Abstract
Purpose of Review
Osteogenesis is a complex process involving the specification of multiple progenitor cells and their maturation and differentiation into matrix-secreting osteoblasts. Osteogenesis occurs not only during embryogenesis but also during growth, after an injury, and in normal homeostatic maintenance. While much is known about osteogenesis-associated regulatory genes, the role of microRNAs (miRNAs), which are epigenetic regulators of protein expression, is just beginning to be explored. While miRNAs do not abrogate all protein expression, their purpose is to finely tune it, allowing for a timely and temporary protein down-regulation.
Recent Findings
The last decade has unveiled a multitude of miRNAs that regulate key proteins within the osteogenic lineage, thus qualifying them as “ostemiRs.” These miRNAs may endogenously target an activator or inhibitor of differentiation, and depending on the target, may either lead to the prolongation of a progenitor maintenance state or to early differentiation. Interestingly, cellular identity seems intimately coupled to the expression of miRNAs, which participate in the suppression of previous and subsequent differentiation steps. In such cases where key osteogenic proteins were identified as direct targets of miRNAs in non-bone cell types, or through bioinformatic prediction, future research illuminating the activity of these miRNAs during osteogenesis will be extremely valuable.
Summary
Many bone-related diseases involve the dysregulation of transcription factors or other proteins found within osteoblasts and their progenitors, and the dysregulation of miRNAs, which target such factors, may play a pivotal role in disease etiology, or even as a possible therapy.
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Introduction
Birth defects that affect the skeleton account for 5% of all infant deaths. In survivors, skeletal defects can result in lifelong burdens ranging from stunted growth and malformations to poor bone density and the need for surgery. The key to understanding normal and abnormal bone formation, in addition to finding possible treatments for bone disorders, lies in studying the genetic regulation of osteogenesis. Recent advances in genomics have led to the discovery of endogenous small RNA molecules, known as microRNAs (miRNAs, miRs), which regulate genetic expression at the transcript level through degradation or a translational halt of the target transcript. Understanding how miRNAs contribute to the regulation of osteogenesis may provide insights into the molecular causality of skeletal defects and disorders.
Faulty Osteogenesis as the Root Cause of Skeletal Defects
Osteogenesis describes the process of bone formation during early development, bone homeostasis in adults, and bone remodeling after an injury. Osteoblasts, the cells that secrete a bone-specific extracellular matrix (ECM), which later becomes calcified and mineralized (Fig. 1), is derived from progenitor cells of a distinct germ layer origin in the embryo. Vertebrae and some craniofacial bones are derived from paraxial mesoderm, while the appendicular skeleton, or long bones, are derived from lateral plate mesoderm [5]. The majority of the craniofacial bones and cartilage however are derived from cranial neural crest cells (NCCs).
Osteogenesis in the long bones and from cranial neural crest cells. In the lateral mesoderm-derived long bones, osteoblasts are created through three different processes: (i) in the perichondrium, they are directly differentiated from mesenchymal cells; (ii) in trabecular bone, they are also created from mesenchymal cells, but with influence from hypertrophic chondrocytes; (iii) trabecular osteoblasts, according to recent lineage tracing evidence, may also derive from transdifferentiating hypertrophic chondrocytes [1,2,3,4]. In contrast, neural crest-derived craniofacial bone is differentiated from cranial neural crest cells that specify at the neural plate border, which later delaminate and become migratory. Through intramembranous ossification, these crest-derived progenitors first differentiate into mesenchyme, which directly converts into pre-osteoblasts without a cartilage intermediate. Developmental trajectory is denoted by encircled numbers
Independent of their origin, bone progenitor cells condense at sites of future skeletal element formation. These condensations are composed of loosely packed mesenchymal cells, which during endochondral ossification differentiate into chondrocytes, undergo hypertrophy, apoptosis, and are later replaced by osteoblasts [5] (Fig. 1). Recent lineage-tracing experiments ended a decade long debate whether the cartilage anlage was necessary for mesodermal progenitors to differentiate into osteoblasts. That it was dispensable was demonstrated by showing that a subset of collagen X expressing hypertrophic chondrocytes survive and can directly transdifferentiate into osteoblasts [1,2,3,4] (Fig. 1), a hypothesis that had long been fueled by the fact that these transitory chondrocytes begin to express Runt-related factor 2 (Runx2) [6,7,8], a master osteogenic transcription factor (TF) [9•]. In contrast, during intramembranous ossification or dermal bone formation, progenitor cells of neural crest cell origin differentiate directly into osteoblasts (reviewed in [10]) (Fig. 1). In cases where mesenchymal cells commit directly to the osteogenic lineage, they first form pre-osteoblasts, which have the ability to proliferate expansively [11]. Osteocytes, the terminally matured osteoblasts, are fully surrounded by ECM and communicate with each other through dendritic processes to sense mechanical stimuli and regulate mineral homeostasis [12•].
