Abstract
MicroRNAs (miRNAs) have become integral nodes of post-transcriptional control of genes that confer cellular identity and regulate differentiation. Cell-specific signaling and transcriptional regulation in skeletal biology are extremely dynamic processes that are highly reliant on dose-dependent responses. As such, skeletal cell-determining genes are ideal targets for quantitative regulation by miRNAs. So far, large amounts of evidence have revealed a characteristic temporal miRNA signature in skeletal cell differentiation and confirmed the essential roles that numerous miRNAs play in bone development and homeostasis. In addition, microarray expression data have provided evidence for their role in several skeletal pathologies. Mouse models in which their expression is altered have provided evidence of causal links between miRNAs and bone abnormalities. Thus, a detailed understanding of the function of miRNAs and their tight relationship with bone diseases would constitute a powerful tool for early diagnosis and future therapeutic approaches.
Introduction
Skeletal development is a process that involves a complex sequence of events, which are regulated by a wide range of signaling pathways (Karsenty 2008). Yet, it mainly involves only three specific types of cells: chondrocytes in cartilage and osteoblasts and osteoclasts in bone. In recent years, considerable efforts have been devoted to understanding the mechanisms that mediate the transition from mesenchymal stem cells (MSCs) to osteoblast and chondroblast lineages. It is well known that osteoprogenitor maturation is controlled by several extracellular signals including bone morphogenetic proteins (BMPs), hedgehogs, WNTs, and fibroblast growth factors, the actions of which lead to the expression of chondroblast- or osteoblast-specific genes (Karsenty 2008). Osteoclasts arise from hematopoietic cells and are essential for bone resorption during skeletal development, homeostasis, and regeneration (Duong & Rodan 2001, Horowitz et al. 2001). Furthermore, there is a strong crosstalk between them: osteoblasts are involved in the regulation of osteoclast differentiation through the receptor activator of nuclear factor κB ligand (RANKL)–RANK pathway, essential for a satisfactory balance between bone deposition and bone resorption throughout life (Duong & Rodan 2001, Karsenty & Wagner 2002).
Recently, numerous studies have shown that microRNAs (miRNAs) are important post-transcriptional regulators in virtually all biological processes (Hobert 2008). The miRNA field has advanced so rapidly that it has become an integral component of the way we think gene expression is regulated in cartilage and bone development. Cell-specific signaling and transcriptional regulation in skeletal biology are extremely dynamic processes that are highly reliant on dose-dependent responses. As such, they are ideal targets for quantitative regulation by miRNAs. Moreover, the multigene regulatory capacity of miRNAs enables them to cooperatively balance the final precursor cell fate. Thus, different miRNAs can act as either positive or negative determinants within multiple pathways involved in skeletal development processes. The expression of miRNAs is finely orchestrated, being upregulated and downregulated to control the differentiation stage of each bone cell, leading to a characteristic temporal miRNA signature in bone development and homeostasis. Nevertheless, despite all the information available about miRNAs and skeletogenesis, few in vivo studies have been conducted to validate each miRNA and it remains unclear how in vivo changes in specific miRNAs compromise normal bone development. The purpose of this review is to summarize the current knowledge of miRNA function in skeletal cell lineages and to discuss the main miRNA-related skeletal disorders and the therapeutic perspectives that they provide.
miRNAs: biogenesis and function
miRNAs are short, single-strand, noncoding RNAs approximately 20–25 nucleotides long that have emerged as novel tools capable of post-transcriptionally modifying the expression of mature mRNAs and proteins (Bartel 2004, Mattick & Makunin 2006, Hobert 2008; Fig. 1).
The transcription of miRNAs is mostly mediated by RNA polymerase II, but it can also be mediated by RNA polymerase III (Borchert et al. 2006). Sequences encoding miRNAs are found around the genome as separate transcriptional units, although a minority of these sequences are located within the introns of coding genes (generally as clustered miRNAs; Kapinas & Delany 2011). miRNAs are first transcribed as long primary units called pri-miRNAs, which contain characteristic secondary loop structures (Starega-Roslan et al. 2011). Various miRNAs can be co-transcribed in a single pri-miRNA, possibly inducing additional effects on a single pathway or gene or allowing crosstalk between different pathways (He et al. 2010). The characteristic hairpin of pri-miRNAs helps the microprocessor complex containing Drosha (RNAse III) and some cofactors, including the double-strand RNA-binding protein DGCR8 (DiGeorge syndrome critical region gene), to recognize them from among similar structures present in the nucleus (Han et al. 2006, Seitz & Zamore 2006). As a result, a 60–80-nucleotide double-strand miRNA precursor (pre-miRNA) is generated. Pre-miRNAs maintain their stem–loop configuration and have a two-nucleotide extension at their 3′-end. However, some precursors arising from short introns (mirtrons) are capable of bypassing Drosha cleavage and are exported (as regular pre-miRNAs) by exportin 5 to the cytoplasm, where they continue canonical miRNA processing (Lund et al. 2004). miRNA precursors are cleaved by a second endonuclease (Dicer), resulting in a double strand of about 21–24 nucleotides. Thanks to argonaute 2 (AGO2), a protein present in the RNA-induced silencing complex (RISC), one of the strands is recruited and guides the complex to its target, whereas the other strand (miRNA*) is degraded.
The 5′-end of mature miRNAs contains the seed region (nucleotide positions 2–7 or 2–8), which has the capacity to identify the complementary bases of the 3′-UTR of the target mRNAs and trigger their cleavage and degradation (Guo et al. 2010). Nevertheless, there is usually an imperfect complementarity, and the final effect of miRNA activity is a decrease in protein expression due to translational suppression. Interestingly, miRNAs have also been found to target the 5′-UTRs of mRNAs (Lytle et al. 2007, Lee et al. 2009) and to induce target translation (Vasudevan et al. 2007).
miRNAs and skeletal cell specification
It is well known that miRNAs play an important role in chondrogenic and osteogenic differentiation during cartilage and bone formation (Hobert 2008, Kapinas & Delany 2011). The first in vivo approach used to study this was implemented through the conditional ablation of the Dicer (Dicer1) gene under the control of the Col2a1 promoter (Kobayashi et al. 2008). Mutant mice were found to display severe skeletal growth defects due to a reduction in the number of proliferating chondrocytes, leading to premature death. Evident skeletal phenotypes were similarly observed in mice with Dicer deficiency in osteoprogenitor cells (using Cre under the control of the 2.3 kb fragment of the Col1a1 promoter). The ablation of Dicer in progenitors prevents their differentiation and compromises fetal survival (Gaur et al. 2010). In addition, Mizoguchi et al. (2010) have demonstrated that osteoclast Dicer is also crucial for normal osteoclast resorption and osteoblast activity. Osteoclast-specific Dicer knockout mice were generated by crossing Cathepsin K-cre mice with Dicer flox mice. These mice were found to exhibit higher bone mass and a decrease in osteoclast surface and number. Additionally, the expression of not only osteoclast-related genes but also osteoblast-related ones (Col1a1, Bglap, and runt-related transcription factor 2 (Runx2)) was found to be downregulated (Mizoguchi et al. 2010). These data indicate that miRNAs are important not only during bone development but also for bone homeostasis throughout life.
miRNAs and osteoblast differentiation
Lineage commitment from MSCs to osteoblasts has been well characterized. During embryogenesis, bone development occurs by either endochondral or intramembranous ossification. In both mechanisms, osteoblasts and chondrocytes arise from mesenchymal cell condensations, and the factors that contribute to the transcriptional control of these differentiation processes have been widely studied (Nakashima & de Crombrugghe 2003, Karsenty 2008). Osterix (Osx), Runx2, distal-less homeodomain-containing family 5 (Dlx5), Msx2, and Atf4, among others, have been identified as essential transcription factors for osteoblast differentiation (Nakashima & de Crombrugghe 2003, Karsenty 2008; Fig. 2).
From a common osteochondroprogenitor, the osteoblast differentiation process encompasses different well-characterized stages. The above-mentioned transcription factors (Osx, Runx2, and Atf4) are indispensable for inducing progression to the osteocyte fate, the most mature form of differentiation. Furthermore, each step of osteoblast progression can be clearly recognized by a cohort of molecules that are differentially expressed. Runx2 plays an essential role in the first step of differentiation into pre-osteoblasts. Pre-osteoblast-specific markers include alkaline phosphatase and low levels of type 1 collagen. Later, they require Osx to reach the mature osteoblast stage and to be able to synthesize extracellular matrix (ECM) proteins. Functional osteoblasts additionally express osteocalcin and bone sialoprotein markers and they are responsible for the future mineralized bone matrix. Although the majority of the cells of the osteoblast population undergo apoptosis, a small fraction will differentiate into osteocytes, the main bone population. Osteocytes are matrix-embedded cells and are important mechanosensors controlling bone formation. Recent studies have also indicated osteocytes to be the main source of RANKL (TNFSF11) and therefore closely related to bone resorption.
Runx2 is the first to be expressed in mesenchymal cell condensations, and from the perspective of molecular biology, it is one of the key transcription factors involved in osteoblast differentiation, together with Osx (Karsenty et al. 1999, Ducy 2000). Runx2 is expressed as early as day E10.5 and is necessary and sufficient to identify cells as osteochondroprogenitors, as Runx2-null mice are unable to produce mature osteoblasts (Ducy et al. 1997, Komori et al. 1997, Nakashima & de Crombrugghe 2003). From this stage to chondroblast commitment, Runx2 levels decrease until it almost disappears at day E16.5, whereas during osteoblast differentiation Runx2 levels remain stable and induce osteocalcin (Bglap) expression at around day E15.5. Thus, Runx2-targeting miRNAs simultaneously modulate osteogenesis and chondrogenesis.
The expression of Runx2 is regulated by several signaling pathways, including vitamin D3 (1,25(OH)2D3), transforming growth factor β (TGFβ)/BMP2, and Wnt, among others, and it regulates the expression of numerous osteoblastic genes such as Osx (Sp7), Alpl (alkaline phosphatase), Col1a1, Spp1 (osteopontin), Ibsp (bone sialoprotein), and Bglap (osteocalcin). Runx2 mRNA has a very long 3′-UTR, which probably contains multiple regulatory elements (Huang et al. 2010), and it is therefore not surprising that several examples of post-transcriptional Runx2 mRNA regulation through miRNAs have been described.
miR-204/-211 specifically binds to the 3′-UTR of Runx2 and inhibits osteoblast differentiation by promoting adipocyte commitment from mesenchymal progenitors (C3H10T1/2, ST2, and hMSCs; Huang et al. 2010). Furthermore, as Runx2 has the capacity to regulate the expression of Bglap from day E15.5 onwards (Ducy & Karsenty 1995), miR-204 accumulation leads to the repression of Bglap expression (Huang et al. 2010).
miR-133 also inhibits Runx2 translation, and its expression is downregulated by BMPs in C2C12 cells (Li et al. 2008). Studies carried out by independent groups have reported controversial results on the function of miR-31 during osteoblast commitment of human MSCs. Gao et al. (2011) have described miR-31 to be a downregulated miRNA during osteoblast differentiation in vitro, indicating that Runx2 is one of its physiological targets. However, miR-31 was later identified as an upregulated miRNA in a similar study of hMSC differentiation and osterix was confirmed to be one of its targets, indicating a regulatory network (Baglio et al. 2013). Other in vitro studies have elucidated a regulatory loop involving miR-31, Runx2, and Satb2 (special AT-rich sequence-binding protein 2): downregulation of miR-31 expression by Runx2 in differentiating bone marrow MSCs (BMMSCs) facilitates osteogenic commitment due to an increase in SATB2 protein expression (Deng et al. 2013). Additionally, miR-30 family members have been widely studied as regulators of osteoblast differentiation, mainly through the suppression of the expression of Smad1 and Runx2 transcription factors (Zhang et al. 2011a, Wu et al. 2012, Eguchi et al. 2013).
