Identification and analysis of type II TGF-β receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells

Abstract

Our previous studies have demonstrated that bone morphogenetic protein 9 (BMP-9) is one of the most efficacious BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanism underlying the BMP-9-induced osteogenic differentiation of MSCs remains to be fully elucidated. In this study, dominant negative (DN) type II TGF-β receptors were constructed and introduced into C3H10T1/2 stem cells, then in vitro and in vivo assays were carried out to analyze and identify the type II TGF-β receptors required for BMP-9-induced osteogenesis. We found that three DN type II TGF-β receptors, DN-BMPRII, DN-ActRII, and DN-ActRIIB, diminished BMP-9-induced alkaline phosphatase (ALP) activity, led to a decrease in BMP-9-induced Smad binding element (SBE)-controled reporter activity, reduced BMP-9-induced expressions of Smad6 and Smad7, and decreased BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, finally resulted in decreased bone masses and immature osteogenesis. These findings strongly suggested that three wild-type II TGF-β receptors, BMPRII, ActRII and ActRIIB, may play a functional role in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells. However, C3H10T1/2 stem cells can express BMPRII and ActRII, but not ActRIIB. Using RNA interference (RNAi), we found that luciferase reporter activity and ALP activity induced by BMP-9 were accordingly inhibited along with the knockdown of BMPRII and ActRII. Taken together, our results demonstrated that BMPRII and ActRII are the functional type II TGF-β receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.

Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells

“…1C, BMP9 simultaneously stimulated the phosphorylation/activation of transcription factors Samd1/5/8, p38 and ERK1/2 MAPKs, without affecting the total amounts of these proteins. Our recently reports have demonstrated that dominant negative (dn) mutant of TGFβ receptors ALK1, ALK2, BMPRII and ActRII, which lack the kinase domain, remarkably inhibited osteoinductive activity and signal transduction of BMP9 (8,9). Here, when exogenous dn-ALK1, dn-ALK2, dn-BMPRII and dn-ActRII were introduced into C3H10T1/2 cells in conjunction with BMP9 ( Fig.…”

“…Recombinant adenoviruses harboring dominant-negative forms of ALK1, ALK2, BMPRII, ActRII were generated as previously described (8,9). Recombinant adenoviruses expressing siRNA targeted Smad4 (AdR-si-Smad4), p38 (AdR-si-p38), ERK1/2 (AdR-si-ERK1/2) were kindly provided by Dr. Tong-chuan He of University of Chicago Medical Center.…”

“…BMP9 conditioned medium were prepared as described (8,9). Briefly, subconfluent HCT116 cells (in 75-cm2 flaks) were infected with an optimal titer of Ad-BMP9.…”

“…Recent studies have indicated that BMP9, one of the least studied BMPs, is a more potent inducer in promoting osteogenic differentiation of MSCs (6)(7)(8)(9). Further expression profiling analysis has identified several important signaling mediators of BMP9 in MSCs (7).…”

“…Our previous study revealed that TGFβ type I receptors ALK1 and ALK2 are essential for BMP9-induced osteoinductive activity (8). A recent study by Wu et al elucidated that TGFβ type II receptors BMPRII and ActRII are the functional receptors necessary to transduce BMP9-induced osteogenic signaling (9). Despite these valuable discoveries, BMP9 remains as the least characterized BMPs, and the signaling mechanism through which BMP9 regulates osteogenic differentiation of MSCs is still largely unknown and warrants extensive studies.…”