NOTCH inhibits osteoblast formation in inflammatory arthritis via noncanonical NF-κB
Hengwei Zhang, … , Brendan F. Boyce, Lianping Xing
Hengwei Zhang, … , Brendan F. Boyce, Lianping Xing
Published June 2, 2014
Citation Information: J Clin Invest. 2014;124(7):3200-3214. https://doi.org/10.1172/JCI68901.
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Research Article Bone Biology

NOTCH inhibits osteoblast formation in inflammatory arthritis via noncanonical NF-κB

Abstract

NOTCH-dependent signaling pathways are critical for normal bone remodeling; however, it is unclear if dysfunctional NOTCH activation contributes to inflammation-mediated bone loss, as observed in rheumatoid arthritis (RA) patients. We performed RNA sequencing and pathway analyses in mesenchymal stem cells (MSCs) isolated from transgenic TNF-expressing mice, a model of RA, to identify pathways responsible for decreased osteoblast differentiation. 53 pathways were dysregulated in MSCs from RA mice, among which expression of genes encoding NOTCH pathway members and members of the noncanonical NF-κB pathway were markedly elevated. Administration of NOTCH inhibitors to RA mice prevented bone loss and osteoblast inhibition, and CFU-fibroblasts from RA mice treated with NOTCH inhibitors formed more new bone in recipient mice with tibial defects. Overexpression of the noncanonical NF-κB subunit p52 and RELB in a murine pluripotent stem cell line increased NOTCH intracellular domain–dependent (NICD-dependent) activation of an RBPjκ reporter and levels of the transcription factor HES1. TNF promoted p52/RELB binding to NICD, which enhanced binding at the RBPjκ site within the Hes1 promoter. Furthermore, MSC-enriched cells from RA patients exhibited elevated levels of HES1, p52, and RELB. Together, these data indicate that persistent NOTCH activation in MSCs contributes to decreased osteoblast differentiation associated with RA and suggest that NOTCH inhibitors could prevent inflammation-mediated bone loss.

Authors

Hengwei Zhang, Matthew J. Hilton, Jennifer H. Anolik, Stephen L. Welle, Chen Zhao, Zhenqiang Yao, Xing Li, Zhiyu Wang, Brendan F. Boyce, Lianping Xing

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Figure 1

Increased expression of NOTCH target genes in MSCs from TNF-Tg mice and TNF-treated MSCs.

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Increased expression of NOTCH target genes in MSCs from TNF-Tg mice and ...
(A) BM cells were isolated from 6-month-old TNF-Tg mice and WT littermates (n = 3 per genotype). CD45–SCA1+CD105+ MSCs were subjected to RNA-Seq using a single-cell protocol. Differentially expressed genes between TNF-Tg and WT cells were subjected to pathway analysis. (B) RNA-Seq reads (top) and qPCR data (bottom) from CD45–SCA1+CD105+ MSCs of TNF-Tg and WT mice. RPKM, reads per kilobase per million. (C and D) Expression of Hes1 and Hey1 in TNF-treated (24 hours) 3rd passage of bone-derived WT MSCs (C) and in the C3H10T1/2 murine MSC line (D) by qPCR. (E) Expression of HES1 in TNF-treated (24 hours) human MSCs by qPCR. (F) 2-month-old TNFR1/2 dKO mice and WT littermates received TNF (0.5 μg/injection/d i.p.) or PBS for 5 days. BM cells were subjected to CD45– or CD45+ cell isolation for Hes1 expression by qPCR. *P < 0.05 vs. respective control.