| BACKGROUND AND OBJECTIVEThere are almost50million fracture patients each year in our country, and the incidence of bone nonunion is about5%-10%, mainly results from infection after open fractures. Pain, limb function and psychological barriers, etc, results from delayed union and ununion of fracture, lead to great suffering to patients and financial burden of patients and society. Bone formation process is unity of opposites of the equilibrium process between osteoblast and osteoclast activity, in which bone matrix synthesis induced by osteoblast from mesenchymal stem cell (MSC) differentiation and bone matrix degradation induced by osteoclast from bone marrow mononuclear macrophages differentiation. Numberous of new osteoblast could accelerate bone synthesis and calcification in fracture healing process to increase bone mass and density. Research that chronic persistent inflammation to stimulate bone resorption has been inconclusive, however the effect of chronic persistent inflammation in osteoblast differentiation especially from MSC is not yet definitive so far, in spite of scholars have been searching for it.Although physiologic inflammation is a defensive reaction of the body and positive effects on fracture healing, persistent inflammation after infection or other factors have obvious negative consequences. Tumor necrosis factor-alpha (TNF-a) were found to express high levels in rheumatoid arthritis, patients with estrogen deficiency or insenescence. TNF-a, one of the most important promoting inflammatory cytokines, is involved in mediated immune response, induced local inflammatory response, regulation for proliferation, differentiation, apoptosis and functional activity of peripheral tissue cells, which impact tissue and organ development and function. TNF-a on the regulation of osteoblast function in the following respects:direct inhibition on osteoblast differentiation, mature and osteogenesis; stimulation on osteoblast to secrete osseous adjustment factor thereby promoting bone absorption; induction osteoblast apoptosis to reduce the number of osteoblast leading to inhibition of bone formation; influence the regulating effect of other differentiation factors or growth factors to osteoblast. In vivo experiment, TNF-a could inhibit the expression of key transcription factors of osteoblast, including Runx2and Osterix. The expression inhibition of Runx2induced TNF-a via loss of stability in mRNA of Runx2and cell apoptosis, and up regulation of Smurfl, Smurf2to result in Runx2degradation in osteoblast. For the inhibition to Osterix by TNF-a through the independent mechanism, that is to inhibit osteoblast differentiation relyed on indirect inhibition of Runx2activity according to multiple signaling pathways including MAPK, ceramide and oxidative stress, rather than NF-KB, c-Jun N-terminal kinase and P38lightning kinase pathways. To date the mechanism of TNF-a in osteoblast inhibition is complex and not very clear. The explore of mechanism which inflammation stimulation effect the osteoblast differentiation, promising potential drug targets to prevent inhibition of inflammatory factors on bone regeneration, is always payed close attention and badly in need of solution in such research field.Proteomics research in osteogenesis differentiationThere is a sophisticated s control system in the process of osteoblast differentiation by inflammatory stimulation, which not just be a sigle signaling pathway, but also be the cross talk among numerous signaling pathways. Possible strategies for circumventing inflammatory inhibition of tissue regeneration can be pursued partly once the signaling pathway(s) involved have been elucidated. It is still not to expound the comprehensive mechanism of osteoblast differentiation inhibition by inflammatory stimulation. Meanwhile, for most early reasearch, intervention which for inflammation inhibits osteoblast differentiation were focus on the single target blocking in sigle signaling pathway. However, to interrupte multiple targets of signaling pathways should be the latest research trends to prevent inhibition of bone regeneration induced by inflammation. Only in-depth exploration for mechanism of osteoblast differentiation by inflammatory stimuli, can we to discovery the potential specificity and medicinal targets, and then to develop new drugs based on composed targets to promote osteoblast differentiation in inflammatory microenvironment.Post-genome era, as the executor of the biological functions, protein becomes a hot research topic in the field of life science. Proteomics, is a high throughput screening and analysis, to the protein expression level, protein modification and function, interaction between proteins of all the organism, tissues or cells. Compared with single gene or protein research, proteomics could increase the reliability of the disease diagnosis and prognosis, and reflect the state of the body.The task is tremendously huge and almost impossible to finish, to study these proteins and detecte new