microRNAs and Cartilage Gary Gibson,1 Hiroshi Asahara2 1 Bone and Joint Center, Henry Ford Hospital, Detroit, Michigan, 2Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California
Received 11 February 2013; accepted 2 May 2013 Published online 10 June 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22397
ABSTRACT: microRNAs are small non-coding RNAs that in the last decade have emerged as overarching regulators of gene expression. Their abundance, ability to repress a large number of target genes and overlapping target specificity indicate a complex network of interactions that is still being defined. A number of studies focused on the role of microRNAs in cartilage have identified a small number, including miR-140 and -675 as playing important roles in regulation of cartilage homeostasis and together with the broader description of the activity of microRNAs in other tissues are beginning to define the function of microRNAs in cartilage development and homeostasis. ß 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1333–1344, 2013. Keywords: microRNA; cartilage
microRNAs are a large family of small (21 nucleotide) non-coding RNAs. They were first identified as sites of mutations affecting the development of the worm, Caenorhabditis elegans.1,2 It took another approximately 7 years to identify similar non-coding RNAs in mammals3,4 but in the subsequent decade and a bit, there have been more than 20,000 publications describing microRNA synthesis and function. More than 2,000 microRNAs have been identified in the human genome based on sequencing and computational methods. Each microRNA recognizes target sequences in many (frequently estimated to be more than 100) genes and each gene contains target sequences for many microRNAs creating huge potential for gene regulation and complexity. Although a clear understanding of microRNA function remains elusive, advances in microRNA identification, biogenesis, target specificity, and target interaction provide promise of a clearer rational understanding of microRNA function.
microRNA BIOSYNTHESIS Larger precursor microRNAs (primary microRNA or pri-miR, several hundred to thousands of nucleotides in length) are transcribed from either introns of protein coding genes (about one-third) or remote, frequently poly-cistronic (clustered microRNAs, such as miR-17-92 or miR-654/376), intergenic sites. PrimiRs are processed in the nucleus by a protein complex that contains the RNase III, Drosha, and a protein that binds double stranded RNA, diGeorge syndrome critical region gene (DGCR-8). Transport of the resulting 60–80 nucleotide pre-miR cleavage product to the cytoplasm is mediated by a GTP-dependent, dsRNA-binding protein, Exportin 5. In the cytoplasm another protein complex containing the RNase III enzyme, Dicer, cleaves pre-miR at the stem loop structure to generate an approximately 22 nucleotide imperfect double stranded miR duplex. Although either strand of the RNA duplex can function as a Correspondence to: Gary Gibson (T: 313 916 2632; F: 313 916 8064; E-mail:
[email protected]) # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
miRNA only one strand (with an overall tendency to favor the 50 strand) is incorporated into the RNA induced silencing complex (RISC)5 (Fig. 1). In some cases both the 50 or 30 strands of the RNA are incorporated into RISC complexes with similar frequency and in some microRNAs the preferred strand is dependent on tissue type or stage of developmental.6 Members of the Argonaute family of proteins are central to the function of the RISC complex and guide microRNA interaction with target mRNA sequences, in some cases cleave target mRNAs and play a critical role in translational repression of target mRNAs.
microRNA NOMENCLATURE The precursor microRNAs are designated mir (lower case r) as in mir-140 whereas the mature microRNAs are designated miR (upper case R) as in miR-140-5p for the mature form of microRNA 140 derived from the 50 strand of the precursor (or miR-140-3p derived from the 30 strand of the precursor). When relative expression levels are known the microRNA with low expression (i.e., the degraded strand) is designated by an asterix, as in miR-140 , which in this case is the same as miR-140-3p. This designation is confusing and although officially retired is unfortunately still commonly used. The designation suggests very low abundance. However, in some microRNAs, including miR140, the abundance of the -3p and -5p stands are similar and in the case of highly expressed microRNAs the low abundant species frequently have higher expression levels than the abundant strand of low abundant microRNAs. Small differences of one or two bases between microRNAs are indicated by additional lower case letters, such as miR-29a and miR-29b. microRNAs that have identical sequences but arise from different parts of the genome are indicated by a dash number suffix, such as miR-29b-2. The species of origin is indicated by a three letter prefix, such as hsamiR-140-5p or mmu-miR-140-5p for the human and mouse microRNAs, respectively.7,8
TARGET REGULATION microRNAs recognize specific target sequences of mRNAs to induce post-transcription repression. This JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
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Figure 1. Pathways to generate miRNAs from intronic intergenic sites. Intronic miRNAs are transcribed from DNA as mRNA. The intron is spliced out from mRNA and pre-miR generated by Drosha cleavage. In some case, when introns are short and have the capacity for hairpin folding (termed mirtrons) pre-miRNAs are generated independent of Drosha cleavage. Intergenic miRNAs, which are often present as clusters, are transcribed as pri-miR and pre-miRNA generated by Drosha cleavage. Pre-miRNAs are trimmed by Dicer to generate mature forms of miRNA that associates with the RISC complex.
