The Mechanisms Of Mechanical Stretching-increased Tenomodulin Expression Promotes Tenocyte Migration

Tendon connects muscle to bone and function as muscle contraction force transmitters,driving and maintaining joint movement.Tendon is prone to injury due to long-term exposure to the microenvironment of tension loading.Upon tendon injuries,tenocytes migrate to the damaged site and facilitate the repair process through tenocyte proliferation and the secretion of extracellar matrix(ECM)components.However,the roles and mechanisms of mechanical and chemical factors that regulate tenocyte migration in the process of injured tendon repair are still unclear.Tenomodulin(Tnmd)is a type II transmembrane glycoprotein,as a tendon and tenocyte maturation-specific marker that contributes to the development of tendon and the repaired of damaged tendon through regulating the proliferation,migration and secretion of collagen protein of TSCs(tendon stem cells)and tenocytes,but the mechanisms of Tnmd-regulated tenocyte migration remain unknown.It has been reported that compared with tenocytes in tendon,the expression of Tnmd in isolated and cultured tenocytes in vitro is sharply down-regulated,and appropriate mechanical loading significantly up-regulates and maintains Tnmd expression in cultured tenocytes.Mechanical loading is a key factor in maintaining the homeostasis and function of tendon and tenocytes,which activated protein kinases and induced mechanosensitive molecules expression to regulate tenocyte responses,such as inflammation,proliferation and migration.However,the mechanisms in which mechanical loading regulate Tnmd expression and its role in tenocyte migration is yet to be defined.Taking the tenocytes of rat Achilles tendon isolated and cultured in vitro as the research object,by simulating the tendon biomechanical microenvironment of tenocytes,investigates the effects of mechanical stretching on Tnmd expression and tenocyte migration and explores the potential mechanotransduction pathway.At the same time,we contrasted a tenocyte line with stable overexpression of Tnmd by gene cloning technology to clarify the effect of Tnmd on tenocyte migration and further explore the mechanism of Tnmd regulating cell migration;The main research contents and results are as follows:(1)Mechanical stretching-induced Tnmd expression promotes tenocyte migrationQuantitative PCR and Western blot(WB)results showed that mechanical stretching(0.5 Hz、10% for 1 h)significantly increased the levels of Tnmd mRNA and protein of cultured tenocytes.The results of transwell assay showed that the migration ability of loaded-tenocytes was significantly increased.Knockdown of Tnmd by shRNA interference significantly inhibited stretching induced Tnmd expression and tenocyte migration,proving that stretching promoted tenocyte migration by up-regulating Tnmd expression.(2)Mechanical stretching induced chromatin decondensation to alters nuclear deformation promoted Tnmd expression and migration of tenocytesConsidering that mechanical affect gene transcription and various tenocyte responses via the deformation of F-actin cytoskeleton and nuclear.Fluorescent stained F-actin and DAPI stained nuclei revealed that stretching-reinforced F-actin stress fibers,increased spindle shape of nuclei and decreased nuclear size.Lamin A/C level and chromatin are important factors for nuclear deformation response.The expression of Lamin A/C in tenocytes was detected by WB and the chromatin decondensation was detected by in situ DNase I sensitivity assay.It was found that stretching did not affect Lamin A/C levels of tenocytes,but significantly increased chromatin deconcentration.Inhibition of chromatin decondensation of tenocytes found that chromatin decondensation,nuclear deformation,Tnmd expression and tenocyte migration were significantly inhibited in loaded-tenocytes.Epigenetic modification of histone 3(H3)is necessary to regulate the condensed state of chromatin.WB results showed that stretching significantly inhibited the expression of H3K27me3 and increased the level of H3K27 ac and H3K9 ac in tenocytes,and inhibition of these protein levels attenuated chromatin decondensation of loaded-tenocytes,suggesting that stretching induces chromatin decondensation by inhibiting the trimethylation of H3K27 and increasing the acetylation of H3K27 and H3K9,which might change nuclear deformation to regulate Tnmd expression and tenocyte migration.Further reults showed that inhibition of F-actin stress fibers formation by latrunculin A(Lat A)significantly inhibited chromatin decondensation and H3k27me3 expression in stretching stimulated-tenocytes.And Tnmd expression and tenocyte migration in loaded-tenocytes was also abrogated,suggesting that mechanical stretching-enhanced F-actin stress fibers inhibited triple methylation of H3K27 to increase chromatin decondensation,resulting in promoted expression of Tnmd and migration of tenocytes.