microRNAs in Cartilage Development, Homeostasis, and Disease

Abstract

microRNAs (miRNAs) regulate gene expression mainly at the posttranscriptional level. Many different miRNAs are expressed in chondrocytes, and each individual miRNA can regulate hundreds of target genes, creating a complex gene regulatory network. Experimental evidence suggests that miRNAs play significant roles in various aspects of cartilage development, homeostasis, and pathology. The possibility that miRNAs can be novel therapeutic targets for cartilage diseases led to vigorous investigations to understand the role of individual miRNAs in skeletal tissues. Here, we summarize our current understanding of miRNAs in chondrocytes and cartilage. In the first part, we discuss roles of miRNAs in growth plate development and chondrocyte differentiation. In the second part, we put a particular focus on articular cartilage and discuss the significance of variety of findings in the context of osteoarthritis, the most common degenerative joint disease.

Introduction

Cartilage-forming chondrocytes derive from mesenchymal progenitor cells. During embryonic development, mesenchymal progenitor cells proliferate, aggregate at mesenchymal condensations, and then start differentiating into chondrocytes to form cartilage templates for future bones and cartilage [1]. Chondrocytes secrete unique extracellular matrix (ECM) proteins, such as type II collagen and aggrecan, to generate cartilage matrix. The most common form of cartilage, hyaline cartilage, provides mechanical support. Hyaline cartilage is found in joints, ribs, and growth plates. Two types of hyaline cartilage play major roles in development and function of long bones. Growth plate cartilage drives long bone growth, and articular cartilage provides shock absorption and mobility in joints.

Distinct groups of chondrocytes exist in the growth plate. Resting chondrocytes, located at the most epiphyseal side of the bone, proliferate slowly and differentiate into proliferating chondrocytes. Proliferating chondrocytes, characterized by their vigorous proliferation, produce cartilage matrix proteins important for cartilage function, such as type II collagen and aggrecan, and then further differentiate into postmitotic hypertrophic chondrocytes that express type X collagen. Hyper-trophic chondrocytes undergo mineralization and are eventually replaced by mineralized bone. This process leads to longitudinal bone growth. Tightly coordinated chondrocyte proliferation and differentiation, regulated by a variety of signaling molecules, such as Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTHrP), bone morphogenetic proteins (BMP), and fibroblast growth factors (FGFs), is essential for normal bone growth. The transcription factor, sex determining region Y (SRY) box 9 (Sox9), plays an essential role for chondrocyte differentiation, function, and survival. Sox9 directly regulates expression of Collagen II and aggrecan [2, 3]. The activity of Sox9 is enhanced by the related molecules Sox5 and Sox6 [4]. Other transcription factors important for chondrocyte differentiation include myocyte enhancer factor 2 (MEF2) family transcription factors and Runx2. MEF2 transcription factors, negatively regulated by histone deacetylase 4 (HDAC4) and also possibly by other class IIa HDACs through direct binding, promote hypertrophic differentiation. Runx2, a transcription factor essential for osteoblast differentiation, is expressed in growth plate chondrocytes and stimulates chondrocyte hypertrophy [5, 6]. These signaling molecules and transcription factors regulate chondrocyte-specific gene expression. In vivo and in vitro evidence has shown that miRNAs also play important roles in chondrocyte biology by suppressing undesired gene expression at the post-transcriptional level.

miRNAs are ∼22 nucleotide-long, noncoding RNAs that directly bind to target RNAs in a sequence-complimentary manner to inhibit translation and facilitate degradation of their target transcripts. miRNAs are encoded in the genome, and are transcribed as long primary transcripts (pri-miRNAs). PrimiRNAs are subsequently processed into small hairpin precursor miRNAs (pre-miRNAs), which are transported to the cytoplasm to be further cleaved by Dicer into mature and functional miRNAs [7].

A global reduction of miRNAs after Dicer deletion in multiple types of bone cells including chondrocytes causes significant defects in vivo, providing the basis for further investigation of individual miRNAs in bone and cartilage.

