Bone morphogenetic proteins 2, 6, and 9 differentially regulate the osteogenic differentiation of immortalized preodontoblasts

Our study attempted to compare the efficacies of bone morphogenetic protein (BMP) 2, 6, and 9 in inducing osteogenic differentiation of preodontoblasts (PDBs). We immortalized PDBs by introducing a reversible SV40 T antigen-based immortalization system. Cell proliferation capability was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The effects of BMP2, 6, and 9 on the osteogenic differentiation of immortalized preodontoblasts (iPDBs) were measured by alkaline phosphatase (ALP) activity assays and alizarin red S staining. The expression of osteogenic markers was evaluated by semiquantitative real-time polymerase chain reaction analysis. To assess ectopic bone formation, rat-derived iPDBs were transfected in culture with adenoviral vectors designated Ad-BMP2, 6, and 9 and subcutaneously or intramuscularly injected into mice. Several BMPs retained endogenous expression in PDBs and regulated the mRNA expression of mineralized tissue-associated proteins. ALP activity and mineralized nodule formation were significantly increased in the Ad-BMP9-transfected group relative to the control group. In addition, the most significant hard tissue formation was in this group. The results indicated that BMP signaling was involved in the osteogenic differentiation of iPDBs. BMP9 could be an efficacious accelerant of the osteogenic differentiation of iPDBs.

Effect of 15-Deoxy-Δ-prostaglandin JNanocapsules on Inflammation and Bone Regeneration in a Rat Bone Defect Model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the major metabolites from prostaglandin D2 in arachidonic acid metabolic pathway, has potential anti-inflammatory properties. The objective of this study was to explore the effects of 15d-PGJ2-loaded poly(D,L-lactide-co-glycolide) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration in local bone defect.</p><p><b>METHODS</b>The study was conducted on 96 Wistar rats from June 2014 to March 2016. Saline, unloaded nanoparticles, free 15d-PGJ2or 15d-PGJ2-NC, were delivered through a collagen vehicle inside surgically created transcortical defects in rat femurs. Interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels in the surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time polymerase chain reaction over 14 days. Simultaneously, bone morphogenetic protein-6 (BMP-6) and platelet-derived growth factor-B (PDGF-B) messenger RNA (mRNA) in the defect were examined. New bone formation and EphrinB2 and osteoprotegerin (OPG) protein expression in the cortical defect were observed by Masson's Trichrome staining and immunohistochemistry over 28 days. Data were analyzed by one-way analysis of variance. Least-significant difference and Dunnett's T3 methods were used with a bilateral P< 0.05.</p><p><b>RESULTS</b>Application of l5d-PGJ2-NC (100 μg/ml) in the local bone defect significantly decreased IL-6, IL-1β, and TNF-α mRNA and protein, compared with saline-treated controls (P < 0.05). l5d-PGJ2-NC upregulated BMP-6 and PDGF-B mRNA (P < 0.05). New bone formation was observed in the cortical defect in l5d-PGJ2-NC-treated animals from 7th day onward (P < 0.001). Expression of EphrinB2 and OPG presented early on day 3 and persisted through day 28 in 15d-PGJ2-NC group (P < 0.05).</p><p><b>CONCLUSION</b>Stable l5d-PGJ2-NC complexes were prepared that could attenuate IL-6, IL-1β, and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.</p>