can lead to decreased translation and/or decreased mRNA levels. Ribosome profiling, a technique measuring the association of mRNAs with ribosomes used as an indirect measure of protein production, has shown that microRNAs act predominantly to decrease mRNA levels.9 The inverse association of microRNA and mRNA levels can thus be used as a simple and efficient means to identify miRNA targets. Target microRNAmRNA recognition is complex and difficult to predict. It has been shown to depend on perfect Watson–Crick pairing between positions 2–7, called the seed region, of the microRNA and the mRNA target sequence, usually in the 30 untranslated region (UTR). Pairing to the 30 region of the microRNA (nucleotides 13–16) can supplement seed matching and compensate to a slight extent for a single nucleotide mismatch in the seed region.10 These features, together with; free energy of the microRNA:mRNA duplex; conservation of microRNA and target sequence; positioning of the target in the 30 UTR and secondary structure of the UTR have been used in the development of algorithms (such as TargetScan, PicTar, Diana, Mirdb) to predict target mRNAs. Unfortunately these have poor overlap and high false positive (40–60%) and false negative (50– 70%) predictions.11,12 Recent crosslinking immunoprecipitation (CLIP) studies of the Argonaute complex used to identify target RNAs has shown that nearly half the identified target sequences reside in coding sequences13 and has identified alternative miRNA target sites containing imperfect pairing at positions 5–6 of the microRNA in a high proportion (15%) of JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
mRNA targets14 further complicating target identification. Target sites in coding sequences, however, may not be functional in highly expressed target genes. Silencing complexes bound to coding sequences would be expected to be displaced during translation by passage of the ribosome.10 Most genes contain multiple microRNA target sites and many contain several target sites for a specific microRNA. microRNAs typically inhibit target genes to a modest extent (less than 50%). It is generally believed that multiple target sites for the same or different microRNAs improve efficiency of regulation. However, most studies are based on bioinformatics15 and experimental demonstration of synergy is sparse. The miRBase website lists 2,042 distinct microRNAs in the human genome (release August 19, 2012, http://www.mirbase.org). Only approximately onethird of these is conserved across species and is generally the focus of more detailed studies of microRNA function. Non-conserved microRNAs have been shown to be expressed at much lower levels than conserved microRNAs and their functional significance in regulation of gene expression has been questioned.5,16 The challenges associated with identifying specific physiological roles of microRNA might be alleviated with the extensive development of microRNA knockout mice. Multiple programs have begun to tackle the development of genome wide gene knockout mice under the International Knockout Mouse Consortium.17 A similar program has been initiated for the
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large-scale generation of conditional reporter-tagged microRNA knockout mice. So far 46 conditional microRNA knockout mice have been reported. The program has also generated lacZ-reporter targeting vectors for nearly 50% of microRNAs conserved in humans and mice. Using these resources and consistent with previous studies of the distribution of microRNA in fish and chickens,18–20 microRNA expression was typically shown to be stage and/or tissue dependent. The miR654/376 cluster, in addition to miR-140, showed strong localization to cartilage21 and it will be interesting to follow more detailed localization studies and the effects of deletion in the knockout mice.21 The studies of a limited number (11) of knockout mice suggested microRNA ablation does not generate severe developmental phenotypes. This is consistent with other microRNA knockout studies in mice and C. elegans. Even though microRNAs were discovered in these worms in a study of a developmental mutation, subsequent deletion of the majority of microRNAs showed they were not essential for development and worms were without gross abnormality.22 The rarity of severe developmental effects has been attributed to the extensive redundancy among microRNAs. Numerous microRNAs are grouped into families based on shared seed sequences and would be expected to have shared target genes. However, redundancy does not explain the mild phenotype of the knockout mice where the microRNAs do not have paralogs. Redundancy can also be provided by other microRNAs that target the same gene. MRNAs usually contain target sequences for many microRNAs. Absence of a specific microRNA would be expected to be compensated by actions of several other microRNAs acting on the same mRNA. A popular concept that accommodates the mild suppressive effects and extensive network of overlapping functions is that microRNA act to maintain cell homeostasis by tuning gene expression. This is consistent with the observation that many microRNA knockouts exhibit phenotypes only when subject to stress or injury. This has been extended to the concept that microRNAs function as a major mediator of stress response and implicates their role in a wide variety of diseases, including osteoarthritis, that are caused or exacerbated by cell stress.23,24 Although less common, several microRNAs with profound effects on development and cell function have been identified. Perhaps the most startling example is the reprogramming of somatic cells to a pluripotent state by transfection of a small number of microRNAs. Expression of the miR302/367 cluster efficiently reprogrammed human fibroblasts to an induced pluripotent stem cell without the requirement for exogenous transcription factors. The mechanism of action is not well defined but the microRNAs appear to stimulate demethylation of the genome as well as damping the expression of differentiation signals utilizing the combinatorial capacity of microRNAs to regulate expression of multiple targets.25,26
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Another example of microRNAs with multiple profound effects is the miR-17-92 cluster. Many microRNAs are situated in polycistronic clusters. The miR17-92 is one of the best-characterized, encoding 6 microRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a). miR-17-92 is an oncogene; a downstream target of the Myc oncogene; it targets the tumor suppressor PTEN, cell cycle inhibitors p21 and p57, and promotes angiogenesis through repression of thrombospondin 1 and connective tissue growth factor suppression.27 Its association with chondrogenesis is particularly interesting. A microdeletion resulting in loss of miR-17-92 has been shown to be the cause of some cases of a rare autosomal dominant condition known as Feingold syndrome. Patients with this condition display an array of skeletal abnormalities including short stature, digital anomalies and microcephaly. Mice with a hemizygous deletion of miR-17-92 showed an almost identical phenotype.28 Thus the relatively modest reduction in miR-17-92 expression has profound phenotypic consequences and demonstrates the essential role of this microRNA cluster in skeletal development. Another intriguing example of a microRNA affecting skeletal development has been demonstrated from studies of a family with an X-linked chondrodysplasia associated with severe short stature, disproportionate limb shortening and hand, head and facial abnormality. A point mutation in the 30 UTR of HDAC6 was demonstrated in all affected patients examined. This mutation altered the target sequence of miR-433 and resulted in increased HDAC6 expression.29 Although the role of HDAC6 and miR-433 remain to be defined, these studies suggest a critical role in skeletal development and chondrogenesis.
