RESUMO
BACKGROUND: Lens degeneration in Fpr1(-/-) mice prompted us to search for functional FPR1 expression directly on lens epithelial cells. RESULTS: FPR1 is functionally expressed on human lens epithelial cells but has atypical properties compared with hematopoietic cell FPR1. CONCLUSION: Lens epithelial cell FPR1 may be involved in development and maintenance of the lens. SIGNIFICANCE: This is the first link between non-hematopoietic expression of FPR1 and an ophthalmologic phenotype. Formyl peptide receptor 1 (FPR1) is a G protein-coupled chemoattractant receptor expressed mainly on leukocytes. Surprisingly, aging Fpr1(-/-) mice develop spontaneous lens degeneration without inflammation or infection (J.-L. Gao et al., manuscript in preparation). Therefore, we hypothesized that FPR1 is functionally expressed directly on lens epithelial cells, the only cell type in the lens. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. Competition binding using FPR1 ligands N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Nle = Norleucine), formylmethionylleucylphenylalanine, and peptide W revealed the same profile for FHL 124 cells, neutrophils, and FPR1-transfected HEK 293 cells. Saturation binding with fluorescein-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys revealed ~2500 specific binding sites on FHL-124 cells (K(D) ~ 0.5 nm) versus ~40,000 sites on neutrophils (K(D) = 3.2 nm). Moreover, formylmethionylleucylphenylalanine induced pertussis toxin-sensitive Ca(2+) flux in FHL 124 cells, consistent with classic G(i)-mediated FPR1 signaling. FHL 124 cell FPR1 was atypical in that it resisted agonist-induced internalization. Expression of FPR1 was additionally supported by detection of the intact full-length open reading frame in sequenced cDNA from FHL 124 cells. Thus, FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr1 knock-out mice.
Assuntos
Células Epiteliais/metabolismo , Cristalino/metabolismo , Receptores de Formil Peptídeo/genética , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Knockout , Receptores de Formil Peptídeo/metabolismoRESUMO
The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Cristalino/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ubiquitina/metabolismo , Western Blotting , Proteína Tirosina Quinase CSK , Células Cultivadas , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Cristalino/citologia , Cristalino/efeitos dos fármacos , Mutação/genética , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Ubiquitinação , Quinases da Família srcRESUMO
Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.
Assuntos
Movimento Celular , Quinase 5 Dependente de Ciclina/metabolismo , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Humanos , Modelos Biológicos , Cadeias Leves de Miosina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Talina/metabolismoRESUMO
Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.
Assuntos
Movimento Celular/fisiologia , Quinase 5 Dependente de Ciclina/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Repressoras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular , Quinase 5 Dependente de Ciclina/genética , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Células Epiteliais/citologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Cinetina/metabolismo , Cadeias Leves de Miosina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Quinases da Família src/genéticaRESUMO
The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body. Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens. Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2. Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers. The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100 ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied. Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques. Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.
Assuntos
Diferenciação Celular/fisiologia , Cristalino/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Células Cultivadas , Células Epiteliais/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , RatosRESUMO
Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Fibras de Estresse/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Quinase 5 Dependente de Ciclina/análise , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Mutação , Fosforilação , RNA Interferente Pequeno/metabolismo , Coelhos , Fibras de Estresse/ultraestrutura , Transfecção , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo. METHODS: Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 microl drop of 15 microM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson's dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting. RESULTS: Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization. CONCLUSIONS: Topical application of olomoucine in 1% DMSO significantly enhances closure of small epithelial debridement wounds without increasing inflammation or impairing reepithelialization.
Assuntos
Córnea/fisiopatologia , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Desbridamento , Inibidores Enzimáticos/farmacologia , Cinetina/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Caderinas/metabolismo , Células Cultivadas , Córnea/metabolismo , Córnea/patologia , Córnea/cirurgia , Células Epiteliais/enzimologia , Humanos , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/enzimologia , Limbo da Córnea/lesões , Limbo da Córnea/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos , Neutrófilos/patologia , Fatores de Tempo , Distribuição TecidualRESUMO
Previous studies implicate cyclin-dependent kinase 5 in cell adhesion and migration of epithelial cells of the cornea and lens. To explore molecular interactions underlying these functions, we performed yeast two-hybrid screening of an embryonic rat lens library for proteins that interact with cyclin-dependent kinase 5 and its regulators, p35 and p39. This screen identified a specific interaction between p39 and muskelin, an intracellular protein known to affect cytoskeletal organization in adherent cells. Immunohistochemistry detected muskelin in the developing lens and in other tissues, including brain and muscle. Glutathione S-transferase pull-down experiments and co-immunoprecipitations confirmed the specificity of the p39-muskelin interaction. Deletion analysis of p39 showed that muskelin binds to the p39 C terminus, which contains a short insertion (amino acids 329-366) absent from p35. Similar analysis of muskelin mapped the interaction with p39 to the fifth kelch repeat. Co-expression of p39 and muskelin in COS1 cells or lens epithelial cells altered the intracellular localization of muskelin, recruiting it to the cell periphery. These findings demonstrate a novel interaction between muskelin and the cyclin-dependent kinase 5 activator p39 and suggest that p39 may regulate the subcellular localization of muskelin.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Actinas/química , Animais , Sítios de Ligação , Encéfalo/metabolismo , Células COS , Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular , Movimento Celular , Quinase 5 Dependente de Ciclina , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Células Epiteliais/metabolismo , Deleção de Genes , Biblioteca Gênica , Glutationa Transferase/metabolismo , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Cristalino/metabolismo , Microscopia de Fluorescência , Músculos/metabolismo , Oligonucleotídeos/química , Peptídeos/química , Fosfotransferases/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , RNA/química , RNA Mensageiro/metabolismo , Coelhos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
