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1.
J Biol Chem ; 293(10): 3758-3769, 2018 03 09.
Article in English | MEDLINE | ID: mdl-29363574

ABSTRACT

Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness because of endothelial dysfunction. We previously reported that IL-1ß stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1), and ZEB2 This, in turn, induced expression of fibronectin, vimentin, and type I collagen, and suppressed E-cadherin in CECs in vitro and ex vivo siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.


Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Endothelium, Corneal/metabolism , Eye Proteins/metabolism , Gene Expression Regulation , Receptor, Fibroblast Growth Factor, Type 2/agonists , Snail Family Transcription Factors/metabolism , Zinc Finger E-box-Binding Homeobox 1/metabolism , Biomarkers/metabolism , Cell Movement , Cell Proliferation , Cell Shape , Cell Transdifferentiation , Cells, Cultured , Collagen Type I/agonists , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/chemistry , Cyclin-Dependent Kinase 2/genetics , Endothelium, Corneal/cytology , Endothelium, Corneal/pathology , Enzyme Activation , Eye Proteins/agonists , Eye Proteins/antagonists & inhibitors , Eye Proteins/genetics , Humans , RNA Interference , Receptor, Fibroblast Growth Factor, Type 2/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Snail Family Transcription Factors/antagonists & inhibitors , Snail Family Transcription Factors/genetics , Wound Healing , Zinc Finger E-box Binding Homeobox 2/agonists , Zinc Finger E-box Binding Homeobox 2/genetics , Zinc Finger E-box Binding Homeobox 2/metabolism , Zinc Finger E-box-Binding Homeobox 1/agonists , Zinc Finger E-box-Binding Homeobox 1/antagonists & inhibitors , Zinc Finger E-box-Binding Homeobox 1/genetics
2.
Int Clin Psychopharmacol ; 31(5): 275-86, 2016 09.
Article in English | MEDLINE | ID: mdl-27487259

ABSTRACT

We evaluated the effectiveness of aripiprazole among bipolar patients who had switched to this medication as a result of difficulty maintaining on their prestudy atypical antipsychotics (AAPs) because of subsyndromal mood symptoms or intolerance. This study included 77 bipolar patients who were in syndromal remission with an AAP as monotherapy or with an AAP combined with a mood stabilizer(s) who needed to switch from their present AAP because of subsyndromal symptoms or intolerance. At 24 weeks after switching to aripiprazole, the remission rates on the Montgomery-Åsberg Depression Rating Scale (MADRS) and on both the MADRS and the Young Mania Rating Scale were increased significantly in the full sample and in the inefficacy subgroup. In the inefficacy subgroup, the MADRS score change was significant during the 24 weeks of study. Total cholesterol and prolactin decreased significantly after switching to aripiprazole. The proportion of patients who had abnormal values for central obesity and hypercholesterolemia decreased significantly from baseline to week 24. These findings suggest that a change from the current AAP to aripiprazole was associated with improvement in subsyndromal mood symptoms and several lipid/metabolic or safety profile parameters in patients with bipolar disorder with tolerability concerns or subsyndromal mood symptoms.


Subject(s)
Antipsychotic Agents/therapeutic use , Aripiprazole/therapeutic use , Bipolar Disorder/diagnosis , Bipolar Disorder/drug therapy , Drug Substitution , Adult , Antipsychotic Agents/adverse effects , Aripiprazole/adverse effects , Body Weight/drug effects , Drug Substitution/trends , Female , Follow-Up Studies , Humans , Hypercholesterolemia/chemically induced , Male , Middle Aged , Prospective Studies , Treatment Outcome
3.
J Biol Chem ; 290(44): 26752-64, 2015 Oct 30.
Article in English | MEDLINE | ID: mdl-26370090

ABSTRACT

The cornea is the anterior, transparent tissue of the human eye that serves as its main refractive element. Corneal endothelial cells are arranged as a monolayer on the posterior surface of the cornea and function as a pump to counteract the leakiness of its basement membrane. Maintaining the cornea in a slightly dehydrated state is critical for the maintenance of corneal transparency. Adult human corneal endothelial cells are G1-arrested, even in response to injury, leading to an age-dependent decline in endothelial cell density. Corneal edema and subsequent vision loss ensues when endothelial cell density decreases below a critical threshold. Vision loss secondary to corneal endothelial dysfunction is a common indication for transplantation in developed nations. An impending increase in demand for and a current global shortage of donor corneas will necessitate the development of treatments for vision loss because of endothelial dysfunction that do not rely on donor corneas. Wnt ligands regulate many critical cellular functions, such as proliferation, making them attractive candidates for modulation in corneal endothelial dysfunction. We show that WNT10B causes nuclear transport and binding of RAC1 and ß-catenin in human corneal endothelial cells, leading to the activation of Cyclin D1 expression and proliferation. Our findings indicate that WNT10B promotes proliferation in human corneal endothelial cells by simultaneously utilizing both ß-catenin-dependent and -independent pathways and suggest that its modulation could be used to treat vision loss secondary to corneal endothelial dysfunction.


Subject(s)
Endothelial Cells/metabolism , Proto-Oncogene Proteins/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , rac1 GTP-Binding Protein/metabolism , Active Transport, Cell Nucleus , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Line, Transformed , Cell Proliferation/drug effects , Cornea/cytology , Cornea/drug effects , Cornea/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , Dishevelled Proteins , Endothelial Cells/cytology , Endothelial Cells/drug effects , G1 Phase Cell Cycle Checkpoints/drug effects , Gene Expression Regulation , Humans , Interleukin-1beta/pharmacology , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Wnt Proteins/antagonists & inhibitors , Wnt Proteins/genetics , beta Catenin/genetics , rac1 GTP-Binding Protein/antagonists & inhibitors , rac1 GTP-Binding Protein/genetics
4.
Neuropsychiatr Dis Treat ; 11: 1561-71, 2015.
Article in English | MEDLINE | ID: mdl-26170669

ABSTRACT

Our goal was to compare the recommendations of the Korean Medication Algorithm Project for Bipolar Disorder 2014 (KMAP-BP 2014) with other recently published guidelines for the treatment of bipolar disorder. We reviewed a total of four recently published global treatment guidelines and compared each treatment recommendation of the KMAP-BP 2014 with those in other guidelines. For the initial treatment of mania, there were no significant differences across treatment guidelines. All recommended mood stabilizer (MS) or atypical antipsychotic (AAP) monotherapy or the combination of an MS with an AAP as a first-line treatment strategy for mania. However, the KMAP-BP 2014 did not prefer monotherapy with MS or AAP for dysphoric/psychotic mania. Aripiprazole, olanzapine, quetiapine, and risperidone were the first-line AAPs in nearly all of the phases of bipolar disorder across the guidelines. Most guidelines advocated newer AAPs as first-line treatment options in all phases, and lamotrigine in depressive and maintenance phases. Lithium and valproic acid were commonly used as MSs in all phases of bipolar disorder. As research evidence accumulated over time, recommendations of newer AAPs - such as asenapine, paliperidone, lurasidone, and long-acting injectable risperidone - became prominent. This comparison identifies that the treatment recommendations of the KMAP-BP 2014 are similar to those of other treatment guidelines and reflect current changes in prescription patterns for bipolar disorder based on accumulated research data. Further studies are needed to address several issues identified in our review.

5.
Mol Cell Biol ; 34(18): 3535-45, 2014 Sep 15.
Article in English | MEDLINE | ID: mdl-25022753

ABSTRACT

Wnt5a can activate ß-catenin-independent pathways for regulation of various cellular functions, such as migration, that play critical roles in wound repair. Investigation of Wnt5a signaling may help identify therapeutic targets for enhancing corneal endothelial wound healing that could provide an alternative to corneal transplantation in patients with blindness from endothelial dysfunction. However, Wnt5a signaling in corneal endothelial cells (CECs) has not been well characterized. In this study, we show transient induction of Wnt5a by interleukin-1ß (IL-1ß) stimulation proceeds through NF-κB in human CECs. This leads to binding of Fzd5 to Ror2, resulting in activation of disheveled protein (Dvl) and subsequently disheveled-associated activator of morphogenesis 1 (DAAM1). This leads to activation of Cdc42 and subsequent inhibition of RhoA. Inhibition of RhoA leads to parallel dephosphorylation and inactivation of LIM domain kinase 2 along with dephosphorylation and activation of slingshot 1, resulting in dephosphorylation and activation of cofilin and leading to enhanced cell migration. These findings suggest that Wnt5a enhances cell migration through activation of Cdc42 and inactivation of RhoA in human CECs.


Subject(s)
Cell Movement , Cornea/metabolism , Endothelial Cells/metabolism , Interleukin-1beta/metabolism , Proto-Oncogene Proteins/metabolism , Wnt Proteins/metabolism , cdc42 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , Binding Sites , Cell Movement/drug effects , Cells, Cultured , Gene Expression Regulation , Humans , NF-kappa B/metabolism , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Wnt-5a Protein , beta Catenin/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/antagonists & inhibitors
6.
Biol Cell ; 105(4): 175-89, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23331079

ABSTRACT

BACKGROUND INFORMATION: Interleukin (IL)-1ß is a major pro-inflammatory cytokine that plays a crucial role in the regulation of inflammation and wound healing in the cornea. Elucidation of IL-1ß signalling may help identify therapeutic targets for corneal wound healing; however, mechanisms such as cell migration, a component of IL-1ß-induced wound healing response in human corneal endothelial cells (CEC), have not been well characterised. RESULTS: Stimulation of human CEC with IL-1ß activated expression of fibroblast growth factor 2 (FGF2) and resulted in enhanced cell migration. This, in turn, was abolished by treatment with either IL-1 receptor antagonist or SU-5402, a pan-fibroblast growth factor signalling inhibitor. Phosphatidyl inositol (PI) 3-kinase or IL receptor-associated kinase 1/4 antagonists demonstrated that IL receptor-associated kinase 1/4 activates PI 3-kinase, which in turn phosphorylates p38 and inhibitor κB kinase α/ß, leading to FGF2 expression through activation of activator protein 1 (AP-1) and nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in human CEC. Treatment of IL-1ß-stimulated human CEC with either AP-1 or NF-κB antagonists decreased FGF2 expression and resulted in reduced IL-1ß-enhanced cell migration. Co-treatment of IL-1ß-stimulated human CEC with both inhibitors completely blocked FGF2 expression and IL-1ß-enhanced cell migration. Chromatin immunoprecipitation assays demonstrated that AP-1 and NF-κB directly bind to the FGF2 promoter following IL-1ß stimulation. CONCLUSIONS: The results show that binding of IL-1ß to its receptor in human CEC leads to parallel activation of AP-1 and NF-κB pathways, leading, in turn, to FGF2 expression and enhanced cell migration.