Delta12-prostaglandinJ2-nano capsule up-regulates growth factor expression and enhances bone regeneration in rats / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of local delivery of delta12-prostaglandinJ2-loaded poly (lactic-co-glycolic acid) (Δ(12)-PGJ2-NC) on growth factors expression and bone formation.</p><p><b>METHODS</b>Δ(12)-PGJ2-NC was prepared by the emulsion solvent diffusion method. The physical and chemical properties of the nanoparticles were evaluated by particle size analysis, transmission electron microscopy, drug-loading ratio and the in vitro release study. Then standardized transcortical defect (5.0 mm × 1.5 mm) was conducted in the femur of 48 male Wistar rats which were randomly divided into four groups (n = 12), S, K, F, and N. Thirty microliter of saline (S), unloaded nanoparticles (K), Δ(12)-PGJ2 (F) and Δ(12)-PGJ2-NC(N) in a collagen vehicle were delivered inside a titanium chamber fixed over the defect. Then, four subgroups were randomly divided in each group named as D3, D7, D14, and D28 (n = 3) according to the days 3, 7, 14, and 28 after the surgery. At days 3, 7, 14, and 28, the mRNA expression of the bone morphogenetic protein-6 (BMP-6), platelet-derived growth factor-B (PDGF-B) in defect aera was analyzed by real time quantitive-polymerase blotting. HE staining was employed to reveal new bone formation in weeks 2 and 4.</p><p><b>RESULTS</b>Δ(12)-PGJ2-NC appeared opalescent white and remained relatively stable, with an average particle size of (135.2 ± 0.85) nm. The images from transmission electron microscopy showed that Δ(12)-PGJ2-NC was spherical in shape and homogeneously distributed. The encapsulation efficiency of Δ(12)-PGJ2 with the poly (lactic-co-glycolic acid) (PLGA) nanocapsules was about 92%. The in vitro release of Δ(12)-PGJ2-NC at 37 °C showed a sustained fashion and the average accumulated amount was 30%, 52%, 77%, 91%, and 98% respectively, at 0.5, 1, 2, 4 and 6 h. Compared with the animals treated with saline, after dose of 100 mg/L Δ(12)-PGJ2 and Δ(12)-PGJ2-NC apllication, the mRNA expression level of BMP-6, PDGF-B increased significantly (P < 0.05, P < 0.001). The protein expression of BMP-6, Ephrin-B2 also was up-regulated. Histomorphometry revealed that new bone formation increased at the same dose of 100 mg/L. But the unloaded nanoparticles did not have the same effect (P > 0.05).</p><p><b>CONCLUSIONS</b>A stable Δ(12)-PGJ2 loaded nanoparticle was successfully prepared. Δ(12)-PGJ2-NC may upregulate the expression of BMP-6, PDGF-B and Ephrin-B2, and promote new bone formation in bone defect area.</p>

Expression of serum FSTL-1 in bone metastasis of prostate cancer and its clinical implication / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the expression of follistatin-like protein 1 (FSTL-1) in bone metastasis of prostate cancer (BMPC), the correlation of serum FSTL-1 with the chronic inflammatory factor interleukin-6 (IL-6) and bone morphogenetic protein 6 (BMP6) , and the clinical application value of serum FSTL-1 in BMPC.</p><p><b>METHODS</b>Using ELISA, we measured the expression levels of serum FSTL-1, IL-6, and BMP6 in 35 patients with BMPC and another 30 with benign prostatic hyperplasia (BPH) and performed correlation analysis on the data obtained.</p><p><b>RESULTS</b>Compared with the BPH controls, the BMPC patients showed a significantly decreased expression of serum FSTL-1 ([34.45 ± 12.35] μg/L vs [20.23 ± 8.69] μg/L, P < 0.01) and increased levels of IL-6 ([11.21 ± 8.62] μg/L vs [23.56 ± 20.12] μg/L, P < 0.05) and BMP6 ([293.50 ± 39.72] μg/L vs [428.30 ± 178.40] μg/L, P < 0.05). There was a significant negative correlation between the level of serum FSTL-1 and those of IL-6 and BMP6 in the BMPC patients, with correlation coefficients of -0.971 and -0.972, respectively (P < 0.05).</p><p><b>CONCLUSION</b>The expression of serum FSTL-1 decreases in patients with bone metastasis of prostate cancer, and it is correlated with the levels of inflammatory factor and cell transformation factor. This finding offers a novel biological marker for the development and progression of prostate cancer as well as a new biological target factor for its intervention.</p>

Chondrogenesis of periodontal ligament stem cells by transforming growth factor-β3 and bone morphogenetic protein-6 in a normal healthy impacted third molar / 国际口腔科学杂志·英文版