Dysregulation of any event during osteogenesis can result in a wide array of skeletal disorders and diseases. While cleft lip and palate may be visibly apparent immediately at birth [13], others, such as osteogenesis imperfecta (OI) or brittle bone disease, present a variety of symptoms from loose joints to weak bones, which cause dysmorphism and premature death later in life [14]. Some rarer diseases, such as fetal skeletal dysplasia, exhibit stunted bone growth, poor mineralization, and limb agenesis [15]. The underlying causes may be genetic, including mutations in genes such as collagen and fibroblast growth factor receptors, or may be environmental such as in the case of phosphate or vitamin D metabolism deficiency [15].
Transcriptional Control of Osteogenesis
The etiology of skeletal malformations is often associated with the misregulation of the genes coding for ECM proteins or master transcription factors. In osteogenesis, master lineage regulatory TFs include Runx2, also known as Core-binding factor α1, and Osterix (Osx) [16]. RUNX2 controls the promoters of all major osteoblast ECM genes, including Col1a1 (type I collagen), Spp1 (osteopontin), Ibsp (bone sialoprotein, BSP), and Bglap (osteocalcin, OCN), the transcription of which contributes to the establishment of an osteoblast phenotype [9•, 17]. Consequently, disrupted Runx2 results in a complete lack of bone [6]. Furthermore, mutations in RUNX2 that diminish protein activity can lead to cleidocranial dysostosis [18], a disease characterized by absent parietal bones and delayed skull ossification [19].
As a central mediator of osteogenesis, RUNX2 executes signals from the Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGFs) signaling pathways [16, 20,21,22]. In Runx2 expressing cells, Osx is responsible for their final commitment to mature osteoblasts [23]. OSX works partially through activating the Wnt signaling pathway [24], downstream of which transcription of Runx2 is activated [25]. Thus, it appears that Runx2 and Osx work in a feed-forward loop that implements the osteogenic phenotype [26•].
microRNAs in Disease Etiology and Development
The molecular underpinnings of osteogenic gene expression have been extensively studied in a variety of model organisms [26•], but only recently have we started to explore the critical roles that miRNAs play in their regulation. After the first miRNA, lin-4, was discovered in Caenorhabditis elegans [27•], it was subsequently found that miRNAs were highly conserved throughout the animal kingdom, as well as in plants [28,29,30]. Furthermore, genetic studies in animal models demonstrated that miRNAs play crucial roles in animal development [31, 32] and disease etiology [33, 34]. Instead of representing all-or-nothing on/off switches, miRNAs fine-tune gene expression by dampening protein expression epigenetically through binding to the 3′ UTR of mRNA transcripts [35]. Accelerated deadenylation and subsequent decapping destabilizes these mRNAs [36], ultimately reducing protein abundance.
Usually found throughout intergenic regions of the genome, miRNA genes often cluster together, such that they can be transcribed as poly-cistronic transcripts [37] (Fig. 2). Polymerase II transcription generates a stem-loop structure containing the primary miRNA (pri-miRNA), which can range in size from hundreds of nucleotides up to kilobases [47]. The pri-miRNA then adopts a secondary stem loop structure, which undergoes nuclear cleavage by a multiprotein complex. The core components of this complex are the RNase III enzyme Drosha and the dsRNA-binding domain protein DGCR8 (DiGeorge syndrome chromosomal [or critical] region 8) [48]. The resulting hairpin-shaped pre-miRNA, which is about 65 nucleotides long, contains a 2-nt 3′ overhang that is recognized by exportin-5, allowing nuclear export via a Ran-GTP-dependent mechanism [40]. microRNAs, which are encoded in introns and are part of the primary mRNA transcript, are processed by the spliceosome, which also produces a pre-miRNA that is then shuttled into the cytoplasm for further processing via the same mechanism [38, 39].