SATB2 belongs to the family of special AT-rich sequence-binding proteins, members of which are present in the nuclear matrix and can bind to AT-rich sequences, activating the transcription of particular genes (Britanova et al. 2005). In vivo studies have shown that Satb2 physically interacts with and enhances the activity of Runx2 and Atf4 (Dobreva et al. 2006, Conner & Hornick 2013). Coupling these osteoblast-specific transcription factors, Satb2 increases the transcription of Bglap by binding to its promoter (Dobreva et al. 2006) and can also increase the expression of Ibsp by direct attachment to an osteoblast-specific promoter element (Dobreva et al. 2006). In addition, although Dobreva et al. did not find changes in Osx expression, others have reported that Satb2 acts in cooperation with Runx2 to upregulate Osx expression (Zhang et al. 2011b). The miR-23a–27a–24-2 cluster inhibits osteogenesis in vitro by downregulating the expression of Satb2 through the direct binding of the three miRNAs to its 3′-UTR. Moreover, Runx2 directly suppresses the expression of the cluster, whereas complementarily miR-23a targets Runx2 (Hassan et al. 2010). Interestingly, other clusters have also been studied, such as the auto-regulatory feedback loops controlling Runx2 expression. miR-3960/-2861 is transactivated by Runx2 in vitro, thereby maintaining its own levels of expression by blocking the expression of Hoxa and Hdac5, negative regulators of osteoblast differentiation (Kanzler et al. 1998, Dobreva et al. 2006, Li et al. 2009a, Hu et al. 2011). Furthermore, an in vivo approach has demonstrated that Satb2 is also targeted by miR-34s, affecting osteoblast proliferation mainly by means of miR-34b and miR-34c. Mice with osteoblast-specific deletion in miR-34bc from day E16.5 (using Cre under the control of the 2.3 kb fragment of Col1a1 promoter) were found to exhibit increased cortical bone volume, bone mineral density, and cortical thickness of long bones (Wei et al. 2012).
Following the expression of Runx2 in osteoprogenitors, Osx further strengthens the establishment of bone cell phenotype. Osx belongs to the Sp/Kruppel-like family of transcription factors because of its characteristic DNA-binding domain consisting of three tandem C2H2-type zinc finger motifs at the C-terminus. Osx is located downstream of Runx2 and, in fact, Runx2 directly binds to the Osx promoter (Nakashima et al. 2002, Nishio et al. 2006). The expression of Osx begins at around day E13.5 and it promotes the expression of osteoblast markers such as Alpl, Ibsp, and Bglap. The expression of Osx has been shown to be positively regulated by BMP, insulin-like growth factor 1 (IGF1), and MAPK signaling pathways in undifferentiated MSCs (Celil & Campbell 2005, Celil et al. 2005, Ortuno et al. 2010), and it can also regulate its own expression by interacting with its own promoter (Yoshida et al. 2012).
Obviously, Osx can also be post-transcriptionally regulated by miRNAs. Shi et al. (2013) described miR-214 as a downregulated miRNA during BMP2-induced osteoblast differentiation in C2C12 cells. miR-214 antagomirs lead to the overexpression of Osx and other related osteoblast markers such as Alpl, Col1a1, and Bglap. In addition, it has also been reported that miR-214 inhibits Twist (which inhibits the activity of Runx2 as a transcription factor) in intrahepatic cholangiocarcinomas and is overexpressed in elderly patients with fractures, in whom it directly targets ATF4 (Li et al. 2012, Wang et al. 2013a). As has been stated above, miR-204/-211 targets Runx2 mRNA in vitro. Furthermore, in vivo studies comparing differentially expressed miRNAs in calvaria from day E18.5 Osx-deficient and WT embryos have revealed that Osx-deficient osteoblasts display miR-204/-211 overexpression. As it is known that Osx-deficient calvaria exhibit an increase in Runx2 expression (Zhou et al. 2010), Chen et al. (2013a) have suggested that miR-204/-211 accumulation would dampen Runx2 overexpression and that Osx coordinately regulates the levels of this miRNA to maintain correct Runx2 expression.
A regulatory loop for Osx expression involves miR-93 in primary osteoblasts. During osteoblast mineralization, Osx can bind to the miR-93 promoter to repress its transcription and, as miR-93 also targets Osx mRNA, this facilitates the maintenance of osterix levels (Yang et al. 2012). Shi et al. (2013) have also reported the downregulation of miR-93 expression during the differentiation of C2C12 cells under BMP stimulation. Fine-tuning of Osx expression by miRNAs is also observed in the miR-322/Tob2 feedback mechanism in vitro. The Tob2 protein specifically controls the decay of Osx mRNA by regulation of its mRNA deadenylation, while BMP2 represses miR-322 expression and reduces miR-322 binding to the Tob2 3′-UTR; thus, higher Tob2 protein levels would control Osx levels (Gamez et al. 2013).
Changes in miR-637 levels have the capacity to maintain the balance between osteoblast and adipocyte differentiation in hMSCs by the inhibition of OSX expression and activation of adipogenic markers such as peroxisome proliferator-activated receptor γ (PPARγ (PPARG)) and CCAAT/enhancer-binding protein α (c/EBPα (CEBPA)) (Zhang et al. 2011c). Other miRNAs have also been shown to determine osteoblast–adipocyte balance in vitro (Li et al. 2013, Liao et al. 2013, Wang et al. 2013b). For instance, miR-3077-5p and miR-705, which work together as negative regulators of osteoblast differentiation through the suppression of Runx2 and Hoxa10 expression, eventually lead to a positive regulation of adipogenic differentiation (Liao et al. 2013). As has been mentioned above, it has been reported that miR-31 forms part of a regulatory loop linking Runx2 and Satb2, but it can also directly regulate the 3′-UTR of Osx. It has been suggested that an increase in Osx expression due to low expression of miR-31 can play a role in osteosarcomas, as observed in the MG-63 osteosarcoma cell line (Baglio et al. 2013).
Dlx5 is a BMP-responsive gene activated through a responsive element in its proximal promoter. Moreover, Dlx5 interacts with the Osx promoter and mediates BMP2-induced Osx expression independently of Runx2 (Lee et al. 2003, Ulsamer et al. 2008). miR-141 and miR-200a have been found to act as repressors of Dlx5 and Osx expression, and it has been demonstrated that both bind to Dlx5 mRNA in vitro (Itoh et al. 2009). Moreover, miR-141 and miR-200a target p38α (Mapk14) in mouse models of ovarian cancer, in agreement with previous screenings in human ovarian adenocarcinomas (Mateescu et al. 2011). Positive effects of p38 phosphorylation have been observed on Dlx5 transcriptional activity and on Osx stability (Ulsamer et al. 2008, Ortuno et al. 2010); consequently, the activity of miR-141 and miR-200a in osteoblast lineages is also probably mediated by changes in p38 phosphorylation of Dlx5 and Osx.
Recent studies on Bglap2–miR-214 transgenic mice overexpressing miR-214 have revealed its inhibitory role in the regulation of bone formation. In vitro manipulation of miR-214 revealed that direct targeting of Atf4 was required to inhibit osteoblast activity (Wang et al. 2013a). As the expression of Osx and Runx2 is tightly related, miRNAs targeting proteins involved in their upstream regulatory pathways will affect the expression of both. This is the case with miR-138, inhibited during human MSC osteoblast differentiation, which targets the focal adhesion kinase (FAK) and ERK1/2 pathways, leading to decreased phosphorylation of Runx2 and a lower expression of Osx (Eskildsen et al. 2011). Other studies have elucidated additional mechanisms connecting miRNAs to osteoblast differentiation by means of targeting different osteoblast differentiation pathways (Mizuno et al. 2008, Inose et al. 2009, Li et al. 2009b, Kapinas et al. 2010, Wang & Xu 2010, Zhang et al. 2012a).
miRNAs and chondroblast differentiation
In contrast to adipogenic and osteogenic differentiation-related miRNAs, fewer studies have been conducted on chondrogenic differentiation-related miRNAs. The Runx2 transcription factor also plays an important role in chondrocyte commitment. Runx2 (and Runx3) are transiently necessary for pre-hypertrophic chondrocytes to reach the hypertrophic state (Yoshida et al. 2004). Sox9 (Sry-related HMG box) is one of the main drivers of chondrocyte differentiation and its absence leads to a failure in chondrocyte commitment in Sox9−/− MSCs or knockout mice (Bi et al. 1999, 2001, Mori-Akiyama et al. 2003). Sox9 is required for the commitment of osteochondroprogenitors and for Runx2 expression in mesenchymal cell condensations (Akiyama et al. 2002, 2005). Other members of the Sry family, such as Sox6 and Sox5, also play important roles (Lefebvre et al. 2001; Fig. 2).
Several miRNAs (miR-1247, miR-145, miR-140, and miR-199a) have been reported to exert an effect on chondrogenesis by eventually affecting Sox9 expression positively (Karlsen et al. 2013) or negatively (Laine et al. 2012, Martinez-Sanchez et al. 2012, Martinez-Sanchez & Murphy 2013). Of all the miRNAs affecting chondroblast differentiation, miR-140 has received the most research attention to date (He et al. 2009, Nakamura et al. 2011, Nicolas et al. 2011, Yang et al. 2011, Gibson & Asahara 2013, Karlsen et al. 2013, Papaioannou et al. 2013). The results of these studies indicate that miR-140 is one of the main regulators of chondroblast differentiation through its effects on the expression of not only Sox9 (Karlsen et al. 2013), but also several other targets (Hdac4, Sp1, Smad3, and aggrecan) (Pais et al. 2010, Nakamura et al. 2011, Yang et al. 2011, Karlsen et al. 2013). Moreover, independent groups have developed miR-140-null mice, which displayed a concordant phenotype with major growth defects of endochondral bones (Miyaki et al. 2010, Nakamura et al. 2011, Papaioannou et al. 2013). Interestingly, Sox9, L-Sox5, and Sox6 have been proved to cooperatively activate the miR-140 promoter in vivo and in vitro (Miyaki et al. 2010, Yang et al. 2011, Yamashita et al. 2012), as well as other chondrogenic differentiation-related miRNAs (Guerit et al. 2013, Martinez-Sanchez & Murphy 2013).
miR-181a is highly expressed in chondrocytes, and it has been suggested that it works as a negative feedback system to preserve the homeostasis of cartilage by targeting Ccn1 (Ccna2; which promotes chondrogenesis) and Acan (encoding the protein aggrecan, the major proteoglycan in the cartilage ECM) (Sumiyoshi et al. 2013). miR-181b has also been reported to regulate Col2a1 expression, and its expression is elevated in human osteoarthritic chondrocytes in vitro (Song et al. 2013a). Other miRNAs regulate cell differentiation by targeting chromatin epigenetic modifiers (Tuddenham et al. 2006, Guan et al. 2011). For instance, miR-365 stimulates chondrocyte differentiation through Hdac4 repression, thereby increasing the levels of Ihh and Col X (markers of pre-hypertrophic chondrocytes and hypertrophic chondrocytes respectively; Guan et al. 2011).
