A microRNA signature for a BMP2-induced osteoblast lineage commitment program

Abstract

Bone morphogenetic proteins (BMPs) are potent morphogens that activate transcriptional programs for lineage determination. How BMP induction of a phenotype is coordinated with microRNAs (miRNAs) that inhibit biological pathways to control cell differentiation, remains unknown. Here, we show by profiling miRNAs during BMP2 induced osteogenesis of C2C12 mesenchymal cells, that 22 of 25 miRNAs which significantly changed in response to BMP2 are down-regulated. These miRNAs are each predicted to target components of multiple osteogenic pathways. We characterize two representative miRNAs and show that miR-133 directly targets Runx2, an early BMP response gene essential for bone formation, and miR-135 targets Smad5, a key transducer of the BMP2 osteogenic signal, controlled through their 3UTR sequences. Both miRNAs functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 pathways that synergistically contribute to bone formation. Although miR-133 is known to promote MEF-2-dependent myogenesis, we have identified a second complementary function to inhibit Runx2-mediated osteogenesis. Our key finding is that BMP2 controls bone cell determination by inducing miRNAs that target muscle genes but mainly by down-regulating multiple miRNAs that constitute an osteogenic program, thereby releasing from inhibition pathway components required for cell lineage commitment. Thus, our studies establish a mechanism for BMP morphogens to selectively induce a tissuespecific phenotype and suppress alternative lineages.S keletal development requires stringent control of a program for gene activation and suppression in response to physiological cues. The potent osteogenic bone morphogenetic proteins (BMPs) (BMP2/7) and canonical Wnt signaling activate skeletal-related genes for formation of cartilage and bone (1-3). BMPs regulate the differentiation of multiple cell types (4-6) but will also drive commitment of mesenchymal stem cells into a specific lineage. BMP2, for example, supports osteoblastogenesis (7, 8). There has been a principal focus on identifying the mechanisms by which only one cell phenotype is activated by BMP2. BMP2 intracellular receptor Smads, particularly Smad5, activates osteoblast-essential genes, including the transcription factors required for in vivo bone formation Runx2 (Cbfa1/ AML3) and Osterix (9-11). The contribution of global mechanisms for suppression of alternative lineages is equally important for understanding tissue development and diseases.MicroRNAs (miRNAs) have emerged as key negative regulators of diverse biological and pathological processes, including developmental timing, organogenesis, apoptosis, cell proliferation, and differentiation (reviewed in ref. 12) and in the control of tumorigenesis (13,14). miRNAs are small (22-nt) endogenous noncoding RNAs that anneal to the 3Ј UTR of target mRNAs to mediate inhibition of translation and lower protein levels. It remains to be established how specific miRNAs contribute to regulate the onset of a tissue-specific phenotype i...

miR-764-5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression

“…(27) miR-133 and miR-135 functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 activities that synergistically contribute to bone formation. (28) miR-204/ 211 acts as negative regulators of osteoblast differentiation and subsequent mineralization in mesenchymal progenitor cells and bone marrow stromal cells through a negative regulation of Runx2 protein translation. (15) All these miRNAs directly target osteoblast differentiation factors.…”

c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104

“…(13,(30)(31)(32) Cells were stimulated with BMP2 in serumfree medium, and the involvement of JNK1 in regulating osteoblastic differentiation was investigated. We found that under such conditions, BMP2 induced ALP activity and JNK inhibition increased it similar to the results in serum-containing conditions.…”

MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

“…In human adipose tissue-derived MSCs (hADSCs), miR-26a has been reported to repress the translation of the osteogenic marker, SMAD1 (15), whereas overexpression of miR-196a regulates HOXC8, enhances osteogenic differentiation, and decreases hADSC proliferation (16). miR-125b has been shown to inhibit osteoblast differentiation in the mouse MSC line ST2 (17), whereas miR-133 and -135 directly target Runx2 and Smad5, respectively, and inhibit differentiation of osteoprogenitors of C2C12 mesenchymal cells (18). Moreover, miR-141 and -200a were recently found to be involved in preosteoblast differentiation through regulation of their common target Dlx5 (19), whereas miR-206 inhibits osteogenesis in vitro and in vivo by targeting Cx43 (20).…”