Background/Purpose: OA is a complex disease not limited to cartilage degeneration. Indeed, several experiments suggest that subchondral bone remodeling could initiate and/or contribute to cartilage loss in OA through a bone/cartilage interplay. We aimed to identify soluble mediators released by loaded osteoblasts/osteocytes that could induce the release of pro-catabolic factors by chondrocytes by using a novel and unique bone/cartilage communication model.

Methods: Thanks to a three dimensional (3D) culture model, murine osteoblasts were submitted to compression in Biopress Flexercell plates (1.7 MPa, 1Htz during 24h). Then, conditioned media from compressed (CM) or uncompressed (UCM) osteoblasts/osteocytes were used to stimulate mouse articular chondrocytes. Chondrocyte expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, tissue inhibitors of metalloproteinases (TIMPs), and cartilage extracellular matrix components type II collagen and aggrecan were assessed by RT-PCR, western blot analysis and ELISA. Then, soluble mediators released by compressed osteoblasts/osteocytes were identified by iTRAQ®, a differential secretomic analysis approach.

Results: Media from compressed osteoblast (CM) strongly induced MMP-3 and -13 chondrocyte mRNA expression (Table). Consistently, CM also significantly stimulated the releases of MMP-3 and -13 by chondrocytes (respectively 10.6±0.75 fold, p<0.001, and 6.5±2.1 fold, p<0.01 compared to control). In addition, CM enhances TIMP-1 expression whereas it strongly inhibited TIMP-2 and -3 expressions (Table). CM also affected cartilage matrix proteins expressions by downregulating aggrecan and type II collagen mRNA levels (Table). Effects of CM on cytosolic type II collagen protein amounts were confirmed by western blot (a decrease of 31±9%, p<0.01). In order to identify osteoblast soluble mediators responsible for this chondrocyte phenotype, osteoblast conditioned media were analyzed by iTRAQ®. This sophisticated proteomic technique allowed identification of 105 proteins secreted by osteoblasts among which only 10% were upregulated in response to compression. Among them, 14-3-3ε dose-dependently induced the release of pro-catabolic factors by chondrocytes, mimicking the effects of CM osteoblasts/osteocytes media. Furthermore, 14-3-3ε was strongly released by human OA subchondral bone and stimulated MMP-3 expression in human OA chondrocytes.

Table:Effects of media from uncompressed or compressed osteoblasts on MMPs, TIMPs and matrix proteins chondrocytes mRNA expression. 

 

       Results are expressed in fold compared to control (non-stimulated chondrocytes).

Conclusion: Therefore, we identify 14-3-3ε as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA leading to MMP expression by chondrocytes. We speculate that 14-3-3ε is a potential target for a future disease-modifying OA drug.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-14-3-3a-as-a-new-subchondral-bone-mediator-involved-in-cartilage-degradation-during-osteoarthritis/