Subject(s)
Cell Movement , Cornea/cytology , Endothelial Cells/metabolism , Fibroblast Growth Factor 2/genetics , Interleukin-1beta/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Cell Line , Cornea/metabolism , Endothelial Cells/cytology , Fibroblast Growth Factor 2/metabolism , Humans , Interleukin-1beta/genetics , NF-kappa B/genetics , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/genetics , Signal Transduction , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Up-Regulation
7.
Invest Ophthalmol Vis Sci ; 53(10): 6389-92, 2012 Sep 21.
Article in English | MEDLINE | ID: mdl-22893673

ABSTRACT

PURPOSE: Neuron-specific enolase (NSE) is a biomarker for neuronal stress. Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease affecting retinal ganglion cells (RGC). These RGCs and their axons in the retinal nerve fiber layer (RNFL) and optic nerve head may show subclinical pathology in unaffected mutation carriers, or undergo cell death in affected patients. We hypothesize that increased levels of blood NSE may characterize LHON carriers as a biomarker of ongoing RGC stress. METHODS: Serum was obtained from 74 members of a Brazilian pedigree with LHON carrying the homoplasmic 11778/ND4 mitochondrial DNA mutation. Classified by symptoms and psychophysical metrics, 46/74 patients were unaffected mutation "carriers," 14/74 were "affected," and 14/74 were "off-pedigree" controls. Serum NSE levels were determined by ELISA specific for the γ subunit of NSE. RESULTS: Serum NSE concentrations in carriers (27.17 ± 39.82 µg/L) were significantly higher than affected (5.66 ± 4.19 µg/L; P = 0.050) and off-pedigree controls (6.20 ± 2.35 µg/L; P = 0.047). Of the 14/46 (30.4 %) carriers with significantly elevated NSE levels (mean = 75.8 ± 42.3 µg/L), 9/14 (64.3%) were male. Furthermore, NSE levels were nearly three times greater in asymptomatic male carriers (40.65 ± 51.21 µg/L) than in asymptomatic female carriers (15.85 ± 22.27 µg/L; P = 0.034). CONCLUSIONS: Serum NSE levels are higher in LHON carriers compared with affected and off-pedigree individuals. A subgroup of mostly male carriers had significantly elevated serum NSE levels. Thus, male carriers are at higher risk for LHON-related neuronal stress.


Subject(s)
Asymptomatic Diseases/epidemiology , Optic Atrophy, Hereditary, Leber/epidemiology , Optic Atrophy, Hereditary, Leber/metabolism , Phosphopyruvate Hydratase/blood , Brazil/epidemiology , DNA, Mitochondrial/genetics , Family Health , Female , Humans , Male , Optic Atrophy, Hereditary, Leber/genetics , Pedigree , Risk Factors , Sex Distribution , Stress, Physiological/physiology
8.
Invest Ophthalmol Vis Sci ; 53(3): 1530-8, 2012 Mar 21.
Article in English | MEDLINE | ID: mdl-22323467

ABSTRACT

PURPOSE: To determine the role of nuclear factor-κB (NF-κB) during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1ß stimulation in corneal endothelial cells (CECs). METHODS: Expression and/or activation of IL-1 receptor-associated protein kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA kit, while binding of NF-κB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. RESULTS: Brief stimulation of CECs with IL-1ß upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after IL-1ß stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/ß and degrades IκB, subsequently leading to activation of NF-κB. The induction of FGF-2 by IL-1ß was completely blocked by inhibitors to NF-κB activation (sulfasalazine) or PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-κB is the transcription factor of FGF-2 as NF-κB binds the putative NF-κB binding site of the FGF-2 promoter. CONCLUSIONS: These data suggest that IL-1ß signaling combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene.


Subject(s)
Endothelium, Corneal/cytology , Epithelial-Mesenchymal Transition/physiology , Fibroblast Growth Factor 2/metabolism , NF-kappa B/physiology , Animals , Cell Proliferation/drug effects , Cells, Cultured , Chromatin/metabolism , Endothelium, Corneal/drug effects , Endothelium, Corneal/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , I-kappa B Kinase/metabolism , Immunoblotting , Interleukin-1 Receptor-Associated Kinases/metabolism , Interleukin-1beta/pharmacology , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Rabbits , TNF Receptor-Associated Factor 6/metabolism
9.
Exp Eye Res ; 95(1): 35-9, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21855543

