Glucocorticoid- and protein kinase A-dependent transcriptome regulation in airway smooth muscle

Abstract

Glucocorticoids (GCs) and protein kinase A (PKA)-activating agents (beta-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma--excessive ASM growth--are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1 beta and TNF-alpha mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2-dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1 beta stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1 beta, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein-coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of beta-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.

Figure 1.

Experimental design of microarray analysis. Human airway smooth muscle (ASM) cells stably expressing GFP or PKI-GFP chimera (generated as outlined in Materials and Methods) were grown to confluence, arrested for 24 hours in 0.1% bovine serum albumin–containing media, pretreated with vehicle or Fluticasone (10 nM) for 30 minutes, and stimulated with epidermal growth factor (EGF) (10 ng/ml), IL-1β (20 U/ml), or both (E+I) for 8 hours. Cells were lysed in TRIzol and total RNA isolated by phenol/isopropanol precipitation. RNA was processed as outlined in Materials and Methods and gene expression was assessed using the Affymetrix U133A microchip. Gene expression was analyzed in four different cultures derived from four individual donors.

Figure 2.

Glucocorticoid (GC) treatment and protein kinase A (PKA) inhibition regulate mitogenic effects of IL-1β. Effect of PKA inhibition via heterologous expression of PKA inhibitor (PKI) or pretreatment with GC on IL-1β-, EGF-, and EGF+IL-1β–stimulated [³H]thymidine incorporation in human ASM. Data represent mean ± SE values, n = 4.

Figure 3.

Clustering analysis of microarray data with dCHIP. Clustering analysis based on similarly regulated genes (variability 0.5–100 and present call 50%) revealed clustering into two groups with the following subgroups: (1a) Flu or Flu- and EGF-stimulated cells; (1b) unstimulated and EGF-stimulated cells; (2a) IL-1β– or E+I-stimulated cells; (2b) IL-1β– or E+I-stimulated cells in presence of Flu. Stars by sample name indicate number of replicates per condition + 1.

Figure 4.

Effects of fluticasone treatment of ASM on chemotactic effect of conditioned media from ASM. Conditioned media were harvested from GFP or PKI-GFP–expressing ASM cells treated for 18 hours with vehicle (basal) or EGF (10 ng/ml) plus IL-1β (20 U/ml) (E+I) ± 10 nM Flu. These media were tested for the ability to promote chemotaxis of human neutrophils purified (∼ 95% purity) from peripheral blood as described in Materials and Methods. Formyl-methionyl-leucyl-phenylalanine (FMLP; 1 μM) and IL-8 (1 μM) were used as positive controls. Data represent mean ± SE values, n = 4.

Figure 5.

Effects of autocrine/juxtacrine factors induced in ASM cells. (A and B) Conditioned media were harvested from GFP- and PKI-expressing cells stimulated for 18 hours with EGF (10 ng/ml) plus IL-1β (20 U/ml) and used to stimulate [Ca²⁺]_i mobilization in GFP- and PKI-expressing cells as described in Materials and Methods. (A) Representative tracing and (B) cumulative results (mean ± SE values, n = 3) are presented. (C) dsRed-expressing cells were co-cultured with GFP- or PKI-GFP–expressing cells, stimulated with EGF (10 ng/ml), IL-1β (20 U/ml), or both in presence of BrdU (60 μM). BrdU incorporation was evaluated in dsRed⁺ and GFP⁺ cell populations. Data represent mean ± SE values, n = 4.