MicroRNA Mediated Regulation of GRβ Expression in Airway Smooth Muscle: Implications for Glucocorticoid Insensitivity and Cell Proliferation

American Journal of Respiratory and Critical Care Medicine · 2025-05-01

Abstract Rationale: Glucocorticoid (GC) insensitivity presents a significant treatment challenge in inflammatory diseases such as asthma, where reduced GC sensitivity is associated with increased disease severity. While GC insensitivity mechanisms in immune cells from severe asthma patients are well-studied, these mechanisms in airway smooth muscle cells (ASMCs) remain relatively poorly understood. ASMCs, key targets for GC therapy, contribute to bronchoconstriction, inflammation, and airway remodeling in asthma. The effects of GC are mediated through the glucocorticoid receptor (GR), which includes GRα and GRβ isoform produced by alternative splicing. These isoforms differentially influence GC cellular responses, with increased GRβ expression lowering the GRα:GRβ ratio, a shift associated with GC insensitivity. MicroRNAs (miRNAs), post-transcriptional regulators of gene expression, may modulate GRβ levels and thus impact GC sensitivity. This study investigates whether specific miRNAs regulate GRβ expression in ASMCs after stimulation with the mitogen EGF. Methods: Primary ASM (ASM) cells isolated from asthma and non-asthma patients were treated with the mitogen EGF (10 ng/mL) at intervals of 1, 2, 3, 6, 18, 24, and 48 hours. GRβ knockdown and miRNA inhibition were achieved by transfecting cells with specific siRNA targeting GRβ and inhibitors for miR-21, miR-33, and miR-144 using FuGENE 6. Cell proliferation was measured using the CyQUANT assay, while GRβ mRNA expression levels were quantified via qRT-PCR. Statistical analyses were performed with GraphPad Prism 10, utilizing two-way ANOVA followed by Tukey's multiple comparisons test. Significance was defined as p < 0.05. Results: Treatment with EGF significantly increased GRβ expression in ASM cells from both asthma and non-asthma patients, with the peak response at 3 hours (p < 0.01). Transfection with GRβ-targeting siRNA effectively reduced GRβ expression by approximately 60% compared to both non-transfected cells and scramble control-transfected cells. Interestingly, GRβ knockdown significantly decreased EGF-induced cell proliferation by 16.6% (33067 vs. 27560 cells) at 24 hours and 13.8% (26264 vs. 23823 cells) at 48 hours. Inhibition of miR-21 led to a significant reduction in GRβ expression in both in both untreated (p < 0.001) and EGF-treated (p < 0.001) cells; however, this effect was seen only in cells derived from non-asthma patients. Conversely, miR-144 inhibition significantly reduced GRβ expression in cells from both asthma and non-asthma patients. MiR-33 inhibition did not affect GRβ expression. Conclusions: These findings indicate that miR-144 may regulate GRβ expression in ASMCs from asthma patients, potentially affecting GC sensitivity and ASMC proliferation. Future studies using gain-of-function approaches are warranted to further these findings.

Kinase-mediated Phosphorylation of Glucocorticoid Receptors in Human Airway Smooth Muscle Cells

