The microRNA-23a cluster regulates the developmental HoxA cluster function during osteoblast differentiation

Abstract

MicroRNAs (miRs) and Hox transcription factors have decisive roles in postnatal bone formation and homeostasis. In silico analysis identified extensive interaction between HOXA cluster mRNA and microRNAs from the miR-23a cluster. However, Hox regulation by the miR-23a cluster during osteoblast differentiation remains undefined. We examined this regulation in preosteoblasts and in a novel miR-23a cluster knockdown mouse model. Overexpression and knockdown of the miR-23a cluster in preosteoblasts decreased and increased, respectively, the expression of the proteins HOXA5, HOXA10, and HOXA11; these proteins' mRNAs exhibited significant binding with the miR-23a cluster miRNAs, and miRNA 3'-UTR reporter assays confirmed repression. Importantly, during periods correlating with development and differentiation of bone cells, we found an inverse pattern of expression between HoxA factors and members of the miR-23a cluster. HOXA5 and HOXA11 bound to bone-specific promoters, physically interacted with transcription factor RUNX2, and regulated bone-specific genes. Depletion of HOXA5 or HOXA11 in preosteoblasts also decreased cellular differentiation. Additionally, stable overexpression of the miR-23a cluster in osteoblasts decreased the recruitment of HOXA5 and HOXA11 to osteoblast gene promoters, significantly inhibiting histone H3 acetylation. Heterozygous miR-23a cluster knockdown female mice (miR-23a Cl^WT/ZIP) had significantly increased trabecular bone mass when compared with WT mice. Furthermore, miR-23a cluster knockdown in calvarial osteoblasts of these mice increased the recruitment of HOXA5 and HOXA11, with a substantial enrichment of promoter histone H3 acetylation. Taken together, these findings demonstrate that the miR-23a cluster is required for maintaining stage-specific HoxA factor expression during osteogenesis.

Keywords: Bone epigenetics; H3K27 acetylation; HoxA cluster; bone; epigenetics; gene regulation; homeobox; miR-23a cluster; microRNA (miRNA); osteoblast; osteoblast differentiation; transcription factor.

Figure 1.

Identification and validation of functional miR-23a cluster binding sites in the 3′-UTR of HoxA5, HoxA10, and HoxA11 mRNA 3′-UTR. A, putative binding sites for miR-23a cluster members were identified in the 3′-UTRs of HoxA5 (nucleotide positions 97–103, 558–564, and 704–710), HoxA10 (nucleotide positions 1032–1039), and HoxA11 (nucleotide positions 38–44, 496–503, and 525–531) mRNA using miRNA bioinformatics programs (Table 1). B and C, quantitative analysis of the total RNA to examine relative HoxA member mRNA expression levels, normalized to Gapdh mRNA levels, in MC3T3-E1 cells stably infected with empty vector control or miR-23a cluster (CMV-driven stem loop for miR-23a cluster) (B) and empty vector control or anti-miR-23a cluster (MIR-ZIP, H1 promoter-driven stem loop for anti-miR-23a cluster)-expressing lentiviral particles, detected by RT-qPCR after 72 h (C). Oligo(dT) primer was used to synthesize cDNA, and gene-specific primers were used to amplify the cDNA (Table S5). D, representative Western blotting of HOXA3, HOXA5, HOXA10, HOXA11, and HOXA13 protein levels, normalized to actin levels, in MC3T3-E1 cells stably infected with control, miR-23a cluster, or anti-miR-23a cluster (MIR-ZIP)-expressing lentiviral particles, after 72 h. Cell lysates (25 μg) were loaded on 10% SDS-PAGE and detected with the indicated antibodies. Molecular masses (kDa) of the indicated HOX factors are shown. E, relative mRNA expression of HoxA5, HoxA10, and HoxA11 in MC3T3-E1 cells stably infected with control (C), miR-23a–expressing, or miR-27a–expressing lentiviral vectors, detected by RT-qPCR after 72 h. Oligo(dT) primer was used to synthesize cDNA, and gene-specific primers were used to amplify the cDNA (Table S5). F, representative RISC-IP assay in polysome lysates of MC3T3-E1 cells overexpressing the miR-23a cluster immunoprecipitated with IgG (control) or anti-AGO2 antibody. Immunoprecipitated RNA was isolated using the TRIzol method. Hexamer oligonucleotide primers were used to synthesize cDNA, and gene-specific primers were used to amplify the cDNA (Table S5). Data are presented as the amount of miR-23a and miR-27a bound to the 3′-UTR of HoxA5, HoxA10, and HoxA11 (black bars) in the RISC relative to total HoxA5, HoxA10, and HoxA11 3′-UTR cDNA input. Normal IgG was used as a nonspecific pulldown control (white bar). miR-23a cluster target Satb2 was used as positive (58) and Gapdh gene was used as negative control. G–I, relative luciferase activity in HEK-293T cells transfected with WT (WT 3′-UTR) or mutated (MT 3′-UTR) HoxA5, HoxA10, and HoxA11 3′-UTR constructs along with nonspecific miRNA control (C), miR-23a, miR-27a, synthetic miRNA (Syn 23a, Syn 27a), miR-23a cluster construct (miR-Cl), or anti-miR-23a cluster construct (MIR-ZIP) for 48 h. Cells were lysed in Passive Lysis Buffer (Promega Corp.) and assayed (20 μl) for luciferase activity. Relative luciferase activity was normalized with Renilla luciferase activity and expressed in relative luminescence units. Statistical significance was determined by Student's t test. *, p ≤ 0.05 versus matched control. Luc, luciferase. Error bars, S.E.

