Supplemental data for: Decreasing miR-433-3p activity in the osteoblast lineage blunts glucocorticoid-mediated bone loss

Glucocorticoid excess causes bone loss due to decreased bone formation and
increased bone resorption; miR-433-3p is a miRNA that negatively regulates
bone formation in male mice by targeting Runx2 as well as RNAs involved in
Wnt, protein kinase A and endogenous glucocorticoid signaling. To examine
the impact of miR-433-3p on glucocorticoid-mediated bone loss, transgenic
mice expressing a miR-433-3p tough decoy inhibitor in the osteoblast
lineage were administered prednisolone via slow-release pellets. Bone loss
was greater in control mice treated with prednisolone compared with
miR-433-3p tough decoy mice due to higher osteoclast activity in the
controls. In whole femurs, Rankl was significantly higher in
prednisolone-treated controls compared with miR-433-3p tough decoy mice.
Surprisingly, negative regulators of Wnt signaling Sost and Dkk1 were
higher in miR-433-3p tough decoy mice and were unaffected by prednisolone.
Luciferase-3’ UTR reporter assays demonstrated that Sost is a novel
miR-433-3p target, whereas Dkk1 is a previously validated miR-433-3p
target. miR-433-3p levels are lower in matrix synthesizing osteoblasts
than in more osteocytic cells, thus the impact of miR-433-3p on the
osteoblast lineage maybe dependent on cell context: it is a negative
regulator in matrix-depositing osteoblasts by targeting RNAs important for
differentiation and function, but a positive regulator in osteocytes, due
to its ability to target prominently expressed negative regulators of Wnt
signaling, Sost and Dkk1. The mechanisms by which miR-433-3p indirectly
regulates glucocorticoid-mediated osteoclastogenesis remain unknown.
However, we speculate that this regulation may be mediated miR-433-3p
activity in osteocytes, which play an important role in controlling
osteoclastogenesis.