Background/Purpose:

Delta/Notch like EGF-related receptor (DNER) is a single-pass transmembrane protein with characteristic EGF-like repeats in the extracellular domain, similar to those of the Notch receptor and its ligand Delta. In our previous study, an overexpression of DNER mRNA was observed in lesional areas of human osteoarthritis (OA) articular cartilage compared to unaffected zones of the same tissue sample. Based on these findings and on the fact that Notch signaling pathway plays a major role in cell fate regulation and differentiation of human articular chondrocytes, we analyzed the role of DNER in the pathogenesis of OA.

Methods:

Human articular cartilage was obtained from knee joint explants of OA patients undergoing total joint replacement surgery. The expression of DNER in human cartilage was analyzed by immunohistochemistry. Isolated primary chondrocytes were nucleofected with siRNA targeting human DNER or with a plasmid for DNER overexpression (pDNER), respectively. Knockdown and overexpression were confirmed by immunocytochemical staining for DNER in the nucleofected chondrocytes at different time points of cultivation. RNA was isolated and the effects of the artificially modified DNER expression on specific cartilage metabolic parameters (collagen type 2 and 1, Sox9, MMP9, ADAMTS4 and 5) analyzed using quantitative real-time PCR.

Results:

Histologically, DNER was significantly increased in the affected cartilage of all patients (n=3) on the protein level compared to the intact zones of the same tissue. An overexpression of DNER in primary human OA chondrocytes peaked 24 h after nucleofection (increase: 50000 fold). 72 h after nucleofection, a down-regulation of collagen type 2 (-7,89 fold) and an up-regulation of collagen type 1 (2,07 fold) as well as Sox 9 (7.39 fold) mRNA was measured.The overexpression of DNER was detectable at different time points on the protein level. In contrast, following knockdown of DNER, its expression was significantly decreased (p≤0.001) but there was no significant change in the expression profile of collagen type 2 (0.94 fold; p≤0.621), collagen type 1 (0.74 fold; p≤0.097), Sox9 (0.83 fold), MMP9 (0.97 fold), ADAMTS4 (0.64 fold) and 5 (1.2 fold). The confirmation of the DNER knockdown on the protein level at different time points identified that DNER is still to be constitutively found in the membrane.

Conclusion:

Since DNER overexpression led to a down-regulation of collagen type 2 in human articular chondrocytes whilst the expression of collagen type 1 and Sox9 were up-regulated, our preliminary results could lead to the assumption that DNER might contribute to the dedifferentiation of human OA chondrocytes within a “regenerative” attempt in order to  de-organize and re-assemble adapted cartilage extracellular matrix to reconstitute physiological homeostasis and the ability to re-sustain to physical load during the course of human OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/investigations-on-the-role-of-deltanotch-like-egf-related-receptor-in-the-pathogenesis-of-osteoarthritis/