Reciprocal Regulation of HSD11B1 and HSD11B2 Predicts Glucocorticoid Sensitivity in Childhood Acute Lymphoblastic Leukemia.

There are few biomarkers to predict efficacy of glucocorticoid treatment in childhood acute lymphoblastic leukemia (ALL) at diagnosis. Here, we demonstrate reciprocal regulation of 11beta-hydroxysteroid dehydrogenase (11ß-HSD), may predict the apoptotic response of ALL to glucocorticoid treatment. Our data may be useful to refine glucocorticoid treatment, to retain benefit while minimizing side effects.

Glucocorticoid regulation of the promoter of 11beta-hydroxysteroid dehydrogenase type 1 is indirect and requires CCAAT/enhancer-binding protein-beta.

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.

Differential effect of regional drug pressure on dihydrofolate reductase and dihydropteroate synthetase mutations in southern Mozambique.

The prevalence and frequency of the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) mutations associated with sulfadoxine-pyrimethamine (SP) resistance at 13 sentinel surveillance sites in southern Mozambique were examined regularly between 1999 and 2004. Frequency of the dhfr triple mutation increased from 0.26 in 1999 to 0.96 in 2003, remaining high in 2004. The dhps double mutation frequency peaked in 2001 (0.22) but declined to baseline levels (0.07) by 2004. Similarly, parasites with both dhfr triple and dhps double mutations had increased in 2001 (0.18) but decreased by 2004 (0.05). The peaking of SP resistance markers in 2001 coincided with a SP-resistant malaria epidemic in neighboring KwaZulu-Natal, South Africa. The decline in dhps (but not dhfr) mutations corresponded with replacement of SP with artemether-lumefantrine as malaria treatment policy in KwaZulu-Natal. Our results show that drug pressure can exert its influence at a regional level rather than merely at a national level.

Proinflammatory cytokine induction of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in human adipocytes is mediated by MEK, C/EBPß, and NF-κB/RelA.

CONTEXT: Levels of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-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. 11ß-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 11ß-HSD1 in human adipocytes. RESULTS: The proinflammatory cytokines IL-1α (10 ng/mL) and TNFα (20 ng/mL) increased 11ß-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) ß phosphorylation at Thr235 and IL-1α/TNFα induction of 11ß-HSD1 (P≤.007). The small interfering RNA-mediated knockdown of C/EBPß and nuclear factor (NF)-κB/RelA or inhibition of NF-κB/RelA also attenuated cytokine induction of 11ß-HSD1 (P≤.001). Moreover, induction of 11ß-HSD1 by IL-1α in SGBS cells was associated with nuclear localization of C/EBPß and NF-κB/RelA. Chromatin immunoprecipitation experiments showed C/EBPß and NF-κB/RelA located to the 11ß-HSD1 promoter in human adipose tissue. Treatment of adipocyte fractions or SGBS adipocytes with metformin or acetylsalicylic acid, which target C/EBPß and NF-κB/RelA signaling, attenuated the IL-1α induction of 11ß-HSD1 (P≤.002). CONCLUSIONS: Increased proinflammatory signaling in inflamed adipose tissue may mediate elevated 11ß-HSD1 expression at this site via MEK, C/EBPß, 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 11ß-HSD1 in human obese/metabolic syndrome adipose tissue.

Pro-inflammatory cytokine induction of 11ß-hydroxysteroid dehydrogenase type 1 in A549 cells requires phosphorylation of C/EBPß at Thr235.

11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts inert glucocorticoids into active forms, thereby increasing intracellular glucocorticoid levels, important to restrain acute inflammation. 11ß-HSD1 is induced by pro-inflammatory cytokines in a variety of cells. Here, we show 11ß-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 11ß-HSD1 as well as that encoding C/EBPß. IL-1α/TNFα-induced phosphorylation of C/EBPß at Thr235 was also attenuated by p38 MAPK inhibition suggesting involvement of a p38 MAPK-C/EBPß pathway. siRNA-mediated knock-down of C/EBPß 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/EBPß 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/EBPß 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/EBPß 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/EBPß at Thr235 is critical in this.

Stable conditional expression and effect of C/ebpß-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 ß (C/ebpß (Cebpb)) (C/EBPß-LIP) in fully differentiated 3T3-L1 adipocytes. Because overexpression of C/ebpß-LIP impairs adipocyte differentiation, the C/ebpß-LIP construct was first integrated in 3T3-L1 preadipocytes but expression was induced only when adipocytes were fully differentiated. Increased C/EBPß-LIP in mature adipocytes down-regulated C/ebpß target genes including 11ß-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/ebpß-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/ebpß-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.

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

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) catalyses intracellular regeneration of active glucocorticoids, notably in liver and adipose tissue. 11ß-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 11ß-HSD1 is down-regulated in mice, plausibly to counteract metabolic disease. Transcription of 11ß-HSD1 is directly regulated by members of the CCAAT/enhancer binding protein (C/EBP) family. Here we show that while total C/EBPß in adipose tissue is unaltered by HF diet, the ratio of the C/EBPß isoforms liver-enriched inhibitor protein (LIP) and liver-enriched activator protein (LAP) (C/EBPß-LIP:LAP) is increased in subcutaneous adipose. This may cause changes in 11ß-HSD1 expression since genetically modified C/EBPß((+/L)) mice, with increased C/EBPß-LIP:LAP ratio, have decreased subcutaneous adipose 11ß-HSD1 mRNA levels, whereas C/EBPß(ΔuORF) mice, with decreased C/EBPß-LIP:LAP ratio, show increased subcutaneous adipose 11ß-HSD1. C/EBPß-LIP:LAP ratio is regulated by endoplasmic reticulum (ER) stress and mTOR signalling, both of which are altered in obesity. In 3T3-L1 adipocytes, 11ß-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/EBPß and its processing to LIP and LAP in transcriptional regulation of 11ß-HSD1 in adipose tissue. Down-regulation of 11ß-HSD1 by increased C/EBPß-LIP:LAP in adipocytes may be part of a nutrient-sensing mechanism counteracting nutritional stress generated by HF diet.

Functional effects of polymorphisms in the human gene encoding 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1): a sequence variant at the translation start of 11 beta-HSD1 alters enzyme levels.

Regeneration of active glucocorticoids within liver and adipose tissue by the enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) may be of pathophysiological importance in obesity and metabolic syndrome and is a therapeutic target in type 2 diabetes. Polymorphisms in HSD11B1, the gene encoding 11 beta-HSD1, have been associated with metabolic phenotype in humans, including type 2 diabetes and hypertension. Here, we have tested the functional consequences of two single nucleotide polymorphisms located in contexts that potentially affect tissue levels of 11 beta-HSD1. We report no effect of allelic variation at rs846910, a polymorphism within the 5'-flanking region of the gene on HSD11B1 promoter activity in vitro. However, compared with the common G allele, the A allele of rs13306421, a polymorphism located two nucleotides 5' to the translation initiation site, gave higher 11 beta-HSD1 expression and activity in vitro and was translated at higher levels in in vitro translation reactions, possibly associated with a lower frequency of "leaky scanning." These data suggest that this polymorphism may have direct functional consequences on levels of 11 beta-HSD1 enzyme activity in vivo. However, the rs13306421 A sequence variant originally reported in other ethnic groups may be of low prevalence because it was not detected in a population of 600 European Caucasian women.