IGF1 potentiates BMP9-induced osteogenic differentiation in mesenchymal stem cells through the enhancement of BMP/Smad signaling

Abstract

Engineered bone tissue is thought to be the ideal alternative for bone grafts in the treatment of related bone diseases. BMP9 has been demonstrated as one of the most osteogenic factors, and enhancement of BMP9-induced osteogenesis will greatly accelerate the development of bone tissue engineering. Here, we investigated the effect of insulin-like growth factor 1 (IGF1) on BMP9-induced osteogenic differentiation, and unveiled a possible molecular mechanism underling this process. We found that IGF1 and BMP9 are both detectable in mesenchymal stem cells (MSCs). Exogenous expression of IGF1 potentiates BMP9-induced alkaline phosphatase (ALP), matrix mineralization, and ectopic bone formation. Similarly, IGF1 enhances BMP9-induced endochondral ossification. Mechanistically, we found that IGF1 increases BMP9-induced activation of BMP/Smad signaling in MSCs. Our findings demonstrate that IGF1 can enhance BMP9-induced osteogenic differentiation in MSCs, and that this effect may be mediated by the enhancement of the BMP/Smad signaling transduction triggered by BMP9. [BMB Reports 2016; 49(2): 122-127].

Fig. 1.. Endogenous expression of BMP9 and IGF1 in progenitor cells and BMP9-induced expression of IGF1 in C3H10T1/2 cells. (A) PCR results show the endogenous expression of BMP9 and IGF1 in the three progenitor cells. GAPDH was used as a loading control. (B) Western blots show the endogenous expression of BMP9 and IGF1 in the three progenitor cells. GAPDH was used as a loading control. (C) Western blots show the BMP9-induced expression of IGF1 and IGF2 in C3H10T1/2 cells. GAPDH was used as a loading control. (D) Representative image and Western blot show the expression of IGF1 mediated by the adenovirus. GAPDH was used as loading control. (E) Representative image and Western blot show the expression of BMP9 mediated by the adenovirus. GAPDH was used as a loading control.

Fig. 2.. Effect of IGF1 on different BMP9-induced osteogenic markers in C3H10T1/2 cells. (A) ALP activity shows the effect of IGF1 on BMP9-induced osteogenic differentiation in C3H10T1/2 cells. (B) ALP staining shows the effect of IGF1 on BMP9-induced osteogenic differentiation in C3H10T1/2 cells. (C) Western blots show the effect of IGF1 on the expression of BMP9-induced OPN in C3H10T1/2 cells. GAPDH was used as a loading control. (D) Alizarin Red S staining shows the effect of IGF1 on BMP9-induced mineralization in C3H10T1/2 cells on day 20. The upper panel shows the scanning results, and the lower panel shows the images taken with the microscope.

Fig. 3.. Effect of IGF1 on BMP9-induced ectopic bone formation. (A) Representative retrieved bone masses show the effect of IGF1 on BMP9-induced ectopic bone formation. (B) H&E staining shows the effect of IGF1 on BMP9-induced ectopic bone formation. Representative images are shown (BM, bone matrix). (C) Masson’s Trichrome staining shows the effect of IGF1 on BMP9-induced ectopic bone formation. Representative images are shown (MBM, matured bone matrix; UBM, unmatured bone matrix).

Fig. 4.. Effect of IGF1 on BMP9-induced activation of BMP/Smad signaling in C3H10T1/2 cells. (A) Luciferase reporter activity shows IGF1 potentiates the activation of BMP9-induced BMP/Smad signaling. (B) Immunocytochemical staining shows the effect of IGF1 on BMP9-induced Smad1/5/8 phosphorylation (blue arrow shows the positive staining of Phosphorylated Smad1/5/8 in C3H10T1/2 cells). (C) Western blots show the effect of IGF1 on the level of BMP9-induced Smad1/5/8 and phosphorylation of Smad1/5/8. GAPDH was used as a loading control. (D) Quantification of Western blots shows the effect of IGF1 on the level of BMP9-induced Smad1/5/8 and phosphorylation of Smad1/5/8.