Mechanism Of Osteogenic And Adipogenic Differentiation Of Tendon Stem Cells By Sirt1

IntroductionTendons are fibrous bands which can connect muscles to bones and transmit muscular forces to the bones, allowing joint motion and subsequent body movement. Cases of tendinopathy are increasing, as it currently accounts for about 50% of all sports injuries. Due to the limited methods of treatment, conservative treatments fail in about 24%–45.5% of patients leading to refractory tendinopathy. Therefore, it is necessary to better understand the pathogenesis of tendinopathy on the cellular and molecular level to develop new and more effective treatment strategies.However, tendons are constantly subjected to large mechanical loading and are thus prone to pathological changes, known as tendinopathy. Tendinopathy is a collective term for tendon disorders involving inflammation and/or degeneration. Although the precise pathogenic mechanisms of tendinopathy remain unclear, the typical histopathological features include accumulation of lipid cells, mucoid degeneration, tissue calcification, or some combination of these, suggesting that tendons contain cells with multi-differentiation potentials.Tendon stem cells(TSCs) have been identified in humans, mice, rabbits and rats. These stem cells can differentiate into non-tenocyte lineages such as adipocytes, chondrocytes, and osteocytes under intensive, repetitive mechanical loading, providing a possible mechanism for the osteogenic and adipogenic changes associated with tendinopathies.PGE2 is a major mediator of pain and acute inflammation. Mechanical stretching of tendon fibroblasts(tenocytes) or tendon explants has been shown to increase the production of PGE2 in in vitro studies. PGE2 treatment may result in degenerative changes of the tendon characterized by lipid accumulation and tissue calcification, partly by inducing the differentiation of TSCs into non-tenocytes, including adipocytes and osteocytes. Moreover, PGE2 treatment of human TSCs(hTSCs) induced the production of bone morphogenetic protein-2(BMP-2) in culture, and BMP-2 may mediate PGE2-induced osteogenic differentiation of hTSCs.Sirt1 is a NAD+-dependent histone deacetylase, which plays an important role in energy metabolism and cell differentiation. Some studies have shown that Sirt1 can regulate osteogenic differentiation by self-deacetylation in mesenchymal stem cells(9). Resveratrol, an activator of Sirt1, can decrease the number of adipocytes and increase the expression of osteoblast markers. Inhibition of Sirt1 can increase the number of adipocytes and lipoblast markers and reduce the expression of osteoblast markers. Thus, the activation of Sirt1 can inhibit adipogenic differentiation and promote osteogenic differentiation. It is unknown that whether Sirt1 is expressed in TSCs and how it regulates osteogenic and adipogenic differentiation in TSCs.This study, based on previous work, was to investigated the involvement of these pathways in PGE2-induced osteogenic differentiation using cultured rat TSCs, and defined a key role for the PI3-kinase-Akt pathway in PGE2-induced BMP-2 production and BMP-2-mediated osteogenic differentiation. we also investigated the roles of IGF-1 and BMP- 2 in PGE2- induced adipogenic differentiation of cultured rat TSCs. Using real-time PCR and western blot analysis, we detected the gene and protein expression of Sirt1 in TSCs at different time points after treatment with Sirt1 activators and inhibitors in this study. We additionally analyzed the resulting changes to osteoblasts and lipoblasts before and after treatment, in order to elucidate the mechanism of Sirt1 regulation on osteogenic and adipogenic differentiation of TSCs, as this may provide some insight for the development of more effective treatments for tendon injury.MethodsThe first part: We isolated and cultured the rat TSCs. And then immunofluorescence analysis was performed using rat TSCs with octamer-binding transcription factor 