As has been mentioned above, it should be noted that one particular miRNA may act as a switch for the selection of different cell commitment processes. miR-96, miR-124, and miR-199a have been studied in human BMMSCs and have been found to be differentially expressed during osteogenic, adipogenic, or chondrogenic induction: whereas miR-124 is expressed exclusively in adipocytes, the expression of miR-199a is upregulated in osteoblasts and chondrocytes (Laine et al. 2012).
miRNAs and osteoclast differentiation
In contrast to osteoblasts and chondrocytes, osteoclasts arise from hematopoietic cells and are the primary bone-resorbing cells. The transition from mononuclear pre-osteoclasts to mature osteoclasts is dependent on cell–cell fusion and is controlled by sequential exposure to signaling molecules (Fig. 3). Macrophage colony-stimulating factor (M-CSF) and RANKL are the two main cytokines involved in osteoclast differentiation (Manolagas 2000). M-CSF activates the c-Fms receptor, present in early osteoclast precursors, and acts as a survival/proliferation factor by activating Akt, microphthalmia transcription factor (Mitf), or the anti-apoptotic protein B-cell leukemia/lymphoma-associated gene 2 (BCL2). Moreover, M-CSF also stimulates the expression of Rank (Tnfrsf11a). Rankl is a member of the tumor necrosis factor α (TNFα) superfamily present in osteoblasts and stromal cells and can be a membrane-anchored molecule but can also be released as a soluble molecule following proteolytic cleavage. Lately, osteocytes have emerged as an important source of RANKL, indicating a key role for osteocytes in osteoclastogenesis (Nakashima et al. 2011, Xiong et al. 2011). The RANK–RANKL signaling system links osteoblast lineage and hematopoiesis-derived cells for osteoclast differentiation and activation. Together with M-CSF, RANK signaling is the main signaling pathway involved in osteoclast maturation (Tanaka et al. 2005). RANK stimulation leads to the recruitment of TNF receptor-associated cytoplasmic factors (TRAFs), mainly TRAF6, leading to the activation of several pathways, including the nuclear factor (NF) and activator of transcription NFATc1. Nfatc1 is widely accepted to be the key transcription factor involved in osteoclast differentiation (Kobayashi et al. 2001, Gohda et al. 2005). Nfatc1 eventually regulates several osteoclast-specific genes in cooperation with other transcription factors: AP1, PU.1, Mitf, and c-Fos (Tondravi et al. 1997, Takayanagi et al. 2002, Crotti et al. 2008). Their transcriptional targets are osteoclast-specific genes such as tartrate-resistant acid phosphatase (TRAP (ACP5)), cathepsin K, calcitonin receptor, and dendritic cell-specific transmembrane protein (Kukita et al. 2004). RANK can be blocked by osteopontegrin (OPG), therefore inhibiting osteoclast differentiation due to the suppression of RANKL stimuli. OPG is produced by osteoblasts and acts as a decoy receptor, preventing the coupling of RANKL to RANK and therefore reducing osteoclast resorption. Thus, the RANKL:OPG ratio must be accurately balanced to control osteoclastogenesis.
There are relatively few reports on the role of miRNAs in osteoclastogenesis. As in the case of osteoblasts, osteoclast-specific Dicer alteration has been shown to profoundly affect osteoclast activity in vivo and in vitro (Sugatani & Hruska 2009, Mizoguchi et al. 2010). In these models, a reduction in the expression of osteoclast markers (the expression of Trap and Nfatc1 mRNA was downregulated) and an increment in the values of bone parameters such as bone volume, trabecular thickness, and trabecular number have been observed, all leading to a mild osteopetrotic phenotype as a consequence of decreased osteoclast number and surface.
Of all the miRNAs involved in osteoclast differentiation, miRNA-223 has been studied the most. It was first identified as being specific to the CD11b-positive myeloid cell line (Chen et al. 2004). Sugatani and colleagues further confirmed miR-223 expression in the mouse osteoclast precursor cell line RAW 264.7 and showed that the modulation of pre-miR-223 alters osteoclast differentiation. Moreover, the levels of miR-223 in mouse bone marrow macrophages (BMMs) were found to be higher than those in osteoclasts, indicating that it must be repressed for appropriate osteoclast differentiation to occur (Sugatani & Hruska 2007). In the same study, miR-223 was shown to target Nfia, an osteoclastogenesis suppressor that eventually negatively regulates the M-CSF receptor. Later, other groups validated these effects of miR-223 on osteoclastogenesis and revealed PU.1-binding sites in the miR-223 promoter (Fukao et al. 2007, Sugatani & Hruska 2009, Shibuya et al. 2013). Sugatani et al. posited the existence of a feed-forward network whereby M-CSF induces PU.1 in osteoclast precursors, and PU.1 stimulates pri-miR-223 transcription, which, by downregulating the expression of Nfia, ultimately increases the levels of M-CSF receptor.
As with several other miRNAs affecting osteoclastogenesis, miRNA-223 has also been studied as a marker gene for rheumatoid arthritis (RA; Shibuya et al. 2013). As a result of the in vitro miR-223 studies mentioned above, it has been suggested that the increased levels of miR-223 found in RA synovium could be related to the inhibition of osteoclastogenesis. Furthermore, miR-21 has also been identified by Sugatani et al. (2011) as a miRNA upregulated during RANKL-induced osteoclastogenesis. Moreover, c-Fos and AP1 were found to be associated with its promoter. miR-21 loss-of-function experiments in a model of RANKL induction of BMMs showed a decrease in c-Fos phosphorylation and lower Nfatc1 and cathepsin K expression, all due to increased levels of programmed cell death 4 (PDCD4). Taken together, these findings indicate the existence of a new positive loop mechanism involving c-Fos/miR-21/PDCD4 (Sugatani et al. 2011). In addition, Mann et al. performed a differential miRNA screening using the RAW 264.7 cell line under RANKL and M-CSF treatment to induce osteoclastic differentiation. miR-155 was described in this study as an early inhibitor of MITF, a nuclear effector that integrates M-CSF/RANKL signals to initiate the expression of osteoclast-specific genes (Mann et al. 2010). The RAW 264.7 cell line can differentiate into either macrophages or osteoclasts, and the results of this study suggest that the upregulation of miR-155 expression facilitates macrophage commitment, therefore inhibiting osteoclast differentiation (by targeting MITF). These data indicate that miR-155 is involved in the commitment switch of hematopoietic precursors (Mann et al. 2010). Zhang et al. (2012b) revealed that miR-155 is inhibited by IFNβ during osteoclast differentiation, and they identified the effect of miR-155 on the 3′-UTR of MITF and suppressor of cytokine signaling 1 (SOCS1). Moreover, miR-155 has been observed to be involved in the pathogenesis of autoimmune arthritis in mice, being proposed as a novel target for the treatment of RA (Bluml et al. 2011). Other examples include miR-124, which has been shown to directly target Nfatc1 expression in BMMs (Lee et al. 2013), and miR-503, which targets Rank expression (Chen et al. 2013b).
Some of the miRNAs involved in osteoclast function have been shown to affect osteoclast cytoskeleton or migration. The expression of miR-31 has been found to increase in BMMs under RANKL stimulation. Moreover, it tightly controls cytoskeleton organization in osteoclasts by targeting Rhoa, essential for actin ring formation and bone resorption (Mizoguchi et al. 2013). Franceschetti et al. (2013) reported that all miR-29 family members (miR-29a, miR-b, and miR-c) are induced during osteoclast differentiation of mouse BMMs and RAW 264.7 cell line. However, Rossi et al. (2013) reported a decrease in miR-29b expression during human osteoclast differentiation from circulating human precursors. They also demonstrated the inhibition of osteoclastogenesis by miR-29b through the downregulation of c-FOS or NFATC1 expression, while Franceschetti et al. (2013) reported new targets such as Nfia, Cdc42, and Srgap2, among others, indicating that miR-29 positively maintains migration and cell commitment to osteoclasts.
Reciprocal interplay between miRNAs and signaling pathways in skeletal biology
Regulation of miRNA expression
Although several osteogenic differentiation-related miRNAs have been identified in the last decade, little is known about their transcriptional regulation. Numerous screenings have been performed to characterize the miRNA expression scenario during different stages of cell differentiation, but fewer studies have attempted to describe the molecular linkage between the stimuli and their regulatory effect on miRNA expression in full detail. miRNA processing and maturation can be regulated through the interaction of additional proteins with the Drosha complex. For instance, SMAD proteins interact with Drosha to specifically regulate the expression of some miRNAs, as in the case of miR-21 (Davis et al. 2008). Specific sequences are required in the loop of pri-miRNAs for them to be post-transcriptionally regulated by the SMAD–Drosha complex (Davis et al. 2010). Using several different models, it has been shown that miRNA expression can be regulated through several mechanisms, including the regulation of pre-miRNA nuclear export and Dicer cleavage, regulation of promoter activity by methylation and histone modification, or direct regulation of RNA polymerase II recruitment (Davis-Dusenbery & Hata 2010). However, much less is known about the influence of osteogenic signaling inputs on these mechanisms. To summarize the main themes in what is known about the interplay between signaling and miRNAs, we focus on two important pathways in skeletal development: BMP and Wnt.
The interplay between miRNAs and BMP signaling
BMPs form the largest subfamily of the TGFβ superfamily and are profoundly involved in skeletogenesis (Shi & Massague 2003, Miyazono et al. 2010). Early events in BMP signaling are initiated through the phosphorylation of specific BMP receptor-regulated SMAD proteins, namely R-SMAD1, R-SMAD5, and R-SMAD8. After phosphorylation, R-SMADs form heteromeric complexes with the common mediator SMAD4, which then migrate to the nucleus and activate the transcription of specific target genes (Shi & Massague 2003). Two additional SMADs are known as inhibitory SMADs, SMAD6 and SMAD7. Furthermore, BMPs can also activate noncanonical, SMAD-independent pathways, mainly MAPK pathways (Erk, p38), the LIMK pathway, or the PI3K pathway, which are also involved in osteoblast differentiation (Shi & Massague 2003, Gamell et al. 2008, Ulsamer et al. 2008, Ortuno et al. 2010).
Several miRNAs negatively or positively regulate BMP signaling (Fig. 4) and, in turn, BMPs coordinate a wide range of changes in miRNA expression (Inose et al. 2009). BMP biology has been widely studied in C2C12 cells, switching the differentiation pathway from a myoblastic to an osteoblastic phenotype (Katagiri et al. 1994), and several miRNA studies have been performed using this model (Li et al. 2008, Inose et al. 2009). For instance, Li et al. (2008) conducted a miRNA screening during BMP2-induced osteogenesis in C2C12 cells and found that the expression of almost all miRNAs was downregulated during osteoblast differentiation. These data have been confirmed by other groups (Gamez et al. 2013). The same authors also described miR-133 and miR-135 as negative regulators of osteogenesis that act by directly targeting Runx2 and Smad5 respectively, thereby inhibiting osteogenic differentiation (Li et al. 2008). In addition, it has recently been reported that under BMP stimuli, C2C12 inhibits the processing of myomiRs (muscle-specific miRNAS and myogenic miRNAs) due to the association of phosphorylated R-SMADs and Co-SMAD with phosphorylated KH-type splicing regulatory protein (KSRP (KHSRP)) in the nucleus (Pasero et al. 2012). KSRP is a single-strand RNA-binding protein that is essential for the maturation of myomiRs and for the establishment of the myogenic lineage in C2C12 cells (Briata et al. 2012); thus, KSRP sequestering by SMADs blocks myogenic differentiation in favor of the osteoblast lineage.