microRNA biogenesis and target recognition. miRNAs encoded in intergenic regions can be transcribed between exons or as polycistronic miRNAs by RNA polymerase II/III [37]. Single transcribed miRNAs are spliced out of the exons by the spliceosome into a hairpin structure known as pre-miRNAs, while polycistronic miRNAs are known as pri-miRNAs before being processed to single pre-miRNAs by DROSHA and DGCR8 proteins [38, 39]. The pre-miRNAs are then exported from the nucleus via Exportin-5 [40]. The hairpin loop of the pre-miRNA is then cleaved off by Dicer, forming a miRNA duplex [41], and loaded onto the RNA induced silencing complex (RISC) via Argonaut proteins [42, 43]. The miRNA along with RISC is then loaded onto the target mRNA transcript and can halt transcription [43] or cause degradation by exonuclease activity [44,45,46]
Next, the pre-miRNA is cleaved by cytoplasmic Dicer to produce the mature ~19–25 nt miRNA duplex [41]. The miRNA strand with the lower relative thermodynamic stability of base-pairing at its 5′ end is then loaded onto an Argonaut protein and incorporated into the RNA-induced silencing complex (RISC). This results in a mature miRNA, which is then directed to the 3′ UTR of the target mRNAs [42]. Based on partial complementarity, the mRNA targets are then blocked from being translated [43] or directed to the cellular 5′-to-3′ mRNA decay pathway. There, mRNAs are first deadenylated, decapped, and ultimately degraded by the cytoplasmic 5′-to-3′ exonuclease XRN1 [44,45,46].
Based on these mechanisms, the regulatory nature of miRNAs is multifold. Due to the partial complementarity described above, one miRNA may have several target sites in the 3′UTR of a single mRNA, and one 3′UTR has tens to hundreds of different binding sites for different miRNAs. Thus, multiple co-expressed miRNAs may act in concert to ensure that the expression of a specific mRNA is repressed as efficiently as possible. In addition, because one miRNA has multiple distinct mRNA targets, a down-regulated miRNA may offset the up-regulation of another in the control of a common target mRNA. Given these considerations, it appears that the complexity of osteogenesis is compounded by miRNA expression as well as by any feedback regulation that may exist between the microRNAs and their targets, a few examples of which will be discussed in this review.
Of the hundreds of miRNAs confirmed to exist, a subset of them associated with terminal osteoblast differentiation and acquisition of osteogenic identity from already committed progenitors have been designated “ostemiR” [49•]. This review discusses the role of microRNAs during early and late specification of progenitor cells with a particular emphasis on osteogenic potential in the craniofacial skeleton. In addition to the known ostemiRs (Table 1), we also discuss miRNAs that silence proteins associated with osteogenesis in non-bone tissues.
microRNAs that Control Osteoblast Identity and Homeostasis
Osteoblasts secrete a variety of unique proteins that comprise an extremely specialized ECM. A subset of these serve as a scaffold upon which mineral is deposited in a final maturation step, while others impart structural flexibility to withstand compressive and tensile stress. Any microRNA targeting the mRNAs for the non-collagenous glycoproteins and proteoglycans such as osteonectin, BSP, and OCN, which are all implicated in calcification of the ECM [80], would execute an important control over matrix mineralization. Consequently, the osteonectin-targeting miR-29a and -29c appear to play a role in osteoblast maturation and indeed, their expression levels increase during late osteogenesis [50]. Similarly, miR-125b, which directly targets the OCN mRNA, Bglap, is highly expressed in primary human osteoblasts isolated from human trabecular bone and thus is implicated in normal bone homeostasis [52].
In bone, vitamin D bound to its receptor, VDR, participates in the mineralization process, and disruption to the VDR pathway can lead to mineralization defects such as those found in Rickets [81]. The importance of miR-125b in osteoblast identity is further illustrated in the fact that exogenous miR-125b blocked differentiation, while in contrast, its inhibition indirectly yielded higher ALP activity [82]. Furthermore, in breast and prostate cancer, which often metastasize to bone [83], down-regulation of miR-125b results in increased expression of its target Vdr [53], again demonstrating the importance of miR-125b in osteogenesis. While the only currently reported miRNAs directly targeting the bone-type Alpl are miR-204/211 [54], 16 additional conserved miRNA binding sites are predicted in its 3′UTR (microrna.org). Since expression of ALP not only maintains, but also initiates matrix mineralization, thusly associating with earlier stages of osteogenesis, the miRNAs post-transcriptionally regulating this enzyme may be expressed by mesenchymal stem cell as was indeed confirmed for miR-204 [71•].
