Toll‐like receptor 3 activation in retinal pigment epithelium cells – Mitogen‐activated protein kinase pathways of cell death and vascular endothelial growth factor secretion

Purpose: Toll‐like receptor 3 (TLR3) is a receptor of the innate immune system, recognizing double‐stranded RNA. TLR3 can lead to cytokine release or apoptosis and has recently been associated with the development of geographical atrophy via cytotoxic effects on the retinal pigment epithelium (RPE). The current study was conducted to elucidate the underlying pathways of TLR3 effects in the RPE. Methods: TLR3 activation via polyinosinic acid/polycytidylic acid (Poly I:C) was investigated in primary porcine RPE cells, focussing on cell death and vascular endothelial growth factor (VEGF) secretion. Primary cells were stimulated with different concentrations of Poly I:C. Cell death was investigated in trypan blue exclusion assay and cell death detection ELISA. VEGF and IFN‐ß secretion were also detected in ELISA. As Mitogen‐activated protein kinases (MAPK) play an important part in TLR3‐mediated signal transduction, we investigated the influence of JNK, ERK1/2 and p38 on cell death and VEGF secretion, using commercially available inhibitors. Results: Activation of TLR3 by Poly I:C induced concentration‐dependent cell death, partly mediated by JNK. ERK1/2 was activated and exerted some protection. Furthermore, higher concentrations of Poly I:C increased VEGF secretion after 4 and 24 hr, which was independent of MAPK. Conclusion: The induction of cell death in RPE cells by TLR3 activation confirms possible involvement of TLR3 activation in GA. As cell death is partly mediated by JNK, more studies should be conducted investigating the role of JNK in RPE cell death to evaluate whether its inhibition might be a new therapeutic opportunity for the treatment of geographical atrophy. Additionally, effects on VEGF secretion can be found.     

Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function

PURPOSE: This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF165-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology. METHODS: C57/Bl6 mice were injected with VEGF165 and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF165 and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability. RESULTS: Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF165. It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF165-mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF165-mediated permeability compared with control (untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF165. CONCLUSIONS: Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF165-induced paracellular permeability.     

LeTx抑制MEK通路对体外血管紧张素诱导的VEGF分泌的影响

目的:体外观察血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)对视网膜色素上皮细胞(retinal pigmented epithelium,RPE)VEGF分泌的影响以及能否通过炭疽毒素致死因子(lethalfactor,LF)通过阻断MEK/MAPK通路对VEGF分泌的影响。方法:体外培养人RPE细胞分为A,L和N组。A组用含100μmol/LAngⅡ及25mL/L小牛血清的低糖DMEM培养液刺激。L组于A组培养液刺激细胞前,先用10μmol/LEF与100nmol/LLF联合处理细胞24h;N组细胞培养于仅含25mL/L小牛血清的DMEM培养液。作用不同时间梯度后收集细胞上清。采用ELISA法测定各组VEGF浓度。结果:AngⅡ刺激RPE45min即能诱导VEGF分泌,这种作用在刺激3h达到最强,约为对照组7.9倍。预先用LeTx处理的RPE细胞并不能够对AngⅡ的刺激产生反应。在任一刺激时段,细胞培养液中VEGF浓度始终维持在较低水平。结论:LeTx通过抑制MEK通路对AngⅡ刺激的VEGF分泌有抑制作用。     

