Bone morphogenetic protein-9 downregulates StAR expression by inducing snail expression via SMAD1/5/8 signaling in human granulosa-lutein cells.

Ovarian steroidogenesis mediated by granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis. Bone morphogenetic protein-9 (BMP-9), also known as growth differentiation factor-2 (GDF-2), is a member of the transforming growth factor-beta (TGF-ß) superfamily. BMP-9 induces epithelial-mesenchymal transition (EMT) that contributes to cancer progression. However, the function of BMP-9 in the female reproductive system remains largely unknown. It has been recently shown that BMP-9 is expressed in human follicular fluid and can downregulate StAR expression in human ovarian granulosa cells. However, the underlying molecular mechanisms warrant investigation. Our results show that treatment of primary granulosa-lutein (hGL) cells with BMP-9 downregulates StAR expression. In addition, two EMT-related transcription factors, Snail and Slug, are upregulated by the treatment of BMP-9. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we show that BMP-9 upregulates Snail and Slug expression by activating SMAD1/5/8 signaling. We also examine the effects of BMP-9 on SMAD-independent signaling pathways, including ERK1/2, p38, JNK, AKT, and CREB. However, none of them is affected by the BMP-9. Moreover, we use gain- and loss-of-function approaches to reveal that only Snail, not Slug, is required for the BMP-9-induced downregulation of StAR expression in hGL cells. This study increases the understanding of the physiology function of BMP-9 in hGL cells and provides important insights into the regulation of StAR expression.

BMP9 functions as a negative regulator in the myogenic differentiation of primary mouse myoblasts.

BMP9, a member of the TGF-ß superfamily, reveals the great translational promise for it has been shown to have the strong effect of osteogenic activity in vitro and in vivo. However, the implantation of certain BMPs (bone morphogenetic proteins) into muscular tissues induces ectopic bone formation. BMPs induce osteoblastic differentiation in skeletal muscle, suggesting that myogenic stem cells, such as myoblasts, are the potential progenitors of osteoblasts during heterotopic bone differentiation. Here, we investigate the role of BMP9 during primary mouse myoblasts differentiation. We found BMP9 enhanced cell proliferation and reduced myogenic differentiation of primary mouse myoblasts. In addition, adenovirus-mediated overexpression of BMP9 delayed muscle regeneration after BaCl2-induced injury. ALK1 knockdown reversed the inhibition of myoblast differentiation induced by BMP9. Our data indicate that BMP9 inhibits myogenic differentiation in primary mouse myoblasts and delays skeletal muscle regeneration after injury.

BMP9 maintains the phenotype of HTR-8/Svneo trophoblast cells by activating the SDF1/CXCR4 pathway.

BACKGROUND: Bone morphogenetic protein 9 (BMP9) has been shown to regulate processes such as angiogenesis, endothelial dysfunction, and tumorigenesis. However, the role of BMP9 in preeclampsia (PE) is unclear. The purpose of this study was to investigate the role and mechanism of BMP9 in PE. METHODS: The effects of BMP9 on the viability, migration and invasion of HTR-8/Svneo cells were investigated by CCK-8 assay, wound healing assay and Transwell invasion assay. The effect of BMP9 on apoptosis of HTR-8/Svneo cells was detected by flow cytometry. Plasma levels of BMP9, SDF1 and CXCR4 were detected by ELISA kit. qRT-PCR and Western blot were used to detect the expression levels of each gene in the cells. RESULTS: Overexpression of BMP9 promoted the proliferation and migration of trophoblast cells and inhibited apoptosis. Knockdown of BMP9 had the opposite effect. The levels of BMP9, SDF1 and CXCR4 in the plasma of PE patients were down-regulated, and BMP9 was positively correlated with the levels of SDF1 and CXCR4. BMP9 also significantly upregulated the mRNA and protein levels of SDF1 and CXCR4 in HTR-8/SVneo cells. Further mechanistic studies found that BMP9 promoted the migration and invasion of HTR-8/SVneo cells and inhibited apoptosis by activating the SDF1/CXCR4 pathway. CONCLUSION: We demonstrate for the first time that BMP9 promoted the migration and invasion of HTR-8/SVneo cells and inhibits apoptosis by activating the SDF1/CXCR4 pathway. This suggests that BMP9 may be a biomarker molecule for PE.

The effects of Gli1 and Gli2 on BMP9-induced osteogenic differentiation of mesenchymal stem cells.

