The Study Of The Effects Of Dex On RTSCsproliferation, Differentiation And Migration And Related Mechanism

BackgroundGlucocorticoid-induced tendon rupture is very common in clinical practice, and the rerupture risk is rather high compared with tendon rupute caused by trauma. The mechanism remains unclear.It is well known that the maintenance and repair of adult tissues relies on smallpopulations of resident stem cells. Recently, a group of adult stem cells has been found in human, mouse, rat and rabbit tendon, and this group of cells was therefore named tendon stem cells. While it only represents 5-6% of total nucleated cells, but it is very significant that TSCs migrate to the injury site and differentiate into tenocytes [8, 9]. So it is very important for illustrating the mechnism of Glucocorticoid-induced tendon rupture and decreased self-repair ability to study the effects of glucocorticoid on proliferation, differentiation and migration of TSCs and related mechnism.ObjectiveIn this study, we choose a typical type of glucocorticoid-dexamethasone(Dex) to study the effects of different concentrations of Dex on rat tendon stem cells(rat tendon stem cells, r TSCs) proliferation, differentiation, and migration and to elucidate the mechnism of Dex inhibiting of differentiation of rTSCs into tenocytes, promoting rTSCs to differentiate into adipocytes and also inhibition of rTSCs migration. And finally this study attempts to clarify the mechnism of glucocorticoid leads to spontaneous rupture of tendon, decreased self-repair capacity and search for a cure.Materials and methods1. Isolation, culture and identification of rTSCs and the effect of Dex on the proliferation of rTSCsFirstly, we isolated rTSCs by collagenase digestion and picking clones under a microscope and then. And then the ability of rTSCs differentiate into osteocytes, chondracytes and adipocytes were identified. We also identified the cell surface markers CD44 and CD90 by flow cytometry and intracellular markers Nucleostemin and SSEA-4 by immunofluorescence。Last, we study the effectes of different concentrations of Dex on r TSCs proliferation by CCK-8 and also further verified the effectes of 1uM Dex on r TSCs proliferation by Ki-67 method.2. The Effects of Dex on the differentiation of r TSCs and related mechnism(1) The Effects of Dex on the differentiation of rTSCsFirstly, we compared the normal human achillis tendon tissue and glucocorticoid-induced ruptured tendon tissue by HE staining and Anti-type I collagen immunohistochemical staining. The effect of 1uM Dex on percentage of CD44 and CD90 positive rTSCs were studied by flow cytometry and by qPCR the effects of different concentration of Dex on the r TSCs Scx and C/EBP α mRNA expression was also invesitgated. Last, type I collagen(Col I) and tenomodulin(TNMD) were regarded as tenocytes markers, and ap2 and C/EBPαas adipocytes markers. Their mRNA and protein expression were detected by q PCR or WB and immunofluorence staining to identify whether r TSCs differentiate into tenocytes and adipocytes. We also observed r TSCs shape change and performed oil red O staining to study rTSCs differentiation.(2) The role of Scx in glucocorticoid inhibiting tendon stem cells differentiation into tenocytesFirstly, immunohistochemical assays were performed to compare the expression of scleraxis between Achilles tendons ruptured by trauma and those ruptured due to long-term glucocorticoid therapy for the treatment of autoimmune diseases. By transfection Scx over expression and interference plasmids, the role of Scx in glucocorticoid inhibiting r TSCs differentiation into tenocytes was investigated. The possible binding sites of upstream 2000 bp from Scx gene start codon with GR were predicted Gene-mapper and ChIP-sequence software, and further verified by Ch IP-PCR.(3) The role of DKK1/GSK-3 β / β-catenin in glucocorticoid promoting r TSCs differentiation into adipocytes and the mechanismAfter treatment with Dex, we investigated the ligands of classical and non-classical WNT signal pathway and found that Dex upregulated DKK1 mRNA and protein expression. After transfection with DKK1 sh RNA plasmids and treatment with Licl, we detected P-GSK-3β(ser9), P-GSK-3β(tyr216)and active β-catenin protein expression by WB. And q PCR and WB were used to detected the mRNA and protein expression of ap2 and C/EBPα. We also used oil red O staining to observe mature adipocytes to study the role of DKK1/GSK-3 β / β-catenin in glucocorticoid promoting rTSCs differentiation into adipocytes.3. The effects of Dex on rTSCs migration and related mechanism(1) The effects of Dex on rTSCs migrationAfter rTSCs treated with 1u M Dex,we investigated the effects of Dex on cytoskeleton by phalloidin staining. We also observed the effects of Dex on r TSCs migration by scratch test and transwell test.