Cell type-specific deletion of Dicer in committed osteoprogenitors results in embryonic lethality at embryonic day (E)14.5 [44
]. The mutant embryos display a deformed cartilaginous skeleton, and a lack of bone formation. This study used the rat 2.3-kb Col1a1
promoter driven-Cre construct to delete the floxed Dicer
alleles, and this Cre construct is transiently expressed during embryonic development, likely causing the embryonic lethality. These data reinforce the concept that Dicer-mediated miRNA processing is critical for osteogenesis.
Targeted deletion of Dicer in mature osteoblasts, using an osteocalcin-Cre construct, led initially to delayed perinatal bone formation, with a subsequent increase in postnatal bone acquisition [44
]. In these mice, significant increases in cortical and trabecular bone volume were evident at 4 months of age, and persisted to 8 months. Long bone from mice lacking Dicer expression in mature osteoblasts had increased levels of mRNA for type I collagens, RANKL, and tartrate-resistant acid phosphatase (TRAP). Although data on osteoblast and osteoclast number were not reported, these results suggest increased bone remodeling in the mutant mice. In contrast, another group deleted Dicer in mature osteoblasts using a mouse 2.3-kb Col1
promoter driven-Cre construct, and reported no effect on bone phenotype [45
]. Comparing these two studies, it is likely that differences in the degree of Cre-mediated recombination and cell type specificity had an impact on the resulting mouse phenotypes. Nevertheless, these data suggest that Dicer activity is important for controlling both early and late bone cell differentiation programs.
knockdown of Dicer or Drosha in human MSCs inhibited osteogenic differentiation, confirming similar effects in human cells [46
]. Indeed, since Dicer is important for the processing of many miRNAs, dramatic effects on cell differentiation with Dicer ablation are not unexpected. However, Dicer-independent mechanisms for miRNA maturation have recently been described [47
]. It will be interesting to consider how these mechanisms may impact skeletal phenotype and to identify which miRNAs are regulated by these alternative pathways.
The BMP signaling pathway plays a prominent role in promoting osteoblast differentiation and bone formation [48
]. Several studies have focused on miRNAs modulated by BMP signaling as a means to understand the role of miRNAs in osteoblasts. From these studies, it is clear that a panel of BMP-regulated miRNAs is important for the regulation of osteoblast differentiation. Since some miRNAs can be co-expressed and/or co-regulated, it is possible that families of miRNAs may promote one phenotype at the expense of another. This appears to be the case for miR-133 and miR-135, which are down-regulated during BMP-2-induced osteoblastic differentiation of C2C12 premyogenic cells, but are up-regulated in these cells during myoblastic differentiation [49
]. Over-expression of miR-133 or miR-135 blocked the BMP-mediated induction of osteoblastic markers, such as alkaline phosphatase (ALP), osteocalcin, and HOXA10. Further studies demonstrated that Runx2 is a target for miR-133, and Smad5 is a target for miR-135. Runx2 is a transcription factor essential for osteoblast differentiation, whereas Smad5 is an intracellular Runx2 co-receptor [50
]. The concomitant down-regulation of these two miRNAs by BMP-2 likely plays an important role in the up-regulation of Runx2 and Smad5 during osteogenic differentiation.
Similarly, miR-206 is decreased in response to BMP-2 in C2C12 cells and during differentiation of primary murine osteoblasts [52
]. Over-expression of miR-206 inhibits ALP activity, and Connexin 43 (Cx43) was shown to be a target of miR-206. Cx43 is a gap junction protein necessary for osteoblast differentiation and function [53
]. In vivo
, miR-206 is highly expressed in muscle and perichondrial osteoblasts at E14.5, is moderately expressed in bone collar cells at E16.5, and is undetectable in bone at E18.5. Transgenic mice expressing miR-206 in mature osteoblasts display a low bone mass phenotype, particularly evident in the trabecular compartment. Bone formation rate was decreased in the transgenic mice, suggesting a defect in osteoblast function, whereas the osteoclast surface was unchanged. These data confirm miR-206 as a negative regulator of osteoblast function.
