Epidermal growth factor stimulation of RPE cell survival: contribution of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways

Epidermal growth factor (EGF) previously has been shown to stimulate short-term survival in vitro of cells derived from the native amphibian retinal pigment epithelium (RPE). In the present experiments, we have examined intracellular signaling pathways responsible for mediating these survival-specific growth factor effects, distinct from proliferative effects, using the human epithelial cell line RPE D407. When maintained as single cells in suspension culture in the absence of serum and exogenous survival factors, RPE D407 cell viability gradually declined over a 3-4 day period as a result of apoptotic cell death, a pattern similar to that seen for eye-derived RPE cells. Exposure to EGF (50 ng ml(-1)) enhanced cell survival by nearly 40% and caused a parallel increase in the tyrosine phosphate content of the EGF receptor (EGFR), as determined by immunoprecipitation and Western blotting. Both effects were completely blocked by 1 microm AG1478, an EGFR-selective tyrosine kinase inhibitor. EGF also stimulated phosphorylation of the phosphatidylinositol 3'-kinase (PI3K)-dependent effector kinase Akt, as well as that of the MEK-dependent mitogen-activated kinase (MAPK), extracellular signal-regulated kinase (ERK). Furthermore, EGF-induced protection was substantially reduced by either the PI3K inhibitor LY294002 (25 microm) or the MEK inhibitor U0126 (10 microm), under conditions in which phosphorylation of Akt and ERK1/2, respectively, was blocked. Our results indicate that EGF-stimulated survival of RPE D407 cells takes place as a result of signaling through both PI3K and ERK/MAPK pathways. Further, residual anti-apoptotic activity stimulated by EGF in the presence of both blockers suggests that additional as yet unidentified growth factor-dependent survival pathways exist.     

Electric fields and MAP kinase signaling can regulate early wound healing in lens epithelium

PURPOSE: To use lens epithelial cell monolayer wounds as a model system for the aberrant cell migration underlying posterior capsule opacification (PCO) and to investigate the effects of an applied physiological electric field (EF) on monolayer wound healing. METHODS: Scratch wounds were made in cultured bovine lens epithelial monolayers, and the wounds were exposed to an EF, with or without U0126 treatment (an inhibitor of active extracellular signal-regulated kinase [ERK 1/2]). Serial wound images were taken and wound areas were measured. Western blot analysis and immunocytochemical staining for ERK 1/2 in the wounded monolayers were performed. RESULTS: An applied EF of a given polarity influenced the healing of lens epithelial monolayer wounds. Wounds facing the anode healed at normal rates, those facing the cathode closed much more slowly. U0126, an inhibitor of mitogen-activated protein kinase (MAPK) signaling, inhibited wound healing, with or without exposure to an EF. Western blot analysis showed that both wounding and application of an EF enhanced the activation of ERK 1/2 independently and that U0126 completely inhibited these activations of ERK 1/2 in monolayers. Immunocytochemical staining showed an asymmetric activation of ERK 1/2 in EF-exposed wounds, with much weaker fluorescence in cathode-facing wounds, which could contribute to differentially directed wound-healing rates in an EF. CONCLUSIONS: Exposure to an EF inhibited the healing of lens epithelial monolayer wounds facing the cathode. ERK signaling pathways were involved in healing of lens epithelial monolayer wounds and in the EF-directed migration of the wound edge. It may be possible to use an applied EF to regulate the aberrant migration of lens epithelial cells that results in PCO after cataract surgery.     

Signal transduction pathways for epidermal growth factor stimulated cyclooxygenase-2 induction in astrocytes

Cyclooxygenase-2 (COX-2) derived prostaglandins (PGs) are pathophysiological mediators in various disease states. Recently, we have demonstrated the rapid, epidermal growth factor receptor (EGFR)-dependent induction of COX-2 and PGE(2) synthesis in astrocytes following optic nerve injury and in culture. We have now investigated the signal transduction pathways activated by EGFR to accomplish the expression of COX-2 in primary optic nerve astrocytes. When astrocytes were exposed to EGF, marked, rapid gene expression of COX-2 was observed. Activation of EGFR caused an increase in the phosphorylation of extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and c-Jun NH (2)-terminal kinase (JNK). Furthermore, U0126, an ERK pathway inhibitor, and SB203580, a p38 MAPK inhibitor, diminished EGF-induced COX-2 expression; whereas, a JNK inhibitor did not suppress COX-2 expression by EGF. Using inhibitors of several other signaling cascades, we found that, unlike epithelial and cancer cells, NF-kappaB, PI 3-kinase/Akt and PKC were not signaling pathways for EGFR-dependent induction of COX-2 in optic nerve astrocytes. Taken together, these data suggest that ERK and p38 are key components of the intracellular signaling switch that transduces EGFR activation into COX-2 induction and PGE(2) biosynthesis in optic nerve astrocytes.     

