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. Author manuscript; available in PMC: 2022 Apr 25.
Published in final edited form as: Pulm Pharmacol Ther. 2018 Sep 8;53:12–19. doi: 10.1016/j.pupt.2018.09.004

Differential Regulation of Cytokine and Chemokine Expression by MK2 and MK3 in Airway Smooth Muscle Cells

Mariam Ba 1, Shanti Rawat 1, Ronna Lao 1, Marilyn Grous 2, Michael Salmon 2, Andrew J Halayko 3, William T Gerthoffer 1, Cherie A Singer 1
PMCID: PMC9036849  NIHMSID: NIHMS1507631  PMID: 30205157

Abstract

Background:

Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role in airway diseases. This is partially regulated by p38 mitogen-activated protein kinase (MAPK), which further activates two closely related isoforms of the MAPKactivated protein kinases (MKs), MK2 and MK3. The MKs have similar substrate specificities but less is known about differences in their functional responses. This study was undertaken to identify differential downstream inflammatory targets of MK2 and MK3 signaling and assess cross-talk between the MAPK pathway and NF-κB signaling relevant to ASM function.

Methods:

Wild-type and kinase-deficient MK2 (MK2WT, MK2KR) and MK3 (MK3WT, MK33A) were expressed in human ASM cells stimulated for 20 hours with 10 ng/ml each interleukin (IL)1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Inflammatory mediator secretion was assessed by Luminex assays and ELISA. Signaling pathway activation was monitored by Western blotting.

Results:

Expression of these MKs and stimulation with 10 ng/ml IL-1β, TNFα and IFNγ for 20 hours did not affect secretion of multiple cytokines including IL-4, IL-5, IL-13 and monocyte chemotactic protein (MCP)-1/CCL2 but did differentially affect the secretion of regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF). RANTES/CCL5 secretion was decreased by MK2WT or MK3WT and stimulated by inhibition of MK2 or MK3 activity with expression of the kinase-deficient enzymes MK2KR or MK33A. IL-6 and GM-CSF secretion was decreased by inhibition of MK2 activity with MK2KR and while MK3WT had no effect, the kinase-deficient MK33A further decreased secretion of these mediators. Cross-talk of the MKs with other signaling pathways was investigated by examining NF-κB activation, which was inhibited by expression of MK3 but not affected by MK2.

Conclusions:

These results suggest an inhibitory role for MK2 and MK3 activity in RANTES/CCL5 secretion and cross-talk of MK3 with NF-κB to regulate IL-6 and GM-CSF. These findings differentiate MK2 and MK3 function in ASM cells and provide insight that may enable selective targeting of MKs in ASM to modulate local inflammation in airway disease.

Keywords: Cell signaling, inflammation, airways, asthma

1. Introduction

There is abundant evidence that airway, vascular and gastrointestinal smooth muscle cells can be induced to secrete bioactive molecules that promote inflammation and serve an immunomodulator function [1–4]. Synthesis and release of cytokines and chemokines by airway smooth muscle (ASM) cells participate in airway hyperreactivity and promote the progression of airway remodeling during pathogenesis of lung disease. Expression of several key inflammatory signals such as interleukin −1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1)/CCL2 depends on activation of the p38 mitogen-activated protein kinases (MAPK) pathway in ASM cells, as it does in lymphoid cells, indicating that MAPK signaling plays a fundamental role in determining the profile of inflammatory genes expressed in response to allergic insult [5, 6].

The MAPK-activated protein kinases (MKs), including MK1/p90Rsk, MK2, MK3, MK4/Mnk and MK5/PRAK, are a family of serine/threonine kinases targeted by MAPKs [7]. MK2 and MK3 are ubiquitously expressed, highly homologous enzymes with 75% amino acid identity and are primarily activated by p38 α/β MAPK [7–9]. MK2 and MK3 share nearly identical substrate recognition sequences and are activated under similar conditions of cellular stress and inflammation [10–12]. MK2 and MK3 are co-expressed in various tissues such as lung and heart; however, MK2 expression and activity in vivo is significantly greater than MK3 [13], a feature that has complicated efforts to dissect substrate specificity for each isoform and the functional outcomes that can be induced.

