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. IntroductionPatients with chronic inflammatory diseases, such as rheumatoid arthritis (RA), often have severe systemic bone loss and increased risk of fracture due to increased bone resorption and decreased bone formation, partially mediated by elevated TNF levels (1). We (1-4) and others (5, 6) have reported that TNF inhibits bone formation by affecting major osteoblast regulatory pathways, including BMP/SMAD/RUNX2 and WNT-β-catenin, but the role of TNF in osteoblast differentiation from MSCs has not been fully defined. The TNF transgenic (TNF-Tg) mouse model we use, line 3647, represents a good model of RA to study the influence of chronically elevated, but relatively low, levels of TNF and TNF-induced inflammation on bone cell function and MSC differentiation into osteoblasts (7). To attempt to identify molecules responsible for reduced differentiation of MSCs into osteoblasts in RA, we performed genome-wide screening and pathway analyses using data from RNA sequencing (RNA-Seq) of MSCs purified from TNF-Tg mice and WT littermates. We found that genes in the NOTCH and noncanonical NF-κB signaling pathways were markedly upregulated in TNF-Tg mouse MSCs, raising the possibility that NOTCH may interact with noncanonical NF-κB proteins in MSCs to inhibit their osteogenic differentiation.NOTCH is a family of evolutionarily conserved receptors that regulate cell fate. NOTCH receptors are activated following direct contact with their ligands expressed on adjacent cells. In mammals, there are 4 NOTCH receptors (NOTCH1-NOTCH4)...

Inhibition of Notch1 promotes hedgehog signalling in a HES1-dependent manner in chondrocytes and exacerbates experimental osteoarthritis

“…Inhibition of Notch signalling promotes osteoblast activity, while activation of Notch signalling inhibits osteoblast function in vitro . 41 Moreover, targeted inactivation of Hes1 in osteoblasts increased the trabecular bone volume and the trabecular number,42 while inhibition of Notch signalling ameliorated tumour necrosis factor alpha-induced bone loss and stimulated osteoblast activity in mice 43. As described previously, subchondral bone formation was also accompanied by locally enhanced osteoclastogenesis 44.…”

NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis

“…Interestingly, a recent study on osteoarthritis (OA) showed that an intra-articular injection of DAPT significantly suppresses joint-destruction in (Col2a1)-Cre ERT / Rbpj fl/fl OA mice (Hosaka et al, 2013). In addition, DAPT treatment of mice overexpressing TNFα that causes inflammatory bone resorption also effectively suppresses systemic osteoporotic phenotype (Zhang et al, 2014). Since DAPT efficiently inhibited the observed bone absorption in the osteoclast-specific Fbw7 conditional knockout mice, Notch signaling could be a potential target for treating various bone absorptic disorders.…”

Transient gamma-secretase inhibition accelerates and enhances fracture repair likely via Notch signaling modulation

“…A recent study may provide some understanding of how transient Notch inhibition enhances chondrogenic and osteogenic differentiation from MSCs in the inflammatory setting of fracture repair. Zhang et al [38] demonstrated that chronic inflammation increased the expression of non-canonical NF-κB proteins, which activated Notch signaling by binding to and promoting nuclear translocation of NICD. Additionally, this inflammation mediated Notch activation in MSCs suppressed osteogenic differentiation, which could be prevented by administration of the Notch inhibitor, DAPT.…”