ABSTRACT

This review describes the molecular mechanism of endothelial mesenchymal transformation (EMT) mediated by fibroblast growth factor-2 (FGF-2) in corneal endothelial cells (CECs). Corneal fibrosis is not frequently observed in corneal endothelium/Descemet's membrane complex; but when this pathologic tissue is produced, it causes a loss of vision by physically blocking light transmittance. Herein, we will address the cellular activities of FGF-2 and its signaling pathways during the EMT process. Furthermore, we will discuss the role of inflammation on FGF-2-mediated EMT. Interleukin-1ß (IL-1ß) greatly upregulates FGF-2 production in CECs, thus leading to FGF-2-mediated EMT; the whole spectrum of the injury-mediated inflammation (IL-1ß pathway) and the subsequent EMT process (FGF-2 pathway) will be briefly discussed. Intervention in the two pathways will provide the means to block EMT before inflammation causes an irreversible change, such as the production of retrocorneal fibrous membrane observed in human eyes.


Subject(s)
Cornea/blood supply , Corneal Diseases/metabolism , Endothelial Cells/metabolism , Epithelial-Mesenchymal Transition , Fibroblast Growth Factor 2/metabolism , Interleukin-1beta/metabolism , Actin Cytoskeleton/metabolism , Animals , Cell Shape , Collagen Type I/metabolism , Corneal Diseases/pathology , Endothelial Cells/pathology , Fibrosis , Humans , Mitosis , Phosphatidylinositol 3-Kinase/metabolism , Signal Transduction , Up-Regulation
10.
Invest Ophthalmol Vis Sci ; 52(11): 8216-23, 2011 Oct 17.
Article in English | MEDLINE | ID: mdl-21948550

ABSTRACT

PURPOSE: FGF-2 stimulates cell proliferation of rabbit corneal endothelial cells (rCECs) by degrading the cyclin-dependent kinase inhibitor p27(Kip1) (p27) through its phosphorylation mechanism. The authors investigated whether the cell proliferation of human CECs (hCECs) is also induced by FGF-2 stimulation through the p27 phosphorylation pathway. METHODS: Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Transfection of hCECs with small interference RNA (siRNA) was performed using a transfection reagent. RESULTS: FGF-2 stimulated cell proliferation in hCECs; the FGF-2 action was completely blocked by pathway-specific inhibitors for PI 3-kinase (LY294002) and MEK1/2 (U0126), respectively. Using immunoblotting, the authors showed that FGF-2 induced phosphorylation of p27 at both serine 10 (Ser10) and threonine 187 (Thr187) sites. These effects were also completely blocked by LY294002 or U0126. The authors then determined cross-talk between PI 3-kinase and extracellular signal-regulated kinase (ERK)1/2; blocking of ERK1/2 activation by LY294002 indicated that in hCECs ERK1/2 works as a downstream effector to PI 3-kinase for cell proliferation induced by FGF-2, whereas the ERK1/2 pathway in rCECs is parallel to the PI 3-kinase pathway. However, the downstream mechanism involved in cell cycle progression in hCECs is identical to that of rCECs: phosphorylation of p27 at Ser10 was mediated by kinase-interacting stathmin (KIS), confirmed with siRNA to KIS, and phosphorylation of p27 at Thr187 was mediated by cell division cycle 25A (Cdc25A), confirmed using Cdc25A inhibitor. CONCLUSIONS; FGF-2 stimulates proliferation of hCECs through PI 3-kinase and its downstream target ERK1/2 pathways. This linear signal transduction significantly downregulates p27 through its phosphorylation at both Ser10 and Thr187 sites mediated by KIS and Cdc25A, respectively.


Subject(s)
Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Endothelium, Corneal/metabolism , Fibroblast Growth Factor 2/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Adolescent , Adult , Aged , Butadienes/pharmacology , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Humans , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/antagonists & inhibitors , MAP Kinase Kinase 2/metabolism , Microscopy, Confocal , Middle Aged , Morpholines/pharmacology , Nitriles/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , RNA, Small Interfering/genetics , Serine/metabolism , Tetrazolium Salts/metabolism , Thiazoles/metabolism , Threonine/metabolism , Tissue Donors , Transfection , Young Adult
11.
Invest Ophthalmol Vis Sci ; 52(1): 417-26, 2011 Jan 21.
Article in English | MEDLINE | ID: mdl-20811053

ABSTRACT

PURPOSE: To determine the mechanism of p27 phosphorylation through common and differential pathways triggered by FGF-2 in corneal endothelial cells (CECs). METHODS: A GTP pull-down assay was performed to identify Rac1-GTP. Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by an MTT assay. Transfection of CECs with kinase-interacting stathmin (KIS) siRNA was performed. RESULTS: FGF-2 activated Rac1 through Akt, and Rac1 inhibitor greatly inhibited the FGF-2-stimulated cell proliferation. Rac1 inhibitor reduced p27 phosphorylation at both serine 10 (Ser10) and threonine 187 (Thr187). ERK1/2 was also involved in FGF-2-stimulated CEC proliferation and phosphorylation of p27 at Ser10 and Thr187 in parallel to phosphatidylinositol (PI) 3-kinase. In both PI 3-kinase/Rac1 and ERK1/2 pathways, Ser10 of p27 is phosphorylated by KIS, confirmed by siRNA to KIS, which subsequently hampered the FGF-2-stimulated cell proliferation, while Thr187 of p27 was phosphorylated through Cdk2 activated by Cdc25A. Cdc25A inhibitor blocked activation of Cdk2, phosphorylation of p27 at Thr187, and cell proliferation. FGF-2 induced both KIS and Cdc25A during the G1 phase; the maximum KIS expression was observed 4 hours after FGF-2 stimulation, while the maximum Cdc25A expression was observed at 12 hours. Blockade of ERK1/2 and Rac1 greatly reduced KIS and Cdc25A expression. CONCLUSIONS: Results suggest that FGF-2 uses both PI 3-kinase/Rac1 and ERK pathways for cell proliferation; two signals employ common pathways for phosphorylating p27 according to the sites (KIS for Ser10 and Cdc25A/Cdk2 for Thr187) with their characteristic kinetics (early G1 for Ser10 and late G1 for Thr187).


