RESUMO
Aberrant regulation of signal transduction pathways can adversely derail biological processes for tissue development. One such process is the embryonic eyelid closure that is dependent on the mitogen-activated protein kinase kinase kinase 1 (MAP3K1). Map3k1 KO in mice results in defective eyelid closure and an autosomal recessive eye-open at birth phenotype. We have shown that in utero exposure to dioxin, a persistent environmental toxicant, induces the same eye defect in Map3k1+/- heterozygous but not WT pups. Here, we explore the mechanisms of the Map3k1 (gene) and dioxin (environment) interactions (GxE) underlying defective eyelid closure. We show that, acting through the aryl hydrocarbon receptor, dioxin activates epidermal growth factor receptor signaling, which in turn depresses MAP3K1-dependent Jun N-terminal kinase (JNK) activity. The dioxin-mediated JNK repression is moderate but is exacerbated by Map3k1 heterozygosity. Therefore, dioxin exposed Map3k1+/- embryonic eyelids have a marked reduction of JNK activity, accelerated differentiation and impeded polarization in the epithelial cells. Knocking out Ahr or Egfr in eyelid epithelium attenuates the open-eye defects in dioxin-treated Map3k1+/- pups, whereas knockout of Jnk1 and S1pr that encodes the sphigosin-1-phosphate (S1P) receptors upstream of the MAP3K1-JNK pathway potentiates the dioxin toxicity. Our novel findings show that the crosstalk of aryl hydrocarbon receptor, epidermal growth factor receptor, and S1P-MAP3K1-JNK pathways determines the outcome of dioxin exposure. Thus, gene mutations targeting these pathways are potential risk factors for the toxicity of environmental chemicals.
Assuntos
Dioxinas , Receptores ErbB , MAP Quinase Quinase Quinase 1 , Receptores de Hidrocarboneto Arílico , Animais , Feminino , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dioxinas/toxicidade , Receptores ErbB/metabolismo , Receptores ErbB/genética , Pálpebras/metabolismo , Pálpebras/anormalidades , Interação Gene-Ambiente , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Knockout , Receptor Cross-Talk , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Morphogenesis is a complex and highly coordinated process orchestrated by temporal spatial activity of developmental pathways. How the different pathways interact to guide the developmental program remains an intriguing and open question. MAP3K1-JNK and Wnt are signaling pathways crucial for embryonic eyelid closure, an epithelial morphogenetic event conserved in mammals. Here we used a mouse model of eyelid development and genetic and biochemistry tools to investigate the relationships between the two pathways. We found that Wnt activation repressed MAP3K1 expression. Using Axin-LacZ reporter mice, spatial Wnt activity was detected in the leading edge of the developing eyelid. Conditional knockout of Wntless (Wls) in ocular surface ectoderm blocked eyelid formation, and significantly increased MAP3K1 expression in eyelid cells at the nasal canthus region. Conversely, knockout of Dkk2, encoding a canonical Wnt antagonist, resulted in an increase of Wnt activity in cells at the upper eyelid margin near the nasal canthus. Up-regulation of Wnt signaling in the Dkk2-knockout embryos corresponded to down-regulation of MAP3K1 expression. In vitro data showed that Wnt3a treatment decreased MAP3K1 promoter activity, whereas activation of Wnt by lithium chloride inhibited MAP3K1 expression, and attenuated MAP3K1-mediated JNK activity. Our data identify a unique signal crosstalk between Wnt signaling and the MAP3K1-JNK pathway in epithelial morphogenesis.
