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 5

Noncanonical NF-κB proteins p52 and RELB mediate TNF-induced NOTCH activation.

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Noncanonical NF-κB proteins p52 and RELB mediate TNF-induced NOTCH activ...
(A) Nfkb mRNA expression in CD45–SCA1+CD105+ MSCs from TNF-Tg mice and WT littermates by RNA-Seq. (B) NF-κB protein expression in CD45– MSC-enriched cells from TNF-Tg mice and WT littermates by Western blot. Fold change in protein level (below) was determined by measuring band intensity. (C) Nfkb2, Relb, Runx2, and Alp mRNA expression in CD45– MSC-enriched cells from DAPT- or vehicle-treated TNF-Tg mice by qPCR. (D) Reporter activity in C3H10T1/2 cells cotransfected with RBPjκ-Luc and with NOTCH2-NICD–, p52-, and/or RELB-expressing vectors. Fold increase versus empty vector was calculated. (E) Expression of Hes1, Runx2, and Alp in cells as in D. (F) Expression of Hes1 and Hey1 in CD45–Ter119– MSCs from p52/RELB dKO mice, Relb–/– mice, and control littermates. Values are mean ± SD of 3 pairs of mice. (G and H) Bone-derived MSCs of p52/RELB dKO and control littermates were treated with TNF. (G) Expression of p52, RELB, and HES1 protein by Western blot. (H) Expression of Runx2 and Alp by qPCR. *P < 0.05, #P < 0.05 vs. control.