Oxidative stress affects retinal pigment epithelial cell survival through epidermal growth factor receptor/AKT signaling pathway

AIM: To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor (EGFR)/AKT signaling pathway in retinal pigment epithelial (RPE) cells. METHODS: Human RPE cell lines (ARPE-19 cell) were treated with different doses of epidermal growth factor (EGF) and hydrogen peroxide (H2O2). Cell viability was determined by a methyl thiazolyl tetrazolium assay. Cell proliferation was examined by a bromodeoxyuridine (BrdU) incorporation assay. EGFR/AKT signaling was detected by Western blot. EGFR localization was also detected by immunofluorescence. In addition, EGFR/AKT signaling was intervened upon by EGFR inhibitor (erlotinib), PI3K inhibitor (A66) and AKT inhibitor (MK-2206), respectively. H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine (NAC). RESULTS: EGF treatment increased ARPE-19 cell viability and proliferation through inducing phosphorylation of EGFR and AKT. H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway. EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT, while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT. EGF-induced phosphorylation and endocytosis of EGFR were also affected by H2O2 treatment. In addition, antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through alleviating reduction of EGFR, and phosphorylated and total AKT proteins. CONCLUSION: Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.     

Hepatocyte growth factor induces proliferation of lens epithelial cells through activation of ERK1/2 and JNK/SAPK

PURPOSE: Posterior capsule opacification (PCO) is caused by proliferation and migration of lens epithelial cells (LECs) remaining after cataract surgery. In this study, the effect of HGF in LECs and the signaling pathways that contribute to HGF-induced proliferation were investigated. METHODS: Capsular bags prepared from porcine eyes were maintained in serum-free DMEM. The human lens epithelial B3 cells (HLE B3) and rat lens epithelial explants were cultured in MEM supplemented with 20% FCS and medium 199 with 0.1% BSA, respectively. Cell proliferation was determined by MTT assay, proliferating cell nuclear antigen (PCNA) expression, or flow cytometry. An antisense oligonucleotide was used to inhibit cyclin D1 expression. Activation of the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways was detected by immunoblot analysis. RESULTS: The proliferation of LECs in a capsular bag culture was significantly inhibited by treatment with the neutralizing antibody for HGF receptor. Stimulation of HLE B3 with hepatocyte growth factor (HGF) activated the MAPKs, ERK, and JNK/SAPK, but not p38. Activation of both ERK and JNK/SAPK was necessary for the HGF-stimulated induction of cyclin D1, which in turn was necessary for the HGF-induced proliferation of LECs. PI3K also participated in the regulation of cyclin D1 expression upstream of ERK and JNK/SAPK. CONCLUSIONS: The data indicate that HGF is a potent growth factor for LECs and may contribute to the development of PCO and suggest that the signaling pathways involved in HGF-stimulated proliferation may constitute potential therapeutic targets in the treatment of PCO.     

Upregulation of phospholipase Cgamma1 activity during EGF-induced proliferation of corneal epithelial cells: effect of phosphoinositide-3 kinase

