engleski

Signaling through Notch 1 decreases osteoclast progenitor activity in the mouse model of rheumatoid arthritis

Background: Periarticular and systemic bone loss in rheumatoid arthritis is mediated by increased osteoclast activity. Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220- NK1.1-CD11blo/+CD115+ cells, are specifically increased in arthritis. We aimed to determine the effects of OCP Notch signaling inhibition and Notch 1 signal activation on OCP activity and arthritis-induced bone resorption in murine collagen-induced arthritis (CIA). Methods: Male C57Bl/6, CX3CR1CreERT2xRBP-J (Notch inhibition) and CX3CR1CreERT2xNICD1 (Notch 1 constitutive activity) mice were immunized with chicken type II collagen, treated with i.p. injections of tamoxifen (75 mg/kg) to induce Cre- mediated recombination and sacrificed at day 35 following immunization. Cre negative littermates were used as controls. Expression of Notch receptors 1 through 4 on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL) and stained for TRAP expression. Murine hindpaws were scanned with Bruker SkyScan 1076 µCT and talar bones were analyzed. Research was approved by the Ethics Committee. Results: We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 45% and 60% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Notch 1 signal activation in OCPs differentiating in vitro leads to reduced numbers of TRAP+ osteoclasts while Notch deletion stimulated osteoclastogenesis. Arthritic CX3CR1CreERT2+xNICD1 mice had lower OCP numbers and an increase in expression of all four Notch receptors on OCPs, while arthritic CX3CR1CreERT2+xRBP-J mice had decreased OCP expression of Notch 1 through 3. Talar bone volume was reduced in arthritic CX3CR1CreERT2+xRBP-J mice. Conclusion: Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo Notch 1 constitutive signal activation suppressed while Notch deletion enhanced osteoclastogenesis in CIA model. Taken together with our previous results of enhanced osteoclast formation by using neutralizing Notch 1 antibodies we confirmed an inhibitory role of Notch 1 signaling in osteoclast differentiation during arthrtis. Acknowledgments: The work has been supported by Croatian Science Foundation projects IP-2018-01- 2414, UIP-2017-05-1965 and DOK- 2018-09-4276.

osteoclast progenitor ; Notch ; rheumatoid arthritis ; collagen-induced arthritis ; Rbpj ; NICD

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