Membrane Receptor Mechanism Of Human Vascular Smooth Muscle Cells Phenotype Switching Regulated By Cellular Repressor Of E1A-stimulated Genes(CREG)

Object Atherosclerosis(AS)and restenosis(RS) post percutaneous coronary intervention(PCI) are most danger to human health as cellular proliferative desease. It is important to study how to prevent AS and RS post-PCI. Recent study suggested mature vascular smooth muscle cells(VSMC) had remarkable plasticity. In physiologic condition, VSMC show the differentiation phenotype. Whereas, when injury occurred, the VSMC would transform its differentiation phenotype to un-differentiation phenotype and gain the abilities to migrate, proliferate and metabolize extracellular matrix. The phynotye transition of VSMC is the pathologic mechanism of AS and RS post-PCI. So, to study the mechanism of phynotype transition is an important aspect to provent hyperplasic vascular desease.In 1998, Gill's study showed the cellular repressor of E1A-stimulated genes(CREG) could promote cell differentiation and maturity. Also, CREG might effect on the maintenance of cellular differentiation status. In 1999, we firstly cloned the CREG genes with mRNA differential display technology from human internal thoracic artery-Shenyang(HITASY-Shenyang)and confirmed that expressing of CREG was highly correlated to VSMC's differentiation, which suggested CREG took part in switching from proliferation phenotype to differentiation phenotype. While, the mechanism of CREG, as one secreted glycoprotein, promotes cellular differentiation and inhibits its proliferation need to be defined. By Far Western assay, Gill et showed the CREG executed the biologic function after binding to the mannose-6-phosphate/insulin-like growth factorⅡ(M6P/IGF2R) depended its glycosylation sites. To confirm the role of M6P/IGF2R in the CREG biologic function, we decreased the quantity of M6P/IGF2R on the cellular membrane using okadaic acid and found the effect of CREG on promoting VSMC differentiation was inhibited, which suggested the M6P/IGF2R might mediate the process of CREG regulating VSMC differentiation.This study intend to clear if CREG directly bind to M6P/IGF2R, if the binding depends on its glycosylation sites, if the binding is on the VSMC'membrane and the mechanism of M6P/IGF2R mediating CREG biologic function.Methods (1)The ORF(open reading frame) fragment of human CREG(wtCREG) with terminator codon mutation was amplificated by RT-PCR and the eukaryotic expression vector, pcDNA3.1 myc-His/wtCREG, was constructed. At the same time, change the Asn residue of the glycosylation sites (160,193,216 Asn residue) with Ala residue and construct expression vector pcDNA3.1 myc-His/mCREG. The human 293F cells were transfected with pcDNA3.1 myc-His/wt/mCREG using Lipofectamine 2000, and stably transfected cell clones were selected by G418. The recombinant secreted wt/mCREG/myc-His protein were purified by Ni-NTA affinity chromatography and characterized with glucosidase and Western blot assay. Measure the concentration compared to the standard albumn.(2)The optimal effect concentration of CREG on VSMC was studied. Add .the wt/mCREG with optimal concentration to HITASY cell (VSMC line gained from human internal thoracic artery) and OB2 cell (HITASY which do not express CREG by retrovirus jamming the shCREG). The effects of CREG on the change of proliferation, migration and differentiation of VMSC were studied by cell cycle, wound-healing, Western blot assay and gelatinase digestion analysis. To clear the role of M6P/IGF2R in the CREG biologic function, we add M6P/IGF2R anti-body (2,4,8nM) to OB2 cell with wt/mCREG at the same time to study the blocking effect. Also, double immunofluorescence staining and co-immunoprecipitation were performed to analyses if CREG peotein directly binded to M6P/IGF2R.(3)Segments of M6P/IGF2R domains were recombined by RT-PCR(IGF2R-1:1~3.domains; IGF2R-2:4~6.domains;.IGF2R-4:7~10.domains).PQE31 IGF2R-1,2,3 expression vectors with His-label were constructed and transfected to E coli JM109. The domains protein was gained by Ni-NTA affinity chromatography and purified. SDS-PAGE and Western blot assay were performed. Measure the concentration compared to the standard albumin. To analyses binding of CREG with M6P/IGF2R domains(IGF2R-4:11~15 domain bought from R&D company) and calculate the dissociation constant (KD value) using solid phase binding experiment. Then, the segment which wt/mCREG most binding to was identified.M6P and G6P blocking test was performed to study if the glycosylated structure of CREG mediated its binding to M6P/IGF2R by M6P. The in vivo competitive binding test was study by adding the segment protein which CREG most binding to into the supernatant with wt/mCREG receptively. The change of CREG biologic effects on VSMC were analyseses to identified the direct binding of CREG to IGF2R domains。 