Wei Shui

Wnt signaling in bone formation and its therapeutic potential for bone diseases

The Wnt signaling pathway plays an important role not only in embryonic development but also in the maintenance and differentiation of the stem cells in adulthood. In particular, Wnt signaling has been shown as an important regulatory pathway in the osteogenic differentiation of mesenchymal stem cells. Induction of the Wnt signaling pathway promotes bone formation while inactivation of the pathway leads to osteopenic states. Our current understanding of Wnt signaling in osteogenesis elucidates the molecular mechanisms of classic osteogenic pathologies. Activating and inactivating aberrations of the canonical Wnt signaling pathway in osteogenesis results in sclerosteosis and osteoporosis respectively. Recent studies have sought to target the Wnt signaling pathway to treat osteogenic disorders. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway. Antibodies against endogenous antagonists, such as sclerostin and dickkopf-1, have demonstrated promising results in promoting bone formation and fracture healing. Lithium, an inhibitor of glycogen synthase kinase 3β, has also been reported to stimulate osteogenesis by stabilizing β catenin. Although manipulating the Wnt signaling pathway has abundant therapeutic potential, it requires cautious approach due to risks of tumorigenesis. The present review discusses the role of the Wnt signaling pathway in osteogenesis and examines its targeted therapeutic potential.

BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells

Summary Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1&agr; (HIF1&agr;)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1&agr; expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1&agr; potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1&agr; or HIF1&agr; inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1&agr; expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1&agr; and BMP9 in osteogenic differentiation. Mechanistically, HIF1&agr; is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9‐induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9‐induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre‐osteoblast C2C12 cells. BMP9‐induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7‐induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9‐induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2‐induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9‐induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9s potent osteogenic capability in MSCs.

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.

Targeting BMP9-Promoted Human Osteosarcoma Growth by Inactivation of Notch Signaling

Osteosarcoma (OS) is the most common primary malignancy of bone and is usually associated with poor prognosis due to its high incidence of metastasis and chemoresistance. Molecular pathogenesis of OS is poorly understood. We previously showed that OS cells are refractory to BMP9-induced osteogenesis and respond favorably to proliferation and tumor growth. Here we investigate if Notch signaling mediates the BMP9-promoted cell proliferation and tumor growth of human osteosarcoma (OS). We find that the expression of Notch1, Notch2, Notch3, DLL1, JAG1 and JAG2 is readily detected in most of the tested OS cell lines. BMP9-promoted OS cell proliferation, migration, and cell cycle S/G2 progression are effectively inhibited by a dominant-negative mutant of Notch1 (dnNotch1) or the γ-secretase inhibitor Compound E (ComE). Furthermore, BMP9-promoted tumor growth and osteolytic lesions in vivo are significantly inhibited by dnNotch1. BMP9 up-regulates the expression of Notch1, Notch3, DLL1, and JAG1 in OS cells. Accordingly, BMP9 stimulation induces a nuclear accumulation of NICD, which is blocked by ComE. Our results demonstrate that BMP9-promoted OS proliferation and tumor growth is at least in part mediated by Notch signaling, suggesting that osteogenic BMPs may function as upstream regulators of Notch signaling in OS tumorigenesis. Thus, pharmacologic intervention of Notch signaling may be explored as a new therapeutic strategy for human OS tumors.

Reversibly Immortalized Mouse Articular Chondrocytes Acquire Long-Term Proliferative Capability While Retaining Chondrogenic Phenotype.

Cartilage tissue engineering holds great promise for treating cartilaginous pathologies including degenerative disorders and traumatic injuries. Effective cartilage regeneration requires an optimal combination of biomaterial scaffolds, chondrogenic seed cells, and biofactors. Obtaining sufficient chondrocytes remains a major challenge due to the limited proliferative capability of primary chondrocytes. Here we investigate if reversibly immortalized mouse articular chondrocytes (iMACs) acquire long-term proliferative capability while retaining the chondrogenic phenotype. Primary mouse articular chondrocytes (MACs) can be efficiently immortalized with a retroviral vector-expressing SV40 large T antigen flanked with Cre/loxP sites. iMACs exhibit long-term proliferation in culture, although the immortalization phenotype can be reversed by Cre recombinase. iMACs express the chondrocyte markers Col2a1 and aggrecan and produce chondroid matrix in micromass culture. iMACs form subcutaneous cartilaginous masses in athymic mice. Histologic analysis and chondroid matrix staining demonstrate that iMACs can survive, proliferate, and produce chondroid matrix. The chondrogenic growth factor BMP2 promotes iMACs to produce more mature chondroid matrix resembling mature articular cartilage. Taken together, our results demonstrate that iMACs acquire long-term proliferative capability without losing the intrinsic chondrogenic features of MACs. Thus, iMACs provide a valuable cellular platform to optimize biomaterial scaffolds for cartilage regeneration, to identify biofactors that promote the proliferation and differentiation of chondrogenic progenitors, and to elucidate the molecular mechanisms underlying chondrogenesis.

Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

Inhibition of Histone Deacetylases Potentiates BMP9-Induced Osteogenic Signaling in Mouse Mesenchymal Stem Cells

Background/Aims: We have demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent BMPs in regulating osteoblast differentiation of mesenchymal stem cells (MSCs) although the molecular mechanism underlying BMP9-induced osteogenesis remains to be fully elucidated. It is known that epigenetic regulations play an important role in regulating the stem cell potency and lineage commitment. Here, we investigate if the inhibition of histone deacetylases (Hdacs) affects BMP9-induced osteogenic differentiation of MSCs. Methods: Using the Hdac inhibitor trichostatin A (TSA), we assess that TSA enhances BMP9-mediated osteogenic markers and matrix mineralization in MSCs, and bone formation in mouse embryonic limb explants. Results: We find that the endogenous expression of most of the 11 Hdacs is readily detectable in MSCs. BMP9 is shown to induce most Hdacs in MSCs. We demonstrate that TSA potentiates BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs, as well as late osteogenic markers osteopontin (OPN) and osteocalcin (OCN) and matrix mineralization. Fetal limb explant culture studies reveal that TSA potentiates BMP9-induced endochondral bone formation, possibly by expanding hypertrophic chondrocyte zone of growth plate. Conclusion: Our findings strongly suggest histone deacetylases may play an important role in fine-tuning BMP9-mediated osteogenic signaling through a negative feedback network in MSCs. Thus, Hdac inhibitors may be used as novel therapeutics for bone fracture healing.

Characterization of chondrocyte scaffold carriers for cell‐based gene therapy in articular cartilage repair

Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in three-dimensional scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and open-cell polylactic acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At 4 weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs.

The Current and Future Therapies for Human Osteosarcoma

Osteosarcoma (OS) is the most common non-hematologic malignant tumor of bone in adults and children. As sarcomas are more common in adolescents and young adults than most other forms of cancer, there are a significant number of years of life lost secondary to these malignancies. OS is associated with a poor prognosis secondary to a high grade at presentation, resistance to chemotherapy and a propensity to metastasize to the lungs. Current OS management involves both chemotherapy and surgery. The incorporation of cytotoxic chemotherapy into therapeutic regimens escalated cure rates from <20% to current levels of 65-75%. Furthermore, limb-salvage surgery is now offered to the majority of OS patients. Despite advances in chemotherapy and surgical techniques over the past three decades, there has been stagnation in patient survival outcome improvement, especially in patients with metastatic OS. Thus, there is a critical need to identify novel and directed therapy for OS. Several Phase I trials for sarcoma therapies currently ongoing or recently completed have shown objective responses in OS. Novel drug delivery mechanisms are currently under phase II and III clinical trials. Furthermore, there is an abundance of preclinical research which holds great promise in the development of future OS-directed therapeutics. Our continuously improving knowledge of the molecular and cell-signaling pathways involved in OS will translate into more effective therapies for OS and ultimately improved patient survival. The present review will provide an overview of current therapies, ongoing clinical trials and therapeutic targets under investigation for OS.