Hypertrophy and hyperplasia of airway smooth muscle cells (ASMCs) are characteristic pathological findings of severe asthma [
1,
39]. ASMCs are increasingly recognized as an important source of inflammatory cytokines and chemokines, as well as the effector cells of bronchoconstriction [
2‐
8,
40]. Bronchial thermoplasty has recently been introduced as a nonpharmacological therapy for moderate-to-severe asthma patients who are uncontrolled despite optimal medical therapy. This treatment could reduce exacerbations and the emergency room visit rate, resulting in improved quality of life [
41‐
43]. Although the mechanism of action is incompletely understood, it has been suggested that bronchial thermoplasty works by reducing ASMCs [
44]. Reduction of ASMCs may decrease the secretion of cytokines and chemokines from ASMCs in patients with severe asthma. Interestingly, it has been demonstrated that increased expressions of CXCL8 and eotaxin in ASMCs, as well as an increase of airway smooth muscle area, are seen in patients with severe asthma compared with those with moderate asthma [
45].
CXCL8 is secreted by ASMCs following various stimuli, such as TNF-α and cigarette smoke [
7,
26‐
34]. Our previous study showed that extracellular acidification induced ASMCs to generate IL-6 and CTGF via OGR1 [
18,
19]. In the present study, the ASMCs, in which the expression of OGR1 was decreased by siRNA targeted for OGR1, released less CXCL8 in response to extracellular pH 6.3 than controlled ASMCs, suggesting that CXCL8 is also generated and released through OGR1-mediated intracellular signal transduction in acidic pH-stimulated ASMCs. The amount of CXCL8 released from pH 6.3-stimulated cells was substantial and about 20–25% of that from TNF-α-stimulated ASMCs. Because we have previously shown that IL-6 release is inhibited by a MEK1/2 inhibitor and a p38 MAPK inhibitor in acidic pH-stimulated ASMCs, the effects of these kinase inhibitors on CXCL8 release in acidic pH-stimulated cells were examined. A MEK1/2 inhibitor, U0126, but not a p38 MAPK inhibitor, SB203580, inhibited CXCL8 release in acidic pH-stimulated ASMCs, suggesting that p38 MAPK activation is not always required for CXCL8 production in ASMCs. In fact, it has been reported that MEK1/2-ERK1/2 activation is required, but p38 MAPK activation is not, for CXCL8 production in poly(I:C)-stimulated human fetal ASMCs [
46] or heat shock protein 22-stimulated aortic smooth muscle cells [
47]. CXCL8 gene transcription seems to be regulated by transcription factors, such as NF-κB, AP-1, or C/EBP, because the binding sites of these transcription factors are present in the CXCL8 promoter region [
47,
48]. Among them, NF-κB is generally involved in CXCL8 gene transcription in many kinds of cells [
49‐
51]. In this study, serine 536 phosphorylation of NF-κB p65 was increased after acidic pH stimulation, in contrast to our previous study [
18]. This discrepancy may depend on individual differences of the primary ASMCs used in the present study. Phosphorylation at Ser536 on the p65 subunit of NF-κB is mediated by IKK during LPS stimulation. Ser536 phosphorylation is responsible for the recruitment of coactivators such as p300, promoting the transcriptional activation of NF-κB and the subsequent production of inflammatory cytokines [
52‐
54]. Interestingly, BAY 11–7082, an inhibitor of the NF-κB pathway, almost completely inhibited the serine 536 phosphorylation of NF-κB p65 and acidic pH-stimulated CXCL8 production in ASMCs. CXCL8 secretion from ASMCs in the presence of BAY 11–7082 was less than that in the pH 7.4-adjusted medium without any stimulation. These results suggest that a certain level of NF-κB activity may be essential for CXCL8 production in ASMCs, and that NF-κB and ERK1/2 are major regulators of CXCL8 production in ASMCs induced by acidic pH stimulation, as well as other stimuli [
30,
46,
55,
56]. Recently, it was reported that OGR1-mediated NF-κB activation is required for acidic pH-induced CXCL8 production in a human pancreatic β-cell line [
51]. This also indicates that NF-κB activity is essential for OGR1-mediated CXCL8 production.
OGR1 has been shown to couple to Gq/11 protein in ASMCs [
17,
18]. It is expected that phospholipase C (PLC) is activated and mediates the hydrolysis of membrane inositol phospholipids to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, which in turn triggers the release of calcium from intracellular stores following Gq/11 activation [
14,
57,
58]. Because the PKC inhibitor bisindolylmaleimide I inhibited OGR1-mediated CXCL8 secretion in ASMCs, PKC activation following PLC may be involved in the pathway to CXCL8 gene transcription. In the present study, DEX inhibited CXCL8 secretion from acidic pH-stimulated ASMCs in a dose-dependent manner. CXCL8 mRNA expression was also inhibited by DEX, as well as CXCL8 protein. Although DEX partially inhibited CXCL8 release in TNF-α-stimulated ASMCs, the inhibition rate was smaller than that in acidic pH-stimulated cells. Glucocorticoids (GCs) may regulate cytokine gene expression in several ways. The glucocorticoid receptor (GR) influences gene expression by physically interacting with other transcription factors without contacting DNA itself. This mechanism is called transrepression [
59]. Activated GRs translocate to the nucleus and bind to coactivators to inhibit histone acetyltransferase activity and recruiting histone deacetylase-2, which reverses histone acetylation, leading to suppression of targeted genes [
60,
61]. Although NF-κB activity was essential for CXCL8 secretion of ASMCs, DEX affected neither serine 536 phosphorylation of NF-κB p65 nor binding of NF-κB p65 to its consensus DNA sequence. These results support the possibility that CXCL8 gene transcription is inhibited by DEX at the step after a dimer of p50 and p65 NF-κB proteins binds to a specific NF-κB recognition site. The detailed mechanism of DEX with respect to CXCL8 gene transcription in ASMCs and the reason why the inhibition rate by DEX is different between acidic pH-stimulated cells and TNF-α-stimulated ones should be elucidated by further research.