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1.
J Biol Chem ; 291(8): 3947-58, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26719333

RESUMO

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.


Assuntos
Cristalinas/biossíntese , Fator 2 de Crescimento de Fibroblastos/metabolismo , Cristalino/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-maf/biossíntese , Animais , Embrião de Galinha , Cristalinas/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Cristalino/citologia , Células MCF-7 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-maf/genética , Elementos de Resposta/fisiologia , Regulação para Cima/fisiologia
2.
Adv Exp Med Biol ; 801: 395-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24664723

RESUMO

LMAN1 is a type I transmembrane protein that selectively transports its cargo proteins from ER to ER-Golgi intermediate compartment (ERGIC) and Golgi. Lman1 is a direct target of the transcription factor NRL in mouse retina. Therefore, we examined the in vivo function of LMAN1 in retina. Although Lman1 (- / -) mouse eyes did not show abnormality in histology and electroretinogram analysis at 3 months, Lman1 (- / -) retina at 6 months showed a decrease in cis-Golgi markers GM130 and GRASP65. We also observed abnormal level and location of Rhodopsin in these mice. Taken together, LMAN1 may play a role in photoreceptor gene transport and homeostasis.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Homeostase/fisiologia , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras de Vertebrados/fisiologia , Animais , Transporte Biológico/fisiologia , Lectinas de Ligação a Manose/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout
3.
Dev Biol ; 362(2): 219-29, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22173065

RESUMO

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation.


Assuntos
Apoptose/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cristalino/embriologia , Morfogênese/fisiologia , Receptor Notch2/metabolismo , Transdução de Sinais/fisiologia , Animais , Ciclo Celular/fisiologia , Primers do DNA/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Cristalino/metabolismo , Cristalino/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real
4.
Mol Vis ; 19: 319-32, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23401660

RESUMO

PURPOSE: Although cyclin-dependent kinase 5 (Cdk5) inhibits the formation of junctions containing N-cadherin, the effect of Cdk5 on junctions containing E-cadherin is less clear. The present study investigates the functional significance of Cdk5 in forming and maintaining cell-cell stability in corneal epithelial cells. METHODS: A Cdk5-deficient human corneal limbal epithelial cell line was generated by lentiviral transduction of small hairpin RNA specific for Cdk5 (shCdk5-HCLE cells). A blasticidin-inducible vector for expression of Cdk5-specific short hairpin RNA (ShCdk5) was generated by recombination and packaged into non-replicative lentiviral particles for transduction of human corneal limbal epithelial (HCLE) cells. Blasticidin-resistant cells were isolated for analysis. Cell aggregations were performed using HCLE, Cdk5 inhibitor olomoucine, ShCdk5, and MDA-MB 231 cells in the presence and absence of calcium, and particle size was measured using image analysis software. Relative protein concentrations were measured with immunoblotting and quantitative densitometry. Total internal reflection fluorescence (TIRF) microscopy was performed on cells transfected with green fluorescent protein (GFP)-E-cadherin or GFP-p120, and internalization of boundary-localized proteins was analyzed with particle tracking software. The stability of surface-exposed proteins was determined by measuring the recovery of biotin-labeled proteins with affinity chromatography. Rho and Rac activity was measured with affinity chromatography and immunoblotting. RESULTS: Examining the effect of Cdk5 on E-cadherin containing epithelial cell-cell adhesions using a corneal epithelial cell line (HCLE), we found that Cdk5 and Cdk5 (pY15) coimmunoprecipitate with E-cadherin and Cdk5 (pY15) colocalizes with E-cadherin at cell-cell junctions. Inhibiting Cdk5 activity in HCLE or suppressing Cdk5 expression in a stable HCLE-derived cell line (ShHCLE) decreased calcium-dependent cell adhesion, promoted the cytoplasmic localization of E-cadherin, and accelerated the loss of surface-biotinylated E-cadherin. TIRF microscopy of GFP-E-cadherin in transfected HCLE cells showed an actively internalized sub-population of E-cadherin, which was not bound to p120 as it was trafficked away from the cell-cell boundary. This population increased in the absence of Cdk5 activity, suggesting that Cdk5 inhibition promotes dissociation of p120/E-cadherin junctional complexes. These effects of Cdk5 inhibition or suppression were accompanied by decreased Rac activity, increased Rho activity, and enhanced binding of E-cadherin to the Rac effector Ras GTPase-activating-like protein (IQGAP1). Cdk5 inhibition also reduced adhesion in a cadherin-deficient cell line (MDA-MB-231) expressing exogenous E-cadherin, although Cdk5 inhibition promoted adhesion when these cells were transfected with N-cadherin, as previous studies of Cdk5 and N-cadherin predicted. Moreover, Cdk5 inhibition induced N-cadherin expression and formation of N-cadherin/p120 complexes in HCLE cells. CONCLUSIONS: These results indicate that loss of Cdk5 activity destabilizes junctional complexes containing E-cadherin, leading to internalization of E-cadherin and upregulation of N-cadherin. Thus, Cdk5 activity promotes stability of E-cadherin-based cell-cell junctions and inhibits the E-cadherin-to-N-cadherin switch typical of epithelial-mesenchymal transitions.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Junções Intercelulares/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Transição Epitelial-Mesenquimal , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/metabolismo , Microscopia de Fluorescência , RNA Interferente Pequeno/genética
5.
Dev Biol ; 332(1): 166-76, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19481073

