The Small Molecule Phenamil Induces Osteoblast Differentiation and Mineralization

Abstract

Stimulation of osteoblast differentiation from mesenchymal stem cells is a potential strategy for bone repair. Bone morphogenetic proteins (BMPs) that induce osteoblastic differentiation have been success-fully used in humans to treat fractures. Here we outline a new approach to the stimulation of osteoblast differentiation using small molecules that stimulate BMP activity. We have identified the amiloride derivative phenamil as a stimulator of osteoblast differentiation and mineralization. Remarkably, phena-mil acts cooperatively with BMPs to induce the expression of BMP target genes, osteogenic markers, and matrix mineralization in both mesenchymal stem cell lines and calvarial organ cultures. Transcriptional profiling of cells treated with phenamil led to the identification of tribbles homolog 3 (Trb3) as a mediator of its effects. Trb3 is induced by phenamil selectively in cells with osteoblastic potential. Both Trb3 and phenamil stabilize the expression of SMAD, the critical transcription factor in BMP signaling, by promoting the degradation of SMAD ubiquitin regulatory factor 1. Small interfering RNA-mediated knockdown of Trb3 blunts the effects of phenamil on BMP signaling and osteogenesis. Thus, phenamil induces osteogenic differentiation, at least in part, through Trb3-dependent promotion of BMP action. The synergistic use of small molecules such as phenamil along with BMPs may provide new strategies for the promotion of bone healing. Bone is continuously remodeled throughout life by a tightly coupled process involving absorption by osteoclasts and for-mation by osteoblasts. Dysregulation of this coupled remodel-ing can lead to diseases such as osteoporosis (18, 19). The precursors of osteoblasts are pluripotent cells known as mes-enchymal stem cells (MSCs) (3, 7, 36). MSCs are viewed as potential tools for therapeutic intervention in diseases related to impaired function of osteoblasts because they can be de-rived from bone marrow, manipulated in culture, and admin-istered back to donor individuals (12, 13, 24, 39, 40). However, the mechanistic pathways that drive differentiation of MSCs along the osteoblast lineage are not completely understood. Therefore, elucidation of molecular mechanisms underlying osteogenesis not only is important for our understanding of bone development but also may advance strategies for bone repair. Targeting therapeutic molecules to bone in order to enhance the bone-forming activity of osteoblast precursors may aid in the treatment of bone disease. Osteoinductive factors are required to drive the lineage-specific differentiation of MSCs into osteoblastic cells in cul-ture. Osteoblast differentiation is influenced by multiple sig-naling pathways, including transforming growth factor ␤1, Hedgehog, Wnt, fibroblast growth factors, insulin-like growth factor 1, and bone morphogenetic proteins (BMPs) (8, 20, 25, 46, 49). Strategies employing BMPs have been successfully used with animals and humans to regenerate bones (16, 22, 27). However, the high cost and supraphysiologic doses of BMPs necessary to achieve osteoinductive activity illustrate the need for additional strategies for the stimulation of osteoblast differentiation and bone formation in vivo (23, 32, 48). Recent studies of zebrafish have validated the con-cept of employing small molecules to modulate BMP activity in vivo (50). Similarly, certain oxysterols have been shown to activate sonic hedgehog (SHH) and to stimulate osteoblastic differentiation and bone formation (1, 15). However, small-molecule BMP stimulators that are able to bypass the need for high doses of BMP and induce bone formation remain to be identified. Here we show that the small molecule phenamil, a derivative of the diuretic amiloride, induces osteoblastic differentiation and mineralization of mouse MSCs. Phenamil and BMPs show additive effects on the expression of BMP target genes, osteo-genic markers, and matrix mineralization in M2-10B4 (M2) MSCs as well as in calvarial organ cultures. We show that phenamil acts, at least in part, by inducing the expression of tribbles homolog 3 (Trb3), a previously identified positive reg-ulator of BMP signaling (9, 33, 51). We further show that phenamil reduces the protein level of SMAD ubiquitin regu-latory factor 1 (Smurf1) and induces expression of SMAD, the critical transcription factor in BMP signaling. These results suggest that phenamil or related small molecules may repre-sent a novel strategy for increasing BMP activity in the clinical setting.