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Pulmonary fibrosis (PF) is an abnormal remodeling of cellular composition and extracellular matrix that results in histological and functional alterations in the lungs. Apoptosis signal-regulating kinase-1 (ASK1) is a member of the mitogen-activated protein (MAP) kinase family that is activated by oxidative stress and promotes inflammation and apoptosis. Here we show that bleomycin-induced PF is reduced in Ask1 knockout mice (Ask1(−/−)) compared with wild-type (WT) mice, with improved survival and histological and functional parameters restored to basal levels. In WT mice, bleomycin caused activation of ASK1, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) in lung tissue, as well as changes in redox indicators (thioredoxin and heme-oxygenase-1), collagen content, and epithelial–mesenchymal transition markers (EMTs). These changes were largely restored toward untreated WT control levels in bleomycin-treated Ask1(−/−) mice. We further investigated whether treatment of WT mice with an ASK1 inhibitor, selonsertib (GS-4997), during the fibrotic phase would attenuate the development of PF. We found that pharmacological inhibition of ASK1 reduced activation of ASK1, p38, and ERK1/2 and promoted the restoration of redox and EMT indicators, as well as improvements in histological parameters. Our results suggest that ASK1 plays a central role in the development of bleomycin-induced PF in mice via p38 and ERK1/2 signaling. Together, these data indicate a possible therapeutic target for PF that involves an ASK1/p38/ERK1/2 axis.

In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming growth factor–β (TGF-β)–stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-β (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-β–stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction–dependent manner. The abundance of ordered α–smooth muscle actin (α-SMA) filaments formed in the presence of TGF-β were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-β–stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-β–stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P > 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml−1), exerted no effect on TGF-β–regulated contractile protein expression (n = 8, P > 0.05), α-SMA organization, or the ratio of F-actin to G-actin (n = 4, P > 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-β treatment, still reduced contractile protein expression, even when the TGF-β–receptor kinase inhibitor, SB431542 (10 μM), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-β is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

Airway smooth muscle cell hyperplasia contributes to airway remodeling and hyperreactivity characteristic of asthma. Changes to potassium channel activity in proliferating human airway smooth muscle (HASM) cells have been described, but no regulatory role in proliferation has been attributed to them. We sought to investigate the expression of the intermediate conductance calcium-activated potassium channel K(Ca)3.1 in HASM cells and investigate its role in proliferation. Smooth muscle cells derived from human airways were grown in vitro and K(Ca)3.1 channel expression was measured using Western blot, RT-PCR, and patch clamp electrophysiology. Pharmacologic inhibitors of the channel were used in assays of cellular proliferation, and flow cytometry was used to identify cell cycle regulation. HASM cells expressed K(Ca)3.1 channel mRNA, protein, and activity with up-regulation evident after transforming growth factor-beta stimulation. Pharmacologic inhibition of K(Ca)3.1 led to growth arrest in cells stimulated to proliferate with mitogens. These inhibitors did not cause cellular toxicity or induce apoptosis. We have demonstrated, for the first time, the expression of K(Ca)3.1 channels in HASM cells. In addition, we have shown that K(Ca)3.1 channels are important in HASM cell proliferation, making these channels a potential therapeutic target in airway remodeling.

Rationale: Chronic inflammatory conditions including asthma, show varying levels of resistance to treatment by glucocorticoids (GC). This resistance may involve multiple mechanisms including: oxidative inactivation of histone deacetylase-2, downregulation of the active glucocorticoid receptor-α (GR-α) and an upregulation of the transactivation-inactive glucocorticoid receptor-β (GR-β). Method: Using A549 human epithelial cell line, GC responsiveness was assessed by measuring dexamethasone (0.1 1000nM) regulation of interleukin-1alpha (IL-1alpha) induced interleukin-8 (IL-8) production. Following preincubation of A549 cells in transforming growth factor beta (TGFbeta), we observed a right shift and significant diminution (from control 79 ± 2% to 34 ± 4%) of inhibition in the presence of 400pM TGFbeta (n=7; P