Background/Purpose: In rheumatoid arthritis (RA), production of TNF promotes osteoclast activity and inhibits osteoblasts (OBs) and bone healing. Schnurri-3 (SHN3) is a potent suppressor of bone formation, and its inhibition protects against systemic bone loss and articular erosion in RA models¹. We investigated the pathways regulating SHN3 expression and subsequent OB function by TNF.

Methods: OBs were differentiated +/- TNF for 3 days to perform bulk RNA sequencing. Gene expression analysis was performed on OBs, treated +/- NF-κB inhibitor Bay 11-7082 prior to the addition of TNF. Mice were generated with a constitutively active (ca) NF-κB pathway in OB lineage cells. To accomplish this, Ikk-ca^fl/flor Ikk-ca^fl/fl;shn3^fl/fl mice were crossed with Prx-1Cre transgenic mice to generate: Ikk-ca;prx1 (ca-IKK specifically in OB lineage cells), and Ikk-ca;shn3;prx1(ca IKK in OB lineage-cells with deletion of SHN3). Bone marrow stromal cells (BMSCs) were isolated from femurs for gene expression analysis. Femur microCT was performed to analyze bone architecture. Finally, we identified four putative NF-κB-binding sites for the RelA (p65) and p50 heterocomplex at sites +10, 120, 360, and 390 within the first 1,000 base pairs in the mouse Shn3 gene promoter and tested those for binding using Chromatin Immunoprecipitation Polymerase Chain Reaction (ChIP-PCR).

Results: From the RNAseq KEGG pathway analysis of OBs treated with TNF, several TNF-responsive pathways were identified including Wnt signaling, MAPK, NF-κB, and osteoclast differentiation pathways. We observed increased mRNA levels for the p65/p50 transcription factors and for SHN3 itself in TNF-treated OBs. Treating OBs with TNF in the presence of the NF-κB inhibitor led to reduced SHN3 mRNA expression. ChIP-PCR in OBs confirmed TNF-induced NF-κB activation is accompanied by binding of the p65/p50 heterodimer to the SHN3 promoter at the +10bp position. Furthermore, SHN3 mRNA expression was significantly higher in OBs expressing Ikk-ca;prx1 compared to controls (Ikk-ca^fl/fl), confirming NF-κB pathway activation regulates SHN3 expression in OBs. Since SHN3 functions as a negative regulator of OB differentiation, activation of the NF-κB pathway by expressing IKK-ca in OB-lineage cells (Ikk-ca;prx1) resulted in a significant reduction in femoral bone mass compared to the controls Ikk-ca^fl/fl;shn3^fl/fl. Importantly, NF-κB-mediated bone loss was reversed by OB lineage-specific deletion of SHN3 in Ikk-ca;shn3;prx1mice. These mice have bone volume that is comparable to Shn3;prx1 mice.

Conclusion: Our findings demonstrate that TNF activates NF-κB signaling in OBs, leading to upregulation of SHN3 gene expression by binding at promoter position +10. In mice, constitutive activation of NF-κB signaling promotes bone loss, at least in part by increasing SHN3 expression. However, inhibiting SHN3 expression in OB lineage cells mitigates NF-κB-induced bone loss, suggesting a potential therapeutic strategy to prevent inflammation-induced bone loss in RA.

Reference:

1: Stavre, Z. et al. Schnurri-3 inhibition suppresses bone and joint damage in models of rheumatoid arthritis. Proc. Natl. Acad. Sci. U. S. A. 120, e2218019120 (2023).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/schnurri-3-inhibition-protects-from-nf-kb-induced-bone-loss-in-inflammatory-arthritis/