Bone Morphogenetic Protein-2 Gene Research Articles

Inhibitory role of REV-ERBα in the expression of bone morphogenetic protein gene family in rat uterus endometrium stromal cells.

Uterus circadian rhythms have been implicated in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes. Bone morphogenetic proteins (BMPs), having clock-controlled regulatory sites in their gene promoters, are expressed in the uterus during decidualization, but the regulation of the Bmp gene expression is poorly understood. The present study was designed to dissect the physiological roles of the uterus oscillators in the Bmp expression using the uterus endometrial stromal cells (UESCs) isolated from Per2-dLuc transgenic rats on day 4.5 of gestation. The in vitro decidualization of UESCs was induced by medroxyprogesterone acetate and 2-O-dibutyryl cAMP. A significant decline of Per2-dLuc bioluminescence activity was induced in decidual cells, and concomitantly, the expression of canonical clock genes was downregulated. Conversely, the expression of the core Bmp genes Bmp2, Bmp4, Bmp6, and Bmp7 was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Bmp genes, such as Bmp2, Bmp4, and Bmp6 were upregulated. However, Bmp1, Bmp7, and Bmp8a were not significantly affected by Bmal1 silencing. The expression of all Bmp genes was enhanced after treatment with the REV-ERBα antagonist (SR8278), although their rhythmic profiles were differed from each other. The binding of REV-ERBα to the proximal regions of the Bmp2 and Bmp4 promoters was revealed by chromatin immunoprecipitation-PCR analysis. Collectively, these results indicate that the Bmp genes are upregulated by the attenuation of the cellular circadian clock; in particular, its core component REV-ERBα functions as a transcriptional silencer in the Bmp gene family.

Bone morphogenetic protein 2 gene transduction enhances the osteogenic potential of human urine-derived stem cells.

IntroductionUrine-derived stem cells (USCs) have the ability to differentiate into osteogenic lineage. Previous studies have raised the possibility that USCs could be used for bone repair. To harness the power of USCs in promoting bone regeneration, methods must be developed to induce USCs to osteogenic lineage efficiently. The present study investigates the effect of lentivirus-encoded bone morphogenetic protein 2 (BMP2) gene transduction on the osteogenic potential of USCs.MethodsUSCs were isolated from voided urine and transduced with Lentiviral vector encoding BMP2. An in vitro study was performed to detect Lentiviral-BMP2 transduced USCs differentiated towards osteogenic lineage. Furthermore, Lentiviral-BMP2 transduced USCs were transplanted in vivo to examine the ectopic bone formation ability. After six weeks, retrieval samples were obtained for immunostaining and histological analysis.ResultsThe results showed that the transduction efficiencies were over 90%, and transduced USCs had high expression levels of the BMP2 gene and secreted BMP2 protein. Alkaline activity and mineral deposition staining demonstrated that transduced USCs differentiate into osteogenic lineages without the addition of osteogenic supplements. Transduced USCs also showed high expression of bone-related markers, including runt-related protein-2 (Runx2) and osteocalcin (OCN), confirming this lentiviral-BMP2 construct provides sufficient stimuli for osteogenic differentiation. Histological analysis indicated that the transduced USCs induced robust new bone formation in nude mice. Six weeks after transplantation, human derived cells were observed to participate in bone formation.ConclusionsThese results demonstrate that BMP2 gene transduction provides an effective method to enhance the osteogenic potential of USCs.

BMP7 Gene involved in nonsyndromic orofacial clefts in Western Han Chinese.

BackgroundNonsyndromic orofacial clefts (NSOCs) are the most common craniofacial birth defects with complex etiology in which multiple genes and environmental exposures are involved. Bone morphogenetic protein 7 (BMP7), as a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to play crucial roles in palate and other orofacial ectodermal appendages development in animal models.Material and MethodsThis study was designed to investigate the possible associations between BMP7 gene and the NSOCs (221 case-parent trios) in Western Han Chinese. Five tagSNPs at BMP7, rs12438, rs6099486, rs6127973, rs230188 and rs6025469 were picked and tried to cover the entire gene. In order to identify the contribution of BMP7 gene to the etiology of NSOCs, we performed several statistical analysis from different aspects including transmission disequilibrium test (TDT), pairwise linkage disequilibrium (LD), parent-of-origin effect and Chi-squared/Fisher’s exact tests.ResultsRs6127973 G allele and G/G homozygotes were over-transmitted for both NSOCs (P=0.005 and P=0.011, respectively) and NSCL/P (P=0.0061 and P=0.011, respectively), rs6127973 G allele was also paternally over-transmitted for both NSOCs (P=0.0061) and NSCL/P (P=0.011).ConclusionsThis study suggested that rs6127973 may be a risk factor of being NSOCs and confirmed the role of BMP7 gene in orofacial deformity from Western Han Chinese, which will also supply scientific evidence for future research and genetic counseling. Key words: Single nucleotide polymorphisms, nonsyndromic orofacial clefts, BMP7.

Adeno-associated virus-mediated BMP-7 and SOX9 in vitro co-transfection of human degenerative intervertebral disc cells.

Bone morphogenetic protein-7 (BMP-7) and SOX9 are important transcription factors in chondrogenesis. In this study, we examined the biological function of the adeno-associated virus (AAV)-mediated BMP-7 and SOX9 double gene in vitro co-transfection of nucleus pulposus cells of human degenerative intervertebral disc. Human intervertebral disc nucleus pulposus cells were cultured in vitro and subcultured for 5 generations. Using rAAV-BMP-7 and rAAV-SOX9 AAV2-type AAV viruses, the cells were divided into 4 groups: blank normal, BMP-7 transfection, SOX9 transfection, and BMP-7 and SOX9 co-transfection. After 48 h, expression of type II collagen and its mRNA in nucleus pulposus cells was determined. The expression of type II collagen in BMP-7 transfection, SOX9 transfection, and co-transfection groups was up-regulated to varying degrees compared to the blank control group. The type II collagen mRNA level expression in the co-transfection group was significantly higher than in other groups (P < 0.05). AAV-mediated BMP-7 and SOX9 in vitro co-transfection can promote the synthesis of type II collagen in nucleus pulposus cells in the human degenerative intervertebral disc. Double-gene therapy has a synergistic effect in the treatment of intervertebral disc degeneration.

Intravenous Transplantation of BMP2-Transduced Endothelial Progenitor Cells Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Rats

Background/Aims: Acute lung injury (ALI) and its aggressive stage, acute respiratory distress syndrome (ARDS), are characterized by diffuse damage and increased permeability of the endothelial barrier, leading to alveolar infiltrates and interstitial edema. Enhancing endothelial integrity represents a novel therapeutic strategy for ALI/ARDS. Endothelial progenitor cells (EPCs) have been reported to participate in endothelial repair of ALI and also serve as a tool for gene therapy. Further, bone morphogenetic protein 2 (BMP2) is an essential signaling molecule that regulates the fate of different cell types. The aim of our study is to explore whether bone marrow-derived EPCs transduced with lentiviral-mediated BMP2 gene might benefit lipopolysaccharide (LPS)-induced ALI in a rat model. Methods: Rats were divided randomly into five groups. The sham group was given normal saline via the trachea and right jugular vein. The other four groups underwent intratracheal-LPS-induced ALI followed after 30 min by treatment with either normal saline, EPCs, EPCs transduced with empty lentiviral vector (EPCs-null), or EPCs transduced with BMP2 (EPCs-BMP2) via the right jugular vein. Results: We found that the lung injury score, oxygenation, and inflammatory response were significantly ameliorated in the three EPC-treated groups (EPCs, EPCs-null, and EPCs-BMP2). In addition, EPCs-BMP2 further improved endothelium repair and capillary permeability, causing markedly reduced wet-to-dry lung-weight ratio and BALF protein content, and increased levels of BMP2 protein, BMP2 mRNA, and eNOS protein in lung tissues. Conclusion: Transplantation of BMP2-transduced EPCs effectively attenuates edema and protein exudation compared with EPCs alone in LPS-induced ALI via enhanced expression of BMP2 and eNOS.

Persistent Noggin arrests cardiomyocyte morphogenesis and results in early in utero lethality.

