Notch signaling pathway promotes osteogenic differentiation of mesenchymal stem cells by enhancing BMP9/Smad signaling

Abstract

Notch is an important pathway in that it regulates cell-to-cell signal transduction, which plays an essential role in skeletal remodeling. Bone morphogenetic protein (BMP)9 has been regarded as one of the most efficient BMPs by which to induce osteogenic differentiation in mesenchymal stem cells (MSCs). Understanding the interaction between Notch and BMP9 signaling is a critical issue for optimizing the application of MSCs and BMPs in bone tissue engineering. In the present study, we investigated the role of Notch signaling in the BMP9‑induced osteogenic differentiation of MSCs. Our data demonstrated that Notch signaling obviously enhanced BMP9‑induced osteogenic differentiation in MSCs in vitro and in vivo. Notch signaling augmented the activity of BMP9‑induced BMP/Smad signaling and increased the gene expression of essential osteogenic factors induced by BMP9 in MSCs, such as runt‑related transcription factor 2 (Runx2), type I collagen (Colla1) and inhibitor of differentiation (Id)1. We also found that Notch signaling promoted the expression of activin‑like kinase 2 (ALK2) induced by BMP9, and the inhibitory effect of dnALK2 on BMP9‑induced osteogenic differentiation was rescued by constitutive overexpression of Delta‑like 1 (DLL1). Notch signaling also exhibited an apparent effect on the proliferation of mouse embryo fibroblasts (MEFs) during BMP9‑induced osteogenic differentiation. These results indicate that Notch plays a significant role in mediating BMP9‑induced osteogenic differentiation in MSCs, which may be partly regulated by upregulation of the expression of ALK2.

Figure 1

Notch signaling enhances BMP9-induced early osteogenic differentiation of MSCs. (A) MEFs were treated with Ad-BMP9 in the presence of various concentrations of DAPT (L=5 μM, M=10 μM and H=15 μM), and the BMP9-induced ALP activity was assessed by quantitative assay and staining assay at 7 day(s) post-treatment. (B) MEFs were infected with various titers of Ad-dnNotch1, followed by treatment with BMP9-CM, ALP activity was measured by quantitative assay and staining assay at 5 day(s) post-treatment. (C) MEFs were exposed to Ad-RFP or Ad-DLL1 in the presence of BMP9-CM, and the BMP9-induced ALP activity was assessed by quantitative assay and staining assay at 7 day(s) post-treatment. (D) MEFs were treated with BMP9-CM and/or Ad-dnNotch1, and the ALP activity was measured by quantitative assay and staining assay at 3, 5, 7 day(s) post-treatment. Magnification, ×100. *P<0.05, ^**P<0.01. ns, no statistical significance; BMP, bone morphogenetic protein; MEFs, mouse embryo fibroblasts; ALP, alkaline phosphatase; dnNotch1, dominant-negative mutant of Notch1; BMP9-CM, BMP9-conditioned media; DLL, Delta-like.

Figure 2

Notch signaling increases BMP9-induced late osteogenic differentiation of MSCs. (A) C2C12 cells were treated with different concentrations of DAPT (M=10 μM, and H=15 μM, respectively) in the presence of BMP9-CM, and the gene expression of OCN was determined by semi-quantitative RT-PCR at 7, 9 and 11 day(s) post-treatment and quantification by densitometry. (B) MEFs were exposed to Ad-dnNotch1 in the presence of BMP9-CM, and the gene expression levels of OPN and OCN were determined by semi-quantitative RT-PCR at 9 day(s) post-treatment and quantification by densitometry. (C) MEFs were infected with Ad-DLL1 in the presence of BMP9-CM, and matrix mineralization was assessed at 11 day(s) post-treatment by Alizarin Red S staining assay. Magnification, ×100. ^*P<0.05. BMP, bone morphogenetic protein; BMP9-CM, BMP9-conditioned media; OCN, osteocalcin; MEFs, mouse embryo fibroblasts; dnNotch1, dominant-negative mutant of Notch1; OPN, osteopontin; DLL, Delta-like.