(3)Mechanical stretching up-regulates Tnmd expression and tenocyte migration by activating MAPKs signalingMitogen-activated protein kinase(MAPKs)are activated by intracellular or external stress stimuli that regulate gene transcription and cell migration.WB result showed that stretching increased the phosphorylation levels of MAPKs extracellular regulated kinase 1/2(ERK1/2),mitogen-activated kinase 14(p38)and c-Jun N-terminal kinase(JNK),Inhibitor of MAPKs inhibited stretching-induced Tnmd expression and tenocyte migration,and F-actin stress fibers of tenocytes and loaded-tenocytes was also significantly inhibited.Moreover,inhibition of ERK1/2,JNK activation significantly reduced stretching-induced H3K27me3 modification and chromatin decondensation.These results suggested that stretching-induced Tnmd expression increased tenocyte migration via ERK1/2/JNK dependent F-actin stress fibers to repress H3K27me3 that increase chromatin decondensation.(4)The mechanisms of Tnmd promotes tenocyte migrationTo further explore the mechanism by which Tnmd regulate tenocyte migration,Tnmd-tenocytes(the tenocyte line with stable overexpression of Tnmd)was constructed and obtained by gene cloning technology.The results of transwell assay showed that the migration ability of Tnmd-tenocytes was significantly increased.Considering nuclear deformability limits tenocyte migration through ECM spaces.Analysis of the morphological characteristics of DAPI-stained nuclei showed that Tnmd overexpression significantly increased the spindle shape of nuclei and decreased the size of nuclei.Further analysis of the distribution characteristics of nuclear size indicated that the high expression of Tnmd decreased nuclear size contributed to the migration of tenocytes.Given that nuclear deformability mainly depends on the level of Lamin A/C.WB results showed that the overexpression of Tnmd significantly increased Lamin A/C expression of tenocytes.and knockdown of Lamin A/C in Tnmd-tenocytes by siRNA-interference technology inhibited Tnmd-tenocyte migration,spindle shape and size of nuclei,demonstrating that Tnmd promotes tenocyte migration through up-regulating Lamin A/C mediated nuclear deformation.Many studies have reported that F-actin and microtubulin cytoskeleton dynamics act independently or coupled on the nucleus to control nuclear strain of migrating cells.Fluorescent staining of F-actin and microtubule cytoskeleton protein α-tubulin of Tnmdtenocytes showed that Tnmd enhanced F-actin stress fibers of tenocytes and induced Factin stress fibers and microtubule cytoskeleton to extend along the long axis of tenocytes.Depolymerization of F-actin and microtubule cytoskeleton showed that the cell migration,nuclear deformation and Lamin A/C expression in Tnmd-tenocytes was significantly inhibited.Moreover,knockdown of Lamin A/C in Tnmd-tenocytes resulted in the arrangement of F-actin stress fibers and microtubule skeleton induced by Tnmd from extending along the long axis to spreading around the cells.These results demonstrate that Lamin A/C is tightly coupled to F-actin/microtubulin skeleton rearrangement to regulate nuclear deformation,which promotes tenocyte migration.Interestingly,Y27632 inhibited F-actin stress fibers of Tnmd-tenocytes further significantly increased Tnmd-tenocyte migration.This result is not consistent with the inhibition of cell migration by depolymerizing F-actin cytoskeleton with Lat A treatment,suggesting that Y27632 may simultaneously activate other signaling pathway that promote tenocyte migration.WB detection showed that Y27632 pretreatment significantly increased the phosphorylation level of ERK1/2 in Tnmd-tenocytes.Inhibition of ERK1/2 activation significantly reduced the migration of Tnmd-tenocytes.These results demonstrated that Y27632 promotes Tnmd-tenocyte migration through activating ERK1/2 pathway.Together,mechanical stretching activates ERK1/2 、JNK signaling enhances Factin stress fibers,thereby inhibiting H3K27me3 to increase chromatin decondensation,which then increase Tnmd expression to promoted tenocyte migration.Moreover,we also demonstrated that the coupling of F-actin /microtubule skeleton rearrangement with Lamin A/C which control nuclear deformation contributes to Tnmd-tenocyte migration.These found providing insight into the role of Tnmd in tendon development and injured tendon repair,and contribute to develop new treatment based on mechanical and chemical coupling-mediated tendon repair.