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. 2010 Nov 16;107(46):19879-84.
doi: 10.1073/pnas.1007698107. Epub 2010 Oct 27.

A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program

Mohammad Q Hassan  1 , Jonathan A R Gordon, Marcio M Beloti, Carlo M Croce, Andre J van Wijnen, Janet L Stein, Gary S Stein, Jane B Lian
Affiliations

A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program

Mohammad Q Hassan et al. Proc Natl Acad Sci U S A. .

Abstract

Induced osteogenesis includes a program of microRNAs (miRs) to repress the translation of genes that act as inhibitors of bone formation. How expression of bone-related miRs is regulated remains a compelling question. Here we report that Runx2, a transcription factor essential for osteoblastogenesis, negatively regulates expression of the miR cluster 23a∼27a∼24-2. Overexpression, reporter, and chromatin immunoprecipitation assays established the presence of a functional Runx binding element that represses expression of these miRs. Consistent with this finding, exogenous expression of each of the miRs suppressed osteoblast differentiation, whereas antagomirs increased bone marker expression. The biological significance of Runx2 repression of this miR cluster is that each miR directly targets the 3' UTR of SATB2, which is known to synergize with Runx2 to facilitate bone formation. The findings suggest Runx2-negative regulation of multiple miRs by a feed-forward mechanism to cause derepression of SATB2 to promote differentiation. We find also that miR-23a represses Runx2 in the terminally differentiated osteocyte, representing a feedback mechanism to attenuate osteoblast maturation. We provide direct evidence for an interdependent relationship among transcriptional inhibition of the miR cluster by Runx2, translational repression of Runx2 and of SATB2 by the cluster miRs during progression of osteoblast differentiation. Furthermore, miR cluster gain of function (i.e., inhibition of osteogenesis) is rescued by the exogenous expression of SATB2. Taken together, we have established a regulatory network with a central role for the miR cluster 23a∼27a∼24-2 in both progression and maintenance of the osteocyte phenotype.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
A functional Runx2 DNA binding site regulates expression of the miR-23a∼27a∼24-2 cluster and miR-23a regulates Runx2. (A) Stem loop structure of the transcript for the murine miR cluster in chromosome 8. (B) Representation of the rat −0.639-kb miR-23a∼27a∼24-2 cluster promoter fragment. Transcription factor analysis of the proximal promoter showing the Runx binding site by TRANSFAC, TESS program (TGTGGT, −185 to −190 for rat), the INR motif (initiator, CCCCACCTCC), and the CT motif (CTCT…) sequence at −56 to −34 (23). (C) EMSA of nuclear proteins from MC3T3-E1 cells using an oligonucleotide WT and mutant probe (Table S1) for the Runx2 site in the miR promoter. Control binding (C, lane 1), self-competitor for WT (miR self, lane 2), Runx2 consensus WT (Runx2, lane 3) or mutant (Runx2 mt, lane 4), and antibody supershift (lane 5) are shown. Open arrow, Runx2 complex; solid arrow, supershifted Runx2 protein–DNA complex; star, NS band. (D) Functional activity of the Runx2 site in the −0.639 kb human miR cluster promoter and its deletion and mutant constructs with luciferase reporter. The reporter constructs were cotransfected with control vector (gray bars) or Runx2 expression construct (solid black bars) in MC3T3-E1 cells (Materials and Methods describes quantification). (E) Northern blot for expression of mature miR-23a, -27a, and -24-2 during primary ROB cell differentiation (days 4–20). U6 was used for control. (F) Expression of the miR cluster by qPCR and Runx2 protein (Inset; Western). (A) Total RNA from ROB cells was assayed for each miR (precursor and mature) normalized to U6 expression as indicated. Runx2 protein detected by a mouse monoclonal antibody (MBL International). Actin was used for control. (G) Upper: Diagram of the 3′ UTR of Runx2 mRNA illustrating miR-23a binding site and a mutation (mT). Lower: Western analysis of Runx2 protein in day 20 ROB cells transduced on day 4 with lentiviral overexpression of miR-23a and -27a. Actin was used as loading control. (H) Runx2 3′ UTR LUC assay demonstrating miR-23a regulation using MC3T3-E1 cells (Materials and Methods).
Fig. 2.
Fig. 2.