(2) The role of ROCKs in Dex inhibiting rTSCs migrationFirstly, we used q PCR and WB to investigate the effects of Dex on ROCK1 和 ROCK2 m RNA and protein expression. ROCKs inhibitor Y-27632 and knockdown lentivirus transfection were used and scratch test, transwell test were also used to study the role of ROCKs in Dex inhibiting r TSCs migration.4. Statistical analysis Statistically significant differences were determined using Student’s t-test or two-way analysis of variance. A P-value of 0.05 was considered significant. All values are presented as the mean±standard deviation(SD).Results1. rTSCs express mesenchymal stem cell surface markers and posses multiple differentiation potentialP0 r TSCs isolated from rat achilles tendon appear cobblestone like. The percentages of CD44 and CD90 positive cells were beyond 90% by flow cytometry, and SSEA-4 was expressed in cytoplasm and Nucleostemin was expressed in nucleus by immunofluorecence staining. The isolated r TSCs have capacity to differentiate into osteocytes, adipocytes and chondrocytes after induction.2. Low concentration of Dex promotes rTSCs proliferation, and high concentration of Dex inhibits rTSCs proliferationrTSCs were treated with different concentration 0nM、1n M、10nM、100nM、1u M of Dex, after 6h、12h、24h、48h it was demonstrated that 10 nM Dex promoted r TSCs proliferation and 1uM Dex inhibited rTSCs proliferation by CCK-8 test. After 12h、24h、48h,1n M、10n M Dex promoted r TSCs proliferation,but 100nM、1uM Dex inhibited rTSCs proliferation。It was further verified by Ki-67 immunofluorencence staining that 1uM Dex inhibited rTSCs proliferation.3. Dex shifted r TSCs differentiation from tenocytes to adipocytesIt is demonstrated by HE staining and anti-Col I immunohistochemical assays that collagen fibers in the Achilles tendon ruptures caused by trauma were arranged in an orderly manner, whereas the collagen fibers in the Achilles tendon ruptures caused by Dex were arranged irregularly and were accompanied by adipose tissue metaplasia. Achilles tendons that ruptured due to the long-term application of glucocorticoids displayed not only structurally disordered tendon fiber bundles but also significantly decreased levels of Col I. After treatment of TSCs with/or without 1u M Dex for 3 days, flowcytometry analysis showed that the number of CD44- and CD90-positive cells decreased. Moreover, the number of CD44 and CD90 positive cells in the Dex treatment group decreased more than that in the control group. Compared with the control, we detected a decrease in the number of long, spindle shaped cells after treatment with Dex for 7 days. TSCs were treated with Dex for 1, 3, 5, or 7 days, and the expression of Col I and TNMD mRNA was analyzed by q PCR. The results showed that there were no significant changes in mRNA expression after treatment with Dex for 1 day, whereas after treatment with Dex for 3 days, the expression of Col I and TNMD mRNA was significantly inhibited. However, regardless of the effect of Dex treatment, the level of Col I gradually increased with time. Western blotting and immunofluorescence analysis showed that Dex significantly decreased the expression of Col I. Dex at 1 n M has no effect on the expression of either scleraxis or C/EBPα, Dex at 10 n M promoted scleraxis expression, and Dex at 100 nM of Dex had no effect on scleraxis expression, while Dex at the high concentration of 1 μM inhibited scleraxis expression. Dex at 10 n M inhibited C/EBPα expression, and the high concentrations of Dex, 100 n M and 1 μm, promoted C/EBPα expression. After TSCs were treated with Dex for 3 days, 5 days, 7 days and 14 days, q PCR showed increases in the adipogenic differentiation markers aP2 and C/EBPα. With time, aP2 and C/EBPα levels were gradually increased in both the control group and the experimental group. Western blotting results showed that after treating TSCs with Dex, aP2 and C/EBPα protein expression levels also increased. Immunofluorescence assays of aP2-positive cells showed