microRNAs AND CARTILAGE BIOLOGY The essential role of microRNAs in normal skeletal development was clearly demonstrated by the conditional, tissue-specific (Col2a1-Cre:Dicerfl/fl) deletion of Dicer. Dicer null mice showed severe growth retardation that was attributed to decreased chondrocyte proliferation and an accelerated differentiation into hypertrophic chondrocytes.30 The cause of reduced chondrocyte proliferation was not clear. There was no indication of the induction of ectopic apoptosis, stress response, or senescence pathways. However, up-regulation of Hmga2 was observed in Dicer-deficient chondrocytes supporting the role previously demonstrated for this gene in skeletal development31 and the role of microRNA let-7 in suppression of Hmga2 expression.32 The role of microRNAs in cartilage development and homeostasis has received a rapid growth in research attention in the last few years. As may be expected from their overarching role in the regulation of cellular homeostasis microRNAs have been shown to play roles in all aspects of cartilage biology and development from regulation of matrix production and turnover, growth factor regulation, chondrocyte differentiation, division and apoptosis, and disease (Fig. 2). Due JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
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primarily to its high expression and close association with the cartilage phenotype miR-140 has been by far most intensely studied microRNA in cartilage.
miR-140 Several studies profiling microRNA expression have revealed that many microRNAs show specific spatial and temporal expression patterns during embryonic and post-natal development.33–35 Systematic whole mount in situ hybridization analysis for miRNAs in zebrafish revealed that many miRNAs showed a tissue specific expression pattern.33 In this study, miR-140 was the only microRNA shown to have a cartilage-specific expression pattern. Further studies showed cartilagespecific expression of miR-140 in mouse embryos36 and demonstrated that miR-140 expression specificity was regulated at the transcription level rather than Dicer dependent processing.37 miR-140 expression, generally at much lower levels, is also observed in a variety of other tissues, consistent with other highly expressed cartilage genes, such as aggrecan.38,39 Function of miR-140 in Cartilage Development Injection of zebrafish embryos with miR-140 duplex had a profound effect on the facial phenotype, including effects resembling cleft palate. This phenotype was similar to one produced by suppression of PDGF signaling and together with the presence of a miR-140 binding site in the 30 UTR of a PDGF receptor (PDGFRa) suggested miR-140 affected palatogenesis by disruption of PDGF signaling.40 In support of this suggestion, patients with cleft palate were recently shown to have a significantly higher number of missense mutations in the coding regions and the 30 UTR of PDGFa (in one case adjacent to and affecting miR140 binding sites) than a matched control population.41 To date, two groups have independently developed miR-140 knockout mice. Kobayashi et.al. reported that miR-140 knockout mice show growth defects of endochondral bones, resulting in dwarfism and craniofacial deformities. Dnpep, a gene encoding the aspartyl aminopeptidase enzyme, was identified as the most likely miR-140 target by comparing profiles of RNAs that associated with the RISC complex, Argonaute 2
Figure 2. Multiple mRNAs and biological pathways can be targeted by each miRNAs. Each miRNAs by binding specific sequences, most frequently in 30 UTR of target genes, represses the expression of many genes. Molecular networks can be regulated for the specific modulation of biological and pathological functions. JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