Multiple bone morphogenetic protein (BMP) genes are expressed in the developing heart from the initiation to late-differentiation stages, and play pivotal roles in cardiovascular development. In this study, we investigated the requirement of BMP activity in heart development by transgenic over-expression of extracellular BMP antagonist Noggin. Using Nkx2.5-Cre to drive lineage-restricted Noggin within cardiomyocyte progenitors, we show persistent Noggin arrests cardiac development at the linear heart stage. This is coupled with a significantly reduced cell proliferation rate, subsequent cardiomyocyte programmed cell death and reduction of downstream intracellular pSMAD1/5/8 expression. Noggin mutants exhibit reduced heartbeat which likely results in subsequent fully penetrant in utero lethality. Significantly, confocal and electron micrographic examination revealed considerably fewer contractile elements, as well as a lack of maturation of actin-myosin microfilaments. Molecular analysis demonstrated that ectopic Noggin-expressing regions in the early heart's pacemaker region, failed to express the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (Hcn4), resulting in an overall decrease in Hcn4 levels. Combined, our results reveal a novel role for BMP signaling in the progression of heart development from the tubular heart stage to the looped stage by means of regulation of proliferation and promotion of maturation of the in utero heart's contractile apparatus and pacemaker.

Molecular characterization of the BMP7 gene and its potential role in shell formation in Pinctada martensii.

Bone morphogenetic protein 7 (BMP7), also called osteogenetic protein-1, can induce bone formation. In this study, the obtained full-length cDNA of BMP7 from Pinctada martensii (Pm-BMP7) was 2972 bp, including a 5'-untranslated region (UTR) of 294 bp, an open reading fragment of 1290 bp encoding a 429 amino acid polypeptide and a 3'-UTR of 1388 bp. The deduced protein sequence of Pm-BMP7 contained a signal peptide, a pro-domain and a mature peptide. The mature peptide consisted of 135 amino acids and included a transforming growth factor β family domain with six shared cysteine residues. The protein sequence of Pm-BMP7 showed 66% identity with that from Crassostrea gigas. Two unigenes encoding Pm-BMPRI (Pm-BMP receptor I) and Pm-BMPRII were obtained from the transcriptome database of P. martensii. Tissue expression analysis demonstrated Pm-BMP7 and Pm-BMPRI were highly expressed in the mantle (shell formation related-tissue), while Pm-BMPRII was highly expressed in the foot. After inhibiting Pm-BMP7 expression using RNA interference (RNAi) technology, Pm-BMP7 mRNA was significantly down-regulated (p < 0.05) in the mantle pallium (nacre formation related-tissue) and the mantle edge (prismatic layer formation related-tissue). The microstructure, observed using a scanning electron microscope, indicated a disordered growth status in the nacre and obvious holes in the prismatic layer in the dsRNA-Pm-BMP7 injected-group. These results suggest that Pm-BMP7 plays a crucial role in the nacre and prismatic layer formation process of the shell.

Associations of polymorphisms in the bone morphogenetic protein-2 gene with risk and prognosis of osteosarcoma in a Chinese population.

Osteosarcoma is the most common type of bone cancer in adolescence. Bone morphogenetic protein-2 (BMP-2) plays important roles in the development of bone and cartilage and in inhibiting the tumorigenicity of cancer stem cells in human osteosarcoma cell line. The aim of this study was to examine whether polymorphisms in the BMP2 gene are associated with osteosarcoma risk and prognosis in Chinese population. Five single nucleotide polymorphisms (SNP) in the BMP2 gene were genotyped in a case-control study, including 203 osteosarcoma patients and 406 cancer-free controls. We found that rs3178250 TT genotype was associated with significant increased osteosarcoma risk (age-adjusted odds ratio (OR) = 2.06, 95% confidence intervals (CI) of 1.23-3.45) compared with CC genotype. Subjects carrying the AA genotype of rs1005464 had significant decreased cancer risk (age-adjusted OR = 0.44, 95% CI of 0.23-0.85) compared with those carrying the GG genotype. Haplotype analysis also showed that carriers of the G-T-T-G and A-T-T-G haplotypes (rs235764-rs3178250-rs235768-rs1005464) had significant increased risks of osteosarcoma (age-adjusted OR = 1.85, 95% CI of 1.28-2.66 and age-adjusted OR = 1.51, 95% CI of 1.06-2.16) compared with the G-C-T-A haplotype carriers. Besides, rs1005464 was an independent prognostic factor for osteosarcoma patients (GA vs. GG: age-adjusted hazard radio (HR) = 0.60, 95% CI of 0.36-0.99). Our data suggest that genetic mutations in the BMP2 gene are associated with osteosarcoma risk and prognosis in a Chinese population.

