同源性磷酸酶-张力蛋白经PI3K/AKT通路调控关节纤维化肌成纤维细胞增殖
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摘要
背景:研究显示大鼠关节纤维化中相关调控肌成纤维细胞表达的基因目前报导较少,对抑癌基因同源性磷酸酶-张力蛋白(phosphatase and tensin homolog,PTEN)在关节纤维化组织的表达目前尚未见报道。目的:探讨关节纤维化大鼠关节囊组织中PTEN基因的表达变化及下游PI3K/AKT信号通路在大鼠关节纤维化中的作用。方法:膝关节固定法建立大鼠膝关节纤维化模型,取膝关节后关节囊,组织块培养法分离培养肌成纤维细胞。采用定量实时PCR检测PTEN mRNA的表达,采用蛋白质印迹Western Blot方法检测肌成纤维细胞中胶原蛋白1-A1、PTEN蛋白的表达情况及下游PI3K,AKT通路的信号分子改变情况。再使用PI3K/AKT通路阻滞剂LY294002对肌成纤维细胞进行处理,观察肌成纤维细胞的增殖改变情况。结果与结论:关节纤维化中PTEN mRNA及蛋白表达水平较对照侧下调(P <0.05)。PTEN表达下调后,下游PI3K/AKT通路的信号分子PI3K,p-AKT的表达水平出现上调(P <0.05),肌成纤维细胞的细胞增殖活跃,使用PI3K/AKT通路阻滞剂LY294002后细胞增殖受到抑制。提示纤维化关节中PTEN表达发生下调,并经由PI3K/AKT通路调控关节纤维化的发展。
BACKGROUND:There are few reports about the genes that regulate the expression of myofibroblasts in rats with arthrofibrosis. The expression of tumor suppressor gene phosphatase and tensin homolog(PTEN) in fibrosis tissues has not yet been reported.OBJECTIVE:To investigate the expression of PTEN gene in articular capsule tissue in rats with arthrofibrosis and the significance of PI3K/AKT signaling pathway in arthrofibrosis.METHODS:The rat model of arthrofibrosis was established by knee fixation, and the posterior articular capsule of knee joint was removed.Myofibroblasts were isolated and cultured by tissue explant method. The expression level of PTEN mR NA was detected by quantitative real-time PCR. The protein expression levels of COL1-A1, and PTEN in myofibroblasts and the signal molecules change in downstream PI3K/AKT signaling pathway were detected by western blot assay. The PI3K/AKT signal pathway specific blocker LY294002 was used to observe the changes of myofibroblast proliferation.RESULTS AND CONCLUSION:The expression levels of PTEN m RNA and protein in arthrofibrosis were significantly down-regulated compared with the normal articular group(P < 0.05). The down-regulated expression level of PTEN could up-regulate the expression levels of PI3K and AKT signaling molecules(P < 0.05) and enhanced the proliferation of myofibroblasts. After application of the PI3K/AKT blocker LY294002, the proliferation of myofibroblasts was inhibited. Our results suggest that there is a down-regulation in PTEN expression in articular capsule tissue of rats with arthrofibrosis, and PTEN regulates the development of articular capsule tissue fibrosis via PI3K/AKT signaling pathway.
BACKGROUND:There are few reports about the genes that regulate the expression of myofibroblasts in rats with arthrofibrosis. The expression of tumor suppressor gene phosphatase and tensin homolog(PTEN) in fibrosis tissues has not yet been reported.OBJECTIVE:To investigate the expression of PTEN gene in articular capsule tissue in rats with arthrofibrosis and the significance of PI3K/AKT signaling pathway in arthrofibrosis.METHODS:The rat model of arthrofibrosis was established by knee fixation, and the posterior articular capsule of knee joint was removed.Myofibroblasts were isolated and cultured by tissue explant method. The expression level of PTEN mR NA was detected by quantitative real-time PCR. The protein expression levels of COL1-A1, and PTEN in myofibroblasts and the signal molecules change in downstream PI3K/AKT signaling pathway were detected by western blot assay. The PI3K/AKT signal pathway specific blocker LY294002 was used to observe the changes of myofibroblast proliferation.RESULTS AND CONCLUSION:The expression levels of PTEN m RNA and protein in arthrofibrosis were significantly down-regulated compared with the normal articular group(P < 0.05). The down-regulated expression level of PTEN could up-regulate the expression levels of PI3K and AKT signaling molecules(P < 0.05) and enhanced the proliferation of myofibroblasts. After application of the PI3K/AKT blocker LY294002, the proliferation of myofibroblasts was inhibited. Our results suggest that there is a down-regulation in PTEN expression in articular capsule tissue of rats with arthrofibrosis, and PTEN regulates the development of articular capsule tissue fibrosis via PI3K/AKT signaling pathway.
引文
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[7]Feng X,Jiang J,Shi S,et al.Knockdown of mi R-25 increases the sensitivity of liver cancer stem cells to TRAIL-induced apoptosis via PTEN/PI3K/Akt/Bad signaling pathway.Int JOncol.2016;49(6):2600-2610.