(Ago 2) protein in wild type and miR-140 null mice.42 Dnpep expression was increased in miR-140-null chondrocytes and this was suggested to cause the skeletal phenotype by reducing basal BMP signaling.42 The phenotype of the miR-140 knockout mice generated by the Asahara group was identical with that generated by Kobayashi et al. In addition the structure of the articular joint cartilage appeared grossly normal in the 1-month-old miR-140 null mice.43 The Role of miR140 in Osteoarthritis Pathogenesis From about 3 months of age miR-140 null mice showed progressive proteoglycan loss and cartilage fibrillation characteristic of early osteoarthritis.43 Adamts-5, an enzyme central to the degradation of articular cartilage in osteoarthritis pathogenesis, was shown to be directly regulated by miR-140, suggesting that miR140 may play independent roles in endochondral ossification and articular cartilage homeostasis, via regulating different sets of genes.38,43 This is consistent with the observation that miR-140 expression was lower in chondrocytes from osteoarthritic articular cartilage compared with those from normal articular cartilage and that inflammatory signals associated with cartilage degeneration repressed miR-140 expression44 in tissue culture. To date, several reports have revealed multiple miR-140 targets, including PDGFa,40 Dnpep,42 ADAMTS5,43SP1,45 HDAC4,36 IGFBP1,46 and MMP-13 (Fig. 3).46 In future it will be interesting to compare targets in the epiphyseal growth plates during developmental with those associated with articular cartilage homeostasis. Identification of the gene network regulated by miR-140 in growth plates and in articular cartilage could identify novel cascades critical for skeletal development and osteoarthritis pathogenesis.39 Regulatory Mechanism of miR-140 Expression in Cartilage miR-140 has an expression pattern in cartilage similar to that of other cartilage-specific genes and recent analysis suggests its expression is similarly regulated. Sox9 acts as an initial chondrogenesis regulator and is known to regulate genes expressing cartilage-specific proteins, such as type II, IX, and XI collagen.47 In zebrafish, Sox9 was shown to be upstream of miR-140 expression.48 Using multiple conditional Sox9 knockout mice and potential enhancer transgenic mice Yamashita et al.37 confirmed the role of Sox 9 in cartilage-specific miR-140 expression and demonstrated Sox9 dependent miR-140 expression was further promoted by L-Sox5 and Sox6 (Fig. 3). miR-140 and its Backbone Gene; WWP2 miR-140 is located in an intron of the WWP2 gene coding for an E3 ubiquitin ligase.37,45 As most of miRNAs located in intron regions are derived during the process of intron excision, miR-140 may be produced from WWP2 primary transcripts, which also show a cartilage-specific expression pattern.45,49
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Figure 3. Molecular network regulated by miRNAs in cartilage. Various aspects of the cartilage gene network are tightly regulated by multiple miRNAs. Green lines and green circles represent anabolic regulation, whereas red lines and red circles show catabolic effects on cartilage.
However, the possibility that the miR-140 primary transcript is generated independently from the WWP2 gene cannot be eliminated. Interestingly, WWP2 knockout mice show a shortened snout and domed skull similar to the miR-140 null mice.43,50 However, WWP2 gene expression was shown in two independent miR-140 knockout mice to be unaltered.42,43 Furthermore the phenotype of the miR-140 null mice is partially rescued by crossing with cartilage specific miR-140 transgenic mice (Asahara, unpublished data) indicating that the phenotype of the miR-140 knockout mice is not caused by loss of WWP2. In this regard, it would be of interest to know how these two distinct molecules share biological functions in cartilage. One of the working hypotheses is that these molecules act together at different levels for the efficient regulation of gene networks. Understanding the interactions of host genes and the mircroRNAs housed within their introns is an important part of the challenge of untangling the web of microRNA biology.39
OTHER microRNAs AFFECTING CARTILAGE EXTRACELLULAR MATRIX
Dudek et al.51 have shown that expression of type II collagen in chondrocytes is stimulated by miR-675.