Uniaxial cyclic stretch promotes osteogenic differentiation and synthesis of BMP2 in the C3H10T1/2 cells with BMP2 gene variant of rs2273073 (T/G).

Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic bone formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of bone morphogenetic protein 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H10T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, 10% stretch). In the presence of mechanical stress, the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (P<0.05). In conclusion, these results suggested that BMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to bone transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.

Single Nucleotide Polymorphisms in Osteogenic Genes in Atrophic Delayed Fracture-Healing: A Preliminary Investigation.

We propose that fracture-healing potential is affected by the patient's genome. This genotype is then phenotypically expressed by the patient at the time of injury. We examined the hypothesis that patients who exhibit delayed or impaired fracture-healing may have one or more single nucleotide polymorphisms (SNPs) within a series of genes related to bone formation. We performed a population-based, case-controlled study of delayed fracture-healing. Sixty-two adults with a long-bone fracture were identified from a surgical database. Thirty-three patients had an atrophic nonunion (delayed healing), and twenty-nine displayed normal fracture-healing. These patients underwent buccal mucosal cell harvesting. SNP genotyping was performed with use of bead array technology. One hundred and forty-four SNPs (selected from HapMap) within thirty genes associated with fracture-healing were investigated. Three SNPs did not segregate in the population and were excluded from the analysis. Eight of the remaining SNPs failed the test for Hardy-Weinberg equilibrium (p value smaller than the Bonferroni-corrected level of 0.05/141 = 0.000355) and were excluded. Five SNPs on four genes were found to have a p value of <0.05 in the additive genetic model. Of these five significant SNPs, three had an odds ratio (OR) of >1, indicating that the presence of the allele increased the risk of nonunion. The rs2853550 SNP, which had the largest effect (OR = 5.9, p = 0.034), was on the IL1B gene, which codes for interleukin 1 beta. The rs2297514 SNP (OR = 3.98, p = 0.015) and the rs2248814 SNP (OR = 2.27, p = 0.038) were on the NOS2 gene coding for nitric oxide synthase. The remaining two SNPs had an OR of <1, indicating that the presence of the allele may be protective against nonunion. The rs3819089 SNP (OR = 0.26, p = 0.026) was on the MMP13 gene for matrix metallopeptidase 13, and the rs270393 SNP (OR = 0.30, p = 0.015) was on the BMP6 gene for bone morphogenetic protein 6. Variations in the IL1B and NOS2 genes may contribute to delayed fracture-healing and warrant further investigation. Impaired fracture union may have genetic contributions.

Proliferation and odontogenic differentiation of BMP2 gene‑transfected stem cells from human tooth apical papilla: an in vitro study.

Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue-derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral-mediated BMP2 gene-transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector-transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK-8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: i) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ii) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; iii) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro.

Osteoblastic differentiation of rabbit adipose-derived stem cells transfected by polyethylenimine-mediated bone morphogenetic protein-2 gene in vitro

Objective To investigate the expression of human bone morphogenetic protein-2 (BMP-2) gene transfected to rabbit adipose-derived stem cells (ADSCs) mediated by polyethylenimine and assess the effect of osteoblastic differentiation of the cells.Methods A total of 10 g adipose tissue was collected from the neck of adult Japanese white rabbits,and after isolation and culture in vitro ADSCs were obtained,and identified phenotypically.BMP-2 was transfected into rabbit ADSCs mediated by polyethylenimine (13 × 103).Positive cell clones were selected with G-418.The expression of BMP-2 mRNA and protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) respectively.The expression of osteocalcin and collagen Ⅰ was detected by Western blotting.Alkaline phosphatase was detected with ALP kit,and stained with alizarin red staining.The untransfected groups served as the control groups.Results ADSCs were successfully isolated from rab bit adipose tissue.ADSCs at passage 3 and 6 highly expressed CD29 and CD44 (the positive rate being more than 90％) and low expressed CD34 and CD45 (the positive rate being less than 5％).The expression of BMP-2 mRNA and protein was detectable and that of alkaline phosphatase,osteocalcin and collagen I increased in transfected ADSCs,and Alizarin red staining was postive.Conclusion It is feasible that polyethylenimine-mediated BMP-2 gene can be transfected into rabbit ADSCs.BMP-2 gene was effectively expressed in ADSCs and induce directional osteogenic differentiation of ADSCs. Key words: Polyethylenimine; Bone morphogenetic protein-2; Adipose-derived stem cells; Gene transfection