Subject(s)
Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Endothelium, Corneal/cytology , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblast Growth Factor 2/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Blotting, Western , Cells, Cultured , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Electrophoresis, Polyacrylamide Gel , Endothelium, Corneal/drug effects , Endothelium, Corneal/enzymology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , RNA, Small Interfering/genetics , Rabbits , Serine/metabolism , Stathmin/genetics , Threonine/metabolism , Transfection , cdc25 Phosphatases/metabolism , rac1 GTP-Binding Protein/antagonists & inhibitors
12.
Invest Ophthalmol Vis Sci ; 51(2): 822-9, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19797202

ABSTRACT

PURPOSE: To determine whether the elevated level of interleukin (IL)-1beta in aqueous humor after transcorneal freezing upregulates FGF-2 synthesis in rabbit corneal endothelium through PI3-kinase and p38 pathways. METHODS: Transcorneal freezing was performed in New Zealand White rabbits to induce an injury-mediated inflammation. The concentration of IL-1beta was measured, and the expression of FGF-2, p38, and Akt underwent Western blot analysis. Intracellular location of FGF-2 and actin cytoskeleton was determined by immunofluorescence staining. RESULTS: Massive infiltration of polymorphonuclear leukocytes (PMNs) to the corneal endothelium was observed after freezing, and IL-1beta concentration in the aqueous humor was elevated in a time-dependent manner after freezing. Similarly, FGF-2 expression was increased in a time-dependent manner. When corneal endothelium was stained with anti-FGF-2 antibody, the nuclear location of FGF-2 was observed primarily in the cornea after cryotreatment, whereas FGF-2 in normal corneal endothelium was localized at the plasma membrane. Treatment of the ex vivo corneal tissue with IL-1beta upregulated FGF-2 and facilitated its nuclear location in corneal endothelium. Transcorneal freezing disrupted the actin cytoskeleton at the cortex, and cell shapes were altered from cobblestone morphology to irregular shape. Topical treatment with LY294002 and SB203580 on the cornea after cryotreatment blocked the phosphorylation of Akt and p38, respectively, in the corneal endothelium. These inhibitors also reduced FGF-2 levels and partially blocked morphologic changes after freezing. CONCLUSIONS: These data suggest that after transcorneal freezing, IL-1beta released by PMNs into the aqueous humor stimulates FGF-2 synthesis in corneal endothelium via PI3-kinase and p38.


Subject(s)
Aqueous Humor/metabolism , Endothelium, Corneal/metabolism , Fibroblast Growth Factor 2/biosynthesis , Interleukin-1beta/physiology , Neutrophil Infiltration/physiology , Phosphatidylinositol 3-Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Actins/metabolism , Animals , Blotting, Western , Chromones/pharmacology , Corneal Opacity/etiology , Corneal Opacity/metabolism , Corneal Opacity/pathology , Cryosurgery , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique, Indirect , Imidazoles/pharmacology , Interleukin-1beta/pharmacology , Microscopy, Fluorescence , Morpholines/pharmacology , Neutrophils/metabolism , Phosphorylation , Pyridines/pharmacology , Rabbits , Time Factors , Up-Regulation
13.
Invest Ophthalmol Vis Sci ; 50(5): 2067-76, 2009 May.
Article in English | MEDLINE | ID: mdl-19136710

ABSTRACT

PURPOSE: To determine the mechanism by which IL-1beta induces FGF-2 and to elucidate the signaling pathways of IL-1beta-induced FGF-2 in corneal endothelial cells (CECs). METHODS: Expression and/or activation of FGF-2, p38, ERK1/2, and Akt was analyzed by immunoblot analysis. Cell proliferation was measured by MTT assay. Pharmacologic inhibitors were used to block PI 3-kinase, p38, or ERK1/2. RESULTS: Brief stimulation of CECs with IL-1beta activated PI 3-kinase and p38 in a biphasic fashion. The first wave of activation, triggered by IL-1beta, involves the inductive activity of IL-1beta on FGF-2 production; the second wave of activation, triggered by the induced FGF-2, involves the promotion of cellular activities. In both pathways, p38 acts downstream to PI 3-kinase. The inductive activity of IL-1beta on FGF-2 is further evidenced by the conditioned medium, which contains a large amount of FGF-2. Stimulation of CECs with IL-1beta also activated ERK1/2 in a delayed fashion. The IL-1beta-induced FGF-2 exerted cellular activities using distinct pathways: the second wave of activation of PI 3-kinase and p38 was involved in cell migration, whereas cell proliferation was simultaneously stimulated by ERK1/2 and the second wave of PI 3-kinase. Likewise, the conditioned medium demonstrated cellular activities and pathways identical with those observed in cells treated with IL-1beta. CONCLUSIONS: These data suggest that CECs produce FGF-2 by IL-1beta stimulation through PI 3-kinase and p38. The IL-1beta-induced FGF-2 facilitates cell migration via PI 3-kinase and p38, whereas it stimulates cell proliferation using PI 3-kinase and ERK1/2 in parallel pathways.


