Growth differentiation factor 5 in cartilage and osteoarthritis: A possible therapeutic candidate

Abstract Growth differentiation factor 5 (GDF‐5) is essential for cartilage development and homeostasis. The expression and function of GDF‐5 are highly associated with the pathogenesis of osteoarthritis (OA). OA, characterized by progressive degeneration of joint, particularly in cartilage, causes severe social burden. However, there is no effective approach to reverse the progression of this disease. Over the past decades, extensive studies have demonstrated the protective effects of GDF‐5 against cartilage degeneration and defects. Here, we summarize the current literature describing the role of GDF‐5 in development of cartilage and joints, and the association between the GDF‐5 gene polymorphisms and OA susceptibility. We also shed light on the protective effects of GDF‐5 against OA in terms of direct GDF‐5 supplementation and modulation of the GDF‐5‐related signalling. Finally, we discuss the current limitations in the application of GDF‐5 for the clinical treatment of OA. This review provides a comprehensive insight into the role of GDF‐5 in cartilage and emphasizes GDF‐5 as a potential therapeutic candidate in OA.


| INTRODUC TI ON
Cartilage is an integral part of the skeletal system that is com-  3 Subsequently, the secondary ossification centre newly develops at the epiphysis and radially spreads within it. The part between the secondary ossification centre and the joint cavity is permanently retained as articular cartilage. 4 Articular cartilage is a highly specialized tissue that covers and protects the ends of long bones. It consists of collagen type II, hyaluronic acid, and a rich proteoglycan matrix secreted by chondrocytes. 5 These cells regulate the homeostasis of articular cartilage through balancing their own anabolism and catabolism. 6 Because of the unique function of cartilage, the interference during chondrogenesis and the loss of cartilage tend to cause joint disorder.
Osteoarthritis (OA) is one of the most common joint diseases, primarily owing to the progressive destruction of cartilage. 7 In addition, features of OA include synovial inflammation and subchondral bone remodelling. 7 The disease severely influences people's quality of life worldwide, and the incidence of OA is increasing because of the ageing population throughout the world. 7 Nevertheless, current treatment options for OA are limited to relieving pain and promoting functional improvement in patients rather than inhibiting OA progression. Therefore, there is an urgent need to develop more effective pharmacologic treatments for OA. Growth factors are a group of bioactive endogenous polypeptides that can stimulate cellular growth, proliferation and differentiation. 8 In articular cartilage, numerous growth factors, such as transforming growth factor-β1 (TGF-β1), insulin growth factor I, bone morphogenetic protein (BMP) and fibroblast growth factors, have been widely investigated regarding the regulation of development and homeostasis of articular cartilage. 9 In addition, growth factors are readily available and recombined with fewer side-effects. Therefore, growth factors offer promising therapeutic approaches for OA. Among these growth factors, growth differentiation factor 5 (GDF-5), a member of the TGFβ family, has attracted wide attention in the field of OA. GDF-5 is one of the earliest markers of joint development. 10 The steps underlying joint development, growth, remodelling and homeostasis require regulation of GDF-5 and relative signalling pathways. 11 Therefore, this growth factor is critical to joint health. Accumulated evidence has revealed a robust and highly reproducible correlation between GDF5 and knee OA susceptibility. 12 GDF-5 deficiency contributes to the pathogenesis of OA, 13 whereas GDF-5 supplementation has a beneficial effect on the experimental model of OA. 14 These studies raise the possibility of manipulating GDF-5 levels for the treatment of OA. In this review, we will provide an overview of GDF-5 and its roles in cartilage development and OA. Importantly, we highlight and discuss the potential applications of GDF-5 for OA treatment.

| OVERVIE W OF G DF-5
GDF-5, also known as cartilage-derived morphogenetic protein-1 (CDMP-1) or bone morphogenetic protein-14, is a member of the TGFβ/BMP superfamily. 15 It was first identified in 1994, 16 and subsequently, human GDF-5 was cloned during the same year. 17,18 The structure of GDF-5 is closely related to the BMPs. 19 GDF-5 is synthesized as a large precursor protein, which comprises two major domains: the N-terminal prodomain with a cleavage site and signal sequence and the active C-terminal domain. The precursor protein then is cleaved at a characteristic RXXR (Arg, X, X, Arg) site to release the active peptide. The active peptide is highly conserved with seven cysteine residues 19,20 and contains two regions: the N-terminal region, which is involved in forming a tail-like structure within GDF-5 dimers, and the C-terminal region, which is responsible for forming of homodimers and heterodimers. 21 GDF-5 transduces signals by binding to two types of the transmembrane serine/threonine kinase receptors, types I and II. 22,23 Among the seven known type I receptors, BMP receptor (BMPR)-IA   and BMPR-IB have been demonstrated to be associated with skeletal patterning. 24-26 GDF-5 has high binding affinity to BMPR-IB,   BMPR-II and Activin type II receptors. 26,27 Upon binding to type I   and type II receptors, the signalling cascade of GDF-5 activates the downstream Smad pathway. The phosphorylated Smad 1/5/8 then forms the complex with Smad 4, a common Smad that translocates into the nucleus to regulate the transcription of multiple genes including COL2A1 and ACAN, and cellular processes, such as proliferation, differentiation and synthesis of the ECM. 28,29

| G DF-5 IN C ARTIL AG E DE VELOPMENT
Chondrogenesis is the differentiation process that leads to the formation of cartilage and bone. 30 This process involves the recruitment, migration and condensation of mesenchymal cells as well as differentiation and maturation of chondrocytes. 31 The GDF-5 expression profile indicates its importance in chondrogenesis, especially in prechondrogenic condensation. 32 In vivo studies have shown that mutations in Gdf-5 may cause the autosomal recessive syndromes such as brachypodism (bp) in mice and Grebe-type and Hunter-Thompson chondrodysplasia in humans, characterized by the shortening of the skeletal elements and abnormal development of some joints. 24,33,34 To investigate how GDF-5 controls skeletogenesis, some in vivo molecular function studies have been conducted, and the results showed that overexpressed Gdf-5 in the embryos and developing chick limb increased the size of the skeletal elements during the condensation stage or later when the skeletal elements have been formed respectively. 35 Gdf-5 overexpression can initiate chondrogenesis, which enhances cell adhesiveness of mesenchymal cells and proliferation of chondrocytes. 35,36 During mesenchymal condensation, the presence of GDF-5 stimulated the activities of N-cadherin and further improved cell-cell adherence, thereby promoting condensation. 37  Intriguingly, the combination of GDF-5, TGF-β1 and BMP-2 promoted the greatest upregulation of chondrogenic genes, including SOX9, COL2A1 and ACAN, and synthesis of cartilage-specific matrix, eventually yielding robust cartilage rich in GAGs and collagen type II after 4 weeks of maturation. 41 In addition, recombinant GDF-5 protein can also improve chondrogenic differentiation in other stem cells, such as foetal human MSCs, 42 human embryonic stem cells, 43 canine MSCs, 44 embryonic chick mesenchymal cells 37 and rabbit adipose-derived stromal cells. 45 Furthermore, adenovirus-mediated overexpression of Gdf-5 exerted similar effects on chondrogenesis in adipose stem cells. 46 The phosphorylation of Smad 1/5/8, the downstream signalling molecule of TGF signalling, is greatly involved in the terminal differentiation of chondrocytes. 47  All these findings demonstrate that GDF-5 is essential for chondrogenesis, which governs the development of cartilage and bone ( Figure 1).

| G DF-5 IN JOINT DE VELOPMENT
Over the past decades, the emergence of the interzone at the presumptive joint site has been considered as an initial indicator of joint development, and the interzone cells have been suggested to serve as joint progenitors. 50 The interzone, derived from mesenchymal tissue, loses the specific gene expression of chondrocytes and, notably, expresses GDF-5, which acts as an indicator of the interzone in the early development of joints. 51,52 It has been found that the gene mutation of the human GDF-5 and its receptor BMPR-1B can lead to skeletal malformations, including brachydactyly and/or chondrodysplasia. 25,33,53,54 In addition, mice with brachypodism exhibit sufficient GDF-5 expression occurring out of the interzone, giving rise to the fusion of digit joints. 55 These findings confirm that GDF-5 is critical for the development of joints.
In constitutively active Gdf-5-Cre mice, the broad labelling of R26R-reporter cells was seen throughout multiple joint tissues. 56 In another study, Decker et al 57

F I G U R E 1
The role of GDF-5 during cartilage development. The process starts with mesenchymal stem cell (MSCs) condensation. Condensed cells are differentiated into chondrocytes that construct a cartilage template (anlagen) by synthesizing ECM. Chondrocytes at the centre of condensation become hypertrophic and hypertrophic cartilage is calcified. These terminally differentiated chondrocytes undergo apoptosis and are replaced by the calcified bone in the primary ossification centre. Later, the secondary ossification centre is newly developed at the epiphysis and radially spreads within it. The part between the secondary ossification centre and the joint cavity is permanently retained as articular cartilage. During cartilage development, GDF-5 promotes mesenchymal cell condensation and differentiation into chondrocytes and further stimulates chondrocytes differentiating into proliferative, prehypertrophic and hypertrophic cells. In articular cartilage, GDF-5 functions to maintain cartilage homeostasis However, recent evidence has suggested that cells from the surrounding area are integrated to form joints during development. 56,58 In a study of Shwartz et al, 51 GDF-5-positive cells were detected in the epiphyses, articular cartilage, meniscus and intraarticular ligaments in the knee from E10.5 to E18.5. Furthermore, by using a knock-in Gdf-5-CreERT2 and Cre-dependent reporter mice, Shwartz et al 51 found that GDF-5 (+) cells from the surrounding tissues continuously flowed into the interzone, where they contributed to the formation of joint tissues. In contrast, the interzone/ early-specific cells lost GDF-5 expression and migrated out to primarily form the epiphyseal cartilage. The similar migration was also observed by Decker et al. 59 The major cells responsible for forming the interzone did not form articular cartilage but formed transient cartilage, meniscus and ligaments. 51 On the other hand, the peripheral cells have been well-recognized to prominently contribute to the formation of joint components. 4 In the late stage of joint development, GDF-5 expression was decreased in the knee region and appeared to be dispensable to joint development. 52 Thus, the role of GDF-5 in the joint/cartilage development may be determined by their spatiotemporal environment. In Gdf5-CreERT2/R26-zsGreen mice, the CreERT2-positive cell labelling was more restricted to cells within the eventual articular cartilage after tamoxifen administration at late embryonic time points. 59 These labelled cells and/or their progeny remained in the articular cartilage until at least 6 months of age 59 (Figure 2). In adulthood, GDF-5 might be related to homeostasis of the articular cartilage because genomewide association studies have revealed that GDF-5 is a susceptible gene for OA. 60

| GDF-5 gene polymorphism is associated with knee OA
Single-nucleotide polymorphisms (SNPs) are common genomic DNA variations within a population. 61 An SNP located within the coding sequence of a gene may give rise to an amino acid substitution and further alter protein function, which might be associated with susceptibility to human diseases. 61 It is widely acknowledged that SNPs in some genes are related to the susceptibility to OA. 62,63 Such genes include those participating in signalling cascades involved in joint and bone biology as well as those involved in inflammatory pathways. 64 SNPs in these genes are closely related to abnormal proliferation and differentiation of chondrocytes, leading to abnormal cartilage development and morphology. 65 Over the past decades, numerous studies have demonstrated that polymorphisms in GDF-5 highly contribute to the pathogenesis of OA. 60,66,67 In particular, the SNP rs143383, a Tto-C transition in the 5ʹ untranslated region (UTR) of the GDF-5 gene and the GDF5 promoter, is a major susceptibility allele for OA in Asian and European populations. 60,68,69 Expression of the OA-associated T allele has been found to be significantly lower than that of the C allele in patients with OA. [68][69][70] Also, a meta-analysis based on 23 995 subjects has shown that the C allele of the GDF5 gene was protective for knee OA susceptibility across different populations. 71 However, there are some differences in the association between genetic variants of GDF-5 and OA of the knee, hip and hand. Using a random effects model, a significant difference was identified between patients with F I G U R E 2 GDF-5 (+) cells are involved in joint formation. The interzone emerges at the presumptive joint site within a pre-cartilage tissue. Mesenchymal GDF-5 (+) cells constitute the interzone area at incipient joint sites in early embryonic limbs, and flanking cells are recruited from surrounding tissues into the GDF-5 lineage. Subsequently, joint cavitation occurs in the centre of the interzone, and the surrounding cells influx into the joint region. The GDF-5 (+) cells in the interzone and its surroundings gradually form articular cartilage and synovial joints. In the late stage of joint development, GDF-5 is decreased in the knee region and GDF-5 (+) cells are restricted within the eventual articular cartilage knee OA and controls for the T allele of rs143383. For hand OA, a moderate association was observed for rs143383 in the combined population. However, non-statistically significant summary odds ratio of hip OA was found in both combined studies and European studies. 72 For age, significant differences between knee OA and the control group were found in individuals aged more than 60 years, and no significant difference was observed for those aged less than 60 years in Asian population. 73 The imbalanced allelic expression could lead to reduced GDF-5 expression in the whole joint, which probably increases individual susceptibility to OA. 74 The trans-acting factors Sp1, Sp3 and DEAF-1 have been identified as transcriptional repressors of GDF-5 expression by modulating the allelic imbalance of rs143383. 74  Greek Caucasians, and no significant differences in allelic and genotypic frequencies were found when the individuals were stratified by sex. 77 The different results regarding rs143383 in different populations might be attributed to ethnic differences in GDF-5 methylation caused by environmental and genetic factors. 74 The existence of regulatory polymorphisms highlights the complexity of the regulation of GDF-5 expression. Importantly, it also provides multiple sites to better understand the susceptibility to OA and develop possible preventive approaches.

| The expression of GDF-5 during OA
GDF-5 is involved in the pathology of the whole joint organ, which could be reflected by altered expression of GDF-5 at the different stages of OA. GDF-5 expression has been found to be upregulated in the cartilage of patients with OA compared with that in control patients. 75 Further, GDF-5 expression was correlated with the expression of WNT9A and SOX11, which are known upstream regulators of GDF-5 expression. 78 In an experimental OA mice model that underwent surgical unilateral destabilization of the medial meniscus (DMM), GDF-5 downstream regulatory elements were activated in articular chondrocytes at 2 weeks, particularly in areas of initial damage. 78 Moreover, GDF-5 was also highly expressed during cartilage repair in DMM mice and was switched on in injured synovium in the prospective areas of cartilage formation, which implies that GDF-5 plays a role in cartilage repair. 78  Chronic low-grade inflammation causes many pathologic changes in OA. 82 This pathological condition is mediated primarily by inflammatory mediators, including tumour necrosis factor α (TNF) and interleukin 1β (IL-1β). 82 Bobacz K et al 83 showed that GDF-5 expression was also reduced in articular cartilage in TNF-transgenic mice with chronic arthritis. Additionally, GDF-5 was expressed in fibroblasts of adult synovial tissue and may counteract macrophage infiltration, which is considered an indicator of inflammatory infiltration. Its expression in the superficial lining layer of normal synovium represents a paracrine of GDF-5 to maintain homeostasis of cartilage, however, the redistribution of GDF-5 expression towards into deeper layers under a chronic pathological conditions in joints suggests a loss in maintenance and repair of intra-articular joint structure. 84 In vitro stimulation of TNF caused a reduction in GDF-5 expression in rheumatoid arthritis and OA fibroblasts compared with unstimulated cells respectively, however, another critical pro-inflammatory factor, IL-1β, revealed no change in GDF-5 expression in OA fibroblasts. 84 These studies indicate that inflammatory conditions might be the reason why GDF-5 expression is reduced in OA. However, the aetiology of OA is complex, and whether other pathological factors, such as oxidative stress, cell senescence and nutrition deprivation, can affect GDF-5 expression requires further investigation.
All these findings indicate that GDF-5 expression is altered in cartilage with the progression of OA, and the alteration of expression might reflect the function of GDF-5 and the stage of this disease.