The sequences of mature miR-141 and miR-200a are very similar, and these two miRNAs are decreased in BMP-2-treated MC3T3-E1 osteoblastic cells. miR-141 and -200a appear to be negative regulators of osteoblast differentiation, since their over-expression inhibits this process. Dlx5 (Distal-less homeobox 5) is a master osteogenic transcription factor, and the 3' UTR for Dlx5 has a potential binding site for miR-141 or -200a [54
]. In cells over expressing miR-141 or -200a, Dlx5 protein levels were decreased. Since Dlx5 activates the transcription of osterix, a transcription factor crucial for osteoblast differentiation, it is possible that the BMP-2-induced repression of miR-141 and -200a expression may indirectly augment the transcription of osterix, to promote osteoblastogenesis [55
BMP-2 was also shown to decrease the expression of miR-208 [56
]. Over-expression of miR-208 antagonized the BMP-2-mediated osteoblastic differentiation of MC3T3-E1 cells and primary mouse osteoblasts. The osteogenic transcription factor Ets1 was shown to be a target of miR-208. Ets1 stimulates the transcription of osteopontin and Runx2, and this was inhibited by miR-208 over-expression [56
]. Thus, BMP-2 decreases miR-208 expression, allowing up-regulation of Ets1 and promoting osteoblastic differentiation.
In ST2 mouse mesenchymal cells, miR-125b expression is decreased after a 6-day treatment with BMP-4. miR-125b has a negative effect on osteoblastic differentiation, as over-expression of miR-125b decreased ALP activity and knockdown of miR-125b increased ALP. Potential targets for miR-125b in osteoblasts have not been validated; however, miR-125b may play a role in regulating cell proliferation [58
]. In contrast, miR-210 was found to be increased during BMP-4-induced osteoblastic differentiation of ST2 cells [59
]. miR-210 plays a positive role in osteoblast differentiation. Transcripts for ALP and osterix were increased in ST2 cells over-expressing miR-210. Activin A receptor type 1B (AcvR1b) was identified as a target of miR-210. AcvR1b is essential for activin signaling, which may play a role in repressing osteoblastic differentiation [60
Commitment of mesenchymal cells to a particular lineage depends on intracellular and extracellular cues to guide differentiation. miR-204 is induced during the adipogenic differentiation of murine C3H10T1/2 cells and human MSCs. Concomitantly, Runx2 is decreased. Runx2 is a target for miR-204. Over-expression of miR-204 negatively regulates Runx2 expression, inhibits osteoblast differentiation, and promotes adipocyte differentiation [61
]. Runx2 can also be negatively regulated by some members of the miR-23a/27a/24-2 cluster in murine osteoblasts [62
]. The cluster is up-regulated during differentiation of rat osteoblasts, but only miR-23 appears to directly target the Runx2 3' UTR.
During terminal osteoblastic differentiation of human adipocyte-derived stem cells, miR-26a expression is increased. Further, as miR-26a increases, SMAD1 levels decrease. SMAD1 was shown to be a target for miR-26a, and knockdown of miR-26a increased the expression of osteoblast maker genes, including those encoding COL1A1, osteopontin, and osteocalcin [63
]. SMAD1 is a critical downstream mediator of BMP signaling, and increased expression of SMAD1 augments osteoblastogenesis.
miR-199a and -346 are up-regulated during osteoblastic or adipogenic differentiation of human MSCs [46
]. These miRNAs negatively regulate leukemia inhibitory factor (LIF), which is a marker for human MSC multipotency, and is associated with the uncommitted state of embryonic and adult stem cells [64
]. Indeed, expression of LIF is decreased as stem cell plasticity is decreased. The inhibition of LIF by miR-199a and -346 in human MSCs likely contributes to the induction of differentiation.