These observations arose from investigation of the function of a non-coding RNA, H19, which is expressed by human and mouse chondrocytes at levels comparable to the most abundant cartilage genes, COL2A1, or ACAN (aggrecan) and whose expression parallels expression of these genes during phenotypic modulation in cell culture. H19 expression was shown to be regulated by the transcription factor Sox 9. Type II collagen expression was blocked in the absence of Sox 9 but expression of miR-675 was able to completely rescue expression of type II collagen in Sox 9 depleted cells suggesting miR-675 mediates the effect of Sox 9 on Col2A1 expression in these articular chondrocytes. The mechanism by which miR-675 stimulates type II collagen expression has not been defined but was suggested to target a Col2A1 repressor. H19 and miR675 lie within a very interesting imprinted domain on the short arm of human chromosome 11. Imprinting is a phenomenon whereby gene expression is restricted to only one allele dependent on the parental origin. The gene encoding H19 and miR-675 is only expressed from the allele inherited from the mother. The allele inherited from the father contains the IGF2 gene and miR-483 (encoded within an IGF2 intron). Non-coding RNAs, including microRNAs are present in all JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
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imprinted domains although their function in silencing parental transcripts remains obscure.52 Although this domain does not appear to be imprinted in mature chondrocyte it is interesting to note that H19, miR675, IGF2,53 and miR-48354 have been shown to be highly up-regulated in studies of differential microRNA expression in osteoarthritis. These studies suggest the domain containing H19, IGF 2, miRs-675 and -483 play important roles in the regulation of the cartilage matrix. Recent studies55 have also shown that expression of miR-675 is tightly regulated by the RNA binding and stress response protein HuR. HuR appears to suppress miR-675 expression by inhibition of Drosher processing. Relocation of HuR from the nucleus to cytoplasm in response to cellular stress is proposed to release Drosher inhibition and thus rapidly stimulate miR-675 expression. This response of miR-675 to cellular stress might also explain its increased expression in chondrocytes from cartilage of osteoarthritic patients. miR-145 has been shown to regulate expression of cartilage matrix genes by directly targeting Sox 9. Over-expression of miR-145 suppresses Sox 9 expression during chondrogenic differentiation of mouse mesenchymal stem cells and in human articular chondrocytes. As a consequence expression of articular cartilage matrix genes, Col2A1, ACAN, Comp, Col9A2, and Col11A1 were suppressed and genes associated with the hypertrophic phenotype, Runx2 and MMP-13 stimulated.56,57 Over-expression or inhibition of several other microRNAs has been shown to influence expression of cartilage matrix genes, although their mechanisms of action are unclear. miR-199a and miR-193 have been shown to inhibit COL2A1 and ACAN expression in
Figure 4. Summary of changes in miRNAs expression in OA samples. Orange circles show increased miRNAs in OA, whereas blue ones show decreased miRNAs in OA. miRNAs that show variability of expression depending on different stages of development or different regions of cartilage are circled by blue/orange gradation. JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
human chondrocytes when over-expressed and to stimulate expression of these genes when inhibited.58 Deletion of a cluster of microRNAs including miR-199a and miR-214 in mice resulted in severe skeletal deformity suggesting a critical role in skeletal development.59 However, the microRNAs are expressed in many tissues, including the heart and indirect effects on skeletal development cannot be excluded. Studies of chick sternal chondrocytes have shown that miR-1 represses ACAN expression but only in hypertrophic chondrocytes. No inhibition was seen in reserve or dividing chondrocytes isolated from the caudal region.60 Loss of miR-1 expression observed with chondrocyte hypertrophy was suggested to support the increased aggrecan expression seen with hypertrophic differentiation. Expression of miR-29a and -29b were shown to be suppressed with chondrogenic differentiation of mouse mesenchymal stem cells and shown to directly target Col2A1 mRNA and suppress type II collagen expression.61 Although a lot can be learned from investigations of other tissues, some caution should be used as microRNA function has been shown to depend on tissue and cell type, stage of development or age, and cellular environments. Tenascin C is associated with the development of articular cartilage but decreases markedly in the adult. It’s expression, typical of many proteins present in immature articular cartilage, is reactivated in the articular cartilage from osteoarthritic patients.62 Studies of breast tumor metastasis have shown that loss of miR-335 (a microRNA highly expressed in cartilage30) stimulates metastasis in part by the stimulation of Tenascin C expression a direct target of miR-33563 suggesting suppression of Tenascin expression with development of mature articular cartilage and its reactivation with development of osteoarthritis might be associated with changes in miR-335 expression. Hyaluronate, synthesized by plasma membrane bound hyaluronate synthase 2 (HAS2), is essential for normal cartilage formation. Studies in heart valve development have shown that miR-23, a microRNA that is also expressed in human chondrocytes,30 directly inhibits HAS2 expression and disrupts extracellular matrix formation by endocardial cells.64 Stimulation of miR-23 expression, as shown in some studies of osteoarthritic cartilage,55 would be expected to inhibit HAS2 expression and exacerbate the cartilage degeneration. Fibrosis is a substantial pathologic feature of several diseases and as a result has been the focus of considerable research attention, including the regulatory role of microRNAs. Fibrosis involves an imbalance of extracellular matrix synthesis and turnover, involving pathways that also regulate matrix homeostasis in chondrocytes. The miR-29 family has been implicated in cardiac and hepatic fibrosis. Loss of expression of these microRNAs is associated with increased fibrosis consistent with their demonstrated target sites on
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multiple collagen mRNAs, including predicted targets on COL2A1, COL9A1, and COL11A1.65 It is interesting to note that miR-29a has been inversely correlated with BMI; however, there was no correlation implicating an association between mechanical load or fat metabolism and cartilage matrix production.54 The function of most of these microRNAs in cartilage has not been studied but similar roles might be predicted.
