11β-Hydroxysteroid dehydrogenase-1: key regulator in oxysterol metabolism?
Tijana Mitic1, Iain McNae2, Scott P Webster1, Malgorzata Wamil1, Brian R Walker1, Patrick W F Hadoke1 & Ruth Andrew1
7-Oxysterols modulate lipid transport and promote oxidative stress and apoptosis in the vascular wall. 7-Ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC) are inter-converted by 11β-hydroxysteroid dehydrogenase-1 (11βHSD1), better known for metabolising glucocorticoids. Pharmacological inhibition of 11βHSD1 protects against atherosclerosis and these beneficial effects may be mediated through changes in glucocorticoids or oxysterols. To establish the kinetics of 7-oxysterol and glucocorticoid metabolism by murine 11βHSD1 and 2, we studied murine recombinant 11βHSD1 protein and liver and kidneys from C57Bl/6 and 11βHSD1−/− and 2−/− mice (male, 9 weeks old, n=6). 7-Oxysterols and glucocorticoids were quantified by HPLC-UV/radiodetection and GCMS.
Hepatic microsomal 11βHSD1 oxidised 7βOHC to 7KC (Km 3.5 mM; Vmax 32.6 pmol/μg/min), but affinity for 7βOHC was 10-fold less than for corticosterone (Km 0.32 mM; Vmax 4.6 nmol/μg/min). Specificity of 11βHSD1 for this reaction was confirmed by lack of similar metabolism in microsomes from 11βHSD1−/− mice. Oxysterols and glucocorticoids were mutual competitive inhibitors (7βOH IC50 2.20±9.85 μM inhibited corticosterone metabolism (10−9−2 M); Lineweaver Burk, Ki 1.65 μM; Dixon and global fit analysis for competitive inhibition, R2=0.68 and 0.79). Renal 11βHSD2 was unable to catalyse 7βOHC oxidation.
Murine 11βHSD1 catalysed NADPH-dependent reduction of 7KC to 7βOHC (Km 1.2 mM), although dehydrogenation of 7βOHC was preferred (Km 0.33 mM). However, turnover was higher for 7KC than 7βOHC (Kcat 1.4 vs 0.11 pmol/s). 7α-Hydroxycholesterol was not a substrate for dehydrogenation, nor was it generated by reduction of 7KC. In silico modelling of 7-oxysterol interaction with 11βHSD1 (1Y5M crystal structure), suggested more favourable binding of the enzymes catalytic triad (Y183-S170-NADP) to 7KC and 7βOHC-epimer than to 7αOHC.
Murine 11βHSD1 catalyses the metabolism of 7-oxysterols as well as glucocorticoids. This competitive interaction may be crucial in understanding the role of vascular 11βHSD1 in the pathophysiology of atherosclerosis.