OBJECTIVETo observe the expression of Runx2 in osteoblasts in response to centrifugation in vitro and discuss the function of bone morphogenetic protein (BMP) signal transduction pathway in this course.

METHODSCells were divided into four groups, group A, B, C and D, pretreated with DMEM containing 10% fetal bovine serum, 10% fetal bovine serum, 100 ng x mL(-1) Noggin and 100 ng x mL(-1) Noggin for 24 hours separately. 271 x g centrifugation was loaded for 5 min to these groups except group A and C, other conditions were the same. The total RNA of each group were extracted, and reversed transcription to cDNA after 30 min. The expression of Runx2 in response to centrifugation in vitro was analyzed by quantitative real time PCR.

RESULTSThe expression of Runx2 mRNA in group B was significantly higher than that in group A (P < 0.05). The expression of Runx2 mRNA in group D was significantly lower than that in group B (P < 0.05). There was no statistically significant difference among group A, C, D (P = 0.692).

CONCLUSIONBMP signal transduction pathway plays an important role in the response of osteoblasts to mechanical stimulations. It may also play a central role in the cascade information dissemination of osteoblasts.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; Cell Differentiation ; Centrifugation ; Core Binding Factor Alpha 1 Subunit ; Gene Expression Regulation ; Humans ; In Vitro Techniques ; Osteoblasts ; RNA, Messenger ; Signal Transduction

Bone morphogenetic proteins(BMPs) are regarded as members of the transforming growth factor-beta superfamily with characteristic features in their amino acid sequences. A number of studies have demonstrated the biologic activities of BMPs, which include the induction of cartilage and bone formation. Recently there was a attempt to overcome a limitation of mass production, and economical efficieny of rh-BMPs. The method producing PTD by using bacteria have advantages of acquiry a mass of proteins. Hences, a new treatment which deliver protein employed by protein transduction domain(PTD) has been tried. The purpose of this study was to evaluate the bone regenerative effect of TATBMP-2 and TAT-HA2-BMP-2 employed by PTD from HIV-1 TAT protein for protein translocation in the rat calvarial model. An 8mm calvarial, critical size osteotomy defect was created in each of 32 male Spraque-Dawley rats(weight 250~300g). The animals were divided into 4 groups of 32 animals each (4 animals/group/healing interval). The defect was treated with TATBMP-2/ACS(Absorbable collagen sponge) (TATBMP-2 0.1mg/ml), TAT-HA2-BMP-2/ACS(TAT-HA2-BMP-2 0.1mg/ml), ACS alone or left untreated for surgical control(negative control). The rats were sacrificed at 2 or 8 weeks postsurgery, and the results were evaluated histologically. The results were as follows: New bone formation were not significantly greater in the TATBMP-2/ACS group relative to negative, and positive control groups. New bone was evident at the defect sites in TAT-HA2-BMP-2/ACS group relative to negative, positive control and TATBMP-2 groups. There were a little bone regeneration in TATBMP-2 groups. While, enhanced local bone formation were observed in TAT-HA2-BMP-2 group. But, The results was not the same in all rat defects. Therefore, further investigations are required to develop a method, which disperse homogenously, and adhere to target cells.
Amino Acid Sequence ; Animals ; Bacteria ; Bone Morphogenetic Proteins ; Bone Regeneration ; Cartilage ; Collagen ; Gene Products, tat ; HIV-1 ; Humans* ; Male ; Osteogenesis ; Osteotomy ; Protein Transport ; Rats

OBJECTIVETo investigate the probabilities of core-biding factor a1 (Cbfa1) expression by human skin fibroblasts induced in vitro.

METHODSThe fibroblasts were isolated, purified from human skin, and were grown in incubation in the media of TNF-alpha, BMP-2, and combined TNF-alpha and BMP-2 at certain concentrations, respectively. The changes in biological features of these fibroblasts correlated with osteogenesis were detected by immunohistochemistry and RT-PCR assay.

RESULTSTNF-alpha could switch phenotype of collagen in fibroblasts from Type I and III to Type I and induce fibroblasts to express Ras and BMP type I receptor (BMPR-IA). TNF-alpha in combination with BMP-2 could induce fibroblasts to express Cbfa1 and osteocalcin mRNA.

CONCLUSIONHuman skin fibroblast could be induced into pro-osteoblast expressing Cbfa1, an osteoblast-specific transcription factor and a regulation of osteoblast differentiation, and combined use of TNF-alpha and BMP-2 was one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; Bone Morphogenetic Proteins ; pharmacology ; Cells, Cultured ; Collagen ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; Core Binding Factors ; Fibroblasts ; metabolism ; Humans ; Neoplasm Proteins ; Osteocalcin ; biosynthesis ; Protein-Serine-Threonine Kinases ; biosynthesis ; RNA, Messenger ; analysis ; Receptors, Growth Factor ; biosynthesis ; Skin ; cytology ; Transcription Factors ; biosynthesis ; genetics ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha ; pharmacology