American Journal of Respiratory and Critical Care Medicine · 2025-05-01

Abstract Rationale: Glucocorticoid (GC) receptors (GR) are nuclear receptors that mediate both non-genomic and genomic effects. Upon ligand binding, the GC-GR complex translocates to the nucleus, binds specific DNA sequences, and modulates transcription. GR is a phosphoprotein, and its phosphorylation is crucial for receptor activation, affecting ligand binding, DNA interaction, subcellular localization, and biological activity. The N-terminal domain (NTD) of GR contains several phosphorylation sites, including Ser211 and Ser226. Our previous studies demonstrated that GR phosphorylation at Ser211 enhances transcriptional activity. Another antecedent related to the direct phosphorylation of Ser211 was the involvement of the cyclin-dependent kinase 5 (CDK5). However, the kinases involved in this process remain unclear. This study investigates the role of kinases in GR phosphorylation in airway smooth muscle (ASM) cells. Methods: We first sought to identify the kinases induced by glucocorticoids using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D) to measure 37 kinases in human ASM cells treated with cortisol (1 µg/mL) for 1, 3, and 5 minutes. Principal component analysis (PCA) was performed with R (version 4.4.1), and statistical analysis was conducted using GraphPad Prism (version 10.4.0) with one-way ANOVA followed by Dunnett's post-test (p<0.05). In separate experiments, ASM cells were treated with the pan-CDK inhibitor Roscovitine (1, 10, and 20 µM) for 2 hours, followed by cortisol stimulation (1 µg/mL) for 1 hour. GR phosphorylation at Ser211 and total GR expression were analyzed by Western blot, and cell viability was assessed using the MTT assay. Results: Kinase profiling showed significant changes in kinase activity following cortisol treatment. After 1 minute, beta-catenin increased by 31%, PLC-γ1 (pY783) by 13%, and Src (pY419) by 24%. At 3 minutes, STAT3 (pY705) increased by 42%, and STAT6 (pY641) peaked at 5 minutes. PCA of the first two components explained 91.5% of the variation and showed a positive correlation between beta-catenin and Src (pY419) and a negative correlation with HSP27 (pS78/pS82). In pharmacological inhibition studies, Roscovitine decreased GC-induced pSer211 phosphorylation by 60%, 72%, and 77% at 1, 10, and 20 µM, respectively, without affecting cell viability. Conclusions: Our results suggest that multiple kinases, particularly the CDK family, regulate GR phosphorylation at Ser211 in human ASM cells. These insights may guide the development of therapeutic strategies targeting these pathways to modulate glucocorticoid responses, especially in glucocorticoid-resistant diseases.

Glucocorticoid Insensitivity in Asthma: The Unique Role for Airway Smooth Muscle Cells

International Journal of Molecular Sciences · 2022-08-11 · 17 citations

Although most patients with asthma symptoms are well controlled by inhaled glucocorticoids (GCs), a subgroup of patients suffering from severe asthma respond poorly to GC therapy. Such GC insensitivity (GCI) represents a profound challenge in managing patients with asthma. Even though GCI in patients with severe asthma has been investigated by several groups using immune cells (peripheral blood mononuclear cells and alveolar macrophages), uncertainty exists regarding the underlying molecular mechanisms in non-immune cells, such as airway smooth cells (ASM) cells. In asthma, ASM cells are among the targets of GC therapy and have emerged as key contributors not only to bronchoconstriction but also to airway inflammation and remodeling, as implied by experimental and clinical evidence. We here summarize the current understanding of the actions/signaling of GCs in asthma, and specifically, GC receptor (GR) "site-specific phosphorylation" and its role in regulating GC actions. We also review some common pitfalls associated with studies investigating GCI and the inflammatory mediators linked to asthma severity. Finally, we discuss and contrast potential molecular mechanisms underlying the impairment of GC actions in immune cells versus non-immune cells such as ASM cells.

Potential Role of Mast Cells in Regulating Corticosteroid Insensitivity in Severe Asthma

Advances in experimental medicine and biology · 2021-01-01 · 5 citations

Glucocorticoid Receptor β (GRβ): Beyond Its Dominant-Negative Function

International Journal of Molecular Sciences · 2021-03-31 · 64 citations

Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRβ, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRβ has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and "directly" regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRβ has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRβ-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRβ and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted.

New Insights on the Role of pentraxin-3 in Allergic Asthma

Frontiers in Allergy · 2021-06-11 · 9 citations

Pentraxins are soluble pattern recognition receptors that play a major role in regulating innate immune responses. Through their interaction with complement components, Fcγ receptors, and different microbial moieties, Pentraxins cause an amplification of the inflammatory response. Pentraxin-3 is of particular interest since it was identified as a biomarker for several immune-pathological diseases. In allergic asthma, pentraxin-3 is produced by immune and structural cells and is up-regulated by pro-asthmatic cytokines such as TNFα and IL-1β. Strikingly, some recent experimental evidence demonstrated a protective role of pentraxin-3 in chronic airway inflammatory diseases such as allergic asthma. Indeed, reduced pentraxin-3 levels have been associated with neutrophilic inflammation, Th17 immune response, insensitivity to standard therapeutics and a severe form of the disease. In this review, we will summarize the current knowledge of the role of pentraxin-3 in innate immune response and discuss the protective role of pentraxin-3 in allergic asthma.

