饰胶蛋白聚糖与转化生长因子β1对大鼠肾系膜细胞生长及相关信号途径的影响

目的探讨饰胶蛋白聚糖（DCN）与转化生长因子β1（TGF-β1）对大鼠肾系膜细胞（MsC）生长及相关信号途径的影响。方法经脂质体介导将DCN基因转染体外培养的大鼠肾MsC,经筛选和鉴定后,收集阳性细胞克隆的培养上清（DCN上清）。采用流式细胞仪检测经TGF-β1（2μg/L）和（或）DCN上清作用后MsC细胞周期的变化。采用Western印迹法分别检测两者单独或联合作用后,MsC磷酸化丝裂原活化蛋白激酶（p-MAPK）、磷酸化Smad2 （p-Smad2）和p21蛋白表达情况。采用免疫共沉淀方法检测阳性细胞克隆上清中DCN与TGF-β1的结合情况。结果TGF-β1或DCN上清单独作用时均可抑制MsC的增殖,G_2-M期细胞数分别为对照组的81%及86.5%,而两者共同作用时G_2-M期细胞数与对照组差异无统计学意义。经TGF-β1和DCN上清单独作用12 h后,p-ERK1/2表达为对照组的4.4、3.7倍和3.2、3.7倍;p-SAPK/JNK表达为对照组的2.0、1.5倍和1.9、1.5倍;而两者共同作用组p-ERK1/2和p-SAPK/JNK表达与对照组差异无统计学意义。TGF-β1单独作用12 h后,MsC p-Smad2的表达为对照组的2.6倍,而DCN单独作用组及2者联合作用组其表达与对照组差异无统计学意义。DCN单独作用12 h后p21蛋白的表达为对照组的2.6倍,而TGF-β1单独作用及2者联合作用组其表达与对照组差异无统计学意义。阳性细胞克隆上清中DCN可以直接与TGF-β1结合。结论TGF-β1与DCN单独作用均可抑制MsC增殖及激活MAPK信号途径,但2者共同作用时其作用相互抵消。TGF-β1激活Smad信号途径作用可被DCN阻断;DCN上调p21蛋白作用可被TGF-β1阻断。2者通过不同信号分子抑制细胞生长,其作用可能与两者相互结合有关。     

PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis

BACKGROUND: Platelet-derived growth factor (PDGF) has been consistently implicated in the cell proliferation and extracellular matrix accumulation, which characterize progressive glomerular disease. In the present study, the effects of a potent and selective inhibitor of PDGF receptor tyrosine kinase, STI 571, were examined in vitro and in vivo. METHODS: Cultured mesangial cells were incubated with PDGF (50 ng/mL) and fibroblast growth factor-2 (FGF-2; 50 ng/mL) and treated with STI 571 (0.13 to 2.0 micromol/L). Experimental mesangial proliferative glomerulonephritis was induced in male Wistar rats with monoclonal OX-7, anti-rat Thy-1.1 antibody with rats randomized to receive either STI 571 (50 mg/kg intraperitoneally daily) or vehicle. Animals were examined six days later. RESULTS: In vitro, both PDGF and FGF-2 induced a threefold increase in mesangial cell 3H-thymidine incorporation. STI 571 reduced PDGF but not FGF-2-stimulated mesangial cell proliferation in a dose-dependent manner, with complete abolition at 0.4 micromol/L. In animals with Thy-1.1 glomerulonephritis, PDGF receptor tyrosine kinase blockade was associated with significant reductions in mesangial cell proliferation (P < 0.001), the number of activated (alpha-smooth muscle positive) mesangial cells, and glomerular type IV collagen deposition (P < 0.001). CONCLUSION: The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by blockade of PDGF receptor activity suggests the potential clinical utility of this approach as a therapeutic strategy in glomerular disease.     