Association of bone morphogenetic protein-2 gene polymorphisms with susceptibility to ossification of the posterior longitudinal ligament of the cervical spine in Chinese patients

Objective To investigate the association of single nucleotide polymorphism (SNPs) of bone morphogenetic protein-2 (BMP-2) gene encoding region with susceptibility to ossification of the posterior longitudinal ligament (OPLL) of cervical spine and its severity in Chinese patients.Methods A total of 420 OPLL patients and 506 age-and sex-matched controls were studied.Genomic DNA was isolated from venous blood and polymerase chain reaction (PCR) was performed with a standerd protocol.The PCR products were analyzed by direct sequencing.To compare the genotypic and allelic distribution between OPLL cases and control subjects.Results The number of subjects in the total study population with the Ser37Ala (T/G)TT,TG and GG genotypes was 752,174 and 0,respectively,with frequencies of 81％,19％ and 0,respectively.There was a statistical difference between OPLL cases and control subjects (P ＜ 0.001),The TG genotype was associated with the occurrence of OPLL.The number of subjects in the total study population with the Arg190Ser (A/T) AA,AT and TT genotypes was 228,464 and 234,respectively,with frequencies of 24％,50％ and 26％,respectively.There was a statistical difference between OPLL cases and control subjects (P ＜ 0.01),The AT genotype was associated with the occurrence of OPLL.For the Ser87Ser (A/G) polymorphism,patients with G allele had a significantly greater number of ossified vertebrae compared with patients without A allele.Condusion The Ser37Ala (T/G) polymorphism in exon2 and the Arg190Ser (A/T) polymorphism in exon3 of BMP-2 gene are associated with the occurrence of OPLL.The Ser87Ser (A/G) polymorphism in exon2 of BMP-2 gene is associated with more extensive OPLL.BMP-2 gene is the susceptibility gene of OPLL. Key words: Ossification of posterior longitudinal ligament; Bone morphogenetic protein-2; Single nucleotide polymorphisms

Mechanical forces induce odontoblastic differentiation of mesenchymal stem cells on three-dimensional biomimetic scaffolds.

The mechanical induction of cell differentiation is well known. However, the effect of mechanical compression on odontoblastic differentiation remains to be elucidated. Thus, we first determined the optimal conditions for the induction of human dental pulp stem cells (hDPSCs) into odontoblastic differentiation in response to mechanical compression of three-dimensional (3D) scaffolds with dentinal tubule-like pores. The odontoblastic differentiation was evaluated by gene expression and confocal laser microscopy. The optimal conditions, which were: cell density, 4.0 × 105 cells/cm2 ; compression magnitude, 19.6 kPa; and loading time, 9 h, significantly increased expression of the odontoblast-specific markers dentine sialophosphoprotein (DSPP) and enamelysin and enhanced the elongation of cellular processes into the pores of the membrane, a typical morphological feature of odontoblasts. In addition, upregulation of bone morphogenetic protein 7 (BMP7) and wingless-type MMTV integration site family member 10a (Wnt10a) was observed. Moreover, the phosphorylation levels of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 were also enhanced by mechanical compression, indicating the involvement of the MAPK signalling pathway. It is noteworthy that human mesenchymal stem cells (MSCs) derived from bone marrow and amnion also differentiated into odontoblasts in response to the optimal mechanical compression, demonstrating the importance of the physical structure of the scaffold in odontoblastic differentiation. Thus, odontoblastic differentiation of hDPSCs is promoted by optimal mechanical compression through the MAPK signalling pathway and expression of the BMP7 and Wnt10a genes. The 3D biomimetic scaffolds with dentinal tubule-like pores were critical for the odontoblastic differentiation of MSCs induced by mechanical compression. Copyright © 2014 John Wiley & Sons, Ltd.

Reduced BMP6 expression by DNA methylation contributes to EMT and drug resistance in breast cancer cells.