Diseases, such as bone nonunion with bone defects, osteoporosis, etc, seriously endanger people's quality of life, and bone tissue engineering based on mesenchymal stem cells is an effective method to solve such problems. Several studies have shown that BMP9 can effectively promote osteogenic differentiation of MSCs, but the underlying molecular mechanisms are still unclear. Gli1 and Gli2 were important transcription factors and play an important role in the Hedgehog signaling pathway. In this study, we investigated the role of Gli1 and Gli2 in BMP9-induced osteogenic differentiation of MSCs. We found that inhibition of Gli1 and Gli2 weakened BMP9-induced osteogenic differentiation of MSCs, and early osteogenic markers (alkaline phosphatase, ALP), late osteogenic markers (calcium salt deposition), the expression of pivotal osteogenic markers were attenuated, and inhibition of Gli1 and Gli2 weakened the expression of p-Smad1/5/8 and p-p38 induced by BMP9. In conclusion, our study shows that Gli1 and Gli2 play an important role in BMP9-induced osteogenic differentiation.

Bone Morphogenetic Protein-9 Promotes Osteogenic Differentiation and Mineralization in Human Stem-Cell-Derived Spheroids.

Background and Objectives: Alkaline phosphatase activity, mineralized matrix, and osteogenic-related gene expression have been shown to increase in response to bone morphogenetic protein-9 (BMP-9). In this study, spheroids derived from human gingival stem cells were used to determine the effects of BMP-9 on cell survival, osteogenesis, and mineralization. Materials and Methods: Human gingival stem cells were used to produce spheroids and then grown to concentrations of 0, 0.1, 1, 10, and 100 ng/mL with BMP-9. On days 1, 3, 5, and 7, morphological examination was carried out. A live/dead assay and Cell Counting Kit-8 was used to assess the vitality of cells. On days 7 and 14, alkaline phosphatase activity assays were carried out using a commercially available kit to examine the osteogenic differentiation of cell spheroids. Alizarin Red Staining was performed on the 7th and 14th days to evaluate mineralization, and RUNX2 and COL1A1 expression levels were evaluated on the 7th and 14th days using real-time polymerase chain reactions. Results: The BMP-9 added at the measured quantities did not appear to alter the shape of the well-formed spheroids produced by stem cells on day 1. In addition, treatment with BMP-9 at doses of 0, 0.1, 1, 10, or 100 ng/mL did not significantly alter cell diameter. Throughout the whole experimental process, viability was maintained. On day 14, the alkaline phosphatase activity in the groups dosed with 0.1, 1, 10, or 100 ng/mL was statistically higher than that in the unloaded control group (p < 0.05). According to qPCR data, the mRNA expression level of RUNX2 with 1 ng/mL dosing was higher on day 7 compared to that of the unloaded control group (p < 0.05). Conclusions: These findings suggest that BMP-9 can be employed to stimulate early osteogenic differentiation in stem cell spheroids.

Bone morphogenetic protein (BMP)9 in cancer development: mechanistic, diagnostic, and therapeutic approaches?

Bone morphogenetic protein (BMP)-9 is considered a member of the transforming growth factor (TGF)ß superfamily. It was first found as an inducer of bone and cartilage formation and then discovered that this factor mediates several physiologic functions and hemostasis. Besides physiological conditions, BMP9 has also been elucidated that it is involved in several pathological situations, especially cancer. In various cancers, dysregulation of BMP9 has raised the issue that BMP9 might play a conflicting role in tumour development. BMP9 binding to its receptors (BMPRs), including ALKs and BMPRII, induces canonical SMAD-dependent and non-canonical PI3K/AKT and MAPK signalling pathways in tumour cells. BMP9, via inducing apoptosis, inhibiting tumour-promoting cell signalling pathways, suppressing epithelial-mesenchymal transition (EMT) process, blocking angiogenesis, and preventing cross-talk in the tumour microenvironment, mainly exerts tumour-suppressive functions. In contrast, BMP9 triggers tumour-supportive signalling pathways, promotes EMT, and enhances angiogenesis, suggesting that BMP9 is also involved in tumour development. It has been demonstrated that modulating BMP9 expression and functions might be a promising approach to cancer treatment. It has also been indicated that evaluating BMP9 expression in cancers might be a biomarker for predicting cancer prognosis. Overall, BMP9 would provide a promising target in cancer management.

Activin A Limits VEGF-Induced Permeability via VE-PTP.