Combination of pre-operative radiotherapy and surgery suppresses local accumulation of collagen and TGF-beta1 in rats

BACKGROUND: The systemic and local tissue repair responses of radiation in combination with surgery are still unclear. We have studied the effect of fractionated pre-operative radiotherapy with or without subsequent laparotomy on collagen accumulation using a rodent model. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats were divided into four groups (eight rats per group): 1) sham radiation and sham laparotomy (control); 2) sham radiation and laparotomy; 3) radiation and sham laparotomy; and 4) radiation followed by laparotomy. Expanded polytetrafluoroethylene (ePTFE) tubes were implanted subcutaneously in the abdominal wall in the radiotherapy field and on the back outside the radiotherapy field day 0. The abdomen (3 cm x 4 cm) was irradiated day 3 (10 Gy) and again day 7 (10 Gy). On day 10, implants were extirpated, laparotomy undertaken in groups 2 and 4 and new ePTFE tubes implanted subcutaneously. The second implants were extirpated on day 20. Implants were analyzed for hydroxyproline, total protein and transforming growth factor-beta1 (TGF-beta1) levels. RESULTS: On day 10, hydroxyproline (P < 0.05) and TGF-beta1 (P < 0.001) were lower in ePTFE tubes in irradiated compared with non-irradiated rats. On day 20, the abdominal ePTFE hydroxyproline remained low (P < 0.001) in animals subjected to laparotomy and pre-operative irradiation while hydroxyproline levels of rats subjected to irradiation only were similar to controls. The effects of radiation on hydroxyproline were confined to the irradiated abdominal area. There was a positive correlation between hydroxyproline and TGF-beta1 levels in the abdominal wall implant day 20 (r = 0.53, P < 0.005). CONCLUSION: A clinically relevant fractionated radiation scheme reduced subcutaneous collagen accumulation pre-operatively and profoundly within the radiation field post-operatively after laparotomy, possibly because of lowered TGF-beta1 levels.     

Aldose reductase regulates TGF-beta1-induced production of fibronectin and type IV collagen in cultured rat mesangial cells

AIM: To study the effects of aldose reductase (AR) on production of fibronectin and type IV collagen in rat mesangial cells (MsC). METHODS: The vector, pcDNA3-AR, was constructed based on pET-15b-AR. Lipofect AMINE was used for stable transfection and G418 was used for selecting positive clones. Sorbinil and zopolrestat were added for suppressing the activity of AR, respectively. The production of fibronectin and type IV collagen and the activation of Smads and MAPK signal transduction pathway were analysed by western blot and AP-1 activity was analysed by electrophoretic mobility shift assays (EMSA). RESULTS: The normal MsC showed increased expression of fibronectin and type IV collagen with stimulation of TGF-beta1. Compared with the normal MsC, the MsC pre-incubated with ARI showed reduced expression (P < 0.05) and the AR-transfected MsC showed increased expression (P < 0.05). The normal MsC showed activation of ERK, JNK and p38 with stimulation of TGF-beta1, while the activation of JNK and p38 was inhibited in the MsC pre-incubated with ARI and only the activation of JNK was enhanced in the AR-transfected MsC. The normal MsC showed enhanced AP-1 activity with the stimulation of TGF-beta1, and similarly the activity was inhibited in the MsC pre-incubated with ARI and was more enhanced in the AR transfected MsC. CONCLUSION: AR can regulate the expression of fibronectin and type IV collagen with the stimulation of TGF-beta1 in MsC, which may have relations with the activation of JNK-MAPK and p38-MAPK signalling pathways and AP-1.     