[8]Su D,Zhang CL,Gao YC,et al.Gene Expression and Correlation of Pten and Fabp4 in Liver,Muscle,and Adipose Tissues of Type 2 Diabetes Rats.Med Sci Monit.2015;21:3616-3621.
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[12]He R,Wang Z,Lu Y,et al.Chaperonin containing T-complex polypeptide subunit eta is a potential marker of joint contracture:an experimental study in the rat.Cell Stress Chaperones.2015;20(6):959-966.
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[15]Piao S,Ryu JK,Shin HY,et al.Repeated intratunical injection of adenovirus expressing transforming growth factor-beta1 in a rat induces penile curvature with tunical fibrotic plaque:a useful model for the study of Peyronie's disease.Int J Androl.2008;31(3):346-353.
[16]Arnott JA,Nuglozeh E,Rico MC,et al.Connective tissue growth factor(CTGF/CCN2)is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.J Cell Physiol.2007;210(3):843-852.
[17]Lee YT,Shao HJ,Wang JH,et al.Hyaluronic acid modulates gene expression of connective tissue growth factor(CTGF),transforming growth factor-beta1(TGF-beta1),and vascular endothelial growth factor(VEGF)in human fibroblast-like synovial cells from advanced-stage osteoarthritis in vitro.JOrthop Res.2010;28(4):492-496.
[18]Venkatesan N,Tsuchiya K,Kolb M,et al.Glycosyltransferases and glycosaminoglycans in bleomycin and transforming growth factor-β1-induced pulmonary fibrosis.Am J Respir Cell Mol Biol.2014;50(3):583-594.
[19]Li J,Zhang W,Jiao R,et al.DIM attenuates TGF-β1-induced myofibroblast differentiation in neonatal rat cardiac fibroblasts.Int J Clin Exp Pathol.2015;8(5):5121-5128.
[20]Wu KL,Wu CA,Wu CW,et al.Redox-sensitive oxidation and phosphorylation of PTEN contribute to enhanced activation of PI3K/Akt signaling in rostral ventrolateral medulla and neurogenic hypertension in spontaneously hypertensive rats.Antioxid Redox Signal.2013;18(1):36-50.
[21]Kitagishi Y,Matsuda S.Redox regulation of tumor suppressor PTEN in cancer and aging(Review).Int J Mol Med.2013;31(3):511-515.
[22]Nozhat Z,Hedayati M.PI3K/AKT Pathway and Its Mediators in Thyroid Carcinomas.Mol Diagn Ther.2016;20(1):13-26.
[23]Martelli AM,Evangelisti C,Chappell W,et al.Targeting the translational apparatus to improve leukemia therapy:roles of the PI3K/PTEN/Akt/mTOR pathway.Leukemia.2011;25(7):1064-1079.
[24]Guo D,Teng Q,Ji C.NOTCH and phosphatidylinositide3-kinase/phosphatase and tensin homolog deleted on chromosome ten/AKT/mammalian target of rapamycin(m TOR)signaling in T-cell development and T-cell acute lymphoblastic leukemia.Leuk Lymphoma.2011;52(7):1200-1210.
[25]Koromilas AE,Mounir Z.Control of oncogenesis by e IF2αphosphorylation:implications in PTEN and PI3K-Akt signaling and tumor treatment.Future Oncol.2013;9(7):1005-1015.
[26]van Diepen MT,Eickholt BJ.Function of PTEN during the formation and maintenance of neuronal circuits in the brain.Dev Neurosci.2008;30(1-3):59-64.
[27]Wang X,Huang H,Young KH.The PTEN tumor suppressor gene and its role in lymphoma pathogenesis.Aging(Albany NY).2015;7(12):1032-1049.
[28]Chetram MA,Hinton CV.PTEN regulation of ERK1/2signaling in cancer.J Recept Signal Transduct Res.2012;32(4):190-195.
[2]Parikh SN,Myer D,Eismann EA.Prevention of arthrofibrosis after arthroscopic screw fixation of tibial spine fracture in children and adolescents.Orthopedics.2014;37(1):e58-65.
[3]Jaiswal SK,Sharma A,Gupta VK,et al.Curcumin Mediated Attenuation of Carbofuran Induced Oxidative Stress in Rat Brain.Biochem Res Int.2016;2016:7637931.
[4]Li X,Xie W,Xie C,et al.Curcumin modulates miR-19/PTEN/AKT/p53 axis to suppress bisphenol A-induced MCF-7 breast cancer cel proliferation.Phytother Res.2014;28(10):1553-1560.
[5]Takashima M,Parsons CJ,Ikejima K,et al.The tumor suppressor protein PTEN inhibits rat hepatic stellate cell activation.J Gastroenterol.2009;44(8):847-855.
[6]Bawa-Khalfe T,Yang FM,Ritho J,et al.SENP1 regulates PTEN stability to dictate prostate cancer development.Oncotarget.2017;8(11):17651-17664.
