TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs. Mesenchymal stem cells (MSCs),2 representing a very small fraction of the total population of nucleated cells in bone marrow are adherent marrow stromal cells that can self-renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages (1-4). Bone morphogenetic proteins (BMPs), members of the TGF␤ superfamily, play an important role in stem cell biology (5, 6) and function to regulate cell proliferation and differentiation during development (7, 8). Several BMPs have been shown to regulate osteoblast differentiation and subsequent bone formation (3, 4, 7-9) and genetic disruptions of these factors have resulted in various skeletal and extraskeletal abnormalities during development (9, 10). We have conducted a comprehensive analysis of the osteogenic activity of 14 human BMPs and demonstrated that BMP9 is one of the most potent BMPs in promoting osteogenic differentiation of MSCs (3,11,12). We also demonstrated that osteogenic BMP9 regulates a distinct set of downstream targets in MSCs (13-16).BMP9 (a.k.a., GDF2) was originally identified from fetal mouse liver cDNA libraries, and is a relatively uncharacterized member of the BMP family (17). BMP9 is highly expressed in the developing mouse liver, and recombinant human BMP9 stimulates hepatocyte proliferation (17,18). It has been reported that BMP9 may play role in regulating glucose and iron homeostasis in liver (19,20). BMP9 has been shown to be a potent synergistic factor for hematopoietic progenitor generation and colony formation (21) and may play a role in the induction and main...

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

“…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.…”

“…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).…”

“…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).…”

“…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.…”

How to use BMP-2 for clinical applications? A review on pros and cons of existing delivery strategies

“…For BMP-2 and -4 -10, signalling is mediated by the type I receptors (BMPR-IA or IB) whereas the BMP-5/-6/-7 subgroup utilizes ActR-I (Alk2) for signalling (for review, see Katagiri et al [34]). For BMP-9 high affinity binding to ALK1 has been reported but signalling can also occur via ActR-I [35]. The obvious discrepancy between the number of ligands (more than 20 BMP members have been identified in mammals to date) and the limited amount of receptors raises important questions especially how these proteins can share so many different cellular functions and furthermore how those can act as morphogens during embryogenesis.…”