A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans

Abstract

MicroRNAs (miRNAs) interfere with translation of specific target mRNAs and are thought to thereby regulate many cellular processes. Recent studies have suggested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify a new miRNA (miR-2861) in primary mouse osteoblasts that promotes osteoblast differentiation by repressing histone deacetylase 5 (HDAC5) expression at the post-transcriptional level. miR-2861 was found to be transcribed in ST2 stromal cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-2861 enhanced BMP2-induced osteoblastogenesis, whereas inhibition of miR-2861 expression attenuated it. HDAC5, an enhancer of runt-related transcription factor 2 (Runx2) degradation, was confirmed to be a target of miR-2861. In vivo silencing of miR-2861 in mice reduced Runx2 protein expression, inhibited bone formation, and decreased bone mass. Importantly, miR-2861 was found to be conserved in humans, and a homozygous mutation in pre-miR-2861 that blocked expression of miR-2861 was shown to cause primary osteoporosis in 2 related adolescents. Consistent with the mouse data, HDAC5 levels were increased and Runx2 levels decreased in bone samples from the 2 affected individuals. Thus, our studies show that miR-2861 plays an important physiological role in osteoblast differentiation and contributes to osteoporosis via its effect on osteoblasts.

Figure 1. miR-2861 is primarily expressed in osteoblasts.

(A) Schematic diagram of the secondary structure of pre–miR-2861. The structures were predicted by mfold, and the mature miR-2861 sequence is underlined. (B) Northern blot analysis of miR-2861 expression in osteoblasts, osteoclasts, and various mouse tissues. (C) miR-2861 expression during BMP2-induced ST2 osteogenic differentiation. ST2 cells were treated with BMP2 (300 ng/ml) for the indicated durations, and miR-2861 expression was measured by Northern blotting. The blots were stripped and rehybridized with ³²P-labeled oligonucleotide probes for U6. U6 snRNA was used as a loading control. Circles represent the percentage of U6 expression level at various time points.

Figure 2. miR-2861 promotes BMP2-induced ST2 osteogenic differentiation.

(A) Northern blot analysis of miR-2861 levels in ST2 cells after transfection with pre–miR-2861 or miR-C. U6 was used as a loading control. (B) Overexpression of miR-2861 enhanced BMP2-induced ST2 osteogenic differentiation. ST2 cells were stably transfected with pre–miR-2861 or miR-C, then treated with BMP2 for 48 hours. ALP activity and osteocalcin secretion were determined. Data are shown as means ± SD. *P < 0.05 vs. miR-C, n = 5. (C) Microscopic view of the effects of miR-2861 on matrix mineralization in ST2 cells. Shown is a representative microscopic view at a magnification of ×200 of cells transfected with pre–miR-2861 or miR-C after 20 days of culture. Quantification of Alizarin Red S stain via extraction with cetyl-pyridinium chloride. Data are shown as means ± SD. *P < 0.05 vs. miR-C, n = 3. (D) Levels of Runx2 mRNA were determined using qRT-PCR and are shown as fold induction relative to control. Runx2 protein expression was determined by Western blot and expressed as densitometry of Runx2/β-actin. Data are shown as means ± SD. *P < 0.05 vs. miR-C, n = 3.

Figure 3. Inhibition of miR-2861 limits BMP2-induced osteogenic differentiation.

ST2 cells were treated with BMP2 and transiently transfected with anti–miR-2861 or anti–miR-C. (A) Northern blotting showed that anti–miR-2861 repressed the expression of miR-2861 in BMP2-induced ST2 cells. (B) ALP activity and osteocalcin secretion were measured at 48 hours as described in Figure 2B. (C) Runx2 mRNA and protein expression were detected as described in Figure 2D. *P < 0.05 vs. anti–miR-C.

Figure 4. miR-2861 functional activity on target genes.

(A) Schematic of the miR-2861 putative target site in mouse HDAC5 CDS and alignment of miR-2861 with WT and MUT CDS regions of HDAC5 showing complementary pairing. The 2 mutated nucleotides are underlined. (B) miR-2861 targeted the HDAC5 CDS. ST2 cells were cotransfected with the luciferase reporter carrying WT-pGL3-HDAC5 or MUT-pGL3-HDAC5, phRL-null (Renilla plasmid), and miR-C or pre–miR-2861. Effects of miR-2861 and miR-C on the reporter constructs were determined 48 hours after transfection. Firefly luciferase values, normalized for Renilla luciferase, are presented. Data are shown as means ± SD. *P < 0.05 vs. MUT-pGL3-HDAC5, n = 3. (C) miR-2861 regulated HDAC5 expression at the post-transcriptional level. ST2 cells were transfected with pre–miR-2861 or miR-C. Northern blot analysis identified miR-2861 level in ST2 cells. Total cell lysates were collected and analyzed 48 hours after transfection. The level of HDAC5 mRNA was determined using qRT-PCR and normalized to β-actin. Data are shown as means ± SD. HDAC5 protein expression was determined by Western blot, using β-actin as a loading control. Results were expressed as densitometry of HDAC5/β-actin. Data are shown as means ± SD. *P < 0.05 vs. miR-C, n = 3.

Figure 5. The mutant HDAC5 CDS construct rescues miR-2861–modulated ST2 osteogenic differentiation.

