As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is highly capable of promoting osteogenic differentiation. However, the underlying mechanism involved remains largely unknown. Recent studies have demonstrated that RUNX1 (runt-related transcription factor 1) is essential in osteoblast/chondrocyte maturation. In this study, we investigated the function of RUNX1 in BMP9-induced osteogenic of murine mesenchymal stem cell line (C3H10T1/2) and murine multi-lineage cell lines (C2C12 and MEFs). Our data showed that BMP9 promoted the endogenous expression of RUNX1 in C3H10T1/2, C2C12 and MEFs. Moreover, RUNX1 was probably a direct target of BMP9/Smad signaling. BMP9-induced osteogenic differentiation was enhanced by overexpression of RUNX1, whereas inhibited by knockdown RUNX1 in C3H10T1/2, C2C12 and MEFs. Further mechanism studies demonstrated that RUNX1 might affect BMP9-induced phosphorylation of Smad1/5/8, but not the phosphorylation of p38 and ERK1/2.Our results suggest that RUNX1 may be an essential modulator in BMP9- induced osteogenic differentiation of MSCs (Mesenchymal stem cells).

Although BMP6 is highly capable of inducing osteogenic differentiation of mesenchymal progenitor cells (MPCs), the molecular mechanism involved remains to be fully elucidated. Using dominant negative (dn) mutant form of type I and type II TGFβ receptors, we demonstrated that three dn-type I receptors (dnALK2, dnALK3, dnALK6), and three dn-type II receptors (dnBMPRII, dnActRII, dnActRIIB), effectively diminished BMP6- induced osteogenic differentiation of MPCs. These findings suggested that ALK2, ALK3, ALK6, BMPRII, ActRII and ActRIIB are essential for BMP6-induced osteogenic differentiation of MPCs. However, MPCs in this study do not express ActRIIB. Moreover, RNA interference of ALK2, ALK3, ALK6, BMPRII and ActRII inhibited BMP6-induced osteogenic differentiation in MPCs. Our results strongly suggested that BMP6-induced osteogenic differentiation of MPCs is mediated by its functional TGFβ receptors including ALK2, ALK3, ALK6, BMPRII, and ActRII.

Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/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/beta-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. beta-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, beta-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 beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.

Osteosarcoma is the most common primary malignant bone neoplasm and is associated with abysmal prognosis. There are limitations of current treatment methods. Therefore, developing new agents to treat osteosarcoma is exceptionally urgent.

Circular RNAs (circRNAs) dysregulation has been revealed to function in the pathological processes of cancers. Herein, the role and mechanisms of hsa_circ_0002082 in breast cancer (BC) progression were elucidated.

Bone morphogenetic proteins (BMPs), particularly BMP9, have been shown to promote the osteogenic differentiation of murine multilineage cells (MMCs) and to promote bone formation in bone diseases; however, the mechanisms involved remain poorly understood. MicroRNAs (miRNAs or miRs) have been proven to regulate mesenchymal stem cell (MSC) differentiation. In this study, we identified a novel mechanism that unravels the functional axis of a key miRNA (miR-21) which contributes to BMP9‑induced osteogenic differentiation. We screened differentially expressed miRNAs in MMCs during BMP9‑induced osteogenic differentiation and found that miR-21 was significantly upregulated by BMP9 during the osteogenesis of MMCs. Furthermore, miR-21 was confirmed to promote the osteogenic differentiation of the MMCs by suppressing Smad7, which negatively regulates the osteogenic differentiation of MMCs. The upregulation of miR-21 may promote the osteogenic differentiation of MMCs in synergy with BMP9. The findings of our study revealed a novel function of miR-21, and suggest that the overexpression of miR-21 contributes to bone formation by promoting BMP9‑induced osteogenic differentiation. Our data may provide a molecular basis for the development of novel therapeutic strategies to treat bone diseases, such as osteoporosis and other inflammatory bone diseases.