RESUMO

Previous studies of the developing lens have shown that Notch signaling regulates differentiation of lens fiber cells by maintaining a proliferating precursor pool in the anterior epithelium. However, whether Notch signaling is further required after the onset of fiber cell differentiation is not clear. This work investigates the role of Notch2 and Jagged1 (Jag1) in secondary fiber cell differentiation using rat lens epithelial explants undergoing FGF-2 dependent differentiation in vitro. FGF induced Jag1 expression and Notch2 signaling (as judged by the appearance of activated Notch2 Intracellular Domain (N2ICD)) within 12-24 h. These changes were correlated with induction of the Notch effector, Hes5, upregulation of N-cadherin (N-cad), and downregulation of E-cadherin (E-cad), a cadherin switch characteristic of fiber cell differentiation. Induction of Jag1 was efficiently blocked by U0126, a specific inhibitor of MAPK/ERK signaling, indicating a requirement for signaling through this pathway downstream of the FGF receptor. Other growth factors that activate MAPK/ERK signaling (EGF, PDGF, IGF) did not induce Jag1. Inhibition of Notch signaling using gamma secretase inhibitors DAPT and L-685,458 or anti-Jag1 antibody markedly decreased FGF-dependent expression of Jag1 demonstrating Notch-dependent lateral induction. In addition, inhibition of Notch signaling reduced expression of N-cad, and the cyclin dependent kinase inhibitor, p57Kip2, indicating a direct role for Notch signaling in secondary fiber cell differentiation. These results demonstrate that Notch-mediated lateral induction of Jag1 is an essential component of FGF-dependent lens fiber cell differentiation.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cristalino/citologia , Cristalino/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Biomarcadores/metabolismo , Caderinas/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Proteína Jagged-1 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microdissecção , Modelos Biológicos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Wistar , Proteínas Serrate-Jagged
6.
Exp Cell Res ; 314(19): 3542-50, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18838073

RESUMO

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/metabolismo
7.
Mol Vis ; 14: 542-9, 2008 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-18385789

RESUMO

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 Tecidual
8.
J Vis Exp ; (31)2009 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-19773734

RESUMO

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 , Ratos
9.
Arthritis Rheum ; 58(6): 1774-83, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18512813

RESUMO

OBJECTIVE: Triptolide and tripdiolide are thought to be active components of the Chinese antirheumatic herbal remedy Tripterygium wilfordii Hook F, which has been shown to be effective in treating murine lupus nephritis. This study was undertaken to examine the therapeutic effect of triptolide and tripdiolide on established lupus nephritis in (NZB x NZW)F1 mice. METHODS: (NZB x NZW)F1 mice were treated with vehicle, triptolide, or tripdiolide for 15 weeks beginning at the age of 29 weeks (after the development of lupus nephritis). Body weight, proteinuria, and anti-double-stranded DNA (anti-dsDNA) antibodies were monitored, and the kidney and spleen were assessed histologically. Culture supernatants of spleen mononuclear cells were assayed for cytokines. RESULTS: By 28 weeks, most (NZB x NZW)F1 mice had developed lupus nephritis. Vehicle-treated mice exhibited progressive proteinuria, hypoalbuminemia, elevated blood urea nitrogen (BUN) levels, and evidence of severe nephritis. In contrast, proteinuria and BUN levels were significantly reduced in mice treated with either triptolide or tripdiolide as compared with those treated with vehicle. There was no hypoalbuminemia or apparent evidence of lupus nephritis in mice treated with either of the 2 diterpenoids. At 44 weeks of age, the survival rate in mice treated with vehicle (35.7%) was markedly lower than that in mice treated with either triptolide (87.5%) or tripdiolide (88.2%). The mean level of anti-dsDNA antibody in mice treated with tripdiolide was lower than that in the vehicle-treated mice upon completion of the treatment course. Production of tumor necrosis factor, interleukin-6, and monocyte chemoattractant protein 1 by spleen cells was also decreased after diterpenoid therapy. CONCLUSION: Therapy with triptolide or tripdiolide significantly ameliorated lupus nephritis in (NZB x NZW)F1 mice, reduced cytokine and chemokine production, and prolonged survival.


