Mechanical Tension Promotes The Osteogenic Differentiation Of Rat Tendon Stem Cells Through The Wnt5a/Wnt5b/JNK Signaling Pathway

IntroductionTendinopathy is a common disease in orthopedics. More than thirty kinds of tendinopathy, include tennis elbow, achilles tendinitis, supraspinatus tendinitis etc. Professional athletes suffer from tendinopathy often means the end of the career. At present, there is no effective treatment for tendinopathy. The main pathological change in tendinopathy is heterotopic ossification and adipose tissue formation in tendon tissue [1]. The majority of scholars believe that tendon fiber injury caused by overuse is the leading cause of tendon disease, but its pathogenic mechanism is not clear enough. In 2007, Bi et al [2] isolated the stem cells in the tendon tissue, named tendon stem cells(TSCs),they have demonstrated that TSCs are the precursors of tendon cells and are capable of differentiating into multiple lineages and possess multiple differentiation capacity. Gulotta et al [3] confirmed that TSCs have a biological function similar to that of mesenchymal stem cells(MSCs) or bone marrow mesenchymal stem cells(BMSCs) and that they are directly involved in and play a critical role in the regeneration and repair of tendon micro-damage. An in vitro studies found that TSCs morphological change can affect the direction of differentiation, under 4% mechanical stress, TSCs mainly differentiated into tenocytes, involved in the repair of tendon injuries and tenocytes regeneration; under 8% mechanical stress, TSCs mainly differentiated into osteoblasts, adipose cells and chondrocytes[4], However, under the condition of mechanical stress, the molecular biology mechanism of osteogenetic differentiation in TSCs is unclear. The mechanism of TSCs differentiate to non-tenocytes under mechanical stress is of great significance to reveal the pathogenesis of tendinopathy.Wnt signaling pathway plays an important role in the process of embryonic development. Wnt signaling pathway is divided into the canonical and the non-canonical signal pathways.The non-canonical Wnt signaling pathways mainly control cell polarity, adhesion and movement. Wnt signaling pathway is closely related to the osteoblasts formation, proliferation and apoptosis[5-6]. Wnt5 a and Wnt5 b belong to the non-canonical Wnt signaling pathway, are sensitive to stress stimulus from extracellular, and are associated with stem cells differentiate to osteogenesis[7-8]. The C-Jun amino N-terminal protein kinases( JNK) was discovered in 1990, is one of the main members of the mitogen activated protein kinase(MAPK) family, JNK signaling pathway can be activated by mechanical stress, cytokine and so on,involved in cell apoptosis, differentiation, and remodel cytoskeleton [9], JNK has the mechanical sensitivity and can raise Runx2 gene expression[10], The study found that the osteogenesis of MC3T3E1-cell in mice were regulated by JNK signaling pathways[11].The non-canonical Wnt/PCP(planar cell polarity) signaling pathways regulate JNK signaling pathways, locate in the upstream of JNK signaling pathways. In vertebrates, Wnt5 a, Wnt5 b, Wnt11 as ligand, couple on Frizzled receptor and its co-receptor Ror2, activate the G protein and Dvl, then assembled into Dvl-Rac1 complex, Dvl-Rac1 complex activate JNK kinases regulate cytoskeleton and cell adhesion[12-15]. The non-canonica Wnt signaling pathway is regulated by the its upstream Rho/Rock molecules, mechanical stress induced osteogenesis differentiation of stem cells was controlled by RhoA/Wnt5 a signaling pathway[8]. How non- canonical Wnt signaling pathways regulate its downstream molecules to promote the osteogenetic differentiation of stem cells is to be proved by further experiments. Whether or not the osteogenetic differentiation of stem cell induced by mechanical tension is regulated by Wnt/JNK signaling pathway need to be proved.We study the ability of osteogenic differentiation of rat tendon stem cells(rTDSCs) under mechanical tension induced, testing the gene and protein levels of Wnt5 a, Wnt5 b under mechanical tension induced, and the regulation relationships between Wnt5 a, Wnt5 b and JNK, and to explore the contribution of the Wnt5a/Wnt5b/JNK signaling pathway to the mechanical tension induced osteoblast differentiation of rTDSCs.Methods:1. Identification of rTDSCs:Isolation and culture cells from rats Achilles, using flow cytometry(FCM) to identify cell surface markers anti-CD31, anti-CD44, anti-CD90, and anti-CD34, using immunofluorescence to detect anti-nucleostemin, anti-Nanog, anti- octamer-binding transcription factor 4(Oct-4).Culture cells with osteogenic differentiation medium, after 21 days, extract RNA, use Real-time Polymerase Chain Reaction(RT-PCR) to detect mRNA expression of Runx2, DIX5, Wnt5 a and Wnt5 b, stain cells by alizarin