Liver has numerous critical metabolic functions including lipid metabolism, which is usually dysregulated in obesity, the metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD). Increasing evidence indicates bone morphogenetic proteins (BMPs) play an important role in adipogenesis and thermogenic balance in adipogenic progenitors and adipose tissue. However, the direct impact of BMPs on hepatic steatosis and possible association with NAFLD are poorly understood. Here, we found that BMP4 was up-regulated in oleic acid-induced steatosis and during the development of high fat diet (HFD)-induced NAFLD. Exogenous BMP4 reduced lipid accumulation and up-regulated the genes involved in lipid synthesis, storage and breakdown in hepatocytes. Exogenous BMP4 inhibited hepatic steatosis, reduced serum triglyceride levels and body weight, and alleviated progression of NAFLD in vivo. Mechanistically, BMP4 overexpression in hepatocytes down-regulated most components of the mTORC1 signaling axis. Collectively, these findings strongly suggest that BMP4 may play an essential role in regulating hepatic lipid metabolism and the molecular pathogenesis of NAFLD. Manipulating BMP4 and/or mTORC1 signaling axis may lead to the development of novel therapeutics for obesity, metabolic syndrome, and NAFLD.

Osteosarcoma (OS) is a primary malignant tumor of bone. Chemotherapy is one of the crucial approaches to prevent its metastasis and improve prognosis. Despite continuous improvements in the clinical treatment of OS, tumor resistance and metastasis remain dominant clinical challenges. Macropinocytosis, a form of non-selective nutrient endocytosis, has received increasing attention as a novel target for cancer therapy, yet its role in OS cells remains obscure. Benzethonium chloride (BZN) is an FDA-approved antiseptic and bactericide with broad-spectrum anticancer effects. Here, we described that BZN suppressed the proliferation, migration, and invasion of OS cells in vitro and in vivo, but simultaneously promoted the massive accumulation of cytoplasmic vacuoles as well. Mechanistically, BZN repressed the ERK1/2 signaling pathway, and the ERK1/2 activator partially neutralized the inhibitory effect of BZN on OS cells. Subsequently, we demonstrated that vacuoles originated from macropinocytosis and indicated that OS cells might employ macropinocytosis as a compensatory survival mechanism in response to BZN. Remarkably, macropinocytosis inhibitors enhanced the anti-OS effect of BZN in vitro and in vivo. In conclusion, our results suggest that BZN may inhibit OS cells by repressing the ERK1/2 signaling pathway and propose a potential strategy to enhance the BZN-induced inhibitory effect by suppressing macropinocytosis.

Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.

S100A8 and S100A9, two members of the S100 protein family, have been reported in association with the tumor cell differentiation and tumor progression. Previous study has showed that their expression in stromal cells of colorectal carcinoma (CRC) is associated with tumor size. Here, we investigated the clinical significances of S100A8 and S100A9 in tumor cells of CRC and their underlying molecular mechanisms.

Osteosarcoma (OS) is a primary bone tumor associated with locally aggressive growth and early metastatic potential that typically occurs in children and adolescents. Chinese traditional medicine Cinnamomum cassia Presl has been shown to have significant tumor-killing effect, in which cinnamaldehyde (CA) is the main active ingredient.

Osteosarcoma (OS) is the most common malignant bone tumor worldwide and is associated with a poor prognosis, often being accompanied by lung metastasis at an early stage. At present, there are several side‑effects associated with the OS clinical treatment of OS, with the treatment effects often being unsatisfactory. Thus, there is an urgent need for the development of safe and effective novel drugs for the treatment of OS. Schisandrin B (Sch B) has been previously demonstrated to exhibit antitumor properties. The present study was focused on the effects of Sch B on OS cells (143B, MG63, Saos2 and U2OS) in vitro and in vivo, and also on its possible antitumor mechanisms. In cell experiments, it was revealed that Sch B inhibited OS cell proliferation, migration and invasion, and increased OS cell apoptosis. As regards its biosafety, no notable effects of Sch B on the vitality of normal cells were observed. Mechanistically, it was demonstrated that Sch B blocked OS cell proliferation in the G1 phase. Subsequently, by using established animal models, it was revealed that Sch B significantly inhibited OS growth and lung metastasis in vivo. In summary, the results of the present study revealed that Sch B inhibited OS cell proliferation, migration and invasion, and promoted apoptosis via the inhibition of the Wnt/β‑catenin and PI3K/Akt signaling pathways, without causing any noticeable toxic effects on healthy cells at the therapeutic concentrations used. These findings suggest that Sch B has potential for use as a novel agent for the clinical treatment of OS.

