Inflammatory regulation of glucocorticoid metabolism in mesenchymal stromal cells

Abstract

Objective: Tissue glucocorticoid (GC) levels are regulated by the GC-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). This enzyme is expressed in cells and tissues arising from mesenchymal stromal cells. Proinflammatory cytokines dramatically increase expression of 11β-HSD1 in stromal cells, an effect that has been implicated in inflammatory arthritis, osteoporosis, obesity, and myopathy. Additionally, GCs act synergistically with proinflammatory cytokines to further increase enzyme expression. The present study was undertaken to investigate the mechanisms underlying this regulation.

Methods: Gene reporter analysis, rapid amplification of complementary DNA ends (RACE), chemical inhibition experiments, and genetic disruption of intracellular signaling pathways in mouse embryonic fibroblasts (MEFs) were used to define the molecular mechanisms underlying the regulation of 11β-HSD1 expression.

Results: Gene reporter, RACE, and chemical inhibitor studies demonstrated that the increase in 11β-HSD1 expression with tumor necrosis factor α (TNFα)/interleukin-1β (IL-1β) occurred via the proximal HSD11B1 gene promoter and depended on NF-κB signaling. These findings were confirmed using MEFs with targeted disruption of NF-κB signaling, in which RelA (p65) deletion prevented TNFα/IL-1β induction of 11β-HSD1. GC treatment did not prevent TNFα-induced NF-κB nuclear translocation. The synergistic enhancement of TNFα-induced 11β-HSD1 expression with GCs was reproduced by specific inhibitors of p38 MAPK. Inhibitor and gene deletion studies indicated that the effects of GCs on p38 MAPK activity occurred primarily through induction of dual-specificity phosphatase 1 expression.

Conclusion: The mechanism by which stromal cell expression of 11β-HSD1 is regulated is novel and distinct from that in other tissues. These findings open new opportunities for development of therapeutic interventions aimed at inhibiting or stimulating local GC levels in cells of mesenchymal stromal lineage during inflammation.

Figure 1

Proinflammatory cytokine and glucocorticoid regulation of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression at the promoter level. A, Effect of the protein synthesis inhibitor cycloheximide (CHX) on HSD11B1 gene transcription in rheumatoid arthritis (RA) synovial fibroblasts. CHX did not affect induction of 11β-HSD1 expression by tumor necrosis factor α (TNFα), but partially reduced the additional expression obtained with TNFα and dexamethasone (DEX) combined. Con = control. B, Rapid amplification of complementary DNA ends (RACE) analysis. Experiments were performed using RA synovial fibroblasts, osteoarthritis (OA) synovial fibroblasts, and MG-63 osteoblasts. Data shown are for RA synovial fibroblasts; similar results were obtained with OA synovial fibroblasts and MG-63 osteoblasts (data not shown). RACE analysis demonstrated that RA and OA synovial fibroblasts and MG-63 osteoblasts use the proximal promoter, and there is no difference in transcription start site according to treatment or disease state. Arrows indicate the transcriptional start site for each transcript. C, Results of dual-luciferase activity assays in MG-63 cells. Interleukin-1β (IL-1β) stimulated proximal promoter activity, but no effect was seen with DEX alone. The combination of IL-1β and DEX also stimulated promoter activity, but not to a greater extent than that obtained with IL-1β alone. P values are versus control. Values in A and C are the mean ± SD.

Figure 2

Effect of NF-κB inhibitors on 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) oxoreductase expression and activity in C2C12 myoblasts. The inhibitors used were parthenolide (Parth) (10 μM), pyrrolidine dithiocarbamate (PDTC) (5 mM), and lactacystin (Lacta) (10 μM). A and B, Effects of NF-κB inhibition on basal expression and activity of 11β-HSD1 (A) and on the increase seen during myoblast differentiation (B). C and D, Effects of NF-κB inhibition on 11β-HSD1 expression (C) and activity (D) in freshly isolated muscle tissue from 4 mice. Values are the mean ± SD. P values in A, C, and D are versus control.

Figure 3

NF-κB is the major mediator of the induction of 11β-HSD1 by TNFα in fibroblasts (normal synovial and dermal; n = 3 independent patient lines), osteoblasts, and myoblasts. The effect of NF-κB inhibition (INH) with parthenolide (10 μM) plus pyrrolidine dithiocarbamate (5 mM) on the induction of 11β-HSD1 activity by TNFα, DEX, and their combination is shown. Values are the mean ± SD. See Figure 1 for other definitions.

Figure 4

Molecular analysis of the role of NF-κB in regulation of 11β-HSD1 expression. A, Effect of treatments (TNFα, IL-1β, DEX, combined TNFα and DEX, or combined IL-1β and DEX) on 11β-HSD1 mRNA expression in mouse embryonic fibroblasts from wild-type (WT), RelB-knockout (KO), and RelA-knockout mice (all on a C57BL/6 background; lines from 9 embryos). B, Effect of TNFα with or without DEX and with or without NF-κB inhibitor (INH) on RelA nuclear translocation in RA synovial fibroblasts. RelA (p65) translocated to the nucleus in response to TNFα treatment. This effect was not blocked by glucocorticoid, but was completely inhibited by NF-κB inhibitor. Values are the mean ± SD. See Figure 1 for other definitions.

Figure 5

Molecular analysis of the role of p38 MAPK signaling in 11β-HSD1 expression. A, Effect of the p38 MAPK inhibitor SB202190 on the induction of 11β-HSD1. Expression of 11β-HSD1 expression with TNFα plus SB202190 treatment was increased to a similar extent as was observed with TNFα plus DEX treatment. B, Effect of dual-specificity phosphatase 1 (DUSP-1) deletion in mouse embryonic fibroblasts (lines from 4 embryos). The results indicate that inhibition of p38 MAPK by DEX occurs primarily through induction of DUSP-1. Values are the mean ± SD. NS = not significant; WT = wild-type; KO = knockout (see Figure 1 for other definitions).

Figure 6

Schematic representation of the pathways by which TNFα/IL-1β and glucocorticoids regulate 11β-HSD1 expression and activity in musculoskeletal cells. TNFα/IL-1β exert both stimulatory and inhibitory effects via NF-κB and p38 MAPK pathways, respectively. The inhibitory pathway can be blocked by glucocorticoids, primarily through a dual-specificity phosphatase 1 (DUSP-1)–dependent mechanism. See Figure 1 for other definitions.