Agnieszka Zielinska

Lack of Significant Metabolic Abnormalities in Mice with Liver-Specific Disruption of 11β-Hydroxysteroid Dehydrogenase Type 1.

Glucocorticoids (GC) are implicated in the development of metabolic syndrome, and patients with GC excess share many clinical features, such as central obesity and glucose intolerance. In patients with obesity or type 2 diabetes, systemic GC concentrations seem to be invariably normal. Tissue GC concentrations determined by the hypothalamic-pituitary-adrenal (HPA) axis and local cortisol (corticosterone in mice) regeneration from cortisone (11-dehydrocorticosterone in mice) by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme, principally expressed in the liver. Transgenic mice have demonstrated the importance of 11β-HSD1 in mediating aspects of the metabolic syndrome, as well as HPA axis control. In order to address the primacy of hepatic 11β-HSD1 in regulating metabolism and the HPA axis, we have generated liver-specific 11β-HSD1 knockout (LKO) mice, assessed biomarkers of GC metabolism, and examined responses to high-fat feeding. LKO mice were able to regenerate cortisol from cortisone to 40% of control and had no discernible difference in a urinary metabolite marker of 11β-HSD1 activity. Although circulating corticosterone was unaltered, adrenal size was increased, indicative of chronic HPA stimulation. There was a mild improvement in glucose tolerance but with insulin sensitivity largely unaffected. Adiposity and body weight were unaffected as were aspects of hepatic lipid homeostasis, triglyceride accumulation, and serum lipids. Additionally, no changes in the expression of genes involved in glucose or lipid homeostasis were observed. Liver-specific deletion of 11β-HSD1 reduces corticosterone regeneration and may be important for setting aspects of HPA axis tone, without impacting upon urinary steroid metabolite profile. These discordant data have significant implications for the use of these biomarkers of 11β-HSD1 activity in clinical studies. The paucity of metabolic abnormalities in LKO points to important compensatory effects by HPA activation and to a crucial role of extrahepatic 11β-HSD1 expression, highlighting the contribution of cross talk between GC target tissues in determining metabolic phenotype.

Direct visualization of repair of oxidative damage by OGG1 in the nuclei of live cells

Oxidative DNA damage caused by intracellular reactive oxygen species (ROS) is widely considered to be important in the pathology of a range of human diseases including cancer as well as in the aging process. A frequently occurring mutagenic base lesion produced by ROS is 8‐oxo deoxyguanine (8‐oxo dG) and the major enzyme for repair of 8‐oxo dG is 8‐oxoguanine‐DNA glycosylase 1 (OGG1). There is now substantial evidence from bulk biochemical studies that a common human polymorphic variant of OGG1 (Ser326Cys) is repair deficient, and this has been linked to individual risk of pathologies related to oxidative stress. In the current study, we have used the technique of multiphoton microscopy to induce highly localized oxidative DNA damage in discrete regions of the nucleus of live cells. Cells transfected with GFP‐tagged OGG1 proteins demonstrated rapid (<2 min) accumulation of OGG1 at sites of laser‐induced damage as indicated by accumulation of GFP‐fluorescence. This was followed by repair as evidenced by loss of the localized fluorescence over time. Quantification of the rate of repair confirmed that the Cys326 variant of OGG1 is repair deficient and that the initial repair rate of damage by Cys326 OGG1 was 3 to 4 fold slower than that observed for Ser326 OGG1. These values are in good agreement with kinetic data comparing the Ser326 and Cys326 proteins obtained by biochemical studies.

Formation of Threohydrobupropion from Bupropion Is Dependent on 11β-Hydroxysteroid Dehydrogenase 1

Bupropion is widely used for treatment of depression and as a smoking-cessation drug. Despite more than 20 years of therapeutic use, its metabolism is not fully understood. While CYP2B6 is known to form hydroxybupropion, the enzyme(s) generating erythro- and threohydrobupropion have long remained unclear. Previous experiments using microsomal preparations and the nonspecific inhibitor glycyrrhetinic acid suggested a role for 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in the formation of both erythro- and threohydrobupropion. 11β-HSD1 catalyzes the conversion of inactive glucocorticoids (cortisone, prednisone) to their active forms (cortisol, prednisolone). Moreover, it accepts several other substrates. Here, we used for the first time recombinant 11β-HSD1 to assess its role in the carbonyl reduction of bupropion. Furthermore, we applied human, rat, and mouse liver microsomes and a selective inhibitor to characterize species-specific differences and to estimate the relative contribution of 11β-HSD1 to bupropion metabolism. The results revealed 11β-HSD1 as the major enzyme responsible for threohydrobupropion formation. The reaction was stereoselective and no erythrohydrobupropion was formed. Human liver microsomes showed 10 and 80 times higher activity than rat and mouse liver microsomes, respectively. The formation of erythrohydrobupropion was not altered in experiments with microsomes from 11β-HSD1-deficient mice or upon incubation with 11β-HSD1 inhibitor, indicating the existence of another carbonyl reductase that generates erythrohydrobupropion. Molecular docking supported the experimental findings and suggested that 11β-HSD1 selectively converts R-bupropion to threohydrobupropion. Enzyme inhibition experiments suggested that exposure to bupropion is not likely to impair 11β-HSD1-dependent glucocorticoid activation but that pharmacological administration of cortisone or prednisone may inhibit 11β-HSD1-dependent bupropion metabolism.

