Extracellular acidification-induced CXCL8 production through a proton-sensing receptor OGR1 in human airway smooth muscle cells: a response inhibited by dexamethasone

Abstract

Background Human 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. Methods ASMCs 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. Results Acidic 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. Conclusions CXCL8 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.

Metal-Stimulated Interleukin-6 Production Through a Proton-Sensing Receptor, Ovarian Cancer G Protein-Coupled Receptor 1, in Human Bronchial Smooth Muscle Cells: A Response Inhibited by Dexamethasone

“…DEX also significantly inhibited IL-8 mRNA expression in pH 6.3-stimulated human BSMCs. 9 DEX inhibited Ni- and Co-stimulated IL-6 production in a dose-dependent manner. This inhibition was significant at a DEX concentration ranging between 1 and 1000 nM ( Figure 5A and B ).…”

“…In our previous study, only OGR1 mRNA was practically detected in human BSMCs among four kinds of GPCRs, namely OGR1, G protein-coupled receptor 4 (GPR4), T-cell death-associated gene 8 (TDAG8), and G2A 8 and the expression level of OGR1 mRNA after treatment with OGR1-siRNA was decreased to about 5% of the level found after treatment with NT-siRNA in BSMCs. 9 Human BSMCs under Co and Ni stimulation were transfected with OGR1-siRNA to decrease OGR1 expression in this study. We confirmed the knockdown efficacy of OGR1-siRNA as well.…”

“…Cells were grown in the complete culture medium (Lonza), which was composed of smooth muscle cell basal medium supplemented with human epidermal growth factor, insulin, human fibroblast growth factor β, 5% fetal bovine serum, gentamicin, and amphotericin B, under a humidified atmosphere of 95% air plus 5% CO 2 at 37°C, as previously reported. 9 , 10 Three to six passages of BSMCs were used in the experiments. Human BSMCs were cultured in collagen I-coated 12-well plates (BioCoat TM , Corning International K.K., Tokyo, Japan) for enzyme-linked immunosorbent assay (ELISA) and in collagen I-coated 6-well plates (BioCoat TM ) for real-time RT-PCR using TaqMan probes.…”

“…The supernatant was then analyzed by Western blotting with specific antibodies for phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), and GAPDH as previously described. 8 , 9 …”

“… 30 These findings suggest that IL-6 may play an important role in the pathogenesis of asthma, particularly in obese or non-allergic asthma. Interestingly, extracellular acidification induces secretion of interleukin-6 (IL-6), 8 interleukin-8 (IL-8)/ C-X-C motif chemokine ligand 8 (CXCL8), 9 and connective tissue growth factor (CTGF) 10 in addition to cell contraction 3 through OGR1 activation in human BSMCs.…”