Differential regulation of cytokine and chemokine expression by MK2 and MK3 in airway smooth muscle cells

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 MAPK-activated 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 (MK2^WT, MK2^KR) and MK3 (MK3^WT, MK3^3A) were expressed in human ASM cells stimulated for 20 h 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 h 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 MK2^WT or MK3^WT and stimulated by inhibition of MK2 or MK3 activity with expression of the kinase-deficient enzymes MK2^KR or MK3^3A. IL-6 and GM-CSF secretion was decreased by inhibition of MK2 activity with MK2^KR and while MK3^WT had no effect, the kinase-deficient MK3^3A 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: Airways; Asthma; Cell signaling; Inflammation.

Figure 1.

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.

Figure 2.

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 Ad^GFP, NT cultures, n = 3 ± SEM.

Figure 3.

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 Ad^GFP. * = p < 0.05 from Ad^GFP; † = p < 0.05 from MK2^WT, ‡ = p < 0.05 from MK3^WT.

Figure 4.

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 Ad^GFP, NT. * = p < 0.05 from Ad^GFP, Cyto; † = p < 0.05 from MK2^WT, Cyto; ‡ = p < 0.05 from MK3^WT, NT.

Figure 5.

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.