Human studies indicate that local glucocorticoid (GC) generation within osteoblasts plays a critical role in various bone diseases. Human osteoblasts express the enzyme 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) that converts inactive GCs (cortisone, dehydrocorticosterone) to their active counterparts (cortisol, corticosterone). This activation capacity critically depends on expression of a cofactor generating enzyme hexose-6-phosphate dehydrogenase. Enzyme expression in mouse bone has however received less attention. We have now characterised local GC metabolism in bone cells derived from C57BL/6 mice and examined the its functional consequences in mice with targeted deletion of H6PDH.
Primary mouse osteoblast cultures were derived from calvaria (n=8 mice) and long bones (n=8) of mice by outgrowth of collagenase treated bone chips. The osteoblastic character of these cells was confirmed by high basal and GC-inducible alkaline phosphatase activity bone restricted gene expression. 11b-HSD1 but not 11b-HSD2 mRNA was detected. Enzyme activity studies revealed predominant glucocorticoid activation (cortisone to cortisol conversion 3.8+2.1; dehydrocorticosterone to corticosterone 6.6+1.1 pmol/mg/hr) further indicating 11b-HSD1 expression. As in human osteoblasts 11b-HSD1 expression increased with IL-1b treatment (2.5+0.2 fold with 10 ng/ml IL-1b). Hexose-6-phosphate dehydrogenase (H6PDH) was also expressed and targeted deletion changed the directionality of 11b-HSD1 activity towards predominant glucocorticoid inactivation. Primary cultures of mouse osteoclasts were established from bone marrow treated with MCSF/RANKL. 11b-HSD1 mRNA expression and activity were initially low but transiently increased (mRNA expression by 120%; activity by 30%) on day 5 coincident with the appearance of multinucleated osteoclasts. Marrow cultures from mice with H6PDH deletion were unexpectedly found to have substantially greater numbers of osteoclasts than the wild type.
These data indicate that mouse osteoblasts and osteoclasts express GC activating enzymes and this activity is dependent on H6PDH. The unexpected finding of increased osteoclastogenesis in H6PDH KO mice suggests that glucocorticoid activation negatively impacts on osteoclast differentiation.