The inappropriate activation of glucocorticoid receptors (GR) has been associated with an increased incidence of metabolic syndrome, osteoporosis, infection and cataracts, whereas impaired mineralocorticoid receptor (MR) action leads to renal salt retention with hypertension, hypokalemia and heart failure. Thus, a tight control of mineralocorticoid and glucocorticoid homeostasis is essential for health. Glucocorticoid and mineralocorticoid action is regulated in a tissue-specific manner by 11b-hydroxysteroid dehydrogenases (11b-HSDs). 11b-HSD1 converts biologically inactive 11-ketoglucocorticoids (cortisone, 11-dehydrocorticosterone) into active 11b-hydroxyglucocorticoids (cortisol, corticosterone). Due to its elevated expression in obese individuals and its association with the development of metabolic syndrome, 11b-HSD1 has emerged as a promising therapeutic target. 11b-HSD2 catalyzes the reverse reaction and prevents cortisol-induced activation of MR. Since recent clinical trials have shown that treatment with MR antagonists reduced the mortality in patients with heart failure by 15-30%, there is a profound interest to uncover the mechanisms of MR activation.

Based on our preliminary results, we hypothesize that oxidized cholesterol products, taken up from processed food or generated in vivo during oxidative stress, can activate the MR. Inhibition of 11b-HSD1, which we have shown recently to metabolize 7KC, may cause adverse effects upon activation of the MR by these alternative ligands, including cardiac fibrosis. In a recent study, we provided evidence for the existence of a bridging protein responsible for the tethering of MR to 11b-HSD2 in the absence of aldosterone. We now identified 5 potential candidate proteins interacting with MR or 11b-HSD2 and hypothesize that they might modulate the localization of MR and its initial step of activation. Such associated proteins represent potential targets for diseases such as hypertension. We also cloned a putative 11b-HSD3 that might play an important role in the modulation of MR and/or GR function in cells not expressing 11b-HSD1 or 11b-HSD2.

Recently, we demonstrated that hexose-6-phosphate dehydrogenase (H6PDH) stimulates the oxoreductase activity of 11b-HSD1. Based on preliminary results I hypothesize that the two enzymes interact directly and that H6PDH represents an alternative target for treating adverse effects of enhanced glucocorticoid levels. Our preliminary results indicate that dehydroepiandrosterone (DHEA) and peroxisome-proliferation activated receptor (PPAR) agonists regulate the expression of 11b-HSD1 and H6PDH. The impact of these compounds and of 11b-HSDs on the proliferation and differentiation of stem cells and cells from specific tissues remains to be investigated. The development of suitable compounds reducing 11b-HSD1 expression or inhibiting its activity may find potential application in the therapeutic treatment of patients with metabolic syndrome. I hypothesize that many of the recently developed inhibitors will fail due to the lack of selectivity and propose to apply a novel approach to identify selective inhibitors and targets of side-effects.

To address physiologically and pharmacologically relevant questions related to MR- and GR-mediated actions and their control by 11b-HSD enzymes, I propose to:

 I.     investigate 7-oxycholesterol metabolism by 11b-HSD1 and its potential role in MR activation and the development of hypertension and cardiac fibrosis in a series of cell-based experiments as well as in an extensive study with rats;

II.    characterize the role of selected interacting proteins, post-translational modification and protein stability for the 11b-HSD2-dependent protection of MR from glucocorticoids using biochemical assays and cell-based systems;

III.   investigate the relevance of the lumenal orientation of 11b-HSD1 regarding its regulation by H6PDH and regarding a potential role in "loading" cortisol-binding globulin (CBG) with cortisol in biochemical and cell-based assays;

IV.  apply a recently developed strategy based on protein structure similarity clustering (PSSC) to identify novel classes of inhibitors selective for specific 11b-HSDs and 17b-HSDs and extend this strategy to identify targets of drug side-effects and environmental chemicals;

             The proposed research is relevant not only for understanding the role of glucocorticoids and mineralocorticoids in diseases such as metabolic syndrome and heart failure, but also for general understanding of steroid hormone action and pre-receptor regulation. Specifically, the knowledge gained from the proposed research may support the development of therapeutic interventions by targeting MR and GR as well as 11b-HSDs and 17b-HSDs in order to control inappropriate steroid hormone action.