One of the in vivo substrates of MK2 and MK3 is the small heat shock protein HSP27 (HSPB1), [8, 14, 15], a chaperone that is involved in actin remodeling, cell migration and survival [15, 16]. MK2 is a major HSP27 kinase that regulates migration of various cell types, including mouse embryonic fibroblasts and smooth muscle cells, in response to cellular stress [13, 17–19]. This function depends on a proline-rich SH3-binding domain in MK2, which is absent from MK3 [8, 20]. MK2 and MK3 also phosphorylate serum-response factor and mRNA binding proteins tristetraprolin (TTP) to mediate de novo transcription of TTP and stabilize the transcription of various inflammatory cytokines, respectively [11, 21]. MK2 has been shown to stabilize cell cycle modulators and cytokine mRNAs by phosphorylating the RNA stabilizing factor protein hnRNP A0 [22], whereas MK3 regulates chromatin remodeling by phosphorylation of the polycomb repressive complex protein Bmi1 [23]. In these ways, the MKs phosphorylate somewhat different sets of target proteins to mediate diverse cellular events including cytokine biosynthesis, cell migration, cell-cycle progression and cytoskeletal remodeling. Despite this insight, the independent contributions of MK2 and MK3 to inflammatory mediator synthesis and secretion are not completely clear because both enzymes are active simultaneously and have overlapping functions. Additionally, MK3 can compensate for the loss of MK2 [12, 24].

In smooth muscle, there are few studies that have specifically addressed differential expression and activity of MK2 and the role of MK3 in smooth muscle cells is not well characterized. It is known that MK2 can be activated and is involved in various aspects of gene regulation in smooth muscle cells. In vascular smooth muscle cells, angiotensin II upregulates MK2 protein expression through production of reactive oxygen species via nicotinamide adenine dinucleotide phosphate oxidase [25]. Further, knockout of MK2 in aortic smooth muscle is associated with downregulation of key smooth muscle cell marker genes, such as smooth muscle actin. myosin heavy chain and smoothelin [26]. In ASM cells, cigarette smoke extract is shown to upregulate IL-8 transcript levels via p38 MAPK/MK2 mediated-signaling [27]. However, the role of MK3 in smooth muscle cells has not been studied in detail.

To discriminate effects of MK2 from those of MK3 in ASM, we expressed wild type and mutant kinases in human ASM cells induced to synthesize inflammatory mediators. The secretion of a panel of Th1/Th2 cytokines and chemokines was evaluated following stimulus with a cytokine cocktail containing IL-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. We further evaluated secretion of regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, MCP-1/CCL2, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) following adenoviral-mediated expression of wild-type or kinase-deficient forms of MK2 and MK3. Effects of the MKs on related signaling pathways were further investigated by examining MAPK and NF-κB activation.

2. Methods

2.1. Generation of Recombinant MK Adenoviruses

Plasmids containing myc-tagged, wild type mouse MK2 (MK2WT) or the K76R MK2 mutant (MK2KR) in pcDNA 3.1 were kindly provided by Dr. M. Gaestel (Medical School of Hannover, Germany) [28]. Plasmids containing hemagglutinin (HA)-tagged wild type human MK3 (MK3WT) or the T210A, S260A, T322A MK3 mutant (MK33A) were provided in collaboration with GlaxoSmithKline. Recombinant adenoviruses were constructed using the AdEasy system (Stratagene, LaJolla, CA). Plasmids containing MK2 and MK3 were subcloned into the pAdTrack-CMV shuttle vector containing green fluorescent protein (GFP) behind a separate CMV promoter. Recombinant pAdTrack-CMV shuttle vectors were digested with PmeI to linearize the plasmid and transfected into the E.coli host strain BJ5183 containing the adenoviral backbone plasmid pAdEasy. Successful MK2 or MK3/pAdEasy recombinants were selected by kanamycin resistance. To produce adenoviruses, 2 μg of DNA from pAdEasy recombinants was digested with PacI and transfected into a viral packaging cell line. Adenoviruses were harvested, plaque-purified, and titered by an agarose overlay plaque assay. In all experiments, a pAdTrackCMV/pAdEasy recombinant containing GFP but no transgene was used as a control for virus infection (AdGFP).

2.2. Airway Smooth Muscle Cell Culture, Adenoviral Transduction and Cytokine Treatment

Primary cultured human ASM cells were obtained from 2nd−4th generation mainstem bronchi of patients undergoing lung resection surgery [29] in accordance with procedures approved by the Human Research Ethics Board at the University of Manitoba. These cells have been screened for human pathogens and characterized as ASM shown to express standard smooth muscle markers [30, 31]. Cells from passages 4–7 were used and grown in a humidified 5% CO2 atmosphere at 37°C in M199 media supplemented with 5% normal calf serum (NCS), 0.5 ng/ml EGF, 5 μg/ml insulin, 2 ng/ml FGF, 50 μg/ml gentamicin and 50 ng/ml amphotericin B. When the cultures reached confluence, cells were infected with adenoviruses in 200 μl of M199 containing 0.1% NCS for 60 minutes. Virus containing media was then removed and transduced cells were incubated in 0.1% NCS media for the duration of the experiment up to 3 days prior to treatment. Where indicated cultures were treated with a cytokine cocktail consisting of 10 ng/ml each IL-1β, TNFα, and IFNγ at the indicated time point or left untreated. This treatment has been shown to induce the expression of various inflammatory cytokines in ASM cells in a manner dependent, in part, on activation of p38 MAPK and NF-κB [1, 31]. In selected experiments, cells were pretreated with a 0.1% DMSO vehicle, 25 μM SB203580 or 1 μM MG-132 for 15 minutes prior to cytokine treatment.