Another well-studied miRNA regulated by BMPs is miR-206 (Inose et al. 2009, Sato et al. 2009). The expression of miR-206 is downregulated by BMPs in C2C12 cells and its overexpression blocks osteoblast differentiation due to its effect on connexin43 mRNA, required for osteoblastic gene expression and function (Lecanda et al. 1998, Plotkin & Bellido 2013). Sato et al. (2009) have also suggested that BMP control of miR-206 occurs post-transcriptionally in C2C12 cells by the repression of pri-miR processing, and other studies have indicated that miR-206 is also required for myogenic differentiation in the same model (Kim et al. 2006). Moreover, miR-206 transgenic mice (2.3 kb Col1a1 promoter) suffer from reduced bone mass because of a decrease in bone formation (Inose et al. 2009). TGFβ inhibits both miR-206 expression and myogenic differentiation in vitro through an increase in HDAC4 protein expression (Winbanks et al. 2011).
miR-125b inhibits the proliferation of ST2 cells (murine MSCs) and BMP4 stimulation attenuates miR-125b expression in these cells. Thus, miR-125b inhibits osteoblast differentiation, possibly regulating the early stages of osteoblastogenesis (Mizuno et al. 2008). miR-141/-200a, mentioned above as Dlx5 and Osx repressors, are also regulated by BMP2 in the MC3T3-E1 cell line. Under BMP treatment, the expression of both miRNAs is downregulated, thus avoiding Dlx5 and Osx miRNA-related repression (Itoh et al. 2009). The expression of miR-322 is also downregulated by BMP2 and has been shown to indirectly repress Osx expression to facilitate further osteogenic differentiation (Gamez et al. 2013).
BMP2 also controls miRNAs involved in chondrogenic differentiation: for instance, the expression of miR-199a* is upregulated by BMP2 treatment, and its overexpression in pre-chondrogenic cells (ATDC5) or in the multipotential murine C3H10T1/2 cell line represses the expression of chondroblast markers Sox9 and Col2a1. miR-199a* also represses the 3′-UTR Smad1 transcript. Taken together, these data indicate that BMP2 reduces the expression of miR-199a*, avoiding Smad1 post-transcriptional miRNA regulation and repressing chondrogenic differentiation-specific markers (Lin et al. 2009).
miRNAs can also regulate R-SMAD expression. In addition to the above-mentioned examples, Smad1 has also emerged as a target of miR-26a in osteogenic differentiation of human adipose-tissue-derived stem cells (Luzi et al. 2008). These studies thus indicate that miR-26a restrains osteoblast commitment when reaching terminal differentiation (Luzi et al. 2008). Other miRNAs also target BMP receptors. For instance, the expression of miR-210 is upregulated during BMP4-induced osteoblast differentiation of mouse mesenchymal ST2 cells. miR-210 positively regulates osteoblast commitment by targeting the Acvr1b receptor (type 1 receptor; Mizuno et al. 2009). The 3′-UTR of the ACVR1/ALK2 gene has recently been studied in vitro to elucidate miRNAs that when expressed induce BMP signaling alterations in fibrodysplasia ossificans progressiva (Mura et al. 2012). Several additional BMP-modulated miRNAs have been identified (Li et al. 2008, 2009a, Lin et al. 2009, Bae et al. 2012, Gamez et al. 2013), generally as a result of high-throughput expression analysis but without precise information on their transcriptional control and function.
The interplay between miRNAs and Wnt/β-catenin signaling
Wnt signaling encompasses canonical and noncanonical pathways depending on the implication of β-catenin. Canonical Wnt/β-catenin signaling initiates by binding WNT1 class family members to Frizzled (Fzd) and the co-receptors LDL receptor-related proteins 5 and 6 (Lrp5/6). In the absence of stimuli, β-catenin is normally retained in the cytoplasm by a protein complex involving GSK3β (GSK3B), casein kinase 1a (CK1A (CSNK1A1)), APC, and axin, which is also finally responsible for its ubiquitination and degradation. When the Fzd–LRP receptor complex is stimulated by Wnt binding, recruitment of Disheveled leads to the inhibition of GSK3β activity. Thus, β-catenin accumulates and eventually enters the nucleus, where it binds to Tcf/Lef1 and regulates transcription. Several molecules negatively affect the Wnt pathway at different points and have been shown to be highly important for bone biology. Dickkopf (Dkk) family members or SOST antagonizes Wnt signaling by binding to LRP5 and 6, whereas SFRPs sequester Wnts away from binding to the receptors (Bafico et al. 2001, Wu & Nusse 2002; Fig. 5).
In vitro studies have generated controversial results about the effects of Wnt signaling on osteoblast differentiation, and theories exist about either a positive or a negative influence depending on the state of specification of the target cell. Most data indicate that β-catenin acts positively to maintain stem cell pluripotency and self-renewal; however, once MSCs reach commitment to osteochondroprogenitors, β-catenin promotes osteoblast progression. Moreover, Wnt signaling leads to Runx2 expression due to a Tcf regulatory element in its promoter (Gaur et al. 2005) and has been proved to work cooperatively with BMP signaling to induce other osteogenic genes such as Osx, Dlx5, and Msx2 (Rodriguez-Carballo et al. 2011).
One of the main mechanisms whereby miRNAs affect Wnt signaling is through the inhibition of Wnt/β-catenin pathway repressors (Fig. 5). The expression of miR-218 is upregulated during osteoblast differentiation, leading to an increase in the expression of osteoblast markers such as Alpl, Runx2, and Bglap. These effects correlate with a decreased expression of Sfrp2, Sost, and Dkk2. Moreover, BMP and Wnt stimuli induce higher miR-218 levels, leading to the upregulation of β-catenin and Tcf1 (Hnf1a) expression and therefore linking miR-218 to a positive loop mechanism involving Wnt signaling (Hassan et al. 2012). Dkk1 is also a miRNA target, leading to enhanced Wnt signaling. For instance, miR-29a negatively regulates the expression of not only Dkk1 but also Kremen2 (a decoy receptor of Wnt signaling) and Sfrp2, thereby potentiating the β-catenin pathway and promoting osteoblast differentiation of hMSCs (Kapinas et al. 2010). The expression of miR-29a and miR-29c is induced after osteoblast differentiation of MC3T3-E1, human fetal osteoblastic cells (hFOBs), and human primary osteoblasts. It has been suggested that TCF/LEF-binding sites present in miR-29 promoter are required for Wnt induction of miR-29 expression (Kapinas et al. 2009, 2010). Dkk1 is also targeted by miR-335-5, which binds directly to its 3′-UTR and decreases Dkk1 protein levels during osteoblast differentiation (Zhang et al. 2011d). It has also been suggested that Dkk1 levels are post-transcriptionally downregulated by miRNAs, allowing Wnt signaling to support lineage commitment. Ultimately, in the most mature form of osteocytes, the levels of miRNAs affecting Dkk1 decrease to allow Dkk1 to fine-tune the intensity of Wnt signaling, thereby avoiding disproportionate mineralization (Zhang et al. 2011d). Another target of miRNA activity is the scaffold protein APC, which is part of the destruction complex of β-catenin. The expression of miR-27 and miR-142-3p is induced in hFOBs and, in turn, these induce osteoblast differentiation. Both promote β-catenin accumulation by targeting APC (Wang & Xu 2010, Hu et al. 2013).
Several other pathways are involved in osteoblast differentiation (Notch, IGFs, hedgehogs, etc.), and these are also post-transcriptionally regulated by miRNAs. It is clear nowadays that not only can the expression of a single miRNA fluctuate during cell fate commitment but also a specific miRNA can target different mRNAs depending on the cellular stage. Moreover, a single miRNA can affect several signaling pathways simultaneously, allowing a cooperative effect or the fine-tuned expression of specific mRNAs. One example is miR-34c. Its effect on osteoblasts has been studied in vitro and in vivo, and it has been shown that it targets not only different factors in the Notch signaling pathway but also Satb2 and Runx2 somehow, in a Notch-independent manner (Bae et al. 2012). Determining how miRNAs regulate and are regulated by different pathways and how they interact to orchestrate a singular scenario for each differentiation step remains a tremendous challenge. In addition, bone-specific in vivo approaches have occasionally yielded controversial results compared with in vitro information, probably due to the influence of the cell environment and alterations in osteoblast–osteoclast communication.
miRNAs and skeletal disorders: therapeutic perspectives
miRNAs have emerged as important players in a wide range of pathologies. Multiple screenings have been performed in an attempt to determine miRNA signatures for several skeletal diseases. Osteoarthritis (OA) is the main degenerative articular disease caused by an imbalance between cartilage synthesis and degradation, leading to a progressive loss of movement, functional disability, and joint pain and inflammation. At present, therapy is usually based on symptomatic treatment, mainly using non-steroidal anti-inflammatory drugs (NSAIDs), but these fail to slow articular degeneration and disease progression. Studies on OA have demonstrated that miRNA expression is regulated during this process, indicating the possibility of future miRNA therapies (Iliopoulos et al. 2008, Jones et al. 2009, Akhtar et al. 2010, Diaz-Prado et al. 2012). One of the main characteristics of osteoarthritic chondrocytes is the secretion of the pro-inflammatory cytokines interleukin 1β (IL1β) and TNFα. Chondrocyte death occurs in OA and the remaining chondrocytes express IL1β (IL1B), leading to the upregulation of matrix degradation enzymes such as metalloproteinases (MMPs), particularly MMP13, and the aggrecanases ADAMTS4 and ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs). MMPs and ADAMTS are involved in ECM degradation and OA progression due to their capacity to cleave type 2 collagen or aggrecan respectively.
Several miRNAs are involved in the regulation of IL1β downstream mediators and, in turn, IL1β has been used in vitro in human chondrocytes as a model for the study of OA. Numerous miRNAs have been shown to be regulated by IL1β action (Miyaki et al. 2009, Dai et al. 2012, Matsukawa et al. 2013). MMP13 is regulated directly by miR-27b and miR-127-5p (Akhtar et al. 2010, Park et al. 2013) or indirectly by miR-27a, miR-9, miR-488, or miR-22 (Iliopoulos et al. 2008, Jones et al. 2009, Tardif et al. 2009, Song et al. 2013b), among others.
As has been mentioned above, ADAMTS aggrecanases are also important miRNA targets in OA patients and in vitro models. Besides its known role in chondrocyte differentiation, miR-140 also plays a central role in OA (Araldi & Schipani 2010). In vivo, miR-140 and miR-146a target ADAMTS5 (Miyaki et al. 2009, 2010, Tardif et al. 2009, Li et al. 2011), while miR-125b targets ADAMTS4 (Matsukawa et al. 2013). Moreover, miR-101 directly inhibits the expression of collagen type II and aggrecan genes through the downregulation of SOX9 expression (Dai et al. 2012). miR-34a is also involved in OA through the regulation of chondrocyte apoptosis and migration (Abouheif et al. 2010, Kim et al. 2011). Inhibition of miR-34 activity leads to a reduction in IL1β-induced apoptosis in osteoarthritic rat chondrocytes (Abouheif et al. 2010). The expression of miR-149 is also downregulated in human primary osteoarthritic chondrocytes and in sw1353 chondrocytes under IL1β/TNFα stimulation and, in turn, miR-149 strongly downregulates the levels of pro-inflammatory cytokines such as IL6, TNFα, and IL1β, thus eventually acting as a feedback loop for cytokine expression (Santini et al. 2013). Moreover, it has been shown that Cox2 (Ptgs2) expression requires p38 activity (Susperregui et al. 2011) and IL1β stimulation induces p38 activation, leading to a negative regulation of miR-199a* that directly controls the expression of Cox2 by binding to its 3′-UTR (Akhtar & Haqqi 2012).
Another bone-related disease with an important impact is osteoporosis (OP). In both men and women, loss of bone mass typically starts between 40 and 50 years of age, but there is a major loss in women due to the decrease in estrogen levels after menopause. More importantly, OP leads to a higher risk of fractures among elderly women due to bone fragility, although surprisingly the risk of mortality after a hip fracture is higher in men.
