Crosstalk between Wnt and bone morphogenetic protein signaling during osteogenic differentiati

Mesenchymal stem cells(MSCs)originate from many sources,including the bone marrow and adipose tissue,and differentiate into various cell types,such as osteoblasts and adipocytes.Recent studies on MSCs have revealed that many transcription factors and signaling pathways control osteogenic development.Osteogenesis is the process by which new bones are formed;it also aids in bone remodeling.Wnt/β-catenin and bone morphogenetic protein(BMP)signaling pathways are involved in many cellular processes and considered to be essential for life.Wnt/β-catenin and BMPs are important for bone formation in mammalian development and various regulatory activities in the body.Recent studies have indicated that these two signaling pathways contribute to osteogenic differen-tiation.Active Wnt signaling pathway promotes osteogenesis by activating the downstream targets of the BMP signaling pathway.Here,we briefly review the molecular processes underlying the crosstalk between these two pathways and explain their participation in osteogenic differentiation,emphasizing the canonical pathways.This review also discusses the crosstalk mechanisms of Wnt/BMP signaling with Notch-and extracellular-regulated kinases in osteogenic differentiation and bone development.

Influence of bone morphogenetic protein type IA receptor conditional knockout in lens on expression of bone morphogenetic protein 4 in lens

AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the vertebrate eye.METHODS: Cre-positive mice were mated with Crenegative mice to generate 50% Cre-positive(conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring(wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm.Removal of paraffin wax and dehydrating for sections,and then the procedure of in situ hybridization was processed, BMP4 MK1784-m(BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P【0.05 showed that the difference was statistically significant.· RESULTS: Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of BMP4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P 【0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5.CONCLUSION: ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.

The biological function of type I receptors of bone morphogenetic protein in bone

Bone morphogenetic proteins （BMPs） have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors （BMPRI and BMPRII）. In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 （ALK2, also called type IA activin receptor）, activin- llke kinase 3 （ALK3, also called BMPRIA）, and activin-like kinase 6 （ALK6, also called BMPRIB）. The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future.

Expression of Vascular Endothelial Growth Factor in Bone Morphogenetic Protein-2 Induced Osteogenesis

An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in bone morphogenetic protein- 2 induced osteogenesis . The experimental results demonstrated that the expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenehymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenehymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14 th day, accompanied by numerons hypertrophic chondrocytes. When mature cartilage calcified and neu, bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. Signals of VEGF mRNA and VEGF can not be detected in the control groups.

Bone morphogenetic proteins:Relationship between molecular structure and their osteogenic activity

Bone morphogenetic proteins(BMPs)are a family of potent,multifunctional growth factors belonging to transforming growth factor-(TGF-).They are highly conservative in structures.Over 20 members of BMPs with varying functions such as embryogenesis,skeletal formation,hematopoiesis and neurogenesis have been identified in human body.BMPs are unique growth factors that can induce the formation of bone tissue individually.BMPs can induce the differentiation of bone marrow mesenchymal stem cells into osteoblastic lineage and promote the proliferation of osteoblasts and chondrocytes.BMPs stimulate the target cells by specific membrane-bound receptors and signal transduced through mothers against decapentaplegic(Smads)and mitogen activated protein kinase(MAPK)pathways.It has been demonstrated that BMP-2,BMP-4,BMP-6,BMP-7,and BMP-9 play an important role in bone formation.This article focuses on the molecular characterization of BMPs family members,mechanism of osteogenesis promotion,related signal pathways of osteogenic function,relationships between structure and osteogenetic activity,and the interactions among family members at bone formation.