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-β3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

Construction of a recombinant adenovirus co-expressing bone morphogenic proteins 9 and 6 and its effect on osteogenesis in C3H10 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus co-expressing bone morphogenic protein (BMP) 9 and BMP6 and observe its effect on the osteogenesis in C3H10 cells.</p><p><b>METHOD</b>The full-length sequences of BMP9 and BMP6 were amplified from AdEasy vector by PCR and cloned into the shuttle plasmid pASG2 vector to construct the co-expression shuttle plasmid pASG2-BMP9, 6 followed by homologous recombination with plasmid pAdeasy-1 in BJ5183. After confirmation by restriction endonuclease digestion, the recombinant vector was transfected into HEK293 cells, and high-titer recombinant adenovirus (Ad-BMP9, 6) was collected after amplification. Ad-BMP9, 6 was then transduced into C3H10 cells in vitro, and the mRNA expression of BMP9 and BMP6 was detected by RT-PCR. The osteogenic capability of the transfected cells was observed by alkaline phosphatase staining and calcium-alizarin red staining.</p><p><b>RESULTS</b>AdBMP9,6 was constructed successfully and effectively infected in C3H10 cells, in which high expressions of BMP6 and BMP9 were detected. C3H10 cells infected with Ad-BMP9,6 showed stronger alkaline phosphatase and calcium-alizarin red staining than the cells transfected by either BMP9 or BMP6 alone.</p><p><b>CONCLUSION</b>The recombinant adenovirus co-expressing BMP9 and BMP6 we constructed shows a more potent effect than the adenoviruses expressing either BMP9 or BMP6 alone in inducing the osteogenic differentiation of C3H10 cells into osteoblasts.</p>

Effects of Murine Dermal Cells on the Regulation of Hair Growth Is Dependent on the Cell Number and Post-Natal Age of Newborn Mice

Dermal cells from neonatal mice can initiate the formation of hair follicles (HFs) when combined with adult mouse epidermal cells and transplanted subcutaneously into athymic mice. In the present study, the effects of dermal cells on HF formation were tested in terms of total cell number and the time course of cell harvest. Results demonstrated that the number of dermal cells is critical to the formation of HF. Furthermore, hair forming ability is rapidly decreasing as the neonatal mice age. To examine potential differences in gene expression, cDNA array was performed. Results demonstrate that numerous molecules which are directly involved in receptor and signaling correlated with decreased hair inductivity in early time points after delivery. It is reported that bone morphogenic protein (BMP)-6 and Wnt3a treatment increased hair inductivity of dermal papilla cells. But in our study, no changes were observed in the expression levels of BMP-6 and Wnt3a. However, several Wnt related genes demonstrate increased or decreased expression levels. Thus, our results suggest that co-ordinated regulation of these molecules will be important in hair neogenesis within our model system.

Effect of human bone morphogenetic protein 2, 3, 6, and 12 on osteosarcoma cell line UMR106 / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of human bone morphogenetic protein (hBMPs) 2/3/6 and 12 on osteosarcoma cell UMR106.@*METHODS@#Adenovirus-BMP2/3/6 and 12 (AdBMP2/3/6 and12) were used to treat the cell line. Their proliferation, apoptosis, and transmigration were detected by Trypan blue exclusion test, TdT-mediated biotinylated-dUTP nick end labeling (TUNEL), acridine orange-ethidium bromide (AO/EB) double fluorescent dye staining, and transwell-room test, respectively. The alkaline phosphatase (ALP) activity was detected to reflect the differentiation of tumors.@*RESULTS@#Compared with the control groups, the cell survival rate of the experimental groups treated with AdBMP2/3/6 and 12 showed a significant time-dependent decrease (P<0.01). The apoptosis indexes were increased significantly (P<0.01) and the results from TUNEL and AO/EB method were consistent. The cell numbers of transmembrane significantly decreased at 24,48, and 72 h (P<0.01). AdBMP2/3/6 and 12 treatment enhanced the activity of ALP activity from day 3 and this effect might still be observed up to day 9 of the treatment (P<0.01).@*CONCLUSION@#hBMPs2/3/6 and 12 can inhibit the proliferation and transmigration, and induce their apoptosis and differentiation in osteosarcoma cell line UMR106.