IGF-1-induced lipid accumulation impairs mesangial cell migration and contractile function

BACKGROUND: Chronic treatment of mesangial cells with insulin-like growth factor-1 (IGF-1) results in intracellular lipid accumulation. These mesangial cells resemble foam cells. METHODS: To determine whether this phenotype affects cell function, lipid-laden mesangial cells were tested for their ability to migrate in response to IGF-binding protein-5 (IGFBP-5) and to contract in response to angiotensin II (Ang II). IGFBP-5 binding and subsequent activation of the signal transduction cascade for migration were examined. To confirm that lipid accumulation was responsible for impaired contractility, lipid was removed from lipid-laden mesangial cells and the cells were re-tested for contractile response to Ang II. RESULTS: In comparison to control mesangial cells, lipid-laden cells failed to migrate in response to IGFBP-5. Although cellular binding of IGFBP-5 was not altered by lipid accumulation, IGFBP-5 failed to activate cdc42, a Rho GTPase required for IGFBP-5-mediated mesangial cell migration. These data indicate that lipid accumulation within the mesangial cell interferes with the signal transduction response to IGFBP-5. In addition, mesangial cells treated with IGF-1 had reduced contraction to Ang II. When lipid accumulation was exaggerated by adding cholesteryl esters to the culture medium, mesangial cells failed to contract in response to Ang II. Following removal of excess lipid from these mesangial cells, the contractile response to Ang II was restored. CONCLUSIONS: IGF-1 induces lipid accumulation in mesangial cells, which impairs their ability to respond to specific migratory and contractile stimuli. These observations are relevant to understanding functional abnormalities in diseases where mesangial foam cells occur, such as focal and segmental glomerulosclerosis and diabetic nephropathy.     

饰胶蛋白聚糖对大鼠肾系膜细胞生长的抑制作用及其信号转导途径的研究

目的 探讨饰胶蛋白聚糖(DCN)对大鼠系膜细胞(MsC)生长的抑制作用及其信号转导分子MAPKs和p21表达的影响&#65377;方法 经脂质体介导将DCN基因转染体外培养的大鼠MsC,筛选和鉴定后,收集阳性细胞克隆的培养上清(DCN上清),将其加入正常MsC的培养液中, 采用流式细胞仪检测细胞周期&#65377;用Western 印迹法分别检测MAPKs,包括细胞外调节激酶(ERK)1/2&#65380;应激活化蛋白激酶(SAPK)/氨基末端激酶(JNK)和p38和p21蛋白表达;用免疫荧光法观察p21在细胞中的表达&#65377;结果 DCN上清明显抑制正常MsC的增殖, G2-M期细胞数明显减少,仅为对照组的35%(P < 0.05); 磷酸化ERK1/2及SAPK/JNK表达增强, 分别为对照组的2.2倍&#65380; 1.4倍及1.7倍&#65380; 1.8倍;磷酸化p38无明显变化&#65377;DCN抗体呈浓度依赖性抑制磷酸化ERK1/2&#65380; SAPK/JNK的表达上调&#65377;DCN上清还可使细胞p21的表达明显增强,而DCN抗体也同样呈浓度依赖性地抑制其上调表达的作用, ERK1/2 及SAPK/JNK通路抑制剂U0126和circumin均可抑制其上调表达的作用,分别为对照组的64%和61%;而p38通路抑制剂SB203580则对其无影响&#65377;结论 DCN对肾MsC的生长有抑制作用,其机制可能经ERK1/2&#65380;SAPK/JNK和p21蛋白介导&#65377;     

Heparin decreases mesangial matrix accumulation after selective antibody-induced mesangial cell injury.

Anti-rat thymocyte antibody-induced injury of glomerular mesangial cells is characterized initially by lysis (1 h) and is followed by proliferation (beginning at 3 to 4 days), with resolution that can include a focal increase in mesangial matrix (by 28 days). Chronic administration (every 12 h) of heparin (anticoagulant or nonanticoagulant) resulted in a decrease in antibody-induced mesangial cell proliferation, which, in turn, was associated with a decrease in the size and number of areas of focal mesangial matrix increase. The effect could not be attributed to the effect of heparin on complement, to alterations in the small numbers of la-positive cells that characterize the lesion, or to binding of antibody to glomeruli. The beneficial effects of heparin in reducing mesangial cell proliferation, with a subsequent reduction in matrix increase, suggest that mesangial cell responses are a major element in the development of at least some forms of glomerulosclerosis. The possible mechanisms by which these effects of heparin may be achieved are discussed.     