Role of miRNAs in Cartilage Development

In Vivo Studies

We previously showed that global reduction in miRNAs in chondrocytes by conditionally deleting the Dicer gene resulted in a significant growth defect and premature death [8]. Proliferation of growth plate chondrocytes was substantially reduced, while the number of hypertrophic chondrocytes was increased. These results suggest that miRNAs regulate proliferation and differentiation of growth plate chondrocytes. Expression of Hmga2, a well-characterized let-7 miRNA target, was upregulated in Dicer-deficient chondrocytes.

let-7 miRNAs are most abundant miRNAs in somatic tissues including chondrocytes. We have shown that let-7 family miRNAs are required for normal chondrocyte proliferation in the growth plate [9•]. Overexpression of the let-7 inhibitor Lin28a in chondrocytes suppressed let-7 miRNA expression. This reduced chondrocyte proliferation and led to mild growth impairment. Lin28a overexpression in chondrocytes upregulated predicted let-7 target genes, including cell division cycle 34 (Cdc34) and E2F transcription factor 5 (E2f5). The skeletal phenotype of Lin28a transgenic mice was mild, but compound miR140 deficient/Lin28a transgenic mice show a dramatic growth defect. As discussed below, miR-140 modulates mainly chondrocyte differentiation. Therefore, let-7 miRNAs and miR-140 coordinately regulate skeletal development by regulating chondrocyte proliferation and differentiation, respectively [9•].

miR-140 is expressed abundantly and relatively specifically in chondrocytes. miR-140 is encoded in an intronic sequence of the Wwp2 gene [10•]. Expression of miR-140 and Wwp2 is regulated by Sox9 [10•, 11•, 12]. An upstream region of the pri-miR-140 gene, present in an intron of the Wwp2 gene, possesses chondrocyte-specific promoter activity and is directly regulated by Sox9 [4].

miR-140 deletion causes a mild skeletal developmental defect. miR-140-null mice show short endochondral bones and reduced longitudinal growth of the skull [13••, 14••]. We have previously shown that miR-140-deficiency causes premature hypertrophic chondrocyte differentiation and delayed differentiation of resting chondrocytes to proliferating chondrocytes [14••]. In chondrocytes, Dnpep, a gene encoding for an aspartyl aminopeptidase, was identified as a direct miR-140 target. Other miR-140 target genes, such as Pdgfra, HDAC4, Smad3, and Rala, have been identified in other studies [15–18]. The in vivo significance of the regulatory role miR-140 for each individual target gene remains to be determined.

miR-199a, miR-199a* and miR-214 are expressed in mesenchymal cells, perichondrial cells and periarticular chondrocytes. These miRNAs are generated from the long RNA transcript, Dnm3os, transcribed from the Dynamin 3 gene (Dnm3) locus. Mutation of the Dnm3os locus by LacZ insertion reduced expression of all three miRNAs [19]. This resulted in growth retardation, craniofacial hypoplasia, dorsal vertebral hypoplasia, and osteopenia, suggesting that these miRNAs play physiologically significant roles in skeletal development. In vitro studies have shown that miR-199 is upregulated upon chondrocyte differentiation in mesenchymal stem cells [20–22].

In Vitro Studies

In addition to the relatively limited in vivo studies discussed above, a large number of studies have investigated roles of specific microRNAs in vitro.

In vitro assays have identified miRNAs that regulate chondrocyte function and differentiation. Several miRNAs are downregulated upon chondrocyte differentiation and negatively regulate the process. miR-145 was found to be downregulated during chondrocyte differentiation of mouse C3H10T1/2 cells [23]. miR-145 inhibited chondrocyte differentiation; miR-145 directly targets and suppresses the expression of Sox9, which leads to reduced mRNA levels of chondrocyte markers such as Col2a1, Col9a1, Col11a1, and Acan (aggrecan).

Ohgawara et al showed that miR-18a targets and suppresses connective tissue growth factor (Ccn2/Ctgf), a molecule important for endochondral bone formation, in human chondrocytic HCS-2/8 cells [24]. miR-18a overexpression also suppressed aggrecan expression in this study.

miR-1 was found to be downregulated upon hypertrophic differentiation of chondrocytes [25]. miR-1 overexpression in HCS-2/8 cells and chicken primary chondrocytes reduced aggrecan expression. However, the mechanism by which miR-1 decreased aggrecan levels was unclear.

In chicken limb mesenchymal cells, miR-375 was also downregulated upon chondrocyte differentiation [26]. miR-375 inhibition increased chondrogenic differentiation in these cells. Additionally, miR-375 inhibition increased migration of chondrocyte progenitor cells and stimulated chondrocyte differentiation in a wound healing assay, which mimics the process of mesenchymal condensation. Cadherin-7 was proposed as the miR-375 target responsible for these effects.

miR-1247 was reported to be expressed abundantly in mouse chondrocytes [27]. Sox9 negatively regulates miR-1247 expression, while miR-1247 itself targets Sox9. This creates a negative feedback loop; therefore, miR-1247 may function as part of the autoregulatory system of Sox9 function.

Sumiyoshi et al found that miR-181a, a miRNA highly expressed in chicken chondrocytes, directly targets and suppresses the pro-chondrogenic gene, CCN family member 1(Ccn1) and aggrecan [28].

In chicken cells, miR-221 expression increases upon inhibition of chondrocyte differentiation [29]. Inhibition of miR-221 increases proliferation of limb mesenchymal cells. miR-221 suppresses chondrocyte proliferation by targeting and suppressing Mdm2, a ubiquitin ligase that facilitates degradation of p53 as well as the transcription factor, Slug, a negative regulator of chondrocyte progenitor proliferation.

In MSCs, miR-449 negatively regulates chondrocyte differentiation [30]. In human bone marrow MSCs and also in human chondrosarcoma cells, miR-449 suppresses expression of Lef1, a critical component of the canonical Wnt signaling pathway. This suppression downregulates Col2a1 and Sox9 expression, and reduces proteoglycan production.

While many miRNAs negatively regulate chondrogenesis as discussed above, there are also miRNAs that promote chondrogenesis in vitro. During chondrogenic differentiation of mesenchymal stem cells (MSCs), miR-574-3p is highly upregulated [31]. miR-574-3p shows an expression pattern similar to that of miR-140. miR-574-3p targets retinoid X receptor alpha (Rxra) whose expression is progressively reduced during chondrocyte differentiation. Sox9 binds to the promoter of the miR-574 gene and increases its transcription in chondrocytes. Since Rxra was previously shown to inhibit Sox9 activity, miR-574-3p may be part of a positive feedback loop that promotes chondrogenesis.

Expression of miR-335, co-expressed with its host gene Mest, increases upon chondrocyte differentiation of mouse MSCs [32]. miR-335 overexpression in MSCs promotes chondrocyte differentiation. miR-335-5p targets Daam1 and Rock1, negative regulators of Sox9. Sox9 downregulates expression of miR-29a and miR-29b, which, in turn, suppress Mest expression. Therefore, this regulatory relay may form a positive feedback loop to enhance chondrogenesis. In another study using human MSCs, miR-29a was identified as one of the most downregulated miRNAs during chondrogenesis [33]. Consistent with the aforementioned study, Sox9 was again found to downregulate miR-29a. Overexpression of miR-29a inhibited chondrogenesis. In this study, Foxo3a was identified as a direct target of miR-29a.

There are several other miRNAs whose expression is regulated upon chondrocyte differentiation of mouse MSCs. miR-24 and miR-199b were upregulated by more than fivefold upon differentiation into chondrocytes and osteoblasts [20]. miR-101, miR-124a and miR-199a were also upregulated, while miR-18 and miR-96 were downregulated. In human MSCs, miR-199a was upregulated by four-fold upon chondrocyte differentiation [21]. These miRNAs may regulate lineage determination during MSC differentiation.

Role of microRNA in Articular Cartilage Homeostasis and Osteoarthritis

In addition to growth plate cartilage, chondrocytes form permanent cartilage in ribs and joints. Permanent cartilage has distinct and unique properties compared with growth plate cartilage. Impairment of function of permanent cartilage, particularly synovial joint cartilage, limits skeletal mobility and poses significant health risks especially to the elderly. In this section, we discuss roles of miRNAs in synovial joint cartilage in relation to osteoarthritis (OA), the most common cartilage disease.

OA is characterized by destruction of the articular cartilage caused by an imbalance between synthesis and degradation of extra-cellular matrix (ECM) components, such as type II collagen and proteoglycans. Age-related chondrocyte dys-function likely reduces ECM synthesis, while various stresses induce aberrant expression of matrix-degrading enzymes, including matrix metalloproteinases (Mmps) and a disintegrin and a metalloproteinase domain with thrombospondin motif (Adamts) family enzymes [34]. It is also recognized that inflammation plays an important role in OA progression. Significant efforts have been made to understand the molecular mechanisms underlying OA pathogenesis. In recent years, studies have revealed that microRNAs are involved in articular cartilage homeostasis and OA. Because there have been published a relatively large number of studies on this subject, we concisely summarize the relevant literature in Table 1. We also individually discuss several miRNAs of particular interest.

Table 1. Summary of microRNAs in OA.

