Abstract
Human amniotic mesenchymal stem cells (hAMSCs) are multiple potent progenitor cells (MPCs) that can differentiate into different lineages (osteogenic, chondrogenic, and adipogenic cells) and have a favorable capacity for angiogenesis. Schnurri-3 (Shn3) is a large zinc finger protein related to Drosophila Shn, which is a critical mediator of postnatal bone formation. Bone morphogenetic protein 9 (BMP9), one of the most potent osteogenic BMPs, can strongly upregulate various osteogenesis- and angiogenesis-related factors in MSCs. It remains unclear how Shn3 is involved in BMP9-induced osteogenic differentiation coupled with angiogenesis in hAMSCs. In this investigation, we conducted a comprehensive study to identify the effect of Shn3 on BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs and analyze the responsible signaling pathway. The results from in vitro and in vivo experimentation show that Shn3 notably inhibits BMP9-induced early and late osteogenic differentiation of hAMSCs, expression of osteogenesis-related factors, and subcutaneous ectopic bone formation from hAMSCs in nude mice. Shn3 also inhibited BMP9-induced angiogenic differentiation, expression of angiogenesis-related factors, and subcutaneous vascular invasion in mice. Mechanistically, we found that Shn3 prominently inhibited the expression of BMP9 and activation of the BMP/Smad and BMP/MAPK signaling pathways. In addition, we further found activity on runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF), and the target genes shared by BMP and Shn3 signaling pathways. Silencing Shn3 could dramatically enhance the expression of Runx2, which directly regulates the downstream target VEGF to couple osteogenic differentiation with angiogenesis. To summarize, our findings suggested that Shn3 significantly inhibited the BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs. The effect of Shn3 was primarily seen through inhibition of the BMP/Smad signaling pathway and depressed expression of Runx2, which directly regulates VEGF, which couples BMP9-induced osteogenic differentiation with angiogenesis.
Highlights
Bone defects occur frequently and have various causes, such as trauma, infections, and tumors[1]
Cell Counting Kit (CCK)-8 results showed that the proliferative curve of Human amniotic mesenchymal stem cells (hAMSCs) exhibited an “S” pattern, and hAMSCs went through a logarithmic growth phase for 4 days after 1 day of latency
Our results showed that alkaline phosphatase (ALP) activity was dramatically decreased in the Bone morphogenetic protein 9 (BMP9)+Shn[3] group compared to the BMP9 group at days 3, 5, and 7, whereas exogenous Shn[3] expression alone did not exhibit any significant effect on ALP activity of hAMSCs at 7 days (Fig. 2c)
Summary
Bone defects occur frequently and have various causes, such as trauma, infections, and tumors[1]. It has been reported that biological treatment modalities contribute to the repair of bone defects. Official journal of the Cell Death Differentiation Association. Li et al Cell Death and Disease (2020)11:72 essential elements, such as seed cells, growth factors, and implant scaffolds, and provide new treatment options for bone defects[3]. Many types of mesenchymal stem cells (MSCs) have been found to originate from various types of tissue, such as bone marrow-derived MSCs (BMSCs), as well as adipose, peripheral blood, and muscle and ligamentderived MSCs4–8. There are many disadvantages in the process of extracting BMSCs; invasive surgery carries a high risk of infection and bleeding and may cause immunological rejection after implantation
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