Osteonectin Regulates The Extracellular Matrix Mineralization Of Osteoblasts Through P38 Signaling Pathway

Background and objective:Extracellular matrix mineralization is an essential and critical step for bone repair and reconstruction.Type ? collagen secreted by osteoblasts is a major component of extracellular matrix and provides the basic framework for mineralization,while the non-collagen proteins are engaged in regulating hydroxylapatite deposition.Osteonectin is one of the important non-collagen proteins,also known as SPARC(secreted protein acidic and rich in cysteine)or BM-40,it has a high affinity with type I collagen and hydroxyapatite and is involved in the whole process of bone matrix mineralization;osteonectin also regulates the synthesis of extracellular Matrix and Collagen,affects the number and function of osteoblasts,it plays an important role in the regulation of mineralization.P38 MAPK(p38 Mitogen-activated protein kinase pathways)is an important signal transmission pathway and regulates alkaline phosphatase activity and extracellular matrix mineralization in response to different osteogenic signals in osteoblasts.Previous studies on the role of SPARC in mineralization have mostly focused on gene knockout animal experiments,the purpose of our research is to provide more direct experimental basis and to investigate whether p38 MAPK is an important signal transduction pathway,which can afford functional sites for the effective intervention of bone matrix mineralization.Methods:Part ?:The isolation,culture and identification of the osteoblasts The osteoblasts were isolated by enzymatic digestion from the calvarium of newborn SD rats(<48 hours)and purified by differential adhesion method.Alkaline phosphatase staining was used to determine the positive rate of osteoblasts and Alizarin red staining was applied for the detection of mineralized nodules in the osteoblasts.Part ?:The effect of different concentrations of osteonectin on the expression of OCN,OPN,BSP and p38 MAPK genes during the mineralization of osteoblastsOsteoblasts were randomly assigned into different groups:A(Control)group;B group,adding 0.01?g/ml osteonectin;C group,adding 0.1?g/ml osteonectin;D group,adding 1?g/ml osteonectin;E group,adding 10?g/ml osteonectin.The gene expressions of non-collagen proteins(OCN:osteocalcin;OPN:osteopontin;BSP:bone sialoprotein)and p38 MAPK were detected by RT-qPCR(reverse transcription quantitative polymerase chain reaction)on the 2nd,5th,and 8th day to clarify the role of osteonectin in the mineralization of osteoblasts,the optima osteonectin concentration and the observation time were determined according to the results.Part ?:The effect of osteonectin on other mineralization indexes of osteoblasts and the blocking effect of SB203580(p38 MAPK specific blocker)According to the results of part ?,we further confirmed the positive regulatory role of the osteonectin and investigated the Specific mechanism of p38 in osteoblasts,the experiment was reclassified as followed:Control group,added no osteonectin;Optima osteonectin group,added with optima concentration osteonectin;p38 Blocker Group,this group was pretreated with SB203580(a specific inhibitor of p38 MAPK signaling pathway)for 2 hours before adding the optima osteonectin.RT-qPCR was also performed on the mRNA expressions of SIBLINGs(Small Integrin-Binding LIgand N-linked Glycoproteins)members:DMP1(Dentine matrix protein 1)?DSPP(Dentin sialophosphoprotein)?MEPE(Matrix extracellular phosphoglycoprotein),the intracellular calcium ion were detected by flow cytometry,the bone formation were identified with Alizarin red staining and the osteoblasts of each group were observed by transmission electron microscopy(TEM);Western blot analysis was used to measure the protein expressions of p38 MAPK and P-p38.Part ?:Construction of recombinant adenovirus vector P38MAPK and lentiviral vector P38MAPK-shRNAConstruction of Ad-p38 MAPK:p38 MAPK primers were designed and added with the restriction site according to the Gene Library;the rat heart cDNA was used as a template for PCR amplification;then the target gene fragment was inserted into the corresponding restriction site of pShuttle-CMV to construct the recombinant adenovirus shuttle plasmid;after digested and linearized by enzymes,it was co-transformed with AdEasyTM into BJ5183 strain,then the AD-p38MAPK DNA were extracted and recombined to transfect cells.Construction of p38 MAPK-shRNA vector:p38 MAPK targeting sequences shRNA1?shRNA2?shRNA3 and the control sequences were chemically synthesized;after digested and linearized by enzymes,the sequences were transfected into the cells by linking