red; culture cells with adipogenic differentiation medium, after 14 days,detect mRNA expression of PPARγ、ap2 mRNA, stain cells by Oil red;culture cells with chondrogenic differentiation medium, after 21 days, stain cartilage tissue by alcian blue.2. Detect the ability of osteogenic differentiation of rTDSCs after uniaxial mechanical tension(UMT)induced. rTDSCs be induced by UMT at 8% elongation and a frequency of 1 HZ for 48, 60, or 72 hours. Detect mRNA expression of Runx2, DIX5, Alpl, Col1a1 by RT-PCR, and detect protein expression of Runx2 by western blot(WB)3. rTDSCs be induced by UMT for 48, 60, or 72 hours, detect mRNA expression of Wnt5 a, Wnt5 b, Ror2 and Rac1 compare with non-loading control(NC), detect Wnt5 a, Wnt5 b protein expression after 48, 60 hours of UMT compared with the NC.4. To explore the contribution of the Wnt5 a, Wnt5 b to the UMT induced osteoblast differentiation of rTDSCs. With short hairpin RNA(short hairpin RNA, ShRNA) lentiviruses interference Wnt5 a and Wnt5 b of rTDSCs respectively, then UMT induce for 60 hours, detect protein expression of Runx2 by WB compare with non- interference control.5. After UMT, detect the change of the cytoskeleton and the level of the phosphatic-JNK(P-JNK). Treat with the JNK excitant anisomycin(AM) or JNK inhibitor SP600125 before UMT induction, to confirm whether JNK regulate UMT-induced Runx2 expression. With ShRNA lentiviruses and cDNA lentiviruses interference JNK of rTDSCs respectively, lead to further confirm the above conclusion.6. With shRNA interference rTDSCs Wnt5 a, Wnt5 b respectively, add JNK stimulant AM simultaneously, after UMT, detect protein expression of Runx2 and P-JNK levels by WB compare with non- interference control.Result:1. We have Isolated cells from Achilles of rats, FCM analysis showed that cells positive staining for the CD90, The immunofluorescence demonstrated that the nucleostemin,Oct-4 and Nanog were expressed in cells. After 21 days of osteogenic induction, the mRNA expression of DIX5 and Runx2 was higher in the osteogenic differentiation medium, mineralized calcium deposits were observed by Alizarin Red staining, however, no mineralized calcium deposits were found in the GM group.The mRNA expression of Wnt5 a and Wnt5 b was higher compared to the GM group; after 14 days of adipogenic induction, the mRNA expression of PPARγ、ap2 was higher in the adipogenic differentiation medium, lipid droplet by oil red staining; after chondrogenic induction, cartilage tissue were observed by alcian blue staining.2. After UMT for 48, 60 and 72 hours,the mRNA expression of the Runx2, Dlx5, Alpl and Col1a1 in rTDSCs was increased compared with the NC group; Runx2 protein levels increased compared with the NC group; the alkaline phosphatase level was also higher compared with the NC group.3. UMT for 48, 60, or 72 hours increased the mRNA expression of Wnt5 a,Wnt5b in rTDSCs as well as that of Ror2 and Rac1 compared with the NC group. The Wnt5 a and Wnt5 b protein levels were higher in rTDSCs exposed to 48 and 60 hours of UMT compared with the NC group.4. Wnt5 a,Wnt5b genes were interfered by shRNA. After 60 hours of UMT, Runx2 protein expression was lower in the Wnt5a-shRNA and Wnt5b-shRNA groups compared with the shRNA-control group.5. Rrhodamine-phalloidin staining showed, after UMT, irreversible cytoskeletal changes had occurred in rTDSCs. After UMT, P-JNK levels were higher in the UMT groups than in the NC group, rTDSCs were treated with the JNK activator anisomycin and the JNK inhibitor SP600125 for 30 minutes before UMT induction, anisomycin increased Runx2 mRNA expression and SP600125 decreased Runx2 mRNA expression in rTDSCs after UMT induction compared with the UMT+no drug group. cDNA-JNK promoted Runx2 protein expression of rTDSCs induced by UMT, and shRNA-JNK reduced Runx2 protein expression of rTDSCs induced by UMT respectively.6. The UMT‐induced upregulation of Runx2 protein and mRNA expression were suppressed in rTDSCs transfected with shRNA‐Wnt5a and shRNA‐Wnt5b compared to the shRNA‐control group, Runx2 protein and mRNA expression were restored by the addition of anisomycin.Conclusion:1. rTDSCs has the property of stem cells,and possess multiple differentiation capacity.2. The osteogenic differentiation of rTDSCs appeared after UMT along with increased mRNA and protein expression of the Runx2.3. Wnt5 a, Wnt5 b mRNA and protein levels were also upregulated after UMT stimulation.4.Wnt5 a and Wnt5 b could modulate UMT-induced osteogenic differentiation of rTDSCs.5. UMT cause irreversible cytoskeletal changes in rTDSCs.JNK also modulate UMTinduced osteogenic differentiation of rTDSCs.6. UMT induced the osteogenic differentiation of rTDSCs via the Wnt5 a /Wnt5b/JNK signaling pathway. Accordingly, this pathway may influence the heterotopic ossification of tendon tissue subjected to excessive tension...