Jinyong Luo

Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery.

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

Regulation of osteogenic differentiation during skeletal development.

Bone formation during skeletal development involves a complex coordination among multiple cell types and tissues. Bone is of crucial importance for the human body, providing skeletal support, and serving as a home for the formation of hematopoietic cells and as a reservoir for calcium and phosphate. Bone is also continuously remodeled in vertebrates throughout life. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption, respectively. Early development of the vertebrate skeleton depends on genes that control the distribution and proliferation of cells from cranial neural crest, sclerotomes, and lateral plate mesoderm into mesenchymal condensations, where cells differentiate to osteoblasts. Significant progress has been made over the past decade in our understanding of the molecular framework that controls osteogenic differentiation. A large number of morphogens, signaling molecules, and transcriptional regulators have been implicated in regulating bone development. A partial list of these factors includes the Wnt/beta-catenin, TGF-beta/BMP, FGF, Notch and Hedgehog signaling pathways, and Runx2, Osterix, ATF4, TAZ, and NFATc1 transcriptional factors. A better understanding of molecular mechanisms behind osteogenic differentiation would not only help us to identify pathogenic causes of bone and skeletal diseases but also lead to the development of targeted therapies for these diseases.

A Comprehensive Analysis of the Dual Roles of BMPs in Regulating Adipogenic and Osteogenic Differentiation of Mesenchymal Progenitor Cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.

Osteosarcoma Development and Stem Cell Differentiation

Osteosarcoma is the most common nonhematologic malignancy of bone in children and adults. The peak incidence occurs in the second decade of life, with a smaller peak after age 50. Osteosarcoma typically arises around the growth plate of long bones. Most osteosarcoma tumors are of high grade and tend to develop pulmonary metastases. Despite clinical improvements, patients with metastatic or recurrent diseases have a poor prognosis. Here, we reviewed the current understanding of human osteosarcoma, with an emphasis on potential links between defective osteogenic differentiation and bone tumorigenesis. Existing data indicate osteosarcoma tumors display a broad range of genetic and molecular alterations, including the gains, losses, or arrangements of chromosomal regions, inactivation of tumor suppressor genes, and the deregulation of major signaling pathways. However, except for p53 and/or RB mutations, most alterations are not constantly detected in the majority of osteosarcoma tumors. With a rapid expansion of our knowledge about stem cell biology, emerging evidence suggests osteosarcoma should be regarded as a differentiation disease caused by genetic and epigenetic changes that interrupt osteoblast differentiation from mesenchymal stem cells. Understanding the molecular pathogenesis of human osteosarcoma could ultimately lead to the development of diagnostic and prognostic markers, as well as targeted therapeutics for osteosarcoma patients.

Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation

Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self‐renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP‐9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin‐like growth factor 2 (IGF‐2) on BMP‐9‐induced bone formation. We have found that endogenous IGF‐2 expression is low in MSCs. Expression of IGF‐2 can potentiate BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF‐2 has been shown to augment BMP‐9‐induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF‐2 enhances BMP‐9‐induced endochondral ossification, whereas IGF‐2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF‐2 effect on BMP‐9‐induced ALP activity and matrix mineralization. Mechanistically, IGF‐2 is further shown to enhance the BMP‐9‐induced BMPR‐Smad reporter activity and Smad1/5/8 nuclear translocation. PI3‐kinase (PI3K) inhibitor LY294002 abolishes the IGF‐2 potentiation effect on BMP‐9‐mediated osteogenic signaling and can directly inhibit BMP‐9 activity. These results demonstrate that BMP‐9 crosstalks with IGF‐2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP‐9 and IGF‐2 may be explored as an effective bone‐regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture.

Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects

Osteosarcoma (OS) is the most common primary malignancy of bone. Here, we investigated a possible role of defective osteoblast differentiation in OS tumorigenesis. We found that basal levels of the early osteogenic marker alkaline phosphatase (ALP) activity were low in OS lines. Osteogenic regulators Runx2 and OSX, and the late marker osteopontin (OPN) expressed at low levels in most OS lines, indicating that most OS cells fail to undergo terminal differentiation. Furthermore, OS cells were refractory to osteogenic BMP-induced increases in ALP activity. Osteogenic BMPs were shown to upregulate early target genes, but not late osteogenic markers OPN and osteocalcin (OC). Furthermore, osteogenic BMPs failed to induce bone formation from human OS cells, rather effectively promoted OS tumor growth in an orthotopic OS model. Exogenous expression of early target genes enhanced BMP-stimulated OS tumor growth, whereas osteogenic BMP-promoted OS tumor growth was inhibited by exogenous Runx2 expression. These results suggest that alterations in osteoprogenitors may disrupt osteogenic differentiation pathway. Thus, identifying potential differentiation defects in OS tumors would allow us to reconstruct the tumorigenic events in osteoprogenitors and to develop rational differentiation therapies for clinical OS management.

BMP‐9‐induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β‐catenin signalling

Bone morphogenetic protein 9 (BMP‐9) is a member of the transforming growth factor (TGF)‐β/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/β‐catenin signalling plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. Wnt3A and BMP‐9 enhanced each other’s ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP‐9‐induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR‐5 or low‐density lipoprotein receptor‐related protein (LRP)‐6 exerted no inhibitory effect on BMP‐9‐induced ALP activity. β‐Catenin knockdown in MSCs and MEFs diminished BMP‐9‐induced ALP activity, and led to a decrease in BMP‐9‐induced osteocalcin reporter activity and BMP‐9‐induced expression of late osteogenic markers. Furthermore, β‐catenin knockdown or FrzB overexpression inhibited BMP‐9‐induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP‐9 induced recruitment of both Runx2 and β‐catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between β‐catenin and Runx2, plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs.

Wnt signaling and human diseases: what are the therapeutic implications?

Wnt signaling plays an important role in regulating cell proliferation and differentiation. De-regulation of these signaling pathways has been implicated in many human diseases, ranging from cancers to skeletal disorders. Wnt proteins are a large family of secreted factors that bind to the Frizzled receptors and LRP5/6 co-receptors and initiate complex signaling cascades. Over the past two decades, our understanding of Wnt signaling has been significantly improved due to the identification of many key regulators and mediators of these pathways. Given that Wnt signaling is tightly regulated at multiple cellular levels, these pathways themselves offer ample nodal points for targeted therapeutics. Here, we focus on our current understanding of these pathways, the associations of Wnt signaling with human disorders, and the opportunities to target key components of Wnt signaling for rational drug discovery.

Mesenchymal stem cells: Molecular characteristics and clinical applications.

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.

Hey1 Basic Helix-Loop-Helix Protein Plays an Important Role in Mediating BMP9-induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.