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.

Methods
Supplemental Table 1: primer sets for qRT-PCR and murine Sost 3' UTR cloning

Supplemental Figure 1:The Col1a1miR-433TuD is not expressed in the osteoclast lineage: Differential interference contrast images overlaid by tdTomato signal in primary bone marrow monocytes cultured with MCSF+RANKL (30 ng/ml each) for 4 days.