TGF-beta 1 stimulates glucose uptake by enhancing GLUT1 expression in mesangial cells

BACKGROUND: An increase in the expression of transforming growth factor-beta 1 (TGF-beta 1) has been proposed to play an important role in the excessive production of extracellular matrix (ECM) proteins seen in diabetes. Because the linkage between glucose metabolism and ECM protein production was found in mesangial cells overexpressed with the brain-type glucose transporter (GLUT1), we hypothesized that TGF-beta 1 could affect glucose metabolism. METHODS: To prove this hypothesis, we examined the effect of TGF-beta 1 on glucose uptake, the first step of glucose metabolism, in mesangial cells. 2-Deoxy-D-glucose (2DOG) uptake and the expression of GLUT1 were measured in mesangial cells exposed to various concentrations of TGF-beta 1. The kinetic constants were determined using 2DOG and 3-O-methyl-D-glucose (3OMG). The effect of anti-TGF-beta neutralizing antibody on 2DOG uptake and GLUT1 mRNA was also examined in mesangial cells cultured under high-glucose (22.2 mM) conditions for 72 hours. RESULTS: TGF-beta 1 stimulated 2DOG uptake in mesangial cells by approximately 2.5-fold in a dose- (1.25 ng/ml maximum) and time-dependent manner, with a peak stimulation at nine hours. The increase in 2DOG uptake by TGF-beta 1 was completely abolished by the addition of 1 microgram/ml cycloheximide, and kinetic analysis of 2DOG or 3OMG uptake revealed an increase in Vmax by TGF-beta 1. Furthermore, TGF-beta 1 enhanced the expression of GLUT1 mRNA from one hour, followed by an enhancement of the expression of GLUT1 protein at nine hours. Finally, 2DOG uptake was significantly enhanced in cells cultured under high-glucose (22.2 mM) conditions as compared with that in cells under normal glucose (5.6 mM) conditions, and this increase in 2DOG uptake in cells under high-glucose conditions was inhibited by the addition of anti-TGF-beta neutralizing antibody. CONCLUSIONS: TGF-beta 1 stimulates glucose uptake by enhancing the expression of GLUT1 in mesangial cells, which leads to the acceleration of intracellular metabolic abnormalities in diabetes.     

Estradiol reverses TGF-beta1-induced mesangial cell apoptosis by a casein kinase 2-dependent mechanism

BACKGROUND: The slower rate of progression of chronic renal disease in women than in men is explained in part by the ability of estradiol to reverse the stimulatory effect of transforming growth factor-beta1 (TGF-beta1) on collagen IV synthesis at the level of casein kinase 2 activation. Casein kinase 2 also phosphorylates and activates the pro-apoptotic protein, p53. We hypothesized that estradiol would reverse TGF-beta1-induced mesangial cell apoptosis by antagonizing the stimulatory effects of TGF-beta1 on casein kinase 2 activity, thereby preventing p53 activation. METHODS: The effects of TGF-beta1 on mesangial cell apoptosis, p53 phosphorylation, Bax and Bcl-2 levels, caspase 9 activity, and cleavage of PARP were examined. The abilities of estradiol and a specific inhibitor of CK2 (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) (DRB) to modulate the effects of TGF-beta1 on these processes were also examined. RESULTS: TGF-beta1 (2 ng/mL), which up-regulates CK2 activity, induces apoptosis in murine mesangial cells together with p53 serine389 phosphorylation, up-regulation of Bax, suppression of Bcl-2, destabilization of mitochondrial permeability transition pores, stimulation of caspase 9 activity and activation of PARP. TGF-beta1-induced p53 activation and all the intermediary steps leading to mesangial cell apoptosis were reversed by estradiol (10-9 mol/L) and by DRB, potent inhibitors of CK2 activity, but not by inhibitors of the p38 MAPK, ERK or JNK signaling cascades. In contrast, TGF-beta1 failed to induce apoptosis in p53 knockout mesangial cells. CONCLUSIONS: Our data suggest that CK2 mediates the stimulatory effects of TGF-beta1 on mesangial cell apoptosis via a p53-dependent mechanism. The ability of estradiol to reverse TGF-beta1-induced apoptosis may contribute to the protective effects of female gender on the course of chronic renal disease.     

