The Changes Of Nuclear Mechanics And Its Involved Moleculear Mechanisms In Osteopontin-promoted Migration Of Bone Marrow-Derived Mesenchymal Stem Cells

Cell migration is a complex and important biological process in multicellular organisms and plays pivotal roles in many physiological and pathological settings,including embryogenesis,wound healing,inflammation and cancer metastasis.Meanwhile cell migration requires collective effects of many internal and external factors,where the former refers to intracellular signal transduction system and skeleton proteins,molecular motor and cell motility structure that control cell movements,the later represents to multiple sets of extracellular signal molecules.Most importantly,cell migration is determined by the morphology,location and biomechanical properties of nucleus,which appears to be the most stiffness organelles in eukaryotic cells.Bone marrow-derived mesenchymal stem cells(BMSCs)are not noly with selfrenewal capability and multi-derectional differentiation potencial,but homing and migrating to wounded sites and plays important roles in tissue repaire and regeneration.In the process of recruiting and migrating form bone marrow to wonded sites the migration capability are regulate by multiple mechanical and chemical factors.Osteopontin(OPN)is a secreted phosphorylated acidic glycoprotein distributed widely in many tissues and cells and is involved in multiple physical and pathological processes by recognization and interaction with its receptors.It is found that OPN is upregulated in woundes sites and play as a chemoattractant that directs cell migration.Our previous research found that OPN promotes significantlly the migration of BMSCs,but the underlying molecular and nuclear biomechanical mechanisms are largely remained uncovered.In our research rat BMSCs are selected as researching object and subject to atomic force microscopy(AFM)to detect the nuclear stiffness change and the involving molecular mechanisms under OPN treatment,further researches concern mainly on 2 main factors that regulate nuclear properties: lamin expression and chromatin structrural changes.Interaction of nucleus and cytoplasm is also detected.At last roles of FAKERK1/2 pathway in mediating OPN regulated nuclear mechanical properties in BMSCs are discussed.Main contents and results are listed as followes:(1)Isolation,cultivation and identification of MSCs.Density gradient centrifugation was applied to isolate and cultivate rat bone marrow originated mesenchymal cells.Morphological observation find that individual cell showed fibroblast-like morphology with high refraction and the cell group exhibited circinate orientation.Surface antigen analysis using flow cytometry indicates that the surface antigen CD44 and CD90 were positive while CD34 was negative,which correlats with typical BMSCs surface antigen properties.In all,the isolated cells are prooved to be BMSCs combining morphological observation and flow cytometry results.(2)OPN promotes BMSs migration via lessening cell nuclear youngs' modulus.Transwell essay has already demonstrated that OPN could promote BMSCs migration,which is closely related to mechanical properties of the nucleus.AFM experiments demonstrated that OPN treatment reduces significantly the young's modulus of nucleus,where the OPN treated is 1.00 ± 0.14 kPa while the untreatd control is about 0.34±0.08 kPa.(3)OPN promotes BMSCs migration by downregulating the expression of lamin A/C proteinTo furthur analyse the mechanism of nuclear mechanical properties alternation,RTPCR and Western blot were applied to detect lamin A/C expression while it is a main factor in determing cell nuclear mechanical properties.Results found a lamin A/C downregulation after 12 h treatment of OPN.To elucidate the relation of lamin A/C expression and BMSC migration,lentiviral LMNA overexpression and knockdown models were established.Results suggest that the overexpression enhances nuclear stiffness but meanwhile retards OPN induced BMSCs migration and knockdown vise versa,indicating that lamin A/C regulates OPN induced BMSCs migration via excerting influence on nuclear stiffness.(4)OPN promotes BMSCs migration by regulating chromotin structure.Chromatin structure is another regulatory factor of nuclear mechanical properties.Using AFM and transwell essay,it is found that reduce of the chromatin fiber compaction might decrease nuclear stiffness of BMSCs and impede the ability of the nucleus to undergo the structural changes required for migration of BMSCs through small space.This result indicates that cell migration is contingent on global condensation of the chromatin fiber.Next,we found that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation(H3K27me3)through the methyltransferase EZH2.While EZH2 supressor DZNep significantly retards the effects of OPN on H3K27me3 and EZH2 and reduces youngs' modulus of nucleus and promotes cell migration.It is found that knocking down of EZH2 using a lentiviral EZH2 knockdown model decreases nuclear stiffness and blocks BMSC migration.Those results indicated that OPN upregulates heterochromatin marker H3K27me3 expression via expression of EZH2 and results in increased compaction of the chromatin fiber and cell migration capability.(5)OPN promotes BMSCs migration via regulating nuclear mechanical properties by F-actinCoupling of the nucleus and cytoplasm is an important factor in determining cell migration.To uncover the mechanism of force signal transducing to nucleus from cytoplasm,Cytochalasin D(cyto D)and Jasplakinolide(Jasp)are separatively applied to depolymerize and polymerize F-actin.Immunostaining were applied to prove their effects on F-Actin in existence of OPN.Immunostaining and AFM tests demonstrated that the polymerization pattern of F-actin regulates the area and young's modulus of nucleus.Knocking down of SUN1 protein that is a main component of LINC complex using siRNA results in significant reduction of both young's moulus of nucleus and migration capability,indicating that the destruction of LINC complex affects cell migration possibly by blocking the force signal transducing from cytoplasm to nucleus.(6)FAK-ERK1/2 mediating OPN promoted BMSCs migrationPrevious research suggests that OPN promotes cell migration by remodling cytoskeleton by FAK-ERK1/2 signaling pathway,while if this pathway is involved in mediating the OPN signal transduction from cytoplasm to the nucleus is to be clarified.Western blot demonstrated that OPN treated cells express significantly higher phospholated FAK and ERK1/2,whereas the suppression of FAK and ERK 1/2 phosphorylation using respectively supressor PF573228 and PD98059 results in supression on OPN induced cell migration in different extent.Further findings showed that OPN induced reduction of Youngs' modulus of cell nuclears and lamin A/C expression is somewhat restored after FAK or ERK1/2 supressor treating.Those results suggest that FAK-ERK1/2 mediates the regulatory effects of OPN on lamin A/C expression and cell nuclear stiffnessThe possible mediation of FAK-ERK1/2 signals in OPN regulating chromatin structure were detected.The results demonstrated that EZH2 and H3K27me3 expression is unaffected after FAK suppression but is significantly reduced after ERK1/2 suppression.ERK suppression also reduced OPN induced chromatin compaction.These results indicate that OPN promotes BMSCs migration by ERK1/2 mediating the chromatin compaction via the upregulation of heterochromatin marker H3K27me3 and its methyltransferase EZH2 expression.In conclusion,OPN could reduces lamin A/C expression and induces chromatin compaction to regulate mechanical properties of cell nucleus,which in turn,promotes directed BMSCs migration.Our results clarified the effects of nuclear mechanical properties on OPN promoted BMSCs migration and the involving regulatory mechanism,providing theoretical direction for clinical application of BMSCs in wounded tissue repairing.