Organotins disrupt the 11beta-hydroxysteroid dehydrogenase type 2-dependent local inactivation of glucocorticoids

Abstract

Organotins, important environmental pollutants widely used in agricultural and industrial applications, accumulate in the food chain and induce imposex in several marine species as well as neurotoxic and immunotoxic effects in higher animals. Reduced birth weight and thymus involution, observed upon exposure to organotins, can also be caused by excessive glucocorticoid levels. We now demonstrate that organotins efficiently inhibit 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), converting active 11beta-hydroxyglucocorticoids into inactive 11-ketoglucocorticoids, but not 11beta-HSD1, which catalyzes the reverse reaction. Di- and tributyltin as well as di- and triphenyltin inhibited recombinant and endogenous 11beta-HSD2 in lysates and intact cells with IC50 values between 500 nM and 3 microM. Dithiothreitol protected 11beta-HSD2 from organotin-dependent inhibition, indicating that organotins act by binding to one or more cysteines. Mutational analysis and 3-D structural modeling revealed several important interactions of cysteines in 11beta-HSD2. Cys90, Cys228, and Cys264 were essential for enzymatic stability and catalytic activity, suggesting that disruption of such interactions by organotins leads to inhibition of 11beta-HSD2. Enhanced glucocorticoid concentrations due to disruption of 11beta-HSD2 function may contribute to the observed organotin-dependent toxicity in some glucocorticoid-sensitive tissues such as thymus and placenta.

Figure 1

DTT prevents organotin-dependent inhibition of 11β-HSD2. The oxidation of cortisol by 11β-HSD2 was determined using cell lysates, as described in “Materials and Methods.” Addition of DTT at a final concentration of 2 mM restored 70–80% of the activity measured in absence of organotins.

Figure 2

Expression of wild-type 11β-HSD2 and cysteine to serine mutants. C-terminally FLAG-epitope tagged wild-type and mutant 11β-HSD2 enzymes were expressed in HEK-293 cells, and protein expression was analyzed by Western blotting as described in “Materials and Methods.” After detection of the FLAG-tagged 11β-HSD2 enzymes, nitrocellulose membranes were stripped, and actin expression was detected as a control for the amount of protein loaded on the SDS gel. A representative blot from three comparable experiments is shown.

Figure 3

Effect of preincubation on 11β-HSD2 activity. The oxidation of cortisol to cortisone was determined after preincubation for 5 or 10 min with vehicle (control), 1.5 μM TPT, or 2 μM TBT in lysates of HEK-293 cells expressing 11β-HSD2. Data are mean ± SD from at least three independent experiments.

Figure 4

Effects of dilution of the enzyme-inhibitor (EI) complex on TBT-dependent inhibition of 11β-HSD2. Lysates from HEK-293 cells expressing 11β-HSD2 were split into two equal aliquots. TBT was added to the first aliquot, and the same volume of vehicle, serving as a control, was added to the second. Both aliquots were incubated for 5 min at 37°C, followed by determination of the activity of the control and EI mixture either undiluted or after a 2- or 4-fold dilution. Data are mean ± SD from at least three independent experiments measured in triplicate. *p < 0.05.

Figure 5

Additive inhibitory effect of a mixture of organotins on 11β-HSD2 activity. Conversion of cortisol to cortisone by 11β-HSD2 stably expressed in intact HEK-293 cells was measured in a volume of 50 μL cell culture medium containing 40 nM cortisol and the corresponding amount of the organotin, as indicated (see “Materials and Methods”). Data were normalized to the control and are mean ± SD from at least three independent experiments measured in triplicate. *Statistical significance of p < 0.01 compared with all other values.

Figure 6

Dose–response curves for TBT-induced inhibition of 11β-HSD2 in intact cells expressing endogenous 11β-HSD2. RCCD-2, rat renal cortical collecting duct cell line; JEG-3, human choriocarcinoma cell line; SW-620, human colon adenocarcinoma cell line. Details on culture conditions and activity assay in intact cells are given in “Materials and Methods.”

Figure 7

Predicted interactions of cysteine residues in the conserved core domain of 11β-HSD2. ( A) Cys¹²⁷, Cys¹²⁸, and Cys²⁴⁸ are oriented into the solvent, away from the catalytic pocket, and have few stabilizing interactions with other residues. ( B) Cys⁹⁰ interacts with amino acids that stabilize Glu¹¹⁵ and Asp⁹¹, which have a critical role by forming hydrogen bonds to the ribose hydroxyl on NAD⁺ that are important to stabilize binding of the cofactor and maintain its orientation to the substrate. ( C) Cys¹⁸⁸ is not directly involved in interactions with NAD⁺ but stabilizes several amino acids that interact with the pyrophosphate segment of the cofactor. ( D) The thiol on Cys²²⁸ stabilizes the position of Pro²²⁷ and Glu²⁷⁷, which are important for positioning of the catalytic tyrosine and the nicotinamide ring and for binding of the steroid substrate. The thiol group of Cys²⁶⁴ has important interactions with Leu²⁸⁴, Ala²⁸⁵, and Pro²⁸⁸ in the helix in the C-terminal region of 11β-HSD2, which is important for substrate binding. Predicted interatomic distances in angstroms are depicted by number. Blue: nitrogen; green: carbon; purple: phosphorus; red: oxygen; and yellow: sulfur.