Hue H. Luu

Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs).

Background:Bone morphogenic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway to evaluate the ability of certain BMPs to promote fracture-healing and spinal fusion. The optimal BMPs to be used in different clinical applications have not been elucidated, an

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.

Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells

Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was β-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells.

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.

CCN1/Cyr61 Is Regulated by the Canonical Wnt Signal and Plays an Important Role in Wnt3A-Induced Osteoblast Differentiation of Mesenchymal Stem Cells

ABSTRACT Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/β-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.

Wnt / β-Catenin Signaling Pathway as Novel Cancer Drug Targets

Wnt proteins are a large family of secreted glycoproteins. Wnt proteins bind to the Frizzled receptors and LRP5/6 co-receptors, and through stabilizing the critical mediator beta-catenin, initiate a complex signaling cascade that plays an important role in regulating cell proliferation and differentiation. Deregulation of the canonical Wnt/beta-catenin signaling pathway, mostly by inactivating mutations of the APC tumor suppressor, or oncogenic mutations of beta-catenin, has been implicated in colorectal tumorigenesis. Although oncogenic mutations of beta-catenin have only been discovered in a small fraction of non-colon cancers, elevated levels of beta-catenin protein, a hallmark of activated canonical Wnt pathway, have been observed in most common forms of human malignancies, indicating that activation of this pathway may play an important role in tumor development. Over the past 15 years, our understanding of this signaling pathway has significantly improved with the identification of key regulatory proteins and the important downstream targets of beta-catenin/Tcf transactivation complex. Given the fact that Wnt/beta-catenin signaling is tightly regulated at multiple cellular levels, the pathway itself offers ample targeting nodal points for cancer drug development. In this review, we discuss some of the strategies that are being used or can be explored to target key components of the Wnt/beta-catenin signaling pathway in rational cancer drug discovery.

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.

Transcriptional Characterization of Bone Morphogenetic Proteins (BMPs)-Mediated Osteogenic Signaling

Bone formation is presumably a complex and well‐orchestrated process of osteoblast lineage‐specific differentiation. As members of the TGFβ superfamily, bone morphogenetic proteins (BMPs) play an important role in regulating osteoblast differentiation and subsequent bone formation. Several BMPs are able to induce de novo bone formation. Although significant progress has recently been made about the transcriptional control of osteoblast differentiation, detailed molecular events underlying the osteogenic process remain to be elucidated. In order to identify potentially important signaling mediators activated by osteogenic BMPs but not by non‐osteogenic BMPs, we sought to determine the transcriptional differences between three osteogenic BMPs (i.e., BMP‐2, BMP‐6, and BMP‐9) and two inhibitory/non‐osteogenic BMPs (i.e., BMP‐3 and BMP‐12). Through the microarray analysis of approximately 12,000 genes in pre‐osteoblast progenitor cells, we found that expression level of 203 genes (105 up‐regulated and 98 down‐regulated) was altered >2‐fold upon osteogenic BMP stimulation. Gene ontology analysis revealed that osteogenic BMPs, but not inhibitory/non‐osteogenic BMPs, activate genes involved in the proliferation of pre‐osteoblast progenitor cells towards osteoblastic differentiation, and simultaneously inhibit myoblast‐specific gene expression. BMP‐regulated expression of the selected target genes was confirmed by RT‐PCR, as well as by the CodeLink Bioarray analysis. Our findings are consistent with the notion that osteogenesis and myogenesis are two divergent processes. Further functional characterization of these downstream target genes should provide important insights into the molecular mechanisms behind BMP‐mediated bone formation.

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.