Runx2 and Runx3 differentially regulate articular chondrocytes during surgically induced osteoarthritis development

The Runt-related transcription factor (Runx) family plays various roles in the homeostasis of cartilage. Here, we examined the role of Runx2 and Runx3 for osteoarthritis development in vivo and in vitro. Runx3-knockout mice exhibited accelerated osteoarthritis following surgical induction, accompanied by decreased expression of lubricin and aggrecan. Meanwhile, Runx2 conditional knockout mice showed biphasic phenotypes: heterozygous knockout inhibited osteoarthritis and decreased matrix metallopeptidase 13 (Mmp13) expression, while homozygous knockout of Runx2 accelerated osteoarthritis and reduced type II collagen (Col2a1) expression. Comprehensive transcriptional analyses revealed lubricin and aggrecan as transcriptional target genes of Runx3, and indicated that Runx2 sustained Col2a1 expression through an intron 6 enhancer when Sox9 was decreased. Intra-articular administration of Runx3 adenovirus ameliorated development of surgically induced osteoarthritis. Runx3 protects adult articular cartilage through extracellular matrix protein production under normal conditions, while Runx2 exerts both catabolic and anabolic effects under the inflammatory condition.


Introduction
Osteoarthritis (OA) is the most prevalent form of joint disease characterized by cartilage degeneration.
Progression of OA eventually leads to disability in older adult patients, which imposes a great socioeconomic burden. 1,2 OA, a multifactorial disease, is caused by aging, in ammation, heavy weight, hard work, and other factors. 3,4 In the early stage of OA, cartilage degeneration is obvious in the super cial zone (SFZ) of articular cartilage, which has a speci c role for joint lubricity by producing lubricin, encoded by the proteoglycan 4 (Prg4) gene. 5,6 Prg4-knockout (KO) mice display irregular surfaces at 2 months, and subsequently exhibit non-in ammatory hyperplastic synovium. 6 The deeper zone (DZ) of articular cartilage consists of chondrocytes and extracellular matrix. 7 Chondrocytes maintain the equilibrium of extracellular matrix and cartilage homeostasis by producing cartilage matrix proteins such as type II collagen α1 (Col2a1) and proteoglycans including Aggrecan (Acan). 8,9 Transcription of Col2a1 is strongly induced by sex determining region Y-box 9 (Sox9) through Sox9 consensus sites in introns1 and 6. 8, [10][11][12][13] Notably, SOX9 overexpression was shown to alleviate the progression of experimental OA. 14 In contrast to such anabolic factors, matrix metalloproteinase 13 (Mmp13) plays essential roles in cartilage degeneration and OA development through degradation of Col2a1. [15][16][17][18][19] MMP13 expression was low in normal and early degenerative cartilage, but strongly upregulated in late-stage OA specimens. 20 Increased MMP13 protein was observed in chondrocytes under in ammatory conditions, e.g. stimulation by interleukin-1 beta (IL-1β). 14 Previous studies have revealed multiple upstream molecules of Mmp13, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) associated factors, 21 hypoxia-inducible factor 2-alpha (HIF-2α), [20][21][22][23][24] and Runt-related transcription factor 2 (Runx2). [17][18][19] The Runx family, including Runx1, Runx2, and Runx3, plays central roles in the development and homeostasis of cartilage and bone. More than twenty years have passed since Runx2 was identi ed as the master regulator of chondrocyte maturation and endochondral ossci cation. 25,26 Runx family members have a Runt homology domain and the DNA recognition consensus motif TG(T/C)GGT. [27][28][29] Runx2 directly induces many kinds of chondrocyte hypertrophy-related genes, such as Mmp13, and positively regulates hypertrophic differentiation of chondrocytes and ossi cation. 29,30 Heterozygous knockout 31 or cartilage-speci c knockout 32 of Runx2 attenuated cartilage degeneration in knee joints of surgically induced OA model mice. Additionally, chondrocyte-speci c Runx2 overexpression led to posttraumatic OA progression. 33 However, chondrocytes from cartilage-speci c Runx2-knockout mice exhibited decreased expression of both Mmp13 and Col2a1. 34 Moreover, recent knee joint transcriptomes of 10-, 62-, and 95-week-old mice revealed increased expression of several in ammatory response-related genes as a result of aging, whereas the expression of several chondrocyte differentiation-related genes, including Sox9 and Runx2, decreased with aging. 35 In contrast to the catabolic effects of Runx2, Runx1 36 and Runx3 37 exert chondrogenic effects. It was previously reported that Runx1 enhances cartilage matrix production, while Runx1 de ciency accelerates OA progression. 36 Although Runx3 has been shown to promote aggrecan expression 37 and involved in regulation of chondrocyte proliferation and differentiation, 38 only a limited number of studies have examined roles of Runx3 in articular cartilage. Considering the diverse effects of Runx1, Runx2 may also have unique anabolic roles in articular chondrocytes, such as the production of cartilage matrix.
In this study, we examined roles of Runx2 and Runx3 in OA development using conditional knockout mice. We employed two surgical OA models in which in ammatory responses are deeply involved, and an aging model exhibiting more natural disease progression. Col2a1-Cre ERT2 and Prg4-Cre ERT2 mice were used for Cre/loxP recombination in chondrocytes of whole articular cartilage and SFZ chondrocytes, respectively. We further analyzed mechanisms underlying regulation of articular chondrocytes by Runx2 and Runx3 using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that Runx3 contributes to maintenance of articular cartilage through induction of extracellular matrix, while Runx2 exerts both catabolic and anabolic effects in a biphasic manner under the in ammatory condition.

Results
Expression of Runx2 and Runx3 in articular cartilage during development of surgically induced OA To examine Runx2, Runx3, and Sox9 expression in articular cartilage during OA development, we prepared an 8-week-old mouse OA model by surgical resection of the medial collateral ligament and medial meniscus (medial model). 39 Immunohistochemistry of normal cartilage showed that Runx2 protein was predominantly located in the DZ of preoperative articular cartilage, while Runx3 and Sox9 was found in all layers (Fig. 1a). After surgery, Runx3 and Sox9 started to decrease in surgically treated knee joints, accompanied by Col2a1. Runx2 was continuously detected in the DZ. Immunohistochemistry revealed enhanced expression of Mmp13, especially 2 weeks after surgery and predominantly in the DZ of articular cartilage at 4, 6, and 8 weeks after surgery (Fig. 1a). We additionally examined changes in Runx2, Runx3, and Sox9 expression in articular cartilage with aging. In the knee joints of WT mice, their expression decreased over time and was markedly weakened at 12 months of age (Fig. 1b).
We performed in vitro analyses to examine roles of Runx2 and Runx3 under in ammation using IL-1β, which is widely employed to model chondrocyte degradation. 14,40-42 Primary chondrocytes 43 were exposed to IL-1β at different concentrations for 24 h (Fig 1c); or 1 ng/mL IL-1β for 0−48 h (Fig 1d). Runx3 was downregulated by IL-1β exposure in dose- (Fig 1c) and time-dependent manners (Fig 1d), but levels of Runx2 mRNA were unchanged following IL-1β exposure (Fig 1c, d). Conversely, exposure to IL-1β elicited dose-and time-dependent upregulation of Mmp13 and suppression of Sox9, followed by a decrease of Col2a1 (Fig 1c, d). These results indicate that exposure of in vitro chondrocyte to 1 ng/mL IL-1β for 24 h reproduces similar expression patterns of marker genes and proteins observed in vivo surgical OA models, including stable expression of Runx2, reduced Runx3, Sox9 and Col2a1, and increased Mmp13.

Runx3 knockout accelerated OA development through Prg4 and Acan suppression
To reveal roles of Runx3 in whole or SFZ articular cartilage after skeletal growth, we compared Runx3 / littermates 44 (R3-Cntl) with Col2a1-Cre ERT2 ;Runx3 / (R3-cKO ER ) 45 and Prg4-Cre ERT2 ;Runx3 / (R3-pKO ER ) 46 mice. We prepared three distinct OA models ( Supplementary Fig. 1). As a result of the medial model, 39 OA development was signi cantly accelerated in R3-cKO ER (Fig. 2a, b) and R3-pKO ER (Fig. 2c, d) knee joints compared with R3-Cntl joints. In the articular cartilage of sham-operated R3-cKO ER joints, Runx3-positive cells decreased by 70% (Fig 2b). Positive areas of Acan and Prg4 decreased with Runx3 knockout (Fig. 2b). In the articular cartilage of sham-operated R3-pKO ER joints, Runx3-positive cells decreased by 40% and the positive area of Prg4 was decreased (Fig. 2d). Additionally, eight weeks after destabilizing the medial meniscus (DMM) 47 as a minor injury-induced OA model, OA development was also signi cantly accelerated in 24-week-old R3-pKO ER mice compared with R3-Cntl mice ( Supplementary   Fig. 2). In the aging model ( Supplementary Fig. 1c), OA development was unchanged in R3-cKO ER mice compared with R3-Cntl mice (Fig. 2e, f). In the articular cartilage of 18-month-old joints, the rates of Runx3-positive cells were not different for either genotype (Fig. 2f), probably because Runx3 expression decreased with aging as shown in Fig.1b. Expression of the marker proteins in both mice was similar as well (Fig. 2f). By Runx3 knockout in the whole articular cartilage, chondrocyte apoptosis was not signi cantly changed in the sham side, the surgical OA model, or with aging, as well as by Runx3 knockout in the SFZ articular cartilage of the sham side ( Supplementary Fig. 3a-d).
We then examined expression of OA associated genes in SFZ and DZ cells from Runx3 knockout and control mice. To enhance knockout e ciency, we prepared Runx3 / (R3-Cntl) and Col2a1-Cre;Runx3 / (R3-cKO) mice, which displayed normal skeletal development ( Supplementary Fig. 4a-g). Prg4, was decreased in R3-cKO SFZ cells and Acan was also decreased in R3-cKO DZ cells (Fig. 2g). Other genes were not changed in SFZ cells or DZ cells (Fig. 2g). We then prepared SFZ and DZ cells from R3-pKO ER and R3-Cntl littermates, and treated them with water-soluble tamoxifen. Only Prg4 was decreased in Runx3-de cient SFZ cells ( Supplementary Fig. 5). We further performed Runx3 overexpression in Runx3 knockout cells using adenoviral transduction. Downregulation of Prg4 in R3-cKO SFZ cells and Acan in R3-cKO DZ cells was recovered to control levels by Runx3 overexpression (Fig. 2h, i). Additionally, we performed dimethylmethylene blue (DMMB) assays 48 to investigate the catabolic effect of Runx3. Runx3 knockout did not signi cantly change the released glycosaminoglycan (GAG) content (Fig. 2j).
Immunohistochemistry showed that Runx2-positive cells were reduced by approximately 40% in R2-Hetero cKO cartilage and 80% in R2-Homo cKO cartilage of sham and OA joints (Fig. 3b, c), respectively. Protein levels of Sox9 and Col2a1 were unchanged in the sham joints of both genotypes (Fig. 3b), but Col2a1 was signi cantly suppressed in OA joints of R2-Homo cKO mice (Fig. 3c). Mmp13 was signi cantly reduced both in sham and OA joints of R2-Hetero and R2-Homo cKO mice (Fig. 3b, c). In the aging model, we further compared OA development between R2-Homo Cntl and R2-Homo cKO mice. At 18 months of age, OA development with aging was unchanged in R2-Homo cKO mice compared with R2-Cntl mice, as well as immunohistochemistry of Runx2, Sox9, Col2a1, or Mmp13 ( Supplementary Fig.  7a, b).
To examine chondrocyte apoptosis, we performed TdT-mediated dUTP nick end labeling (TUNEL) staining of medial and DMM model knee joints. Percentages of TUNEL-positive cells were unchanged by Runx2 deletion in both models ( Supplementary Fig. 8a). Conversely, TUNEL staining revealed upregulated chondrocyte apoptosis in the sham joints of R2-Homo KO mice ( Supplementary Fig. 8b). Cell numbers in articular cartilage were decreased in R2-Homo cKO mice ( Supplementary Fig. 8b). To examine short-term effects of Runx2 knockout, we injected tamoxifen into 7-week-old male littermate mice of the four genotypes daily for ve days, and sacri ced 1 week after injection without any surgery. Chondrocyte apoptosis was enhanced in the articular cartilage of R2-Homo cKO mice with decreased cell numbers ( Supplementary Fig. 8c).

Col2a1 expression was suppressed in homozygous Runx2-knockout chondrocytes under in ammatory conditions
Surgical induction differently altered OA development in R2-Hetero and Homo cKO mice, but had no effect on aging-model mice, indicating that Runx2 may be involved in cartilage degeneration by severe in ammation rather than aging with mild in ammation. To reveal roles of Runx2 in chondrocytes under in ammation, we performed ex vivo and in vitro analyses. We employed IL-1β because it is one of the essential cytokines responsible for human OA, increased in surgical OA models, and has been widely used in ex vivo and in vitro experiments for OA. [49][50][51] Safranin-O staining of femoral heads cultured for 2 weeks with or without IL-1β displayed consistent decreases in proteoglycans for each genotype (Fig. 4a).
Immunohistochemistry con rmed e cient knockdown of Runx2 in R2-Hetero and R2-Homo cKO femoral heads (Fig. 4a). Col2a1 protein levels were unchanged in normal cultures for both genotypes, but decreased following IL-1β exposure, especially in the R2-Homo cKO group (Fig. 4a). qRT-PCR using mRNA from the homogenized femoral heads con rmed that Runx2 was decreased according to genotype, but unchanged by IL-1β exposure (Fig. 4b). Mmp13 increased in all genotypes following IL-1β exposure, and the expression pattern of Mmp13 between genotypes was similar to that of Runx2 (Fig. 4b). Sox9 and Col2a1was decreased by IL-1β and unchanged between genotypes with a marked decrease in Col2a1 in R2-Homo cKO femoral heads following IL-1β exposure (Fig. 4b).
We next examined temporal changes in mRNA levels of marker genes in chondrocytes from the four genotypes exposed to 1 ng/mL IL-1β from 0−48 h. mRNA levels of Runx2 were e ciently decreased in R2-Hetero and R2-Homo cKO chondrocytes, and stable in each genotype following IL-1β exposure (Fig.  4c). Mmp13 increased with time and was suppressed in R2-Hetero and R2-Homo cKO chondrocytes compared with their respective controls at each time point. Sox9 gradually decreased, and there was no difference between genotypes at any point examined. Col2a1 was signi cantly suppressed in R2-Homo cKO more than 24 h after IL-1β exposure (Fig. 4c).
Genome-wide analysis of Runx2 and Runx3 association pro les in chondrocytes determined by ChIP-seq and RNA-seq To investigate mechanisms underlying chondrocyte regulation by Runx3 and Runx2, we planned ChIP-seq with an anti-FLAG antibody. For Runx3, we prepared SFZ cells from WT mice transfected with the FLAGtagged Runx3 expression vector. For the ChIP-seq, 21,730 raw peaks met the peak calling criterion and approximately 50% of all peaks mapped to an interval between ±50 and 500 kb from the transcriptional start sites (TSS) (Fig. 5a). Genomic Regions Enrichment of Annotations Tool (GREAT) Gene Ontology (GO) analysis 52 indicated that the extracellular structure organization and collagen bril organization terms were the most signi cantly enriched in the gene set (Fig. 5a). De novo motif analysis of the top 1,000 speci c peaks using MEME-ChIP 53 identi ed a previously predicted Runx motif, TG(T/C)GG(T/C) (Fig. 5b). ChIP-seq data around genes encoding Prg4 and Acan showed several peaks for Runx3 binding (Supplementary Fig. 9). Relative luciferase activities of the regions around Prg4 and Acan were increased by Runx3 overexpression (Supplementary Fig. 9). To further investigate the alterations of gene expression pro les by Runx3 knockout, we performed RNA-seq using SFZ and DZ cells from R3-cKO and R3-Cntl mice ( Supplementary Fig. 10, Supplementary Table 1−4). Among 18 genes up-or downregulated by more than 2-fold in the R3-cKO SFZ cells, nine were the top one-third peak nearest genes as shown in the ChIPseq, including Prg4 and Mmp9 (Fig. 5c). For Runx2, we performed ChIP-seq in accordance with previous reports, 12,54 using chondrocytes treated with vehicle control (primary chondrocytes) or exposed to 1 ng/mL IL-1β (in amed chondrocytes) derived from Runx2-FLAG mice (Hojo H., et al. in prep.). For the ChIP-seq, 37,163 raw peaks in primary chondrocytes and 14,571 raw peaks in in amed chondrocytes met the peak calling criteria (Fig. 5d). Peak distributions between primary chondrocytes and in amed chondrocytes were similar. In both groups, a striking enrichment was observed around the TSS; approximately 24% of all peaks from two ChIP-seq data are within ± 500 bp of the TSS, even though this region represents only 0.001% of the genome 52 ( Fig. 5d). GREAT GO analysis 52 identi ed "collagen bril organization" as the fth and second most signi cantly enriched term in the gene sets for primary chondrocytes and in amed chondrocytes, respectively (Fig. 5d). We hypothesized that there were functional differences between the TSSassociated dataset and dataset excluding data ± 500 bp from the TSS, similar to Sox9. 12 GREAT GO analyses showed cell cycle associated terms in TSS-associated Runx2 datasets of both primary and in amed chondrocytes and terms for a Runx2-regulated skeletal program in the dataset excluding data ± 500 bp from TSS ( Supplementary Fig. 11). A Venn diagram of GREAT GO analyses indicated that Runx2 was associated with collagen bril organization in chondrocytes with or without in ammation (Fig. 5d).
We next performed de novo motif analysis of the top 1,000 speci c peaks using MEME-ChIP. 53 As expected, primary motifs were similar to the previously predicted Runx motif, TG(T/C)GGT, even in the dataset excluding data ± 500 bp from the TSS (Fig. 5e). Interestingly, we observed that poly-A sequence, reported as a SOX9 consensus sequences, 55 was enriched in the TSS-associated Runx2 dataset. To identify the functional relationship between Sox9 and Runx2, we further analyzed our present dataset combined with previous Sox9 and related ChIP-seq datasets. 12 The TG(T/C)GGT motif was highly enriched at the predicted center of Runx2 ChIP-seq peaks in primary and in amed chondrocytes. Interestingly, the TG(T/C)GGT motif was slightly enriched at the predicted center of Sox9 ChIP-seq peaks (Fig. 5f). A poly-A sequence was also recovered, but there was no centering within the Runx2 and Sox9 peaks (Fig. 5f). Thus, the poly-A motif is unlikely to be the preferred primary site for Runx2 or Sox9 engagement in chondrocytes.
To test the integration of multiple regulatory inputs through Runx2-and Sox9-directed enhancer modules, we analyzed peak intensity associations. There were strong associations between Sox9-12 and Runx2-peak regions, which were notably enhanced in in amed chondrocytes (Fig. 5g). Clear associations of Runx2-peak regions with enhancer signatures were evident, speci cally: (1) bi-modal patterns of H3K4 dimethylation (H3K4me2) peaks at Runx2-peak center regions, which indicates both promoters and  Fig. 8b, c), we examined apoptosis-related genes. Among the top 200 genes downregulated by Runx2 knockout in primary chondrocytes, eleven genes (including Ihh, Igf1, and Wnt11) were associated with "negative regulation of apoptosis" (Fig. 5h). Among the top 200 genes downregulated by Runx2 knockout in in amed chondrocytes, ten (including Col2a1) matched with the top 500 genes nearest to Runx2 peaks detected by ChIP-seq (Fig. 5i).

Transcriptional regulation of Col2a1 and Mmp13 by Runx2 and Sox9
We mapped Runx2-FLAG, Sox9-FLAG, 60 and active histone marks (H3K4me2 or H3K27Ac in chondrocytes 12 or osteoblasts 54 from 1-day-old mice) in ChIP-seq data using CisGenome browser. Sox9 and Runx2 sequence data showed similar patterns in introns 1 and 6 of Col2a1; speci cally, minor peaks at intron 1 and major peaks at intron 6 (Fig. 6a). These peaks are identical to the functional enhancers of Col2a1 containing Sox9 motifs. 8 Magni ed views indicated that the peak centers of Runx2 and Sox9, containing their consensus motifs, were 300−400 bp apart (Supplementary Fig. 13). Based on the CisGenome browser, we prepared luciferase reporter vectors containing the Runx2 motifs around Col2a1 and Mmp13 genes shown in Fig. 6a. Relative luciferase activity of Col2a1 intron 1 and 6 fragments decreased in chondrocytes exposed to ≥ 1 ng/mL IL-1β (Fig. 6b), which exhibited decreased Sox9 mRNA while that of Runx2 remained stable (Fig. 1c, d). However, luciferase activities of Col2a1 and of Mmp13 TSS upstream regions were unchanged compared with controls (Fig. 6b). We next examined the effect of Sox9 on activity of each reporter. Sox9 overexpression increased activities of reporters containing the enhancers in Col2a1 introns 1 and 6 in a dose-dependent manner (Fig. 6c), but did not affect activity of the region upstream of the Col2a1 TSS and decreased activity of the region upstream of the Mmp13 TSS (Fig. 6c). Additionally, we co-transfected Runx2 and Sox9 (Fig. 6d). Notably, when the amount of Sox9 was decreased, luciferase activities of the Col2a1 intron 6 enhancer were increased by Runx2 cotransfection (Fig. 6d). In contrast, when Sox9 was overexpressed, luciferase activities of the Col2a1 intron 1 enhancer were not affected by Runx2 transfection (Fig. 6d). Activity of the region upstream of the Col2a1 TSS was increased by Runx2 transfection without Sox9 transfection (Fig. 6d).
We further performed luciferase assays using chondrocytes from the four genotypes with or without IL-1β exposure. Relative luciferase activities of Col2a1 intron 1 and 6 enhancers were decreased in in amed chondrocytes compared with primary chondrocytes for each genotype (Fig. 6e), similar to the results shown in Fig. 6b. Additionally, luciferase activities of Col2a1 intron 1 and 6 enhancers, and the region upstream of the Col2a1 TSS, were suppressed in in amed chondrocytes of R2-Homo cKO mice compared with other genotypes (Fig. 6e). Compared with controls, Runx2 heterozygous or homozygous knockout decreased the activities of reporters containing the region upstream of the Mmp13 TSS in both primary and in amed chondrocytes (Fig. 6d). Taken together, the present results indicate that Runx2 could activate Col2a1 transcription through the enhancer in intron 6, in addition to the enhancer in intron 1 and region upstream of the Col2a1 TSS.
Enhanced expression of Runx3 inhibited surgically induced OA development Finally, we investigated effects of adenoviral Runx2 or Runx3 overexpression in vitro and in vivo. Transduced of a Runx2 adenovirus (Ad-Runx2) at different doses to WT chondrocytes increased Runx2 expression in a dose-dependent manner (Fig. 7a). Although mRNA levels of Sox9 and Col2a1 were unchanged, Mmp13 was upregulated in Runx2 overexpressing chondrocytes (Fig. 7a).
When we transduced Runx3 adenovirus at different doses to primary chondrocytes from WT mice, Prg4 was increased in a dose-dependent manner (Fig. 7b). Acan and Sox9 were also increased by Runx3 overexpression (Fig. 7b). Catabolic factors were not changed, except for slightly increased Hif2a (Fig. 7b). We next introduced GFP or Runx3 adenoviral vectors into knee joints of WT mice that received surgical induction for the medial model at eight weeks of age. Runx3 expression was e ciently enhanced by the intra-articular injection of adenovirus, resulting in signi cant suppression of OA progression (Fig. 7c−d).

Discussion
The present study revealed that Runx3 protects articular cartilage degradation in mouse surgically induced OA models through extracellular matrix protein production, while Runx2 exerts both catabolic and anabolic effects under in ammatory conditions. In vitro and comprehensive analyses of transcriptional regulation by Runx3 indicate its association with organization of extracellular matrix, and identi ed Prg4 in SFZ as a transcriptional target gene, as well as Acan in DZ as a potential target (Fig. 2,  5). The present study also showed that homozygous knockout of Runx2 in articular chondrocytes markedly accelerated OA development, which was accompanied by suppressed Col2a1 protein level; in contrast, heterozygous knockout inhibited OA and decreased Mmp13 expression (Fig. 3). Loss-offunction of Runx2 in vitro downregulated Mmp13 expression in both primary and in amed chondrocytes, but decreased Col2a1 only in in amed chondrocytes in which Sox9 was decreased (Fig. 4). ChIP-seq revealed that overlapping of Runx2 and Sox9 peaks was enhanced by exposure to IL-1β (Fig. 5). Luciferase assays indicated that Runx2 can compensate for Col2a1 transcription in in amed chondrocytes (Fig. 6). Overexpression of Runx2 increased Mmp13, but not Col2a1. In contrast, overexpression of Runx3 increased Prg4 and Acan, and intra-articular administration of Runx3 adenovirus suppresses the development of OA induced by the medial model surgery (Fig. 7).
ChIP-seq and RNA-seq results of this study displayed the different target genes of Runx2 and Runx3. However, because Runx family members have a common consensus motif, we could not identify the molecular mechanisms underlying how Runx2 and Runx3 distinguish their own target genes. Notably, expression patterns of these two proteins in articular cartilage during OA development are different (Fig. 1). Runx3 is expressed in all cartilage layers and decreased during OA development, whereas Runx2 expression is observed in the DZ and sustained during OA development (Fig. 1). These differences in expression are consistent with our hypothesis, but do not explain differences in their target gene speci city. Various factors, such as epigenomic regulation of genes and cofactors of each Runx protein, probably de ne transcriptional activation of Runx2-or Runx3-speci c target genes.
In articular chondrocytes, Runx3 induces representative cartilage matrix gene, Prg4. Lubricin/Prg4, a mucin-like O-linked glycosylated protein, functions as a boundary lubricant in articular cartilage to decrease wear and friction, and the accumulation of lubricin at the surface of cartilage is important for joint homeostasis. 5,61 In the present study, both in vivo and in vitro experiments demonstrated that Prg4 is downregulated by Runx3 de ciency and Aggrecan/Acan, working as a shock absorber 62 and mainly expressed in DZ, 63 is also a candidate of Runx3 transcriptional target (Fig. 2). ChIP-seq and RNA-seq indicate that Prg4 gene is the transcriptional target of Runx3 (Fig. 5). Considering that Prg4 expression is regulated according to mechanical loading 64 and that joint instability and subsequent excessive mechanical loading to articular cartilage are caused by the medial model or DMM model surgery, the decrease in Prg4 may be more responsible for the accelerated OA development in cKO ER and pKO ER mice than other molecules. In contrast to these anabolic effects, Runx3 de ciency did not change the expression of Mmp13 or proteoglycan release from articular cartilage (Fig. 2). Chondrocyte hypertrophy and apoptosis were also not changed by Runx3 knockout (Supplementary Fig. 3, 4) These data indicate that Runx3 does not directly affect regulation of cartilage degradation or chondrocyte survival. ChIP-seq and RNA-seq results identi ed Mmp9 as a target of Runx3 (Fig. 5c); however, consistent data for Mmp9 could not be obtained by qPCR and immunohistochemistry (Fig. 2). Although we did not examine other candidate genes, such as Ccl and Ibsp, they may play some roles in regulation of articular cartilage as downstream molecules of Runx3. In the present study, OA development was enhanced in homozygous Runx2 knockout mice, which challenges the results of previous studies. 31,32 General heterozygous knockout of Runx2 decreased cartilage destruction and Mmp13 expression 31 and OA was suppressed with signi cantly reduced expression of Mmp13 in Aggrecan-Cre ERT2 ;Runx2 / mice. However, the e ciency of Runx2 knockdown was approximately 50%. 32 Considering the expression level of Runx2, the present results of R2-Hetero cKO mice (Fig. 3) were consistent with these previous ndings. In R2-Homo cKO mice, the e ciency of Runx2 suppression was approximately 80% in all experiments (Fig. 3, 4). When the knockout e ciency reached more than 50%, both Col2a1 and Mmp13 were suppressed, 34 which was consistent with our results for R2-Homo cKO mice (Fig. 3, 4).
Col2a1 expression was unchanged by Runx2 homozygous knockout in primary chondrocytes, but decreased in in amed chondrocytes (Fig. 4). These data were also supported by in vivo experiments showing that Col2a1 protein was not decreased in sham joints of R2-Homo cKO mice, but was signi cantly decreased in OA joints (Fig. 3d, e). A number of previous studies showed that Sox9 is most responsible for transcriptional induction of Col2a1. 8,10−13 These data evoked our hypothesis that Runx2 may sustain Col2a1 expression under in ammatory condition where Sox9 is decreased. The results of ChIP-seq and luciferase assays showed that the enhancer in Col2a1 intron 6, which contains both Sox9 and Runx2 consensus motifs, was activated by both transcription factors (Fig. 6c). The luciferase activity of the enhancer was tightly regulated by the amount of Sox9 and suppressed in accordance with decreased expression of Sox9 (Fig. 6c). Interestingly, although co-transfection of Runx2 and Sox9 did not enhance activity of the intron 6 enhancer, Runx2 signi cantly upregulated its activity when Sox9 was decreased (Fig. 6c). Accordingly, the intron 6 enhancer may be more important for transcriptional regulation of Col2a1 by Sox9 and Runx2 than the intron 1 enhancer. Previous studies also showed that the intron 6 region is a functional enhancer for the Col2a1 gene, 8 and transactivity of the intron 6 enhancer by Sox9 is stronger than that of the intron 1 enhancer. 8,12 All these data are consistent with our hypothesis; however, the underlying molecular mechanisms remain unknown.
In the present study, we could not further clarify how Runx2 protein sustains Col2a1 transcription when Sox9 protein decreases. A previous report displayed signi cant enrichment of TG(T/C)GGT motif in the top 2,000 Sox9 peaks in rib chondrocytes by ChIP-seq, 12  CisGeonme browser magni ed view analysis supported the second idea, as Runx2 peak centers in Col2a1 introns 1 and 6 are 300−400 bp away from Sox9 binding sites 8,12 (Supplementary Fig. 13). Overlap of Runx2 and Sox9 peaks was enhanced in in amed chondrocytes (Fig. 5g). In the in ammatory condition, decreased expression of endogenous Sox9 and subsequent decreased binding of Sox9 with its consensus motif may induce Runx2 to approach Runx motifs located near around the Sox9 motif. This hypothesis may also explain why Runx2 overexpression did not increase mRNA levels of Col2a1 when endogenous Sox9 was su ciently expressed (Fig. 7a). In contrast to Runx3, Runx2 may support the cartilage anabolism only in pathogenic conditions.
Mmp13 induction seems to be more straightforward. In the present study, Mmp13 induction was consistently suppressed both by homozygous and heterozygous knockout of Runx2 (Fig. 3, 4), meanwhile it was not affected by Runx3. These results indicate that Runx2 directly regulated Mmp13 transcription, as indicated in previous reports. [17][18][19]65 Although Runx2 activated luciferase activity upstream of the Mmp13 TSS, the Runx2 peak was not as large as we expected (Fig. 6). Obviously, Mmp13 transcription is regulated by various signaling pathways and molecules other than Runx2. Mmp13 was increased by IL-1β exposure in a dose-and time-dependent manner without increasing endogenous Runx2 expression (Fig. 1c, d). We previously reported that NF-κB, HIF-2α, Notch signaling, and its downstream factor Hes1 are potent inducers of Mmp13. 24,66,67 Runx2 overexpression also indirectly induces MMP13 through mitogen-activated protein kinase pathways. 17 Considering that Mmp13 suppression by Runx2 knockout was partial (Fig. 3, 4), Runx2 must be associated with induction of Mmp13 transcription; however, its effect size remains unknown.
In conclusion, the present study revealed that Runx3 protects articular cartilage against post-traumatic OA, and that Runx2 exerts both catabolic and anabolic effects on articular cartilage during surgically induced OA development. Prg4 and Acan may be the main mediators of the anabolic effects of Runx3, Runx2, and Sox9 in knee joints of 4-, 6-, 12-, and 18-month-old mice. Scale bars, 100 μm. c,d mRNA levels of marker genes in mouse primary chondrocytes exposed to interleukin 1 beta (IL-1β) at concentrations ranging from 0.01 to 2 ng/mL for 24 h (c), or 1 ng/mL IL-1β for 0−48 h (d   Cntl, and R2-Homo cKO mice exposed to 1 ng per mL IL-1β. n = 3 biologically independent experiments.