Insulin-like Growth Factor 1(IGF1) Potentiates Bmp9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells And Bone Formation

Bone is one of the few organs that retains the potential for regeneration into adult life, and is the only tissue that can undergo continual remodeling throughout life. Efficacious bone regeneration would have an important impact on the clinical management of many bone and musculoskeletal disorders, such as with segmental bone loss, fracture non-union, and failed spinal fusion. Osteogenesis is a sequential cascade that recapitulates most, if not all, of the cellular events occurring during embryonic skeletal development. During skeletogenesis, bone formation can occur through two different pathways, intramembranous ossification or endochondral ossification. Bone regeneration following a fracture progresses through sequential phases similar to endochondral ossification, starting with chemotaxis and proliferation of mesenchymal stem cells. Mesenchymal stem cells (MSCs) hold great promise for tissue bioengineering and regenerative medicine. MSCs are adherent marrow stromal cells that can self-renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been implicated in regulating stem cell self-renewal and lineage commitment. Bone morphogenetic proteins (BMPs) play an important role in regulating cell proliferation and differentiation during development and have been shown to play an important role in stem cell biology. BMPs belong to the TGF superfamily and consist of at least 15 members in humans. Genetic disruptions of BMPs have resulted in various skeletal and extraskeletal abnormalities during development. BMPs fulfill their signaling activity by interacting with the heterodimeric complex of two transmembrane serine/threonine kinase receptors, BMPR type I and BMPR type II. The activated receptor kinases phosphorylate the transcription factors Smads 1, 5, or 8, which in turn form a heterodimeric complex with Smad4 in the nucleus and activate the expression of target genes in concert with other co-activators. Upon analyzing the 14 types of BMPs, we found that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. We further demonstrated that BMP9 regulates a distinct set of downstream targets that may play a role in regulating BMP-induced osteoblast differentiation of MSCs.BMP9 (also known as growth differentiation factor 2, or GDF-2) was first identified in the developing mouse liver, and its possible roles include inducing and maintaining the cholinergic phenotype of embryonic basal forebrain cholinergic neurons, inhibiting hepatic glucose production and inducing the expression of key enzymes of lipid metabolism, and stimulating murine hepcidin 1 expression. Although the functional role of BMP9 in the skeletal system remains to be fully understood, the potent osteogenic activity of BMP9 suggests that it may be used as an efficacious bone regeneration agent. It is conceivable that other growth factors may act synergistically or enhance BMP9-induced bone formation.Here, we sought to investigate the effect of insulin-like growth factor 1 (IGF1) on BMP9-induced bone formation. As a member of the IGF signaling system, IGF1 plays an important role in prenatal growth and development. IGF1 transduces its signaling through IGF-receptors and activates the phosphatidylinositol-3-kinase (PI3K)/Akt pathway or the mitogen-activated protein kinase (MAPK) pathway. The Igf1 null mice exhibit a 40% decrease in birth weight compared to their wild-type littermates, suggesting an important role of IGF1 in development. We have found that endogenous IGF1 expression is relatively low in MSCs. Exogenous expression of IGF1 can potentiate BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers, such as osteocalcin (OCN) and osteopontin (OPN) in MSCs. IGF1 is shown to augment BMP9-induced ectopic bone formation in stem cell implantation studies. Using perinatal limb explant culture, we have demonstrated that IGF1 enhances BMP9-induced endochondral ossification, while IGF1 itself can promote the expansion of hypertropic chondrocyte zone of the cultured limb explants. Exogenous expression of IGFBP3, IGFBP4 and IGFBP5, leads to inhibition of the IGF1 effect on BMP9-induced ALP activity and matrix mineralization in MSCs. Furthermore, IGF1 is shown to enhance the BMP9-induced BMPR-Smad reporter activity and the nuclear translocation of Smad1/5/8. While BMP9 stimulation does not significantly induce AKT phosphorylation, PI3K inhibitor LY294002 abolishes the IGF1 potentiation effect on BMP9-mediated osteogenic signaling, and can directly inhibit BMP9 activity, suggesting that BMP9 may cross-talk with IGF1 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs.