miRNA-34c regulates Notch signaling during bone development

Abstract

During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.

Figure 1.

miR-34b and -34c are induced during BMP2-mediated C2C12 osteoblast differentiation and directly target 3′-UTR of Notch1. (A) miRNA microarray (Exiqon, miRCURY LNA array v.9.2) was performed with total RNA isolated from BMP2-treated and vehicle-treated C2C12 cells (n = 2 biological replicates). The heat map diagram shows the result of two-way hierarchial clustering of miRNAs and samples. The color scale shown at the bottom illustrates the relative expression level of miRNA across samples. Cr indicates a dyeswap. (B) Validation of the array data for miR-34b and -34c was performed with RNA used for the array using a Taqman miRNA assay, normalized to sno202 (n = 3). *P < 0.05 compared with C2C12 control (con). Also RNA from undifferentiated (Undiff) and differentiated (Diff) of BMSCs was analyzed by Taqman miRNA assay (n = 3). *P < 0.05 compared with Undiff-BMSC. (C) C2C12 cells were transfected with miR-34b, miR-34c, miR-C (control) or transfection reagent only (Mock). After 48 h, Notch1 transcription level was quantified by qRT–PCR (normalized to B2m) (n = 4).*P < 0.05, **P < 0.01 compared with miR-C. (D) Western blots for Notch1 and β-tubulin (control) were performed on total cell lysates. Numbers indicate the quantification of relative Notch1/β-tubulin band intensity ratios. (E) HeLa cells were co-transfected with the luciferase reporter containing either wild-type (WT) 3′-UTR or mutant (Mut1, Mut2 or Mut1 + 2) 3′-UTR Notch1 and miR-34b, -34c or miR-C (n = 3). ***P < 0.001. Three independent experiments were performed.

Figure 2.

Osteoblast-specific miR-34c transgenic mice manifest osteoporosis. (A) Micro-CT analysis of 6-month-old miR-34c Tg mice show decreased BV/TV, Tb.N and Tb.Th and increased Tb.Sp compared with WT (n = 10–13 per group). **P < 0.01, ***P < 0.001. (B) Micro-CT reconstruction of distal femur from 6-month-old miR-34c Tg mice show decreased trabecular bone. Scale bar, 1 mm. (C) Histomorphometric analysis of the vertebrae of 6-month-old miR-34c Tg and WT mice (n = 8–12 per group) reveal decreased Ob.S/BS and N.Ob/B.Pm. *P < 0.05. Error bars are SD. Toludine blue staining of 6-month-old vertebrae section from miR-34c Tg and WT showed decreased osteoblast staining. Osteoblasts are underlined in red. Scale bar, 100 μm. (D) BFR/BS, MS/BS and MAR were decreased in miR-34c Tg. *P < 0.05, **P < 0.01. Error bars are SEM. (E) Oc.S/BS, N.Oc/B. Pm and ES/BS were increased in miR-34c Tg. *P < 0.05. Error bars are SD. TRAP staining (red) of 6-month-old vertebrae section from miR-34c Tg and WT, indicating increased osteoclast staining. Scale bar, 50 μm.

Figure 3.

miR-34c inhibits osteoblast differentiation and increases osteoclastogenesis. (A) Induction of miR-34c was quantified by qRT–PCR after osteoblast differentiation from both WT and miR-34c Tg BMSC (n = 5 per group). *P < 0.05, **P < 0.01. (B) Alizarin Red and Von Kossa staining were performed weekly and quantified using ImageJ (n = 6 per group).*P < 0.05. (C) Co-cultures of WT or miR-34c Tg mesenchymal-derived osteoblasts and WT splenocytes were performed in the presence of 10⁻⁸ m of Vit.D3 and stained for TRAP-positive cells at day 10 (n = 18 per group). ***P < 0.001. (D) Representative stained image. Error bar is SEM.

Figure 4.

miR-34c targets the Notch signaling pathway during osteoblast development in vivo. Total RNA was extracted from P2 calvarial tissues of WT and miR-34c Tg mice. The expression level of Notch receptors (A), Notch ligands (Jag1 and Dll4), Rbpjk and Hey1 (B), osteoblast markers (C) and Opg and Rankl (D) were quantified by qRT–PCR and normalized to B2m (n = 6). *P < 0.05, **P < 0.01. Error bar is SEM. (E) Diagram of miR-34c-mediated regulation of bone development. MiR-34c regulates both osteoclasts and osteoblasts via targeting multiple factors in the Notch signaling pathway and Runx2 and Satb2, respectively, during skeletal development. The direct targets of miR-34c are in bold.