2.3. Western blots

Whole cell lysates were prepared from cultures infected with the MK2 or MK3 adenoviruses and separated by 10% SDS-PAGE for Western blots using previously described methods [31]. α-MK2 (1:3500) and α-MK3 (1:5000) antibodies were obtained from Upstate Biotechnologies (Lake Placid, NY); α-myc (1:2000); α-HA (1:000) and α-cyclooxgenase-1 (COX-1, 1:1000) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). To characterize MK2 and MK3 overexpression with the adenoviral constructs, 5 μg total protein was loaded in each lane and following electrophoresis membranes were blocked with 0.5% gelatin with primary antibody at the dilutions noted above. All secondary antibodies were conjugated to alkaline phosphatase (1:10,000). Images of immunoblots were scanned with a UMAX Powerlook flatbed scanner. COX-1 was visualized as described below.

Western analysis of COX-1, ERK MAPK, p38 MAPK or IκBα took place using the Odyssey infrared imaging system (Licor Biosciences, Lincoln, NE). Blots were blocked in Odyssey blocking buffer diluted 1:1 in PBS. ERK MAPK blots (5 μg total protein) were probed with a dual anti-phosphotyrosine-threonine-p42/p44 (1:5000) and a p44 primary antibody that cross-reacts with p42 (1:1000). p38 MAPK blots (15 μg total protein) were probed with a dual antiphosphotyrosine-threonine-p38 MAPK (1:1000) and p38 MAPK primary antibodies (1:1000). IκBα immunoreactivity (1:000) was normalized to COX-1 (1:1000), which has been shown to remain stable in human ASM cells treated with cytokines [31]. Secondary antibodies were conjugated to AlexaFluor® 680 (Molecular Probes, Eugene, OR) or IRDye™800 (Rockland Immunochemicals, Gilbertsville, PA) for fluorometric detection. All analyses took place within the linear range of the immunoreactive signal using Odyssey imaging software. Phosphoantibodies were obtained from Cell Signaling Technology (Beverly, MA). All other antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA).

2.4. MK Activity Assay

Cultures were stimulated at the times indicated and cell lysates prepared in 30 mM MOPS, 80 mM β-glycerophosphate, 2 mM EGTA, 25mM MgCl2, 40 mM KCl, 0.1mM NaF, 0.1 mM Na3VO4, 1 mM AEBSF, 1 μM leupeptin; pH 7.0. The 40 μl kinase reaction contained 5 μg protein extract, 0.15 mg/ml recombinant human HSP27 and 5 μCi of 250 μM [γ−32P] ATP in 25mM MOPS, 25mM β-glycerophosphate, 15mM MgCl2, 1mM EGTA, 0.1mM NaF, 1mM Na3VO4, 4mM dithiothreitol, pH 7.2. After incubation at 30°C for 30 min, the reactions were terminated by addition of SDS sample buffer and the entire reaction loaded onto a 12% SDSPAGE gel. Following electrophoresis, the gels were stained with Coomassie blue to ensure equal sample loading and phosphorylated HSP27 was visualized and quantified by phosphorimaging on a Bio-Rad 525 Molecular Imager.

2.5. Luminex Assays and ELISA

Fifty μl of sample was analyzed on the Luminex 100, according to the manufacturer’s instructions using the Beadlyte human multi-cytokine detection system consisting of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, MCP-1/CCL2, RANTES/CCL5 (Upstate Biotechnology, Lake Placid, NY). RANTES/CCL5, IL-6 and GM-CSF were also measured by ELISA performed according the manufacturer’s instructions (R&D Systems, Minneapolis, MN). For each assay optimal dilutions were determined to ensure detection within the linear range of the standard curve (1:100, RANTES/CCL5; 1:1000, IL-6 and 1:10, GM-CSF). The minimum detectable levels of RANTES/CCL5, IL-6 and GM-CSF with this assay were approximately 6.6, 3.0 and 0.7 pg/ml, respectively.

2.6. Statistical Analysis

Statistical analysis was performed by one-way ANOVA followed by post-hoc testing with the Student-Newman-Keuls method or t-tests using SigmaStat software (Jandel Scientific, San Rafael, CA).

3. Results

3.1. Characterization of Adenoviral-Mediated MK2 and MK3 Overexpression

To examine differential functions of MK2 and MK3 in ASM cells, recombinant adenovirus vectors were constructed encoding wild-type murine MK2 (MK2WT) or catalytically inactive MK2 containing a K76R mutation in the ATP binding site (MK2KR), and wild-type human MK3 (MK3WT) or catalytically inactive MK3 enzyme containing alanine substitutions at T210, T260 and S322 (MK33A). We first characterized and optimized conditions for expression of each protein after adenoviral infection in ASM cells (Fig. 1). Initial experiments were performed to determine the optimum multiplicity of infection (MOI) for each construct (Fig. 1A, C) by following expression of the respective epitope tags (myc or HA) using 5 μg total protein. Expression of COX-1 is shown as a control in these samples.

Figure 1.

Figure 1.

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Adenoviral-mediated overexpression of MK2 and MK3 in ASMC. Cultures were transduced with adenoviruses at the MOI shown for 2 days (A, C) or transduced at 20 MOI and protein isolated 1 to 6 days postinfection (B, D). In non-infected cultures, protein was isolated day = 0.

Further experiments were performed to determine the time course at which maximum MK2 and MK3 expression levels are seen (Fig. 1B, D). Due to the small amount of total protein loaded (5 μg) to detect exogenously expressed proteins, endogenous levels of MK2 and MK3 were very low or undetectable in control cultures (non-infected; MOI = 0). There was no significant difference in expression levels at least 3 days post-infection, so all further experiments were performed using 20 MOI for 3 days.

3.2. Effect of MK overexpression on cytokine stimulated HSP27 phosphorylation

We assessed the effect of a complex pro-inflammatory cytokine stimulus on MK activity using a combination of 10 ng/ml IL-1β, TNFα and IFNγ previously shown to induce the expression of multiple inflammatory cytokines in ASM cells in a manner dependent, in part, on activation of p38 MAPK [1]. It has further been shown that activated p38 phosphorylates MK2/MK3 which in turn phosphorylates the downstream target HSP27 [32]. MK activity was evaluated by measuring the phosphorylation status of recombinant HSP27 (rHSP27) in response to MK2WT, MK2KR, MK3WT or MK33A expression. To analyze the effect of cytokine stimulation on rHSP27 phosphorylation, we performed a time course phosphorylation assay in non-infected cytokine treated cells. Cytokine stimulation of uninfected cells resulted in a 1.5-fold increase in rHSP27 phosphorylation within 5 minutes that remained above basal levels for up to 20 minutes (Fig. 2A). Similar data was obtained in AdGFP infected cultures, indicating adenoviral infection alone does not affect MK activity (Fig. 2B). Expression of MK2WT alone produced a marked 15fold increase in basal rHSP27 phosphorylation, and with cytokine stimulation MK activity levels were 34-fold higher than AdGFP. In contrast, MK2KR dramatically suppressed both basal and cytokine-stimulated MK activity when compared to MK2WT, consistent with a kinase-dead enzyme.

Figure 2.

Figure 2.

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Effect of MK2 and MK3 on HSP27 phosphorylation. A. Time course phosphorylation of rHSP27 in non-infected cytokine stimulated cells and B. MK activity was assessed in adenoviral infected cultures by measuring rHSP27 phosphorylation (shown in the representative phosphor image) in non-treated (NT) or cytokine treated cells for 5 min. Data are expressed relative to AdGFP, NT cultures, n = 3 ± SEM.

MK3WT also increased both basal and cytokine-stimulated levels of MK activity compared to AdGFP, but this induction, 2.8-fold and 4.6-fold respectively, was not nearly as robust as that seen with MK2WT. MK33A expression reduced both basal and cytokine stimulated MK activation below AdGFP levels, suggesting that the MK33A confers a less active enzyme that appears to be acting as a dominant negative.

3.3. Effect of MK2 and MK3 over expression on cytokine and chemokine secretion

We sought to identify differential inflammatory targets of MK2 and MK3 activation following transduction with MK2WT or MK3WT adenoviruses in response to the proinflammatory stimulus of IL-1β, TNFα and IFNγ.