Prolonged propagation of rat neural stem cells relies on inhibiting autocrine/paracrine bone morphogenetic protein and platelet derived growth factor signals

Continuous expansion of rat neural stem cell lines has not been achieved due to proliferation arrest and spontaneous differentiation in vitro. In the current study, neural precursor cells derived from the subventricular zone of adult rats spontaneously underwent astroglial and oligodendroglial differentiation after limited propagation. This differentiation was largely induced by autocrine or paracrine bone morphogenetic protein and platelet derived growth factor signals. The results showed that, by inhibiting bone morphogenetic protein and platelet derived growth factor signals, adult rat neural precursor cells could be extensively cultured in vitro as tripotent stem cell lines. In addition to adult rat neural stem cells, we found that bone morphogenetic protein antagonists can promote the proliferation of human neural stem cells. Therefore, the present findings illustrated the role of autocrine or paracrine bone morphogenetic protein and platelet derived growth factor signaling in determining neural stem cell self-renewal and differentiation. By antagonizing both signals, the long-term propagation of rat neural stem cell lines can be achieved.

Bone morphogenetic protein 7 mediates stem cells migration and angiogenesis:therapeutic potential for endogenous pulp regeneration

Pulp loss is accompanied by the functional impairment of defense,sensory,and nutrition supply.The approach based on endogenous stem cells is a potential strategy for pulp regeneration.However,endogenous stem cell sources,exogenous regenerative signals,and neovascularization are major difficulties for pulp regeneration based on endogenous stem cells.Therefore,the purpose of our research is to seek an effective cytokines delivery strategy and bioactive materials to reestablish an ideal regenerative microenvironment for pulp regeneration.In in vitro study,we investigated the effects of Wnt3a,transforming growth factor-beta 1,and bone morphogenetic protein 7(BMP7)on human dental pulp stem cells(h-DPSCs)and human umbilical vein endothelial cells.2D and 3D culture systems based on collagen gel,matrigel,and gelatin methacryloyl were fabricated to evaluate the morphology and viability of h-DPSCs.In in vivo study,an ectopic nude mouse model and an in situ beagle dog model were established to investigate the possibility of pulp regeneration by implanting collagen gel loading BMP7.We concluded that BMP7promoted the migration and odontogenic differentiation of h-DPSCs and vessel formation.Collagen gel maintained the cell adhesion,cell spreading,and cell viability of h-DPSCs in 2D or 3D culture.The transplantation of collagen gel loading BMP7 induced vascularized pulp-like tissue regeneration in vivo.The injectable approach based on collagen gel loading BMP7 might exert promising therapeutic application in endogenous pulp regeneration.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

Bone morphogenetic protein 4 induces cholinergic differentiation of brain-derived neural stem cells in vivo and in vitro

In vitro studies have demonstrated that many factors of bone morphogenetic proteins （BMPs） induce cholinergic differentiation of neural stem cells. However, BMP retains the potential to induce increased numbers of cholinergic neurons in central nervous system regions that are rich in cholinergic cells, which is an important determinant of BMP. Therefore, BMP-4 was added to neural stem cell culture medium or the adult rat hippocampal dentate gyrus. Results demonstrated that BMP-4 induced cholinergic differentiation of neural stem cells in vitro and increased the number of cholinergic neurons in the adult rat hippocampal dentate gyrus.

Expression of bone morphogenetic protein 7 in the cerebral cortex of rats after ischemic-hypoxic injury

