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.
View: Text | PDF
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

×

Figure 6

p52 and RELB bind to NICD and are recruited to the Hes1 promoter.

Options: View larger image (or click on image) Download as PowerPoint
p52 and RELB bind to NICD and are recruited to the Hes1 promoter. (A–E) ...
(A–E) C3H10T1/2 cells were treated with TNF for 24 hours in some experiments. (A) Cells were cotransfected with Flag-tagged NOTCH2-NICD, p52, and RELB expression vectors. Whole-cell lysates were subjected to IP with anti-Flag or anti-p52 antibodies and blotted with anti-RELB or anti-Flag antibodies. Experiments were repeated independently 4 times. (B) Colocalization of p52 or RELB with NICD was determined by immunofluorescent staining using anti-p52, -RELB, and –NOTCH2-NICD antibodies under confocal microscopy. (C) Nuclear and cytoplasm proteins were isolated. Protein lysates were subjected to IP with anti-p52 or anti-RELB antibodies. Immunocomplexes were blotted with anti–NOTCH2-NICD. Expression of p52, RELB and NICD proteins were examined in cytoplasmic and nuclear fractions. Experiments were repeated independently 4 times. (D) The ChIP assay was performed on immunocomplex subjected to IP with anti–NOTCH2-NICD, anti-RELB or anti-p52 antibodies. Rat IgG was used as control. Precipitated DNA was measured by qPCR using sequence-specific primers. (E) Total protein lysates were subjected to IP with anti-p52 or anti-RELB antibodies. Immunocomplexes were washed with concentration gradient of NaCl, then blotted with anti–NOTCH2-NICD. Experiments were repeated independently 3 times. Lanes were run on the same gel but were noncontiguous. (F) Bone-derived MSCs from p52/RELB dKO and WT mice were treated with TNF or PBS and subjected to ChIP as in E. Values are mean ± SD of triplicate determinants. *P < 0.05 vs. respective control.