It is well established that the use of therapeutic glucocorticoids to treat inflammatory disease has detrimental effects on bone and we have proposed that these clinical effects are determined by intracellular glucocorticoid generation (inactive cortisone/prednisone to cortisol/prednisolone) by 11beta-hydroxysteroid dehydrogenase type 1 (11b-HSD1). We have recently shown that glucocorticoids and cytokines are able to act cooperatively to upregulate the action of 11b-HSD1, a finding that would partly explain why patients without inflammation do not suffer the same rapid bone loss with GC treatment.
Our previous studies using luciferase reporter constructs failed to identify the regulatory promoter regions involved in this synergistic upregulation of 11b-HSD1 mRNA and enzyme activity. To ascertain whether novel promoter elements or unique transcriptional start sites were involved, we carried out 5′ RACE on total RNA isolated from human osteoblast cells treated with dexamethasone and IL-1 alone or in combination. Using a series of 5′ RACE and gene specific primers we identified a previously described transcript initiating from a well-documented promoter upstream of exon 1 and producing mRNA that would translate into a full length 11b-HSD1 protein. This transcript was not seen in untreated cells but was present in single and combination treatments. However, in RNA from cells treated with IL-1 and dexamethasone in combination, we identified an additional transcript that accounted for 2530% of total mRNA transcribed. Sequence analysis showed that the 5′ cap for the novel transcript originates +2 base pairs into exon 2 using a promoter in intron 1 and would theoretically result in translation initiating from Met49. This would have the consequence of truncating the 11b-HSD1 protein by 48 amino acids at the N terminus, removing the signal sequence directing the protein to its usual location in the endoplasmic reticulum and loss of the transmembrane domain. We are currently investigating possible functionality of this 11b-HSD1 variant using in vitro expression and activity studies and identifying promoter elements in intron 1 that may be responsible for the expression pattern of this novel transcript. This extremely novel finding may have significant implications for the pathogenesis of inflammatory bone disease and also the expression and function of 11b-HSD1 in tissues where glucocorticoids play an important role.