Subject(s)
Endothelium, Corneal/drug effects , Fibroblast Growth Factor 2/metabolism , Interleukin-1beta/pharmacology , Signal Transduction , Animals , Cell Culture Techniques , Cell Movement/drug effects , Cell Proliferation , Endothelium, Corneal/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Immunoblotting , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rabbits , p38 Mitogen-Activated Protein Kinases/metabolism
14.
Invest Ophthalmol Vis Sci ; 49(1): 189-96, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18172092

ABSTRACT

PURPOSE: p27(Kip1) (p27) is an important regulator of G(1) progression. For cells to proliferate, p27 must undergo proteolysis. FGF-2 enables phosphorylation of p27 at both the Thr-187 and Ser-10 sites, an event that is prerequisite for polyubiquitination. This study was undertaken to determine whether degradation of the two phosphorylated p27s is mediated by a distinct ubiquitin E3 ligase complex at different subcellular locations. METHODS: Expression of p27, KPC1, KPC2, Skp1, Skp2, and Cul1 was analyzed by immunoblot analysis. Association of p27 with ubiquitin E3 ligase was determined with coimmunoprecipitation followed by immunoblot analysis. Inhibitors were used to inhibit proteasomal degradation and nuclear export of the phosphorylated p27. DNA synthesis was measured by BrdU incorporation into DNA. RESULTS: Among ubiquitin ligase complex proteins, Cul1, KPC1, and KPC2 were constitutively expressed, whereas expression of Skp1 and Skp2 was temporally induced by FGF-2. Skp1, Skp2, and Cul1 were involved in polyubiquitination of phosphorylated p27 at Thr-187 (pp27Thr187) in nuclei. Maximum association of pp27Thr187 with the ubiquitin E3 ligase occurred 24 hours after FGF-2 stimulation. pp27Ser10 used the cytoplasmic ubiquitin E3 ligases KPC1 and KPC2, with maximum protein interaction observed at 8 hours. MG132 effectively blocked degradation of both pp27Thr187 and pp27Ser10, whereas leptomycin B blocked the nuclear export of pp27Ser10. Both inhibitors blocked BrdU incorporation into DNA. CONCLUSIONS: The findings demonstrate distinct polyubiquitination pathways for pp27Thr187 and pp27Ser10; the former is ubiquitinated through the nuclear ubiquitin E3 ligase system during late G(1) phase; the latter by cytosolic ubiquitin E3 ligase during early G(1) phase.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/metabolism , Endothelium, Corneal/metabolism , Ubiquitin-Protein Ligases/physiology , Ubiquitination/physiology , Animals , Blotting, Western , Cell Nucleus/metabolism , Cell Proliferation , Cells, Cultured , Cullin Proteins/physiology , Cysteine Proteinase Inhibitors/pharmacology , Cytoplasm/metabolism , DNA/biosynthesis , DNA Replication/drug effects , Electrophoresis, Polyacrylamide Gel , Endothelium, Corneal/drug effects , Fatty Acids, Unsaturated/pharmacology , Fibroblast Growth Factor 2/pharmacology , Immunoprecipitation , Leupeptins/pharmacology , Phosphorylation , Rabbits , S-Phase Kinase-Associated Proteins/physiology , SKP Cullin F-Box Protein Ligases/physiology
15.
J Biol Chem ; 282(9): 6444-54, 2007 Mar 02.
Article in English | MEDLINE | ID: mdl-17209046

ABSTRACT

The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G(1)/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G(1)/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G(1) phase of the cell cycle.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/metabolism , Fibroblast Growth Factor 2/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Animals , Cell Proliferation/drug effects , Endothelium, Corneal/cytology , Eye/cytology , Fibroblast Growth Factor 2/pharmacology , Interphase/drug effects , Kinetics , Nuclear Proteins/metabolism , Phosphorylation , Rabbits , Serine/metabolism , Threonine/metabolism
16.
J Biochem ; 140(1): 121-33, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16877773