Once fully embedded in the matrix, osteoblasts take on new functions as they terminally differentiate into osteocytes, which sense and respond to mechanical stimulation in a bone-anabolic manner. This response is mediated by cyclooxygenase-2 (COX2) [84, 85], which produces proliferation-stimulating prostaglandins [86]. While no Cox2-regulating microRNAs have been identified in the context of osteoblasts or mechanical loading, miR-101a and miR-199a down-regulate Cox2 in early mouse embryos during implantation [55]. Identified in several miRNA screens, miR-199a associates with osteoblast differentiation [87, 88], suggesting that its identification as an ostemiR may be linked to its control of Cox2. Additionally, while not directly linked to Cox2, miR-218, miR-191*, miR-3010a, and miR-33 were recently identified in MC3T3-E1 osteoblastic cells to be responsive to mechanical strain [89].
miRNAs Expressed at the Preosteoblast Stage
Prior to matrix calcification, proliferating osteoprogenitors secrete OPN [90], encoded by Spp1. While several microRNAs have been associated with Spp1 regulation (i.e., miR-541 [49•]; miR-21 [91]), no miRNA directly silencing Spp1 has been identified in osteoblasts or their precursors. However, miR-299-5p targets Spp1 in breast cancer cells [57] and miR-127-5p targets Spp1 in chondrocytes [58] and thus they may also do so in pre-osteoblasts.
The non-collagenous component of the ECM constitutes only a small portion. In fact, over 90% of the secreted ECM proteins are collagenous fibrils, primarily those of type I collagen. Since collagen is so abundant, it is not surprising that perturbation of collagen production is a root cause of OI [92, 93]. Any miRNA regulating collagen genes may thus not only be important for normal osteogenesis, but their misregulation could potentially be implicated in the etiology of OI. For example, miR-29b directly regulates Col1a1, Col1a2, and Col3a1 in hepatic cells [59], but this has not yet been replicated in cells of the osteogenic lineage. Secretion of ECM proteins decreases in association with prolonged differentiation, and the function of miR-29b in osteoblasts could be to suppress the expression of collagen proteins allowing the collagen fibril matrix to mature for mineral deposition [94]. Consequently, the expression of miRNAs similar to miR-29b would be expected to be higher as osteoblasts transition to the calcification step.
Rat mesenchymal stem cells overexpressing miR-21 exhibit enhanced performance in a fracture healing model in vivo [91]. The pro-osteogenic effect of miR-21 may occur through an indirect regulation of Col1a1, since miR-21 indirectly causes elevated expression of Col1a1 and in turn deposition of type I collagen, in a murine model of lung fibrosis [95]. Interestingly, recombinant COL1A1 positively regulates miR-21 expression, illustrating an unknown mechanistic feedback loop between protein and miRNA that likely prevents complete transcript shut-off [95]. miR-21 is also increased by the pro-osteogenic TGFβ and BMPs, but surprisingly not through transcriptional control, but rather through enhanced processing of the pri-miRNA [96•]. The designation of miR-21 as an ostemiR is however controversial, because miR-21 is up-regulated in most types of cancer [97].
Another ECM protein produced in pre-osteoblasts is fibronectin, an adhesion protein, which binds to integrins to influence cell proliferation and tissue development [98, 99]. Although miR-200b and miR-377 regulate fibronectin in kidney proximal tubular cells and in diabetic neuropathy respectively, a role for these miRNAs in osteogenesis remains to be determined [60, 61]. Interestingly, miR-377 is expressed during osteogenic differentiation of human dental pulp stem cells [100] and is up-regulated in response to contact of human osteoblast-like MG-63 cells with osteo-inductive biomaterials used for surgical bone restoration [101, 102].
microRNAs Associated with the Mesenchymal State and Mesenchymal Commitment
Mesenchymal stem or stromal cells (MSCs) have the potential to generate skeletal as well as connective tissue [103]. Markers that define MSC identity have been agreed on by the International Society for Cellular Therapy to include the surface expression of CD90, CD73, and CD105 [104]. In terms of microRNAs however, none are known to directly target the corresponding mRNAs during osteogenesis. The only existing evidence comes from cancer cell lines, in which miR-422 targets Nt5e (codes for CD73) [62] and miR-370 negatively regulates Eng (codes for CD105) [63].
Another surface glycoprotein shared by MSCs is CD44, which functions in adhesion and migration and binds hyaluronan and OPN [105]. In both human renal and prostate cancer cells, CD44 has been identified as a direct target of miR-34a [64], a miRNA that promotes osteogenesis in human adipose-derived stem cells [106]. In addition, the 3′UTR of CD146, which has been associated with a higher potential for osteogenic differentiation [107], is directly targeted by miR-329 in endothelial cells [65]. Also, in endothelial cells, expression of Vcam1, which encodes the cell-cell adhesion molecule CD106 and reduces the migratory ability of MSCs [108], is controlled by miR-126 [109]. However, again, direct regulation has yet to be confirmed in osteoblasts or their precursors. Potentiating the MSC state is miR-140-5p, which inhibits osteogenic lineage commitment and is commonly enriched in undifferentiated human MSCs from various tissue sources [110]. In zebrafish, injection of miR-140-5p phenocopies Bmp2 repression, resulting in aberrant embryonic bone development (short stature, curved trunk, craniofacial malformations) and confirming a direct relationship between the two [111, 112].
For expression of pre-osteoblastic matrix genes to occur, which turn MSCs into pre-osteoblasts and later mature osteoblasts, the master TFs RUNX2 and OSX must be genetically activated. miR-125b, a miRNA identified in MSCs throughout many genetic screens [100, 113], is predicted to target Osx. However, a relationship has not been confirmed beyond the finding of reduced Osx mRNA [114]. In contrast, miR-143 [66, 67], miR-145 [66], miR-214 [68], and miR-637 [69] suppress osteogenic differentiation by directly targeting Osx. Due to the ability of miR-322 to directly target Tob2, which normally helps to degrade Osx, Osx mRNA is stabilized, allowing osteoblast differentiation to occur [70].
Genetic manipulation of Runx2 in vivo indicated that its expression is both necessary and sufficient for mesenchymal cell differentiation towards the osteoblast lineage [115, 116]. Discovered as endogenous attenuators of Runx2 expression, which prevent cells from differentiating into osteoblasts, the inhibition of miR-23a, miR-34c, miR-628-3p miR-137, miR-204, miR-205, miR-338-3p, miR-433, miR-375, and miR-135 promoted osteoblast differentiation along with an increase in bone-specific markers [56, 71•, 72,73,74,75,76,77, 79, 117]. A recent publication bioinformatically identified additional microRNAs predicted to target Runx2 [118]. These include miR-141, miR-200a, and miR-200b, whose expression is expected to be down-regulated as differentiation progresses, but remain high in mesenchymal cells thus representing MSC markers. Alternatively, a microRNA may qualify as an ostemiR if its expression is up-regulated and it targets inhibitors of RUNX2, as is the case for miR-129-5p, which targets the signal transducer and activator of transcription 1 [119] that normally sequesters RUNX2 in the cytoplasm to prevent its nuclear activity [78].
In the nucleus, RUNX2 may regulate transcription not only of genes, but also of miRNAs, such as the pro-osteogenic miR-690 [88] and miR-1192 [120]. In contrast, RUNX2 represses the promoter of the miR23a~27a~24–2 cluster [76]. The consequence of this negative feed-forward loop is to cause depression of special AT-rich sequence-binding protein 2 (Satb2), a scaffold protein that increases RUNX2 activity to promote differentiation. This is an interesting example of a cluster, in which all three miRNAs share Satb2 as a common target, but only one of them, miR-23a, targets Runx2, the attenuation of which seems to fine tune the pace of progression of the osteoblast phenotype rather than switching it off completely.
Two miRNAs that directly target Satb2, miR-34b and miR-34c, were shown to affect osteoblast proliferation and differentiation in vivo [79]. SATB2 itself has recently been implicated as a major osteogenic TF, since Satb2 −/− mice exhibit craniofacial abnormalities [121]. Satb2 is expressed in cells of the osteoblast lineage in developing mice [121] and was reported to function both upstream and downstream of RUNX2 and OSX [121,122,123,124]. Due to its participation in regulatory feedback loops together with RUNX2 and miRNAs, this implicates Satb2 in the acquisition of an osteoprogenitor fate from both the NC and the mesoderm-derived progenitor cells.
One such feedback loop comprises miR-31, whereby in bone marrow MSCs, RUNX2 occupies and activates transcription from the miR-31 promoter, which in turn lowers Satb2 mRNA and protein expression [123]. While a more recent paper suggests the same feedback loop directs dental follicle cells towards osteogenesis [125], neither of the two studies provides evidence for the direct binding of miR-31 to the 3′UTR of Satb2. Other studies place SATB2 upstream of RUNX2 protein and under inflammatory conditions, TNFα-activated miR-33-5p can reduce RUNX2 by directly targeting Satb2 [124].
Due to the negative regulation of their target transcripts, it would be expected that any miRNA targeting Satb2 directly would be down-regulated to induce osteogenic commitment. miR-205, for instance, exhibits this expression pattern during bone marrow MSC differentiation, and its inhibition promotes osteogenesis [77]. However, this is another example of a miRNA for which Satb2 regulation is predicted, but has not yet been experimentally confirmed.
microRNAs Involved in Neural Crest Induction and Differentiation
Some osteomiRs participate in the direct conversion of a mesenchymal cell into an osteoblast, such as in the cases of intramembranous ossification or in the perichondrium. Yet others, which are not discussed here, may modulate endochondral bone formation through tuning chondrogenic differentiation from mesenchymal cells. In addition, microRNAs may also take part in the newly discovered transdifferentiation of hypertrophic chondrocytes into osteoblasts [1,2,3,4].
The general importance of microRNAs in the specification of neural crest cell (NCC)-derived bone in the skull, which is formed via intramembranous ossification, became apparent in conditional knockouts of Dgcr8 (which processes nuclear miRNAs) in cranial NCCs [126, 127•]. Although the role of individual miRNAs during NCC development has not been thoroughly analyzed, miRNAs likely contribute to the intricate, multi-step process of craniofacial cartilage and bone formation. This developmental process is controlled by a number of transcription factors that interact in a so-called gene-regulatory network (GRN), governed by feedback loops and repetitive gene expression (Table 2, see also [128•]). However, virtually nothing is known about the miRNAs governing most of the genes in the GRN that regulate NCC specification and development.
The classical theory of NCC specification proposes a partition of a dorsal subset of neuroepithelial cells from the neural tube as the source for NCCs [130]. More recently, it has been proposed that specification occurs during gastrulation and is initiated by neural plate border specifier genes such as Pax7 [131•, 132]. Originally identified as being required for muscle cell differentiation, homeostasis, and repair [133], Pax7 is targeted directly by miR-431 in muscle satellite cells, the overexpression of which increases myogenic differentiation [134]. Thus, misexpression of miR-431 could negatively impact NCC development.
Once the expression of neural plate border specifiers is initiated, Pax7 engages with other border specifiers such as Gbx2, Zic1/3, and Tfap2, in a mutual cross regulatory network (reviewed in [128•]). Only one silencing miRNA has been identified each for Zic3 and Tfap2 mRNAs, namely, miR-564 [135] and miR-214 [136], respectively. Since the cross-regulation of border specifier genes ensures the stabilization of NCC identity, allowing for continued expression of this gene set through subsequent developmental stages, it is highly likely that the misexpression of regulatory miRNAs would disrupt this network. In addition, the neural plate border genes not only control genes within their own network, but also inhibit neural transcription factors (and vice versa) to sharply define cellular identity in the border region [137]. Hence, overexpression of the neural specifiers Sox2/3, which can occur through an absence of microRNA control (i.e., miR-126 [138]), would result in the repression of NCC identity.
Presumptive NCCs reside at the junction between the neural plate and the preplacodal ectoderm and begin to express of a set of NCC specifier genes, among them Snai1/2, FoxD3, and Sox10 [127•]. The only miRNA identified to regulate Foxd3, miR-429, is currently only known to participate in skin pigmentation [139]. In human hepatocellular carcinoma, FOXD3 activates miR-137 transcription, which decreases proliferation and migration in tumor cells [117]. The role of miR-137 in hepatocellular carcinoma may mirror a similar role in NCCs to slow down proliferation and migration and ultimately maintain or expand the NCC pool.