[Designing of human cartilage tissue, by differentiation of adipose-derived stem cells with BMP-6 in alginate scaffold]

In the present study the effect of BMP-6 was investigated on chondrogenesis of adipose-derived stem cells. Mesenchymal stem cells derived from subcutaneous adipose tissue were cultured on alginate scaffold to induce chondrogenesis in experimental group, with chondrogenic medium having BMP-6 growth factor for 3weeks. In control group medium without BMP-6 was applied. The harvested constructs were examined with immunohistochemical and RT-PCR methods for assessment of cartilage-specific characteristics. The results of immunohistochemical method revealed the presence of typical cartilage extracellular matrix components such as type II collagen and aggrecan in constructs induced by BMP-6 growth factor on alginate scaffold. In addition evaluation of the results of RT-PCR analysis confirmed the expression of cartilage- specific genes, such as type II collagen and aggrecan, in the differentiated cells under the influence of growth factor BMP-6. It can be concluded that BMP-6 promotes chondrogenesis of ADSC in 3-D and adipose-derived stem cells could be used for cartilage tissue engineering

Expression and significance of inflammatory factors and bone formation mediators in carotid atherosclerotic plaque / 中南大学学报(医学版)

OBJECTIVE@#To compare protein levels of pro-inflammatory factors and bone formation mediators in the fibrous cap and shoulder region of non-calcified and calcified carotid endarterectomy (CEA) plaques.@*METHODS@#Twenty-two CEA plaques were classified as non-calcified and calcified groups (n=11 each) in accordance with the American Heart Association (AHA) consensus in 1995. To make frozen sections and H&E staining using plaque, the mean percent of carotid stenosis and calcification area was determined by quantitative histomorphometry. The protein levels of pro-inflammatory interleukin-8 (IL-8), monocyte chematactic protein-1 (MCP-1), bone formation mediators bone morphogenetic protein-6 (BMP-6), and osteocalcin in the fibrous cap and shoulder region of plaques were determined by western blot and were quantified using ImageJ software.@*RESULTS@#MCP-1 and IL-8 protein were 1.3 (P>0.05) and 1.5 (P0.05) and 2.1 (P<0.01) folds greater in the calcified plaques compared with those of the non-calcified plaques.@*CONCLUSION@#Inflammation is more likely to occur in non-calcified carotid plaques, and calcification in the plaques may be associated with bone formation, which indicates that decreased inflammation may be the beginning of calcification in carotid atherosclerotic plaques.

Soluble expression of recombinant human BMP6 in Escherichia coli and its purification and bioassay in vitro / 生物工程学报

BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.

Expression of recombinant human BMP6 in CHO cells by fused to the signal peptide and propeptide of another homologue protein / 生物工程学报

BMP6 is a member of TGF-beta superfamily, represent more effective osteogenic activity. Two recombinant plasmids were constructed to expression rhBMP6 in mammalian cells, one contained the cDNA encoding the signal peptide, propeptide and mature peptide of human BMP6, wich was named pcDNA-BMP6, the other contained the recombinant DNA encoding the signal peptide, propeptide of human BMP2 and the mature peptide of BMP6, which was named pcDNA-BMP2/6. Transient expression in Cos7 cells demonstrated that the pcDNA-BMP2/6 produced more rhBMP6 than pcDNA-BMP6. For stable expression, the CHO-dhfr- cells were transfected with pcDNA-BMP2/6 and pSV2-dhfr, then screened by G418 and treated with MTX for targeting gene amplification. The partially purified rhBMP6 by heparin affinity chromatography was shown to possess bone induction activity tested by the induction of alkaline phosphatase activity in C2C12 cells.