ABSTRACT

We previously found that the peroxisomal citrate synthase of Saccharomyces cerevisiae, Cit2p, contains a cryptic targeting signal for both peroxisomes (PTS) and mitochondria (MTS) within its 20-amino acid N-terminal segment [Lee et al. (2000) J. Biochem. 128, 1059-1072]. In the present study, the fine structure of the cryptic signal was scrutinized using green fluorescent protein fusions led by variants of the N-terminal segment. The minimum ranges of the cryptic signals for mitochondrial and peroxisomal targeting were shown to consist of the first 15- and 10-amino acid N-terminal segments, respectively. Substitution of the 3rd Val, 6th Leu, 7th Asn, or 8th Ser with Ala abolished the cryptic MTS function, however, no single substitution causing an obvious defect in PTS function was found. Neither the 15-amino acid N-terminal segment nor the C-terminal SKL sequence (PTS1) was necessary for Cit2p to restore the glutamate auxotrophy caused by the double Deltacit1 Deltacit2 mutation. The Cit2p variant lacking PTS1 [Cit2(DeltaSKL)p] partially restored the growth of both the Deltacit1 Deltacit2 and Deltacit1 mutants on acetate, while that carrying intact PTS1 or lacking the N-terminal segment [Cit2p, Cit2((DeltaNDeltaSKL))p, and Cit2((DeltaN))p] did not. It is thus suggested that the potential of the N-terminal segment as an ambidextrous targeting signal can be unmasked by deletion of PTS1.


Subject(s)
Citrate (si)-Synthase/metabolism , Peroxisomes/enzymology , Protein Sorting Signals/genetics , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Citrate (si)-Synthase/chemistry , Glutamic Acid/metabolism , Green Fluorescent Proteins/genetics , Mitochondria/enzymology , Recombinant Fusion Proteins/genetics
17.
Exp Eye Res ; 83(6): 1309-16, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16769055

ABSTRACT

This review describes the molecular mechanism of endothelial mesenchymal transformation (EMT) mediated by fibroblast growth factor 2 (FGF-2) in corneal endothelial cells. Corneal fibrosis is rarely observed in corneal endothelium/Descemet's membrane complex; but when this pathologic tissue occurs, it causes a loss of vision. Herein, we will address the cellular activities of FGF-2 and its signaling pathways during EMT. FGF-2 has 5 isoforms: 4 nuclear high molecular weight isoforms and 1 extracellular matrix (ECM) isoform. The vast majority of studies published in the field to date have described the effect of the ECM isoform that is released into the extracellular space, from which it can access plasma membrane receptors. Our discussion will focus on the ECM isoform and its receptor-mediated signal transduction.


Subject(s)
Endothelium, Corneal/cytology , Fibroblast Growth Factor 2/physiology , Mesoderm/cytology , Collagen Type I/metabolism , Cornea/pathology , Corneal Injuries , Cytoskeleton/metabolism , Endothelium, Corneal/metabolism , Fibrosis , Humans , Phosphatidylinositol 3-Kinases/physiology , Signal Transduction/physiology
18.
Invest Ophthalmol Vis Sci ; 47(6): 2358-68, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16723445

ABSTRACT

PURPOSE: Endothelial-mesenchymal transformation (EMT), in which the contact-inhibited corneal endothelial cells (CECs) become multilayers of spindle-shaped cells containing protrusive processes, is mediated by fibroblast growth factor (FGF)-2. The involvement in EMT of cross-talk among Rho GTPases mediated by FGF-2 was also investigated. METHODS: GTP pull-down assays were performed to identify the activated Rho GTPases. Transfection of CECs with either constitutively active (ca) or dominant negative (dn) Rho GTPase vectors was performed. Protein-protein interaction was investigated by coimmunoprecipitation and a yeast two-hybrid assay. RESULTS: The alteration of morphology and actin cytoskeleton caused by FGF-2 was mediated by active Rac and inactive Rho. Prolonged treatment of CECs with FGF-2 induced formation of protrusive processes through activated Cdc42. All FGF-2 actions were blocked by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002. Cells transfected with caRacG12V acquired elongated morphology; the actin cytoskeleton was reorganized to the cortex. Formation of protrusive processes was observed in the elongated cells expressing caCdc42G12V or dominant negative (dn)RhoT19N, whereas polygonal cells expressing dnRacT17N, caRhoG14V, or dnCdc42T17N had stress fibers. Further analysis demonstrated that Rac was associated with Cdc42 or Rho through a 32- or 30-kDa Dbl homology/pleckstrin homology-containing protein. CONCLUSIONS: These findings suggest that alteration of cell shape and actin cytoskeleton are closely linked to the sequential activation of Rho GTPases through PI 3-kinase in response to FGF-2 stimulation. Cortical actin is formed via active Rac and inactive Rho followed by formation of protrusive processes mediated by active Cdc42 and inactive Rho.