Following specification, NCCs undergo delamination and leave the neural tube, where they migrate dorsolaterally and differentiate into diverse cell types. This progression requires frequent switches between epithelial and mesenchymal states [140,141,142] similarly observed in cancer metastasis and involves the same reiterated use of a TF machinery including TWIST1 and MSX1/2 [143,144,145,146]. Interestingly, Twist1, which is downregulated by miR-1271 in pancreatic cancer cells [147], is a NCC-specifier gene [128•], which is also necessary for maintaining NCCs after migration, facilitating the formation of craniofacial bone [148]. In turn, Msx1/2 are expressed at the plate border as well as in migrating NCCs, which later re-express Msx2 as well as Dlx5/6 as they transition into mesenchymal osteoprogenitors [149, 150]. Due to their co-regulation of RUNX2 [151], deficiencies in Msx1/2 cause severe cranioskeletal abnormalities, and deletion of both Msx1 and Msx2 cause late gestation lethality [152]. miR-322 indirectly increases Msx2 expression [70] and therefore it is possible that this regulatory mechanism occurs during the migration of NCCs. In turn, the Dlx5 silencing miR-141 and miR-200a [150] may control both the early induction as well as the differentiation of NCCs.
Due to the limited information available on miRNAs that regulate NCC development, we conducted an elementary search for predicted miRNAs using the published GRN and the miRanda algorithm (microRNA.org). Additional helpful hypothetical lists of candidate miRNAs with potential involvement in NC development can be found in a recent review [153]. In total, 201 candidate miRNAs were identified (Table 2), 126 of which (62.7%) targeted more than 1 GRN gene and 25 of which targeted more than 5 genes (Fig. 3). Four of these miRNAs are predicted to silence 10 of the 22 GRN genes across multiple steps of NCC development (Fig. 3 and Table 2). These, we consider the most powerful miRNAs as they may possess the potential to completely block the NC developmental program. Of the 120 candidate miRNAs that specifically target NC specifiers, 8 target 5 or more mRNAs, possibly explaining the co- and inter-regulation of neural crest specifier genes that is necessary to maintain the NC in an undifferentiated state [154]. Intriguingly, seven of these overlap with miRNAs that regulate multiple stages, except for miR-489, which seems specific to neural crest specifiers.
microRNAs potentially implicated in neural crest osteogenesis. Candidate miRNAs predicted to target neural crest GRN genes categorized according to the potential stages of neural crest development they may regulate. Candidate microRNAs that are predicted to target genes in the neural crest gene regulatory network (adopted from [128•]) were assembled with the miRanda algorithm available at microRNA.org [129] and categorized according to the expression of their targets during neural crest development. Based on mouse miRNAs, only conserved microRNAs with a good miRSVR score were taken into account
One of the miRNAs potentially regulating nine different GRN genes is miR-1192, a miRNA induced by RUNX2. This interesting coincidence suggests that once a cell has committed to a specific lineage, it ensures it does not revert back in developmental time. Moreover, 15 of the candidate GRN miRNAs target Runx2, Osx, and/or Satb2, which could be interpreted as a safeguard that helps lock-in a specific cell fate. For instance, considering that miR-204 expression would block the neural plate border state and simultaneously prevent Runx2 expression, it may therefore be expressed by NCCs to preserve NCC identity. A slightly different scenario may occur with miR-217, which targets both border and NC specifier genes. Here, additional targeting of Runx2 may take place in a more mature osteoblast to block all prior cell states.
Conclusion
This review discussed the different stages of osteogenesis bringing together different miRNAs and their targets, the disruption of which may potentially cause severe malformations or deformities. Interestingly, cellular identity is intimately coupled to the expression of miRNAs suppressing previous and subsequent differentiation steps. miRNAs may promote osteogenic differentiation and matrix mineralization through targeting genes with functions in osteoclastogenesis and chondrogenesis and/or signaling, and thus may also qualify as ostemiRs. Although some miRNA target identification has occurred in non-bone cell types, or by bioinformatic prediction, future research illuminating the activity of these miRNAs during (NC-) osteogenesis will be extremely valuable.
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Acknowledgments
We apologize to the authors of many excellent research studies who we could not cite here due to space constraints. This work was supported by a grant from the National Institute of Dental and Craniofacial Research of the National Institutes of Health (R01 DE025330-01A1) and a grant from the Tobacco Related Disease Research Program (25IP-0018, award no. 392351) to NzN.
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Sera, S.R., zur Nieden, N.I. microRNA Regulation of Skeletal Development. Curr Osteoporos Rep 15, 353–366 (2017). https://doi.org/10.1007/s11914-017-0379-7
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DOI: https://doi.org/10.1007/s11914-017-0379-7