Val Kelly

Glucocorticoid Regulation of the Promoter of 11β-Hydroxysteroid Dehydrogenase Type 1 Is Indirect and Requires CCAAT/Enhancer-Binding Protein-β

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inert 11keto-glucocorticoids to active 11beta-hydroxy forms, thereby amplifying intracellular glucocorticoid action. Up-regulation of 11beta-HSD1 in adipose tissue and liver is of pathogenic importance in metabolic syndrome. However, the mechanisms controlling 11beta-HSD1 transcription are poorly understood. Glucocorticoids themselves potently increase 11beta-HSD1 expression in many cells, providing a potential feed-forward system to pathology. We have investigated the molecular mechanisms by which glucocorticoids regulate transcription of 11beta-HSD1, exploiting an A549 cell model system in which endogenous 11beta-HSD1 is expressed and is induced by dexamethasone. We show that glucocorticoid induction of 11beta-HSD1 is indirect and requires new protein synthesis. A glucocorticoid-responsive region maps to between -196 and -88 with respect to the transcription start site. This region contains two binding sites for CCAAT/enhancer-binding protein (C/EBP) that together are essential for the glucocorticoid response and that bind predominantly C/EBPbeta, with C/EBPdelta present in a minority of the complexes. Both C/EBPbeta and C/EBPdelta are rapidly induced by glucocorticoids in A549 cells, but small interfering RNA-mediated knockdown shows that only C/EBPbeta reduction attenuates the glucocorticoid induction of 11beta-HSD1. Chromatin immunoprecipitation studies demonstrated increased binding of C/EBPbeta to the 11beta-HSD1 promoter in A549 cells after glucocorticoid treatment. A similar mechanism may apply in adipose tissue in vivo where increased C/EBPbeta mRNA levels after glucocorticoid treatment were associated with increased 11beta-HSD1 expression. C/EBPbeta is a key mediator of metabolic and inflammatory signaling. Positive regulation of 11beta-HSD1 by C/EBPbeta may link amplification of glucocorticoid action with metabolic and inflammatory pathways and may represent an endogenous innate host-defense mechanism.

Regulation of adipocyte 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) by CCAAT/enhancer-binding protein (C/EBP) β isoforms, LIP and LAP.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses intracellular regeneration of active glucocorticoids, notably in liver and adipose tissue. 11β-HSD1 is increased selectively in adipose tissue in human obesity, a change implicated in the pathogenesis of metabolic syndrome. With high fat (HF)-feeding, adipose tissue 11β-HSD1 is down-regulated in mice, plausibly to counteract metabolic disease. Transcription of 11β-HSD1 is directly regulated by members of the CCAAT/enhancer binding protein (C/EBP) family. Here we show that while total C/EBPβ in adipose tissue is unaltered by HF diet, the ratio of the C/EBPβ isoforms liver-enriched inhibitor protein (LIP) and liver-enriched activator protein (LAP) (C/EBPβ-LIP:LAP) is increased in subcutaneous adipose. This may cause changes in 11β-HSD1 expression since genetically modified C/EBPβ((+/L)) mice, with increased C/EBPβ-LIP:LAP ratio, have decreased subcutaneous adipose 11β-HSD1 mRNA levels, whereas C/EBPβ(ΔuORF) mice, with decreased C/EBPβ-LIP:LAP ratio, show increased subcutaneous adipose 11β-HSD1. C/EBPβ-LIP:LAP ratio is regulated by endoplasmic reticulum (ER) stress and mTOR signalling, both of which are altered in obesity. In 3T3-L1 adipocytes, 11β-HSD1 mRNA levels were down-regulated following induction of ER stress by tunicamycin but were up-regulated following inhibition of mTOR by rapamycin. These data point to a central role for C/EBPβ and its processing to LIP and LAP in transcriptional regulation of 11β-HSD1 in adipose tissue. Down-regulation of 11β-HSD1 by increased C/EBPβ-LIP:LAP in adipocytes may be part of a nutrient-sensing mechanism counteracting nutritional stress generated by HF diet.

Proinflammatory Cytokine Induction of 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) in Human Adipocytes Is Mediated by MEK, C/EBPβ, and NF-κB/RelA

CONTEXT Levels of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which regenerates active glucocorticoids, are selectively elevated in adipose tissue in human obesity and metabolic syndrome, both conditions associated with chronic low-grade inflammation. 11β-HSD1 expression is induced by proinflammatory cytokines in a variety of cell types, including in human adipocytes differentiated in vitro. OBJECTIVE Our objective was to determine the mechanisms by which proinflammatory cytokines induce 11β-HSD1 in human adipocytes. RESULTS The proinflammatory cytokines IL-1α (10 ng/mL) and TNFα (20 ng/mL) increased 11β-HSD1 mRNA levels in human primary adipocyte fractions and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes (P<.001). Inhibition of the MAPK/ERK kinase (MEK) attenuated CCAAT/enhancer binding protein (C/EBP) β phosphorylation at Thr235 and IL-1α/TNFα induction of 11β-HSD1 (P≤.007). The small interfering RNA-mediated knockdown of C/EBPβ and nuclear factor (NF)-κB/RelA or inhibition of NF-κB/RelA also attenuated cytokine induction of 11β-HSD1 (P≤.001). Moreover, induction of 11β-HSD1 by IL-1α in SGBS cells was associated with nuclear localization of C/EBPβ and NF-κB/RelA. Chromatin immunoprecipitation experiments showed C/EBPβ and NF-κB/RelA located to the 11β-HSD1 promoter in human adipose tissue. Treatment of adipocyte fractions or SGBS adipocytes with metformin or acetylsalicylic acid, which target C/EBPβ and NF-κB/RelA signaling, attenuated the IL-1α induction of 11β-HSD1 (P≤.002). CONCLUSIONS Increased proinflammatory signaling in inflamed adipose tissue may mediate elevated 11β-HSD1 expression at this site via MEK, C/EBPβ, and NF-κB/RelA. These molecules/signaling pathways are, therefore, potential targets for drugs, including metformin and acetylsalicylic acid, to prevent/decreased up-regulation of 11β-HSD1 in human obese/metabolic syndrome adipose tissue.