Mechanism of Runx2 regulation of each miR within a cellular context. (A) In vivo occupancy of Runx2 proteins on the miR promoter during ROB cell differentiation. Upper: Arrow indicates position of primers (Table S1) used in ChIP. Lower: ChIP was performed at the indicated days of ROB differentiation using H3K9me3, H4 Penta acetyl, Runx2, and control IgG antibodies. Inset, Top: Runx2 protein expression during days 4, 12, and 20 of ROB cell differentiation. (B) Expression (real-time PCR analysis) of the precursor miR cluster members normalized by U6 at the indicated time points. (C) Overexpression for 24 h (Left, qPCR) and knockdown by siRNA for 72 h (Right; Western) of Runx2 in MC3T3-E1 cells to detect Runx2 levels. (D) Consequences of overexpression of Runx2 for 24 h on precursor miR cluster RNA by qRT-PCR. (E) Precursor miRNA analysis of miR-23a, -27a, and -24-2 upon Runx2 knockdown. (F) Expression analysis of precursors of miR-23a∼27a∼24-2 cluster members by real-time qPCR in genetically deleted Runx2−/− mouse calvarial osteoblast cells.
Fig. 3.
Fig. 3.
Expression of miRNAs 23a and 27a inhibit primary osteoblast differentiation through targeting SATB2. (A) Northern blot showing levels of lentiviral mediated overexpressed miR-23a and -27a in primary ROB during differentiation. (B) Histochemical staining: primary ROB cells were infected with control, miR-23a, and miR-27a lentivirus at day 4 and cultured in differentiation medium for 21 d. ALP activity and von Kossa (VK) for mineral deposition at days 12 and 21 is shown. (C) mRNA expression profile of bone marker genes as indicated on days 4, 12, and 21. (D) Each miR in the cluster, as indicated, down-regulates SATB2 in LUC reporter assay, but not mutant miRs (Results). Location of miR sites are in Fig. S4. (E) Overexpression of miR-23a and -27a decrease SATB2 and Runx2 protein: Western blot after 72 h in MC3T3-E1 cells. Actin used as loading control. (F) Stability of SATB2 mRNA upon overexpression of miR-23a and -27a for 72 h (quantitative RT-PCR), normalized to U6. (G) Biological rescue of miR inhibition of osteoblast differentiation by SATAB2 in MC3T3-E1, miR cluster (miR-Cl), SATB2, or transfected together for 72 h. Total RNA was analyzed for Runx2 and ALP expression normalized to GAPDH.
Fig. 4.
Fig. 4.
Feed-forward control by Runx2 and feedback regulation by miR-23a maintains the physiology of bone formation. (A) In vivo identification of miR-23a, -27a, and target SATB2 by RNP-IP. The miR-23a and 27a-SATB2 mRNA that is complexed with the AGO2 (Materials and Methods) was analyzed for the presence of SATB2 mRNA association. The 3′ UTR of SATB2 mRNA was amplified with primers specific to the binding sites (Table S1). The miR-23a and -27a seed lanes shows SATB2 3′ UTR fragment with the miR-23a and -27a binding. No RT, normal IgG and 2% input were used as negative and positive controls, respectively. (B) Anti-miR (miRZIP) construct and knockdown of miR-23a, -27a, and -24-2. H1 promoter drives the synthesis of all three miRs. (C) miRZIP specific for miR-23a, -27a, and -24-2 knockdown increases SATB2 and Runx2 protein shown by Western blot with antibodies described in Materials and Methods and actin protein as loading control. (D) Biological effect of miRZIP expression increases expression of osteoblast differentiation markers normalized to GAPDH (real-time qPCR). (E) miRZIP does not affect SATB2 mRNA. (F) Coordinated expression of Runx2 and SATB2 in primary ROB cells at indicated days of differentiation by Western blot. Actin was used as loading control. (G) Illustrated is the reciprocal relationship of SATB2 and Runx2 protein with miR-23a∼27a∼24-2 expression (qPCR average of Fig. 1F) in ROB cell differentiation. In the same experiment, Runx2 and SATB2 protein (F) were densitometrically quantified and plotted. (H) Summary of the central role of miR cluster regulation of osteoblastogenesis. Left: Model demonstrates that Runx2 controls the SATB2 functions by down-regulating miR cluster expression in a feed-forward mechanism at day 12, whereas miR-23a down-regulation of Runx2 at day 20 in a feedback loop attenuates Runx2 and target genes for terminal osteoblast differentiation.
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References

    1. Jensen ED, Gopalakrishnan R, Westendorf JJ. Regulation of gene expression in osteoblasts. Biofactors. 2010;36:25–32. - PMC - PubMed
    1. Soltanoff CS, Yang S, Chen W, Li YP. Signaling networks that control the lineage commitment and differentiation of bone cells. Crit Rev Eukaryot Gene Expr. 2009;19:1–46. - PMC - PubMed
    1. Zaidi SK, et al. Mitotic bookmarking of genes: A novel dimension to epigenetic control. Nat Rev Genet. 2010;11:583–589. - PMC - PubMed
    1. Li Z, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci USA. 2008;105:13906–13911. - PMC - PubMed
    1. Li Z, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem. 2009;284:15676–15684. - PMC - PubMed

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