Microarray Analysis of Thyroid Stimulating Hormone, Insulin-Like Growth Factor-1, and Insulin-Induced Gene Expression in FRTL-5 Thyroid Cells

To determine which genes are regulated by thyroid stimulating hormone (thyrotropin, TSH), insulin and insulin-like growth factor-1 (IGF-1) in the rat thyroid, we used the microarray technology and observed the changes in gene expression. The expressions of genes for bone morphogenetic protein 6, the glucagon receptor, and cyclin D1 were increased by both TSH and IGF-1; for cytochrome P450, 2c37, the expression was decreased by both. Genes for cholecystokinin, glucuronidase, beta, demethyl-Q 7, and cytochrome c oxidase, subunit VIIIa, were up-regulated; the genes for ribosomal protein L37 and ribosomal protein L4 were down-regulated by TSH and insulin. However, there was no gene observed to be regulated by all three: TSH, IGF-1, and insulin molecules studied. These findings suggest that TSH, IGF-1, and insulin stimulate different signal pathways, which can interact with one another to regulate the proliferation of thyrocytes, and thereby provide additional influence on the process of cellular proliferation.

Activation of bone morphogenetic protein-6 gene transcription in MCF-7 cells by estrogen / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17beta-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER+) breast cancer cell line MCF-7.</p><p><b>METHODS</b>After the treatment of MCF-7 cells with E2 at different concentrations (10(-11) mol/L, 10(-9) mol/L, 10(-7) mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0.7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10(-7) mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half-site (1/2 ERE) element and Sp1 sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (ChIP) assay was also used to confirm the binding of estrogen receptor alpha (ERalpha) on BMP-6 promoter in the presence of E2.</p><p><b>RESULTS</b>E2 dose dependently increased BMP-6 mRNA expression in human ER+ breast cancer cell line MCF-7. At a dose of 10(-7) mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P < 0.05). Both the 1/2 ERE response element mutant form and the Sp1 site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter's response to E2. Through ChIP assay, the binding of ERalpha on 1/2 ERE response element in BMP-6 promoter was further validated.</p><p><b>CONCLUSION</b>Estrogen induces BMP-6 expression in human ER+ breast cancer cell line MCF-7 through its receptor ERalpha binding on 1/2 ERE element in the BMP-6 promoter.</p>

Chondrogenic Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord Blood / 대한정형외과학회잡지

PURPOSE: The aim of this study was to demonstrate the existence of circulating mesenchymal stem cells (MSC) in the human umbilical cord blood (hUCB) and to evaluate the chondrogenic differentiation potential of hUCB-derived MSC in vitro. MATERIALS AND METHODS: Fifty hUCB harvests were cultured in media supplemented with 10% fetal bovine serum. The adherent fibroblast-like cells were characterized by immunophenotyping and induced to differentiate into chondrocytes in the pellet culture with and without BMP-6. This study performed RTPCR of the chondrogenic markers, Safranin-O stain and type II collagen immunohistochemical stain. RESULTS: The mononuclear cells isolated from hUCB formed adherent colonies with an attached wellspread fibroblast-like morphology. The cells positively expressed the MSC-related antigens, but did not express the hematopoietic, HLA-DR, endothelial, or osteoclast antigens and could be induced to differentiate into chondrocytes under proper stimulation. BMP-6 increased the size of the pellet and the mRNA levels for aggrecan, type II collagen and type IX collagen and enhanced the levels of proteoglycan synthesis during chondrogenic differentiation. CONCLUSION: The homogenous fibroblast-like cells developed in cultures from hUCB with chondrogenic differentiation potential were considered to be MSC. Furthermore, it was found that BMP-6 enhanced chondrogenic differentiation of the hUCB-derived MSC in the pellet culture.