Notch is an important pathway in that it regulates cell-to-cell signal transduction, which plays an essential role in skeletal remodeling. Bone morphogenetic protein (BMP)9 has been regarded as one of the most efficient BMPs by which to induce osteogenic differentiation in mesenchymal stem cells (MSCs). Understanding the interaction between Notch and BMP9 signaling is a critical issue for optimizing the application of MSCs and BMPs in bone tissue engineering. In the present study, we investigated the role of Notch signaling in the BMP9‑induced osteogenic differentiation of MSCs. Our data demonstrated that Notch signaling obviously enhanced BMP9‑induced osteogenic differentiation in MSCs in vitro and in vivo. Notch signaling augmented the activity of BMP9‑induced BMP/Smad signaling and increased the gene expression of essential osteogenic factors induced by BMP9 in MSCs, such as runt‑related transcription factor 2 (Runx2), type I collagen (Colla1) and inhibitor of differentiation (Id)1. We also found that Notch signaling promoted the expression of activin‑like kinase 2 (ALK2) induced by BMP9, and the inhibitory effect of dnALK2 on BMP9‑induced osteogenic differentiation was rescued by constitutive overexpression of Delta‑like 1 (DLL1). Notch signaling also exhibited an apparent effect on the proliferation of mouse embryo fibroblasts (MEFs) during BMP9‑induced osteogenic differentiation. These results indicate that Notch plays a significant role in mediating BMP9‑induced osteogenic differentiation in MSCs, which may be partly regulated by upregulation of the expression of ALK2.

Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation and bone formation, the precise molecular mechanism involved remains to be fully elucidated. In this current study, we explore the possible involvement and detail effects of p38 and ERK1/2 MAPKs on BMP9-induced osteogenic differentiation of mesenchymal progenitor cell (MPCs). We find that BMP9 simultaneously stimulates the activation of p38 and ERK1/2 in MPCs. BMP9-induced early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as matrix mineralization and osteocalcin (OC) are inhibited by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. BMP9-induced activation of Runx2 and Smads signaling are reduced by SB203580, and yet increased by PD98059 in MPCs. The in vitro effects of inhibitors are reproduced with adenoviruses expressing siRNA targeted p38 and ERK1/2, respectively. Using mouse calvarial organ culture and subcutaneous MPCs implantation, we find that inhibition of p38 activity leads to significant decrease in BMP9-induced osteogenic differentiation and bone formation, however, blockage of ERK1/2 results in effective increase in BMP9-indcued osteogenic differentiation in vivo. Together, our results reveal that p38 and ERK1/2 MAPKs are activated in BMP9-induced osteogenic differentiation of MPCs. What is most noteworthy, however, is that p38 and ERK1/2 act in opposition to regulate BMP9-induced osteogenic differentiation of MPCs.

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.

Liver plays an essential role in regulating lipid metabolism, and chronically disturbed hepatic metabolism may cause obesity and metabolic syndrome, which may lead to non-alcoholic fatty liver disease (NAFLD). Increasing evidence indicates long non-coding RNAs (lncRNAs) play an important role in energy metabolism. Here, we investigated the role of lncRNA H19 in hepatic lipid metabolism and its potential association with NAFLD. We found that H19 was up-regulated in oleic acid-induced steatosis and during the development of high-fat diet (HFD)-induced NAFLD. Exogenous overexpression of H19 in hepatocytes induced lipid accumulation and up-regulated the expression of numerous genes involved in lipid synthesis, storage and breakdown, while silencing endogenous H19 led to a decreased lipid accumulation in hepatocytes. Mechanistically, H19 was shown to promote hepatic steatosis by up-regulating lipogenic transcription factor MLXIPL. Silencing Mlxipl diminished H19-induced lipid accumulation in hepatocytes. Furthermore, H19-induced lipid accumulation was effectively inhibited by PI3K/mTOR inhibitor PF-04691502. Accordingly, H19 overexpression in hepatocytes up-regulated most components of the mTORC1 signalling axis, which were inhibited by silencing endogenous H19. In vivo hepatocyte implantation studies further confirm that H19 promoted hepatic steatosis by up-regulating both mTORC1 signalling axis and MLXIPL transcriptional network. Collectively, these findings strongly suggest that H19 may play an important role in regulating hepatic lipid metabolism and may serve as a potential therapeutic target for NAFLD.