4(Oct-4), stage-specific embryonic antigen-4(SSEA-4), and nucleostemin as stem cell markers. TSCs culture medium with or without PGE2, BMP-2, noggin, Akt Inhibitor IV(10um), U0126(10um) and LY294002(20um), was changed every 3 days throughout the experiments. Alizarin red staining and alkaline phosphatase staining assays were used to detect osteogenic differentiation of TSCs when TSCs were treated with PGE2 at a different concentration for 7 days or PGE2 at a concentration of 100 ng/ml for 1day, 3days, 7days and 14 days, respectively. The BMP2 level of cells before and after treated was determined by using ELISA assay, real-time PCR and western blot. We also detected osteogenic differentiation of TSCs when TSCs were treated with BMP2 at a concentration of 200 ng/ml for 1day, 3days, 7days and 14 days, respectively. We isolated and cultured the rat TSCs. And then immunofluorescence analysis was performed using rat TSCs with octamer-binding transcription factor 4(Oct-4), stage-specific embryonic antigen-4(SSEA-4), and nucleostemin as stem cell markers. TSCs culture medium with or without PGE2, BMP-2, IGF-1 was changed every 3 days throughout the experiments. Oil Red O Staining assay was used to detect adipogenic differentiation of TSCs when TSCs were treated with PGE2 at a different concentration for 7 days or PGE2 at a concentration of 100 ng/ml for 3days, 7days and 10 days, respectively. The expression of PPARγwhich was related with adipogenic differentiation was determined by using real-time PCR and western blot. We also detected adipogenic differentiation of TSCs and the expression of IGF-1 when TSCs were treated with PGE2 at a concentration of 200 ng/ml for 1day, 3days, 7days and 14 days, respectively. The stably transfected cell lines with low BMP-2, IGF-1, CEBPδ, CREB and Smad1 gene and protein expression was generated. Real time PCR and western blot were used to test protein expression of BMP-2, IGF-1, CEBPδ, CREB and Smad1 before and after gene transfected cells.The second part: TSCs were treated with the activiator of sirt1 at the concentration of 0.2um and 0.4um. The expression of sirt1 was determined by using real-time PCR and western blot. Then TSCs were treated with the inhibitor of sirt1 at a concentration of 0.2um and the activator of sirt1 at a concentration of 0.4um. The expression of BMP2 and Runx2 which were relatedwith osteogenic differentiation and the expression of PPARγ which were relatedwith adipogenic differentiation were determined by using real-time PCR, western blot, alizarin red staining and Oil Red O Staining assays. We also detected the expression of some signal pathway protein, such as β-catenin, CEBP?, PPAR?. We construct tendinopathy rats using PGE2 at a concentration of 100ng/ml for 4 weeks. And then we observe the osteogenic and adipogenic differentiation of TSCs after treating with SRT1720 at a concentration of 0.4um for 10 days.ResultsThe first part: The stem cell markers Oct-4, SSEA-4, and nucleostemin were expressed in TSCs, as detected by immunofluorescence. After culture of TSCs in PGE2-containing growth medium for 14 days, mineralization of the extracellular matrix and bone-specific alkaline phosphatase activity were significantly increased, compared to the control group without PGE2 treatment. Rat TSCs were incubated with PGE2(100 ng/ml) for different times, and BMP-2 mRNA expression levels in TSCs and in the culture medium were significantly increased on days 7 and 14. PGE2 treatment activated Akt phosphorylation, but had no obvious effect on ERK phosphorylation in TSCs. LY294002 and Akt inhibitor markedly inhibited Akt phosphorylation and significantly reduced BMP-2 mRNA expression and BMP-2 levels in TSCs. In contrast, U0126 had no effect on BMP-2 mRNA expression or BMP-2 levels. BMP-2(200 ng/ml) was added to the TSC culture medium. Then alkaline phosphatase activity and mineralization were increased slightly at day 3, and then increased progressively to reach a significant level by day 14. LY294002 