The Effects And Mechanisms Of PTP1B Phosphorylation Level On The Adhesion And Osteogenesis Of MSCs In SCR Technology

Background:Selective cell retention(SCR)technology,is of an improvement on the construction strategy of bone tissue engineering,has been employed by clinical settings to rapidly manufacture instant bioactive tissue-engineered bone to satisfy the need of bone grafts for spine or joint fusion and the treatment of bone defects.Cell-matrix adhesion is the primary and first step in constructing artificial transplants.Especially in SCR technology,enhancement of adhesion between the osteogenic-related cells and the scaffold would be crucial to further improve the in vivo osteogenesis of SCR-prepared grafts.Non-receptor protein-tyrosine phosphatase 1B(PTP1B)has emerged as a pivotal player in the crosstalk between the Integrin adhesion pathway and the cadherin adhesion pathway.It is speculated the phosphorylation state of PTP1B tyrosine-152(Y152)serves as a central fulcrum in balancing the two different cell adhesion pathways.Thus,inhibition of phosphorylation of PTP1B Y152 via a specific manner would weaken the cell-cell adhesion that mediated by the Cadherin pathway meanwhile strengthen the cell-matrix adhesion that mediated by the Integrin pathway.Method:1)We designed a PTP1B Y152 region-mimicking(152RM)peptide which consists of a sequence of fifteen amino acid residues that covers the domain of and centered by PTP1B Y152 residue,and a cell-penetrable TAT sequence of eleven amino acid residues.By using a solid-phase synthesis method,the 152RM peptide and its Y152/153f mutant control peptide,and the FITC-labeled version of the above peptides were obtained and identified.2)By using a laser confocal microscope,the endocytosis of FITC-152RM peptide and FITC-mutant 152RM peptide was observed.An orthogonal experiment was designed to determine the time-dose-effect relationship of the 152RM peptide on inhibiting the phosphorylation of PTP1B Y152 in suspended human bone marrow-derived MSCs,as detected by Western Blot.3)Western Blot and Co-IP assay were used to determine the phosphorylation changes,and the binding between related proteins,respectively,upon treatment with the 152RM peptide or the mutant 152RM peptide in the presence or absence of different inhibitors.The biocompatibility of the 152RM peptide was determined by LIVE/DEAD cell staining kit.4)We designed a cell sedimentation-driven adhesion(CSDA)method to uniform the initial adhesion time of MSCs to determine the early adhesion of MSCs,and allowed the collection of total proteins in each time point during early MSCs adhesion to analyze the changing profiles of phosphorylation of FAK and Src by Western Blot,and the active profiles of Rho family small GTPases Rac1,Cdc42 and RhoA.5)The centrifugal cell adhesion assay that based on the CSDA method was used to determine the matrix-anchor ability of MSCs in early adhesion.Also,based on the CSDA method,the adhesion morphology of MSCs in mid-term and late adhesion was observed.Base on another optimized method of cell plating,the cytoskeleton reconstruction,filopodia and lamellipodia formation of MSCs in early adhesion were observed by rhodamine-labeled phalloidin;the adhesion areas of MSCs in mid-term and late adhesion were observed and determined by a laser confocal microscope after staining the cells with Integrinβ1-Alexa Fluor 647 antibody.6)In SCR technology,the 152RM peptide pre-cultured bone marrow was used to enrich the DBM scaffold,and that the number and percentage of retained CD271~+cells,CD90~+/CD105~+cells and karyocytes were analyzed by flow cytometry.7)qPCR,Western Blot and ALP activity assay were used to determine the mRNA and protein expression of osteogenic markers of MSCs that effected by the 152RM peptide in an osteogenic environment;the proliferation of MSCs that effected by the 152RM peptide in a growth medium environment was detected by the CCK-8 method.8)The arithmetic mean intensity,as determined by a laser confocal microscope,was served as an index to evaluate the remaining FITC-labeled 152RM peptide that was modified to DBM by mimic SCR or soak at different concentration.qPCR was employed to confirm if the remaining peptide on DBM,via transient modification with mimic SCR,has certain effects on the osteogenic differentiation of MSCs.9)The nuclear protein of MSCs was extracted to analyze the nucleic localization ofβ-catenin;Different inhibitors were employed to interfere the Wnt/β-catenin pathway and the Integrin pathway to explore the mechanism of the positive effect of the 152RM peptide on MSCs,as detected by Western Blot.10)Both the subcutaneous ectopic osteogenesis model and the femur-adjacent osteogenesis model established with athymic mice were employed to evaluate the in vivo osteogenesis of SCR-prepared grafts,as directly determined by a micro-CT,H&E staining and Masson’s trichrome