GROWTH FACTORS microRNAs regulate and are regulated by growth factor networks. Some of the best studied are members of the TGF-b family that are also critical factors in cartilage development and homeostasis. Mature articular cartilage contains very low levels of TGF-b1 and 2; however, these are expressed at high levels in response to cartilage injury and have been associated with the development of osteoarthritis. microRNAs have an interesting and complex interaction responding to and regulating TGF-b family members and signaling intermediates. Upon ligand binding TGF-b receptor dimerization stimulates phosphorylation and binding of receptor regulated Smads (rSmad), Smad 2 or Smad 3 (canonical TGF-b pathway), Smad 1, Smad 5, or Smad 8 (BMP pathway and alternative TGF-b pathway). Phosphorylated rSmad binds the co-Smad, Smad 4. This complex enters the nucleus where it interacts with a DNA recognition sequence and in combination with other factors initiates transcription. There are two inhibitory Smads, Smad 6, and Smad 7 that play an important role in TGF-b signaling by competing with rSmads for Smad 4 or receptor binding. It has been suggested that in cartilage signaling via Smad 2/3 has anabolic consequences (expression of matrix genes, Col2a1 and ACAN) whereas signaling via Smad 1/5/8 has catabolic consequences (expression of Mmp-13 and the hypertrophic marker Col10a1). Also changes in receptor abundance with age contribute to a transition in TGF-b signaling from anabolic to catabolic and cartilage degeneration associated with osteoarthritis.66 microRNAs play an integral part in regulating the expression of several TGF-b signaling molecules and in turn Smad proteins play a role in the regulation of microRNA expression and activity. microRNAs Regulating TGF-b Signaling miR-455-3p is located in an intron of col27a1 the gene encoding type XXVII collagen a minor cartilage collagen. miR-455-3p was shown by in situ hybridization to be expressed in developing mouse and chick long bones and became more restricted to joints with development and articular cartilage maturation. It has been shown to have a similar expression profile to miR-140 during chondrogenic differentiation of the ATDC5 cell line. miR-455-3p directly targets translation of Smad2 and the activin receptor gene ACVR2B, which signals through the Smad 2/3 pathway.67 Studies by the same group had previously shown that miR-140 targets Smad 3 expression68 and suggest that the increased miR-455 and -140 expression they observe in osteoarthritic
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cartilage (this contrasts the decreased miR-140 expression observed by Asahara et al. and exemplifies the difficulty in comparing gene expression in normal and osteoarthritic cartilage discussed below) promotes the TGF-b Smad 1/5/8 pathway by suppressing the Smad 2/ 3 pathway thereby promoting a degradative chondrocyte response.67 Iliopoulos et al.54 demonstrated that miR-22 suppressed the expression of BMP-7 by direct interaction. Overexpression of miR-22 resulted in increased expression of MMP-13 and decreased aggrecan expression. In addition inhibition of miR-22 upregulated BMP-7 expression, blocked inflammatory processes through inhibition of Il-1b, blocked MMP-13 expression and stimulated aggrecan expression suggesting the potential involvement of miR-22 in cartilage degradation. Expression of miR 146a in chondrocytes was shown to lead to a diminished response to TGF-b and increased apoptosis explained by direct binding to the 30 UTR of Smad 4. miR-146a expression was stimulated by Il-1b and increased in a rat model of osteoarthritis.69 miR-146a has also been shown to suppress Il-1b induced MMP-13 and ADAMTS-5 expression in human chondrocytes and to suppress inflammatory cytokines in synovial cells.70 These studies emphasize the complex and often apparently paradoxical effects of microRNAs on chondrocyte activity and caution against predicting their role in cartilage pathology associated with osteoarthritis. As discussed previously the miR-17-92 cluster has been shown to be essential for normal skeletal development. A comprehensive analysis of the effects of miR-17-92 cluster activation on the proteome of neuroblastoma71 and mesenchymal stem cells72 have demonstrated potent inhibition of TGF-b at multiple levels of the signaling pathway, including inhibition of TGF-b2 receptor2, Smad2, and Smad4. Individual and additive effects of microRNA components of the miR17-92 cluster were observed suggesting a context and celltype specific regulation of activity. TGF-b Regulation of microRNA Activity The regulation of microRNA expression by TGF-b has also been demonstrated. In addition to stimulating TGF-b as described above, miR-455 in turn is stimulated by TGF-b. This appears to be due to direct interaction of Smads with the promoter sequence of miR-455. Similar interaction of Smads with promoter sequences has been demonstrated to positively or negatively regulate expression of a number of microRNAs in other cell types.73 Stimulation of microRNA expression by novel post-transcriptional regulation of microRNA processing by Smads has also recently been described. Smads were shown to bind to a target sequence (similar to the Smad binding element found in the promoters of target genes) near the stem region of the hairpin structure of a subset of microRNAs, including miRs -21, -199a, and -140. Enhanced interJOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
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action with the Drosha complex provided by Smad binding was necessary for efficient processing of the pri-miR and expression of the mature microRNA.74 The TGF-b family contains a diverse group of growth factors and downstream signaling molecules that have major influences on chondrogenesis and cartilage homeostasis. A detailed description of the interaction of microRNAs with the TGF-b pathway is expected to provide new understanding of the regulation of these pathways and rational targets for the manipulation of their complex effects on chondrogenesis and chondrocyte activity. microRNAs Regulating the Permanent Cartilage Phenotype Growth cartilage (growth plate and fracture callus) and permanent cartilages (articular, tracheal, and laryngeal cartilages) to a large extent share similar histological features and have very similar gene expression. Definition of the molecular features that distinguish these tissues will provide a critical new understanding of skeletal development; the pathways contributing to degradation of permanent cartilage and information necessary to engineer