PURPOSE: Previously, the authors showed that epidermal growth factor (EGF) stimulates phospholipase Cgamma1 (PLCgamma1) and phosphoinositide-3 kinase (PI3K) activities in confluent rabbit corneal epithelial cells (RCECs). The purpose of this study was to investigate whether PLCgamma1 activity is upregulated during EGF-induced proliferation of RCECs and to determine whether there is any cross-talk between PLCgamma1 and PI3K in these cells. METHODS: Simian virus (SV)-40-immortalized RCECs were cultured in the presence and absence of EGF and other agents. At prescribed time intervals, the cultures were terminated and the cells counted. PLCgamma1 activity in intact cells was assessed by measuring the production of [(3)H]IP(3) in [(3)H]myoinositol-labeled cells. The in vitro enzyme activity was assayed using immunoprecipitated PLCgamma1 and [(3)H]PI(4,5)P(2) as substrate. [(3)H]IP(3), the product of PLCgamma1, was analyzed by anion-exchange chromatography. The changes in protein content and level of phosphorylation of PLCgamma1 were determined by Western immunoblot analysis, with the appropriate antibodies. RESULTS: Addition of EGF (50 ng/ml) caused a time-dependent increase in proliferation of RCECS. The effect of EGF peaked at approximately 36 hours. Under the same experimental conditions, EGF stimulated PLCgamma1 activity with a time course similar to that of cell proliferation. Data from Western immunoblot analysis revealed that the EGF-stimulated PLCgamma1 activity was due to increased synthesis of the enzyme. Furthermore, during cell proliferation, tyrosine phosphorylation of PLCgamma1 increased in a time-dependent manner that corresponded closely with the expression of PLCgamma1. EGF exerted its effects both on cell proliferation and PLCgamma1 activation in a dose-dependent manner. Treatment of the cells with U-73122, a PLC inhibitor, or myr-GLYRKAMRLRY, a myristoylated PLCgamma1 inhibitor peptide, caused attenuation of both the EGF-stimulated cell proliferation and PLCgamma1 activity. Treatment of the cells with the PI3K inhibitors, wortmannin or LY294002, caused inhibition of both EGF-stimulated cell proliferation and PLCgamma1 activation. Addition of PI(3,4,5)P(3) to the in vitro PLCgamma1 assay mixture stimulated the enzyme activity in a dose-dependent manner. CONCLUSIONS: The data suggest a positive correlation between EGF-stimulated PLCgamma1 activation and cell proliferation in RCECS. The EGF-stimulated PLCgamma1 activity was mirrored by increased synthesis and tyrosine phosphorylation of the enzyme. The data also show that PLCgamma1 activation and cell proliferation were inhibited by PI3K inhibitors, suggesting a role for PI3K in EGF-stimulated proliferation of corneal epithelial cells.     

MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule

PURPOSE: The development of posterior capsule contraction following cataract surgery is caused by the activity of residual lens epithelial cells. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which are essential for cell migration and cell mediated contraction following wound healing. The authors investigated whether inhibiting MMP activity can reduce lens epithelial cell migration and as a result, lead to a reduction in cell mediated capsule contraction. METHODS: Human donor lens capsules were cultured and treated with a broad spectrum MMP inhibitor, Ilomastat (GM6001). MMP-2 and MMP-9 production were determined by ELISA. Cell migration onto the posterior capsule and capsule contraction were digitally measured. RESULTS: MMP inhibition significantly reduced lens epithelial cell migration onto the posterior capsule (p<0.05), and a reduction in capsule contraction was observed (p<0.05). CONCLUSIONS: Ilomastat significantly reduced lens epithelial cell migration onto the posterior capsule surface and inhibited capsule contraction. MMP inhibition may have a role in the therapeutic treatment of posterior capsule opacification.     

Activation of SRC kinases signals induction of posterior capsule opacification

PURPOSE: Posterior capsule opacification (PCO) is a complication of cataract surgery resulting from the proliferation, migration, and epithelial-to-mesenchymal transition (EMT) of lens epithelial cells that remain associated with the lens capsule. These changes cause a loss of vision. The authors developed a chick embryo lens capsular bag model to study mechanisms involved in the onset of PCO. Because Src family kinases (SFKs) signal cell proliferation, migration, and EMT, the authors examined whether the inhibition of SFKs can prevent PCO. METHODS: After mock cataract surgery, chick lens capsular bags were pinned to a culture dish and grown in the presence or absence of the SFK inhibitor PP1. Cell movement was followed by photomicroscopy. Progression of proliferation and EMT in the PCO cultures was determined by Western blot analysis and immunofluorescence staining. RESULTS: As occurs in PCO, lens cells in this model proliferated, migrated across the posterior capsule, and expressed EMT markers, alpha-smooth muscle actin (alpha-SMA), and fibronectin (FN). Lens cells treated with PP1 maintained an epithelial phenotype, accumulated cadherin junctions, and did not migrate to the posterior capsule, increase proliferation, or express EMT markers. Therefore, exposure to PP1 prevented PCO. Short-term inhibition of SFKs was sufficient to prevent EMT, but longer inhibition was necessary to prevent lens cell migration. CONCLUSIONS: Progression of PCO involved early activation of SFKs. Lens cell migration preceded EMT, and each of these two events required activation of an SFK signaling pathway. Suppression of SFK activation blocked PCO, suggesting SFKs as a therapeutic target for the prevention of PCO.     

