A program of microRNAs controls osteogenic lineage progression by targeting transcription factor Runx2

Abstract

Lineage progression in osteoblasts and chondrocytes is stringently controlled by the cell-fate-determining transcription factor Runx2. In this study, we directly addressed whether microRNAs (miRNAs) can control the osteogenic activity of Runx2 and affect osteoblast maturation. A panel of 11 Runx2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR-218, and miR-338) is expressed in a lineage-related pattern in mesenchymal cell types. During both osteogenic and chondrogenic differentiation, these miRNAs, in general, are inversely expressed relative to Runx2. Based on 3′UTR luciferase reporter, immunoblot, and mRNA stability assays, each miRNA directly attenuates Runx2 protein accumulation. Runx2-targeting miRNAs differentially inhibit Runx2 protein expression in osteoblasts and chondrocytes and display different efficacies. Thus, cellular context contributes to miRNA-mediated regulation of Runx2. All Runx2-targeting miRNAs (except miR-218) significantly impede osteoblast differentiation, and their effects can be reversed by the corresponding anti-miRNAs. These findings demonstrate that osteoblastogenesis is limited by an elaborate network of functionally tested miRNAs that directly target the osteogenic master regulator Runx2.osteogenesis | chondrogenesis | post-transcriptional regulation C ell-fate determination and subsequent lineage progression of phenotype-committed cells are mediated by master regulatory transcription factors that integrate multiple cell-signaling inputs and generate epigenetic changes in chromatin to modulate gene expression. Transcription factors are components of positive and negative feedback loops that initiate or maintain the acquisition of distinct biological states. Epigenomic mechanisms, including attenuation of mRNA and protein expression by small noncoding microRNAs (miRNAs) (1), permit effective control of gene expression beyond genomic interactions between transcription factors and their cognate elements in gene promoters. The biological potency of miRNAs, which are generated by the RNA processing enzyme Dicer, is based on their ability to control mRNA accumulation and/or protein synthesis through specific interactions with the 3′UTRs of target genes (1). Gene regulatory networks involving transcription factors and miRNAs may mutually reinforce cell fates and support phenotypic maturation of lineage-committed cells.Osteogenic differentiation provides an effective cell model in which to define both epigenetic and epigenomic mechanisms required for cell-fate determination and phenotypic differentiation. Differentiation of multipotent mesenchymal stem cells into the osteoblast lineage and maturation of osteoprogenitors are controlled by multiple extracellular ligands [e.g., BMPs, WNTs, and FGFs] (2-4) that direct the activities of key transcription factors, including Runx2, Osterix, and different classes of homeodomain proteins (5-8). Runx2 is a critical regulator of the osteogenic lineage, and its epigenetic functions modulate ...

miR-34s inhibit osteoblast proliferation and differentiation in the mouse by targeting SATB2

“…Accordingly, it has been suggested that miRNAs may be involved in osteoblast proliferation and/or differentiation (Hassan et al, 2010;Zhang et al, 2011). However, these observations were derived from gain-of-function experiments performed in cell culture.…”

MiR‐455‐3p regulates early chondrogenic differentiation via inhibiting Runx2

“…The Runx2-dependent expression of sphingomyelin phosphodiesterase 3 (Smpd3) was increased by Bone morphogenic protein 2 (BMP-2) stimulation, and Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes [33]. Furthermore, Runx2 regulated the differentiation of MSCs as the target of miRNAs [22,23], such as miR-199a ⁄ , a BMP2-responsive miRNA that regulates chondrogenesis via the direct targeting of Smad1 in murine MSCs [35]. In this study, we showed that Runx2 was expressed more steadily before 21 d, then quickly increased after 21 d, which indicated the involvement of Runx2 in hypertrophic differentiation.…”

“…Runx2 also mediates chondrogenesis and ossification as a co-regulator with Smad, TGF-b, and CCAAT/enhancer binding protein beta (C/EBPb) [5,21]. Furthermore, Runx2 regulates the differentiation of MSCs as the target of miRNAs [22,23]. Recently, miR-455-3p was found to be expressed in the ATDC5 cell model of chondrogenesis and in adult articular cartilage, as shown in human MSCs, and it regulated TGFb signaling by suppressing the Smad2/3 pathway, which contributes to cartilage destruction [5].…”

miRNA Control of Tissue Repair and Regeneration

“…104 Addition of miR-133 prevents such unwanted ossification. 105 Furthermore, addition of miR-675 and miR-221 promotes expression of collagen II and proliferation of mature chondrocytes. In bone engineering, seeding MSCs with miR-148b mimics and miR-489 inhibitor to the osteoinductive scaffold results in increased matrix deposition and calcification.…”