Assuntos
Diterpenos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Imunossupressores/uso terapêutico , Nefrite Lúpica/tratamento farmacológico , Fenantrenos/uso terapêutico , Tripterygium/química , Animais , Modelos Animais de Doenças , Compostos de Epóxi/uso terapêutico , Feminino , Nefrite Lúpica/sangue , Nefrite Lúpica/patologia , Camundongos , Baço/efeitos dos fármacos , Baço/patologia
10.
EMBO J ; 25(10): 2107-18, 2006 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-16675956

RESUMO

Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis. A DCR1/alphaA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of alphaA-crystallin in lens epithelium and fibers. In contrast, the DCR3/alphaA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the alphaA-crystallin promoter in lens chromatin and demonstrated that high levels of alphaA-crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the alphaA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin-remodeling enzymes, and suggest a multistage model for the activation of alphaA-crystallin during lens differentiation.


Assuntos
Cromatina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Cristalino/crescimento & desenvolvimento , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Proteínas Repressoras/metabolismo , Cadeia A de alfa-Cristalina/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , DNA Helicases , Proteínas do Olho/genética , Genes Reporter , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Cristalino/anatomia & histologia , Cristalino/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-maf/genética , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Técnicas de Cultura de Tecidos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Cadeia A de alfa-Cristalina/genética
11.
J Biol Chem ; 280(22): 21376-83, 2005 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-15797862

RESUMO

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-Híbrido
12.
J Cell Sci ; 117(Pt 18): 4089-98, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15280426

RESUMO

Recent studies have shown that Cdk5, a member of the cyclin-dependent-kinase family, regulates adhesion and migration in a mouse corneal epithelial cell line. Here, we extend these findings to corneal wound healing in vivo and examine the mechanism linking Cdk5 to cytoskeletal reorganization and migration. Cdk5 was overexpressed in the corneal epithelium of transgenic mice under control of the ALDH3 promoter. Elevated Cdk5 expression retarded corneal debridement wound closure in these animals and suppressed remodeling of the actin cytoskeleton. Conversely, the Cdk5 inhibitor, olomoucine, accelerated debridement wound healing in organ cultured eyes of normal mice, caused migrating cells to separate from the epithelial cell sheet, and increased the level of activated Src(pY416) along the wound edge. To explore the relationship between Cdk5 and Src in greater detail, we examined scratch-wounded cultures of corneal epithelial cells. Src was activated in cells along the wound edge and blocking this activation with the Src kinase inhibitor, PP1, inhibited wound closure by 85%. Inhibiting Cdk5 activity with olomoucine or a dominant negative construct, Cdk5T33, increased the concentration of Src(pY416), shifted its subcellular localization to the cell periphery and enhanced wound closure. Cdk5(pY15), an activated form of Cdk5, also appeared along the wound edge. Inhibiting Src activity with PP1 blocked the appearance of Cdk5(pY15), suggesting that Cdk5 phosphorylation is Src dependent. Cdk5 and Src co-immunoprecipitated from scratch-wounded cultures, demonstrating that both kinases are part of an intracellular protein complex. These findings indicate that Cdk5 exerts its effects on cell migration during corneal epithelial wound healing by regulating the activation and localization of Src.


Assuntos
Movimento Celular/fisiologia , Doenças da Córnea/metabolismo , Quinases Ciclina-Dependentes/fisiologia , Epitélio Corneano/enzimologia , Cicatrização/fisiologia , Quinases da Família src/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Compartimento Celular/genética , Doenças da Córnea/genética , Quinase 5 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Citoesqueleto/metabolismo , Inibidores Enzimáticos/farmacologia , Epitélio Corneano/citologia , Traumatismos Oculares/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Fosforilação/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética
13.
Transgenic Res ; 11(4): 397-410, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12212842

RESUMO

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.


Assuntos
Cristalinas/genética , Cristalino/fisiologia , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Galinhas , Sequência Consenso , Primers do DNA , Eletroforese em Gel Bidimensional , Humanos , Cristalino/citologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Cadeia B de beta-Cristalina
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