Figure 2.

Temporal expression of the miR-23a cluster and HoxA5 and HoxA11 maintains the physiology of osteoblast differentiation in vitro and in vivo. A, relative expression of the miR-23a cluster miRNA compared with HoxA5 and HoxA11 mRNA during MC3T3-E1 (preosteoblast) cell differentiation at the indicated days with n = 3 time course experiments, normalized to Gapdh levels. Oligo(dT) primer was used to synthesize cDNA, and gene-specific primers were used to amplify the cDNA (Table S5). B, densitometry quantitation (ImageJ) of Western blots (n = 3) showing total protein from MC3T3-E1 cells assayed for HOXA5 and HOXA10 expression at the indicated days of osteoblast differentiation. A very similar pattern of protein profiles was observed from multiple osteoblast time course experiments. C, total RNA isolated from mouse long bones at the indicated times of embryonic development was analyzed for the expression of the miR-23a cluster and HoxA5 and HoxA11 from n = 3 experiments by RT-qPCR. Oligo(dT) primer was used to synthesize cDNA, and gene-specific primers were used to amplify the cDNA (Table S5). U6 expression was used as the experimental control. E, embryonic. Error bars, S.E.

Figure 3.

HOXA5 and HOXA11 functionally interact with RUNX2 to promote bone-specific gene expression. Luciferase reporter assays with Runx2 and Ocn promoters were used to measure promoter activity. Forty-eight hours after transfection, cells were lysed in Passive Lysis Buffer (Promega Corp., Madison, WI) and assayed (20 μl) for luciferase activity. A and B, relative luciferase activity of the −600 bp Runx2 proximal promoter (A) and the −285 bp Ocn promoter (B) 48 h after transient knockdown of HOXA5 or HOXA11 expression using shRNA in HEK-293 cells. C, −285 bp Ocn promoter activity 48 h after transient HOXA5 or HOXA11 overexpression and/or knockdown by respective shRNA transfection. D, relative promoter activity of the −285 bp Ocn promoter 48 h after transient overexpression of RUNX2, HOXA5, or HOXA11 alone or in combination. E, MC3T3-E1 cell lysate at day 7 was immunoprecipitated with anti-RUNX2 antibody followed by Western blot analysis with antibodies against RUNX2 (top), HOXA5 (middle) and HOXA11 (bottom). Immunoprecipitates were loaded on 10% SDS-PAGE and detected with indicated antibodies. Molecular masses (kDa) of the indicated HOX factors and RUNX2 are shown. F, transcription factor search analysis of the Ocn promoter identified putative RUNX2 and HOXA5/10/11 binding sites. G, ChIP assays were performed in MC3T3-E1 cells at day 12 of differentiation using antibodies against HOXA5, HOXA10, HOXA11, RUNX2, and H3K4me3 identified occupancy of the Ocn proximal promoter (−121 to −7) and distal promoter (−620 to −460). Cross-linked soluble chromatin (400–500 μg) were immunoprecipitated overnight with the indicated antibodies. Purified immunoprecipitated DNA were amplified using promoter-specific primers documented in Table S5. H, ChIP–re-ChIP assays were performed to identify the association of HOXA5, HOXA11, and RUNX2 with Ocn promoters. First, a RUNX2 ChIP was performed with anti-RUNX2 antibody, and the resulting chromatin immunoprecipitate (second input) was subjected to re-ChIP with anti-HOXA5 and anti-HOXA11 antibodies (secondary pulldown). Empty vector (Con) and scramble shRNA (Con shRNA) were used in transfection experiments; normal IgG was used as control in co-immunoprecipitation and ChIP assays. Relative luciferase activity was normalized with Renilla luciferase activity and expressed in relative luminescence units. Statistical significance was determined by Student's t test. *, p ≤ 0.05 versus matched control. Error bars, S.E.

Figure 4.

HOXA5 and HOXA11 control osteogenic genes that are essential for osteoblast maturation. A and B, representative Western blotting showing HOXA5 (A) and HOXA11 (B) protein from MC3T3-E1 cells with transient knockdown of HoxA5 or HoxA11 by respective shRNA. Cells were differentiated for 4 days (proliferative phase) or 12 days (matrix formation phase) using osteogenic medium. Total RNA and cell lysates were isolated to examine the effect of HoxA5 or HoxA11 knockdown on osteogenesis (C), and ALP staining of MC3T3-E1 cells with transient knockdown of HoxA5 or HoxA11 by shRNA (day 12 of differentiation). D and E, relative mRNA expression of HoxA5, HoxA11, Runx2, osterix, Alp, Col1A1, osteopontin (Opn), and Ocn after transient knockdown of HOXA5 (D) or HOXA11 (E) in MC3T3-E1 cells at days 4 and 12 of differentiation by real-time RT-qPCR. Gapdh expression was used as the experimental control to calculate δδCT. Statistical significance was determined by Student's t test. *, p ≤ 0.05 versus matched control. Error bars, S.E.

Figure 5.

The miR-23a cluster reduces HOXA5 and HOXA11 recruitment and inhibits epigenetic activation of stage-specific osteogenic genes. ChIP assays (n = 3) were performed with antibodies against HOXA5 or HOXA11 to determine the occupancy of HOXA5 and HOXA11 at the Alp (A), Runx2 (B), and Ocn (C) promoters in control and miR-23a cluster–overexpressing MC3T3-E1 cells at day 12 of differentiation. ChIP assays (n = 3) were performed with antibodies against H3K18ac or H3K27ac (indicative of open chromatin) to determine the occupancy at the Alp (D), Runx2 (E), and Ocn (F) promoters in control and miR-23a cluster–overexpressing MC3T3-E1 cells at day 12 of differentiation. Cross-linked soluble chromatin (400–500 μg) was immunoprecipitated overnight with the indicated antibodies. Purified immunoprecipitated DNA was amplified using promoter-specific primers documented in Table S5. Anti-luciferase antibody was used as a nonspecific control. Percentage occupancy over input is plotted on the y axis as measured by RT-qPCR. Statistical significance was determined by Student's t test. *, p ≤ 0.05 versus matched control. Error bars, S.E.

Figure 6.

Generation of an inducible the MIR-ZIP mouse model. A, illustration depicting the anti-miR-23a cluster MIR-ZIP (∼160-bp) sequence. The stem loop clone was purchased from System Biosciences (Mountain View, CA). Three anti-miRNA sequences were intervened by a TTCCTGTCAGG loop, and a termination signal (TTTTTT) was added. B, overexpression of the anti-miR-23a cluster significantly reduces miR-23a, miR-27a, and miR-24-2 expression as measured by RT-qPCR. Poly(A) tailing of miRNA was performed using a poly(A) polymerase, and cDNA was synthesized with SuperScript III reverse transcriptase using oligo(dT) adapter primer. RT-qPCR with miRNA-specific and universal primer (Table S5) was used to determine miRNA expression. C, schematic of the targeted recombination, selection, generation of KH2 ES cell clones, and induction of anti-miR-23a cluster. D, Southern blot analysis of hygromycin-resistant flp-in ES cell clones. Genomic DNA from ES cell clones was digested with SpeI and probed with a 3′ internal probe from the Col1A1 locus. All three clones were 100% correctly targeted (6.2-kb WT band and 4.1-kb flp-in band). E, validation of hygromycin-resistant flp-in ES cell clone 12 by Dox (2 μg/ml) induction and expression of anti-miRs as quantified by RT-qPCR (according to Fig. 6A). F, genotyping of F1 miR-23a Cl^ZIP mice using allele-specific PCR primers. A 331 bp band was evident with the WT allele, 331 bp and 551 bp bands were evident with the flp-in heterozygous allele, and a 551 bp band was evident with the flp-in homozygous allele. Error bars, S.E.

Figure 7.

Knockdown of the miR-23a cluster significantly increases cortical and trabecular bone mass. MicroCT of cortical (A) and trabecular section (B) from miR-23a Cl^ZIP heterozygous male mice treated with or without Dox. Heterozygous miR-23a Cl^ZIP (n = 6, −Dox) and age-matched (n = 6) Dox-treated mice of 2 months were fed a Dox diet (625 mg/kg) for 2 weeks. Mice were euthanized, and femurs were excised and stored in ethanol. C, microCT analysis of cortical bone from femurs of heterozygous miR-23a Cl^ZIP female mice treated with (n = 3) and without (n = 6) Dox. E, microCT analysis of trabecular bone from femurs of heterozygous miR-23a Cl^ZIP female mice treated with (n = 3) or without (n = 6) Dox. D, F, and G, data were collected by microCT. Significance was determined using Student's t test: *, p < 0.05. Het, heterozygous miR-23a Cl^ZIP; BV, bone volume (mm³); TV, tissue volume (mm³); Tb. Th, trabecular thickness (μm); Tb. Sp, trabecular space (μm); Tb. No, trabecular number (number/mm); BS, bone surface; Conn-Dens, connectivity density (mg/cm³). Error bars, S.E.

Figure 8.

miR-23a cluster controls the post-transcriptional output of HoxA5 and HoxA11 to alter the chromatin status of the Runx2 and Ocn genes. A, ALP staining of calvarial cells derived from female miR-23a Cl^ZIP mice treated with or without Dox and induced to differentiate for 12 days. B and C, miR-23a and miR-27a (B) along with HoxA5 and HoxA11 (C) expression obtained by RT-qPCR of MIR-ZIP (with/without Dox)-derived calvarial cells induced to differentiate for 12 days. D and F, ChIP assays: in vivo occupancy of HOXA5, HOXA11, H3K18ac, and H3K27ac proteins on the osteoblast-specific promoters in MIR-ZIP calvarial cells (with/without Dox) differentiated for 12 days, using antibodies against HOXA5, HOXA11, H3K18ac, and H3K27ac. DNA from immunoprecipitates was amplified by RT-qPCR with Runx2 (+282/+376) and Ocn (+22/−198) gene-specific promoter primers (Table S5). E and G, Runx2 and Ocn expression measured by RT-qPCR of MIR-ZIP (with/without Dox)-derived calvarial cells induced to differentiate for 12 days. Ac, acetylation. Normal IgG was used as control. Significance was determined using Student′s t test: *, p < 0.05; Error bars, S.E.

Figure 9.

miR-23a cluster–HOXA factor regulatory axis controls bone formation. The model demonstrates that members of the miR-23a cluster directly target HoxA transcription factors, disrupting their recruitment and functional interaction with RUNX2 on osteoblast-specific chromatin and leading to gene inactivation. Additionally, the miR-23a cluster inhibits HOXA-RUNX2–driven acetylation of osteoblast-specific nucleosomal histone H3 at lysine 27 (H3K27) and lysine 18 (H3K18).