肿瘤多药耐药与细胞信号转导的研究进展

肿瘤多药耐药是肿瘤治疗失败的重要原因,最近研究发现细胞信号转导通路在肿瘤细胞耐药形成的过程中具有重要作用,主要包括凋亡、NF-κB、MAPK、PI3K/Akt通路及肿瘤干细胞等方面,而应用相应的通路阻断剂逆转耐药将成为提高肿瘤治疗效果的亮点。本文主要对以上与肿瘤多药耐药相关的细胞信号转导通路作一综述。     

白蛋白诱导的肾小管细胞凋亡与丝裂素激活蛋白激酶信号通路

目的 探讨白蛋白诱导肾小管上皮细胞凋亡以及诱导凋亡的信号传导机制&#65377; 方法 将培养的大鼠肾小管细胞NRK-52E分别与不同浓度(10&#65380; 20&#65380; 30 mg/ml)的去脂无内毒素牛血清白蛋白(BSA)共同孵育6&#65380; 12&#65380; 18和24 h&#65377;透射电镜&#65380;共聚焦激光显微镜和流式细胞仪检测细胞凋亡&#65377;BSA 20 mg/ml刺激NRK-52E细胞15&#65380; 30&#65380; 60和120 min后, Westen印迹测定p38&#65380;氨基末端激酶(JNK)和细胞外信号调节激酶(ERK)活性&#65377;将SB202190(20 μmol/L, p38抑制剂)&#65380;SP600125(10 μmol/L, JNK抑制剂)和PD98059(20 μmol/L, ERK抑制剂)分别与白蛋白和NRK-52E细胞共同孵育24 h后检测细胞凋亡&#65377;结果 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡&#65377;白蛋白与NRK-52E细胞共孵育后,p38和JNK活性明显升高,ERK活性显著降低&#65377;SB202190和SP600125可分别抑制白蛋白诱导NRK-52E细胞凋亡,而PD98059促进白蛋白诱导的NRK-52E细胞凋亡&#65377;结论 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡,而p38和JNK激活与ERK抑制介导了白蛋白诱导的肾小管细胞凋亡&#65377;     

Renin-stimulated TGF-beta1 expression is regulated by a mitogen-activated protein kinase in mesangial cells

Recent evidence indicates that renin itself might be profibrotic, independent of angiotensin II; however, the signaling system by which renin exerts a direct effect is not known. We tested the hypothesis that renin receptor activation, in turn, activates the extracellular-signal regulated kinase 1 and 2 (ERK1/2) of the mitogen-activated protein kinase system in mesangial cells. Recombinant rat renin induced a rapid phosphorylation of ERK1/2 and subsequent cell proliferation in a dose- and time-dependent manner. ERK1/2 activation by renin addition was not altered by angiotensin-converting enzyme inhibition or angiotensin receptor blockade. An ERK kinase inhibitor significantly reduced the renin-induced ERK1/2 phosphorylation and the subsequent increase in transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor-1 mRNA expression. A small-inhibiting RNA, siRNA, against the renin receptor completely blocked ERK1/2 activation by rat renin. We conclude that renin induces ERK1/2 activation though a receptor-mediated, angiotensin II-independent mechanism in mesangial cells. This renin-activated pathway triggers cell proliferation along with TGF-beta1 and plasminogen activator inhibitor-1 gene expression. This system may play an important role in the overall profibrotic actions of renin.     