Colorectal cancer (CRC) is a life‑threatening malignant tumor of the digestive tract. Diverse gene mutations and complicated alterations to the signaling pathways in CRC lead to heterogeneity in response to chemotherapy. Moreover, anticancer drugs for CRC chemotherapy are limited due to adverse events. Therefore, developing more effective, tolerable and safe drugs for the treatment of CRC is important. The present study aimed to investigate the effect of lycorine on human CRC cell proliferation, migration, invasion, apoptosis, cell cycle distribution, as well as the underlying molecular mechanism. The crystal violet staining and MTT assay results demonstrated that lycorine suppressed cell proliferation in a dose‑ and time‑dependent manner in the three CRC cell lines, HCT116, LoVo and SW480. Similarly, verified by performing wound healing and Transwell assays, lycorine significantly inhibited HCT116 and LoVo cell migration and invasion in vitro compared with the control group. In LoVo cells, the protein expression levels of matrix metallopeptidases, snail family transcriptional repressor 1, Vimentin and N‑cadherin were significantly downregulated, whereas the protein expression levels of E‑cadherin were significantly upregulated by lycorine treatment compared with the control group. The Hoechst 33258 staining and flow cytometry assay results indicated that lycorine mediated its cytostatic effect on CRC cells potentially via inducing cell cycle arrest, but not apoptosis. Compared with the control group, lycorine significantly induced HCT116 cell cycle arrest at the G2/M phase, but significantly induced LoVo cell cycle arrest at the S and G2/M phases. Furthermore, lycorine significantly downregulated the protein expression levels of cyclin D1 and cyclin E1, but significantly increased p21 and Smad4 protein expression levels in HCT116 and LoVo cells compared with the control group. The intracellular reactive oxygen species (ROS) measurement results also indicated that compared with the control group, lycorine significantly induced ROS accumulation, and increased phosphorylated‑p38 expression levels and AKT phosphorylation. Collectively, the present study suggested that lycorine might induce cell cycle arrest and exert cytostatic effects potentially via activating ROS/p38 and AKT signaling pathways in CRC cells.

Bladder cancer (BC) is one of the most common malignant tumors in the urinary system. Due to the poor prognosis and high mortality rate of the disease, it is urgent to develop new drugs with high efficacy and low toxicity to treat BC. Echinatin (Ecn) is a bioactive natural flavonoid oflicorice that has attracted special attention for its promising anti-tumor potential. Herein, we explored the inhibitory effects of Echinatin on BC cells and probed the possible molecular mechanism. We found that Ecnin vitro inhibited the proliferation, migration, and invasion, arrested the cell cycle at the G2/M phase, and promoted apoptosis in BC cells. Besides, Ecn had no notable cytotoxicity towards human normal cells. We subsequently confirmed that Ecn restrained xenograft tumor growth and metastasis of BC cells in vivo. Mechanistically, Ecn activated the p38 signaling pathway but inactivated the Wnt/β-catenin signaling pathway, while over-expression of β-catenin and the p38 inhibitor both attenuated the inhibitory effects of Ecn on BC cells. Remarkably, Ecn combined with cisplatin (DDP) or gemcitabine (Gem) had synergistic inhibitory effects on BC cells. In summary, our results validate that Ecn inhibits the tumor growth of human BC cells via p38 and Wnt/β-catenin signaling pathways. More meaningfully, our results suggest a potential strategy to enhance DDP- or Gem-induced inhibitory effects on BC cells by combining with Ecn.

Bone morphogenetic protein 9 (BMP9), also named as growth differentiation factor 2 (GDF-2), is the strongest cytokine that promotes osteogenic differentiation in the BMP family, and has broad clinical application value. Nevertheless, the mechanism of BMP9 promotes osteogenic differentiation remain unclear. TAZ, a transcriptional co-activator, has great effects on cell proliferation, differentiation, and stem cell self-renewal. In this research, we investigated the effects of TAZ in BMP9-induced osteogenic differentiation of mesenchymal stem cell line C3H10T1/2 (MSCs) and murine multi-lineage cell lines C2C12 and MEFs (MMCs) and explored its possible mechanisms. This study has found that BMP9 induces the expression of TAZ and promotes its nuclear translocation. Meanwhile, our study found that Ad-TAZ and TM-25659, a TAZ agonist, can enhance the osteogenic differentiation of MSCs and MMCs induced by BMP9. Conversely, Ad-si-TAZ and verteporfin, an inhibitor of TAZ, have the contradictory effect. Likewise, the promotion of TAZ to the BMP9-induced ectopic bone formation in vivo was confirmed by the subcutaneous transplantation of MSCs in nude mice. Furthermore, we have detected that TAZ might increase the levels of the phosphorylation of Smad1/5/8, p38, ERK1/2, and JNK induced by BMP9. Additionally, we also found that TAZ increased the total protein level of β-catenin induced by BMP9. In summary, our results strongly indicated that TAZ will promote the osteogenic differentiation in MSCs and MMCs induced by BMP9 through multiple signal pathways.

Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications.