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Updated: Mar 19, 2026
Stimulation of Notch Signaling in Mouse Osteoclast Precursors
Published on: February 28, 2017
[EFFECT OF Hey1 EXPRESSION ON OSTEOGENIC DIFFERENTIATION AND PROLIFERATION OF C3H10T1/2 CELLS INDUCED BY BONE
Objective:
To investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9 (BMP-9).
Methods:
Hey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank (empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, real-time fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium (BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-shHey1 group); the cells of control groups (C3H10T1/2 group and G3H10T1/2-Blank group) were cultured with normal medium. The mRNA and protein expression levels of osteogenesis related transcription factors (Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase (ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days.
Results:
C3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA and protein expressions of transcription factors (Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker (ALP) (P < 0.05); however, inhibition of Hey1 expression significantly decreased the above indexes (P < 0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance (A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes (P < 0.05).
Conclusion:
Expression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.
Insights
Hey1 expression significantly impacts bone morphogenetic protein 9-induced osteogenic differentiation and proliferation in C3H10T1/2 cells. Overexpression enhances differentiation and proliferation, while inhibition decreases these processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Bone morphogenetic protein 9 (BMP-9) is crucial for osteogenic differentiation.
- The Notch signaling pathway plays a role in cell differentiation and proliferation.
- Hey1 is a key target gene within the Notch signaling pathway.
Purpose of the Study:
- To investigate the role of Hey1 expression in BMP-9-induced osteogenic differentiation and proliferation of C3H10T1/2 cells.
- To elucidate the regulatory mechanism of Hey1 in the early stages of osteogenesis.
Main Methods:
- C3H10T1/2 cells were genetically modified to overexpress or inhibit Hey1 using lentiviral vectors.
- Osteogenic differentiation was assessed by measuring mRNA and protein levels of Runx2, osteopontin, osteocalcin, and alkaline phosphatase (ALP) activity.
- Cell proliferation and cell cycle progression were evaluated using MTT assays and flow cytometry.
Main Results:
- Successful establishment of C3H10T1/2 cell lines with altered Hey1 expression.
- Hey1 overexpression enhanced BMP-9-induced osteogenic differentiation markers (Runx2, osteopontin, osteocalcin, ALP).
- Hey1 inhibition significantly reduced osteogenic differentiation markers.
- Hey1 overexpression promoted cell proliferation and G2+S phase transition, while inhibition decreased these effects.
Conclusions:
- Hey1 is a critical mediator in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.
- Hey1 plays a significant role in regulating early-stage cell proliferation during osteogenesis.
- Targeting Hey1 may offer a therapeutic strategy for bone regeneration.