Peroxisome proliferator-activated receptor-gamma ligands inhibit TGF-beta 1-induced fibronectin expression in glomerular mesangial cells

The thiazolidinedione (TZD) class of antidiabetic drugs, which are ligands for peroxisome proliferator-activated receptor (PPAR)-gamma, has been shown to possess potent anti-inflammatory and antineoplastic actions. Here, we show in mesangial cells that PPAR-gamma agonists inhibit fibronectin expression by transforming growth factor (TGF)-beta 1. TGF-beta 1 enhanced fibronectin mRNA expression, and this enhancement was abrogated by pretreatment with pioglitazone. Electrophoretic mobility shift assay identified that pioglitazone inhibited TGF-beta 1-induced DNA binding of activator protein-1 (AP-1). Pioglitazone inhibited AP-1 reporter activity but not Smad binding elements reporter activity without affecting TGF-beta 1-induced activation of mitogen-activated protein kinases (MAPKs) or Smad2. PPAR-gamma overexpression inhibited TGF-beta 1-induced fibronectin expression as well as the activation of AP-1. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural PPAR-gamma ligand, also inhibited TGF-beta1-induced fibronectin expression by suppressing AP-1 activation by TGF-beta 1. 15d-PGJ(2) inhibited the TGF-beta 1-induced MAPK activation. Dominant-negative PPAR-gamma (Delta PPAR-gamma) completely abrogated the inhibitory effect of pioglitazone and incompletely blocked its effect of 15d-PGJ(2) on TGF-beta 1-induced AP-1 reporter activity. Delta PPAR-gamma overexpression did not affect the inhibitory effect of 15d-PGJ(2) on TGF-beta 1-induced MAPK activation. In conclusion, pioglitazone inhibits TGF-beta 1-induced fibronectin expression by inhibiting AP-1 activation dependent on PPAR-gamma, while 15d-PGJ(2) acts through a dual mechanism independent of and dependent on PPAR-gamma activation in mouse mesangial cells.     

缺血/再灌注过程中信号转导通路的磷酸化调节

器官移植、中风、心肌梗死等会引发局部缺血/再灌注（Ischemia／Reperfusion,I／R）。炎性细胞的活化和细胞毒素因子的表达与再灌注损伤密切相关。这些炎性介质的活化会启动相关下游磷酸化或去磷酸化的级联反应。该文仅综述I／R损伤过程中信号转导通路磷酸化调节机制的研究进展。     

丝裂素活化蛋白激酶信号转导途径对血小板源生长因子所致鼠肾系膜细胞表型转化的调控作用

目的 研究血小板源生长因子（PDGF）对肾小球系膜细胞（MC）表型转化的影响,并探讨丝裂素活化蛋白激酶（MAPK）信号转导途径在其作用中的调控机制。方法 以体外培养的大鼠MC为对象,观察PDGF对细胞增殖、α-平滑肌肌动蛋白（SMA）表达和细胞内c-Jun氨基末端激酶（JNK）活性的作用;观察细胞外信号调节激酶（ERK）途径上游信号MEK-1阻断剂PD98059对PDGF所致上术作用的影响;应用raf-1基因转染并稳定高表达的MC观察其对α-SMA表达,细胞形态及其超微结构的影响。结果 PDGF可刺激MC持续增殖;刺激6～48h可使α-SMA表达增加,但持续刺激72h以上则可导致其表达被抑制近50%,PDGF对JNK活性无明显影响,阻断ERK活化不影响JNK活性及PDGF对α-SMA表达的短时刺激作用。Raf-1蛋白激酶稳定高表达的MC细胞内α-SMA表达缺失,同时细胞内微丝、微管结构减少,细胞形态改变。结论 PDGF对系膜细胞表型转化的影响表现为促进细胞持续增殖、短时作用刺激α-SMA表达而持续作用则抑制α-SMA表达,提示其为具有调控MC增殖和分化双重作用的细胞因子。PDGF的作用可能与MAPK不同亚类的调控有关,其中Raf-1和JNK活性可能对细胞表型具有关键调控作用。