BACKGROUND： Some researches demonstrate that exogenous bone morphogenetic protein 7 （BMP-7） can protect ischemic cerebral nerve tissue and promote recovery of motor energy function; however, there is lack of direct evidences of endogenous BMP-7 effect. OBJECTIVE： To observe the expression of endogenous BMP-7 in nerve tissue with ischemic-hypoxic injury and investigate the possible effects on damaged nerve tissue. DESIGN： Observational contrast animal study. SETTING： Department of Anatomy and Histoembryology, Peking University Health Science Center. MATERIALS： The experiment was carried out in the Nerve Researching Laboratory of Anatomy Department, Peking University Health Science Center from October 2006 to March 2007. A total of 25 adult male SD rats weighing 250 - 300 g and several newborn SD rats were selected from Experimental Animal Center, Peking University Health Science Center. Rabbit-anti-BMP-7 polyclonal antibody was provided by Wuhan Boster Company. METHODS： ① Adult rats were randomly divided into ischemia group （n =10）, sham operation group （n = 10） and normal group （n =5）. Right external-internal carotid artery occlusion was used to infarct middle cerebral artery of adult rats in the ischemia group so as to copy focal cerebral infarction models. Line cork was inserted in crotch of internal and external carotid artery of adult rats in the sham operation group, while adult rats in the normal group were not given any treatments. ② Cerebral cortex of newborn rats was separated to obtain cell suspension. Cells which were cultured for 10 days were divided into control group and hypoxia/reoxygenation group. And then, cells in the hypoxia/reoxygenation group were cultured in hypoxic incubator for 4 hours and given reoxygenation for 24 hours. MAIN OUTCOME MEASURES： Immunohistochemical method was used to measure expression of BMP-7 in cerebral cortex at 24 hours after ischemia/reperfusion culture and in primary hypoxic culture. RESULTS： ① At 24 hours after cerebral ischemia, expression of BMP-7 in cerebral cortex on ischemic side was stronger than that on non-ischemic side in adult rats; meanwhile, numbers of cell expression were increased. However, expression of BMP-7 was not detected in bilateral cerebral cortex of adult rats in both control group and sham operation group. ② After hypoxia of cerebral cortex in primary culture, positive products of BMP-7 were observed in plasma of neuron, but expression of BMP-7 was not found in normal cerebral cortex. CONCLUSION： Endogenous BMP-7 has protective effects on nerve tissue induced by ischemic-hypoxic injury.

Experimental Study of Porous Beta-tricalcium Phosphate and Bone Morphogenetic Protein/porous Beta-tricalcium Phosphate Complex in Rabbit Mandibular Reconstruction

We investigated the osteogenic characteristic and biodegradation behavior of porous beta-tricalcitum phosphate （ β- TCP ） and bone morphogenetic protein/beta-tricalcium phosphate （ BMP/ β- TCP ） complex in mandibular reconstruction and evaluated the advantages of BMP / β- TCP complex in repairing bone defects. Bone defects created in the lower margin of bilateral mandible bodies in 12 rabbits were repaired with β- TCP （ control group ） and BMP/ β- TCP complex （ experimental group ） respectively. The rabbits were euthanized after 2, 4, 8 and 12 weeks and examined by macroscopy, radiography, histology, histomorphometry and image analysis. 2 weeks after surgery, ossftcation of newly-generated tissue in BMP/ β- TCP complex group appeared and after 12 weeks, massive new bone and ossification maturation were seen. However, in β- TCP group without BMP , ossification was not found until 12 weeks after operation. Image analysis showed that bone regeneration rate of BMP/ β- TCP was 30%-40% higher and the degradation rate was 20%- 30% higher than that of β- TCP. Therefore, as a reconstructive material for bone defects, BMP/ β-TCP complex is superior to β-TCP and can be used in oral and maxillofacial surgery.

Bone morphogenetic protein signaling：a promising target for white matter protection in perinatal brain injury

Prematurely born newborns,as well as those born at term,may suffer from several types of brain injury including hypoxic-ischemic injury,intracranial hemorrhage,both intraventricular and parenchymal,and injury that is the consequence of intrauterine growth restriction（IUGR）.Injury of all types can impact the motor and cognitive abilities of survivors.The mechanisms leading to disability are not completely understood.

Establishment and identification of fibroblast clones expressing human bone morphogenetic protein 2

Objective:To establish fibroblasts stably expressing human bone morphogenetic protein 2(hBMP2). Methods:Eukaryonic expression vector(pcDNA3.1-B2) was transduced into NIH3T3 cells using SofastTM, a new generation cationic polymer gene transfection reagent. The positive cell clones were selected with G418. The stable transfection and expression of BMP2 in the NIH3T3 cells were determined by RT-PCR and immunohistochemical stain. Results: BMP2 mRNA was transcripted and expressed in the transfected NIH3T3 cells. Conclusion: With positive compound transfection, outside human BMP2 gene can be successfully transducted into NIH3T3 cells, which is the key step to induce periodontal cells to osseous phenotypes.