Role for extracellular signal-responsive kinase-1 and -2 in retinal angiogenesis

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent mitogen for micro- and macrovascular endothelial cells (ECs). Evidence points to a possible role for two mitogen-activated protein (MAP) kinases, the extracellular-signal responsive kinases (ERK)-1 and -2, in VEGF signaling in ECs. This study was undertaken to begin to define the precise role of MAP kinases in VEGF signal transduction related to angiogenesis. METHODS: Bovine retinal microvascular endothelial cells (BRMECs) and a well-established rat model of retinopathy of prematurity (ROP) were used to investigate the role of ERK-1/2 in EC proliferation and tube formation and in retinal angiogenesis in vivo. RESULTS: Administration of VEGF to BRMEC cultures increased ERK-1/2 phosphorylation, cell proliferation, and tube formation in a dose-dependent manner. Phosphorylation of retinal ERK-1/2 also was increased in the ROP model. An inhibitor of ERK, AG126, and an inhibitor of ERK kinase (MEK), PD98059, exhibited a dose-dependent reduction of ERK phosphorylation and EC proliferation, but not tube formation, in VEGF-stimulated BRMECs. In the ROP model, intravitreous injection of 10 micro M AG126 or PD98059 reduced the retinal neovascular area by 71% and 48%, respectfully. No effect was seen on intraretinal blood vessel growth. CONCLUSIONS: These experiments point to a critical role for ERK and MEK in proliferation of ECs, but not in tube formation. Furthermore, inhibition of either of these two signal intermediates can significantly retard retinal neovascularization. This suggests that the MAPK pathway may provide rational targets for therapeutic intervention in ocular and other diseases with an angiogenic component.     

Activation and role of MAP kinase-dependent pathways in retinal pigment epithelial cells: ERK and RPE cell proliferation

PURPOSE: Retinal pigment epithelial (RPE) cell proliferation plays a key role in the pathogenesis of ocular diseases involving the posterior segment. Mitogen-activated protein kinases (MAPKs) are involved in the control of cell proliferation. This study was conducted to investigate the involvement of the extracellular signal-regulated kinase (ERK) pathway, the major MAPK pathway implicated in cell growth during the induction of RPE cell proliferation. METHODS: RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the Ras/Raf/MAP kinase-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was detected by Western blot analysis and immunochemistry with specific anti-phosphosignaling protein antibodies. Pharmacologic and antisense (AS) oligonucleotide (ODN) strategies were used to analyze the ERK signaling involved in serum-induced cell proliferation. RESULTS: FCS (10%) induced more vigorous RPE cell proliferation than did FGF2, VEGF, platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), alone or in combination. Pharmacologic inhibition of Ras and Raf-1 reduced cell proliferation by 67% to 100% and by 62% to 79%, respectively, demonstrating that activation of the Ras/Raf-1 pathway was essential for FCS-induced RPE cell proliferation. MEK1/2, ERK2, and P90 ribosomal S6 kinase (P90(RSK)), the kinases downstream from ERK2, were strongly activated during cell proliferation. Pharmacologic inhibition of MEK1/2 abolished activation of ERK2, but reduced cell proliferation by only 32%, showing that MEK/ERK participates in the signaling involved in RPE cell growth. Both inhibition of ERK2 activation, which reduced cyclin D1 production, and inhibition of cyclin D1 by AS ODN decreased cell proliferation, suggesting that RPE cell proliferation is mediated by cyclin D1 through ERK2. CONCLUSIONS: The requirement for Ras and the regulatory role of ERK2 in cyclin D1 production and in cell proliferation suggest that the Ras/Raf/MEK/ERK pathway plays a key role in the control of RPE cell proliferation. These data may have important implications for the development of more selective methods for the inhibition of retinal proliferation.     

P2Y receptor-mediated stimulation of Müller glial cell DNA synthesis: dependence on EGF and PDGF receptor transactivation

PURPOSE: To determine whether P2Y receptor-evoked proliferation of Müller glial cells depends on transactivation of receptor tyrosine kinases. METHODS: Primary cultures of Müller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine (BrdU) immunoassay, and the phosphorylation states of the extracellular signal-regulated kinase (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) were determined by Western blot analysis. RESULTS: In Müller cells, the mitogenic effect of P2Y receptor activation by extracellular adenosine triphosphate (ATP) depended on transactivation of both the platelet-derived growth factor (PDGF) and the epidermal growth factor (EGF) receptor tyrosine kinases, as suggested by the blocking effects of the tyrphostins AG1296 and AG1478 on the ATP-induced proliferation and phosphorylation of ERK1/2. Moreover, the PDGF-induced proliferation may depend on transactivation of the EGF receptor kinase. Antibodies against heparin-binding EGF (HB-EGF) or PDGF, as well as inhibition of matrix metalloproteinases (MMPs) blocked ATP-evoked proliferation. At least one metalloproteinase (MMP-9), was implicated in the signal transfer from P2Y to EGF receptors. In contrast, the mitogenic effect of fetal calf serum was independent of growth factor receptor activity. P2Y receptor activation stimulated Müller cell proliferation by activating the ERK1/2 and the phosphatidylinositol 3 (PI3) kinase signaling pathways, whereas the p38 MAPK pathway was not involved in mitogenic signaling. CONCLUSIONS: The present data suggest that P2Y-receptor-induced mitogenic signaling in Müller cells is mediated by transactivation of the PDGF and EGF receptor tyrosine kinases. The transactivation may be mediated by release of PDGF and MMP-dependent shedding of HB-EGF from the Müller cell matrix, respectively. The transactivation of the receptor tyrosine kinases may result in activation of ERK1/2 and PI3 kinase and an increase in the proliferation rate.     

