MicroRNAs and their roles in osteoclast differentiation

Abstract

Osteoclasts, which are derived from monocyte-macrophage precursors, are exclusive bone resorption cells. Limited evidence indicates that a class of small non-coding single-stranded RNAs known as “microRNAs” (miRNAs) is also involved in bone resorption. Although various miRNAs regulate cell proliferation and differentiation, few miRNAs have been reported to play a key role in the regulation of osteoclast differentiation. In this short review, the biology and functional mechanisms of miRNAs in osteoclastogenesis are summarized. The profiling, function, and target prediction of miRNAs are discussed as well.

Keywords

miRNA / osteoclast differentiation / antagomirs

Introduction

Osteoclasts, exclusive bone resorptive cells, are derived from monocyte-macrophage precursors, which are differentiated from myeloid and multipotent hematopoietic stem cells [1]. Osteoclastogenesis is regulated by cytokines, such as the macrophage colony-stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL), and exogenous hormones, such as sex steroids, parathyroid hormone (PTH), vitamin D, insulin-like growth factor-1 (IGF-1), calcitonin, and prostaglandins [2,3]. Many transcription factors, including c-Fos, PU.1, NFκB (p50 and p52), and NFATc1 (nuclear factor of activated T cells, cytoplasmic 1), have been identified to be involved in osteoclastogenesis [4-8]. Moreover, recent studies indicate that a class of small non-coding RNAs known as “microRNAs” (miRNAs) is necessary for osteoclast differentiation, function, and survival. MiRNAs are single-stranded RNAs approximately 19 nt to 25 nt in length that regulate various pathways, including development timing, hematopoiesis, organogenesis, apoptosis, cell proliferation, and tumorigenesis [9]. miRNAs, including miR-125b [10], miR-26a [11], miR-133 and miR-135 [12], miR-204/211 [13], miR-29a [14], miR-141 and miR-200a [15], miR-206 [16], miR-29b [17], miR-210 [18], miR-196a [19], and miR-2861 [20], among others, are involved in osteoblast differentiation and bone formation. In particular, miR-2861 promotes BMP2-induced ST2 osteogenic differentiation by inhibiting histone deacetylase 5 expression and contributing to bone formation. Moreover, the mutation of miR-2861 causes osteoporosis in humans, suggesting that miR-2861 plays a critical role in the pathogenesis of bone disease [20]. Although various miRNAs have been reported to regulate osteoblastic differentiation and bone formation, limited information about the regulatory mechanism of miRNAs in osteoclastic differentiation is available.

Osteoporosis is becoming a serious public health problem as the aging of the global population accelerates. It results from excessive bone loss, which is largely due to increased bone resorption by osteoclasts and/or decreased bone formation by osteoblasts [21]. Evidence has demonstrated the crucial role of miRNAs in osteoblastic differentiation and bone formation. Both processes have been shown to be related to osteoporosis. However, the function of miRNAs in osteoporosis, which is related to osteoclasts, remains unclear.

Here, we review the biology and functions of miRNAs and their role in osteoclastic differentiation.

miRNAs: definition, biology, and function

miRNAs have been reported to be involved in almost every biological process, including development timing, cell differentiation, cell proliferation, cell death, metabolic control, transposon silencing, and antiviral defense [22]. Hundreds of miRNAs, which are evolutionarily conserved [23], have been identified in humans. To date, more than 3% of the genes in humans have been found to encode for miRNAs. About 40 % to 90 % of the human protein encoding genes are under miRNA-mediated gene regulation [24]. miRNA genes are found as single or clustered transcription units [25,26] and expressed from the intron regions of protein-coding or non-protein-coding genes; in some cases, they are expressed as independent transcription units [9,27]. They mediate post-transcriptional gene silencing by binding to sites of antisense complementarity in 3′ untranslated regions (UTRs) of target mRNAs [28].

miRNAs are synthesized through multiple steps. Initially, a miRNA is transcribed into a primary miRNA (pri-miRNA) in the nucleus by RNA polymerase II. pri-miRNA is processed by the double-stranded RNA binding protein DiGeorge syndrome critical region gene 8 and the nuclear RNase Ш enzyme Drosha into stem-loop-structured pre-miRNAs [29]. The pre-miRNA is further transported to the cytoplasm by the Ras-related nuclear protein-guanosine-5′-triphosphate-dependent export receptor Exportin-5 and then cleaved by a second RNase III endonuclease Dicer. This process forms a transient, double-stranded miRNA of 22 nt. The miRNA duplex is incorporated into a multicomponent protein complex known as RNA-induced silencing complex (RISC), which contains the Argonuate 2 protein [30,31]. During the functional process, one strand of the miRNA duplex is rapidly removed and degraded, while the other strand is selected as a mature miRNA. The mature miRNA negatively regulates gene expression through translational repression or mRNA cleavage, which depends on the extent of complementarity between the miRNA and its target. If the target mRNA has perfect complementarity to the miRNA-armed RISC, the mRNA will be cleaved and degraded [32,33]. However, similar to the most common finding in mammalian cells, miRNAs target mRNAs with imperfect complementarity and suppress their translation, resulting in the reduced expression of the corresponding proteins [34,35]. Fig. 1 shows the biogenesis and function of miRNAs.

miRNA profiling

Recent research has applied different methodologies to profile miRNA expression, including Northern blots with radio-labeled probes, oligonucleotide microarrays, quantitative polymerase chain reaction-based amplification of precursor or mature, bead-based profiling methods, and DNA microarrays spotted onto glass surfaces [36]. In contrast to most mRNA profiling technologies, miRNA profiling must address the short nature (~22 nt) of miRNAs and should be able to distinguish between miRNAs that differ by as little as a single nucleotide. Here, we demonstrate three such technologies [37].

Microarray technology has been widely and successfully used in genomic and biologic research, [38]. The application of locked nucleic acid (LNA), one of many successful examples of microarray technology adopted into miRNA studies, modifies oligonucleotide probes by modifying the LNA contents in the probe and eliminating the diversity of melting temperature (T_m) values in individuals. Thus, microarray technology enhances binding affinity and leads to the improvement of miRNA detection specificity and sensitivity [37].

Bead-based arrays, which carry uniquely colored (up to 100 colors), five-micron polystyrene beads and are covered with oligonucleotide capture probes specific to a single miRNA, are hybridized to biotinylated miRNA and then stained with streptavidin-phycoerythrin. Bead-based arrays allow for the inclusion of many combinations of miRNA-capturing beads into a single pool, which are adjusted based on the interaction of bead-coupled probes and provide greater flexibility over time as more miRNAs are discovered and their corresponding beads are created [39].

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is a rapid and reproducible method that has been widely applied in miRNA research over the years; the procedure is recognized as a gold standard for miRNA determination [40]. TaqMan technology, a relatively mature technology for the qRT-PCR application, has been adopted into miRNA research; it utilizes a stem-loop structure specific for binding mature miRNA. An innovative design of TaqMan Low Density Arrays (TLDA), developed from TaqMan technology, has become a medium-throughput method for real-time RT-PCR that uses 384-well microfluidics cards. A single TLDA card may assay up to 384 miRNAs [41].

To study miRNAs in osteoclast differentiation, Sugatani et al. isolated miRNAs in bone marrow-derived monocyte/macrophage precursors (BMMs) after RANKL-induced osteoclastogenesis. They used microarrays performed with probes for 617 mature mouse miRs that present in miRBase. Result showed that 33 miRs were downregulated by RANKL stimulation, 38 were upregulated, and 2 were robustly stimulated. One of the two miRs was miR-21, which has been demonstrated to be a mediating factor in RANKL-induced osteoclastogenesis [42].