pathogenesis based on the traditional biochemical technology. Proteomic technology, in detecting mechanism of inflammation on osteoblast differentiation, provides a powerful tool and method. In The study of osteoblast, it is concentrated two following respects:study the differences expression protein of MSCs to osteoblast and related mechanism, and detectet various reaction mechanisms of osteoblast after various biological and mechanical stimulation in vivo. There has be some progress and breakthrough results including the molecular mechanisms of osteoblasts formation and associated proteins related with osteoblast differentiation. Nonetheless, it has yet to get comprehensive information of proteins change, because of above proteomics research of osteoblast were based on single time points and lack of dynamic study. Furthermore, proteomics research of inflammation on osteoblast differentiation has not yet been reported.Therefore, base on the biological differences between normal osteoblast differentiation and differentiation in the inflammatory microenvironment, we used high-throughput iTRAQ technology combined with mass spectrometry identification method, determined and screened related proteins of osteoblast differentiation in mormal and inflammatory microenvironment, and then verified the functions of the differential proteins. The results of our study may delineate the mechanisms of osteoblast differentiation responsible for the inflammation stimulation, and shed new light on development of specific and medicinal multiple drug targets.The paper includes the following four parts:Part1:Cell model establishment and validation of osteoblast differentiation by inflammatory stimulationC2C12cell line were induced with BMP-2to osteoblast as cell model incubated with TNF-a as inflammatory stimulation. ALP activity and fluorescence quantitative analysis is a relatively late marker for osteoblast differentiation that is used to monitor the progression of osteoblast differentiation and validation of cell model.Part2:Proteome analysis of inflammation on osteoblast differentiation by iTRAQ To detect the mechanisms of inflammation on osteoblast differentiation and shed new light on development of specific and medicinal multiple drug targets. Total protein was taken out of each sample solution and then the protein was digested with Trypsin Gold. After trypsin digestion, peptides were dried by vacuum centrifugation and processed according to the manufacture’s protocol for8-plex iTRAQ reagent. Samples were labeled with the iTRAQ tags and the labeled peptide mixtures were then pooled and dried by vacuum centrifugation. SCX chromatography was performed and data acquisition was performed with a TripleTOF5600System. Related differential proteins of osteoblast differentiation in mormal and inflammatory microenvironment were screened and biological analysis, then dected the various possible functions of the differential proteins.Part3:Expression verification of differential proteins of inflammation on osteoblast differentiationDifferent levels of expression verification contribute to detect the mechanisms of inflammation on osteoblast differentiation and shed new light on development of specific and medicinal multiple drug targets. The interest proteins of ELP2and SERCA3were further expression verified by multiple reaction monitoring (MRM), Q PCR and Western Blotting, in order to provide evidence that above proteins paly important roles in inflammatory on osteoblast differentiation.Part4:The preliminary functional studies of ELP2in inflammation on osteoblast differentiationELP2also named as STAT3-interacting protein1(StIPl), regulating the ligand-dependent activation of STAT3. ELP2was found to be downregulated in BMP-2and then upregulated in BMP-2+TNF-a treated cells by iTRAQ quantitation in our study. There was no report about ELP2in inflammation on osteoblast differentiation. We constructed targeted ELP2siRNA and to investigate the effect of ELP2on the biological characteristics. Results demonstrate that down-regulation of ELP2can effectively ameliorate TNF-a inhibition of osteoblast differentiation which may delineate the mechanisms of osteoblast differentiation responsible for the inflammation stimulation, and shed new light on development of specific and medicinal multiple drug targets.METHOD1. Cell cultureThe mouse myoblast cell line C2C12was obtained from American Type Culture Collection. C2C12cells were cultured in DMEM supplemented with10%FCS and penicillin-streptomycin solution at37℃under a humid atmosphere of95%air and5%CO2.2. Establishment of cell modelsAfter preculture with serum-free DMEM, cells were treated with BMP-2(100ng/ml), TNF-a (5ng/ml), or at concentrations indicated in the figures.1) control: C2C12cells in DMEM;2) BMP-2:C2C12cells treated with BMP-2(100ng/ml) for7d;3) TNF-α:C2C12cells treated with TNF-a (5ng/ml) for7d;4) BMP-2+TNF-a:C2C12cells treated with BMP-2(100ng/ml) and TNF-a (5ng/ml) for7d.3. Alkaline phosphatase (ALP) stainingFor histochemical staining, ALP detection Kit was used to demonstrate the ALP activity. After culturing for7days with medium changed every3days, cells were washed with PBS and incubated with a mixture of naphthol AS-BI alkaline solution with fast red violet LB. The resulting red insoluble deposit indicates site and level of ALP activity. For quantitative analysis, cells were lysed after3days of culture, and cellular ALP activity was measured with a fluorometric detection kit using4-methylumbelliferyl phosphate disodium substrate following manufacturer’s instructions.4. Differential proteins identified by ITRAQAfter protein preparation including cell protein extraction and quantification for ITRAQ, the peptides were labeled with the isobaric tags. The iTRAQ-labeled peptide mixtures were reconstituted with4mL buffer A and loaded onto a4.6x250mm Ultremex SCX column containing5-μm particles. After SCX chromatography was performed with a LC-20AB HPLC Pump system, LC-ESI-MS/MS analysis were conducted based on Triple TOF5600. Protein analysis and identification by Mass Spectrometry and differential proteins were identified.5. Bioinformatics analysis of differential proteinsBioinformatics analysis for the differentially expressed proteins including Molecular Function, Cellular Component and Biological Process, then the Gene Ontology (GO) annotation, Cluster of Orthologous Groups of proteins (COG) and Cluster of GO, in order to detect the biological function of differential proteins. Protein-protein interaction analysis and enrichment analysis of pathway were conducted to determine the metabolic pathway and the signal transduction pathways which differential proteins involved.6.Differential proteins identified involved in osteoblast differentiation by inflammation stimulationDifferential proteins involved in osteoblast differentiation by inflammation stimulation were identified based on pattern recognition, clustering analysis and intersection processing for overall differential proteins above.7. Data processing and statistical methods of MRMThe raw data of MRM was processed using Multiquant software2.0.2. Signal Finderl (AB SCIEX, Foster City, CA, U.S.) with saturation correction at3.5e6was applied to calculate the corresponding peak areas of MRM signals. By setting S/N>10 and LOQ as a threshold, the filtered MRM signals were qualified for quantitative calculation.8. Western BlottingAfter incubation for7d, cells were lysed by RIP A buffer. Equal amounts of total cell lysate were then subjected to SDS-PAGE and immunoblotting analysis using antibody and anti-β-actin. β-actin was used as a loading control. The intensity of protein fragments was quantified with the Quantity One software.9. Quantitative real-time PCR (Q PCR) analysis for mRNA expressionTotal RNAs from cells were extracted using Trizol following the manufacturer’s instruction. Quantification of mRNAs was performed using the Quantitect SYBR Green PCR Kit with a MX3005P multiplex quantitative PCR system according to the manusfacturers’recommendations. GAPDH mRNA was used as a housekeeping gene. The relative expression levels of mRNAs were calculated using the comparative△△CT method. The fold-changes were calculated by the equation2-△△CT.10. RNA oligos and transfection3siRNA of ELP2(ELP2siRNA) and negative siRNA (siRNA NC) were purchased from GenePharma (Shanghai, China). Cells in the exponential growth phase were plated in24-well plates at a density of30-50%confluence, followed by incubation for24h. The cells were then transfected with RNA oligos according to the manufacturer’s instruction.11. Statistical analysisSPSS13.0software (SPSS, San Rafael, CA) was used for statistical analysis. Data were presented as mean±SD of at least3independent experiments. Multiple comparisons using LSD method and Dunnett’s T3method for the unequal variances. Statistical significance was set at a=0.05.RESULTS 1. Cell model establishment and validationThere were significance difference in ALP activity of C2C12cells incubated with BMP-2alone or combined with TNF-a (F=134.18, P=0.00). Compared with normal control group, C2C12cells incubated with BMP-2alone demonstrated strong ALP activity and there were significance difference (P=0.01); compared with BMP-2group, C2C12cells incubated with TNF-a alone markedly reduced the ALP activity and there were significance difference (P=0.00); compared with BMP-2group, there were also significance difference of C2C12cells incubated with BMP-2and TNF-a in ALP activity (P=0.01); meanwhile, compared with TNF-a group, there were also significance difference of C2C12cells incubated with BMP-2and TNF-a in ALP activity (P=0.02). The results of fluorometric quantitative analysis indicated that TNF-a could markedly reduced the osteoblast differentiation induced by BMP-2.2. Differential proteins identified by ITRAQQuantitative proteomic analysis of the C2C12cells proteome of control, BMP-2and BMP-2+TNF-a total of365,226MS/MS spectra acquired from the LC MS/MS analysis resulted in the identification of21,807unique peptides corresponding to4,822proteins. Out of these4,822proteins,22were differentially expressed between N vs BMP-2by≥1.5-fold,7were up and15were down in BMP-2;33were differentially expressed between N vs BMP-2by≥1.5-fold,25were up and8were down in BMP-2;71were differentially expressed between BMP-2vs BMP-2+TNF-a by≥1.5-fold,56were up and15were down in BMP-2+TNF-a.Finally,5were the intersection of differentially expressed proteins between N vs BMP-2, N vs TNF-a and BMP-2vs BMP-2+TNF-a, those were protein S100-A4, ATPase inhibitor (mitochondrial), Cytochrome b5,SERCA3and ELP2. S100-A4, ATPase inhibitor (mitochondrial), Cytochrome b5and ELP2were found to be downregulated in BMP-2and then upregulated in cells with BMP-2+TNF-a treated by iTRAQ quantitation in our study. SERCA3was found to be upregulated in BMP-2and then downregulated in cells with BMP-2+TNF-a treated. 3. Expression validation of ELP2and SERCA3Expression validation of ELP2and SERCA3were explored by the methods of MRM, Western Blotting and Q PCR. ELP2was found to be downregulated in BMP-2and then upregulated in cells with BMP-2+TNF-αtreated. SERCA3was found to be upregulated in BMP-2and then downregulated in cells with BMP-2+TNF-a treated. As demonstrated by Q PCR, there were significance difference in expression of ELP2incubated with BMP-2alone or combined with TNF-a (F=94.33, P=0.00). Compared with normal control group, C2C12cells incubated with BMP-2alone demonstrated lower ELP2expression and there were significance difference (P=0.00); compared with BMP-2group, C2C12cells incubated with BMP-2and TNF-a markedly improved the ELP2expression and there were significance difference (P=0.00). Meanwhile, there were significance difference in expression of SERCA3incubated with BMP-2alone or combined with TNF-a (F=88.54, P=0.00). Compared with normal control group, C2C12cells incubated with BMP-2alone demonstrated higher SERCA3expression and there were significance difference (P=0.00); compared with BMP-2group, C2C12cells incubated with BMP-2and TNF-a markedly reduced the SERCA3expression and there were significance difference (P=0.00). Basically, the results for differential expression of ELP2and SERCA3by the methods of MRM, Western Blotting and Q PCR were consistent with the iTRAQ data.4Knock-down of ELP2ameliorates TNF-a inhibition of osteoblast differentiationELP2was down-regulated in C2C12cells by RNA interference (RNAi) to explore the effect of ELP2on the osteoblast differentiation. Three siRNA sequences were synthesized to inhibit the ELP2expression of C2C12cell. The results showed after transfection for24h, all of these siRNAs inhibited the ELP2expression. The most effective one was chosen in this experiment. Colorimetric staining of ALP in C2C12cells treated with BMP-2and/or TNF-a with addition of either the ELP2siRNA or the siRNA NC showed that ELP2siRNA was able to block most of the TNF-a inhibition of BMP-2activity while siRNA NC had almost no effect. Quantitative fluorometric analysis showed that there were significance difference in ALP activity of C2C12cells following siRNA NC or ELP2siRNA transfection (F=33922.40, P=0.00). After multiple comparisons, the results showed that, compared with normal control group, C2C12cells incubated with BMP-2alone demonstrated strong ALP activity and there were significance difference (P=0.00); compared with BMP-2group, the ALP activity reduced from9.8to2.8-fold (with basal ALP of one sample as1) and there were also significance difference of C2C12cells incubated with TNF-a and siRNA NC in ALP activity (P=0.01); compared with TNF-a+siRNA NC group, the ALP activity improved from2.8to5.7-fold (with basal ALP of one sample as1) and there were also significance difference of C2C12cells incubated with TNF-a and ELP2siRNA in ALP activity (P=0.00).Conclusions1. The osteoblast differentiation of BMP-2-induced could be inhibited by TNF-a, and cell model of osteoblast differentiation by inflammatory stimulation in our study is valid.2. Protein S100-A4, ATPase inhibitor (mitochondrial), Cytochrome b5and ELP2were found to be downregulated in osteoblast differentiation and then upregulated in osteoblast differentiation by inflammatory stimulation. SERCA3was found to be upregulated in osteoblast differentiation and then downregulated in in osteoblast differentiation by inflammatory stimulation.3. After expression validation of ELP2and SERCA3were explored by the methods of MRM, Western Blotting and RT-PCR, the expression ELP2was found to be downregulated in osteoblast differentiation and then upregulated in osteoblast differentiation by inflammatory stimulation. SERCA3was found to be upregulated in osteoblast differentiation and then downregulated in in osteoblast differentiation by inflammatory stimulation.4. Knock-down of ELP2ameliorates TNF-a inhibition of osteoblast differentiation, ELP2may be a specific and medicinal drug target to intevent the inhibition of osteoblast differentiation by inflammation. |
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