Decreased Npas4 and Arc mRNA levels in the hippocampus of aged memory-impaired wild type but not memory preserved 11β-HSD1 deficient mice

Mice deficient in the glucocorticoid‐regenerating enzyme 11β‐HSD1 resist age‐related spatial memory impairment. To investigate the mechanisms and pathways involved, we used microarrays to identify differentially expressed hippocampal genes that associate with cognitive ageing and 11β‐HSD1. Aged wild‐type mice were separated into memory‐impaired and unimpaired relative to young controls according to their performance in the Y‐maze. All individual aged 11β‐HSD1‐deficient mice showed intact spatial memory. The majority of differentially expressed hippocampal genes were increased with ageing (e.g. immune/inflammatory response genes) with no genotype differences. However, the neuronal‐specific transcription factor, Npas4, and immediate early gene, Arc, were reduced (relative to young) in the hippocampus of memory‐impaired but not unimpaired aged wild‐type or aged 11β‐HSD1‐deficient mice. A quantitative reverse transcriptase‐polymerase chain reaction and in situ hybridisation confirmed reduced Npas4 and Arc mRNA expression in memory‐impaired aged wild‐type mice. These findings suggest that 11β‐HSD1 may contribute to the decline in Npas4 and Arc mRNA levels associated with memory impairment during ageing, and that decreased activity of synaptic plasticity pathways involving Npas4 and Arc may, in part, underlie the memory deficits seen in cognitively‐impaired aged wild‐type mice.

Pro-Inflammatory Cytokine Induction of 11β- hydroxysteroid Dehydrogenase Type 1 in A549 Cells Requires Phosphorylation of C/EBPβ at Thr235

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inert glucocorticoids into active forms, thereby increasing intracellular glucocorticoid levels, important to restrain acute inflammation. 11β-HSD1 is induced by pro-inflammatory cytokines in a variety of cells. Here, we show 11β-HSD1 expression in human A549 epithelial cells is increased by pro-inflammatory cytokines (IL-1α/TNFα) via the P2 promoter of the HSD11B1 gene. Inhibition of p38 MAPK attenuated the pro-inflammatory cytokine induction of mRNA encoding 11β-HSD1 as well as that encoding C/EBPβ. IL-1α/TNFα-induced phosphorylation of C/EBPβ at Thr235 was also attenuated by p38 MAPK inhibition suggesting involvement of a p38 MAPK-C/EBPβ pathway. siRNA-mediated knock-down of C/EBPβ and NF-κB/RelA implicated both transcription factors in the IL-1α/TNFα induction of HSD11B1 mRNA. Transient transfections of HSD11B1 promoter-reporter constructs identified the proximal region of the P2 promoter of HSD11B1 as essential for this induction. IL-1α increased binding of C/EBPβ to the HSD11B1 P2 promoter, but this was not observed for NF-κB/RelA, suggesting indirect regulation by NF-κB/RelA. Ectopic expression of mutant chicken C/EBPβ constructs unable to undergo phosphorylation at the threonine equivalent to Thr235 attenuated the IL-1α-induction of HSD11B1, whereas mimicking constitutive phosphorylation of Thr235 (by mutation to aspartate) increased basal expression of HSD11B1 mRNA without affecting IL-1α-induced levels. These data clearly demonstrate a role for both C/EBPβ and NF-κB/RelA in the pro-inflammatory cytokine induction of HSD11B1 in human epithelial cells and show that p38 MAPK-induced phosphorylation of C/EBPβ at Thr235 is critical in this.

Stable conditional expression and effect of C/EBPβ-LIP in adipocytes using the pSLIK system

Murine 3T3-L1 adipocytes are widely used as a cellular model of obesity. However, whereas transfection of 3T3-L1 preadipocytes is straightforward, ectopic gene expression in mature 3T3-L1 adipocytes has proved challenging. Here, we used the pSLIK vector system to generate stable doxycycline-inducible expression of the liver-enriched inhibitor protein isoform of CCAAT/enhancer binding protein β (CEPB (C/EBPβ-LIP)) in fully differentiated 3T3-L1 adipocytes. Because overexpression of C/EBPβ-LIP impairs adipocyte differentiation, the C/EBPβ-LIP construct was first integrated in 3T3-L1 preadipocytes but expression was induced only when adipocytes were fully differentiated. Increased C/EBPβ-LIP in mature adipocytes down-regulated C/EBPβ target genes including 11β-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4 but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/EBPβ-LIP in 3T3-L1 adipocytes is not a general effect. Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/EBPβ-LIP levels. The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology.