Wwp2 maintains cartilage homeostasis through regulation of Adamts5.

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.

Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression.

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses.

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

Sox9 directly promotes Bapx1 gene expression to repress Runx2 in chondrocytes.

The transcription factor, Sry-related High Mobility Group (HMG) box containing gene 9 (Sox9), plays a critical role in cartilage development by initiating chondrogenesis and preventing the subsequent maturation process called chondrocyte hypertrophy. This suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation. However, the precise mechanism by which Sox9 regulates late chondrogenesis is poorly understood. In the present study, the transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes. We identified a critical Sox9 responsive region in the Bapx1 promoter via a luciferase reporter assay. Analysis by chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that Sox9 physically bound to this region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect. Our findings reveal that the molecular pathway modulated by Bapx1 links two major regulators in chondrogenesis, Sox9 and Runx2, to coordinate skeletal formation.