DOI: 10.5061/dryad.1g1jwsv6s
title: Supplemental data for: Decreasing miR-433-3p activity in the osteoblast lineage blunts glucocorticoid-mediated bone loss

description: 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.
description: Supplemental Table 1: primer sets for qRT-PCR and murine Sost
 3' UTR cloning Supplemental Figure 1:The
 Col1a1<sup>miR-433TuD</sup> 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.
description: # Supplemental data for "Decreasing miR-433-3p activity in the
 osteoblast lineage blunts glucocorticoid-mediated bone loss"
 [https://doi.org/10.5061/dryad.1g1jwsv6s](https://doi.org/10.5061/dryad.1g1jwsv6s) ## Description of the data and file structure To examine the impact of miR-433-3p on glucocorticoid-mediated bone loss, transgenic mice expressing a miR-433-3p tough decoy competitive inhibitor in the osteoblast lineage were administered prednisolone via slow-release pellets. Histomorphometry, microCT, histology, serum and RNA analyses were used to examine the impact of the glucocorticoid on bone structure, bone formation and resorption parameters and gene expression.   To determine whether the miR-433 tough decoy was expressed in the osteoclast lineage, primary bone marrow monocytes from control or miR-433 tough decoy mice were cultured with MCSF+RANKL (30 ng/ml each) for 4 days, and imaged for tdTomato fluorescence, which would signify expression of the tough decoy. As a positive control, we also examined bone marrow monocytes from mice expressing LysM-cre and a cre-activated tdTomato-miR-29 TuD cassette knocked into the Rosa26 locus. ### Files and variables The pdf file contains supplemental data (Table S1 and Figure S1) for the Endocrinology manuscript titled "Decreasing miR-433-3p activity in the osteoblast lineage blunts glucocorticoid-mediated bone loss". ## Code/software NA