microRNAs	Expression change in OA	Target gene(s)	Function	Study models
miR140	Decreased	Adamts5	ECM homeostasis	Human articular chondrocytes and human bone marrow-derived mesenchymal stem cells (MSCs) [43].
	Decreased	Adamts5	ECM homeostasis	Cartilage-specific miR-140 overexpression and miR-140 deletion in mice. Mouse models with surgically induced OA and antigen-induced arthritis (AIA) [13].
	Decreased	Igfbp5	ECM homeostasis	Human OA cartilage from patients undergoing total knee arthroplasty (TKA) [37].
	Decreased	Mmp13	ECM homeostasis	The human cartilage cell line C28/I2 treated with IL-1β to mimic an osteoarthritic environment [42].
	Increased	Adamts5, Dnpep	Maintenance of chondrocytes and ECM homeostasis	Human articular cartilage of femoral heads from patients undergone total hip replacement surgery [39].
	Increased	ND	Maintenance of chondrocytes and ECM homeostasis	Human articular chondrocytes from OA patients with knee replacement and biopsy samples of normal cartilage [38].
	Increased	Mmp13	ECM homeostasis	Human articular chondrocyte C28/I2 cells [42].
miR-145	Increased	Sox9	Maintenance of chondrocytes and ECM homeostasis	OA articular cartilage from the knee [44].
	Increased	Smad3	Maintenance of chondrocytes and ECM homeostasis	Human articular cartilage [45].
miR-27b	Decreased	Mmp13	ECM homeostasis	Full-thickness cartilage and subchondral bone from OA patients [41].
miR-27a	Decreased	Igfbp5	ECM homeostasis	Human OA cartilage of the knee [36].
miR-21	Increased	ND	aging	Normal cartilage of the metacarpophalangeal joints of eight horses; four young donors (4 yr old) and four old donors (>15 yr old) [48].
	Increased	Gdf5	ECM homeostasis	Human articular chondrocytes (CH8 cell lines). Ten paired osteoarthritic tissues and matched normal tissues from traumatic amputees [64].
miR-23b	Increased	ND	Chondrocyte differentiation	Synovial fluid samples obtained from 10 osteoarthritis patients by joint puncture. SFMSCs (synovial fluid-derived mesen-chymal stem cells) were isolated from synovial fluid [51].
miR-9	Decreased (61), Increased (54)	Protogenin (61), Mmp13 (54)	Chondrocyte survival	Human articular cartilage. Overexpression of miR-9 in cartilage tissue by lentivirus injection into mouse knee joints [61].
miR-34a	Increased with IL-1β stimulation	Col2a1 and iNOS (indirect effect)	Chondrocyte apoptosis	Articular chondrocytes isolated from rat knee joints [62].
miR-101	Decreased	Dnmt3b	Chondrocyte differentiation	Wing mesenchymal cells. Overexpression of miR-101 in cartilage tissue by injecting miR-101-expressing lentiviruses into mouse knee joints [47].
	Increased with IL-1β stimulation	Sox9	Maintenance of chondrocytes and ECM homeostasis	IL-1β-stimulated rat primary chondrocytes [46].
miR-199a	Decreased	Cox-2	Inflammatory response	Cartilage samples from patients with OA [63].
miR-199a-3p	Increased with age	ND (Not detected)	Maintenance of chondrocytes and ECM homeostasis	Human articular cartilage [50].
miR-146	Decreased	Traf6, Irak1	Inflammatory response	Human OA cartilage [53, 54].
	Decreased (late stage OA), Increased with IL-1β stimulation	Mmp13	ECM homeostasis	Human OA cartilage [55].
miR-146a	Increased	Smad4	Inflammatory response and mechanical stress	Rat OA cartilage, surgically induced OA rats [56], human chondrocytes with pressure-mediated mechanical stress [57].
	Increased	IL-1β, Col13a1, Smad4	Inflammatory response	miRNA expression profiles in the plasma of patients with OA of the knee in comparison with those of healthy subjects [59].
miR-149	Decreased	ND	Inflammatory response	Human OA cartilage [60].
miR-22	Increased	PPARα, Bmp7	ECM homeostasis and BMI	OA knee articular cartilage obtained from patients [43].
miR-455	Increased	Smad2, Acvr2b, Chrdl1	Maintenance of chondrocytes and ECM homeostasis	SW-1353, C3H10T1/2, and 3 T3 cells, human articular cartilage obtained from femoral head [39].
miR-885-5p	Increased	ND	ND	miRNA expression profiles in the plasma of patients with OA [59]. Primary human articular chondrocytes [65].
miR-24	Decreased	p16INK4a (Cdkn2a)	aging	IL-1beta-treated primary OA chondrocytes, mesenchymal stem cells differentiated into chondrocytes [66].
miR-148a	Decreased	Col10a1, Mmp13, Adamts5	ECM homeostasis	Chondrocytes isolated from OA articular cartilage of the knee [67].
miR-127-5p	Decreased	Mmp13	ECM homeostasis	Normal and osteoarthritic (OA) human cartilage [68].
miR-29c	Increased	Col3a1	ECM homeostasis	miRNA expression profiles in the plasma of patients with OA of the knee in comparison with those of healthy subjects [59].
miR-16	Increased	Vegfa, Fgfr1	Inflammatory response	miRNA expression profiles in the plasma of patients with OA of the knee in comparison with those of healthy subjects [59].
miR-93	Increased	Vegfa	Inflammatory response	miRNA expression profiles in the plasma of patients with OA of the knee in comparison with those of healthy subjects [59].
miR-483	Increased	Mmp13, Sox5 and Bmp1	Maintenance of chondrocytes and ECM homeostasis	The surgical OA mouse models by resection of the medial collateral ligament and medial meniscus of the knee joint [69].
miR-483*	Increased	Igf1, Igf2 and Il1r1	Inflammatory response	The surgical OA mouse models [69].
miR-558	Decreased	Cox2, NF-κB,	Inflammatory response	OA cartilage obtained from knee joints from patients, human chondrogenic SW1353 cells [70].
miR-125b	Decreased	Adamts4	ECM homeostasis	Chondrocytes isolated from knee cartilage and treated with interleukin-1 beta [71].
miR-181b	Increased	Mmp13 (indirect effect)	Cartilage development	Human chondrocytes prepared from macroscopically severely damaged zones of osteoarthritic knee joints or biopsy specimens of normal cartilages, surgically induced mouse OA [72].
MiR-675	Increased	Col2a1 (indirect effect)	Chondrocyte differentiation and maintenance	OA cartilage isolated from knee joints of patients with primary or secondary osteoarthritis [73].
miR-194	Increased	Sox5	Chondrocyte differentiation and maintenance	Articular chondrocytes isolated from the knee joint of juvenile rats [74].
miR-488	Decreased	ZIP8/Slc39a, Mmp13 (indirect)	ECM homeostasis	Human articular chondrocytes isolated from biopsy samples of normal cartilages [38].
miR-455	Increased	Smad2, Acvr2b, Chrdl1	Chondrocyte differentiation and maintenance	ATDC5 cells [39].
miR-203	ND	Trpv4	Inflammatory response	Mandibular condylar cartilage [75].
miR-320c	Decreased	Adamts5	Aging	Cartilaginous tissue obtained from 17 OA patients [50].
miR-193b	Increased	Col2a1, aggrecan, Sox9	Maintenance of chondrocytes and ECM homeostasis	Human articular cartilage [50].
miR-222	Increased	ND	Articular cartilage mechanotransduction	Bovine articular cartilage [76].
miR-221	Increased	ND	Articular cartilage mechanotransduction	Bovine articular cartilage [76].
miR-365	ND	Hdac4	Mechanical stimulation of chondrocyte differentiation	Primary chicken chondrocytes cultured in three-dimensional collagen scaffoldings under cyclic loading (1 Hz, 5 % elongation) [77].

