Abstract
Micro-RNAs (miRNAs) are important in regulating cell fate determination because many of their target mRNA transcripts are engaged in cell proliferation, differentiation, and apoptosis. DGCR8, Dicer, and Ago2 are essential factors for miRNA homeostasis. Here we show that these three factors have critical roles in osteoclast differentiation and function. Gene silencing of DGCR8, Dicer, or Ago2 by small interfering RNA revealed global inhibition of osteoclast transcription factor expression and function, decreased osteoclastogenesis, and decreased bone resorption in vitro. In vivo, CD11b(+)-cre/Dicer-null mice had mild osteopetrosis caused by decreased osteoclast number and bone resorption. These results suggest that miRNAs play important roles in differentiation and function of osteoclasts in vitro and in vivo. We found a novel mechanism mediating these results in which PU.1, miRNA-223, NFI-A, and the macrophage colony-stimulating factor receptor (M-CSFR) are closely linked through a positive feedback loop. PU.1 stimulates miRNA-223 expression, and this up-regulation is implicated in stimulating differentiation and function of osteoclasts through negative regulation of NFI-A levels. Down-regulation of NFI-A levels is important for expression of the M-CSFR, which is critical for osteoclast differentiation and function. NFI-A overexpression decreased osteoclast formation and function with down-regulation of M-CSFR levels. Forced expression of the M-CSFR in M-CSF-dependent bone marrow macrophages from Dicer-deficient mice rescued osteoclast differentiation with up-regulation of PU.1 levels. Our studies provide new molecular mechanisms controlling osteoclast differentiation and function by the miRNA system and specifically by miRNA-223, which regulates NFI-A and the M-CSFR levels.
Highlights
NFI-A expression levels were not altered (Fig. 7J). These results demonstrate that the action of miRNA-223 to suppress NFI-A levels in osteoclast precursors is a prerequisite for induction of macrophage colony-stimulating factor receptor (M-CSFR) expression and osteoclastogenesis
Our data are in agreement with a recent report using Ago2Ϫ/Ϫ hematopoietic bone marrow cells concluding that the Slicer endonuclease activity is dispensable for hematopoiesis and that Ago2 is an important regulator of miRNA homeostasis [31]
PU.1, MITF, c-Fos, and NFATc1 are crucial transcription factors in osteoclastogenesis [1, 2], and these transcription factors bind to the tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase 9 (MMP9), cathepsin K (Ctsk), calcitonin receptor (CTR), or integrin 3 promoters for expression of osteoclast-specific markers in activated osteoclasts [33,34,35,36,37,38,39,40,41]
Summary
M-CSF, macrophage colony-stimulating factor; miRNA, micro-RNA; pri-miRNA, primary micro-RNA; pre-miRNA, precursor micro-RNA; DGCR8, DiGeorge syndrome critical region gene 8; RISC, RNAinduced silencing complex; Ago, Argonaute; siRNA, small interfering RNA; RANKL, receptor activator of NFB ligand; MITF, microphthalmia-associated transcription factor; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; TRAP, tartrate-resistant acid phosphatase; MMP9, matrix metalloproteinase 9; Ctsk, cathepsin K; CTR, calcitonin receptor; NFI-A, nuclear factor I-A; M-CSFR, M-CSF receptor; ␣MEM, ␣-minimum essential medium; FBS, fetal bovine serum; RT, reverse transcription; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Pri-miRNAs are processed by the double-stranded RNA-binding protein DiGeorge syndrome critical region gene 8 (DGCR8) and the nuclear RNase III enzyme Drosha into stem-loopstructured miRNA precursors (pre-miRNAs) [16]. Expression of miRNA-26a is up-regulated in late osteoblast differentiation of human adipose tissuederived stem cells, and miRNA-26a modulates this process by targeting the SMAD1 transcription factor [25]. To study the global functions of miRNAs in osteoclast differentiation and bone resorbing activity, we employed a retroviral system for delivery of DGCR8, Dicer, or Ago small interfering RNA into osteoclast precursors and CD11bϩ-cre/ Dicer-null mice and found that the miRNA pathway for generation of miRNAs including miRNA-223 is critical for osteoclast differentiation and function, and miRNAs may be implicated in regulation of the bone remodeling cycle by osteoclasts. 5Ј-CTC CTG CCG ACG ACC CAT T-3Ј 5Ј-GCA TGC TAT CAC CAC ATA T-3Ј 5Ј-GCG AAG TGC AAG TTT CAA T-3Ј
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