protein 3, epidermal growth factor receptor pathway substrate 15, transcription factor CP2, CDC20 cell division cycle 20 homolog, BRCA1 associated protein, and glutaminase were increased their expression after the administration of FGF-5. The pinin, ribosomal protein S29, proliferation-associated 2G4, protein phosphatase 1G, PICTAIRE protein kinase 1, cell division cycle 25A, keratin 7, 15, and 17, bone morphogenetic protein 1 and 7, and placental growth factor were In conclusion, FGF-5 was a potent mitogen for human fibroblasts, but FGF-7 was not. FGF-5 could induce FGF-7 expression, but FGF-7 inhibited FGF-5 expression. Thus, the gingival hyperplasia in the immunosuppressed patients seemed to be occurred via the action of FGF-5. The role of FGF-7 that was expressed in these patients might be late events after the expression of FGF-5.]]>
Bone Morphogenetic Protein 1 ; Carrier Proteins ; Cell Count ; Cell Cycle ; DNA, Complementary ; Fibroblasts ; Gingival Hyperplasia ; Glutaminase ; Humans ; Keratin-7 ; Oligonucleotide Array Sequence Analysis ; Organ Transplantation ; Protein Kinases ; Protein Phosphatase 1 ; Receptor, Epidermal Growth Factor ; Ribosomal Proteins ; Transcription Factors ; Transplants

OBJECTIVE: To explore the effect of bone morphogenetic protein-2 (BMP-2 ) on the expression of membrane Type 1-matrix metalloproteinase (MT1-MMP) and the activation of matrix metalloproteinase-2 (MMP-2) in human A549 lung carcinoma cells. METHODS: Western blot was used to analyse the expression of MT1-MMP protein levels in A549 cells. Enzyme-linked immunosorbentassay was used to show the actions of BMP-2 on the activation of MMP-2 in A549 cells. RESULTS: Treatment with BMP-2 in A549 cells caused a dose- and time-dependent increase in the expression of MT1-MMP protein. BMP-2 dose-dependently activated MMP-2 in A549 cells. CONCLUSION BMP-2 might promote MT1-MMP expression and MMP-2 activation in lung carcinoma cells, which can lead to the increase of extracellular matrix followed by acceleration of tumor cell migration and invasion. It is probably another key mechanism of BMP-2 upregulated lung carcinoma cell migration.
Bone Morphogenetic Protein 2 ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 1 ; biosynthesis ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; Tumor Cells, Cultured

Bone Morphogenetic Protein 1 ; metabolism ; Carcinoma ; metabolism ; pathology ; Carcinoma, Papillary ; metabolism ; pathology ; Cell Adhesion Molecules ; metabolism ; Epithelial-Mesenchymal Transition ; Humans ; Survival Rate ; Thyroid Neoplasms ; metabolism ; pathology

Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of alpha(1A)-adrenergic receptor (alpha(1A)-AR) and a segment of bone morphogenetic protein-1 (BMP-1). In the present study binding between the two proteins was further determined in human embryonic cell 293 (HEK293), a mammalian expression system. Mammalian expression vector PCP3HA was constructed by PCR and consisted of segments of BMP-1 cDNA, and vector PDT-alpha(1A) consisted of the full-length cDNA of human alpha(1A)-AR. They were transfected to HEK293 cells and examined by Western blot. alpha(1A)-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding between alpha(1A)-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked immunosorbent assays (ELISA) and co-immunoprecipitation. In ELISA experiment, the ELISA microwell plate was first coated with anti-FLAG M2 antibody, which recognizes the FLAG-tagged alpha(1A)-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD(490) values among the control group, PDT-alpha(1A) transfection group and PCP3HA transfection group, exhibited no significant difference (0.034+/-0.027, 0.042+/-0.019, 0.030+/-0.0096), but the OD(490) values of PDT-alpha(1A) and PCP3HA co-transfection group (0.57+/-0.12) were significantly higher than those of the other groups (P<0.001, respectively). In co-immunoprecipitation experiments, HEK293 cells expressing alpha(1A)-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immunoprecipitation pellet was immunoblotted with either the HRP conjugated anti-FLAG antibody or the anti-HA antibody, which recognizes the HA-tagged segment of BMP-1. Segment of BMP-1 was present in the pellet immunoprecipitation of PDT-alpha(1A) and PCP3HA co-transfected group. In conclusion, the results indicate that alpha(1A)-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.
Adrenergic alpha-Agonists ; pharmacology ; Bone Morphogenetic Protein 1 ; Bone Morphogenetic Proteins ; genetics ; metabolism ; Cells, Cultured ; Embryo, Mammalian ; Enzyme-Linked Immunosorbent Assay ; Humans ; Kidney ; cytology ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Protein Binding ; Receptors, Adrenergic, alpha-1 ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques

OBJECTIVEThis study evaluates the biological effects of naringin (NAR) joint bone morphogenetic protein (BMP)-2 on the proliferation, alkaline phosphatase (ALP) activity, and expression of osteoblastogenic genes, such as Runt-related transcription factor 2 (Runx2), collagen Ⅰ (ColⅠ), ALP, and osteocalcin (OCN) of pre-osteoblasts.

METHODSThree different NAR concentrations (10, 100, and 1 000 μmol·L⁻&sup1;) were applied, alone or combined with BMP-2(50 ng·mL⁻&sup1;), to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line). Cell numbers (proliferation) were evaluated at first, fourth, and seventh days by Alamar blue assay. ALP activity and the expression of osteoblastogenic genes, such as Runx2, ColⅠ, ALP, and OCN were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) at fourth and seventh day.

RESULTSStimulation by NAR alone and in combination with BMP-2 for 1 day and 4 days could promote cell proliferation, which peaked at a concentration of 100 μmol·L⁻&sup1; NAR combined with BMP-2 could promote cell proliferation significantly (P<0.05). Stimulation by NAR alone and in combination with BMP-2 for 4 and 7 days could promote ALP activity and bone-related gene(ALP, OCN, Runx2, ColⅠ) expression. ALP expression was significantly promoted after stimulation of 100 μmol·L⁻&sup1; NAR and BMP-2 (P<0.05).

CONCLUSIONSNAR exhibits promising potential for improving MC3T3-E1 proliferation and differentiation, and appropriate concentrations of NAR and BMP-2 show synergistic effect.
.

Bone Morphogenetic Protein 2 ; Bone and Bones ; Cell Count ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Collagen Type I ; Core Binding Factor Alpha 1 Subunit ; Flavanones ; pharmacology ; Osteoblasts ; Osteocalcin ; Real-Time Polymerase Chain Reaction

OBJECTIVETo evaluate the effect of bone morphogenetic protein(BMP7)on the differentiation of adipose derived mesenchymal stem cells(AD-MSCs)isolated from different adipose tissues into brown adipocytes in rats.

METHODSPrimary AD-MSCs were isolated from rate interscapular brown adipose tissue(iBAT),inguinal subcutaneous white adipose tissue(sWAT),and epididymal white adipose tissue(eWAT),respectively,and then cultivated in vitro. Differentiation of AD-MSCs into brown adipocytes was induced by BMP7. The characteristics of brown adipocytes were detected by immunofluorescence staining and oil red staining of cells. The expression levels of brown adipocyte-related genes were detected by polymerase chain reaction.

RESULTSAD-MSCs from iBAT and sWAT were differentiated into cluster multilocular cells,which were stained red by oil red "O"staining and showed uncoupling protein 1-positive by immunofluorescent staining method. AD-MSCs from eWAT had a small number of scattered multilocular cells and showed uncoupling protein 1-negative. The results of reverse transcription-polymerase chain reaction showed that the uncoupling protein 1 gene was highly expressed in the iBAT group and sWAT group but was negative in the eWAT group.

CONCLUSIONAD-MSCs isolated from different adipose tissues in rats have different gene expression profiles and differentiation potentials.

Adipocytes, Brown ; physiology ; Adipose Tissue ; metabolism ; Adipose Tissue, Brown ; physiology ; Animals ; Bone Morphogenetic Protein 7 ; metabolism ; Cell Differentiation ; physiology ; Ion Channels ; metabolism ; Mesenchymal Stromal Cells ; physiology ; Mitochondrial Proteins ; metabolism ; Obesity ; metabolism ; Rats ; Uncoupling Protein 1

OBJECTIVE: To evaluate the effect of biglycan on the signaling of cytokines (epidermal growth factor, osteogenic protein-1, and interleukin-1) in bovine intervertebral disc cells. METHODS: Nucleoplasty (NP) and annulus fibrosus (AF) cells of the intervertebral disc tissues were isolated from the tails of young adult bovine. First, the cells were treated in 3 ways: Biglycan alone, cytokines alone (epidermal growth factor, osteogenic protein-1, or interleukin-1), and biglycan combined with cytonkines. Western blot was used to observe the singling of biglycan and cytokines in bovine intervertebral disc cells, and to identify the effect of biglycan on cytokines mentioned above. RESULTS: Biglycan upregulated the signaling (3- 4 folds) with the optimal effect at 10 min and 20 μmol/L both in the AF cells and NP cells. Epidermal growth factor, osteogenic protein-1, or interleukin-1 also upregulated the protein expression in the extracellular matrix of intervertebral disc cells. When combined different biglycan concentrations with epidermal growth factor, osteogenic protein-1, or interleukin-1 to treat the intervertebral disc cells, the concentration of biglycan rose, whereas the cytokine signal decreased both in the bovine AF and NP cells (P<0.01). There was no significant difference between the AF and NP cells. CONCLUSION Biglycan can adhere to the intervertebral disc cells to activate the extracellular signal-regulated kinase (ERK) pathway and this effect is time and concentration dependent. Byglycan can decrease not only the anabolism effect of epidermal growth factor and osteogenic protein-1, but also the catabolism effect of interleukin-1. This regulatory role of biglycan may be very important to maintain the metabolism balance. Biglycan may be good for the repair of intervertebral disc.
Animals ; Biglycan ; physiology ; Bone Morphogenetic Protein 7 ; metabolism ; physiology ; Cattle ; Cells, Cultured ; Cytokines ; metabolism ; physiology ; Epidermal Growth Factor ; metabolism ; physiology ; Interleukin-1 ; metabolism ; physiology ; Intervertebral Disc ; cytology ; metabolism ; Signal Transduction