Maintenance of Articular Chondrocytes and Cartilage

As in the growth plate chondrocytes, miR-140 is highly expressed in human articular chondrocytes. miRNA profiling of patient-derived osteoarthritic chondrocytes has demonstrated downregulation of miR-140 in chondrocytes of osteoarthritic cartilage compared with normal human cartilage [35–37], although some studies also show increased miR-140 expression in OA cartilage [38, 39]. A recent study also reported that the expression level of miR-140 in the knee joint synovial fluid was inversely correlated with the severity of OA in patients [40].

miR-140 is expressed in the surface and middle zones of articular cartilage of the mouse knee joint, exhibiting an expression pattern similar to that of Col2a1 [11•]. Miyaki et al showed that miR-140-deficiency caused early onset of OA in aging mice and accelerated development of OA in OA-induced mice [13••]. Conversely, transgenic mice overex-pressing miR-140 in articular cartilage under a Col2a1 promoter were resistant to proteoglycan and type II collagen loss after antigen-induced joint inflammation. In this study, Adamts5, an aggrecan-degrading enzyme, was identified as a direct target of miR-140 in articular cartilage.

Tardif et al reported significantly decreased miR-140 expression in chondrocytes isolated from patients with OA [36]. Insulin-like growth factor binding protein-5 (Igfbp5) appears to be a direct target of miR-140. In this study, miR-27a was also modestly decreased in OA chondrocytes. Inhibition of miR-27a also significantly increased Igfbp5 as well as Mmp13 expression. A separate study showed that treatment of chondrocytes with Interleukin-1 beta (IL-1β), a major mediator of inflammation in OA, significantly downregulated miR-27b expression. These authors found that miR-27b directly targeted Mmp13 to regulate its expression [41]. miR-140 was also shown to suppress IL-1β-induced Mmp13 expression in the human chondrocytic C28/I2 cells by directly targeting Mmp13 mRNA [42]. Mechanisms controlling miR-140 expression were studied in OA chondrocytes [37]. Smad3, a downstream mediator of the transforming growth factor beta (TGF-β) pathway, reduces expression of miR-140, but not its host gene, Wwp2. This regulation is mediated by direct binding of Smad3 to a regulatory sequence unique to the miR-140 gene. This study also showed that nuclear factor of activated T-cells (NFAT) transcription factors, NFAT5 and 3, regulated miR-140 expression likely through the same regulatory element.

IL-1β treatment suppresses miR-140 expression and increases Adamts5 expression in OA chondrocytes [43]. miR-140 overexpression downregulated IL-1β-induced Adamts5 expression.

