Basal p38 Mitogen-Activated Protein Kinase Regulates Unliganded Glucocorticoid Receptor Function in Airway Smooth Muscle Cells

Abstract

Like many steroid receptors, the glucocorticoid (GC) receptor (GR) is a phosphoprotein. Although there are multiple phosphorylation sites critical for GR transcriptional activity (i.e., serine [S]203, S211, and S226), their respective role in driving GR functions is highly cell specific. We have recently identified protein phosphatase 5 as an essential Ser/Thr phosphatase responsible for impairing GR function via S211 dephosphorylation in airway smooth muscle (ASM) cells. Because p38 mitogen-activated protein kinase (MAPK) directly phosphorylates GR in different cell types in a stimulus-and celldependent manner, we investigated the role of p38 MAPK on GR phosphorylation and function in ASM cells. Cells were transfected with 100 nM p38 MAPK small interfering RNA or 2 mg MAPK kinase 3 expression vector (a specific kinase that directly activates p38 MAPK) in the presence or absence of fluticasone (100 nM) and/or p38 MAPK pharmacological inhibitor SB203580. We found that p38 MAPK blockade positively regulates GR nuclear translocation and GR-dependent induction of the steroid-target gene GC-induced leucine zipper in a hormone-independent manner. We also found that p38 MAPK-dependent regulation of GR functions was associated with a differential action on GR phosphorylation at S203 and S211 residues. This study demonstrated that the inactive state of GR in resting conditions is not only ensured by the absence of the GC ligand but also by p38 MAPK-dependent phosphorylation of unliganded GR at specific residues, which appears to be important in determining the overall GC responsiveness of ASM cells.

Glucocorticoid and TNF signaling converge at A20 (TNFAIP3) to repress airway smooth muscle cytokine expression

“…1). Moreover, aligned with prior ASM studies (8,26), this process appears to be enhanced at certain loci by TNF. For example, GR occupancy levels were markedly increased by TNF treatment compared with vehicle at several tested loci, including IL1A and CXCL8 (see Fig.…”

“…2, C and D, top). In some cases there was detectable GR occupancy with vehicle or TNF treatment alone, consistent with other recent reports of nuclear localization of so-called unliganded GR in ASM (8,26). Regardless of the magnitude of inductive effects of TNF or dex alone on GR occupancy, however, TNF ϩ dex cotreatment resulted in generally similar patterns and levels of GR binding at interro- gated regions within the pro-and anti-inflammatory gene sets.…”

Cistrome-based Cooperation between Airway Epithelial Glucocorticoid Receptor and NF-κB Orchestrates Anti-inflammatory Effects

“…occupancy in the absence of supplemental dex treatment)? We previously attributed this phenomenon, which has been observed in earlier studies by us and other groups (36,53,55,78,79), to the activity of glucocorticoid-like molecules presumed to be present at low levels within the culture medium. However, several aspects of our ChIP-seq data are incompatible with this notion.…”

Decreased adipogenesis and adipose tissue in mice with inactivated protein phosphatase 5

“…Our data show that Ppp5 plays a key role in keeping the unliganded (glucocorticoid-free) GR dephosphorylated at Ser212 and Ser220, which appears to be essential for GR-mediated repression of DLK1, a process required for the initiation of differentiation in proliferating preadipocytes. Other recent studies have provided evidence that the unliganded form of the GR may be crucial for some GR functions [45,46]. The decreased body mass of the Ppp5 D274A/D274A mice compared with Ppp5…”