| Therapeutic potential of GDF-5 in OA and cartilage repair
As mentioned above, GDF-5 is critical for joint homeostasis, especially for cartilage homeostasis. In an in vivo study performed by Parrish et al, 14 87 The β1 integrin family is a major receptor of the ECM, 88 and decreased expression of α5 integrin, a subunits of β1 integrin family, in articular cartilage is related to chondrocyte dedifferentiation during OA progression. 89 Interestingly, GDF-5 can induce the expression of the α5 subunit and the abundant expression of ACAN, collagen type II and Indian hedgehog. 89 Thus, the chondrocyte phenotype in articular cartilage is preserved because of the presence of GDF-5, α5 integrin and Indian hedgehog to maintain articular cartilage and prevent hypertrophy. 89 With respect to catabolism, GDF-5 supplementation could inhibit the expression of the ECM-degrading enzymes, including a disintegrin and metalloproteinase with thrombospondin motifs-4 and matrix metalloproteinase 13 (MMP13) in human chondrocytes. The likely mechanism is that GDF5 stimulation increases the expression of canonical Wnt inhibitors Dickkopf 1 (DKK1) and frizzled-related protein, thereby inhibiting the canonical Wnt signalling. 85 Enochson L et al 85 also indicated that this inhibition was DKK1-dependent. The DKK1-mediated inhibition of the canonical Wnt signalling was responsible for the downregulation of MMP13 expression. This finding demonstrates the molecular mechanism of the anti-catabolic effects of GDF-5 and could, to some extent, contribute to the understanding of the close link between the GDF-5 deficiency and OA development.
Osteophytes are a key feature of OA and are associated with pain and functional disability. 90 Identifying cells that form osteophytes in OA is important to target the cell population for the treatment of OA. It has been shown that platelet-derived growth factor receptor alpha-expressing stem/progenitor cells, present in the periosteum and synovium near the articular cartilage, were descendants of the Gdf5-expressing embryonic joint interzone. 91 These platelet-derived growth factor receptor alpha + GDF-5-lineage cells were activated in OA to form both the cartilage and bone of the osteophyte. 91 The expanding mineralizing fibrocartilage has been considered an earlier indicator of OA onset. 92 GDF-5 progenitors participated in the formation of fibrocartilage, which could develop into osteophytes post-injury. 92 GDF-5 + cells were also found in the enthesis and ligaments, indicating that these GDF-5 progenitors contribute to tendon enthesis and ligament development. 93 In addition, these progenitors could produce zonal enthesis following anterior cruciate ligament reconstruction. 94 The above results suggest that GDF-5-lineage cells present fibrocartilage and enthesis where osteophytes commonly occur during OA, and these cell populations could be targeted for the prevention of osteophyte occurrence.
GDF-5 is also related to cartilage repair and has been wellstudied in this field. 95 Additionally, SOX11, zinc finger E-box-binding homeobox 1 (ZEB1) and paired-like homeodomain transcription factor 1 (PITX1) are considered candidate molecules that could regulate GDF-5. 78 SOX11 could activate GDF-5 expression because of its direct binding to the 5'-UTR of the Gdf-5. 79 Kan et al 79  of OA, miR-449a and miR-21, upregulated in human OA cartilage, are highlighted due to their direct modulatory effects on GDF-5.
Overexpression of miR-21 suppressed the process of chondrogenesis and significantly promoted the levels of catabolic factors by inducing Gdf-5 mRNA decay to repress its expression. 104 Unfortunately, the therapeutic effects of miR-21 inhibition remain unclear. The upregulated miR-449a significantly suppressed ECM synthesis and aggravated chondrocyte ECM degradation by inhibiting GDF-5 expression. 81 Intriguingly, knock-down of miR-449a exhibited the opposite effects with increased mRNA and protein levels of 81 which provides an insight that upregulating GDF-5 via miR-449a might be a promising therapeutic approach for OA.
Osmolarity has been demonstrated as a critical parameter of the OA pathobiology, and the proteoglycan loss during OA could reduce the osmolarity of joint tissue to 270 mOsm, 105,106 whereas the osmolarity of healthy cartilage ranges from 350 to 480 mOsm. 107 Mang and his colleagues found that increasing the medium osmolarity reduced cytokine release and increased matrix production. 108 Interestingly, the response of chondrocytes to GDF-5 at 380 mOsm was more robust than that at 340 mOsm in 3D culture. 108 These results indicate that osmolarity is involved in OA pathogenesis at least partly by affecting cellular responsiveness to GDF-5. Nevertheless, modulating osmolarity to improve responsiveness to GDF-5 in cartilage might be very challenging for OA treatment (Figure 3).

| FUTURE PROS PEC TS
Current evidence has indicated that GDF-5 is essential for the de- In addition, based on extensive studies and meta-analyses, GDF-5 polymorphisms were demonstrated to be highly associated with OA susceptibility, primarily in European and Asian cohorts. 68,109,110 However, there exists some controversy about this association in some populations, which should be further investigated in broader populations. An exact association of those will partly explain the aetiology of OA at a genetic level. In addition, the molecules or alleles controlling GDF-5 expression are potential targets to prevent the onset of OA.
With respect to therapeutic potential, GDF-5 supplementation shows a significant effect on the chondrogenic process and maintenance of cartilage homeostasis, which provides a promising approach for the treatment of OA and cartilage defects. Moreover, the regulation of GDF-5 expression also provides some targets to restore the level of GDF-5 to fulfil its function (Table 1).
Currently, the application of this growth factor is still in infancy.
The possible limitations could be speculated as follows. First, GDF-5, a functional protein, is inclined to serve for local tissues instead to systemic use, which suggests that intra-articular supplementation or regulation appears to be a more appropriate approach. Second, GDF-5 may have limited ability to permeate into cartilage because of its size and the effective pore size of ~6 nm of cartilage for molecular diffusion. 111 Third, human OA chondrocytes respond discordantly to the exogenous GDF5. 112 All these limitations suggest that single GDF-5 treatment might not reach its full effects. Accordingly, how to fully exert its anti-arthritic potential is a critical issue. Cartilage tissue engineering, for example, the combination of GDF-5 and MSCs for cartilage repair, and the manipulation of the tissue microenvironment allowing chondrocytes to better respond to GDF-5 provide new insights into the application of this growth factor. Moreover, any method and approach to improve the safety, feasibility and efficacy of GDF-5 should be considered.

| CON CLUS ION
GDF-5 is essential for cartilage and joint development. GDF-5 polymorphisms are significantly associated with OA susceptibility.
Importantly, supplementation with GDF-5 exhibits prominent curative effects in experimental OA, and regulation of GDF-5 expression and signalling also provides promising therapeutic approaches for this disease. To initially establish this 'star molecule' in the clinical treatment of OA, it is necessary to further explore maximizing the availability and efficacy of GDF-5 in future studies.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Foundation of China [no. 81874020].

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Fengjing Guo worked on the design and conception of this review.
Kai Sun drafted the paper. Jiachao Guo, Xudong Yao and Zhou Guo contributed to revising the paper and all authors approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no data sets were generated or analysed during the current study.