Center for Molecular Oncology, UConn Health, Farmington, Connecticut, USA.
Department of Orthopaedics, Emory University and Atlanta VA Medical Center, Decatur, Georgia, USA.
J Bone Miner Res. 2021 Sep;36(9):1808-1822. doi: 10.1002/jbmr.4339. Epub 2021 Jun 4.
MicroRNAs (miRNAs) are key posttranscriptional regulators of osteoblastic commitment and differentiation. miR-433-3p was previously shown to target Runt-related transcription factor 2 (Runx2) and to be repressed by bone morphogenetic protein (BMP) signaling. Here, we show that miR-433-3p is progressively decreased during osteoblastic differentiation of primary mouse bone marrow stromal cells in vitro, and we confirm its negative regulation of this process. Although repressors of osteoblastic differentiation often promote adipogenesis, inhibition of miR-433-3p did not affect adipocyte differentiation in vitro. Multiple pathways regulate osteogenesis. Using luciferase-3' untranslated region (UTR) reporter assays, five novel miR-433-3p targets involved in parathyroid hormone (PTH), mitogen-activated protein kinase (MAPK), Wnt, and glucocorticoid signaling pathways were validated. We show that Creb1 is a miR-433-3p target, and this transcription factor mediates key signaling downstream of PTH receptor activation. We also show that miR-433-3p targets hydroxysteroid 11-β dehydrogenase 1 (Hsd11b1), the enzyme that locally converts inactive glucocorticoids to their active form. miR-433-3p dampens glucocorticoid signaling, and targeting of Hsd11b1 could contribute to this phenomenon. Moreover, miR-433-3p targets R-spondin 3 (Rspo3), a leucine-rich repeat-containing G-protein coupled receptor (LGR) ligand that enhances Wnt signaling. Notably, Wnt canonical signaling is also blunted by miR-433-3p activity. In vivo, expression of a miR-433-3p inhibitor or tough decoy in the osteoblastic lineage increased trabecular bone volume. Mice expressing the miR-433-3p tough decoy displayed increased bone formation without alterations in osteoblast or osteoclast numbers or surface, indicating that miR-433-3p decreases osteoblast activity. Overall, we showed that miR-433-3p is a negative regulator of bone formation in vivo, targeting key bone-anabolic pathways including those involved in PTH signaling, Wnt, and endogenous glucocorticoids. Local delivery of miR-433-3p inhibitor could present a strategy for the management of bone loss disorders and bone defect repair. © 2021 American Society for Bone and Mineral Research (ASBMR).
微小 RNA（miRNAs）是成骨细胞分化和分化的关键转录后调节因子。miR-433-3p 先前被证明靶向 runt 相关转录因子 2（Runx2），并受骨形态发生蛋白（BMP）信号的抑制。在这里，我们显示 miR-433-3p 在体外小鼠骨髓基质细胞的成骨细胞分化过程中逐渐降低，并且我们确认其对该过程的负调控。尽管成骨细胞分化的抑制剂通常促进脂肪生成，但抑制 miR-433-3p 并不影响体外脂肪细胞分化。多种途径调节成骨作用。使用荧光素酶-3'非翻译区（UTR）报告测定，验证了涉及甲状旁腺激素（PTH）、丝裂原激活蛋白激酶（MAPK）、Wnt 和糖皮质激素信号通路的五个新的 miR-433-3p 靶标。我们显示 Creb1 是 miR-433-3p 的靶标，并且该转录因子介导 PTH 受体激活下游的关键信号。我们还表明，miR-433-3p 靶向羟甾体 11-β 脱氢酶 1（Hsd11b1），该酶将无活性的糖皮质激素局部转化为其活性形式。miR-433-3p 抑制糖皮质激素信号，并且靶向 Hsd11b1 可能有助于这种现象。此外，miR-433-3p 靶向富含亮氨酸重复的 G 蛋白偶联受体（LGR）配体 R 分泌蛋白 3（Rspo3），该配体增强 Wnt 信号。值得注意的是，Wnt 经典信号也被 miR-433-3p 活性减弱。在体内，成骨细胞谱系中 miR-433-3p 抑制剂或坚硬诱饵的表达增加了小梁骨体积。表达 miR-433-3p 坚硬诱饵的小鼠表现出增加的骨形成，而不改变成骨细胞或破骨细胞的数量或表面，表明 miR-433-3p 降低成骨细胞活性。总体而言，我们表明 miR-433-3p 是体内骨形成的负调节剂，靶向包括甲状旁腺激素信号、Wnt 和内源性糖皮质激素在内的关键骨合成途径。miR-433-3p 抑制剂的局部递送可能为管理骨质疏松症和骨缺损修复提供一种策略。