We initially surveyed the effect of this stimulus on secretion of a panel of Th1/Th2 cytokines and chemokines, summarized in Table 1. This stimulus induced IL-1β and RANTES/CCL5, further stimulated secretion of IL-6 and MCP-1/CCL2 but had no detectable effects on levels of IL-2, IL-4, IL-5, IL-10, IL-12 (p70) and IL-13. With exogenous expression of MK2 or MK3, no further changes were seen in cytokine-stimulated secretion of IL-1β (data not shown) or MCP-1/CCL2 (Fig. 3A). However, RANTES/CCL5 secretion decreased 48% with MK2WT expression and 25% with MK3WT (Fig 3B).This effect was reversed by inhibition of MK2 and MK3 activity with exogenous expression of MK2KR and MK33A, which also stimulated RANTES/CCL5 secretion above that measured in control AdGFP cultures. These results indicate that MK2 or MK3 activity inhibits RANTES/CCL5 secretion and suggests an inhibitory role for p38 MAPK signaling in RANTES/CCL5 secretion.

Table 1:

Effect of cytokine stimulation on a panel of Th1/Th2 cytokines and chemokines in ASM cells. Culture media was collected after 20 hours of treatment with 10 ng/ml IL-1β, TNFα and IFNγ for Luminex. Data is reported in pg/ml from 2 donors, n = 3 ± SEM.

Inflammatory Mediator (pg/ml) No Treatment IL-1β, TNFα and IFNγ Stimulation
IL-1β N.D. 6751 + 217
IL-2 N.D. N.D.
IL-4 N.D. N.D.
IL-5 N.D. N.D.
IL-6 1287+ 316.83 40000*
IL-10 N.D. N.D.
IL-12 (p70) N.D. N.D.
IL-13 N.D. N.D.
TNFα N.D. 3680 + 105
IFNγ 104 + 26.05 29685 + 2503
MCP-1/CCL2 950.17 + 147 6808 + 361
RANTES/CCL5 N.D. 12231 + 4620
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N.D. = not detected;

*

beyond detection limits of assay.

Figure 3.

Figure 3.

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Effect of MK2 and MK3 overexpression on cytokine and chemokine secretion.

Adenoviral infected cultures were treated with 10 ng/ml IL-1β, TNFα and IFNγ, 20 hrs. A. MCP-1/CCL2, B. RANTES/CCL5, C. IL-6 secretion was analyzed by Luminex in 50 μl media from 2 donors, n = 3 ± SEM. D. GMCSF secretion was analyzed by ELISA from 3 donors, n = 3 ± SEM. Data expressed as % change from AdGFP. * = p < 0.05 from AdGFP; † = p < 0.05 from MK2WT, ‡ = p < 0.05 from MK3WT.

The effects of MK2 and MK3 on IL-6 secretion were measured by ELISA (Fig. 3C) as cytokine-stimulated secretion reported in Table 1 exceeded assay detection limits. Expression of inactive MK2KR or MK33A inhibited cytokine-stimulated IL-6 secretion to levels below that seen in AdGFP cultures. Indeed, inactive MK2KR over expression reduced IL-6 secretion by 38% compared to that measured in cultures where MK2WT was over expressed, and inactive MK33A reduced IL-6 secretion by 23% compared to that measured during MK3WT expression. These findings further confirm that activation of p38 MAPK/MK2 signaling is required for cytokineinduced IL-6 secretion [1, 33]. In contrast, expression of both MK3WT and MK33A did not further stimulate IL-6 secretion above that seen with AdGFP and in fact, expression of MK33A significantly decreased cytokine-stimulated expression of IL-6. Thus, while MK3 does not directly participate in stimulation of IL-6, the absence of MK3 activity may affect cross-talk with other pathways that mediate IL-6 secretion.

We completed additional studies on the role of MK2 and MK3 on GM-CSF secretion, as this cytokine was not included in our original Luminex assay and is regulated by MAPK signaling in many different cells types, including ASM cells [2, 34]. Basal levels of GM-CSF were not detected by ELISA in the ASM cells used in these experiments but GM-CSF secretion was markedly induced by the addition of IL-1β, TNFα and IFNγ (Fig. 3D). Importantly, expression of MK2WT increased GM-CSF expression by 43% above control levels with AdGFP infection. This response was reversed by expression of MK2KR, which decreased GM-CSF 66% from that seen with MK2WT and 33% below levels seen with AdGFP. In contrast, expression of MK3WT was not sufficient to further induce cytokine-stimulated GM-CSF secretion, suggesting a selective role for MK2 in initiating expression and release of this pro-eosinophilic cytokine. Notably expression of inactive MK33A did decrease GM-CSF secretion by 51%, thus mimicking results obtained for IL-6 and confirming a requirement for MK3 activity for maximum induction of GM-CSF, and again suggesting that cross-talk with other pathways may be important.