Toxoplasma gondii protects against H2O2‐induced apoptosis in ARPE‐19 cells through the transcriptional regulation of apoptotic elements and downregulation of the p38 MAPK pathway

Purpose: Toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, can lead to severe visual impairment. T. gondii inhibits or delays programmed cell death caused by various apoptotic triggers; however, the mechanisms involved in the T. gondii‐induced suppression of apoptosis in retinal cells have not been analysed in detail. Methods: We investigated the role of T. gondii infection in H₂O₂‐induced apoptosis in human retinal pigment epithelial cells (ARPE‐19) by monitoring the activities of apoptosis‐regulating molecules and mitogen‐activated protein kinases (MAPKs), including p38 MAPK. We also examined the gene downstream from p38 MAPK. Results: T. gondii infection significantly inhibited the cellular toxicity of H₂O₂ (500 μm ) and increased cell viability in a multiplicity of infection (MOI)‐dependent manner by reducing DNA fragmentation and reactive oxygen species (ROS) generation in ARPE‐19 cells. Western blot analysis also showed that T. gondii infection prevented the host cell expression of pro‐apoptotic factors, such as Bad and Bax, and the activation of caspase‐3. Infection with T. gondii increased the expression of the anti‐apoptotic factor Bcl‐2 in ARPE‐19 cells under oxidative stress. In accordance with these findings, Toxoplasma infection was protective enough to suppress the phosphorylation of p38 MAPK following H₂O₂ treatment. Exposure to H₂O₂ increased the expression of heme oxygenase‐1 (HO‐1) in ARPE‐19 cells, and its expression was significantly inhibited in H₂O₂‐treated infected cells. Conclusion: The protective function of T. gondii infection against ROS‐induced apoptosis results from changes in the expression of apoptotic molecules and the downregulation of stress‐induced intracellular signalling.     

Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells

AIM: To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5), and pig choroidal endothelial cells (CEC). METHODS: Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab (0.008-2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5'-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The "Live/Dead" viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors (VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry. RESULTS: No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity (maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor. CONCLUSIONS: These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses (2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium.     

Vascular endothelial growth factor expression and secretion by retinal pigment epithelial cells in high glucose and hypoxia is protein kinase C-dependent

Retinal pigment epithelial (RPE) cells express vascular endothelial growth factor (VEGF) in response to high glucose or hypoxia. We hypothesised that VEGF expression and secretion by RPE cells in high glucose and hypoxia are regulated by protein kinase C (PKC). Primary cultured RPE cells from Sprague-Dawley rats were growth-arrested for 48 hr in 0.5% FBS in 5.6 or 30 mm D-glucose. Cells were exposed to hypoxic conditions (<1% O(2), 5% CO(2)) for the last 15-18 hr of growth-arrest. PKC -alpha, -beta(1), -delta, -epsilon, and -zeta were expressed by RPE cells and exposure to high glucose for 48 hr had no effect on expression as demonstrated by Western immunoblotting. High glucose, hypoxia or VEGF stimulated translocation of a number of the PKC isozymes to the membrane or particulate fractions implying activation. In response to high glucose or acute phorbol myristate acetate (PMA) stimulation, VEGF mRNA analysed by RT-PCR was increased. Intracellular VEGF protein identified by immunoblotting and confocal immunofluorescence imaging was significantly increased by high glucose, hypoxia or acute PMA stimulation. Calphostin C or a specific inhibitor of PKC-zeta prevented high glucose-stimulated VEGF expression in high glucose. VEGF secretion, as measured by ELISA in the culture medium, was enhanced in hypoxia but not in high glucose. Following exposure of RPE cells to PMA for 24 hr, PKC-delta was significantly down regulated, whereas PKC-alpha, -beta, -epsilon and -zeta remained unchanged. Secretion of VEGF in normal or high glucose, or hypoxia was significantly reduced following treatment with PMA for 24 hr but not with the PKC-zeta inhibitor. We conclude that in high glucose and hypoxia PKC isozymes are activated and are necessary for VEGF expression. Secretion of VEGF is enhanced in hypoxia and appears to be regulated by PKC-delta. RPE cells may contribute to the pathogenesis of retinopathy caused by high glucose and hypoxia through the expression and secretion of VEGF that are regulated by PKC isozymes.     