Studies of miR-503 inhibition in mouse models of ovariectomy have shown that miR-503 regulates bone resorption in vivo, inhibiting osteoclastogenesis by targeting Rank (Chen et al. 2013b). It has also been suggested that a decrease in miR-2861 expression contributes to OP. miR-2861 has already been shown to be a BMP-induced miRNA, targeting Hdac5 and therefore involved in Runx2 degradation (Li et al. 2009a, Hu et al. 2011). Li et al. (2009a) have shown that inhibition of miR-2861, using a specific antisense oligonucleotide introduced by a single tail vein injection, leads to decreased bone mass, reduced osteoblast activity, and osteoblast marker alteration in mice. miR-3077-5p and miR-705 are overexpressed in osteoporotic MSCs. The levels of both miRNAs decrease normally during osteogenic induction to allow the expression of their targets HOXA10 and RUNX2. Liao et al. (2013) have shown that knockdown of miR-705 and miR-3077-5p in osteoporotic MSCs is sufficient to restore osteoblast differentiation and mineralization. Moreover, 17β-estradiol injections in ovariectomized mice were found to lead to the recovery of the osteoporotic phenotype and a reduction in miR-705/-3077-5p expression (Liao et al. 2013).
miRNAs are also involved in tumor emergence and progression. Nowadays, the use of miRNAs constitutes a novel strategy to improve tumor detection and to predict patient prognosis. One example is miR-132, which was detected as a miRNA expressed at lower levels in osteosarcoma samples. Lower expression of miR-132 is observed in patients with advanced-stage cancer and presenting a poor response to chemotherapy, identifying miR-132 as an indicator of osteosarcoma prognosis (Yang et al. 2013). The downregulation of miR-145 expression is also related to poor prognosis in osteosarcoma patients (Tang et al. 2013). However, the molecular mechanisms of miR-145 and miR-132 in relation to osteosarcomas are unknown, and further studies are needed to fully understand their involvement in carcinogenesis. miRNA screening has been performed in chondrosarcoma biopsies (Yoshitaka et al. 2013), and recently, a study elucidating miRNA changes in osteolytic bone metastasis has been published (Ell et al. 2013). During osteolytic bone metastasis, downregulation of the expression of specific miRNAs leads to increased expression of important osteoclastic genes such as MITF, CALCR, TRAF6, and MMP14. Furthermore, ectopic overexpression of some miRNAs (pre-miR-141 and pre-miR-219) leads to a reduction in osteolytic bone metastasis (Ell et al. 2013).
Thus, a detailed understanding of the function of miRNAs and their tight relationship with bone diseases would constitute a powerful tool for early diagnosis and future therapeutic approaches. Pre-miR or antago-miR therapies have emerged as a novel way to target dysregulated pathways; however, several questions about safety as well as tissue-specific targeting still remain to be answered before clinical applications can be developed.
Concluding remarks
Microarray expression data have provided evidence for the role of miRNAs in several skeletal pathologies. Moreover, mouse models in which their expression is altered have provided evidence of causal links between miRNAs and bone abnormalities. The current identification of a vast number of skeletal-cell-specific miRNAs, each of them with a list of putative targets, has yielded the present challenge of understanding their biological functions. From the data obtained to date, we know that most miRNAs exert their functional effects via multiple target mRNAs, usually by cooperatively targeting genes in the same pathway. Similarly, there is also redundancy, as the same mRNA is targeted by different miRNAs. The very sensitive nature of developmental programs and signaling pathways renders them the perfect candidates for techniques utilizing the dose-dependent effects of miRNAs. In bone physiology, miRNAs are extremely useful nodes, acting as feedback or feed-forward devices that allow buffering effects that confer robustness to skeletal development programs. miRNAs serve as finely tuned precision regulators of the expression of those genes that confer cellular identity and promote differentiation. Moreover, individual miRNAs may even operate as switches to induce differential cell fates. These properties clearly support the notion that the mutation or dysregulation of miRNAs would profoundly affect expression patterns and contribute to skeletal pathologies. Fortunately, these properties also open up opportunities for designing smart therapies based on miRNAs and small RNAs. miRNAs, which are easy to deliver into cells, are intrinsically highly specific and could regulate several targets at the same time. Thus, it is highly feasible that miRNAs, as well as antago-miRNAs, may be used in the future as drugs to treat skeletal pathologies.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review.
Funding
This research was supported by grants from the Ministry of Education of Spain (BFU2011-24254), Fundació La Marató de TV3, and Instituto de Salud Carlos III (ISCIII) (RETIC RD06/0020).
References
Abouheif MM, Nakasa T, Shibuya H, Niimoto T, Kongcharoensombat W & Ochi M 2010 Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro. Rheumatology 49 2054–2060. (doi:10.1093/rheumatology/keq247).
Akhtar N & Haqqi TM 2012 MicroRNA-199a* regulates the expression of cyclooxygenase-2 in human chondrocytes. Annals of the Rheumatic Diseases 71 1073–1080. (doi:10.1136/annrheumdis-2011-200519).
Akhtar N, Rasheed Z, Ramamurthy S, Anbazhagan AN, Voss FR & Haqqi TM 2010 MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes. Arthritis and Rheumatism 62 1361–1371. (doi:10.1002/art.27329).
Akiyama H, Chaboissier MC, Martin JF, Schedl A & de Crombrugghe B 2002 The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes and Development 16 2813–2828. (doi:10.1101/gad.1017802).
Akiyama H, Kim JE, Nakashima K, Balmes G, Iwai N, Deng JM, Zhang Z, Martin JF, Behringer RR & Nakamura T et al. 2005 Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. PNAS 102 14665–14670. (doi:10.1073/pnas.0504750102).
Araldi E & Schipani E 2010 MicroRNA-140 and the silencing of osteoarthritis. Genes and Development 24 1075–1080. (doi:10.1101/gad.1939310).
Bae Y, Yang T, Zeng HC, Campeau PM, Chen Y, Bertin T, Dawson BC, Munivez E, Tao J & Lee BH 2012 miRNA-34c regulates Notch signaling during bone development. Human Molecular Genetics 21 2991–3000. (doi:10.1093/hmg/dds129).
Bafico A, Liu G, Yaniv A, Gazit A & Aaronson SA 2001 Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nature Cell Biology 3 683–686. (doi:10.1038/35083081).
Baglio SR, Devescovi V, Granchi D & Baldini N 2013 MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31. Gene 527 321–331. (doi:10.1016/j.gene.2013.06.021).
Bartel DP 2004 MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116 281–297. (doi:10.1016/S0092-8674(04)00045-5).
Bi W, Deng JM, Zhang Z, Behringer RR & de Crombrugghe B 1999 Sox9 is required for cartilage formation. Nature Genetics 22 85–89. (doi:10.1038/8792).
Bi W, Huang W, Whitworth DJ, Deng JM, Zhang Z, Behringer RR & de Crombrugghe B 2001 Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. PNAS 98 6698–6703. (doi:10.1073/pnas.111092198).
Bluml S, Bonelli M, Niederreiter B, Puchner A, Mayr G, Hayer S, Koenders MI, van den Berg WB, Smolen J & Redlich K 2011 Essential role of microRNA-155 in the pathogenesis of autoimmune arthritis in mice. Arthritis and Rheumatism 63 1281–1288. (doi:10.1002/art.30281).
Borchert GM, Lanier W & Davidson BL 2006 RNA polymerase III transcribes human microRNAs. Nature Structural & Molecular Biology 13 1097–1101. (doi:10.1038/nsmb1167).
Briata P, Lin WJ, Giovarelli M, Pasero M, Chou CF, Trabucchi M, Rosenfeld MG, Chen CY & Gherzi R 2012 PI3K/AKT signaling determines a dynamic switch between distinct KSRP functions favoring skeletal myogenesis. Cell Death and Differentiation 19 478–487. (doi:10.1038/cdd.2011.117).
Britanova O, Akopov S, Lukyanov S, Gruss P & Tarabykin V 2005 Novel transcription factor Satb2 interacts with matrix attachment region DNA elements in a tissue-specific manner and demonstrates cell-type-dependent expression in the developing mouse CNS. European Journal of Neuroscience 21 658–668. (doi:10.1111/j.1460-9568.2005.03897.x).
Celil AB & Campbell PG 2005 BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. Journal of Biological Chemistry 280 31353–31359. (doi:10.1074/jbc.M503845200).
Celil AB, Hollinger JO & Campbell PG 2005 Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling. Journal of Cellular Biochemistry 95 518–528. (doi:10.1002/jcb.20429).
Chen CZ, Li L, Lodish HF & Bartel DP 2004 MicroRNAs modulate hematopoietic lineage differentiation. Science 303 83–86. (doi:10.1126/science.1091903).
Chen Q, Liu W, Sinha KM, Yasuda H & de Crombrugghe B 2013a Identification and characterization of microRNAs controlled by the osteoblast-specific transcription factor Osterix. PLoS ONE 8 e58104. (doi:10.1371/journal.pone.0058104).
Chen C, Cheng P, Xie H, Zhou HD, Wu XP, Liao EY & Luo XH 2013b MiR-503 regulates osteoclastogenesis via targeting RANK. Journal of Bone and Mineral Research 29 338–347. (doi:10.1002/jbmr.2032).
Conner JR & Hornick JL 2013 SATB2 is a novel marker of osteoblastic differentiation in bone and soft tissue tumours. Histopathology 63 36–49. (doi:10.1111/his.12138).
Crotti TN, Sharma SM, Fleming JD, Flannery MR, Ostrowski MC, Goldring SR & McHugh KP 2008 PU.1 and NFATc1 mediate osteoclastic induction of the mouse β3 integrin promoter. Journal of Cellular Physiology 215 636–644. (doi:10.1002/jcp.21344).
Dai L, Zhang X, Hu X, Zhou C & Ao Y 2012 Silencing of microRNA-101 prevents IL-1β-induced extracellular matrix degradation in chondrocytes. Arthritis Research & Therapy 14 R268. (doi:10.1186/ar4114).
Davis BN, Hilyard AC, Lagna G & Hata A 2008 SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454 56–61. (doi:10.1038/nature07086).
Davis BN, Hilyard AC, Nguyen PH, Lagna G & Hata A 2010 Smad proteins bind a conserved RNA sequence to promote microRNA maturation by Drosha. Molecular Cell 39 373–384. (doi:10.1016/j.molcel.2010.07.011).
Davis-Dusenbery BN & Hata A 2010 Mechanisms of control of microRNA biogenesis. Journal of Biochemistry 148 381–392. (doi:10.1093/jb/mvq096).
Deng Y, Wu S, Zhou H, Bi X, Wang Y, Hu Y, Gu P & Fan X 2013 Effects of a miR-31, Runx2, and Satb2 regulatory loop on the osteogenic differentiation of bone mesenchymal stem cells. Stem Cells and Development 22 2278–2286. (doi:10.1089/scd.2012.0686).
Diaz-Prado S, Cicione C, Muinos-Lopez E, Hermida-Gomez T, Oreiro N, Fernandez-Lopez C & Blanco FJ 2012 Characterization of microRNA expression profiles in normal and osteoarthritic human chondrocytes. BMC Musculoskeletal Disorders 13 144. (doi:10.1186/1471-2474-13-144).
Dobreva G, Chahrour M, Dautzenberg M, Chirivella L, Kanzler B, Farinas I, Karsenty G & Grosschedl R 2006 SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation. Cell 125 971–986. (doi:10.1016/j.cell.2006.05.012).
Ducy P 2000 Cbfa1: a molecular switch in osteoblast biology. Developmental Dynamics 219 461–471. (doi:10.1002/1097-0177(2000)9999:9999<::AID-DVDY1074>3.0.CO;2-C).
Ducy P & Karsenty G 1995 Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Molecular and Cellular Biology 15 1858–1869.
Ducy P, Zhang R, Geoffroy V, Ridall AL & Karsenty G 1997 Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89 747–754. (doi:10.1016/S0092-8674(00)80257-3).
Duong LT & Rodan GA 2001 Regulation of osteoclast formation and function. Reviews in Endocrine & Metabolic Disorders 2 95–104. (doi:10.1023/A:1010063225902).
Eguchi T, Watanabe K, Hara ES, Ono M, Kuboki T & Calderwood SK 2013 OstemiR: a novel panel of microRNA biomarkers in osteoblastic and osteocytic differentiation from mesencymal stem cells. PLoS ONE 8 e58796. (doi:10.1371/journal.pone.0058796).
Ell B, Mercatali L, Ibrahim T, Campbell N, Schwarzenbach H, Pantel K, Amadori D & Kang Y 2013 Tumor-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bone metastasis. Cancer Cell 24 542–556. (doi:10.1016/j.ccr.2013.09.008).
Eskildsen T, Taipaleenmaki H, Stenvang J, Abdallah BM, Ditzel N, Nossent AY, Bak M, Kauppinen S & Kassem M 2011 MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. PNAS 108 6139–6144. (doi:10.1073/pnas.1016758108).
Franceschetti T, Kessler CB, Lee SK & Delany AM 2013 miR-29 promotes murine osteoclastogenesis by regulating osteoclast commitment and migration. Journal of Biological Chemistry 288 33347–33360. (doi:10.1074/jbc.M113.484568).
Fukao T, Fukuda Y, Kiga K, Sharif J, Hino K, Enomoto Y, Kawamura A, Nakamura K, Takeuchi T & Tanabe M 2007 An evolutionarily conserved mechanism for microRNA-223 expression revealed by microRNA gene profiling. Cell 129 617–631. (doi:10.1016/j.cell.2007.02.048).
Gamell C, Osses N, Bartrons R, Ruckle T, Camps M, Rosa JL & Ventura F 2008 BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity. Journal of Cell Science 121 3960–3970. (doi:10.1242/jcs.031286).
Gamez B, Rodriguez-Carballo E, Bartrons R, Rosa JL & Ventura F 2013 MicroRNA-322 (miR-322) and its target protein Tob2 modulate Osterix (Osx) mRNA stability. Journal of Biological Chemistry 288 14264–14275. (doi:10.1074/jbc.M112.432104).
Gao J, Yang T, Han J, Yan K, Qiu X, Zhou Y, Fan Q & Ma B 2011 MicroRNA expression during osteogenic differentiation of human multipotent mesenchymal stromal cells from bone marrow. Journal of Cellular Biochemistry 112 1844–1856. (doi:10.1002/jcb.23106).
Gaur T, Lengner CJ, Hovhannisyan H, Bhat RA, Bodine PV, Komm BS, Javed A, van Wijnen AJ, Stein JL & Stein GS et al. 2005 Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. Journal of Biological Chemistry 280 33132–33140. (doi:10.1074/jbc.M500608200).
Gaur T, Hussain S, Mudhasani R, Parulkar I, Colby JL, Frederick D, Kream BE, van Wijnen AJ, Stein JL & Stein GS et al. 2010 Dicer inactivation in osteoprogenitor cells compromises fetal survival and bone formation, while excision in differentiated osteoblasts increases bone mass in the adult mouse. Developmental Biology 340 10–21. (doi:10.1016/j.ydbio.2010.01.008).
Gibson G & Asahara H 2013 MicroRNAs and cartilage. Journal of Orthopaedic Research 31 1333–1344. (doi:10.1002/jor.22397).
Gohda J, Akiyama T, Koga T, Takayanagi H, Tanaka S & Inoue J 2005 RANK-mediated amplification of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis. EMBO Journal 24 790–799. (doi:10.1038/sj.emboj.7600564).
Guan YJ, Yang X, Wei L & Chen Q 2011 MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4. FASEB Journal 25 4457–4466. (doi:10.1096/fj.11-185132).
Guerit D, Philipot D, Chuchana P, Toupet K, Brondello JM, Mathieu M, Jorgensen C & Noel D 2013 Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells. PLoS ONE 8 e62582. (doi:10.1371/journal.pone.0062582).
Guo H, Ingolia NT, Weissman JS & Bartel DP 2010 Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466 835–840. (doi:10.1038/nature09267).
Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT & Kim VN 2006 Molecular basis for the recognition of primary microRNAs by the Drosha–DGCR8 complex. Cell 125 887–901. (doi:10.1016/j.cell.2006.03.043).
Hassan MQ, Gordon JA, Beloti MM, Croce CM, van Wijnen AJ, Stein JL, Stein GS & Lian JB 2010 A network connecting Runx2, SATB2, and the miR-23a–27a–24-2 cluster regulates the osteoblast differentiation program. PNAS 107 19879–19884. (doi:10.1073/pnas.1007698107).
Hassan MQ, Maeda Y, Taipaleenmaki H, Zhang W, Jafferji M, Gordon JA, Li Z, Croce CM, van Wijnen AJ & Stein JL et al. 2012 miR-218 directs a Wnt signaling circuit to promote differentiation of osteoblasts and osteomimicry of metastatic cancer cells. Journal of Biological Chemistry 287 42084–42092. (doi:10.1074/jbc.M112.377515).
He X, Eberhart JK & Postlethwait JH 2009 MicroRNAs and micromanaging the skeleton in disease, development and evolution. Journal of Cellular and Molecular Medicine 13 606–618. (doi:10.1111/j.1582-4934.2009.00696.x).
He J, Zhang JF, Yi C, Lv Q, Xie WD, Li JN, Wan G, Cui K, Kung HF & Yang J et al. 2010 miRNA-mediated functional changes through co-regulating function related genes. PLoS ONE 5 e13558. (doi:10.1371/journal.pone.0013558).
Hobert O 2008 Gene regulation by transcription factors and microRNAs. Science 319 1785–1786. (doi:10.1126/science.1151651).
Horowitz MC, Xi Y, Wilson K & Kacena MA 2001 Control of osteoclastogenesis and bone resorption by members of the TNF family of receptors and ligands. Cytokine & Growth Factor Reviews 12 9–18. (doi:10.1016/S1359-6101(00)00030-7).
Hu R, Liu W, Li H, Yang L, Chen C, Xia ZY, Guo LJ, Xie H, Zhou HD & Wu XP et al. 2011 A Runx2/miR-3960/miR-2861 regulatory feedback loop during mouse osteoblast differentiation. Journal of Biological Chemistry 286 12328–12339. (doi:10.1074/jbc.M110.176099).
Hu W, Ye Y, Zhang W, Wang J, Chen A & Guo F 2013 miR1423p promotes osteoblast differentiation by modulating Wnt signaling. Molecular Medicine Reports 7 689–693. (doi:10.3892/mmr.2012.1207).
Huang J, Zhao L, Xing L & Chen D 2010 MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells 28 357–364. (doi:10.1002/stem.288).
Iliopoulos D, Malizos KN, Oikonomou P & Tsezou A 2008 Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks. PLoS ONE 3 e3740. (doi:10.1371/journal.pone.0003740).
Inose H, Ochi H, Kimura A, Fujita K, Xu R, Sato S, Iwasaki M, Sunamura S, Takeuchi Y & Fukumoto S et al. 2009 A microRNA regulatory mechanism of osteoblast differentiation. PNAS 106 20794–20799. (doi:10.1073/pnas.0909311106).
Itoh T, Nozawa Y & Akao Y 2009 MicroRNA-141 and -200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5. Journal of Biological Chemistry 284 19272–19279. (doi:10.1074/jbc.M109.014001).
Jones SW, Watkins G, Le Good N, Roberts S, Murphy CL, Brockbank SM, Needham MR, Read SJ & Newham P 2009 The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-α and MMP13. Osteoarthritis and Cartilage 17 464–472. (doi:10.1016/j.joca.2008.09.012).
Kanzler B, Kuschert SJ, Liu YH & Mallo M 1998 Hoxa-2 restricts the chondrogenic domain and inhibits bone formation during development of the branchial area. Development 125 2587–2597.
Kapinas K & Delany AM 2011 MicroRNA biogenesis and regulation of bone remodeling. Arthritis Research & Therapy 13 220. (doi:10.1186/ar3325).
Kapinas K, Kessler CB & Delany AM 2009 miR-29 suppression of osteonectin in osteoblasts: regulation during differentiation and by canonical Wnt signaling. Journal of Cellular Biochemistry 108 216–224. (doi:10.1002/jcb.22243).
Kapinas K, Kessler C, Ricks T, Gronowicz G & Delany AM 2010 miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop. Journal of Biological Chemistry 285 25221–25231. (doi:10.1074/jbc.M110.116137).
Karlsen TA, Jakobsen RB, Mikkelsen TS & Brinchmann JE 2013 MicroRNA-140 targets RALA and regulates chondrogenic differentiation of human mesenchymal stem cells by translational enhancement of SOX9 and ACAN. Stem Cells and Development 23 290–304. (doi:10.1089/scd.2013.0209).
Karsenty G 2008 Transcriptional control of skeletogenesis. Annual Review of Genomics and Human Genetics 9 183–196. (doi:10.1146/annurev.genom.9.081307.164437).
Karsenty G & Wagner EF 2002 Reaching a genetic and molecular understanding of skeletal development. Developmental Cell 2 389–406. (doi:10.1016/S1534-5807(02)00157-0).
Karsenty G, Ducy P, Starbuck M, Priemel M, Shen J, Geoffroy V & Amling M 1999 Cbfa1 as a regulator of osteoblast differentiation and function. Bone 25 107–108. (doi:10.1016/S8756-3282(99)00111-8).
Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, Rosen V, Wozney JM, Fujisawa-Sehara A & Suda T 1994 Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. Journal of Cell Biology 127 1755–1766. (doi:10.1083/jcb.127.6.1755).
Kim HK, Lee YS, Sivaprasad U, Malhotra A & Dutta A 2006 Muscle-specific microRNA miR-206 promotes muscle differentiation. Journal of Cell Biology 174 677–687. (doi:10.1083/jcb.200603008).
Kim D, Song J, Kim S, Chun CH & Jin EJ 2011 MicroRNA-34a regulates migration of chondroblast and IL-1β-induced degeneration of chondrocytes by targeting EphA5. Biochemical and Biophysical Research Communications 415 551–557. (doi:10.1016/j.bbrc.2011.10.087).
Kobayashi N, Kadono Y, Naito A, Matsumoto K, Yamamoto T, Tanaka S & Inoue J 2001 Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis. EMBO Journal 20 1271–1280. (doi:10.1093/emboj/20.6.1271).
Kobayashi T, Lu J, Cobb BS, Rodda SJ, McMahon AP, Schipani E, Merkenschlager M & Kronenberg HM 2008 Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. PNAS 105 1949–1954. (doi:10.1073/pnas.0707900105).
Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH & Inada M et al. 1997 Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89 755–764. (doi:10.1016/S0092-8674(00)80258-5).
Kukita T, Wada N, Kukita A, Kakimoto T, Sandra F, Toh K, Nagata K, Iijima T, Horiuchi M & Matsusaki H et al. 2004 RANKL-induced DC-STAMP is essential for osteoclastogenesis. Journal of Experimental Medicine 200 941–946. (doi:10.1084/jem.20040518).