3.4. Effect of MK2 and MK3 over expression on MAPK and NF-κB signaling

In ASM cells, the expression and secretion of RANTES/CCL5, IL-6 and GM-CSF is mediated by multiple signaling pathways, most notably those involving MAPKs and NF-κB, but the relative contribution of each signaling pathway is stimulus-dependent [1, 2, 5, 34]. Results reported here suggest that expression of the MK2 and MK3 may interact with other pathways to modify chemokine secretion. Therefore, we examined the effects of the MK2 and MK3 adenoviruses on MAPK and NF-κB signaling by Western analysis of p38 MAPK and ERK MAPK phosphorylation, and IκBα abundance (Fig. 4). We have previously reported that stimulation with IL-1β, TNFα and IFNγ induces p38 and ERK MAPK phosphorylation [1], and in the present study we confirm this treatment stimulates p38 and ERK MAPK phosphorylation 6.5-fold above basal levels in control cultures transduced with AdGFP, indicating that adenoviral infection does not alter previously reported responses of ASM cells. Basal levels of p38 MAPK phosphorylation are dramatically reduced with expression of all the MK constructs (Fig. 4A) while basal levels of ERK MAPK phosphorylation remain unchanged (Fig. 4B). Cytokinestimulated p38 MAPK phosphorylation is reduced 4.2-fold from basal levels with expression of MK2WT while expression of MK2KR reduces p38 MAPK phosphorylation by 2.5-fold from basal levels. Interestingly, expression of both MK3WT and MK33A reduce cytokine-stimulated p38 MAPK phosphorylation to near the basal levels seen in AdGFP cultures. Thus, p38 MAPK phosphorylation is preferentially affected by MK2 expression. Neither the MK2 nor MK3 constructs appeared to affect cytokine-stimulated ERK MAPK phosphorylation, indicating that perturbations of the MAPK signaling pathway with MK over expression do not result in crosstalk with ERK MAPK.

Figure 4.

Figure 4.

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Effect of MK2 and MK3 on MAPK and NF-κB signaling pathways. Adenoviral infected cultures were treated for 15 min with 10 ng/ml IL-1β, TNFα and IFNγ (Cyto) or non-treated (NT). Densitometric analysis of A: p38 MAPK phosphorylation B: ERK MAPK phosphorylation or C: IκBα immunoreactivity was performed and normalized as described, n = 3 ± SEM, reported as a fold change from AdGFP, NT. * = p < 0.05 from AdGFP, Cyto; † = p < 0.05 from MK2WT, Cyto; ‡ = p < 0.05 from MK3WT, NT.

We also analyzed IκB abundance in the cytosol as a correlative to NF-κB-dependent DNA binding [31].

In AdGFP control cultures stimulated with cytokines, we observed a 50% reduction in cytosolic IκBα (Fig. 4C). Expression of the MK2 and MK3 wild-type constructs increased basal levels of IκBα, suggesting an overall inhibition of basal NF-κB signaling. However, cytokine stimulation did not markedly change cytosolic IκBα abundance in MK2WT or MK2KR cultures. This agrees with earlier data demonstrating that p38 MAPK and NK-κB are both activated by IL-1β, TNFα and IFNγ in ASM cells. However, this previous study demonstrated that IL-1β, TNFα and IFNγ stimulation of NF-κB does not result in cross-talk with p38 MAPK [31]. In contrast, cytokine-stimulated IκBα was reduced with MK3WT expression, and over expression of inactive MK33A reverses these effects, indicating a selective requirement for MK3, but not MK2, in cytokineinduced NF-κB activation.

Based on our findings, we next sought to verify the functional contributions of p38 MAPK and NF-κB to cytokine and chemokine secretion (Fig. 5). Addition of the NF-κB inhibitor MG-132 (1μM), blocked cytokine stimulated secretion of RANTES/CCL5, GM-CSF and IL-6. The p38 MAPK inhibitor SB203580 (25 μM) also blocked GM-CSF (Fig. 5B) and IL6 (Fig. 5C) secretion but increased cytokine-stimulated secretion of RANTES/CCL5 (Fig. 5A), an observation consistent with the effects we observed using MK2KR and MK33A on RANTES/CCL5 expression (Fig. 3) and demonstrating an inhibitory role for p38 MAPK activation in regulating RANTES/CCL5.

Figure 5.

Figure 5.

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Effect of NF-κB and p38 MAPK inhibition on chemokine secretion.

Adenoviral infected cultures were treated with treated for 15 min with 10 ng/ml IL-1β, TNFα and IFNγ (Cyto) ± 0.1% DMSO (+DMSO), 1 μM MG-132 (+MG) or 25 μM SB203580 (+SB). A: RANTES/CCL5, B: GM-CSF and C: IL-6 secretion. Cytokine secretion was measured by ELISA from 3 donors, n = 3 ± SEM. * = p < 0.05 from MG-132 or SB203580; † = p < 0.05 from MG-132. N.D. = not detected.

