Yanhong Gao

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.

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.

TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells.

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.

Retinoic Acids Potentiate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

Background As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs). Methodology/Principal Findings Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARα and RXRα into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRα or RARα significantly increases trabecular bone and osteoid matrix formation. Conclusion/Significance Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT–PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

Tetrandrine Inhibits Wnt/β-Catenin Signaling and Suppresses Tumor Growth of Human Colorectal Cancer

As one of the most common malignancies, colon cancer is initiated by abnormal activation of the Wnt/β-catenin pathway. Although the treatment options have increased for some patients, overall progress has been modest. Thus, there is a great need to develop new treatments. We have found that bisbenzylisoquinoline alkaloid tetrandrine (TET) exhibits anticancer activity. TET is used as a calcium channel blocker to treat hypertensive and arrhythmic conditions in Chinese medicine. Here, we investigate the molecular basis underlying TETs anticancer activity. We compare TET with six chemotherapy drugs in eight cancer lines and find that TET exhibits comparable anticancer activities with camptothecin, vincristine, paclitaxel, and doxorubicin, and better than that of 5-fluorouracil (5-FU) and carboplatin. TET IC50 is ≤5 μM in most of the tested cancer lines. TET exhibits synergistic anticancer activity with 5-FU and reduces migration and invasion capabilities of HCT116 cells. Furthermore, TET induces apoptosis and inhibits xenograft tumor growth of colon cancer. TET treatment leads to a decrease in β-catenin protein level in xenograft tumors, which is confirmed by T-cell factor/lymphocyte enhancer factor and c-Myc reporter assays. It is noteworthy that HCT116 cells with allelic oncogenic β-catenin deleted are less sensitive to TET-mediated inhibition of proliferation, viability, and xenograft tumor growth. Thus, our findings strongly suggest that the anticancer effect of TET in colon cancer may be at least in part mediated by targeting β-catenin activity. Therefore, TET may be used alone or in combination as an effective anticancer agent.

Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells.

Growth hormone (GH) is usually released by somatotrophs in the anterior pituitary in response to the GH‐releasing hormone and plays an important role in skeleton development and postnatal growth. However, it is unclear if extrapituitary GH exerts any effect on murine multilineage cells (MMCs). MMCs are multipotent progenitors that give rise to several lineages, including bone, cartilage, and fat. We have identified bone morphogenic protein 9 (BMP9) as one of the most osteogenic BMPs in MMCs by regulating a distinct set of downstream mediators. In this study, we find that GH is one of the most significantly upregulated genes by BMP9 in mouse MMCs through expression‐profiling analysis. We confirm that GH is a direct early target of and upregulated by BMP9 signaling. Exogenous GH synergizes with BMP9 on inducing early and late osteogenic markers in MMCs. Furthermore, BMP9 and GH costimulation leads to a significant expansion of growth plate in cultured limb explants. Although GH alone does not induce de novo bone formation in an ectopic bone formation model, BMP9 and GH costimulated MMCs form more mature bone, which can be inhibited by silencing GH expression. The synergistic osteogenic activity between BMP9 and GH can be significantly blunted by JAK/STAT inhibitors, leading to a decrease in GH‐regulated insulin‐like growth factor 1 (IGF1) expression in MMCs. Our results strongly suggest that BMP9 may effectively regulate extrapituitary GH expression in MMCs. Thus, it is conceivable that the BMP9‐GH‐IGF axis may be exploited as an innovative strategy to enhance osteogenesis in regenerative medicine.

The Therapeutic Potential of the Wnt Signaling Pathway in Bone Disorders

The Wnt pathway plays a critical role in development and differentiation of many tissues, such as the gut, hair follicles, and bone. Increasing evidence indicates that Wnts may function as key regulators in osteogenic differentiation of mesenchymal stem cells and bone formation. Conversely, aberrant Wnt signaling is associated with many osteogenic pathologies. For example, genetic alterations in the Wnt signaling pathway lead to osteoporosis and osteopenia, while inactivating mutations of Wnt inhibitors result in a hyperostotic skeleton with increased bone mineral density. Hyperparathyroidism causes osteopenia via induction of the Wnt signaling pathway. Lithium, often used to treat bipolar disorder, blocks a Wnt antagonist, decreasing the patients risk of fractures. Thus, manipulating the Wnt pathway may offer plenty therapeutic opportunities in treating bone disorders. In fact, induction of the Wnt signaling pathway or inhibition of Wnt antagonists has shown promise in treating bone metabolic disorders, including osteoporosis. For example, antibodies targeting the Wnt inhibitor Sclerostin lead to increased bone mineral density in post-menopausal women. However, such therapies targeting the Wnt pathway are not without risk, as genetic alternations may lead to over-activation of Wnt/β-catenin and its association with many tumors. It is conceivable that targeting Wnt inhibitors may predispose the individuals to tumorigenic phenotypes, at least in bone. Here, we review the roles of Wnt signaling in bone metabolic and pathologic processes, as well as the therapeutic potential for targeting Wnt pathway and its associated risks in bone diseases.

Crosstalk between Wnt/β-catenin and estrogen receptor signaling synergistically promotes osteogenic differentiation of mesenchymal progenitor cells.

Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.

Defective Osteogenic Differentiation in the Development of Osteosarcoma

Osteosarcoma (OS) is associated with poor prognosis due to its high incidence of metastasis and chemoresistance. It often arises in areas of rapid bone growth in long bones during the adolescent growth spurt. Although certain genetic conditions and alterations increase the risk of developing OS, the molecular pathogenesis is poorly understood. Recently, defects in differentiation have been linked to cancers, as they are associated with high cell proliferation. Treatments overcoming these defects enable terminal differentiation and subsequent tumor inhibition. OS development may be associated with defects in osteogenic differentiation. While early regulators of osteogenesis are unable to bypass these defects, late osteogenic regulators, including Runx2 and Osterix, are able to overcome some of the defects and inhibit tumor propagation through promoting osteogenic differentiation. Further understanding of the relationship between defects in osteogenic differentiation and tumor development holds tremendous potential in treating OS.