A recent study has shown that miR-145 is increased in OA chondrocytes and in response to IL-1β stimulation [44]. miR-145 inhibition was able to reverse the downregulation of type II collagen and aggrecan expression caused by IL-1β treatment. This study identified Smad3 as a direct miR-145 target. An independent study using human articular chondrocytes has shown that miR-145 targets Sox9 [45]. miR-145 overexpression decreased Sox9 expression and subsequently reduced the cellular phenotype of differentiated chondrocytes and decreased miR-140 expression.

Sox9 is also targeted by miR-101 [46]. Silencing of miR-101 in rat articular chondrocytes reversed the IL-1β-induced downregulation of collagen type II and aggrecan. Kim, et al. reported that miR-101 was expressed at a lower level in severely damaged OA regions than in non-OA regions of human articular cartilage [47]. Lentivirus-mediated miR-101 overexpression significantly reduced cartilage destruction in OA-induced mouse knee joints. miR-101 targeted DNA methyltransferase 3 beta (Dnmt3b); this appeared to suppress integrin-α1 expression. Using a surgically-induced mouse OA model, this group also demonstrated that overexpression of miR-101 or integrin-α1 prevented OA progression.

In a study to identify genes associated with aging, miR-21 was found increased in aged horse articular cartilage [48]. Since miR-21 promotes proliferation and matrix synthesis in rat chondrocytes embedded in atelocollagen gels [49], miR-21 upregulation upon aging could be a counter-regulatory mechanism protecting against aging-associated impairment of chondrocyte function. By microarray analysis on human articular chondrocytes, miR-199a-3p and miR-193b were found to be upregulated with age, and miR-320c was found to be downregulated with age [50]. Overexpression of miR-199a-3p or miR-193b in chondrocytes downregulated type II collagen, aggrecan, and Sox9 expression, and decreased proliferation. Therefore, miR-199a-3p and miR-193b may accelerate the aging process of chondrocytes. However, the precise mechanisms by which these miRNAs regulate chondrocyte genes are unclear.

Song et al found that miR-23b was upregulated in human OA chondrocytes [38]. miRNA-23b overexpression induced chondrogenic differentiation by downregulating protein kinase A (PKA) signaling by targeting Prkacb that encodes a catalytic subunit of PKA, in synovial fluid-derived mesenchymal stem cells [51]; miR-23b may contribute to OA progression by inducing chondrocyte differentiation.

Inflammatory Response

miR-146a and miR-146b appear to be key microRNAs in the inflammatory response [52]. miR-146a and miR-146b were regulated by the transcription factor NF-κB, a critical mediator of inflammatory cytokines. These miRNAs were shown to directly target IL-1 receptor-associated kinase 1 (Irak1) and TNF receptor-associated factor 6 (Traf6), upstream regulators of NF-kB [53]. Therefore, miR-146a and miR-146b may be components of a negative feedback loop that regulates cytokine signaling. Jones et al determined the expression profile of 157 miRNAs in late-stage human OA cartilage [54]. Among 17 miRNAs that showed more than 4-fold differential expression between OA and normal cartilage, miR-146a and miR-149 were the top two most downregulated miRNAs in late-stage OA cartilage. Overexpression of miR-146a in isolated human chondrocytes reduced IL-1β-induced TNF-α production, suggesting that this miRNA is a negative regulator of inflammation in OA.

Yamasaki et al found that miR-146a was expressed abundantly in early-stage human OA cartilage and that its expression was reduced as OA advances [55]. In situ hybridization analysis revealed that miR-146a was expressed in the superficial layer with degenerative changes in early stage OA. Consistent with previous reports that miR-146a expression was regulated by cytokine signaling, in vitro stimulation of human normal chondrocytes by IL-1β markedly increased miR-146a expression [55]. Another study also shows upregulation of mIR-146a by IL-1β treatment in primary rat chondrocytes [56]. Surgically-induced instability of rat knee joints also increased miR-146a expression. miR-146a was found to target and downregulate Smad4, which subsequently induced VEGF expression and increased articular chondrocytes apoptosis. Another in vitro study using human articular chondrocytes has also shown that miR-146a is induced in chondrocytes by pressure-mediated mechanical stress [57]. This was accompanied by Smad4 downregulation, VEGF upregulation, and increased chondrocyte apoptosis. miR-146a expression is also induced by HDAC inhibitor treatment in synovial fibroblasts isolated from OA patients [58]. This study showed that HDAC inhibitor treatment increased NF-κB binding to the promoter region of the miR-146a gene. It is therefore possible that miR-146a expression is also epigenetically regulated. Interestingly, miRNA expression profiling showed increased levels of miR-146 in OA patients [59]. This finding suggests that plasma miR-146 can be used as a marker for assessing OA severity.

miR-149 is downregulated in OA chondrocytes [60]. miR-149 targets TNFα, IL1β, and IL6; miR-149 downregulation might contribute to OA progression. Iliopoulos et al analyzed miRNA and protein expression profiles in OA cartilage [35]. They found that 16 miRNAs and 76 proteins were differentially expressed between OA and control cartilage. Many upregulated proteins were involved in inflammatory pathways, such as IL-1β and IL-6, while downregulated proteins were involved in lipid metabolism, such as peroxisome proliferator-activated receptor α (Pparα). This study also found that two upregulated miRNAs (miR-22 and miR-103) were highly correlated with body mass index (BMI) in OA patients. They showed that miR-22 directly targeted Pparα and Bmp7. miR-22 overexpression, or suppression of either Pparα or Bmp7 increased IL-1β and Mmp13 levels in chondrocytes, suggesting that miR-22 may promote OA-related inflammation by modulating lipid metabolism.

Jones et al found that several miRNAs, including miR-9, miR-98, and miR-34, were upregulated in OA cartilage [54]. Overexpression of miR-9 and miR-98 in isolated human chondrocytes reduced IL-1β-induced TNF-α production, and inhibited secretion of Mmp13. Interestingly, another study reported a significant decrease of miR-9 expression in OA chondrocytes [61]. The reason for this contradiction is unclear; possible sample heterogeneity may be to blame because both studies used human OA samples. In the latter study, Protogenin (Prtg), that activates caspase-3 signaling and promotes chondrocyte apoptosis, was found as a direct target of miR-9.

Abouheif et al reported that expression of the p53-regulated miRNA, miR-34a, was upregulated in rat primary chondrocytes upon IL-1β stimulation [62]. Silencing of miR-34a significantly prevented the IL-1β-induced Col2a1 down-regulation, iNOS (inducible nitric oxide synthase) upregulation, and apoptosis in rat chondrocytes.

IL-1β increases cyclooxygenase-2 (Cox2) expression in OA, and IL-1β signaling is mediated also by mitogen activated protein kinases (MAPKs). Akhtar et al showed that IL-1β-induced activation of p38-MAPK signaling reduced miR-199a expression in OA chondrocytes [63]. They showed that miR-199a directly targeted Cox2. Treatment of OA chondrocytes with the p38-MAPK inhibitor, SB202190, increased miR-199a levels and significantly decreased Cox2 expression.

In addition to the miRNAs that we have discussed, many more miRNAs appear to play important roles in various aspects of cartilage homeostasis and OA pathogenesis (Table 1). Understanding the complex miRNA regulatory networks is expected to provide scientific bases for developing novel diagnostic methods and therapeutic strategies for OA.

Conclusions

These studies have demonstrated that various miRNAs regulate many aspects of cartilage development, homeostasis and pathology through diverse mechanisms. Our understanding about roles of miRNAs in chondrocytes and cartilage has significantly increased in the past decade. However, arriving at a comprehensive view on the miRNA-mediated regulatory network in cartilage from the plethora of data obtained from highly heterogeneous systems still requires further coordinated efforts. Testing these findings in more uniform and physiologic settings will clarify the roles of individual miRNAs and their targets, and serve as a strong foundation for translating these findings to help patients with osteoarthritis.