Figure 3

Notch signaling promotes mineralization in BMP9-stimulated MSC implantation in vivo. (A) At 4 week(s), animals were sacrificed, and the ectopic bone masses were retrieved. (B) Histologic analysis of the retrieved samples. The samples were decalcified and paraffin-embedded and sectioned for H&E and Masson's trichrome staining. Arrows, mineralized matrix. (C) Retrieved samples were subjected to micro-CT imaging analysis, and representative three-dimensional reconstructed images are shown. The color bar indicates the BMD from low (green) to high (red). BMP, bone morphogenetic protein; bone mineral density (BMD).

Figure 4

Notch signaling augments the activity of BMP9-induced BMP/Smad signaling. (A and B) C2C12 cells, MEFs and C3H10T1/2 cells were infected with Ad-DLL1 or Ad-dnNotch1 or different concentrations of DAPT (M=10 μM, and H=15 μM, respectively) in the presence of BMP9-CM; total amount and phosphorylated forms of Smad1/5/8 were analyzed by western blot analysis. (C) MEFs, C2C12 cells and C3H10T1/2 cells were transfected with p12xSBE-luc and co-infected with Ad-DLL1 or Ad-dnNotch1 in the presence of BMP9-CM; luciferase activity was assessed at 36 h post-treatment. ^*P<0.05. BMP, bone mor-phogenetic protein; MEFs, mouse embryo fibroblasts; DLL, Delta-like; dnNotch1, dominant-negative mutant of Notch1; BMP9-CM, BMP9-conditioned media.

Figure 5

Notch signaling increases the expression of essential osteogenic factors induced by BMP9 in MSCs. (A) MEFs and C2C12 cells were treated with Ad-dnNotch1 or Ad-DLL1 or different concentrations of DAPT (M=10 μM, and H=15 μM, respectively) in the presence of BMP9-CM. The gene expression of Runx2 and Colla1 was detected by semi-quantitative RT-PCR at indicated time-points and quantification by densitometry. (B) MEFs and C2C12 cells were treated with DAPT (M=10 μM) or Ad-dnNotch1 in the presence of BMP9-CM, and the gene expression levels of Id, Id2, Id3 were detected by semi-quantitative RT-PCR at 24 h post-treatment and quantification by densitometry. ^*P<0.05. BMP, bone morphogenetic protein; MEFs, mouse embryo fibroblasts; dnNotch1, dominant-negative mutant of Notch1; DLL, Delta-like; BMP9-CM, BMP9-conditioned media; Runx2, runt-related transcription factor 2; Colla1, type I collagen; Id, inhibitor of differentiation.

Figure 6

Notch signaling promotes BMP9-induced ALK2 gene expression, and the inhibitory effect of dnALK2 on BMP9-induced osteogenic differentiation is rescued by overexpression of DLL1. (A) MEFs were treated with Ad-DLL1 or Ad-dnNotch1 in the presence of BMP9-CM, and the gene expression of ALK1 and ALK2 was detected by qPCR at 3 day(s) post-treatment. (B) C3H10T1/2 cells, C2C12 cells and MEFs were infected with Ad-DLL1 and/or Ad-dnALK1, Ad-dnALK2, or Ad-RFP for 24 h, and were stimulated with BMP9-CM. ALP activity was assessed by staining assay at 5 day(s) post-treatment. (C) Matrix mineralization was assessed at 9 day(s) post-treatment by Alizarin Red S staining assay. Magnification, ×100. ^*P<0.05. BMP, bone morphogenetic protein; ALK, activin-like kinase; DLL, Delta-like; MEFs, mouse embryo fibroblasts; dnNotch1, dominant-negative mutant of Notch1; BMP9-CM, BMP9-conditioned media; ALP, alkaline phosphatase.

Figure 7

Notch signaling exhibits an apparent effect on proliferation in MEFs during BMP9-induced osteogenic differentiation. (A and B) Cell cycle analysis was detected by FCM in MEFs. MEFs, mouse embryo fibroblasts; BMP, bone morphogenetic protein; FCM, flow cytometry.