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole(Cynoglossus semilaevis)

BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp,which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR（qRT-PCR）. The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages（egg, larva, juvenile and fingerling stages）.The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch（dph）; then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to ＋109. The predicted transcription factor binding sites（E-box binding factors, zinc finger transcription factor, etc.） in this region might participate in the transcriptional regulation of the bmp2 gene.

AB004. Regulation of retinal angiogenesis and vascular permeability by bone morphogenetic protein signaling

The bone morphogenetic protein(BMP)family of proteins has a multitude of roles throughout the body.It plays important roles in development and in the adult vascular endothelium,by modulating the angiogenic response.The endothelial-specific receptor BMP receptor Alk1 is of particular importance in the proper remodeling of the vasculature and its ligand BMP9 has been shown to be a potent inhibitor of neovascularization.Dysregulated BMP signaling has been linked to multiple vascular diseases and can lead to the abnormal angiogenesis.We therefore investigated the role of BMP9/Alk1 signaling in retinal angiogenesis,and its therapeutic implications for vascular pathologies of the eye.

EFFECTS OF TRANSFORMING GROWTH FACTOR β AND RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 ON HUMAN PERIODONTAL LIGAMENT FIBROBLASTS

Objective To evaluate the effects of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP2) on human periodontal ligament fibroblasts (HPDLFs). Methods HPDLFs were done primary culture to detect the distinct concentrations of TGF-P and rhBMF2 on its proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) synthesis and formation of the minerali-zed nodules, respectively. Results TGF-β (5～100ng/ml) significantly stimulated the proliferation of HPDLFs. The ALP activity of HPDLFs was evaluated evidently by 5ng/ml TGF-β. TGF-β( 0. 5 ～ 100ng/ml) had no effects on OC synthesis and formation of the mineralized nodules of HPDLFs. rhBMP2 (0. 25～2mg/ ml) had no remarkable effect on the proliferation of HPDLFs. The ALP activity, OC synthesis and forma-tion of the mineralized nodules of HPDLFs were significantly stimulated by 0. 5～ 2mg /ml rhBMP2. Conclusion The effects of TGF-β and rhBMP2 on HPDLFs are dose-dependent. TGF-P can stimulate HPDLFs to express the early marker of osteoblastic phenotype, and it lacks the ability to promote maturation of the osteogenic phenotype. rhBMP2 can not only stimulate the expression but also promote the maturation of osteoblas-tic phenotype of HPDLFs.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Acupotomy ameliorates subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis by regulating bone morphogenetic protein 2-Smad1 pathway

OBJECTIVE:To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis(KOA).METHODS:The rabbits were divided into blank control,model,acupotomy and electroacupuncture(EA)groups,with 12 rabbits in each.Modified Videman's method was used to prepare KOA model.The acupotomy and EA group were given indicated intervention for 3 weeks.The behavior of rabbits in each group was recorded.Subsequently,cartilage–subchondral bone units were obtained and morphological changes were observed by optical microscope and micro computed tomography.Compression test was used to detect the mechanical properties of subchondral bone,Western blot and real-time polymerase chain reaction(RT-PCR)were applied to detect the expression of bone morphogenetic protein 2-Smad1(BMP2-Smad1)pathway in subchondral bone.RESULTS:Compared with the control group,rabbits in the KOA group showed lameness,knee pain,and cartilage degradation;the subchondral bone showed active resorption,the mechanical properties decreased significantly and the BMP2-Smad1 pathway downregulated significantly.Both acupotomy and EA intervention could increase the thickness of trabecular bone(Tb.Th),the bone volume fraction(BV/TV)and the thickness of subchondral bone plate,reduce the separation of trabecular bone(Tb.Sp),improve the maximum load and elastic modulus of subchondral bone,and effectively delay cartilage degeneration in KOA rabbits.This process may be achieved through upregulation the related proteins of BMP2-Smad1 pathway.The maximum load and elastic modulus of subchondral bone in the acupotomy group were slightly better than those in the EA group.CONCLUSIONS:Acupotomy could effectively protect cartilage by inhibiting abnormal bone resorption and improving mechanical properties of subchondral bone thorough the related proteins of BMP2-Smad1 pathway in KOA rabbits.