Characterization of the basic fibroblast growth factor-evoked proliferation of the human Müller cell line,MIO-M1

Background Basic fibroblast growth factor (bFGF) has been suggested to mediate activation of Müller glial cells in the ischemic–hypoxic retina. However, the intracellular pathways activated by bFGF in human Müller cells have been little explored. We characterized the signaling transduction pathways which are involved in the control and growth factor-evoked proliferation of a recently described human Müller cell line, MIO-M1. In addition, we investigated whether bFGF evoked the release of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) from the cells.Methods The growth factor-evoked proliferation of cultured MIO-M1 cells was estimated by means of a bromodeoxyuridine immunoassay, in the absence and presence of blockers of mitogen-activated protein kinases (MAPKs) and of the phosphatidylinositol-3 kinase (PI3K). The activation state of the p44/p42 MAPK was determined by Western blotting, and the bFGF-evoked release of VEGF and HGF was evaluated by ELISA.Results bFGF evoked a concentration-dependent increase of the cell proliferation, with an EC₅₀ of ~1 ng/ml, via activation of both the p44/p42 MAPK and the p38 MAPK. In contrast, the mitogenic effects of the platelet-derived and the heparin-binding epidermal growth factors were dependent on p44/p42 MAPK activation and independent of activation of p38 MAPK. The transforming growth factors 1 and 2 also evoked cell proliferation which was independent of activation of the MAPKs investigated. bFGF evoked a release of VEGF and of HGF by the cells; these effects were independent of MAPK activation and were possibly mediated by activation of the PI3K signaling pathway.Conclusion bFGF evokes multiple intracellular signaling pathways in human Müller cells which underlie the gliotic cell responses upon ischemic–hypoxic insults in the retina. Beside the stimulation of cell proliferation, which is dependent on activation of p44/p42 and p38 MAPKs, bFGF induces the secretion of VEGF and HGF by Müller cells.     

人参皂苷Rg1治疗高糖损伤大鼠视神经细胞的机制研究:基于生物信息学分析

目的　通过SwissTarget及PubMed 数据库预测人参皂苷Rg1治疗高糖损伤大鼠视神经细胞的潜在靶点及作用机制。方法　利用PubMed compound 网站获取人参皂苷Rg1的分子结构式并于SwissTarget数据库进行Rg1治疗全部疾病的靶点预测, 通过高糖损伤视神经相关PubMed数据库数据集GSE 27382分析视神经损伤患者与健康人员差异基因的表达情况,将SwissTarget数据库的预测结果与PubMed数据库分析结果进行集合,最终筛选Rg1治疗高糖损伤大鼠视神经细胞的潜在靶点IL-2、 PTAFR、PSEN2、PSENEN、NCSTN、APH1A、PSEN1、APH1B、HSP90AA1,将获得的靶点进行KEGG及GO富集分析,明确相关的生物过程及分子功能、信号通路,将Notch/MAPK信号通路选定为机制研究的内容。镜下摘取饲养的16只Wistar大鼠双侧视神经,使用含新生胎牛血清的DMEM/F12 培养基进行原代培养2周,经过形态学验证后,将细胞分为对照组、高糖损伤组（高糖DMEM/F12培养24 h）、Rg1治疗组(10 μg·L^-1 Rg1及高糖DMEM/F12同时培养24 h)及Notch1抑制剂组（10 μg·L^-1 Tangerintin及高糖DMEM/F12同时培养24 h）。采用CCK-8法检测不同组别大鼠视神经细胞培养24 h、 48 h、 72 h的增殖能力,采用免疫印迹法验证Notch/MAPK信号通路蛋白表达的变化。结果　细胞增殖实验结果显示,与对照组相比,高糖损伤组大鼠视神经细胞培养24 h、 48 h、 72 h后吸光度均明显下降（均为P<0.05);与高糖损伤组相比,Rg1治疗组大鼠视神经细胞培养24 h、 48 h、 72 h后吸光度均增加（均为P<0.05)。免疫印记法结果显示,与对照组相比,高糖损伤组大鼠视神经细胞Notch1受体蛋白表达下降,MAPK、JNK、ERK1、P38蛋白表达均增加;而与高糖损伤组相比,Rg1治疗组和Notch1抑制剂组大鼠视神经细胞中Notch1受体蛋白表达均升高,MAPK、ERK1、JNK、P38蛋白表达均下降。结论　人参皂苷Rg1通过激活Notch1受体影响Notch/MAPK信号通路,进而缓解高糖引起的大鼠视神经损伤。     

Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells

PURPOSE: To evaluate the effects of TGF-beta1 and doxycycline on production of gelatinase MMP-9 and activation of Smad, c-Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways in human corneal epithelial cells. METHODS: Primary human corneal epithelial cells were cultured to confluence. The cells were treated with different concentrations of TGF-beta1 (0.1, 1, or 10 ng/mL), with or without TGF-beta1-neutralizing mAb (5 microg/mL), SP600125 (30 microM), PD98059 (40 microM), SB202190 (20 microM), or doxycycline (5-40 microg/mL) for different lengths of time. Conditioned media were collected from cultures treated for 24 to 48 hours to evaluate the MMP-9 production by zymography and activity assay. Total RNA was isolated from cells treated for 6 to 24 hours to evaluate MMP-9 expression by semiquantitative RT-PCR and Northern hybridization. Cells treated for 5 to 60 minutes were lysed in RIPA buffer for Western blot with phospho-specific antibodies against Smad2, JNK1/2, ERK1/2, or p38. RESULTS: TGF-beta1 increased expression, production, and activity of MMP-9 by human corneal epithelial cells in a concentration-dependent fashion. TGF-beta1 also induced activation of Smad2, JNK1/2, ERK1/2, and p38 within 5 to 15 minutes, with peak activation at 15 to 60 minutes. Doxycycline markedly inhibited the TGF-beta1-induced production of MMP-9 and activation of the Smad, JNK1/2, ERK1/2, and p38 signaling pathways. Its inhibitory effects were of a magnitude similar to SP600125, PD98059, and SB202190, specific inhibitors of the JNK1/2, ERK1/2, and p38 pathways, respectively. CONCLUSIONS: These findings demonstrated that doxycycline inhibits TGF-beta1-induced MMP-9 production and activity, perhaps through the Smad and MAPK signaling pathways. These inhibitory effects may explain the reported efficacy of doxycycline in treating MMP-9-mediated ocular surface diseases.     

Protection of RPE cells from oxidative injury by 15-deoxy-delta12,14-prostaglandin J2 by augmenting GSH and activating MAPK