The clinical success of neutralizing vascular endothelial growth factor (VEGF) has unequivocally identified VEGF as a driver of retinal edema that underlies a variety of blinding conditions. VEGF is not the only input that is received and integrated by the endothelium. For instance, the permeability of blood vessels is also regulated by the large and ubiquitously expressed transforming growth factor beta (TGF-ß) family. In this project, we tested the hypothesis that members of the TGF-ß family influence the VEGF-mediated control of the endothelial cell barrier. To this end, we compared the effect of bone morphogenetic protein-9 (BMP-9), TGF-ß1, and activin A on the VEGF-driven permeability of primary human retinal endothelial cells. While BMP-9 and TGF-ß1 had no effect on VEGF-induced permeability, activin A limited the extent to which VEGF relaxed the barrier. This activin A effect was associated with the reduced activation of VEGFR2 and its downstream effectors and an increased expression of vascular endothelial tyrosine phosphatase (VE-PTP). Attenuating the expression or activity of VE-PTP overcame the effect of activin A. Taken together, these observations indicate that the TGF-ß superfamily governed VEGF-mediated responsiveness in a ligand-specific manner. Furthermore, activin A suppressed the responsiveness of cells to VEGF, and the underlying mechanism involved the VE-PTP-mediated dephosphorylation of VEGFR2.

The effect of BMP9 on inflammation in the early stage of pulpitis.

BACKGROUND: Bone morphogenetic protein 9 (BMP9) tends to be associated with various inflammatory responses of diseases, but its relationship with pulpitis remains unknown. OBJECTIVE: This study aimed to evaluate the effects and mechanisms of BMP9 in pulpitis. METHODOLOGY: A rat model of pulpitis was used to evaluate the expression of BMP9, which was also analysed in Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-stimulated human dental pulp cells (hDPCs). The effects and mechanism of BMP9 on the regulation of inflammatory factors and matrix metalloproteinase-2 (MMP2) were evaluated using real-time quantitative PCR, western blotting, and immunocytofluorescence. Moreover, the migration ability of THP-1 monocyte-macrophages, treated with inflammatory supernate inhibited by BMP9, was previously tested by a transwell migration assay. Finally, a direct rat pulp capping model was used to evaluate in vivo the influence of the overexpression of BMP9 in pulpitis. RESULTS: The expression of BMP9 decreased after 24 h and increased after 3 and 7 d in rat pulpitis and inflammatory hDPCs. The overexpression of BMP9 inhibited the gene expression of inflammatory factors (IL-6, IL-8, and CCL2) and the secretion of IL-6 and MMP2 in Pg-LPS-stimulated hDPCs. The level of phosphorylated Smad1/5 was upregulated and the levels of phosphorylated ERK and JNK were downregulated. The inflammatory supernate of hDPCs inhibited by BMP9 reduced the migration of THP-1 cells. In rat pulp capping models, overexpressed BMP9 could partially restrain the development of dental pulp inflammation. CONCLUSION: This is the first study to confirm that BMP9 is involved in the occurrence and development of pulpitis and can partially inhibit its severity in the early stage. These findings provided a theoretical reference for future studies on the mechanism of pulpitis and application of bioactive molecules in vital pulp therapy.

The SIRT1 activator SRT2104 promotes BMP9-induced osteogenic and angiogenic differentiation in mesenchymal stem cells.

Bone defects resulting from trauma, bone tumors, infections and skeletal abnormalities are a common osteoporotic condition with respect to clinical treatment. Of the known bone morphogenetic proteins (BMPs), BMP9 has the strongest osteogenic differentiation potential, which could be beneficial in the construction of tissue-engineered bone. Silent mating type information regulator 2 homolog-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide-dependent deacetylase that deacetylates and modulates histone or non-histone substrates. However, the role of SIRT1 in BMP9-induced osteogenic differentiation of stem cells has not been studied. Furthermore, it is unclear whether SIRT1 interacts with the BMP/Smad and BMP/MAPK pathways in stem cells. We found that SIRT1 expression decreased gradually in a time-dependent manner during BMP9-induced osteogenic differentiation of MSCs. Interactions between SIRT1 and Smad7 promoted degradation of Smad7 and increased Smad1/5/8 phosphorylation. SRT2104, an activator of SIRT, enhanced the expression of osteogenic- and angiogenic-related proteins in BMP9-induced MSCs. In addition, we found that activation of the BMP/MAPK pathway led to osteogenic and angiogenic differentiation of MSCs. Our study demonstrated that SIRT1 expression decreased during BMP9-induced differentiation. The SIRT1 activator SRT2104 promoted BMP9-induced osteogenic and angiogenic differentiation of MSCs through the BMP/Smad and BMP/MAPK signaling pathways.