Action of miRNAs on osteoclast differentiation and their relationship with osteoporosis

Osteoclasts are derived from monocyte-macrophage precursors, which rise from multipotent hematopoietic stem cells. Multipotent hematopoietic stem cells give rise to myeloid stem cells, which further differentiate into megakaryocytes, granulocytes, and monocytes/macrophages [3]. Osteoclasts are unique cells that resorb bone [3]. The degeneration of these cells leads to either failed or increased bone resorption [43]. Osteoclastogenesis is regulated by cytokines and exogenous hormones through several transcription factors that positively or negatively modulate osteoclast proliferation, survival, differentiation, and function [44]. These cytokines include macrophage M-CSF, RANKL, tumor necrosis factor-α, interleukin-1, and interleukin-6 [2,3]. Compared with other cytokines, M-CSF and RANKL, which are produced by osteoblasts or activated T cells, are significantly important. Both M-CSF and M-CSF receptor deficiencies are characterized by osteopetrosis and the lack of macrophages and osteoclasts [2,3]. Exogenous hormones include sex steroids, parathyroid hormone, vitamin D, insulin-like growth factor-1, calcitonin, and prostaglandins [2,3]. A number of transcription factors, including c-Src, c-Fos, PU.1, NFκB (p50 and p52), and NFATc1, have been identified to be involved in osteoclastogenesis [4-8]. In particular, c-Fos and NFκB (p50 and p52) are required for the differentiation of monocyte precursors into osteoclasts [5,45]; c-Fos also induces a second transcription factor, NFATc1, that is essential for osteoclastogenesis [3,46]. NFATc1 was discovered in early RANKL-inducible gene-by-gene expression profiling after RANKL stimulation [47]. NFATc1 molecules act as cofactors with activator protein-1 (AP-1) composed of Fos/Jun proteins to bind to regulatory cis DNA elements. Osteoclast-specific markers, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, have multiple sites recognized by NFATc1, as well as its partner AP-1 [48]. Other transcription factors, such as microphthalmia-associated transcription factor, are also involved in osteoclastogenesis [49].

The function of miRs on osteoclastogenesis, however, remains incompletely understood, and limited information on the regulatory mechanism of miRNAs in the osteoclastic differentiation is available. Sugatani found the miR signature of RANKL-induced osteoclastogenesis, showing 38 elevated miRs and 33 downregulated ones [40]. Specifically, miR-21 was robustly stimulated and identified to be involved in osteoclastogenesis in cultures. The expression of miR-21 was reported to be controlled by the transcription factors for osteoclastogenesis, particularly by c-Fos [40]. RANKL-induced c-Fos upregulates miR-21, which downregulates programmed cell death protein 4 (PDCD4) expression levels so that the transcription factors for osteoclastogensis and osteoclast-specific markers are transcribed. This process showed a positive feedback loop of c-Fos/miR-21/PDCD4, regulating osteoclastogenesis [40].

miR-223 has been proven to be expressed in the RAW264.7 osteoclast precursor cell line and plays a crucial role in osteoclast differentiation. PU.1 induced by M-CSF stimulates the expression of miRNA-223, which downregulates the NFI-A levels required for upregulating macrophage colony-stimulating factor receptor (M-CSFR) levels in cells. As a result, the expression levels of transcription factors, such as PU.1, MITF and c-Fos, are increased. The increases are induced by M-CSF and RANKL through upregulated M-CSFR and RANK. Consequently, cells differentiate into activated osteoclasts by the upregulated osteoclast-specific markers [50]. Whether or not other miRNAs play important roles in osteoclastic differentiation has yet to be reported.

Osteoporosis is a common disorder that leads to reduction in bone mass, deterioration in bone microarchitecture, susceptibility to skeletal fragility, and increased risk of fracture [51]. It results from excessive bone loss, which is largely due to increased bone resorption by osteoclasts and/or decreased bone formation by osteoblasts [21]. Bone tissue arises from mesenchymal stromal cells (MSCs) and differentiates into the osteoblast lineage by genetic and epigenetic mechanisms. MSCs are multipotent cells that have the potential to self-renew and differentiate into various lineages of mesenchymal tissues, such as bones, cartilages, adipose tissues, tendons, and muscles. The role of miRNA in these processes has been revealed in recent studies [52]. Various miRNAs have been reported to regulate osteoblastic differentiation, proliferation, and bone formation, and are related to osteoporosis. However, the function of miRNAs in osteoporosis related to osteoclasts remains unclear.

miRNAs target prediction and validation

The 5′ proximal end of a mature miRNA (nucleotide positions 2nd to 7th, or 2nd to 8th) is referred to as the seed region. This region is important for target recognition by binding to the seed (nucleus) complementary sequence in the 3′ UTR of target mRNAs. Computational and experimental techniques for predicting miRNA targets are mainly based on programming alignment to identify conserved complementary motifs in the 3′ UTR with the seed sequence of the miRNA [53]. These sophisticated bioinformatic approaches reveal that a miRNA is estimated to target, on average, hundreds of mRNAs [54].