and Akt inhibitor did not inhibit BMP2-activated Smad phosphorylation, but significantly down-regulated the mRNA expression levels of Runx2 and osteocalcin, while U0126 had no effect. Adipocyte numbers and PPARγexpression were increased after PGE2 treatment in time- and dose-dependent manners, levelling off at a concentration of 100 ng/ml and duration of 7 days, respectively. These results indicated that PGE2 induced adipogenic differentiation of TSCs. BMP-2 had no significant effect on adipocyte number or on PPARγexpression when we assessed the differentiation of TSCs incubated with increasing concentrations of BMP-2, or with 100 ng/ml BMP-2 for the indicated days. This indicated that BMP-2 alone was unable to mediate PGE2-induced adipogenic differentiation. IGF-1 mRNA and protein levels, measured by qRT-PCR and ELISA, respectively, increased in dose-dependent manners in response to PGE2 treatment when cells were incubated with PGE2(0–200 ng/ml) for 7 days. PGE2 increased intracellular cAMP in TSCs, activated cAMP-dependent protein kinase PKA, and stimulated nuclear translocation of CEBPδ. IGF-1 in the absence of BMP-2 failed to induce adipogenic differentiation in TSCs. However, IGF-1 together with BMP-2 significantly induced adipogenic differentiation in TSCs, as demonstrated by Oil Red O staining. CREB and Smad were activated by phosphorylation in the presence of PGE2 or IGF-1+BMP-2. PGE2 or IGF-1, not BMP-2, increased the phosphorylation of CREB, and the effect of IGF-1 was blocked by CREB shRNA. Either CREB shRNA or Smad shRNA markedly inhibited IGF-1+BMP-2-induced adipogenic differentiation, as indicated by Oil Red O Staining. CREB shRNA and Smad shRNA also inhibited expression of PPARγ2 at the mRNA level.The second part: Our study confirmed the expression of Sirt1 in TSCs and revealed that their osteogenic differentiation capacity, as well as the gene and protein levels of osteogenesis marker BMP2, was elevated with increasing Sirt1 activator concentration and treatment time. Peak values of BMP2 expression were observed on day 10 followed by a marked decrease in osteogenic capacity of TSCs during prolonged treatment with Sirt1 activator. Conversely, the differentiation capability of TSCs was decreased after being treated with Sirt1 inhibitor. We also tested the adipogenic capability of TSCs under the impact of the Sirt1 activator and Sirt1 inhibitor. Sirt1 activator treatment of TSCs induced the opposite effect on TSC adipogenic capability as increases to Sirt1 activator concentration and treatment time gradually weakened the adipogenic potential of TSCs. However, after day 10, adipogenic capability gradually began to increase with prolonged Sirt1 activator treatment. We successfully constructed tendinopathy rats using PGE2 at a concentration of 100ng/ml for 4 weeks. And then we detected that the osteogenic and adipogenic differentiation of TSCs are decreasing after treating with SRT1720 at a concentration of 0.4um for 10 days.ConclusionsThe first part: The PI3K-Akt signaling pathway is involved in PGE2-induced osteogenic differentiation of rat TSCs. Furthermore, PGE2 activates Akt through PI3 K, and then induces BMP-2 mRNA expression. BMP-2 subsequently activates Smad phosphorylation, inducing Runx2 and osteocalcin expression and osteogenic differentiation. IGF-1 and BMP-2 together mediate PGE-2-induced adipogenic differentiation of TSCs. IGF-1 expression is up-regulated via the cAMP/PKA/CEBPδ pathway. IGF-1 and BMP2 phosphorylate and activate downstream CREB and Smad, respectively, which subsequently upregulate PPARγ expression, thus enhancing adipogenic differentiation.The second part: Sirt1 is endogenously expressed in TSCs. We have confirmed that Sirt1 can promote the osteogenic differentiation of TSCs through Sirt1/β-catenin/Runx2 pathway and that it can inhibit the adipogenic differentiation of TSCs through the PI3K/ AKT/ CEBPα/ PPARγ pathway. Sirt1 can inhibit the osteogenic and adipogenic differentiation of TSCs.