Results (1) Produce and purify the recombinant wt/mCREG protein: 1) The ORF(open reading frame) fragment of human CREG with terminator codon mutation was amplified by RT-PCR and inserted into PMD-18T vector, which was confirmed by restricted endonuclease digestion and DNA sequencing. Subclone the CREG cDNA fragment into pcDNA myc-His vector and the eukaryotic expression vector pcDNA3.1 myc-His/wtCREG was constructed which was confirmed by DNA sequencing. The designed CREG cDNA(combined by biotechnique company) with amino acid residue mutated was subcloned into pcDNA myc-His vector by the BamH I/EcoR digestion, which was confirmed by DNA sequencing.2)The recombinant expression vector, wt/mCREG, were transfected to 293F engineering cell sand selected by G418 to obtain the cell clone with CREG high expressing. The cell clone were amplified and harvested. After that, the two kind recombinant fusion proteins, wt/mCREG with myc/His label, in 293 cell lysates were purified by Ni-NTA affinity chromatography and tested by PNGaseF digestion and Western blot assay. SDS-PAGE showed the molecular mass. The concentration were 0.893μg/μl and 0.972μg/μl and the purity were 92% and 93% respectively compared to standard protein.(2)CREG protein biologic function study and the membrane surface receptor mechanism study:1)the optimal effect concentration analysis: add different concentration (200,400,800,1600 nM) wt/mCREG to supernatant of the synchronized HITASY and OB2 cells which were cultured with free serum medium for 72h. After eight hours, the cells were harvested and cell cycle was analysesed showed the ratio of cell in GO/G1 period was increased in all of group. The effect of wtCREG is more powerful than that of mCREG..The optimal concentration of two kinds of CREG proteins was 400nM. The study showed the recombinant wt/mCREG protein depressed the VSMC proliferation depending on dose and the optimal concentration was 400nM;2)biologic function of CREG protein and the membrance receptor mechanism:①effect on VSMC migration: the wound healing experiment showed the OB2 cells migration was slower significantly after added wt/mCREG(400Nm) in supernatant. The HITASY cells migration were very slowly and no remarkable change. The gelatinase digestion and Western blot analysis showed the matrix metalloproteinase(MMP) was decreased and TIMPs was increased;②effect on differentiation: after added wt/mCREG(400nM), the expression of myocardin, SMα-actin, MHC and caldesmin were increased and that of LM-1 and FN were decreased in OB2 cells. These effects were more significant when adding wtCREG.;③effect on VSMC proliferation: Cell cycle assay and BrDU stain showed: after added the wtCREG and mCREG protein, the ratio of cell in G0/G1 phase increased to 0.5773 and 0.5572 from 0.5308 respectively in OB2 group, which increased to 0.7369 and 0.7034 respectively from 0.6297 in HITASY group;3)Role of M6P/IGF2R in CREG biologic function:①ELISA and co-immunoprecipitation showed the wt/mCREG binding to M6P/IGF2R directly.②antibody blocking test: when the anti-IGF2R was added to medium at the same time with wt/mCREG at different concentration(2μg/mL,4μg/mL,8μg/mL),the effects of CREG protein which depressing proliferation, migration, secretion and promoting differentiation were blocked, which had the positive correlation to the concentration of added anti body. The studies showed two combinant CREG promoted VSMC switch to differentiation phaenotype, at the same time, depress VSMC proliferation, migration and secreting extracellular matrix. The function of wt/mCREG can be blocked by anti-M6P/IGF2R. CREG protein binded to M6P/IGF2R directly;(3)analysis of which M6P/IGF2R domain CREG most likely bind to:1) The ORF(open reading frame) fragment of human M6P/IGF2R domains with terminator codon mutation was amplified by RT-PCR and were inserted into PMD-18T vector which were confirmed by DNA sequencing. The M6P/IGF2R cDNA were subcloned to the prokaryotic expression vector PQE31-His and construct the PQE31 IGF2R-1,2,3 recombinant plasmid which were confirmed by DNA sequencing. The recombinant expression vectors were tranfected into Eo1 JM109 and amplified. The expressed M6P/IGF2R domain proteins were gained by Ni-NTA affinity chromatography and analysesed by SDS-PAGE and Western blot. The SDS-PAGE showed the molecular mass were 54kD(IGF2R-1),44kD(IGF2R-2),62kD(IGF2R-3). Adjust the concentration to 10nM. ELISA analysis suggested the wtCREG had the most affinity to IGF2R-3 and IGF2R-4, while the mCREG to IGF2R-4.The KD value were 0.1063pM, 0.2106pM and 6.488pM respectively;The binding of CREG protein to M6P/IGF2R-3 was depressed by M6P; 3) Add the M6P/IGF2R-3 and M6P/IGF2R-4 to OB2 cell with wt/mCREG at the same time by the different concentration. The study showed M6P/IGF2R-4 blocked the effects of wt/mCREG depressing VSMC proliferation and migration, while M6P/IGF2R-3 only blocked the effects of wt/CREG was blocked by M6P/IGF2R-4.Conclution Binding to cytomembrane M6P/IGF2R, CREG protein can down-regulate the proliferation, migration and up-regulate differentiation of VSMC promoting the cells'phaenotype transform from undifferentiation to differentiation no matter if glycosylation structures exist. But , the biologic function is more remarkable when the glycosylation structures exist. CREG protein has different affinity to M6P/IGF2R domain depend on glycosylation structures existing or not. The wtCREG showed high affinity to the 7-10 domains but the mCREG to 11-15 domains.