Bone morphogenetic protein 6 (BMP6) is an important regulator of cell growth, differentiation and apoptosis in various types of tumor. In breast cancer, it was considered as a tumor suppressor. Our previous study also confirmed that BMP6 was a critical regulator of breast cancer drug resistance. However, little is known about how its expression is regulated and its mechanisms in breast cancer drug resistance. In the present study, we assessed the DNA methylation regulation of BMP6 based on the presence of a large CpG island in the BMP6 gene promoter. Quantitative DNA methylation analyses showed a significantly increased DNA methylation level in the drug-resistant cell line MCF-7/ADR compared to their parental cells MCF-7. Moreover, the drug-resistant cell line MCF-7/ADR showed an EMT phenotype confirmed by morphology and the expression of EMT marker gene. MCF-7 cells transfected with BMP6-specific shRNA vector also showed an EMT phenotype. The MCF-7/ADR cells treated with the recombinant BMP6 proteins reversed their EMT phenotype. These data indicated that hypermethylation modifications contributed to the regulation of BMP6 and induced an EMT phenotype of breast cancer during the acquisition of drug resistance.

Efficiently engineered cell sheet using a complex of polyethylenimine-alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation.

Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration.

Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences.

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10(-8)/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10(-8)/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.

Ex vivo bone morphogenetic protein 2 gene delivery using periodontal ligament stem cells for enhanced re-osseointegration in the regenerative treatment of peri-implantitis

Peri-implantitis is a chronic inflammatory process with advanced bone loss and impaired healing potential. For peri-implantitis treatment, tissue engineering can be applied to enhance bone regeneration of peri-implant defects. This study aimed to evaluate ex vivo bone morphogenetic protein 2 (BMP2) gene delivery using canine periodontal ligament stem cells (PDLSCs) for regeneration of peri-implantitis defects. Canine PDLSCs were transduced with adenoviral vectors containing BMP2 (BMP2/PDLSCs). After peri-implantitis was induced by ligature placement in six beagle dogs, regenerative procedures were performed; hydroxyapatite (HA) particles and collagen gel with autologous canine PDLSCs (PDLSC group) or BMP2/PDLSCs (BMP/PDLSC group) or without cells (control group) were grafted into the defects and covered by an absorbable membrane. Three months later, the animals were sacrificed. In vitro, BMP2/PDLSCs showed similar levels of stem cell properties to PDLSCs, such as colony-forming efficiency and expression of MSC markers STRO-1 and CD 146. BMP2/PDLSCs produced BMP-2 until day 21 at a concentration of 4-8 ng/mL. In vivo, the BMP2/PDLSC group showed significantly more new bone formation and re-osseointegration in peri-implantitis defects compared to the other groups. In conclusion, ex vivo BMP2 gene delivery using PDLSCs enhanced new bone formation and re-osseointegration in peri-implantitis defects.

Disruption of the temporally regulated cloaca endodermal β-catenin signaling causes anorectal malformations

The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (ShhCreERT2). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The ShhCreERT2/+; β-cateninflox(ex3)/+; BmprIAflox/− mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.

Identification of BMP2 as an epigenetically silenced growth inhibitor in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of infancy and although therapy has improved over the years, mortality is still fairly high. The establishment of new treatments has been hampered by the limited knowledge of the molecular mechanisms driving development of RMS. One characteristic of cancer cells is aberrant DNA methylation, which could lead to silencing of tumor suppressor genes. However, only a few epigenetically silenced genes have been described in RMS so far. We performed an expression profiling analysis of three RMS cell lines that were treated with the demethylating agent 5'-aza-2'-deoxycytidine (5-Aza‑dC) facilitating re-expression of epigenetically silenced genes. This treatment induced the gene BMP2 (bone morphogenetic protein 2) throughout all cell lines. Detailed methylation analysis of CpG sites in the BMP2 promoter region by bisulfite sequencing and methylation-specific PCR revealed that a high degree of DNA methylation is causatively associated with the suppression of BMP2 in RMS cells. Consequently, treatment of the RMS cell lines with 5-Aza-dC resulted in DNA demethylation of the BMP2 promoter, most prominently in alveolar RMS. Supplementation of recombinant human BMP2 (rhBMP2) led to a reduced viability of RMS cells. Altogether, these findings suggest that suppression of BMP2 by epigenetic silencing may play a critical role in the genesis of RMS, thereby providing a rationale for the development of a new treatment strategy for RMS.