Background/Purpose:

Notch signaling is triggered by binding of ligands such as Jagged-1 (Jag-1) to Notch receptors, which results in cleavage of Notch receptors by the γ-secretase complex and subsequent release of the active Notch intracellular domain (NICD). The NICD is the central regulator of the pathway, which translocation to nucleus where it interacts with the DNA binding factor RBP-J. Aberrant Notch signaling has been implicated into the pathogenesis of various diseases including cancer, Alzheimer’s disease and fibrosis. Of note, increased expression of Notch signaling pathway has recently been observed in OA chondrocytes. However, the implications of the findings for the pathogenesis of OA and potential therapeutic implications have not been investigated so far.

Here, we aimed to further elucidate the role of Notch signaling pathway in OA using genetic and pharmacological inhibition of Notch signaling in the mouse model of destabilization of the medial meniscus (DMM) induced OA.

Methods: To analyze the activation status of the Notch cascade, we quantified the mRNA expression of Notch receptor family by real-time PCR and also analyzed the expression of NICD and Jag-1 protein by immunohistochemistry. To characterize the effects of Notch inhibition on osteoarthritis development in vivo, we evaluated the outcome of Notch anti-sense transgenic mice or mice treated with the γ-secretase inhibitor DAPT in the DMM (destabilization of the medial meniscus) model of OA. We used a modified Mankin score to analyze the histological changes. Hypertrophic differentiation of chondrocytes was analyzed by staining for Collagen X. We also analyzed the mRNA level of Epas1 and Aggrecanase-1 by real-time PCR.

Results:

We demonstrated that the mRNA levels of Notch1 are increased by 200 ± 79 % in human OA cartilage (p<0.05). Moreover, the protein levels of Jag-1 and of the NICD are upregulated in human OA cartilage. Similar results were also obtained in murine OA induced by DMM.

However, genetic or pharmacologic inhibition of Notch signaling exacerbated the osteoarthritic changes in the DMM model. The modified Mankin score upon surgical DMM was significantly increased in mice expressing a Notch-1 anti-sense construct or upon treatment with DAPT (56 ± 6 % increase in Notch as tg and 60% ± 5 increase in DAPT treated mice compared to non-transgenic or sham-treated DMM controls, respectively, p<0.01 for both). Immunostaining for Col X was also strongly increased upon inhibition of Notch signaling indicating increased differentiation into hypertrophic chondrocytes. Genetic or pharmacologic inactivation of Notch signaling also enhanced the mRNA expression of OA markers, such as Epas1 (increases by up to 94 ± 10 % (p<0.05)), Aggrecanase-1 (increases by up to 150 ± 20 % (p<0.01)).

Conclusion: We demonstrate that Notch signaling is hyperactivated in chondrocytes of OA patients. However, inhibition of Notch signaling pathway enhances chondrocyte hypertrophy and exacerbates osteoarthritis in the DMM model, indicating that the activation of Notch signaling in OA is a counter-regulatory mechanism to ameliorate chondrocyte dedifferentiation and cartilage damage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-notch-signaling-increases-the-severity-of-experimental-osteoarthritis/