BMP2-induced ST2 cells were cotransfected with the WT or mutant HDAC5 CDS construct and pre–miR-2861 or miR-C. (A) Transfection of mutant HDAC5 into BMP2-induced ST2 cells rescued the pre–miR-2861–induced increase of ALP activity. Data are shown as means ± SD. *P < 0.05 vs. pre–miR-2861 + WT HDAC5, n = 5. (B) Transfection of the mutant HDAC5 into BMP2-induced ST2 cells rescued the pre–miR-2861–induced downregulation of HDAC5 protein.

Figure 6. Runx2 mutant rescues anti–miR-2861–inhibited ALP activity.

BMP2-induced ST2 cells were cotransfected with anti–miR-C or anti–miR-2861 and Myc-tagged WT type II Runx2 or a Runx2 mutant containing KR substitutions in the HDAC5 deacetylation targets (Runx2-KR-240/245/365/366 mutant). (A) Transfection of the Runx2-KR-240/245/365/366 mutant into BMP2-induced ST2 cells rescued the anti–miR-2861 inhibition of ALP activity. Data are shown as means ± SD. *P < 0.05 vs. anti–miR-2861 + WT Runx2, n = 5. (B) Transfection of the Runx2 mutant into BMP2-induced ST2 cells rescued the anti–miR-2861–inhibited Runx2 protein levels. The abundance of Runx2 or Runx2 mutant was determined by Western blot using an anti-Myc antibody.

Figure 7. Antagomir-2861–treated mice have decreased bone mass and reduced osteoblast activity.

(A) No expression of miR-2861 was detected in bones of antagomir-2861–treated mice. Mice were injected intravenously with PBS, mutant (Mut) antagomir-2861, or antagomir-2861 (3 injections of 80 mg/kg/d in the first week, and another injection on days 1–3 of the fourth week) and bones were harvested at 4 days, 3 weeks, and 6 weeks after the first injection. Expression of miR-2861 was analyzed by Northern blot. (B) Silencing of miR-2861 resulted in decreased BMD. Structural parameters of femurs were measured by micro-CT. (C) micro-CT section of distal and midfemoral diaphyses. (D) Bone formation parameters of femurs measured by histological analysis. (E) Bone resorption parameters of mouse femur. Data are means ± SD of 8 mice per group. *P < 0.05.

Figure 8. Osteoblast markers but not osteoclast markers are affected by the absence of miR-2861 in mice.

(A) qRT-PCR analysis of osteoblast and osteoclast markers in bone extracts from mice. (B) Serum concentrations of bone-specific ALP and TRAP5b. (C) Bone mRNA and protein expression levels of HDAC5 and Runx2. Level of HDAC5 and Runx2 mRNA were determined by qRT-PCR, and the results are expressed as a percentage of the PBS-treated SHAM group. HDAC5 and Runx2 protein expression was determined using Western blotting and normalized to total protein. β-Actin was used as a loading control. Data are means ± SD of 8 mice per group. *P < 0.05.

Figure 9. Identification of a mutation in pre–miR-2861.

(A) miR-2861 expression in adolescent primary osteoporosis patients and controls. miR-2861 expression was analyzed by Northern blotting with total RNA isolated from bones of patients and controls. U6 was used as a loading control. Representative results are presented. (B) Chromatograms for the normal genome and the mutated miR-2861 samples. A fragment of an approximately 600-bp genomic sequence flanking the pre–miR-2861 at both the 5′ and 3′ ends was amplified, purified, and sequenced. Arrows indicate the location of the mutation. (C) Schematic representation of the miR-2861 genotypes of the studied kindred, consisting of 49 members from 4 generations. The proband is indicated by an arrow. Subjects with the homozygous C-G mutation are indicated by closed symbols; heterozygous carriers are indicated by half-shaded symbols.

Figure 10. The miR-2861 mutation represses miRNA expression and attenuates miRNA-mediated translational suppression.

(A) The stem-loop structures of pre–miR-C and pre–miR-G, as predicted by mfold. Mature miRNAs are highlighted in red, and the mutated nucleotide is highlighted in green (and indicated by arrows). The free energy (ΔG) calculated by mfold is indicated. (B) The C-G mutation in the stem of pre–miR-2861 inhibited mature miR-2861 expression. Human 293 cells were transfected with pcDNA3-miR-2861-C, pcDNA3-miR-2861-G, or empty vector. The cells were harvested 48 hours after transfection, and miR-2861 expression was determined by Northern blotting. U6 was used as a loading control. (C) The miR-2861 mutation attenuated miRNA-mediated translational suppression. 293 cells were cotransfected with the luciferase reporter carrying HDAC5 CDS and pcDNA3-miR-2861-C, pcDNA3-miR-2861-G, or miR-C. Effects of the WT, MUT pre–miR-2861, or control on the reporter constructs were determined 48 hours after transfection. Firefly luciferase values, normalized for Renilla luciferase, are presented. Data are shown as means ± SD. *P < 0.05 vs. control, n = 3. (D) HDAC5 and Runx2 protein levels in human bone. HDAC5 and Runx2 protein expression levels were determined by Western blotting with proteins extracted from bone of patients and controls and normalized to total protein. β-Actin was used as a loading control. Representative results are presented.