staining.Results:1)The 152RM peptide,mutant 152RM peptide and the FITC-labeled version of the above peptides were commercially synthesized and identified.The FITC-152RM peptide and FITC-mutant 152RM peptide were able to be greatly endocytosed by suspended MSCs within 60 min;the lowest concentration of 152RM peptide that could decrease the Y152phosphorylation of full-length PTP1B and truncated PTP1B was shown to be 0.33μM upon treatment of 152RM to MSCs for 60 min.Importantly,the 152RM peptide did not show cytotoxicity to MSCs.2)By treating suspended MSCs with 152RM peptide for 60 min,along with the great decrease of the Y152 phosphorylation of full-length PTP1B and truncated PTP1B,the phosphorylation ofβ-catenin Y654 and Src Y419 were significantly increased,and the phosphorylation of Src Y530 was greatly decreased.Meanwhile,the binding between total Cadherin and PTP1B andβ-catenin was greatly inhibited,respectively.Nevertheless,the mutant 152RM peptide did not show similar effects,and those effects can be blunted or reversed by PTP1B inhibitor TCS-401.3)Based on the CSDA method,the centrifugal cell adhesion assay showed the 152RM peptide-treated MSCs had a greater ability to anchor the matrix as compared with that of vehicle-treated MSCs.Moreover,during the mid-term and late adhesion,the 152RM peptide-treated MSCs showed a more active adhesion pattern than those of vehicle control.4)Based on another optimized method we designed to plate the cells,the 152RM peptide-treated MSCs were active to initiate adhesion and exhibited more filopodia and lamellipodia,and began to exhibit a near anisotropic spreading pattern in an earlier time than vehicle-treated MSCs did in the early stage of adhesion.Nevertheless,the PTP1B inhibitor TCS-401 and Src inhibitor Bosutinib reversed the above effects.In the mid-term and late adhesion,152RM peptide-treated MSCs formed higher spreading areas as compared to that of vehicle-treated MSCs during 20 min to 60 min;however,this phenomenon disappeared during 80 min to 100 min.5)The activities of Src and FAK,and small GTPases Rac1 and Cdc42 were greatly higher,and the activity of RhoA was greatly lower,in 152RM peptide-treated MSCs than in vehicle-treated MSCs before the initiation of adhesion and during the early stage adhesion of 2 min to 10 min.6)The number of SCR-retained karyocytes,CD271+cells and CD90+/CD105+cells in 152RM peptide group were higher or greatly higher than in vehicle group;importantly,the percentages of CD271~+cells and CD90~+/CD105~+cells in total karyocytes of 152RM peptide group were greatly higher than those of vehicle group.Moreover,the PTP1B inhibitor TCS-401 and Src inhibitor Bosutinib were able to reverse the above effects7)In the osteogenic environment,the treatment of 152RM peptide to MSCs promoted the mRNA and protein expression of osteogenic markers including RUNX2,OCN,and COL-1,accompanied by the activation of ERK 1/2.Moreover,the 152RM peptide effected on the phosphorylation of PTP1B Y152,β-catenin Y654,Src Y530 and Src Y419 of MSCs in an osteogenic environment which were similar to those of suspended MSCs in a growth medium condition.8)152RM peptide did not show a promotion on MSCs proliferation,although it was shown to disassemble the Cadherin/β-catenin complex,the nucleic localization ofβ-catenin of MSCs did not significantly change under the condition of osteogenic environment or growth medium environment.The remained 152RM peptide on the DBM,came from the mimic SCR,was able to promote the osteogenic differentiation of MSCs.The PTP1B inhibitor,Src inhibitor,and ERk1/2 inhibitor,but not Wnt/β-catenin inhibitor,were able to reverse the positive effect of 152RM peptide on the osteogenic differentiation of MSCs.9)By pre-culturing the heparinized iliac marrow with 152RM peptide for one hour,the SCR-prepared grafts of 152RM peptide-pretreated group exhibited a better in vivo osteogenesis in both of the subcutaneous ectopic osteogenesis model and the femur-adjacent osteogenesis model established with athymic mice,as revealed by a micro-CT,H&E staining,and Masson’s trichrome staining.Conclusion:Specific competitive inhibition of phosphorylation of PTP1B Y152 by the 152RM peptide weakened the cadherin adhesion pathway,meanwhile caused enhancement of the Integrin adhesion pathway,which facilitated adhesion and anchoring of MSCs to the matrix,and enhanced the osteogenic differentiation of MSCs.Importantly,152RM peptide caused higher MSC retention within DBM in the SCR procedure,thus improving in vivo osteogenesis of SCR-prepared grafts.Our research provides evidence that demonstrates the enhancement of the adhesion ability of MSCs themselves to promote their adhesion effectiveness in SCR technology is efficacious and feasible.Moreover,our research also provides new methods and theoretical basis to the research field of cell adhesion,especially,the early stage of cell adhesion.