Abstract
Notch signaling pathway is one of the most important pathways to regulate intercellular signal transduction and is crucial in the regulation of bone regeneration. Nephroblastoma overexpressed (NOV or CCN3) serves as a non-canonical secreted ligand of Notch signaling pathway and its role in the process of osteogenic differentiation of mesenchymal stem cells (MSCs) was undefined. Here we conducted a comprehensive study on this issue. In vivo and in vitro studies have shown that CCN3 significantly inhibited the early and late osteogenic differentiation of mouse embryonic fibroblasts (MEFs), the expression of osteogenesis-related factors, and the subcutaneous ectopic osteogenesis of MEFs in nude mice. In mechanism studies, we found that CCN3 significantly inhibited the expression of BMP9 and the activation of BMP/Smad and BMP/MAPK signaling pathways. There was also a mutual inhibition between CCN3 and DLL1, one of the classic membrane protein ligands of Notch signaling pathway. Additionally, we further found that Hey1, the target gene shared by BMP and Notch signaling pathways, partially reversed the inhibitory effect of CCN3 on osteoblastic differentiation of MEFs. In summary, our findings suggested that CCN3 significantly inhibited the osteogenic differentiation of MEFs. The inhibitory effect of CCN3 was mainly through the inhibition of BMP signaling and the mutual inhibition with DLL1, so as to inhibit the expression of Hey1, the target gene shared by BMP and Notch signaling pathways.
Highlights
Notch signaling pathway is one of the highly conserved signaling pathways in the process of biological evolution[1]
The results showed that Alkaline phosphatase (ALP) activity and calcium deposition were obviously reduced in the BMP9+CCN3 group compared to BMP9+GFP group (Fig. 2a, b, e); whereas compared with the BMP9+RFP group, these indicators were increased significantly in the BMP9+siCCN3 group
It has been proved to play a crucial role in cell proliferation, differentiation, adhesion, Fig. 8 CCN3 inhibits subcutaneous ectopic osteogenesis of mouse embryonic fibroblasts (MEFs) in nude mice by suppressing Hey1. a The general observation of the subcutaneous mass of ectopic osteogenesis in nude mice. b Subcutaneous osteoblast mass for micro-CT scanning to get a representative reconstructed 3D image, scaling 1 mm. c Quantitative analysis of bone tissue and the values of BV/TV, Tb.N, Tb.Sp, Tb.Th, CT, and SMI were analyzed. d hematoxylin& eosin (H&E) staining and Masson’s Trichrome staining to detect the formation of trabecular bone and bone matrix under the treatment as shown. e Immunohistochemistry to determine the expression of osteogenic-related markers under the treatment as shown
Summary
Notch signaling pathway is one of the highly conserved signaling pathways in the process of biological evolution[1]. In the process of mammalian growth and cell differentiation, proliferation, and apoptosis, Notch signaling pathway plays an irreplaceable role[2]. It is crucial in the process of bone regeneration[3,4,5,6]. CCN3 is a member of the CCN family and is widely expressed in the nervous system, musculoskeletal system, and so on It is found in high expression levels both during bone development and fracture healing[14,16,17,18,19,20,21].
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