In the last few years, many distinct computational methods and algorithms have been developed to predict miRNA targets. Among them, TargetScanS [55], PicTar [56], and miRanda [57] are the most common target prediction programs, whereas miRBase [58], Argonaute, miRNAMap [59], and miRGen [60] are databases that combine a compilation of miRNAs with target prediction modules.

Prediction of miRNA targets by computational approaches is based mainly on the following steps. The first step is the identification of potential miRNA binding sites in the mRNA 3′ UTR according to specific base-pairing rules, especially strong Watson-Crick base-pairing of the seed region (6 or 7 nt at the 5′ end) of the miRNA to a complementary site in the 3′ UTR of the mRNA. The second step involves the implementation of cross-species conservation requirements. The third step requires local miRNA-mRNA interactions with a positive balance of minimum free energy.

One direct method for validating targets is the use of a luciferase reporter plasmid with a replaceable 3′ UTR. By cloning sequences corresponding to individually predicted target sites into the 3′ UTR of this reporter and cotransfecting it with miRNA precursors, the inhibition of luciferase activity can be measured.

Antagomirs

To develop an investigative and therapeutic approach for silencing miRNAs in vivo, a class of chemically modified antisense oligonucleotides (ASOs) complementary to specific miRNAs, also known as “antagomirs,” has been designed. These cholesterol-conjugated single-stranded RNA analogs have been shown to transiently interfere with the miRNA function in cell culture reporter assays and mice [61-63]. Antagomirs are specific, efficient, and long-lasting silencers of endogenous miRNAs. An effective ASO is resistant to both non-specific cellular ribonucleases and miRNA-directed cleavage by RISC and binds miRNAs in RISC with high affinity [64]. Inhibition studies with ASOs have been widely used in evaluating gene functions in vitro and in vivo [65,66] and in several antisense therapeutics in clinical trials [67,68].

Research works have used the overexpression or inhibition of miRNAs with corresponding antisense miRNA oligoribonucleotides to further study the miRNA functions in osteoclast differentiation. To investigate whether or not the expression of miR-21 is of critical importance in osteoclastogenesis, Sugatani et al. transduced BMMs with antisense miR-21-containing lentivirus. Levels of PDCD4 protein, a target of miR-21, were found to be extremely upregulated. RANKL-induction of c-Fos phosphorylation and NFATc1 and cathepsin K protein expression were remarkably downregulated in cells harboring reduced levels of miR-21. These findings indicate the relevance of miR-21 expression in the mechanism of osteoclast development [42].

To further investigate whether or not miRNA-223 is essential for osteoclastogenesis, Sugatani et al. performed osteoclast formation assays using antisense-miRNA-223 oligonucleotides. Inhibition of miRNA-223 induced the downregulation of TRAP-positive osteoclast formation, and Western blot analysis showed the upregulation of NFI-A levels, which had a subsequent negative effect on M-CSFR levels. These findings suggest that miRNA-223 expression is critically important for osteoclastogenesis through the M-CSFR expression [50].

Concluding remarks

In recent years, an increasing number of advanced research works on miRNA have provided better understanding of and new insights into the biogenesis and functions of miRNAs, particularly regarding their association with the molecular pathogenesis of a variety of complex diseases. However, information regarding the role of miRNAs in osteoclastogensis and the function of miRNAs in osteoporosis, which is related to osteoclasts, remains limited. Further investigations on miRNA functions in osteoclast differentiation and possible therapeutic strategies for the treatment of osteoporosis need to be conducted.

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