that after treatment of TSCs with Dex for 7 days, large numbers of a P2-positive cells had formed. After r TSCs were treated with 1 μM Dex for 21 days, the formation of lipid droplets in the cytosol was observed through oil red staining.4. Dex inhibited r TSCs differentiation into tenocytes by targeting Scx geneDex significantly inhibited Scx expression in rTSCs after treatment for 1, 3, 5, and 7 days, whereas mifepristone antagonized the inhibitory effect of Dex on Scx expression. Regardless of the effects of Dex and/or mifepristone, Scx expression increased progressively over time. Dex inhibited the expression of Scx protein and that mifepristone could rescue the inhibition of Scx. Achilles tendons that ruptured due to the long-term application of glucocorticoids expressed significantly decreased levels of Scx. To explore how the binding of Dex to the GR affects Scx transcription, we predicted 3 potential GR binding sites within the Scx gene promoter at the region approximately 2000 bp upstream from the start codon using TIFF searches and GeneMapper software. They are-726~-734 bp GGCAAGGT,-1711~-1729 bp ATCCTGTCCTACAAGACT,-1710~-1723 bp ATCCTGTCCTACA relatively. Then, ChIP-PCR assays were conducted and demonstrated that after forming a complex with Dex, GR bound to the TGGAAGCC sequence located between 734 and 726 bp upstream of the start codon of the Scx gene.5. Dex promoted rTSCs differentiating into adipocytes by DKK1/GSK-3β/active β-catenin pathwayFirstly, we excluded the posibility that Dex promoted r TSCs differentiating into adipocytesby BMPs or Hedgehog pathways. When TSCs were treated with Dex for 3, 5, 7 or 14 days, both m RNA and protein expression levels of DKK1 were increased. Through Western blotting, we found that with 3 days of Dex treatment, both P-GSK-3β(ser9) and P-GSK-3β(tyr216) showed little change, whereas with Dex treatment for 5 days or 7 days, P-GSK-3β(ser9) decreased, and P-GSK-3β(tyr216) increased. When TSCs were treated with Dex for 3 days, β-catenin levels did not show a significant decrease, whereas with treatment for 5 days or 7 days, the expression of β-catenin decreased significantly. Immunohistochemistry showed that treatment of TSCs with Dex for 7 days significantly decreased the expression of β-catenin in the cytoplasm and nucleus. DKK1 knockdown attenuated the inhibition of Dex on the canonical WNT pathway and induced adipogenic differentiation of TSCs. Li Cl attenuated the Dex-induced inhibition of the canonical WNT pathway in TSCs and the induction of adipogenic differentiation of TSCs6. Dex inhibits r TSCs migrationDex significantly inhibited r TSCs migration by scratch test and transwell test after 24 h treatment with 1uM Dex. Meantime we found that ROCK1 and ROCK2 m RNA and protein expression in Dex group are higher than control group after 1, 3, 5, 7 days treatment with 1u M Dex, but Dex didn’t upregulated Rho A mRNA and protein expression.7. Dex inhibits rTSCs migration by upregulation of ROCKsY27632 promoted r TSCs lateral migration and also atenuated Dex inhibiting r TSCs lateral migration. Transwell test demonstrated that Y27632 also promoted rTSCs longitudinal migration and atenuated Dex inhibiting r TSCs longitudinal migration. ROCK2 knockdown by lentivirus promoted r TSCs lateral and longitudinal migration and also atenuated Dex inhibiting r TSCs migration.Conclusion1. In this study, r TSCs were isolated successfully by the enzyme digestion method. The morphology, immunophenotypic Prolife and multiple differentiation capacity of obtained r TSCs comform to MSCs characteristics. Low concentration(1n M, 10nM) of Dex promoted r TSCs proliferation, otherwise high concentraiton(100n M, 1uM) of Dex inhibited r TSCs proliferation.2. Dex inhibited rTSCs differentiating into tenocytes by binding to GR followed by GR bingding to Scx promoter. This study demonstrated that low concentration of Dex promoted Scx expression and inhibited C/EBPα expression, otherwise high concentration of Dex promoted C/EBPα expression and inhibited Scx expression. And this study also demonstrated that Dex inducted rTSCs differentiating into adipocytes by DKK1/GSK-3β/β-catenin.3. this study demonstrated that Dex change the rTSCs’ cytoskeletonandinhibited r TSCs migration and ROCKs upregulation play an important role in Dex inhibiting rTSCs migration,but it needs further investigation that how ROCKs upregulation affects rTSCs migration.