image: (A) C3H10T1/2 cells were transfected with AdGFP, AdBMP2, AdDnNotch1, and AdBMP2+DnNotch1, respectively. On day 7, quantitative reverse transcription PCR was used to detect the expression of chondrogenic differentiation markers (Sox9 and Col2a1) and osteoblastic differentiation markers (Runx2, Col1a1, and OPN) of MSCs. (B) To detect the expression of sulfated glycosaminoglycan during C3H10T1/2 cell differentiation, alcian blue staining was performed on day 7 after cells were transfected with recombinant adenovirus. (C) Alkaline phosphatase (ALP) staining experiments were used to determine ALP activity on day 3 (a) and day 7 (b) respectively. (D) For matrix mineralization, alizarin red S staining was performed on day 14 (a); microscopic (b) observations showed that down-regulation of Notch1 signaling inhibited BMP2-induced calcium deposition. (E) Quantitative analysis of ALP activities and calcium deposition. The ALP activity was quantified at OD 405 nm and normalized by protein concentration per well (unit/mg protein) on day 3 (a) and day 7 (b). Alizarin red staining was quantified at OD 405 nm and normalized to total DNA per well (OD405 nm/μg DNA) (c). (F) Western blot analysis for the chondrogenic differentiation markers Sox9 and Col2a1 and the osteogenic markers Runx2, Col1a1, and OPN. Protein bands (a) and quantitative analysis (b). The relative expression of Sox9, Col2a1, Runx2, Col1a1, and OPN proteins were analyzed using GAPDH as control (b). One-way analysis of variance; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 versus the AdGFP group; ####p < 0.0001, ###p < 0.001, ##p < 0.01, and #p < 0.05 versus the indicated group; ns, p > 0.05. BMP2, bone morphogenetic protein 2; Col1a1, collagen type I alpha 1 chain; Col2a1, collagen type II alpha 1 chain; MSC, mesenchymal stem cell; Notch1, Notch receptor 1; OPN, osteopontin; Runx2, RUNX family transcription factor 2; Sox9; SRY-box transcription factor 9.
Credit: Genes & Diseases
Bone morphogenetic protein 2 (BMP-2) can induce the differentiation of mesenchymal stem cells (MSCs) into different osteocyte cells, including chondrocytes, adipocytes, and endothelial cells. While BMP-2 can effectively induce chondrogenic differentiation and inhibit osteogenic differentiation of MSCs, it can also initiate hypertrophic differentiation and endochondral ossification. Previous studies have shown that Notch1 plays a significant role during the early stages of chondrogenic lineage determination and that Notch1 signaling is crucial for the chondrogenic differentiation and endochondral ossification of MSCs.
In a recent study published in the Genes & Diseases journal, researchers at Chongqing Medical University, The University of Chicago Medical Center, and the Chinese Academy of Medical Sciences & Peking Union Medical College used both in vivo and in vitro MSC differentiation models to investigate the role and significance of Notch1 in regulating BMP2-induced chondrogenic differentiation and endochondral ossification.
The authors showed that BMP-2 stimulation induced chondrogenic differentiation of MSCs in the early stages, followed by osteogenic differentiation in the later stages. There was also a simultaneous increase in the expression of the Notch receptor in the early stages, and Notch1 and Jagged1 during the mid-to-late stages. BMP-2 further up-regulated the protein expression of NICD1 and RBPjk, suggesting that Notch1 signaling participates in BMP2-induced endochondral ossification of MSCs.
Furthermore, the down-regulation of Notch1 signaling i) promoted BMP2-induced chondrogenic differentiation of MSCs, as evident from the increased levels of the key chondrogenic differentiation transcription factor Sox9 and the chondrogenic differentiation marker Col2a1; ii) inhibited BMP2-induced osteogenic differentiation, which was associated with decreased levels of Runx2, Col1a1, and OPN; and iii) inhibited BMP2-induced blood vessels and trabecular bone formation. The up-regulation of Notch1 signaling exerted the opposite effect, however, highlighting its regulatory role in BMP2-induced endochondral ossification.
Mechanistically, activated Notch1 signaling promotes endochondral ossification by i) suppressing Sox9 expression via RBPjk-dependent Sox9 promoter inactivation, resulting in the inhibition of osteogenic differentiation and the promotion of chondrogenic differentiation; and ii) promoting VEGFA-mediated angiogenesis.
In conclusion, this study shows that Notch1 signaling regulates BMP2-induced osteogenic, chondrogenic, and angiogenic differentiation of MSCs. Furthermore, Notch1-mediated regulation of Sox9 determines the chondrogenic or osteogenic outcome in MSCs, highlighting its pivotal role in mediating the maintenance of BMP2-induced cartilage regeneration.
Reference
Title of the original paper: Notch1 signaling regulates Sox9 and VEGFA expression and governs BMP2-induced endochondral ossification of mesenchymal stem cells
Journal: Genes & Diseases
Genes & Diseases is a journal for molecular and translational medicine. The journal primarily focuses on publishing investigations on the molecular bases and experimental therapeutics of human diseases. Publication formats include full length research article, review article, short communication, correspondence, perspectives, commentary, views on news, and research watch.
DOI: https://doi.org/10.1016/j.gendis.2024.101336
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