[7]Feng X,Jiang J,Shi S,et al.Knockdown of mi R-25 increases the sensitivity of liver cancer stem cells to TRAIL-induced apoptosis via PTEN/PI3K/Akt/Bad signaling pathway.Int JOncol.2016;49(6):2600-2610.
[8]Su D,Zhang CL,Gao YC,et al.Gene Expression and Correlation of Pten and Fabp4 in Liver,Muscle,and Adipose Tissues of Type 2 Diabetes Rats.Med Sci Monit.2015;21:3616-3621.
[9]Parapuram SK,Shi-wen X,Elliott C,et al.Loss of PTENexpression by dermal fibroblasts causes skin fibrosis.J Invest Dermatol.2011;131(10):1996-2003.
[10]卢国强.创伤后的关节挛缩[J].中国组织工程究,2012,16(39):7345-7349.
[11]He RH,Hu XF,Tan HC,et al.Surface modidication of titanium with curcumin:a promising strategy to combat fibrous encapsulation.J Mater Chem B.2015;3(10):2137-2146.
[12]He R,Wang Z,Lu Y,et al.Chaperonin containing T-complex polypeptide subunit eta is a potential marker of joint contracture:an experimental study in the rat.Cell Stress Chaperones.2015;20(6):959-966.
[13]Watson RS,Gouze E,Levings PP,et al.Gene delivery of TGF-β1 induces arthrofibrosis and chondrometaplasia of synovium in vivo.Lab Invest.2010;90(11):1615-1627.
[14]Hagiwara Y,Chimoto E,Takahashi I,et al.Expression of transforming growth factor-beta1 and connective tissue growth factor in the capsule in a rat immobilized knee model.Ups J Med Sci.2008;113(2):221-234.
[15]Piao S,Ryu JK,Shin HY,et al.Repeated intratunical injection of adenovirus expressing transforming growth factor-beta1 in a rat induces penile curvature with tunical fibrotic plaque:a useful model for the study of Peyronie's disease.Int J Androl.2008;31(3):346-353.
[16]Arnott JA,Nuglozeh E,Rico MC,et al.Connective tissue growth factor(CTGF/CCN2)is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.J Cell Physiol.2007;210(3):843-852.
[17]Lee YT,Shao HJ,Wang JH,et al.Hyaluronic acid modulates gene expression of connective tissue growth factor(CTGF),transforming growth factor-beta1(TGF-beta1),and vascular endothelial growth factor(VEGF)in human fibroblast-like synovial cells from advanced-stage osteoarthritis in vitro.JOrthop Res.2010;28(4):492-496.
[18]Venkatesan N,Tsuchiya K,Kolb M,et al.Glycosyltransferases and glycosaminoglycans in bleomycin and transforming growth factor-β1-induced pulmonary fibrosis.Am J Respir Cell Mol Biol.2014;50(3):583-594.
[19]Li J,Zhang W,Jiao R,et al.DIM attenuates TGF-β1-induced myofibroblast differentiation in neonatal rat cardiac fibroblasts.Int J Clin Exp Pathol.2015;8(5):5121-5128.
[20]Wu KL,Wu CA,Wu CW,et al.Redox-sensitive oxidation and phosphorylation of PTEN contribute to enhanced activation of PI3K/Akt signaling in rostral ventrolateral medulla and neurogenic hypertension in spontaneously hypertensive rats.Antioxid Redox Signal.2013;18(1):36-50.
[21]Kitagishi Y,Matsuda S.Redox regulation of tumor suppressor PTEN in cancer and aging(Review).Int J Mol Med.2013;31(3):511-515.
[22]Nozhat Z,Hedayati M.PI3K/AKT Pathway and Its Mediators in Thyroid Carcinomas.Mol Diagn Ther.2016;20(1):13-26.
[23]Martelli AM,Evangelisti C,Chappell W,et al.Targeting the translational apparatus to improve leukemia therapy:roles of the PI3K/PTEN/Akt/mTOR pathway.Leukemia.2011;25(7):1064-1079.
[24]Guo D,Teng Q,Ji C.NOTCH and phosphatidylinositide3-kinase/phosphatase and tensin homolog deleted on chromosome ten/AKT/mammalian target of rapamycin(m TOR)signaling in T-cell development and T-cell acute lymphoblastic leukemia.Leuk Lymphoma.2011;52(7):1200-1210.
[25]Koromilas AE,Mounir Z.Control of oncogenesis by e IF2αphosphorylation:implications in PTEN and PI3K-Akt signaling and tumor treatment.Future Oncol.2013;9(7):1005-1015.
[26]van Diepen MT,Eickholt BJ.Function of PTEN during the formation and maintenance of neuronal circuits in the brain.Dev Neurosci.2008;30(1-3):59-64.
[27]Wang X,Huang H,Young KH.The PTEN tumor suppressor gene and its role in lymphoma pathogenesis.Aging(Albany NY).2015;7(12):1032-1049.
[28]Chetram MA,Hinton CV.PTEN regulation of ERK1/2signaling in cancer.J Recept Signal Transduct Res.2012;32(4):190-195.