Biochemistry and physiology of hexose-6-phosphate knockout mice.

Hexose-6-phosphate dehydrogenase (H6PDH) has emerged as an important factor in setting the redox status of the endoplasmic reticulum (ER) lumen. An important role of H6PDH is to generate a high NADPH/NADP(+) ratio which permits 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to act as an oxo-reductase, catalyzing the activation of glucocorticoids (GCs). In H6PDH knockout mice 11β-HSD1 assumes dehydrogenase activity and inactivates GCs, rendering the target cell relatively GC insensitive. Consequently, H6PDHKO mice have a phenotype consistent with defects in the permissive and adaptive actions of GCs upon physiology. H6PDHKO mice have also offered an insight into muscle physiology as they also present with a severe vacuolating myopathy, abnormalities of glucose homeostasis and activation of the unfolded protein response due to ER stress, and a number of mechanisms driving this phenotype are thought to be involved. This article will review what we understand of the redox control of GC hormone metabolism regulated by H6PDH, and how H6PDHKO mice have allowed an in-depth understanding of its potentially novel, GC-independent roles in muscle physiology.

Carbonyl reduction of triadimefon by human and rodent 11β-hydroxysteroid dehydrogenase 1.

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the conversion of inactive 11-oxo glucocorticoids (endogenous cortisone, 11-dehydrocorticosterone and synthetic prednisone) to their potent 11β-hydroxyl forms (cortisol, corticosterone and prednisolone). Besides, 11β-HSD1 accepts several other substrates. Using rodent liver microsomes and the unspecific inhibitor glycyrrhetinic acid, it has been proposed earlier that 11β-HSD1 catalyzes the reversible conversion of the fungicide triadimefon to triadimenol. In the present study, recombinant human, rat and mouse enzymes together with a highly selective 11β-HSD1 inhibitor were applied to assess the role of 11β-HSD1 in the reduction of triadimefon and to uncover species-specific differences. To further demonstrate the role of 11β-HSD1 in the carbonyl reduction of triadimefon, microsomes from liver-specific 11β-HSD1-deficient mice were employed. Molecular docking was applied to investigate substrate binding. The results revealed important species differences and demonstrated the irreversible 11β-HSD1-dependent reduction of triadimefon. Human liver microsomes showed 4 and 8 times higher activity than rat and mouse liver microsomes. The apparent Vmax/Km of recombinant human 11β-HSD1 was 5 and 15 times higher than that of mouse and rat 11β-HSD1, respectively, indicating isoform-specific differences and different expression levels for the three species. Experiments using inhibitors and microsomes from 11β-HSD1-deficient mice indicated that 11β-HSD1 is the major if not only enzyme responsible for triadimenol formation. The IC50 values of triadimefon and triadimenol for cortisone reduction suggested that exposure to these xenobiotica unlikely impairs the 11β-HSD1-dependent glucocorticoid activation. However, elevated glucocorticoids during stress or upon pharmacological administration likely inhibit 11β-HSD1-dependent metabolism of triadimefon in humans.

TNFα-mediated Hsd11b1 binding of NF-κB p65 is associated with suppression of 11β-HSD1 in muscle

The activity of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive cortisone (11-dehydrocorticosterone (11-DHC)) (in mice) into the active glucocorticoid (GC) cortisol (corticosterone in mice), can amplify tissue GC exposure. Elevated TNFα is a common feature in a range of inflammatory disorders and is detrimental to muscle function in diseases such as rheumatoid arthritis and chronic obstructive pulmonary disease. We have previously demonstrated that 11β-HSD1 activity is increased in the mesenchymal stromal cells (MSCs) by TNFα treatment and suggested that this is an autoregulatory anti-inflammatory mechanism. This upregulation was mediated by the P2 promoter of the Hsd11b1 gene and was dependent on the NF-κB signalling pathway. In this study, we show that in contrast to MSCs, in differentiated C2C12 and primary murine myotubes, TNFα suppresses Hsd11b1 mRNA expression and activity through the utilization of the alternative P1 promoter. As with MSCs, in response to TNFα treatment, NF-κB p65 was translocated to the nucleus. However, ChIP analysis demonstrated that the direct binding was seen at position −218 to −245 bp of the Hsd11b1 genes P1 promoter but not at the P2 promoter. These studies demonstrate the existence of differential regulation of 11β-HSD1 expression in muscle cells through TNFα/p65 signalling and the P1 promoter, further enhancing our understanding of the role of 11β-HSD1 in the context of inflammatory disease.