PURPOSE: The goal of this study was to identify the mechanisms by which 15-deoxy-Delta(12,14)-prostaglandin J(2) (dPGJ(2)) protects RPE cells from oxidative injury. METHODS: Cell viability was determined by MTT assay. Protein expression and activation of signaling molecules were detected by Western blot. Reduced glutathione (GSH) was determined by a colorimetric assay kit. PPARgamma expression was knockdown by small interfering (si)RNA technique. RESULTS: dPGJ(2) protected ARPE19 cells from oxidative injury, whereas the synthetic PPARgamma agonists AGN195037 and rosiglitazone had no effect. PPARgamma knockdown also did not affect dPGJ(2)'s protective activity. dPGJ(2) upregulated GSH synthesis via induction of glutamylcysteine ligase. GSH depletion sensitized cells to oxidative stress and completely reversed the protective effect of dPGJ(2). dPGJ(2) activated ERK, JNK, and p38; GSH induction by dPGJ(2) depended partially on JNK and p38. In addition, dPGJ(2) significantly extended hydrogen peroxide-induced activation of JNK and p38, but not of Akt. Inhibition of MEK, JNK, and p38 abolished dPGJ(2)'s protection of ARPE19 cells from oxidative injury, whereas inhibiting PI3K/Akt pathway failed to affect dPGJ(2)'s protective effect. Heme oxygenase-1 was strongly induced by dPGJ(2) but was not associated with protection. CONCLUSIONS: Independent of its PPARgamma activity, dPGJ(2) protected cells from oxidative stress by elevating GSH and enhancing MAPK activation. Thus, dPGJ(2) may delay the development of dry-type age-related macular degeneration.     

Activation of extracellular signal-regulated kinase in trabecular meshwork cells.

A number of different agents, such as growth factors, cytokines and phorbol esters have been shown to modulate trabecular meshwork cell function. These studies were designed to evaluate the role extracellular signal-regulated kinase (ERK) pathway plays in mediating the responses to platelet-derived growth factor-BB (PDGF-BB) and phorbol 12-myristate 13-acetate (PMA) in trabecular meshwork cells. The human trabecular meshwork cell line, HTM-3, and the bovine trabecular meshwork (BTM) cells were treated with either PDGF-BB or PMA and the activation of ERK 1/2 evaluated. The effects of the MAP kinase kinase (MEK) inhibitor U0126, and the PKC inhibitor chelerythrine on ERK 1/2 were also determined. In a separate group of experiments, cells were treated with PDGF-BB or PMA and the secretion of matrix metalloproteinase-2 (MMP-2) evaluated. The addition of PDGF-BB or PMA produced time- and dose-dependent activation of ERK 1/2. Pretreatment with U0126 or chelerythrine significantly reduced ERK 1/2 activation induced by PDGF-BB or PMA. The addition of PDGF-BB or PMA stimulated the secretion of MMP-2. This secretory response was inhibited by pretreatment with the MEK inhibitor U0126. In trabecular meshwork cells, PDGF-BB and PMA activate ERK 1/2 by a PKC-dependent mechanism. Activation of ERK 1/2 by these agents in trabecular meshwork cells leads to the secretion of MMP-2. These studies provide evidence that ERK pathway is an important mechanism for integrating various signals that regulate trabecular function.     

Tumstatin肽对视网膜微血管内皮细胞迁移及P38MAPK蛋白表达的影响

目的:观察tumstatin肽对体外培养的视网膜微血管内皮细胞迁移及P38MAPK蛋白表达的影响,初步探讨tumstatin肽抗视网膜内皮细胞迁移的机制。方法:采用细胞划痕实验测定tumstatin肽(T8肽)对血管内皮生长因子(VEGF)诱导下RF/6A细胞(恒河猴视网膜微血管内皮细胞)迁移的影响;Western blotting检测T8肽对VEGF刺激后15,30,45,60min的RF/6A细胞P38MAPK蛋白水平的变化。结果:Tumstatin肽对RF/6A细胞迁移具有抑制作用,且可抑制VEGF对RF/6A细胞的促迁移作用,呈剂量依赖性。正常情况下,RF/6A细胞无P38MAPK蛋白的表达,但VEGF可诱导其表达P38MAPK蛋白,而tumstatin可抑制VEGF诱导的RF/6A细胞P38MAPK蛋白的表达(加入20mg/LT8肽30,45,60min时蛋白表达受到显著抑制,差异有显著性意义,P<0.01)。结论:Tumstatin抑制视网膜微血管内皮细胞的迁移,其作用可能与P38MAPK通路有关。     

Role of PKCepsilon in PGF2alpha-stimulated MMP-2 secretion from human ciliary muscle cells.

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.