Subject(s)
Endothelium, Corneal/cytology , Fibroblast Growth Factor 2/pharmacology , Mesoderm/cytology , Phosphatidylinositol 3-Kinases/metabolism , rho GTP-Binding Proteins/metabolism , Actins/metabolism , Animals , Cell Shape/drug effects , Cells, Cultured , Chromones/pharmacology , Cytoskeleton/drug effects , Endothelium, Corneal/drug effects , Endothelium, Corneal/enzymology , Enzyme Inhibitors/pharmacology , Genetic Vectors , Immunoenzyme Techniques , Mesoderm/enzymology , Microscopy, Confocal , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Binding , Protein Interaction Mapping , Rabbits , Transfection , Two-Hybrid System Techniques , cdc42 GTP-Binding Protein/metabolism , rac GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/genetics
19.
Invest Ophthalmol Vis Sci ; 47(4): 1376-86, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16565371

ABSTRACT

PURPOSE: Acquisition of elongated cells with pseudopodia is observed when corneal endothelial cells (CECs) are simultaneously treated with basic fibroblast growth factor (FGF)-2 and RhoA inhibitors. This study was designed to determine whether these phenotypes are migratory and whether Cdc42 activation and RhoA inactivation are involved in cell migration. METHODS: A scratch-induced directional migration assay was used to measure migratory rates. Activation of Cdc42 was determined by GTP pull-down assay. Transfection was performed using constitutively active (ca) or dominant negative (dn) Rho guanosine triphosphatase (GTPase) vectors. RESULTS: Stimulation with basic FGF-2 alone resulted in a 43% recovery of the wound area, whereas CECs treated with FGF-2 and Y27632 (inhibitor of Rho-associated kinase) achieved an 84% recovery of the wound area with a fast migratory speed (0.72 microm/min). The synergistic effects of FGF-2 and Y27632 were completely blocked by LY294002 (PI 3-kinase inhibitor). Under these conditions, activation of PI 3-kinase and Cdc42 were observed in the migratory cells. The involvement of activated Cdc42 and inactivated Rho in endothelial migration was determined by transfecting CECs with ca- or dnRho GTPase vectors. A high migratory rate (0.52 microm/min) was seen in CECs expressing caCdc42, whereas endothelial migration was completely inhibited in CECs expressing caRho. When cells expressing caCdc42 were treated with FGF-2, migration reached the maximum rate (0.69 microm/min), similar to that observed in cells treated with FGF-2 and Y27632. CONCLUSIONS: These findings suggest that endothelial migration is induced by activated Cdc42 and inactivated Rho via PI 3-kinase after FGF-2 stimulation and that Cdc42 activation is crucial for CECs to acquire the characteristic migratory phenotypes.


Subject(s)
Endothelium, Corneal/drug effects , Fibroblast Growth Factor 2/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Wound Healing/drug effects , cdc42 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/physiology , Animals , Cell Culture Techniques , Cell Movement/physiology , Cell Proliferation , Electrophoresis, Polyacrylamide Gel , Endothelium, Corneal/cytology , Endothelium, Corneal/metabolism , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique, Indirect , Genetic Vectors , Microscopy, Confocal , Rabbits , Transfection , rhoA GTP-Binding Protein/antagonists & inhibitors
20.
J Biol Chem ; 279(31): 32325-32, 2004 Jul 30.
Article in English | MEDLINE | ID: mdl-15173165

ABSTRACT

Our previous work demonstrated that both polymorphonuclear leukocytes (PMNs) and protein fractions released from PMNs induced de novo synthesis of fibroblast growth factor 2 (FGF-2), which in turn becomes the direct mediator of endothelial mesenchymal transformation observed in corneal endothelial cells (CECs). To identify the protein factor, we used ProteinChip Array technology. Protein fractions obtained from the conditioned medium released by PMNs were resolved by two-dimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 17 kDa, were sequentially subjected to in-gel trypsin digestion and mass spectrometry. The 17-kDa peptide band was identified as interleukin-1 beta (IL-1 beta). Biological activities of IL-1 beta were further determined; IL-1 beta altered the shape of CECs from polygonal to fibroblastic morphologies in a time- and dose-dependent manner, whereas neutralizing IL-1 beta antibody, neutralizing antibody to FGF-2, and LY294002 blocked the action of IL-1 beta. IL-1 beta greatly increased the levels of FGF-2 mRNA in a time- and dose-dependent manner; IL-1 beta stimulated expression of all isoforms of FGF-2. IL-1 beta initially induced nuclear accumulation of FGF-2 and facilitated translocation of FGF-2 to plasma membrane and extracellular matrix. IL-1 beta activated phosphatidylinositol (PI) 3-kinase, the enzyme activity of which was greatly stimulated after a 5-min exposure to IL-1 beta. This early and rapid activation of PI 3-kinase greatly enhanced FGF-2 production in CECs; pretreatment with LY294002 hampered the induction activity of IL-1 beta. These observations suggest that IL-1 beta takes part in endothelial to mesenchymal transformation of CECs through its inductive potential on FGF-2 via the action of PI 3-kinase.


Subject(s)
Cornea/metabolism , Endothelium/metabolism , Fibroblast Growth Factor 2/metabolism , Interleukin-1/metabolism , Mesoderm/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Cells, Cultured , Chromones/pharmacology , Culture Media, Conditioned/pharmacology , Cytoskeleton/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Extracellular Matrix/metabolism , Fibroblasts/metabolism , Hydrogen-Ion Concentration , Immunoblotting , Inflammation , Mass Spectrometry , Microscopy, Confocal , Microscopy, Fluorescence , Models, Biological , Molecular Sequence Data , Morpholines/pharmacology , Neutrophils/metabolism , Phenotype , Protein Array Analysis , Protein Isoforms , RNA/metabolism , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription, Genetic , Trypsin/pharmacology , Up-Regulation , Wound Healing
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