Bone morphogenic protein 9 is a novel thermogenic hepatokine secreted in response to cold exposure.

OBJECTIVE: Maintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. METHODS: To investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. RESULTS: Hepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. CONCLUSIONS: BMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.

NELL1 augments osteogenesis and inhibits inflammation of human periodontal ligament stem cells induced by BMP9.

BACKGROUND: Periodontitis could lead to periodontal destruction such as the loss of alveolar bone. The issue that how to achieve the regeneration of alveolar bone and periodontal tissues under the inflammatory environment needs to be solved urgently. Bone morphogenetic protein 9 (BMP9) is one of the most potent osteoinductive BMPs and induces osteogenic differentiation of mesenchymal stem cells. The aim of this study is to explore the possible effect of BMP9 on the osteogenic differentiation of inflammatory periodontal ligament stem cells (PDLSCs). METHODS: Human PDLSCs were cultured in osteoinductive medium with 1 µg/mL lipopolysaccharide Porphyromonas gingivitis (LPS-PG). Adenoviral vector expressing system was used to overexpress target genes. In vitro expression of osteogenic markers was assessed by quantitative reverse transcription polymerase chain reaction, Western blotting, alkaline phosphatase assay, and alizarin red staining. Subcutaneous implantation nude mice models were used to evaluate the effects of BMP9 on PDLSCs in vivo. Microcomputed tomography, hematoxylin & eosin staining, and trichrome staining were performed to assess ectopic bone formation. RESULTS: In the LPS-PG induced inflammatory environment, BMP9 promoted osteogenic differentiation of PDLSCs, but upregulated the expression of inflammatory markers (P > 0.05); NEL-like protein 1 (NELL1) downregulated the expression of inflammation genes in PDLSCs induced by BMP9, while augmenting BMP9-induced osteogenesis of the cells both in vitro and in vivo. In the above process, the MAPK/p38/ERK signaling pathway was triggered by NELL1. CONCLUSION: The combination use of BMP9 and NELL1 might have the potential to promote the regeneration of alveolar bone in periodontitis.

Osteoinductive potential of recombinant BMP-9 in bone defects of mice treated with antiresorptive agents.

The aim of the present study was to investigate the effects of recombinant human (rh)BMP-9 on bone regenerative potential in a mouse model of antibody-mediated antiresorptive therapy (AMART). A monoclonal anti-murine receptor activator of nuclear factor-kappa B ligand (RANKL) antibody (mAb) was used to create an AMART model in mice. rhBMP-9 combined with collagen membrane was implanted in calvarial defects in mAb-treated mice. After 4 weeks, the bone formative potential in the defects was evaluated by micro-computed tomography and histological approaches. The groups implanted with rhBMP-9-containing collagen membranes demonstrated substantial osteopromotive potential, with significantly greater new bone volume (Sham + BMP-9 group; 0.86 ± 0.29 mm3 and mAb + BMP-9 group; 0.64 ± 0.16 mm3) than control PBS-membranes (Sham + PBS group; 0.44 ± 0.29 mm3 and mAb + PBS group; 0.24 ± 0.12 mm3) in both sham and mAb-treated mice. In line with in vivo study, bone marrow cells isolated from both sham and mAb-treated mice confirmed greater osteogenic potential upon stimulation with rhBMP-9 in vitro. These findings suggest for the first time that local rhBMP-9 administration might be a strategy to accelerate bone regeneration in the context of AMART.

Bone morphogenetic protein 9 enhances osteogenic and angiogenic responses of human amniotic mesenchymal stem cells cocultured with umbilical vein endothelial cells through the PI3K/AKT/m-TOR signaling pathway.