PRMT5 Is a Critical Regulator of Breast Cancer Stem Cell Function via Histone Methylation and FOXP1 Expression

Summary Breast cancer progression, treatment resistance, and relapse are thought to originate from a small population of tumor cells, breast cancer stem cells (BCSCs). Identification of factors critical for BCSC function is therefore vital for the development of therapies. Here, we identify the arginine methyltransferase PRMT5 as a key in vitro and in vivo regulator of BCSC proliferation and self-renewal and establish FOXP1, a winged helix/forkhead transcription factor, as a critical effector of PRMT5-induced BCSC function. Mechanistically, PRMT5 recruitment to the FOXP1 promoter facilitates H3R2me2s, SET1 recruitment, H3K4me3, and gene expression. Our findings are clinically significant, as PRMT5 depletion within established tumor xenografts or treatment of patient-derived BCSCs with a pre-clinical PRMT5 inhibitor substantially reduces BCSC numbers. Together, our findings highlight the importance of PRMT5 in BCSC maintenance and suggest that small-molecule inhibitors of PRMT5 or downstream targets could be an effective strategy eliminating this cancer-causing population.

Biomarkers of hypothalamic-pituitary-adrenal axis activity in mice lacking 11β-HSD1 and H6PDH

Glucocorticoid concentrations are a balance between production under the negative feedback control and diurnal rhythm of the hypothalamic–pituitary–adrenal (HPA) axis and peripheral metabolism, for example by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyses the reduction of inactive cortisone (11-dehydrocorticosterone (11-DHC) in mice) to cortisol (corticosterone in mice). Reductase activity is conferred upon 11β-HSD1 by hexose-6-phosphate dehydrogenase (H6PDH). 11β-HSD1 is implicated in the development of obesity, and selective 11β-HSD1 inhibitors are currently under development. We sought to address the concern regarding potential up-regulation of the HPA axis associated with inhibition of 11β-HSD1. We assessed biomarkers for allele combinations of 11β-HSD1 and H6PDH derived from double heterozygous mouse crosses. H6PDH knock out (KO) adrenals were 69% larger than WT while 11β-HSD1 KO and double KO (DKO) adrenals were ∼30% larger than WT – indicative of increased HPA axis drive in KO animals. ACTH-stimulated circulating corticosterone concentrations were 2·2-fold higher in H6PDH KO animals and ∼1·5-fold higher in 11β-HSD1 KO and DKO animals compared with WT, proportional to the observed adrenal hypertrophy. KO of H6PDH resulted in a substantial increase in urinary DHC metabolites in males (65%) and females (61%). KO of 11β-HSD1 alone or in combination with H6PDH led to significant increases (36 and 42% respectively) in urinary DHC metabolites in females only. Intermediate 11β-HSD1/H6PDH heterozygotes maintained a normal HPA axis. Urinary steroid metabolite profile by gas chromatography/mass spectrometry as a biomarker assay may be beneficial in assaying HPA axis status clinically in cases of congenital and acquired 11β-HSD1/H6PDH deficiency.

Cellular and genetic models of H6PDH and 11β-HSD1 function in skeletal muscle

Glucocorticoids are important for skeletal muscle energy metabolism, regulating glucose utilization, insulin sensitivity, and muscle mass. Nicotinamide adenine dinucleotide phosphate‐dependent 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1)‐mediated glucocorticoid activation in the sarcoplasmic reticulum (SR) is integral to mediating the detrimental effects of glucocorticoid excess in muscle. 11β‐Hydroxysteroid dehydrogenase type 1 activity requires glucose‐6‐phosphate transporter (G6PT)‐mediated G6P transport into the SR for its metabolism by hexose‐6‐phosphate dehydrogenase (H6PDH) for NADPH generation. Here, we examine the G6PT/H6PDH/11β‐HSD1 triad in differentiating myotubes and explore the consequences of muscle‐specific knockout of 11β‐HSD1 and H6PDH. 11β‐Hydroxysteroid dehydrogenase type 1 expression and activity increase with myotube differentiation and in response to glucocorticoids. Hexose‐6‐phosphate dehydrogenase shows some elevation in expression with differentiation and in response to glucocorticoid, while G6PT appears largely unresponsive to these particular conditions. When examining 11β‐HSD1 muscle‐knockout mice, we were unable to detect significant decrements in activity, despite using a well‐validated muscle‐specific Cre transgene and confirming high‐level recombination of the floxed HSD11B1 allele. We propose that the level of recombination at the HSD11B1 locus may be insufficient to negate basal 11β‐HSD1 activity for a protein with a long half‐life. Hexose‐6‐phosphate dehydrogenase was undetectable in H6PDH muscle‐knockout mice, which display the myopathic phenotype seen in global KO mice, validating the importance of SR NADPH generation. We envisage these data and models finding utility when investigating the muscle‐specific functions of the 11β‐HSD1/G6PT/H6PDH triad.