permanent cartilage from stem cells. In an elegant series of experiments Gradus et al.75 have shown that microRNAs are essential for the maintenance of the permanent phenotype of tracheal cartilage. The trachea of cartilage-specific, Dicer knockout mice develop normally and express the characteristic Sox genes. However, the expression of matrix genes was severely reduced. In a search for inhibitors of matrix expression the authors identified enhanced Snail 1 expression in the trachea of Dicer knockout mice suggesting loss of Snail inhibitors. Snail 1 is a transcription factor downstream of the FGF receptor. FGF signaling plays an essential role in chondrogenesis and in prehypertrophic chondrocytes Snail 1 suppresses cell division and Col2a1 and ACAN expression.76 miRs-125b and -30a/c were shown to suppress Snail 1 expression by direct binding to the 30 UTR of Snail 1 mRNA. Snail 1 knockdown in a background of miR-125b/-30/a/c inhibition completely restored Col2a1 and ACAN expression. Thus it appears that suppression of Snail 1 expression by miRs-125b and -30a/c is essential for the maintenance of cartilage matrix production in tracheal chondrocytes and preservation of a permanent cartilage phenotype. Similar effects of Snail 1 loss of repression on cartilage matrix production were observed in neurocranial cartilage but not in articular cartilage of newborn mice suggesting this mechanism might not operate in articular chondrocytes to preserve their permanent cartilage phenotype. However, it is possible that this was misinterpreted as a major proportion of the cartilage continuous with the articular in newborn mouse pups is devoted to generation of the secondary ossification center by an endochondral ossification process. It is not permanent articular cartilage and like growth cartilage would not be expected to be affected by de-repression of Snail 1. It will be interesting to see if the permanent JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
articular cartilage in older mice is also similarly affected by loss of miRs-125b/30ac. Maintenance of the specific chondrocyte phenotype is critical to stability of articular cartilage. Expression of genes associated with the hypertrophic phenotype such as Runx2 is believed to contribute to articular cartilage degeneration associated with osteoarthritis.77 Runx2 is a primary regulator of the hypertrophic differentiation of chondrocytes and has been shown to be regulated by multiple microRNAs. A panel of 11 microRNAs, miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR218, and miR-338, were shown to target Runx 2 in chondrogenic and osteogenic cell lines.78 The microRNAs were expressed in an inverse relationship with Runx2 and overexpression resulted in suppression of Runx 2 protein expression. All (except miR-208) attenuated osteoblastic differentiation and would be expected to attenuate chondrocyte hypertrophy. It was observed that 7 of the 11 microRNAs studied also targeted the chondrogenic transcription factor TRPS1 explaining suppression of chondrocyte gene expression when overexpressed in the chondrogenic cell line ATDC5.79 Fine tuning of gene expression associated with the maintenance of tissue homeostasis is a frequently observed function of microRNAs and one critical to those aging diseases associated with slow loss of tissue function. It is expected that further definition of microRNAs associated with maintenance of tissue homeostasis will provide new targets for the development of therapeutic strategies to prevent degenerative changes in cell phenotype, like that observed in osteoarthritis.
INTERACTION OF microRNAS AND CHONDROCYTE ENVIRONMENT Chondrocytes develop and exist in a unique hypoxic and mechanical environment that plays a critical role in regulating their molecular behavior. microRNAs play a role in many aspects of this regulatory network. The role of hypoxia and the transcription factor, hypoxia inducible factor (HIF) has been extensively investigated. Under conditions of normoxia HIF is hydroxylated by HIF prolyl hydroxylase (PHD) and is rapidly degraded by the ubiquitin pathway. Under low oxygen concentrations, such as found in cartilage, HIF is stable, is transported to the nucleus and activates transcription by binding response elements of hypoxia inducible genes.80 Studies have shown that a spectrum of microRNAs is induced under hypoxic condition and some including miRs-103, -210, and -213 were HIF dependant81 suggesting a downstream role in HIF signaling. More recently a study has shown that surprisingly most (103 of 107) of the micoRNAs induced by hypoxia were dependent on type I collagen prolyl 4 hydroxylase (C-P4H) rather than PHD. This is an apparently paradoxical result because C-P4H has a higher affinity for oxygen than PHD expression and is induced rather than repressed under low oxygen
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conditions. C-P4H mediated hydroxylation of Ago2, a critical component of the RISC complex, increases its stability and activity resulting in increased levels of microRNAs and enhanced silencing of target mRNAs.82 It is not surprisingly that many of the CP4H responsive microRNAs, including miR-140, are highly expressed in cartilage. C-P4H-mediated hydroxylation of Ago2 and stimulation of specific microRNA expression under hypoxic conditions provides another pathway regulating cartilage homeostasis. TGF-b well known as a stimulator of collagen expression has also been shown to increase expression of C-P4H,83 which suggests a co-regulation of TGF-b-responsive matrix genes and C-P4HD-regulated microRNAs. miR-365 was the first miRNA demonstrated to respond to mechanical load. Three dimensional cultures of chick chondrocytes were shown to upregulate miR-365 in response to mechanical load and consequently stimulate chondrocyte proliferation and differentiation by targeting HDAC 4, an inhibitor of Ihh and Runx2.84 Mechano-responsive microRNAs have also been deduced from array analysis of RNA extracted from weight bearing and non-weight bearing regions of bovine articular cartilage. miRs-221, -222, -146, -143, -145, -34c, and -155 were highly expressed in weight bearing compared with non-weight bearing regions.85 In separate studies miR-146 was shown to target SMAD 4, repress TGF-b responsiveness and stimulate VEGF in chondrocytes.69 A decreased anabolic response resulting from suppression of TGF-b by miR-146 is contrary to what might be expected in response to high mechanical load. However, this might also reflect the complexity of TGF-b signaling and a repression of the degradative TGF-b pathway as discussed above.
