transcription. CONCLUSIONS AND IMPLICATIONSTaken together, these studies allow us a greater understanding of the molecular basis of MKP-1 regulation by corticosteroids and b2-agonists and this new knowledge may lead to elucidation of optimized corticosteroid-sparing therapies in the future. AbbreviationsAd5, adenoviral serotype 5; ASM, airway smooth muscle; CREB, cyclic-AMP response element binding protein; GC, glucocorticoid; GRE, GC-responsive element; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; MKP-1, MAPK phosphatase 1; MOI, multiplicity of infection; 3'-UTR, 3′-untranslated region
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.
Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.
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