lentivirus shuttle plasmids and auxiliary packaging;the virus titer and the protein expression was detected to select the most disturbing one for the follow-up test.Part ?:Further study on the role of p38 signaling pathway in the regulation of osteoblast mineralization by osteonectinIn order to further investigate the role of p38 MAPK,AD-p38 and p38-rhRNA virus vectors were constructed,the groups were divided as followed:Optima osteonectin group;adding optima osteonectin;p38 Blocker Group,adding optima osteonectin with 20.0 ?mol/l SB203580;AD-p38 MAPK group,the osteoblasts were transfected with AD-p38 MAPK and added with optima osteonectin;p38 MAPK-shRNA group,the osteoblasts were transfected with p38 MAPK-shRNA and added with optima osteonectin;the mRNA and protein expressions of BSP?OCN?OPN?DMP1?MEPE?DSPP and p3 8 MAPK were measured by RT-qPCR and western blot analysis.Results:Part ?:The positive rate detected by Alkaline phosphatase staining of the isolated osteoblasts was close to 100%and the mineralized nodules were observed by Alizarin Red staining and tetracycline culture;the results were satisfactory.Part ?:Different concentrations of osteonectin had different effects on the gene expressions of non-collagen protein(OCN,OPN,BSP)and p38 in osteoblasts;1?g/ml osteonectin was chosen as the optima one and the 5th day was the observation time,which significantly elevated the mRNA expressions of BSP,OCN,OPN and p38 MAPK in comparison with the control group(group A)(OCN,p<0.01;OPN,p<0.05;BSP,p<0.05;p38,p<0.001);these results indicated that osteonectin had a positive effect on mineralization and had activated the p38 MAPK pathway in osteoblasts.Part ?:The results further confirmed the positive regulation of osteonectin on osteoblast mineralization,and the p38 MAPK was its important signal transduction pathway.Compared with control group,the proteins expressions of p38 and P-p38 in the Optima SPARC group were significantly elevated(p<0.001),the expressions of DMP1?DSPP and MEPE genes in the optima osteonectin group were all significantly increased(DMP1,p<0.01;DSPP,p<0.01;MEPE,p<0.001),the intracellular calcium ion in the optima SPARC group was also significantly increased(versus control group,p<0.05);there were more mineralized nodules stained with alizarin red and the osteoblasts were more active containing more mineral-containing vesicles in the optima osteonectin group.While added with SB203580(p38 MAPK specific blocker),the protein expression of P-p38 was decreased significantly(versus optima osteonectin group,p<0.01),and the gene expressions of the BSP?OCN?OPN?DMP1?MEPE?DSPP were also decreased significantly(versus optima osteonectin group:BSP,p<0.01;OCN,p<0.05;OPN,p<0.05;DMP1,p<0.05;DSPP,p<0.05;MEPE,p<0.05),the activity of osteoblasts was inhibited observed by TEM.Part ?:The gene fragment electrophoresis and sequence analysis of the p38MAPK recombinant adenovirus vector were consistent with Gene Bank;compared with the control sequence,p38mapk-shRNA3 was selected to transfect osteoblasts(shRNA1,shRNA2,p<0.01;shRNA 3,p<0.001),the virus titer in the transfected group was 1.7×108IU/ml(the control group:2×108 IU/ml),the result is up to standard.Part ?:The important role of p38 MAPK in the regulation of osteoblast mineralization by osteonectin was further tested;compared with the optima group,the mRNA and protein expressions of BSP?OCN?OPN?DMP1?MEPE?DSPP and the protein expression of P-p38 were all significantly elevated in the Ad-p38 group,but all significantly decreased in p38 MAPK-shRNA group and P38 Blocker Group.Conclusion:Osteonectin regulates the extracellular matrix mineralization of osteoblasts through P38 signaling pathway:?Osteonectin has a positive regulatory effect on osteoblast mineralization:while added with 1?g/ml osteonectin,the expressions of non-collagenous proteins(OCN,OPN,BSP)and Sibling Family Proteins(DMP1,DSPP,MEPE)were significantly increased,the concentration of intracellular calcium were obviously elevated,the formation of mineralized nodules and the osteogenesis activity were promoted in osteoblasts.?The p38 MAPK signaling pathway can be activated by osteonectin in mineralization:the gene and protein expressions of p38 MAPK were significantly increased by 1?g/ml of osteonectin in osteoblasts.?The p3 8 MAPK is an important signaling pathway for the regulation of osteoblast mineralization by osteonectin:added with p38 blockers or transfected with p38-shRNA,the positive regulation of osteonectin on mineralization was significantly decreased in osteoblasts;while transfected with AD-p38,the positive regulation of osteonectin on osteoblast mineralization was obviously upregulated.