microRNAS EXPRESSION IN OSTEOARTHRITIS Indirect evidence for a role for microRNAs in the degeneration of articular cartilage characteristic of osteoarthritis has been suggested throughout this review (Fig. 4). More direct comparison of microRNA expression in normal and osteoarthritic articular cartilage has been described is several publications. Like many studies comparing gene expression in normal cartilage and cartilage from patients undergoing joint replacement surgery, the analyses are inconsistent and consequently very difficult to compare and interpret. Three studies have reported microRNA array analysis (two used the same PCR based arrays and the third used a hybridization based array) of normal and osteoarthritic articular cartilage. Of the thirty microRNAs showing significantly different expression comparing cartilage from normal and osteoarthritic patients only two were observed in more than one study. Of these one microRNA, miR-25, was observed to increase in osteoarthritic cartilage eightfold in one study54 and decrease threefold in the second.86 In addition Iliopoulos et al. observed an approximately fourfold decrease in miR-140 expression in osteoarthrit-
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ic cartilage that was confirmed by Tardif et al.46 and Miyaki et al.44 but Swingler et. al.67 reported increased expression in osteoarthritic cartilage. One microRNA, miR-483-5p, was observed to have eightfold54 and 2.4fold87 increased expression in osteoarthritic cartilage compared to normal cartilage. We have also shown increased expression of miR-483-5p in osteoarthritic compared with normal articular cartilage (Gibson and Zhang, unpublished data). Although technical differences in analysis and data processing can generate discrepancies in microRNA expression, the primary source of variability in these analyses probably arises from the variation in the osteoarthritic and normal tissue analyzed. Osteoarthritis is a slow degenerative disease and the duration of disease in samples obtained cannot be determined but can vary from years to many decades. Also the cartilage used as normal controls although age-matched in these studies can also show considerable variability. The sampling site within a joint and the joint sampled can also generate variability.
CONCLUSIONS The rapidly growing body of research suggests microRNAs play a role in the differentiation and homeostasis of all tissues. This is clearly the case in both growth and permanent cartilages. However, we have only just begun to unravel their role. Investigations to date have studied a fraction of the microRNAs and very few of the potential target interactions have been described in any detail. It is expected that future studies will employ a combination of microRNA array, mRNA array, and proteomics in well controlled experimental systems to begin to define the complex networks of microRNAs and target genes that participate in the precise regulation of chondrocyte differentiation in growth cartilage during our first two decades and in permanent cartilage homeostasis later in life. A broad focus employing array and sequencing approaches is most likely to be productive. Studies of a few microRNAs and target genes selected on known function are unlikely to define microRNA regulation of chondrocyte function. Description of target genes, gene pathways and disease associations using bioinformatics has great potential. However, as discussed previously, reliance on data bases for target gene identification alone is prone to error. It is tempting to associate microRNAs with gene pathways but evidence for specific association of microRNAs with gene pathways is scarce and predictions based on non-validated target genes is likely to be misleading and of little value. Description of microRNA activity and function will require tissue-specific validation of target genes and associated pathways. Difficulties encountered in the interpretation of data from human osteoarthritic cartilage highlights the need to examine microRNA function in well-controlled animal studies. Model systems, such as the medial meniscus destabilization model of post-traumatic osteoarthritis, offer great potential for deciphering the role of microRNAs in osteoarthritis. JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2013
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The non-coding RNA world has opened a new and exciting vista of gene regulation that was unexplored until a few years ago. Ongoing description of the function of non-coding RNAs, including microRNAs, in the regulation of cartilage function offers a new understanding of the mechanisms controlling its development, homeostasis, and degeneration in diseases, like osteoarthritis and the potential to provide rational targets for new therapeutic approaches.
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