11β-HSD1 plays a critical role in trabecular bone loss associated with systemic glucocorticoid therapy

Abstract

Background: Despite their efficacy in the treatment of chronic inflammation, the prolonged application of therapeutic glucocorticoids (GCs) is limited by significant systemic side effects including glucocorticoid-induced osteoporosis (GIOP). 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional enzyme that primarily activates GCs in vivo, regulating tissue-specific exposure to active GC. We aimed to determine the contribution of 11β-HSD1 to GIOP.

Methods: Wild type (WT) and 11β-HSD1 knockout (KO) mice were treated with corticosterone (100 μg/ml, 0.66% ethanol) or vehicle (0.66% ethanol) in drinking water over 4 weeks (six animals per group). Bone parameters were assessed by micro-CT, sub-micron absorption tomography and serum markers of bone metabolism. Osteoblast and osteoclast gene expression was assessed by quantitative RT-PCR.

Results: Wild type mice receiving corticosterone developed marked trabecular bone loss with reduced bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N). Histomorphometric analysis revealed a dramatic reduction in osteoblast numbers. This was matched by a significant reduction in the serum marker of osteoblast bone formation P1NP and gene expression of the osteoblast markers Alp and Bglap. In contrast, 11β-HSD1 KO mice receiving corticosterone demonstrated almost complete protection from trabecular bone loss, with partial protection from the decrease in osteoblast numbers and markers of bone formation relative to WT counterparts receiving corticosterone.

Conclusions: This study demonstrates that 11β-HSD1 plays a critical role in GIOP, mediating GC suppression of anabolic bone formation and reduced bone volume secondary to a decrease in osteoblast numbers. This raises the intriguing possibility that therapeutic inhibitors of 11β-HSD1 may be effective in preventing GIOP in patients receiving therapeutic steroids.

Keywords: 11β-HSD1; Glucocorticoids; Osteoporosis; Trabecular bone.

Fig. 1

a Corticosterone generation in tibia ex vivo biopsies isolated from WT and 11β-HSD1 KO mice determined by scanning thin-layer chromatography. b Serum corticosterone levels determined by ELISA in WT and 11β-HSD1 KO receiving either vehicle or oral corticosterone (100 μg/ml). c Adrenal weights (mg) from WT and 11β-HSD1 KO mice receiving either vehicle or oral corticosterone (100 μg/ml) and d representative paraffin-embedded sections of the liver taken from WT mice receiving either vehicle or oral corticosterone (100 μg/ml) (× 20), stained with haematoxylin and eosin. Values are expressed as mean ± standard error of six animals per group. Statistical significance was determined using two-way ANOVA with a Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

a Representative images of 3D reconstructions of tibia trabecular bone using micro-CT from WT and 11β-HSD1 KO receiving either vehicle or oral corticosterone (100 μg/ml). b Bone volume to tissue volume (BV/TV), c trabecular number (Tb.N), d trabecular thickness (Tb.Th) and e trabecular separation (Tb.Sp) determined by Meshlab software analysis of micro-CT in WT and 11β-HSD1 KO receiving either vehicle or oral corticosterone (100 μg/ml). Values are expressed as mean ± standard error of six animals per group. Statistical significance was determined using two-way ANOVA with a Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001. Black arrows represent regions of mesh surface trabecular thinning

Fig. 3

Histomorphometric analysis of numbers of (a) osteoblasts (N.Ob/B.Pm) and (b) osteoclasts (N.Oc/B.Pm) at the bone perimeter per square millimetre from vertebrae L3 and L4. c Serum P1NP (ng/ml) (d) and serum CTX-1 (ng/ml) were determined by ELISA in WT and 11β-HSD1 KO mice receiving either vehicle or oral corticosterone (100 μg/ml). e Representative images of osteoblasts and f representative images of osteoclasts on trabecular bone surface. g The ratio of RANKL/OPG gene expression in the tibia from WT and 11β-HSD1 KO mice receiving either vehicle or oral corticosterone (100 μg/ml) was determined by quantitative RT-PCR. Values are expressed as mean ± standard error of six animals per group. Statistical significance was determined using two-way ANOVA with a Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001. Black arrows indicate osteoblasts and osteoclasts

Fig. 4

a–f Gene expression (AU) of Bglap, Alp, Ctsk, Runx2, Sost and Dkk1 in tibias taken from WT and 11β-HSD1 KO receiving either vehicle or oral corticosterone (100 μg/ml) determined by quantitative RT-PCR. Values are expressed as mean ± standard error of six animals per group. Statistical significance was determined using two-way ANOVA with a Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001