Adiponectin/AdipoR1 Axis Promotes IL-10 Release by Human Regulatory T Cells

Frontiers in Immunology · 2021-05-18 · 37 citations

Background Adiponectin is an important immunomodulatory mediator in inflammatory conditions. While we previously showed that adiponectin receptor 1 (AdipoR1) is expressed in murine regulatory T cells (Tregs), its expression in human Tregs remain unknown. Here, we examined the expression of AdipoR1 in human Tregs and whether its ligand, globular adiponectin (gAd) affects the Treg ability to secrete IL-10 and the role of Type 2 (T2) inflammation in such process. Methods Human Tregs from peripheral blood were analyzed by flow cytometry for AdipoR1, Helios and IL-10 expression. CD4 + T cells enriched from peripheral blood mononuclear cells (PBMCs) were cultured in the presence or the absence of gAd or the chemical adiponectin receptor agonist, AdipoRon, or in a T2 cytokine milieu. Flow cytometry was then used to assess intracellular IL-10, IL-10 secreting cells, FOXP3 and Helios expression, and phosphorylated p38 MAP kinase (MAPK). IL-10 levels in CD4 + T cell supernatants were quantified by ELISA. Results We found that a subset of human Tregs expressed AdipoR1. Importantly, more Helios - cells expressed AdipoR1 than Helios + cells. Likewise, there was a higher frequency of IL-10 + cells within Helios - AdipoR1 + Tregs compared to Helios + AdipoR1 + Tregs. In contrast, the IL-10 mean fluorescence intensity (MFI) was higher in Helios + AdipoR1 + Tregs compared to Helios - AdipoR1 + Tregs. When human CD4 + T cells were treated with gAd or AdipoRon, a significant increase in IL-10 secretion, FOXP3 expression, and p38 MAPK phosphorylation was observed in Helios - AdipoR1 + Tregs. Interestingly, gAd under T2 cytokine milieu significantly increased the intracellular levels of IL-10, mainly in Helios + AdipoR1 + Tregs, and IL-10 levels in supernatants of CD4 + T cells. Conclusions Collectively, our findings suggest that adiponectin/AdipoR1 axis promotes IL-10 release by Tregs, mainly in Helios - Tregs, and the effect was amplified by T2 inflammation in Helios + Tregs.

Glucocorticoid Receptor Site-Specific Phosphorylation at Serine 134 Differentially Modulates Glucocorticoid Anti-inflammatory Actions in Human Airway Smooth Muscle Cells

2021-05-01

Human Lung Mast Cells Impair Corticosteroid Responsiveness in Human Airway Smooth Muscle Cells

Frontiers in Allergy · 2021-12-29 · 6 citations

The mechanisms underlying corticosteroid insensitivity in severe asthma have not been elucidated although some indirect clinical evidence points toward a role of mast cells. Here, we tested the hypothesis that mast cells can drive corticosteroid insensitivity in airway smooth muscle cells, a key player in asthma pathogenesis. Conditioned media from resting or FcεR1-activated human lung mast cells were incubated with serum-deprived ASM cells (1:4 dilution, 24 h) to determine their impact on the anti-inflammatory action of fluticasone on ASM cell chemokine expression induced by TNFα (10 ng/ml). Conditioned media from FcεR1-activated mast cells (but not that from non-activated mast cells or control media) significantly reduced the ability of 100 nM fluticasone to suppress ASM TNFα-dependent CCL5 and CXCL10 production at both mRNA and protein levels. In contrast, fluticasone inhibition of CXCL-8 production by TNFα was still preserved in the presence of activated mast cell conditioned media. Transcriptomic analysis validated by individual qPCR assays revealed that activated mast cell conditioned media dramatically reduced the number of anti-inflammatory genes induced by fluticasone in ASM cells. Our study demonstrates for the first time that conditioned media from FcεR1-activated mast cells blunt the anti-inflammatory action of corticosteroids in ASM cells by altering their transactivation properties. Because infiltration of mast cells within the ASM bundles is a defining feature of asthma, mast cell-derived mediators may contribute to the glucocorticoid insensitivity present in severe asthma.

Glucocorticoid Receptor (GR)-Beta Modulates PDGF-Induced Airway Smooth Muscle Proliferation

2020-05-01