DNA methyltransferase 3b regulates articular cartilage homeostasis by altering metabolism
Jie Shen，Audrey McAlinden, 1,7 and Regis J. O’Keefe
Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a complex disease affecting the whole joint but is generally characterized by progressive degradation of articular cartilage. Recent genome-wide association screens have implicated distinct DNA methylation signatures in OA patients. We show that the de novo DNA methyltransferase (Dnmt) 3b, but not Dnmt3a, is present in healthy murine and human articular chondrocytes and its expression decreases in OA mouse models and in chondrocytes from human OA patients. Targeted deletion of Dnmt3b in murine articular chondrocytes results in an early-onset and progressive postnatal OA-like pathology. RNA-Seq and methylC-Seq analyses of Dnmt3b loss-of-function chondrocytes show that cellular metabolic processes are affected. Specifically, TCA metabolites and mitochondrial respiration are elevated. Importantly, a chondroprotective effect was found following Dnmt3b gain of function in murine articular chondrocytes in vitro and in vivo. This study shows that Dnmt3b plays a significant role in regulating postnatal articular cartilage homeostasis. Cellular pathways regulated by Dnmt3b in chondrocytes may provide novel targets for therapeutic approaches to treat OA.To determine protein localization of the de novo Dnmt3 enzymes in murine articular cartilage, IHC staining was carried out on tissue sections of 3-month-old WT murine knee joints. Expression of Dnmt3a was undetectable in articular and growth plate cartilage (Figure 1A). However, Dnmt3b was abundantly expressed in chondrocytes of articular cartilage yet almost undetectable in chondrocytes of the underlying growth plate (Figure 1A). During murine cartilage development, Dnmt3b was ubiquitously expressed in proliferating chondrocytes of developing limbs at E18.5, while expression became restricted to articular chondrocytes during postnatal development (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.93612DS1).We next examined the effects of aging and OA on Dnmt3b expression in cartilage. Analysis of an age range of articular cartilage tissue sections revealed a trend toward decreased Dnmt3b expression with increasing age as shown by 2 representative IHC images (at 3 and 27 months old) (Figure 1B). Surgical induction of OA in mice was carried out by meniscal ligament injury (MLI) (8) to destabilize the joint and induce articular cartilage degradation over time. Lower Dnmt3b expression levels were noted in articular chondrocytes 4 weeks following MLI compared with sham controls (Figure 1C). It has been reported that mice fed a high-fat diet (HFD) develop accelerated OA (27). Here we also show reduced expression of Dnmt3b in chondrocytes from mice fed a HFD for 4 weeks compared with control mice fed a normal diet (Figure 1D).In human nondiseased articular cartilage tissue sections, abundant DNMT3B expression was consistently detected, whereas expression was either undetectable or restricted to articular chondrocytes of the deep zone. However, DNMT3B expression in cells from OA tissues ranged from undetectable to some positively labeled cells in the deeper zones of articular cartilage (Figure 1E). Decreased DNMT3B expression in OA cartilage was further confirmed by real-time quantitative PCR (qPCR) from human primary articular chondrocytes (PACs) (Figure 1F). Treatment of human PACs with the proinflammatory cytokine IL-1β resulted in decreased DNMT3B mRNA and protein expression (Figure 1G). These PACs were obtained from knee joints following total knee replacement surgery and were found to be responsive to IL-1β based on the expected changes in COL2A1 and MMP-13 expression (Supplemental Figure 2). Similarly, Dnmt3b expression was also decreased in murine PACs in response to IL-1β (Supplemental Figure 3, E and F). These data suggest that inflammatory mediators (known to increase in the context of OA) may regulate DNMT3B. In fact, we identified an NF-κB binding site in the promoter region of the murine Dnmt3b gene (also present in the human DNMT3B promoter) (Supplemental Figure 3A). Luciferase reporter assays showed functional utilization of the NF-κB binding site following IL-1β treatment of murine ATDC-5 cells; this affect was attenuated following mutation of the binding site (Supplemental Figure 3B). NF-κB interaction with its binding site was also shown by ChIP assays (Supplemental Figure 3, C and D).Given the trend toward decreased Dnmt3b expression with age and OA, the effect of Dnmt3b knockdown in murine PACs (Supplemental Figure 4, A and B) was examined. Knockdown resulted in decreased expression of the anabolic cartilage gene Col2a1 and increased expression of markers associated with catabolism or terminal hypertrophic chondrocyte differentiation (Col10a1, Runx2, and Mmp13) (Supplemental Figure 4C). Alkaline phosphatase (ALP) activity also increased in PACs following Dnmt3b knockdown, albeit not to the level induced by BMP-2 (Supplemental Figure 4D). Since TGF-β and BMP signaling can have opposing effects on chondrocyte catabolism (2), Smad signaling was analyzed following Dnmt3b knockdown. Phospho-Smad2 (p-Smad2) levels decreased while p-Smad1/5 levels increased following Dnmt3b siRNA treatment (Supplemental Figure 4, E and F). This suggests that increased BMP-2 signaling may, in part, explain the increase in chondrocyte catabolism.Two separate IRB-approved protocols were executed at the University of Rochester to collect articular cartilage from normal, OA, and injured joints. Normal cartilage was collected from amputation patients (talus or knee, n = 11) and OA cartilage was harvested from total knee arthroplasty patients (n = 57). Injured cartilage, debrided from patients that underwent arthroscopic knee surgery to treat a recent meniscal injury (within 1 month, n = 14) was collected. At harvest, tissues were immediately fixed in 10% neutral buffered formalin and processed for embedding in paraffin. After facing the cartilage blocks on a microtome, between 1 and 4 biopsy punches were recovered from each block (1 mm diameter, 3 mm deep) and transferred to a receiver block to create a tissue microarray containing 176 biopsies. PACs were also isolated from human knee articular cartilage tissue obtained from surgeons at Washington University Department of Orthopaedic Surgery following total knee replacement surgery.This work is supported by the NIH R01 grant AR069605 to RJO and AM. We would like to acknowledge the technical expertise and assistance of Sarah Mack, Kathy Maltby, and Michael Thullen within the Histology, Biochemistry, and Molecular Imaging Core in the Center for Musculoskeletal Research at the URMC.Conflict of interest: The authors have declared that no conflict of interest exists.Reference information:JCI Insight. 2017;2(12):e93612. https://doi.org/10.1172/jci.insight.93612.