Laine SK, Alm JJ, Virtanen SP, Aro HT & Laitala-Leinonen TK 2012 MicroRNAs miR-96, miR-124, and miR-199a regulate gene expression in human bone marrow-derived mesenchymal stem cells. Journal of Cellular Biochemistry 113 2687–2695. (doi:10.1002/jcb.24144).
Lecanda F, Towler DA, Ziambaras K, Cheng SL, Koval M, Steinberg TH & Civitelli R 1998 Gap junctional communication modulates gene expression in osteoblastic cells. Molecular Biology of the Cell 9 2249–2258. (doi:10.1091/mbc.9.8.2249).
Lee MH, Kwon TG, Park HS, Wozney JM & Ryoo HM 2003 BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochemical and Biophysical Research Communications 309 689–694. (doi:10.1016/j.bbrc.2003.08.058).
Lee I, Ajay SS, Yook JI, Kim HS, Hong SH, Kim NH, Dhanasekaran SM, Chinnaiyan AM & Athey BD 2009 New class of microRNA targets containing simultaneous 5′-UTR and 3′-UTR interaction sites. Genome Research 19 1175–1183. (doi:10.1101/gr.089367.108).
Lee Y, Kim HJ, Park CK, Kim YG, Lee HJ, Kim JY & Kim HH 2013 MicroRNA-124 regulates osteoclast differentiation. Bone 56 383–389. (doi:10.1016/j.bone.2013.07.007).
Lefebvre V, Behringer RR & de Crombrugghe B 2001 L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway. Osteoarthritis and Cartilage 9 (Suppl A) S69–S75. (doi:10.1053/joca.2001.0447).
Li Z, Hassan MQ, Volinia S, van Wijnen AJ, Stein JL, Croce CM, Lian JB & Stein GS 2008 A microRNA signature for a BMP2-induced osteoblast lineage commitment program. PNAS 105 13906–13911. (doi:10.1073/pnas.0804438105).
Li H, Xie H, Liu W, Hu R, Huang B, Tan YF, Xu K, Sheng ZF, Zhou HD & Wu XP et al. 2009a A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. Journal of Clinical Investigation 119 3666–3677. (doi:10.1172/JCI39832).
Li Z, Hassan MQ, Jafferji M, Aqeilan RI, Garzon R, Croce CM, van Wijnen AJ, Stein JL, Stein GS & Lian JB 2009b Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. Journal of Biological Chemistry 284 15676–15684. (doi:10.1074/jbc.M809787200).
Li X, Gibson G, Kim JS, Kroin J, Xu S, van Wijnen AJ & Im HJ 2011 MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis. Gene 480 34–41. (doi:10.1016/j.gene.2011.03.003).
Li B, Han Q, Zhu Y, Yu Y, Wang J & Jiang X 2012 Down-regulation of miR-214 contributes to intrahepatic cholangiocarcinoma metastasis by targeting Twist. FEBS Journal 279 2393–2398. (doi:10.1111/j.1742-4658.2012.08618.x).
Li H, Li T, Wang S, Wei J, Fan J, Li J, Han Q, Liao L, Shao C & Zhao RC 2013 miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells. Stem Cell Research 10 313–324. (doi:10.1016/j.scr.2012.11.007).
Liao L, Yang X, Su X, Hu C, Zhu X, Yang N, Chen X, Shi S, Shi S & Jin Y 2013 Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow. Cell Death & Disease 4 e600. (doi:10.1038/cddis.2013.130).
Lin EA, Kong L, Bai XH, Luan Y & Liu CJ 2009 miR-199a, a bone morphogenic protein 2-responsive microRNA, regulates chondrogenesis via direct targeting to Smad1. Journal of Biological Chemistry 284 11326–11335. (doi:10.1074/jbc.M807709200).
Lund E, Guttinger S, Calado A, Dahlberg JE & Kutay U 2004 Nuclear export of microRNA precursors. Science 303 95–98. (doi:10.1126/science.1090599).
Luzi E, Marini F, Sala SC, Tognarini I, Galli G & Brandi ML 2008 Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. Journal of Bone and Mineral Research 23 287–295. (doi:10.1359/jbmr.071011).
Lytle JR, Yario TA & Steitz JA 2007 Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR. PNAS 104 9667–9672. (doi:10.1073/pnas.0703820104).
Mann M, Barad O, Agami R, Geiger B & Hornstein E 2010 miRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate. PNAS 107 15804–15809. (doi:10.1073/pnas.0915022107).
Manolagas SC 2000 Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocrine Reviews 21 115–137. (doi:10.1210/edrv.21.2.0395).
Martinez-Sanchez A & Murphy CL 2013 miR-1247 functions by targeting cartilage transcription factor SOX9. Journal of Biological Chemistry 288 30802–30814. (doi:10.1074/jbc.M113.496729).
Martinez-Sanchez A, Dudek KA & Murphy CL 2012 Regulation of human chondrocyte function through direct inhibition of cartilage master regulator SOX9 by microRNA-145 (miRNA-145). Journal of Biological Chemistry 287 916–924. (doi:10.1074/jbc.M111.302430).
Mateescu B, Batista L, Cardon M, Gruosso T, de Feraudy Y, Mariani O, Nicolas A, Meyniel JP, Cottu P & Sastre-Garau X et al. 2011 miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response. Nature Medicine 17 1627–1635. (doi:10.1038/nm.2512).
Matsukawa T, Sakai T, Yonezawa T, Hiraiwa H, Hamada T, Nakashima M, Ono Y, Ishizuka S, Nakahara H & Lotz MK et al. 2013 MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes. Arthritis Research & Therapy 15 R28. (doi:10.1186/ar4164).
Mattick JS & Makunin IV 2006 Non-coding RNA. Human Molecular Genetics 15 R17–R29. (doi:10.1093/hmg/ddl046).
Miyaki S, Nakasa T, Otsuki S, Grogan SP, Higashiyama R, Inoue A, Kato Y, Sato T, Lotz MK & Asahara H 2009 MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses. Arthritis and Rheumatism 60 2723–2730. (doi:10.1002/art.24745).
Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Kato Y, Takemoto F, Nakasa T & Yamashita S et al. 2010 MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes and Development 24 1173–1185. (doi:10.1101/gad.1915510).
Miyazono K, Kamiya Y & Morikawa M 2010 Bone morphogenetic protein receptors and signal transduction. Journal of Biochemistry 147 35–51. (doi:10.1093/jb/mvp148).
Mizoguchi F, Izu Y, Hayata T, Hemmi H, Nakashima K, Nakamura T, Kato S, Miyasaka N, Ezura Y & Noda M 2010 Osteoclast-specific Dicer gene deficiency suppresses osteoclastic bone resorption. Journal of Cellular Biochemistry 109 866–875. (doi:10.1002/jcb.22228).
Mizoguchi F, Murakami Y, Saito T, Miyasaka N & Kohsaka H 2013 miR-31 controls osteoclast formation and bone resorption by targeting RhoA. Arthritis Research & Therapy 15 R102. (doi:10.1186/ar4282).
Mizuno Y, Yagi K, Tokuzawa Y, Kanesaki-Yatsuka Y, Suda T, Katagiri T, Fukuda T, Maruyama M, Okuda A & Amemiya T et al. 2008 miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochemical and Biophysical Research Communications 368 267–272. (doi:10.1016/j.bbrc.2008.01.073).
Mizuno Y, Tokuzawa Y, Ninomiya Y, Yagi K, Yatsuka-Kanesaki Y, Suda T, Fukuda T, Katagiri T, Kondoh Y & Amemiya T et al. 2009 miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Letters 583 2263–2268. (doi:10.1016/j.febslet.2009.06.006).
Mori-Akiyama Y, Akiyama H, Rowitch DH & de Crombrugghe B 2003 Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest. PNAS 100 9360–9365. (doi:10.1073/pnas.1631288100).
Mura M, Cappato S, Giacopelli F, Ravazzolo R & Bocciardi R 2012 The role of the 3′UTR region in the regulation of the ACVR1/Alk-2 gene expression. PLoS ONE 7 e50958. (doi:10.1371/journal.pone.0050958).
Nakamura Y, Inloes JB, Katagiri T & Kobayashi T 2011 Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling. Molecular and Cellular Biology 31 3019–3028. (doi:10.1128/MCB.05178-11).
Nakashima K & de Crombrugghe B 2003 Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends in Genetics 19 458–466. (doi:10.1016/S0168-9525(03)00176-8).
Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR & de Crombrugghe B 2002 The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108 17–29. (doi:10.1016/S0092-8674(01)00622-5).
Nakashima T, Hayashi M, Fukunaga T, Kurata K, Oh-Hora M, Feng JQ, Bonewald LF, Kodama T, Wutz A & Wagner EF et al. 2011 Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nature Medicine 17 1231–1234. (doi:10.1038/nm.2452).
Nicolas FE, Pais H, Schwach F, Lindow M, Kauppinen S, Moulton V & Dalmay T 2011 mRNA expression profiling reveals conserved and non-conserved miR-140 targets. RNA Biology 8 607–615. (doi:10.4161/rna.8.4.15390).
Nishio Y, Dong Y, Paris M, O'Keefe RJ, Schwarz EM & Drissi H 2006 Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene. Gene 372 62–70. (doi:10.1016/j.gene.2005.12.022).
Ortuno MJ, Ruiz-Gaspa S, Rodriguez-Carballo E, Susperregui AR, Bartrons R, Rosa JL & Ventura F 2010 p38 regulates expression of osteoblast-specific genes by phosphorylation of osterix. Journal of Biological Chemistry 285 31985–31994. (doi:10.1074/jbc.M110.123612).
Pais H, Nicolas FE, Soond SM, Swingler TE, Clark IM, Chantry A, Moulton V & Dalmay T 2010 Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level. RNA 16 489–494. (doi:10.1261/rna.1701210).
Papaioannou G, Inloes JB, Nakamura Y, Paltrinieri E & Kobayashi T 2013 let-7 and miR-140 microRNAs coordinately regulate skeletal development. PNAS 110 E3291–E3300. (doi:10.1073/pnas.1302797110).
Park SJ, Cheon EJ, Lee MH & Kim HA 2013 MicroRNA-127-5p regulates matrix metalloproteinase 13 expression and interleukin-1β-induced catabolic effects in human chondrocytes. Arthritis and Rheumatism 65 3141–3152. (doi:10.1002/art.38188).
Pasero M, Giovarelli M, Bucci G, Gherzi R & Briata P 2012 Bone morphogenetic protein/SMAD signaling orients cell fate decision by impairing KSRP-dependent microRNA maturation. Cell Reports 2 1159–1168. (doi:10.1016/j.celrep.2012.10.020).
Plotkin LI & Bellido T 2013 Beyond gap junctions: connexin43 and bone cell signaling. Bone 52 157–166. (doi:10.1016/j.bone.2012.09.030).
Rodriguez-Carballo E, Ulsamer A, Susperregui AR, Manzanares-Cespedes C, Sanchez-Garcia E, Bartrons R, Rosa JL & Ventura F 2011 Conserved regulatory motifs in osteogenic gene promoters integrate cooperative effects of canonical Wnt and BMP pathways. Journal of Bone and Mineral Research 26 718–729. (doi:10.1002/jbmr.260).
Rossi M, Pitari MR, Amodio N, Di Martino MT, Conforti F, Leone E, Botta C, Paolino FM, Del Giudice T & Iuliano E et al. 2013 miR-29b negatively regulates human osteoclastic cell differentiation and function: implications for the treatment of multiple myeloma-related bone disease. Journal of Cellular Physiology 228 1506–1515. (doi:10.1002/jcp.24306).