RNA干扰技术干扰信号通路在后发性白内障防治中的应用

后发性白内障即后囊膜混浊(PCO),是白内障超声乳化摘出联合人工晶状体植入术后常见的并发症.白内障术后残留的晶状体上皮细胞(LECs)向后囊膜表面移行、增生及上皮-间质转化(EMT)等细胞生物学行为的改变可引起LECs纤维化、PCO及囊袋皱缩.转化生长因子β2(TGF-β2)是目前已知的参与诱导LECs的EMT及组织病理性纤维化关键的细胞因子,它可通过Smad经典通路参与诱导LECs的EMT过程.除此之外,PI3 K/AKT/mTOR信号通路也参与了TGF-β2诱导的EMT过程.RNA干扰(RNAi)技术作为基因调控手段之一,在通过干扰信号通路而抑制LECs的纤维化及增生、迁移等生物学行为中有一定的应用前景.本文就RNAi技术通过干扰PI3 K/AKT/mTOR信号通路和TGF-β2/Smad信号通路对LECs的生物学行为的影响,及其在PCO防治中的作用进行综述.     

MAPK/ERK1/2 and PI3-kinase signalling pathways are required for vitreous-induced lens fibre cell differentiation

Lens epithelial cells withdraw from the cell cycle to differentiate into secondary fibre cells in response to vitreal factors. Fibroblast growth factor (FGF) in the vitreous has been shown to induce lens fibre differentiation in vivo and in vitro through the activation of defined intracellular signalling, namely via MAPK/ERK1/2 and PI3-K/Akt pathways. To better understand the role of these growth factor-activated signalling pathways in lens fibre differentiation, FGF- and vitreous-induced lens fibre differentiation was examined in primary rat lens epithelial cell explants. The induction of cell elongation and fibre specific β- and γ-crystallin expression in lens explants was accompanied by distinct phosphorylation profiles for ERK1/2 and Akt. Using selective inhibitors (U0126 and LY294002) in blocking studies, these pathways were shown to be required for different aspects of lens fibre differentiation. Furthermore, a short ‘pulse’ treatment of explants with FGF showed that the activation of ERK1/2 over 24 h was not sufficient for the progression of lens fibre differentiation and that cyclic ERK1/2 phosphorylation was required throughout the extended differentiation process. In conclusion, these results support a key role for both ERK1/2 and PI3-kinase/Akt signalling pathways in FGF- and vitreous-induced lens fibre differentiation.     

Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells

PURPOSE: To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS: Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS: P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. CONCLUSIONS: Bacterial infection of HCECs induces EGFR transactivation through HB-EGF ectodomain shedding. EGFR and its downstream ERK and PI3K signaling pathways play a role in preventing epithelial apoptosis in the early stage of bacterial infection.     

基质金属蛋白酶抑制剂对培养的人晶状体上皮细胞移行抑制作用的研究

目的 通过体外人晶状体囊袋模型的建立,探讨基质金属蛋白酶抑制剂GM6001对白内障患者术后晶状体上皮细胞移行的抑制作用及其对细胞活性的影响,以寻找一种安全有效地防治晶状体后囊膜混浊的药物.方法 本文为实验研究.16例供体人眼行模拟白内障手术,建立人晶状体囊袋培养模型,确认赤道部晶状体上皮细胞开始移行后,将晶状体囊袋置于不同浓度(1 μmol/L、10 μmol/L和100 μmol/L)的GM6001及其阴性对照液(100 μmol/L)中培养(每组4个囊袋).相差显微镜下分别测量培养10、20及30 d时各组晶状体上皮细胞在后囊膜移行的距离;酶联免疫吸附实验测定囊袋培养液中MMP-2和MMP-9的浓度.四甲基偶氮唑盐法(MTT)测定不同实验浓度GM6001对细胞活性的影响.多组间及组间计数资料的比较,采用随机区组设计的方差分析.结果 培养的人晶状体囊袋中赤道部晶状体上皮细胞第4天开始移行.GM6001明显抑制了晶状体上皮细胞在后囊膜的移行,呈剂量依赖性(F=53.79,P<0.01):实验第20天,10μmol/L GM6001组晶状体上皮细胞移行距离较对照组减少70%(P<0.01),100μmoL/L组减少98%(P<0.01);GM6001降低了MMP-2、MMP-9表达水平,浓度越高,抑制作用越明显(F=86.59,72.96;P<0.01):实验第20天,10 μmol/L GM6001组MMP-2和MMP-9表达水平较对照组均降低70%(P<0.01),100 μmol/LGM6001组MMP-2水平降低了90%(P<0.01),MMP-9水平降低了87%(P<0.01);MTT法显示,晶状体上皮细胞在GM6001中仍保持增殖活性,各处理浓度组光密度(A)值与对照组比较差异无统计学意义(F=0.62,P>0.05).结论 MMP抑制剂有效地抑制了体外囊袋培养的人晶状体上皮细胞在后囊膜的移行,且对细胞活性无明显影响.因此,MMP抑制剂可能对预防后囊混浊有一定作用.     

Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing

EGF is an essential growth factor needed for epithelial cell proliferation and wound healing of the cornea, but the molecular mechanism is not understood. Although studies have shown that EGF in some non-phagocytic cells induces ROS generation, little is known about the role of ROS in corneal epithelial cells. Therefore, we examined the potential physiological role of ROS in corneal cell proliferation, adhesion and wound healing using rabbit or human corneal epithelial cells, and pig whole cornea organ culture as models. EGF (5 ng/ml)-induced ROS in serum-starved RCE or HCE cells were captured as DCFH fluorescence and detected by confocal microscopy. The elevation of ROS was eradicated when the cells were pretreated with an antioxidant N-acetylcysteine (NAC) or mannitol, or with inhibitor to NADPH oxidase (DPI), or to lipoxygenase (NDGA). EGF-induced ROS generation correlated with cell growth and activation of Akt and MAPK signaling pathways, while NAC eliminated all these effects. EGF-stimulated cell adhesion or migration in cell culture was greatly suppressed in the presence of NAC while EGF-facilitated epithelial cell wound healing in corneal organ culture was also blocked by NAC. This is the first demonstration of a novel ROS physiological function in corneal wound healing.     

SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells

PURPOSE: The authors have previously demonstrated that wounding of human corneal epithelial cells (HCECs) transactivates epidermal growth factor (EGF) receptor (EGFR) and its downstream signaling pathways and that this EGFR signaling is required for epithelial wound healing. In this study, the authors sought to identify the underlying mechanisms for EGFR transactivation in response to wounding in HCECs. METHODS: SV40-immortalized HCEC (THCE) monolayer was wounded and allowed to heal in the presence or absence of a selective inhibitor of the Src family kinases PP2 and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Wound closure was monitored by photographing of the injury immediately or 24 hours after wounding. Activation of EGFR in THCE cells and in primary HCECs was analyzed by immunoprecipitation of EGFR, followed by Western blotting with phosphotyrosine antibody. Phosphorylation of extracellular signal-regulated kinase (ERK), AKT (a major substrate of phosphatidylinositol 3'-kinase [PI3K]), Src at tyrosine Y416, and EGFR at Y845 was analyzed by Western blotting with antibodies specific to phosphorylated proteins. Effects of PP2 on THCE cell migration were determined by Boyden chamber migration assay. RESULTS: Among several inhibitors tested, PP2 blocked wound-induced EGFR phosphorylation in THCE cells. PP2 at 12.5 microM effectively inhibited EGFR transactivation in response to wounding and to the phosphorylation of ERK and AKT in THCE cells and primary HCECs. Consistent with the inhibition of EGFR transactivation, PP2 also attenuated epithelial migration and wound closure with or without exogenously added HB-EGF. PP2 at a concentration as high as 50 microM exhibited no effects on HB-EGF induced ERK phosphorylation. On the other hand, AKT phosphorylation was much more sensitive to PP2 than ERK or EGFR phosphorylation because 3.13 microM PP2 effectively inhibited wound- or HB-EGF-induced AKT phosphorylation. CONCLUSIONS: These results suggest that Src kinase mediates wound-induced EGFR transactivation and participates in a pathway to activate the PI3K-AKT pathway downstream of EGFR in HCECs.     

Attenuation of human lens epithelial cell spreading,migration and contraction via downregulation of the PI3K/Akt pathway

Background

Posterior capsule opacification (PCO) represents a major challenge in the postoperative management of cataract patients. Spreading, migration and contraction of residual human lens epithelial cells play a pivotal role in the pathogenesis of PCO. Therefore, we analyzed the effect of the alkylphosphocholine (APC) erufosine on these cellular features as well as on PI3K/Akt, a crucial pathway in PCO pathogenesis.

Methods

Human lens epithelial cells were cultured under standard cell culture conditions. Cell spreading was analyzed on fibronectin-coated wells and chemokinetic migration was assessed by time-lapse microscopy. For evaluation of cell-mediated collagen matrix contraction, the cells were seeded into collagen gels and incubated with an APC in different non-toxic concentrations before the surface area was measured on day 6. The activity of PI3K/Akt was assessed by an ELISA kit after incubation of the cells with different APC concentrations.