PURPOSE: To demonstrate the interactions of PKCgamma with caveolin (Cav)-1 and connexin(Cx)43 in lipid rafts and its regulation of gap junctions. METHODS: N/N1003A lens epithelial cells, bovine primary lens epithelial cells, and stably transfected N/N1003A lens epithelial cells were used. Coimmunoprecipitation and Western blot analysis were used to detect protein expression and their interactions. Cav-1-containing lipid rafts and redistribution of Cav-1, PKCgamma, and Cx43 were analyzed by sucrose gradients and by consequent Western blot analysis. Cell surface gap junction Cx43 plaques were detected by confocal microscopy. PKCgamma activity was measured with a PKC assay kit. RESULTS: Cav-1 and -2 were found in N/N1003A and bovine primary lens epithelial cells. Cx43 was associated with Cav-1 in lipid rafts. Phorbol ester (TPA) and insulin-like growth factor (IGF)-1 recruited PKCgamma into Cav-1-containing lipid rafts and stimulated the interactions of PKCgamma with Cav-1 and Cx43. TPA and IGF-1 induced redistribution of Cav-1 and Cx43 from light-density fractions to higher density fractions on sucrose gradients. PKCgamma redistributed with Cav-1- and Cx43-containing fractions on stimulation with TPA or IGF-1. Overexpression of PKCgamma-enhanced green fluorescent protein (EGFP) increased the interaction of PKCgamma-EGFP with Cav-1 and Cx43 and decreased gap junction Cx43 plaques without exogenous growth factors. Overexpression of a loss-of-function PKCgamma mutant did not decrease gap junction Cx43 plaques or cause redistribution in lipid rafts, even though the PKCgamma mutant still interacted with Cav-1 and Cx43. CONCLUSIONS: Activation of PKCgamma by TPA or IGF-1 stimulated the interaction of PKCgamma with Cav-1 and Cx43 in lipid rafts, causing Cx43, Cav-1, and PKCgamma to redistribute within the lipid rafts, and this resulted in a decrease in gap junction plaques.
Assuntos
Caveolinas/metabolismo , Células Epiteliais/metabolismo , Junções Comunicantes/metabolismo , Cristalino/metabolismo , Metabolismo dos Lipídeos , Proteína Quinase C/metabolismo , Animais , Western Blotting , Bovinos , Caveolina 1 , Células Cultivadas , Centrifugação com Gradiente de Concentração , Conexina 43/metabolismo , Células Epiteliais/efeitos dos fármacos , Proteínas de Fluorescência Verde , Fator de Crescimento Insulin-Like I/farmacologia , Isoenzimas/metabolismo , Cristalino/efeitos dos fármacos , Proteínas Luminescentes , Microscopia Confocal , Mutagênese Sítio-Dirigida , Coelhos , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
12(S)-hydroxyeicosatetraenoic acid (12(S)HETE) is a bioactive metabolite of arachidonic acid synthesized by 12-lipoxygenase. The 12-lipoxygenase blocker, baicalein, prevents epidermal growth factor (EGF)-induced activation of protein kinase C (PKC) alpha and beta in lens epithelial cells, whereas supplementation with 12(S)HETE reverses this effect, suggesting that EGF and 12(S)HETE may work together to activate PKC. This study investigates the mechanism of PKCbeta activation by EGF and 12(S)HETE. 12(S)HETE alone directed translocation of PKCbeta through the C1 rather than the C2 domain, without activating phosphoinositide 3-kinase (PI3K) or MAPK signaling or increasing intracellular calcium concentration. In the presence of baicalein, EGF triggered an asymmetric phosphorylation of the EGF receptor initiating signaling through PI3K and MAPK, but not PLCgamma. Together, 12(S)HETE and EGF synergistically increased phosphorylation of PKCbeta in the activation loop and C terminus as well as PKCbeta-specific activity. PI3K inhibitors blocked phosphorylation, but MEK1 inhibitors did not. Microvesicles containing phosphatidylinositol 3,4,5-trisphosphate mimicked the action of EGF on PKCbeta activity in the presence of 12(S)HETE. Kinase-inactive PKCbeta mutations in either activation loop or C terminus were effectively translocated by 12(S)HETE, as was PKCbeta in the presence of chelerythrine or Gö-6983. These findings indicate that unphosphorylated PKCbeta is translocated to the membrane by 12(S)HETE and phosphorylated by EGF-dependent PI3K signaling, to generate catalytically competent PKCbeta.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/enzimologia , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Cristalino/citologia , Cristalino/enzimologia , CoelhosRESUMO
Transgenic mouse technology has been very valuable for the study of lens fiber cells since they can not be propagated in cell culture. The targeting of transgenes to the lens has traditionally been done with the alphaA-crystallin promoter. However, while lens-specific, transgenic lines made with the alphaA-crystallin promoter express the transgene at levels 100-300-fold lower than endogenous alphaA-crystallin. Here we propose an alternative, the chicken betaB1-crystallin promoter (-432/+30). Transgenic mice made with this promoter have successfully expressed CAT, d/n m-calpain, Weel, and betaB2-crystallin mRNA at levels comparable to the endogenous betaB1-crystallin gene and no eye abnormalities such as cataracts, have resulted. All of the transgenic lines made with the chicken betaB1-crystallin promoter have expressed the transgene in the lens fiber cells, and the best lines express at levels close to endogenous betaB1-crystallin. While RNA expression is very high, only moderate protein expression has been achieved, implying that the high protein expression of the crystallins is partially controlled at the level of translation. Thus, the chicken betaB1-crystallin promoter directs high level RNA expression to lens fiber cells, which may be especially useful for the expression of ribozyme and anti-sense RNAs in addition to ectopic proteins.