Santini P, Politi L, Vedova PD, Scandurra R & Scotto d'Abusco A The inflammatory circuitry of miR-149 as a pathological mechanism in osteoarthritis Rheumatology International 2013 [in press] doi:10.1007/s00296-013-2754-8).
Sato MM, Nashimoto M, Katagiri T, Yawaka Y & Tamura M 2009 Bone morphogenetic protein-2 down-regulates miR-206 expression by blocking its maturation process. Biochemical and Biophysical Research Communications 383 125–129. (doi:10.1016/j.bbrc.2009.03.142).
Seitz H & Zamore PD 2006 Rethinking the microprocessor. Cell 125 827–829. (doi:10.1016/j.cell.2006.05.018).
Shi Y & Massague J 2003 Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell 113 685–700. (doi:10.1016/S0092-8674(03)00432-X).
Shi K, Lu J, Zhao Y, Wang L, Li J, Qi B, Li H & Ma C 2013 MicroRNA-214 suppresses osteogenic differentiation of C2C12 myoblast cells by targeting Osterix. Bone 55 487–494. (doi:10.1016/j.bone.2013.04.002).
Shibuya H, Nakasa T, Adachi N, Nagata Y, Ishikawa M, Deie M, Suzuki O & Ochi M 2013 Overexpression of microRNA-223 in rheumatoid arthritis synovium controls osteoclast differentiation. Modern Rheumatology 23 674–685. (doi:10.3109/s10165-012-0710-1).
Song J, Lee M, Kim D, Han J, Chun CH & Jin EJ 2013a MicroRNA-181b regulates articular chondrocytes differentiation and cartilage integrity. Biochemical and Biophysical Research Communications 431 210–214. (doi:10.1016/j.bbrc.2012.12.133).
Song J, Kim D, Lee CH, Lee MS, Chun CH & Jin EJ 2013b MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis. Journal of Biomedical Science 20 31. (doi:10.1186/1423-0127-20-31).
Starega-Roslan J, Koscianska E, Kozlowski P & Krzyzosiak WJ 2011 The role of the precursor structure in the biogenesis of microRNA. Cellular and Molecular Life Sciences 68 2859–2871. (doi:10.1007/s00018-011-0726-2).
Sugatani T & Hruska KA 2007 MicroRNA-223 is a key factor in osteoclast differentiation. Journal of Cellular Biochemistry 101 996–999. (doi:10.1002/jcb.21335).
Sugatani T & Hruska KA 2009 Impaired micro-RNA pathways diminish osteoclast differentiation and function. Journal of Biological Chemistry 284 4667–4678. (doi:10.1074/jbc.M805777200).
Sugatani T, Vacher J & Hruska KA 2011 A microRNA expression signature of osteoclastogenesis. Blood 117 3648–3657. (doi:10.1182/blood-2010-10-311415).
Sumiyoshi K, Kubota S, Ohgawara T, Kawata K, Abd El Kader T, Nishida T, Ikeda N, Shimo T, Yamashiro T & Takigawa M 2013 Novel role of miR-181a in cartilage metabolism. Journal of Cellular Biochemistry 114 2094–2100. (doi:10.1002/jcb.24556).
Susperregui AR, Gamell C, Rodriguez-Carballo E, Ortuno MJ, Bartrons R, Rosa JL & Ventura F 2011 Noncanonical BMP signaling regulates cyclooxygenase-2 transcription. Molecular Endocrinology 25 1006–1017. (doi:10.1210/me.2010-0515).
Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T & Inoue J et al. 2002 Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Developmental Cell 3 889–901. (doi:10.1016/S1534-5807(02)00369-6).
Tanaka S, Nakamura K, Takahasi N & Suda T 2005 Role of RANKL in physiological and pathological bone resorption and therapeutics targeting the RANKL–RANK signaling system. Immunological Reviews 208 30–49. (doi:10.1111/j.0105-2896.2005.00327.x).
Tang M, Lin L, Cai H, Tang J & Zhou Z 2013 MicroRNA-145 downregulation associates with advanced tumor progression and poor prognosis in patients suffering osteosarcoma. OncoTargets and Therapy 6 833–838. (doi:10.2147/OTT.S40080).
Tardif G, Hum D, Pelletier JP, Duval N & Martel-Pelletier J 2009 Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes. BMC Musculoskeletal Disorders 10 148. (doi:10.1186/1471-2474-10-148).
Tondravi MM, McKercher SR, Anderson K, Erdmann JM, Quiroz M, Maki R & Teitelbaum SL 1997 Osteopetrosis in mice lacking haematopoietic transcription factor PU.1. Nature 386 81–84. (doi:10.1038/386081a0).
Tuddenham L, Wheeler G, Ntounia-Fousara S, Waters J, Hajihosseini MK, Clark I & Dalmay T 2006 The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Letters 580 4214–4217. (doi:10.1016/j.febslet.2006.06.080).
Ulsamer A, Ortuno MJ, Ruiz S, Susperregui AR, Osses N, Rosa JL & Ventura F 2008 BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38. Journal of Biological Chemistry 283 3816–3826. (doi:10.1074/jbc.M704724200).
Vasudevan S, Tong Y & Steitz JA 2007 Switching from repression to activation: microRNAs can up-regulate translation. Science 318 1931–1934. (doi:10.1126/science.1149460).
Wang T & Xu Z 2010 miR-27 promotes osteoblast differentiation by modulating Wnt signaling. Biochemical and Biophysical Research Communications 402 186–189. (doi:10.1016/j.bbrc.2010.08.031).
Wang X, Guo B, Li Q, Peng J, Yang Z, Wang A, Li D, Hou Z, Lv K & Kan G et al. 2013a miR-214 targets ATF4 to inhibit bone formation. Nature Medicine 19 93–100. (doi:10.1038/nm.3026).
Wang J, Guan X, Guo F, Zhou J, Chang A, Sun B, Cai Y, Ma Z, Dai C & Li X et al. 2013b miR-30e reciprocally regulates the differentiation of adipocytes and osteoblasts by directly targeting low-density lipoprotein receptor-related protein 6. Cell Death & Disease 4 e845. (doi:10.1038/cddis.2013.356).
Wei J, Shi Y, Zheng L, Zhou B, Inose H, Wang J, Guo XE, Grosschedl R & Karsenty G 2012 miR-34s inhibit osteoblast proliferation and differentiation in the mouse by targeting SATB2. Journal of Cell Biology 197 509–521. (doi:10.1083/jcb.201201057).
Winbanks CE, Wang B, Beyer C, Koh P, White L, Kantharidis P & Gregorevic P 2011 TGF-β regulates miR-206 and miR-29 to control myogenic differentiation through regulation of HDAC4. Journal of Biological Chemistry 286 13805–13814. (doi:10.1074/jbc.M110.192625).
Wu CH & Nusse R 2002 Ligand receptor interactions in the Wnt signaling pathway in Drosophila. Journal of Biological Chemistry 277 41762–41769. (doi:10.1074/jbc.M207850200).
Wu T, Zhou H, Hong Y, Li J, Jiang X & Huang H 2012 miR-30 family members negatively regulate osteoblast differentiation. Journal of Biological Chemistry 287 7503–7511. (doi:10.1074/jbc.M111.292722).
Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC & O'Brien CA 2011 Matrix-embedded cells control osteoclast formation. Nature Medicine 17 1235–1241. (doi:10.1038/nm.2448).
Yamashita S, Miyaki S, Kato Y, Yokoyama S, Sato T, Barrionuevo F, Akiyama H, Scherer G, Takada S & Asahara H 2012 L-Sox5 and Sox6 proteins enhance chondrogenic miR-140 microRNA expression by strengthening dimeric Sox9 activity. Journal of Biological Chemistry 287 22206–22215. (doi:10.1074/jbc.M112.343194).
Yang J, Qin S, Yi C, Ma G, Zhu H, Zhou W, Xiong Y, Zhu X, Wang Y & He L et al. 2011 MiR-140 is co-expressed with Wwp2-C transcript and activated by Sox9 to target Sp1 in maintaining the chondrocyte proliferation. FEBS Letters 585 2992–2997. (doi:10.1016/j.febslet.2011.08.013).
Yang L, Cheng P, Chen C, He HB, Xie GQ, Zhou HD, Xie H, Wu XP & Luo XH 2012 miR-93/Sp7 function loop mediates osteoblast mineralization. Journal of Bone and Mineral Research 27 1598–1606. (doi:10.1002/jbmr.1621).
Yang J, Gao T, Tang J, Cai H, Lin L & Fu S 2013 Loss of microRNA-132 predicts poor prognosis in patients with primary osteosarcoma. Molecular and Cellular Biochemistry 381 9–15. (doi:10.1007/s11010-013-1677-8).
Yoshida CA, Yamamoto H, Fujita T, Furuichi T, Ito K, Inoue K, Yamana K, Zanma A, Takada K & Ito Y et al. 2004 Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog. Genes and Development 18 952–963. (doi:10.1101/gad.1174704).
Yoshida CA, Komori H, Maruyama Z, Miyazaki T, Kawasaki K, Furuichi T, Fukuyama R, Mori M, Yamana K & Nakamura K et al. 2012 SP7 inhibits osteoblast differentiation at a late stage in mice. PLoS ONE 7 e32364. (doi:10.1371/journal.pone.0032364).
Yoshitaka T, Kawai A, Miyaki S, Numoto K, Kikuta K, Ozaki T, Lotz M & Asahara H 2013 Analysis of microRNAs expressions in chondrosarcoma. Journal of Orthopaedic Research 31 1992–1998. (doi:10.1002/jor.22457).
Zhang Y, Xie RL, Croce CM, Stein JL, Lian JB, van Wijnen AJ & Stein GS 2011a A program of microRNAs controls osteogenic lineage progression by targeting transcription factor Runx2. PNAS 108 9863–9868. (doi:10.1073/pnas.1018493108).
Zhang J, Tu Q, Grosschedl R, Kim MS, Griffin T, Drissi H, Yang P & Chen J 2011b Roles of SATB2 in osteogenic differentiation and bone regeneration. Tissue Engineering. Part A 17 1767–1776. (doi:10.1089/ten.tea.2010.0503).
Zhang JF, Fu WM, He ML, Wang H, Wang WM, Yu SC, Bian XW, Zhou J, Lin MC & Lu G et al. 2011c MiR-637 maintains the balance between adipocytes and osteoblasts by directly targeting Osterix. Molecular Biology of the Cell 22 3955–3961. (doi:10.1091/mbc.E11-04-0356).
Zhang J, Tu Q, Bonewald LF, He X, Stein G, Lian J & Chen J 2011d Effects of miR-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1. Journal of Bone and Mineral Research 26 1953–1963. (doi:10.1002/jbmr.377).
Zhang Y, Xie RL, Gordon J, LeBlanc K, Stein JL, Lian JB, van Wijnen AJ & Stein GS 2012a Control of mesenchymal lineage progression by microRNAs targeting skeletal gene regulators Trps1 and Runx2. Journal of Biological Chemistry 287 21926–21935. (doi:10.1074/jbc.M112.340398).
Zhang J, Zhao H, Chen J, Xia B, Jin Y, Wei W, Shen J & Huang Y 2012b Interferon-β-induced miR-155 inhibits osteoclast differentiation by targeting SOCS1 and MITF. FEBS Letters 586 3255–3262. (doi:10.1016/j.febslet.2012.06.047).
Zhou X, Zhang Z, Feng JQ, Dusevich VM, Sinha K, Zhang H, Darnay BG & de Crombrugghe B 2010 Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. PNAS 107 12919–12924. (doi:10.1073/pnas.0912855107).