Abstract
BackgroundHuman airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma. They also generate cytokines, chemokines, and matricellular proteins. Ovarian cancer G protein-coupled receptor 1 (OGR1) senses extracellular protons and mediates the production of interleukin-6 (IL-6) and connective tissue growth factor (CTGF) in ASMCs.MethodsASMCs were stimulated for the indicated time by pH 6.3 or pH 7.4-adjusted Dulbecco’s Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM). As a control stimulant, pH 7.4-adjusted 0.1% BSA-DMEM containing 10 ng/mL tumor necrosis factor-α (TNF-α) was used. Interleukin-8/C-X-C motif chemokine ligand 8 (CXCL8) mRNA expression in ASMCs was quantified by RT-PCR using real-time TaqMan technology. CXCL8 secreted from ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Phosphorylation at serine 536 of NF-κB p65 and binding of p65 to oligonucleotide containing an NF-κB consensus binding site were analyzed by Western blotting and an ELISA-based kit.ResultsAcidic pH induced a significant increase of CXCL8 mRNA expression and CXCL8 protein secretion in ASMCs. ASMCs transfected with small interfering RNA (siRNA) targeted for OGR1 produced less CXCL8 compared with those transfected with non-targeting siRNA. Protein kinase C (PKC) inhibitor, MEK1/2 inhibitor, and the inhibitor of IκB phosphorylation reduced acidic pH-stimulated CXCL8 production in ASMCs. Dexamethasone also inhibited acidic pH-stimulated CXCL8 production of ASMCs in a dose-dependent manner. Dexamethasone did not affect either phosphorylation or binding to the consensus DNA site of NF-κB p65.ConclusionsCXCL8 released from ASMCs by extracellular acidification may play a pivotal role in airway accumulation of neutrophils. Glucocorticoids inhibit acidic pH-stimulated CXCL8 production independent of serine 536 phosphorylation and the binding to DNA of NF-κB p65, although NF-κB activity is essential for CXCL8 production in ASMCs.
Highlights
Human airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma
It has been demonstrated that ovarian cancer G protein-coupled receptor 1 (OGR1) family G protein-coupled receptors (GPCRs), including Ovarian cancer G protein-coupled receptor 1 (OGR1), G protein-coupled receptor 4 (GPR4), and T-cell death-associated gene 8 (TDAG8 or GPR65), sense extracellular protons and mediate cellular actions induced by alkaline and acidic pHs of 8 to 6 through histidine residues in a variety of cell types [14,15,16,17]. Among these proton-sensing GPCRs, only OGR1 is expressed in ASMCs, and that IL-6 and connective tissue growth factor (CTGF) are secreted from ASMCs through OGR1-mediated stimulation of intracellular signaling pathways in response to extracellular acidification [18, 19]
C-X-C motif chemokine ligand 8 (CXCL8) production was observed in incubation with pH 7.4-adjusted medium, it was increased about 5-fold in incubation with pH 6.3-adjusted medium compared with pH 7.4 at 24 h (Fig. 1a)
Summary
Human airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma. They generate cytokines, chemokines, and matricellular proteins. Ovarian cancer G protein-coupled receptor 1 (OGR1) senses extracellular protons and mediates the production of interleukin-6 (IL-6) and connective tissue growth factor (CTGF) in ASMCs. Bronchial asthma is a disease characterized by chronic airway inflammation, airway hyperresponsiveness, and reversible airway obstruction. The degree of acidification depends on the severity of asthma, and airway pH seems to reach 5.2. It is normalized by corticosteroid therapy [9, 11]
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