4. Discussion

Previous work in our laboratory and others has demonstrated that p38 MAPK plays a key role in the regulation of inflammatory gene expression in ASM cells. These studies have demonstrated that inhibition of p38 MAPK with SB203580 decreases synthesis of IL-1β, IL-6, and IL-8 [1] and also plays a role in expression of RANTES/CCL5, eotaxin/CCL11, GM-CSF [2] and cyclooxygenase-2 [31]. We undertook our current studies to specifically investigate whether MK2 and MK3, downstream mediators of p38 MAPK activity, differentially affect secretion of inflammatory mediators. We chose to stimulate ASM cells with a combination of IL-1β, TNFα and IFNγ, pro-inflammatory cytokines used to elicit cytokine secretion in this cell type [1, 31, 35]. It has been documented that human ASM cells synthesize IL-5 and increase expression of IL-2 and IL-12 in response to sensitization by atopic asthmatic serum [3]. However, in these studies, we observed no effect on synthesis of Th1 cytokines including IL-2 and IL-12 or Th2 cytokines such as IL-4, IL-5, IL-10 or IL-13. An increase in IL-1β can be attributed to detection of the exogenous IL-1β added with the stimulus in the media. Although previous studies have determined that IL-1β expression, measured as intracellular protein, can be stimulated with this cytokine mixture [1], this was not pursued further since expression of MK2 or MK3 did not affect IL-1β secretion in subsequent experiments (data not shown). Thus, the pro-inflammatory stimulus used here is not sufficient to induce classical Th1/Th2 cytokine expression in cultured human ASM cells but does stimulate IL-6, MCP-1/CCL2, RANTES/CCL5 and GM-CSF.

To investigate the contribution of MK2 and MK3 in the expression of inflammatory mediators, we transduced ASM cells with adenoviruses expressing wild-type or kinase-deficient enzymes. Importantly, the control AdGFP virus did not significantly affect cytokine synthesis compared to non-infected cultures (data not shown), indicating adenovirus infection per se did not impart significant effects on ASM cell function. Transduction of ASM cells with MK2 or MK3 adenoviruses did affect expression of IL-6, RANTES/CCL5 and GM-CSF, but was without effect on cytokine-stimulated MCP-1/CCL2 secretion. MCP-1/CCL2 is a chemoattractant for monocytes and lymphocytes shown to be elevated in bronchoalveolar lavage fluid and serum from asthmatics, and it is a potential biomarker in allergic asthma [36–39]. Synthesis and release of MCP-1/CCL2 can be reduced by inhibition of p38 MAPK with SB203580 [40]. The lack of effect seen in the current study suggests over expression of MK2 or MK3 is not sufficient to regulate MCP-1/CCL2 and other signaling pathways such as NF-κB, ERK and possibly JNK MAPK [40] in ASM cells.

A comparison of substrate specificity for MK2 and MK3 in the presence of the p38 MAPK inhibitor SB203580 previously established that p38 MAPK regulates MK activity in response to inflammatory signals [10, 11]. Additionally, work in tracheal smooth muscle tissues has confirmed that SB203580 blocks MK activity in ASM [41]. In our studies, transduction of ASM cells with either wild-type or catalytically inactive MK2 or MK3 attenuated basal and stimulated p38 MAPK activity. This observation is somewhat paradoxical but suggests the possibility that exogenous expression of MK may result in negative feedback of upstream p38MAPK signaling such as PAK, Ask1, or MKK3/6. Disruption of normal supramolecular signaling complexes might also explain the paradoxical inhibition since MK2 serves as a chaperone for p38 MAPK and activation p38 MAPK leads to phosphorylation and conformational changes in MK2 [13]. Thus, excess MK2 expression likely alters the stoichiometry of the p38 MAPK/MK2 interaction, while MK3 does not. Additionally, inactivation of MAPK signaling by the MAPK phosphatases is emerging as critical negative regulatory mechanism in cytokine biosynthesis [42]. Thus, driving MK expression and activity could indirectly lead to increased expression of phosphatases that ultimately blunt activation of p38 MAPK in our studies. Clarification of such mechanisms will be an avenue for further work.

Understanding the role of MAPK signaling on cytokine and chemokine expression and secretion in ASM cells has previously relied on the use of chemical inhibitors that have stimulusdependent effects. For example, TNFα increases IL-6 and RANTES/CCL5 secretion in a manner partially blocked by p38 MAPK inhibitor SB203580. Further, SB203580 partially inhibited IL-1β and TNFα induced GM-CSF secretion, whereas inhibition of ERK MAPK signaling with PD98059 had no significant effect on IL-6, RANTES or GM-CSF production [34, 43]. When the combination of IL-1β, TNFα and IFNγ is used, addition of SB203580 or PD98059 partially block IL-6 secretion, but both inhibitors in tandem virtually abrogate IL-6 stimulation [1]. This combination stimulus also increases GM-CSF, which can be significantly decreased with PD98059 [44]. The present study did not examine the effect of SB203580 on GM-CSF secretion but in monocytes, both PD98059 and SB203580 inhibit LPS-induced GM-CSF secretion [44]. Thus, this stimulus recruits both ERK and p38 MAPK signaling pathways to drive IL-6 and GMCSF expression, which is further confirmed by the results from this study whereupon expression of MK2WT drives IL-6 and GM-CSF secretion but MK2KR can only partially inhibit this effect. IL-6 and GM-CSF also bear AU-rich sequences in their 3’-untranslated regions that have been identified as crucial cis elements mediating RNA stability by p38 MAPK acting through MK2 [28]. It is likely that additional effects of MK2 on stabilization of mRNA transcripts is another mechanism in which p38 MAPK signaling contributes [9, 12].

Activation of NF-κB signaling is another important inflammatory signaling pathway and is a potential target for cross-talk with MAPK. Stimulation with LPS [45, 46], TNFα [43, 47] or the combination of cytokines used herein activates NF-κB [31]. However, treatment with SB203580 or PD98059 had little effect on NF-κB activity. As reported here, expression of MK2 appears to inhibit basal levels of NF-κB signaling with little additional effect upon cytokine stimulation. The same is not true for MK3. Cytokine stimulation in the presence of MK3WT activates NF-κB signaling in a manner reversed by expression of MK33A. It has been reported that MK2 can act to control the levels of negative feedback in NF-κB signaling by sequestering p38 MAPK in the cytosol [6]. A spatial model of MK2 regulation of NF-κB feedback was proposed by which MK2 moderates the duration of NF-κB signaling. It has further been reported that p38 MAPK phosphorylates histone H3 on cytokine and chemokine genes and that p38 MAPK activity is required by recruitment of NF-κB to these H3 phosphorylated promoters [48]. This occurs without any effects of p38 MAPK on NF-κB activity and suggests that this may be another way in which the p38 MAPK/MK pathway cooperates with NF-κB to affect cytokine expression.

Targeted-deletions of the MK genes in mice have demonstrated their importance in the inflammatory response. MK2 knockout mice are more resistant to LPS-induced endotoxic shock due to decreased production of TNFα, IL-6 and IFNγ [13, 49]. The contribution of MK2 in the production of inflammatory mediators is confirmed in various cell types acquired from MK2 knockout mice [50]. Deletion of MK3 alone, however, does not lead to any notable changes in inflammatory response. Double knockout of both MK2 and MK3 results in further reduction of LPS-induced TNFα and overexpression of MK3 can rescue the deficiencies seen with knockout of MK2 [12, 13]. This suggests that MK2 and MK3 function additively in stress-induced cytokine production and may share the same physiological functions. However, this does not exclude the possibility that stimuli or signals from other signaling pathways may result in differential activation of either enzyme. Also, even though the phosphorylation site recognition motifs of MK2 and MK3 are highly similar [10, 20], substrates of MK2 are often assumed to also be MK3 substrates without any experimental evidence to the contrary. The results presented here demonstrate that while MK2 and MK3 do have similar effects on cytokine and chemokine expression, the signaling and regulatory mechanisms likely differ. This is in agreement with previous study that demonstrated distinct regulatory roles of MK2 and MK3 in macrophages stimulated with LPS [51]. The results presented here suggest that MK3, but not MK2, may be a target for cross-talk with NF-κB signaling and may play an underappreciated role in p38 MAPK signaling.

5. Conclusions

Our data demonstrates that tissue and stimulus-specific analysis of the regulatory differences between MK2 and MK3 enzymes are needed in order to further our understanding of the role of MKs in inflammatory signaling pathways. This will benefit further targeting of the MK pathway in the search of more potent anti-inflammatory therapeutics.

Acknowledgements:

The authors thank Lisa Hanson for technical expertise in preparing reagents for this study.

Funding Resources: C.A.S. is supported by a Career Development Award HL080960. W.T.G. is supported by HL48183. A.J.H is supported through the Canada Research Chairs Program and Canadian Institutes for Health Research. This work was funded in part as a collaboration with GlaxoSmithKline.

Footnotes

Declaration of Interests: The authors declare no competing interests related to this work.

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