Results

Human lens epithelial cell spreading and migration were attenuated by APCs as follows: 7 % spreading, 48 % migration (0.1 μM APC), and 32 % spreading, 68 % migration (1.0 μM APC). APC concentrations of 0.1 μM reduced collagen gel diameter by 5 %, and 1.0 μM by less than 1 %, compared to untreated, cell-populated gels that resulted in a cell diameter contraction of 36 %. PI3K was downregulated in a concentration-dependent manner.

Conclusions

The crucial cellular features of PCO pathogenesis are attenuated by the APC erufosine via downregulation of the PI3K pathway. Thus, erufosine might become a valuable tool for pharmacologic PCO prophylaxis in the future.     

Studies of the mitogen-activated protein kinases and phosphatidylinositol-3 kinase in the lens. 2. The intercommunications

The lens possesses comprehensive mitogen-activated signal transduction pathways (MAPK), which include the mitogen response pathway (Raf-MEK-ERK cascade), the stress-response pathways (p38 and SAPK/JNK cascades) and also the survival pathway (PI-3K-Akt). To understand the cross-cascade intercommunication among signal transduction pathways in the lens, we used specific protein kinase inhibitors and cultured the lenses under unstimulated, basic fibroblast growth factor (bFGF)- or galactose-treated conditions. Inhibitors included genistein (tyrosine kinases inhibitor), U0126 (MEK inhibitor), SB203580 or SB202190 (p38 inhibitor), FTS (Ras inhibitor), wortmannin (PI-3K inhibitor) or phorbol ester (protein kinase C down-regulator following long-term exposure). The results showed that genistein inhibited the activations of the members of the MAPK superfamily and the activation of PI-3K. FTS suppressed the activation of Raf and PI-3K but stimulated the other members of MAPKs. MEK inhibitor restrained the activations of ERK, SAPK/JNK (under bFGF-stimulated condition) and p38 (under galactose-stimulated condition) while p38 inhibitor suppressed ERK but stimulated SAPK/JNK. Both MEK and p38 inhibitors stimulated PI-3K. Wortmannin had a strong inhibitory effect on Raf but little effect on its downstream target proteins. Down-regulating PKC suppressed Raf and PI-3K but stimulated ERK. Taken together, these data suggest that all the stimuli responses are mediated through phosphorylation and that the signaling among the mitogenic and stress response pathways is integrated through 'cross-talk' to process the most appropriate response. The survival signaling pathway appears to communicate well with the mitogenic and stress response pathways. In addition to Ras, both Raf and MEK emerge to be the diverging or regulatory points for signal integration, amplification, suppression or compensatory action in the lens.     

Expression of matrix metalloproteinases of human lens epithelial cells in the cultured lens capsule bag

AIM OF PURPOSE: To observe the different expression of matrix metalloproteinases (MMPs) between pre- and postoperation of sham cataract surgery in vitrohuman lens capsule bag model from the same donor eye in order to investigate a possible role of MMPs in posterior capsule opacification (PCO).Methods: Sham cataract surgeries were performed in six human donor eyes. Immunohistochemical staining was used to detect the expression of MMP-2 and -9 of human lens epithelial cells (LECs) on the anterior capsulorhexis. LEC migration on posterior capsule of human lens from the same donor eye was observed in a modified capsule bag model without pin. Total MMP-2 and -9 protein production were determined by enzyme-linked immunosorbent assay at days 2, 10, 20, and 30 postoperation, respectively. RESULTS: MMP-2 and -9 could not be detected immunohistochemically on the anterior capsulorhexis preoperation of cataract. Lens epithelia cells at the equator began to migrate by day 4. A confluent monolayer of lens epithelia cells was present on the posterior capsule at day 20. Total MMP-2 and -9 protein production increased with time with maximum levels reached on day 30. CONCLUSION: MMP-2 and -9 were showed to be upregulated following sham cataract surgery. MMP expression could play an important role in PCO.     

Signaling-transduction pathways required for ex vivo expansion of human limbal explants on intact amniotic membrane

PURPOSE: Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways. METHODS: The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis. RESULTS: In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached approximately 80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 microM LY294002 or 50 microM SR13668 and was significantly suppressed by 10 microM U0126, but was not affected by 10 microM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion. CONCLUSIONS: Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.     

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P<0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.