Response of Human Renal Cell Carcinoma Cells to Bone Morphogenetic Proteins, and the Expression of Bone Morphogenetic Protein Receptors / 대한비뇨기과학회지

PURPOSE: Bone morphogenetic protein (BMP) is a pleiotropic growth factor, which has been suggested to play a critical role during the development and homeostasis of the kidney. We evaluated the response of the human renal cell carcinoma (RCC) cell lines to BMPs. MATERIALS AND METHODS: We evaluated the growth rate of the human RCC cell lines, 112, 117 and 181, according to the concentrations of BMP-4, -6 and -7, and detected the levels of the BMP receptors (BMPRs) expressed in the cell lines. To demonstrate that the defect in BMP-6 signaling is at the receptor level, BMP-6 resistant cell lines were transfected, with adenovirus containing the constitutively active form of the BMP receptor types II (BMPR-II). After transfection, the cells were transfected with pSBE4, the construct of the BMP-6-responsive luciferase reporter gene, and a luciferase assay performed. RESULTS: The BMP-6 inhibited the proliferation of the 112, but not those of the 117 and 181 cells, in a dose-dependent manner. From Northern blot and immunoblot analyses, it was demonstrated that the 117 and 181 cells had undetectable levels of BMPR-II expression. The levels of luciferase activity, following adenovirus infections, was elevated in both the 117 and 181 cells, suggesting that the down-stream signaling molecules of the BMP-6 was intact in these cell lines. CONCLUSIONS: Taken together, these results demonstrate that the human RCC cell lines 117 and 181 are resistant to the growth inhibitory effects of the BMP-6 due to their decreased levels of BMPR-II expression.

Expression of recombinant human BMP-6 in Escherichia coli and its purification and bioassay in vitro / 生物工程学报

To purify the recombinant human BMP-6 protein and to establish its in vitro bioassay method. The cDNA encoding the mature peptide of hBMP-6 protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), using human placental mRNA as template, and subcloned into the high-expression vector pET-15b under the control of T7 lac promoter. The resulting construct, pET-BMP6, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant hBMP-6 protein (rhBMP-6). After 4 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), rhBMP-6 (approximately 15kD) was expressed and formed inclusion bodies, contributing up to 10% of the total bacterial protein. The inclusion bodies were isolated and redissolved in 8mol/L urea, and the denatured rhBMP-6 was purified to 95% purity by CM-Cellulose 32 ion exchange chromatography (IEC). The osteoinductivity of rhBMP-6 was measured by the expression of some of the osteoblast differentiation marker genes in rhBMP-6-treated C3H10T1/2 cells as reflected by determinations of alkaline phosphatase (ALPase) activity and semi-quantitative RT-PCR. At the end of the purification process, about 80% of rhBMP-6 formed disulphide-linked homodimers after refolding during renaturation. The apparent size of the protein was 30kD on non-reducing SDS-PAGE, similar to that of the native form of hBMP-6. The enzyme assays showed that the ALPase activity was increased in a dose-dependent manner with the treatment of rhBMP-6. After the addition of 300ng/mL of rhBMP-6, the ALPase activity of C3H10T1/2 cells increased significantly. The activity of rhBMP-6 used was comparable to about 70% of that of the standard hBMP-6 derived from eukaryotic cells. RNA extraction data also showed rhBMP-6 stimulated expression of osteoblast marker genes, including type I collagen, osterix, and osteocalcin in a time-dependent manner. After 5 days of treatment, their level of expression was increased to 3 times that of controls. Bone morphogenetic protein (BMP)-6, a member of the 60A subgroup of the bone morphogenetic protein (BMPs) family, plays a pivotal role in bone formation. Previous evidence showed that BMP-6 is selectively up-regulated by estrogen, suggesting its potential role in the treatment of osteoporotic fractures, especially for menopausal osteoporosis. Our present study demonstrates that the recombinant hBMP-6 produced in Escherichia coli is able to induce pre-osteoblastic cells to differentiate into osteoblasts in vitro, and analysis of mRNA expression of type I collagen, osterix, and osteocalcin can be also a method for measuring the osteoinductivity of BMP. This provides the basis for further studies on ectopic bone formation in the body and for the development of auxiliary drugs for the treatment of osteoporotic fractures.