BACKGROUND: Neovascularization plays an essential part in bone fracture and defect healing, constructing tissue engineered bone that targets bone regeneration. Bone morphogenetic protein 9 (BMP9) is a regular indicator that potentiates osteogenic and angiogenic differentiation of MSCs. OBJECTIVES: To investigate the effects of BMP9 on osteogenesis and angiogenesis of human amniotic mesenchymal stem cells (hAMSCs) cocultured with human umbilical vein endothelial cells (HUVECs) and determine the possible underlying molecular mechanism. RESULTS: The isolated hAMSCs expressed surface markers of MSCs. hAMSCs cocultured with HUVECs enhance osteogenic differentiation and upregulate the expression of angiogenic factors. BMP9 not only potentiates angiogenic signaling of hAMSCs cocultured with HUVECs also increases ectopic bone formation and subcutaneous vessel invasion. Mechanically, the coupling effect between osteogenesis and angiogenesis induced by BMP9 was activated by the BMP/Smad and PI3K/AKT/m-TOR signaling pathways. CONCLUSIONS: BMP9-enhanced osteoblastic and angiogenic differentiation in cocultivation with hAMSCs and HUVECs in vitro and in vivo also provide a chance to harness the BMP9-regulated coordinated effect between osteogenic and angiogenic pathways through BMP/Smad and PI3K/AKT/m-TOR signalings. MATERIALS AND METHODS: The ALP and Alizarin Red S staining assay to determine the effects of osteoblastic differentiation. RT-qPCR and western blot was measured the expression of angiogenesis-related factors. Ectopic bone formation was established and retrieved bony masses were subjected to histochemical staining. The angiogenesis ability and vessel invasion were subsequently determined by immunofluorescence staining. Molecular mechanisms such as the BMP/Smad and PI3K/AKT/m-TOR signaling pathways were detected by ELISA and western blot analysis.

Evaluation of the optimal dosage of BMP-9 through the comparison of bone regeneration induced by BMP-9 versus BMP-2 using an injectable microparticle embedded thermosensitive polymeric carrier in a rat cranial defect model.

Bone morphogenetic proteins (BMPs) are well known as enhancers and facilitators of osteogenesis during bone regeneration. The use of recombinant BMP-2 (rhBMP-2) in bone defect healing has drawbacks, which has driven the scouting for alternatives, such as recombinant BMP-9 (rhBMP-9), to provide comparable new bone formation. However, the dosage of rhBMP-9 is quintessential for the facilitation of adequate bone defect healing. Therefore, this study has been designed to evaluate the optimal dosage of BMP-9 by comparing the bone defect healing induced by rhBMP-9 over rhBMP-2. The chitosan (CS) microparticles (MPs), coated with BMPs, were embedded in a thermoresponsive methylcellulose (MC) and calcium alginate (Alg) based injectable delivery system containing a dosage of either 0.5 µg or 1.5 µg of the respective rhBMP per bone defect. A 5 mm critical-sized cranial defect rat model has been used in this study, and bone tissues were harvested at eight weeks post-surgery. The standard tools for comparing the new bone regeneration included micro computerized tomography (micro-CT) and histological analysis. A novel perspective of analyzing the new bone quality and crystallinity was employed by using Raman spectroscopy, along with its elastic modulus quantified through Atomic Force Microscopy (AFM). Results showed that the rhBMP-9 administered at a dosage of 1.5 µg per bone defect, using this delivery system, can adequately facilitate the bone void filling with ample new bone mineralization and crystallinity as compared to rhBMP-2, thus approving the hypothesis for a viable rhBMP-2 alternative.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

Long non-coding RNA (LncRNA) HOTAIR regulates BMP9-induced osteogenic differentiation by targeting the proliferation of mesenchymal stem cells (MSCs).

Long non-coding RNAs are important regulators of biological processes, but their roles in the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. Here we investigated the role of murine HOX transcript antisense RNA (mHotair) in BMP9-induced osteogenic differentiation of MSCs using immortalized mouse adipose-derived cells (iMADs). Touchdown quantitative polymerase chain reaction analysis found increased mHotair expression in bones in comparison with most other tissues. Moreover, the level of mHotair in femurs peaked at the age of week-4, a period of fast skeleton development. BMP9 could induce earlier peak expression of mHotair during in vitro iMAD osteogenesis. Silencing mHotair diminished BMP9-induced ALP activity, matrix mineralization, and expression of osteogenic, chondrogenic and adipogenic markers. Cell implantation experiments further confirmed that knockdown of mHotair attenuated BMP9-induced ectopic bone formation and mineralization of iMADs, leading to more undifferentiated cells. Crystal violet staining and cell cycle analysis revealed that silencing of mHotair promoted the proliferation of iMAD cells regardless of BMP9 induction. Moreover, ectopic bone masses developed from mHotair-knockdown iMAD cells exhibited higher expression of PCNA than the control group. Taken together, our results demonstrated that murine mHotair is an important regulator of BMP9-induced MSC osteogenesis by targeting cell cycle and proliferation.

