Expression And Function Of METEORIN-like Secreted Protein In Bone

Bankgroud:periosteal bone apposition increase the bone diameter,cortical bone thickness,and is important for the bone strength,reducing the bone fracture;and Compared to endosteal osteoblasts,periosteal osteoblasts exhibit greater mechanosensitivity to strain,a lower threshold of responsiveness to parathyroid hormone,higher levels of expression of estrogenαreceptors.So the new drug targeting the periosteum may be an alternate strategy to protect human bones from fracture.But for a long time,the molecular mechanism of periosteal osteoblasts differentiation and function is unclear.The response of periosteam to anti-osteoporosis drug is unknown.We have paid attention to the periosteal osteoblast for a long time.Previous we established establish the in vitro culture model ofperiosteal cells,and studied the response of periosteal osteoblasts to estrogen.We construct periosteum cDNA library and the. differently expressing genes library(SSH library) of human periosteal cells between estrogen and control treatment.Objective:Now we used Dual-luciferase reporter system to screen for unknown function genes expression in periosteum,and studied the expression pattern of the interesting gene in bone and explored the gene's function in osteoblasts.Methods:Part 1:high-throughput screening of novel gene expressing in perioteal cells that could influence the signal pathway(1) Gene clone:The target gene sequence originates from the periosteum eDNA library and the SSH library.PCR methods were used to expand the ORFs area and cloned into eukaryotic expression vector pCDNA3.1(2) Using dual luciferase reporter gene system to screen new function gene that c ould influence signal pathway:the Reporter plasmid(pAP1-luc/pNFAT-luc/pCRE -luc/pNFkB-luc/pStat-luc),which codes a firefly luciferase under the control of pAP1-luc/pNFAT-luc/pCRE-luc/pNFkB-luc/pStat-luc consensus sequence in the p romoter region:an internal control vector pRL-TK and the target gene in pCD NA3.1 were co-transfected into 293T cell,activity of both Firefly and Renilla luciferase are measured to identify the new gene that could influence the sig nal pathwayPart 2:Bioinformatics analysis of METRNLPart 3:the location in MG63 and the expression pattern of METRNL(1)Prokaryotic expression of the polypeptide 83-311 and its antibody preparation(2) cell localization:the pEGFP fluorescence reported gene and Indirect Immunofluorescence methods were used to study the localization of METRNL in MG63; western blot was used to exam the protein in CHO cell transfected with METRNL expression vector(3)using northern blot to exam the mRNA level of METRNL in multiple tissue membranePart 4:the expression pattern of METRNL in bone and its function in osteoblast(1)Immunohistochemistry was used to identify the location and distribution of METRNL in the bone of different age SD rat(2)MG63 cells were stable transfected with pEGFP-METRNL to overexpress the METRNL,the number of mineralization node in osteogenic supplement was compaired with control.And the difference of ALP,OPG was also compared.Result:part 1:we succeeded in cloning five new genes expressing in periosteum osteoblasts into pCDNA3.1.After screening with Dual-luciferase reporter system,we find that METRNL cotransfected with cis-reporter vector can significantly inhibit the transcription activity of AP-1 by 74%when without stimulating as compared with control;after stimulated with PMA and ionomycin,the activity of AP-1 are also inhibited.In addition,the transcription activity of CRE and NFAT are also inhibitedpart 2:Bioinformatics analysis show that human METRNL mRNA contains an open reading frame that encodes a protein of 311 amino acids.The encoded amino-acid sequence showed no significant homology to the sequence of any other previously described protein except the meteorin with 42%homology,suggesting that METRNL is an novel protein.The predicted protein contains a putative signal peptide in its NH-terminal region.Part 3:(1)both the fusion protein EGFP-METRNL and the Indirect Immunofluorescence result shows that the METRNL located in cytoplasm of MG63.After purified the protein from CHO culture medium,we can detected the METRNL by western blot,so METRNL is a secreted protein.(2)Northen blot show that the METRNL mRNA is about 1500 bp,expressing in adult brain,heart,liver,kidney,spleed,ovary,muscle and uterus,but the expression level is very lowPart 4:(1)osteoblasts lining the trabecular and periosteal bone surface express high level of METRNL.In bone,expression of METRNL is highest in embryos and neonates,and decreasing steadily with age to very low levels in 12 month-old long bones.(2) the MG63 cells overexpressing METRNL shows lower mineralization node after 14 days inducing by osteogenic supplement,but the ALP level is higher and the OPG level is lower as compared with control after inducedConclusion:METRNL is a novel secreted proteinMETRNL maybe display its function through AP1,NFAT and CRE.METRNL mainly express in actcive osteoblasts,maybe involve in controlling bone formation.METRNL can significantly enhance the matrix production,but overexpression can inhibit the further differentiation...