The Role Of Noncanonical Wnt5a Signaling In Tendon Stem/Progenitor Cells Senescence

Tendon aging is an inevitable physiological process that results in declines in the structural and functional properties of tendon.Tendon aging is one of the main causes of chronic pain,limited joint mobility and tendon rapture among elderly patients.Tendon tissue contains terminally differentiated tenocytes and a small resident tendon stem cell population,known as tendon stem/progenitor cells(TSPCs).TSPCs play a critical role in tendon repair,regeneration and homeostasis maintaining.Studies have showed that TSPCs senescence is closely associated with the age-related tendon disorder.As compared with young cells,aged TSPCs display deficient self-renewal,migration and tenogenic differentiation capacity,as well as substantial changes in transcriptome,leading to impaired tendon healing and regeneration capacity.Although the decline of TSPCs function on senescence has been well recognized,the molecular mechanisms of this process is still largely unknown.Wnt5a is a prototypical ligand for the noncanonical Wnt pathway and has been reported to play a critical role in aging and regulation of tendon stem cells.To date,there were no studies focused on the role of Wnt5a in TSPCs senescence during tendon aging.In present study,we aimed to identify the specific role of noncanonical Wnt5a in TSPCs senescence,senescence-associated secretory phenotype(SASP)expression and age-related dysfunctions.Furthermore,the possible mechanisms involved in this process were also investigated.In present study,we conducted a natural murine aging model,TSPCs were obtained from 2-month-old and 20-month-old male C57BL/6 mouse.The noncanonical Wnt5a expression pattern was determined by Immunofluorescence,q RT-PCR and western blotting.TSPCs were then treatment with Wnt5a-sh RNA or recombinant Wnt5a.Microarray analysis,?-gal staining,western blotting,q RT-PCR,immunofluorescence and cell cycle analysis were used for confirming the role of Wnt5a in TSPCs senescence or SASP.CFU assay,CCK-8 and PDT assay were used for confirming the role of Wnt5a in TSPCs self-renewal.Scratch assay and actin dynamics analysis were used for investigating the role of Wnt5a in TSPCs migration.q RT-PCR was used for confirming the role of Wnt5a in TSPCs tenogenic differentiation.Besides,the molecular mechanisms involved in regulation of TSPCs senescence were investigated by The GSEA KEGG PATHYWAY analysis,q RT-PCR and western blotting.Unpaired t-test and ANOVAs were used for statistical comparisons,with significance set at P<0.05.All data are shown as mean±SD.In the first part,H&E staining showed irregular collagen fibers and loss of collagen stainability in aged tendon.Alizarin red staining showed a significant calcification site in the aged tendon.Safranin O staining revealed a reduction of proteoglycan content in aged tendon.Moreover,colony forming unit(CFU)assay showed the clonogenicity of mouse TSPCs.TSPCs expressed mesenchymal stem cells markers(CD73 and CD105),fibroblast marker(CD90.2),and were negative for hematopoietic stem cell marker(CD34).TSPCs could differentiate into osteogenic,chondrogenic and adipogenic lineages after cultured in induction medium.Furthermore,RNA-seq analysis revealed substantial changes in transcriptome.GO analysis showed that differential genes distributed majorly in proliferation,migration,motility,secretion,actin cytoskeleton,stem cell differentiation.The GSEA KEGG PATHYWAY analysis suggested that the JAK-STAT signaling pathway may play an important role in TSPCs senescence.In the second part,we analyzed Wnt family microarray data and found a marked increase in the expression of Wnt5a,while other members of the canonical Wnt family did not present with significant changes in expression on TSPCs senescence.The q RT-PCR and Western blotting results confirmed that Wnt5a expression was marked increased in aged TSPCs.Recombinant Wnt5a treatment reduced the expression of?-catenin in young TSPCs,indicating a direct action of Wnt5a on?-catenin expression in TSPCs.Recombinant Wnt5a treatment also reduced the direct downstream target genes of canonical Wnt signaling,Axin2 and Lgr5.In addition,Wnt5a sh RNA treatment significantly reduced?-gal positive senescence cells in aged TSPCs,this was also coupled with a significant repression of senescence marker p16^INK4A in aged TSPCs.Cell cycle analysis suggested that silencing Wnt5a blocked the accumulation of aged TSPCs at G1 phase.Immunofluorescent staining showed that Wnt5a sh RNA treatment significantly increased the frequency of polarized cells in aged TSPCs.On the contrary,recombinant Wnt5a treatment promoted cell senescence phenotype in young TSPCs.Moreover,Wnt5a sh RNA treatment could rescue the increased level of the SASP genes(IL6,IL16,Cxcl1,Cxcl5,Cxcl12,Ereg,Tnfsf11,Ccl2)in aged TSPCs.Colony forming unit assays showed that Wnt5a knockdown significantly increased the colony number.The population doubling time(PDT)and CCK-8 assay demonstrated the Wnt5a knockdown enhanced the proliferative potential of aged TSPCs.The scratch assay revealed a decelerated migration and longer scratch bridging time in the aged TSPCs while Wnt5a sh RNA restored the age-related migration deficit.Wnt5a knockdown also improved actin turnover after treatment with latrunculin A in aged TSPCs.Finally,Wnt5a knockdown rescued the decreased levels of tendon-related markers in aged TSPCs,including Tnmd,Col1A1,Nestin,Scx,Bgn.In the third part,we analyzed gene sets using the GSEA KEGG PATHYWAY analysis to identify enriched signaling pathways in aged and Wnt5a-knockdown aged.The GSEA KEGG PATHYWAY analysis showed a significant decrease in enrichment of genes associated with JAK-STAT signaling pathway in Wnt5a-knockdown aged TSPCs.Western blotting results indicated that the protein levels of p-JAK2 and p-STAT3 were significantly increased in aged TSPCs,which suggested the activation of JAK-STAT signaling,and Wnt5a knockdown inhibited the phosphorylation of JAK2 and STAT3.IFN-?treatment abolished the inhibition effects of Wnt5a sh RNA on JAK-STAT signaling pathway and TSPCs senescence hallmarks.In addition,Wnt5a-induced increases in p-JAK2 and p-STAT3 levels were diminished by Ror2 knockdown in young TSPCs,which suggested that Wnt5a activates JAK-STAT signaling pathway depend on Ror2.Knockdown of Ror2 also suppressed Wnt5a induced increases of p16^INK4A expression,the number of?-gal positive senescent cells,as well as SASP genes.In conclusion,our study demonstrated that aberrant expression of noncanonical Wnt5a is an important contributor to cell senescence in TSPCs.Functionally,inhibition of Wnt5a attenuated TSPCs senescence,age-related cell apolarity and the SASP expression in aged TSPCs.Wnt5a knockdown also restored the age-related dysfunction of self-renewal,migration and tenogenic differentiation.We further found that Wnt5a modulates TSPCs senescence via a previously unknown mechanism to potentiate JAK-STAT signaling.Moreover,we showed that Ror2 acts as the functional receptor of Wnt5a in TSPCs senescence.Our findings suggest a novel essential mechanism involved in TSPCs senescence,which could be an ideal therapeutic agent for age related tendon disorders.