A Review of Recent Developments in the Molecular Mechanisms of Bone Healing.

Between 5 and 10 percent of fractures do not heal, a condition known as nonunion. In clinical practice, stable fracture fixation associated with autologous iliac crest bone graft placement is the gold standard for treatment. However, some recalcitrant nonunions do not resolve satisfactorily with this technique. For these cases, biological alternatives are sought based on the molecular mechanisms of bone healing, whose most recent findings are reviewed in this article. The pro-osteogenic efficacy of morin (a pale yellow crystalline flavonoid pigment found in old fustic and osage orange trees) has recently been reported, and the combined use of bone morphogenetic protein-9 (BMP9) and leptin might improve fracture healing. Inhibition with methyl-piperidino-pyrazole of estrogen receptor alpha signaling delays bone regeneration. Smoking causes a chondrogenic disorder, aberrant activity of the skeleton's stem and progenitor cells, and an intense initial inflammatory response. Smoking cessation 4 weeks before surgery is therefore highly recommended. The delay in fracture consolidation in diabetic animals is related to BMP6 deficiency (35 kDa). The combination of bioceramics and expanded autologous human mesenchymal stem cells from bone marrow is a new and encouraging alternative for treating recalcitrant nonunions.

Endothelial BMPR2 Loss Drives a Proliferative Response to BMP (Bone Morphogenetic Protein) 9 via Prolonged Canonical Signaling.

OBJECTIVE: Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in BMPR2-the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact of BMPR2 loss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients with BMPR2 mutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediated BMPR2 silencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditional Bmpr2 knockout mice (Bmpr2EC-/-). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFß (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP target ID1 in the context of BMPR2 loss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice (Bmpr2EC+/+) but had no measurable effect on mice bearing a heterozygous endothelial Bmpr2 deletion (Bmpr2EC+/-) and caused excessive angiogenesis in both vascular beds for Bmpr2EC-/- mice. CONCLUSIONS: BMPR2 loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials.

SGK1 is a signalling hub that controls protein synthesis and proliferation in endothelial cells.

BMP9 is a cytokine involved in the maturation phase of the angiogenic process that signals through its serine/threonine receptor ALK1 and its coreceptor endoglin. In this paper, we explain how BMP9 directs the regulation of endothelial cell proliferation blockage while in turn stimulating protein synthesis. To achieve this, BMP9 promotes SGK1 synthesis and activation through mTORC2 in order to stimulate the mTORC1/S6K/S6 axis. Moreover, BMP9 blocks proliferation also through SGK1 by reducing the activity of the MEK/ERK signalling pathway. Inhibition of SGK1 activity is sufficient to prevent BMP9-mediated inhibition of ERK, leading to an increase in endothelial cell proliferation. Overall, our findings reveal that SGK1 is a key player during angiogenesis, mediating the pro-quiescent and maturation effects of BMP9/ALK1.

The p53/miR-145a Axis Promotes Cellular Senescence and Inhibits Osteogenic Differentiation by Targeting Cbfb in Mesenchymal Stem Cells.

The osteogenic differentiation capacity of senescent bone marrow mesenchymal stem cells (MSCs) is reduced. p53 not only regulates cellular senescence but also functions as a negative regulator in bone formation. However, the role of p53 in MSCs senescence and differentiation has not been extensively explored. In the present study, we investigated the molecular mechanism of p53 in MSCs senescence and osteogenic differentiation. We found that p53 was upregulated during cellular senescence and osteogenic differentiation of MSCs respectively induced by H2O2 and BMP9. Similarly, the expression of p53-induced miR-145a was increased significantly. Furthermore, Overexpression of miR-145a in MSCs promoted cellular senescence and inhibited osteogenic differentiation. Then, we identified that p53-induced miR-145a inhibited osteogenic differentiation by targeting core binding factor beta (Cbfb), and the restoration of Cbfb expression rescued the inhibitory effects of miRNA-145a. In summary, our results indicate that p53/miR-145a axis exert its functions both in promoting senescence and inhibiting osteogenesis of MSCs, and the novel p53/miR-145a/Cbfb axis in osteogenic differentiation of MSCs may represent new targets in the treatment of osteoporosis.