The miR-29 family is one of the best characterized miRNA families with regard to osteoblast function, and these miRNAs are important positive regulators of osteoblast differentiation. The expression of miR-29 family members is low during the early, matrix deposition phases of osteoblastogenesis. A low level of miR-29 expression is important at this time since miR-29 targets bone matrix RNAs, including COL1A1
, and osteonectin (ON
(secreted protein acidic and rich in cysteine) [65
]. The co-regulation of osteonectin and fibrillar collagens is not unexpected, since osteonectin plays a critical role in regulating collagen fibril formation [67
Later, the expression of miR-29 is increased as the matrix matures and the osteoblast achieves terminal maturation. This increase in miR-29 likely plays a role in the time-dependent suppression of collagen synthesis, which may be necessary to prevent fibrosis, and to allow for subsequent mineral deposition and proper fibril alignment, both of which are necessary for normal bone turnover [68
]. Over-expression of miR-29 family members promotes osteoblastic differentiation, whereas knockdown of miR-29 decreases differentiation markers. It is interesting to note that other validated targets for miR-29, important in osteoblast function, include several proteins that are inhibitors of osteoblast differentiation, such as histone deacetylase (HDAC)4, TGFβ3, AcvR2A, CTNNBIP1 (catenin beta interacting protein I), and DUSP2 (dual specific phosphatase 2) [70
It was recently reported that canonical Wnt signaling, a critical positive regulator of osteoblast differentiation, rapidly induces the expression of miR-29a and -29c [65
]. Since activation of canonical Wnt signaling tends to increase during osteoblast differentiation, it is possible that Wnt signaling plays a role in the increased expression of miR-29 observed during osteoblast differentiation [71
]. Further, miR-29a modulates canonical Wnt signaling in a positive feedback loop, to promote human osteoblast differentiation [72
]. In human osteoblasts, transcription of miR-29a is induced by canonical Wnt signaling, and two T-cell factor/lymphoid enhancer factor (TCF/LEF) binding sites in the miR-29a promoter region are necessary for this induction. miR-29a was also found to target three inhibitors of Wnt signaling, Dkk1 (dikkopf-1), Kremen2 (kringle domain-containing transmembrane protein), and sFRP2 (secreted frizzled related protein 2). Thus, the induction of miR-29a transcription, in response to canonical Wnt signaling, results in decreased Dkk1, Kremen2, and sFRP2 levels, which potentiates Wnt signaling. This loop provides an additional mechanism by which miR-29 can promote human osteoblast differentiation, and a mechanism for fine tuning the expression of specific components in the Wnt signaling pathway [71
A similar study demonstrated that miR-27, which is increased during osteoblast differentiation, positively regulates this process by targeting adenomatous polyposis coli (APC) [73
]. In the absence of canonical Wnt signaling, APC is part of an inhibitor complex that binds β-catenin, preventing β-catenin translocation to the nucleus. This study suggests miR-27 promotes osteoblast differentiation by down-regulating APC, thus allowing for Wnt signaling. Indeed, over-expression of miR-27 increased ALP and osteocalcin, whereas its inhibition decreased these markers.
In the first study assessing miRNAs and osteoporosis in humans, an inactivating mutation in miR-2861 was found to be associated with decreased serum markers of osteoblast activity in a kindred of osteoporosis patients [74
]. Interestingly, miR-2861 appears to be a bone-enriched miRNA. It is highly expressed in osteoblasts, with a lower level of expression in liver. Knockdown of miR-2861 in mice resulted in decreased bone volume, bone formation rate and osteoblast surface. Conversely, increased expression of this miRNA in vitro
augmented osteoblast differentiation. miR-2861 was shown to target the protein coding region of HDAC5. HDAC5 mediates deacetylation of Runx2 and deacetylated Runx2 undergoes Smurf1-mediated degradation [75
]. Therefore, HDAC5 inhibition by miR-2861 likely increases the abundance of acetylated Runx2, allowing for augmentation of osteoblast differentiation. This key study demonstrates the importance of miRNA genes and their contribution to human disease phenotypes.
Transcribed in the same pri-miRNA as miR-2861 is another novel miRNA, miR-3960. These miRNAs likely work in concert, promoting osteoblastic differentiation by indirectly up-regulating Runx2 [76
]. When miR-3960 is over-expressed, ALP, osteocalcin, and Runx2 are increased. Conversely, these differentiation markers are decreased when miR-3960 is inhibited. miR-3960 was found to directly target Hoxa2, a negative regulator of Runx2. Thus, the down-regulation of Hoxa2 by miR-3960 results in increased Runx2 expression. Further, Runx2 can bind the miR-3960 promoter, suggesting regulation of this miRNA cluster.
Overall, it is clear that miRNAs play an important role in osteoblast function and differentiation (summarized in Table ). Future work, defining the function of particular miRNAs in osteoblast commitment and differentiation, could allow for the assembly of a panel of miRNAs that could be used as differentiation markers, similar to the panel of markers presently used to annotate the progression of differentiation, such as Runx2, ALP, osteocalcin, and osterix.
Summary of microRNAs, their targets, expression, and effects on osteoblast differentiation