Assuntos
Pálpebras/embriologia , MAP Quinase Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Via de Sinalização Wnt , Animais , Ectoderma/metabolismo , Pálpebras/enzimologia , Pálpebras/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MAP Quinase Quinase Quinase 1/genética , Camundongos , Morfogênese/genética , Transdução de SinaisRESUMO
PURPOSE: Embryonic eyelid closure is a well-documented morphogenetic episode in mammalian eye development. Detection of eyelid closure defect in humans is a major challenge because eyelid closure and reopen occur entirely in utero. As a consequence, congenital eye defects that are associated with failure of embryonic eyelid closure remain unknown. To fill the gap, we developed a mouse model of defective eyelid closure. This preliminary work demonstrates that the magnetic resonance imaging (MRI) approach can be used for the detection of extraocular muscle abnormalities in the mouse model. METHODS: Mice with either normal (Map3k1+/- ) or defective (Map3k1-/- ) embryonic eyelid closure were used in this study. Images of the extraocular muscles were obtained with a 9.4 T high resolution microimaging MRI system. The extraocular muscles were identified, segmented, and measured in each imaging slice using an in-house program. RESULTS: In agreement with histological findings, the imaging data show that mice with defective embryonic eyelid closure develop less extraocular muscle than normal mice. In addition, the size of the eyeballs was noticeably reduced in mice with defective embryonic eyelid closure. CONCLUSIONS: We demonstrated that MRI can potentially be used for the study of extraocular muscle in the mouse model of the eye open-at-birth defect, despite the lack of specificity of muscle group provided by the current imaging resolution.
Assuntos
Modelos Animais de Doenças , Anormalidades do Olho/diagnóstico por imagem , Doenças Palpebrais/diagnóstico por imagem , Imageamento por Ressonância Magnética , Músculos Oculomotores/anormalidades , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculos Oculomotores/diagnóstico por imagemRESUMO
Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure.
Assuntos
Dioxinas/toxicidade , Poluentes Ambientais/toxicidade , Pálpebras/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase Quinase 1/genética , Receptores de Hidrocarboneto Arílico/genética , Animais , Embrião de Mamíferos , Epitélio/anormalidades , Epitélio/efeitos dos fármacos , Epitélio/embriologia , Epitélio/metabolismo , Pálpebras/anormalidades , Pálpebras/efeitos dos fármacos , Pálpebras/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Interação Gene-Ambiente , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinase 1/metabolismo , Camundongos , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Fosforilação , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de SinaisRESUMO
Epithelial sheet movement is an essential morphogenetic process during mouse embryonic eyelid closure in which Mitogen-Activated Protein 3 Kinase 1 (MAP3K1) and c-Jun play a critical role. Here we show that MAP3K1 associates with the cytoskeleton, activates Jun N-terminal kinase (JNK) and actin polymerization, and promotes the eyelid inferior epithelial cell elongation and epithelium protrusion. Following epithelium protrusion, c-Jun begins to express and acts to promote ERK phosphorylation and migration of the protruding epithelial cells. Homozygous deletion of either gene causes defective eyelid closure, but non-allelic non-complementation does not occur between Map3k1 and c-Jun and the double heterozygotes have normal eyelid closure. Results from this study suggest that MAP3K1 and c-Jun signal through distinct temporal-spatial pathways and that productive epithelium movement for eyelid closure requires the consecutive action of MAP3K1-dependent cytoskeleton reorganization followed by c-Jun-mediated migration.
Assuntos
Epitélio/embriologia , Pálpebras/embriologia , MAP Quinase Quinase Quinase 1/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Western Blotting , Linhagem Celular , Movimento Celular/genética , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células HEK293 , Células HeLa , Humanos , MAP Quinase Quinase Quinase 1/genética , Células MCF-7 , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfogênese/genética , Fosforilação , Proteínas Proto-Oncogênicas c-jun/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Precise coordination of progenitor cell proliferation and differentiation is essential for proper organ morphogenesis and function during mammalian development. The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) has a well-established role in anterior eyelid development, as Map3k1-knockout mice have defective embryonic eyelid closure and an `eye-open at birth' (EOB) phenotype. Here, we show that MAP3K1 is highly expressed in the posterior of the developing eye and is required for retina development. The MAP3K1-deficient mice exhibit increased proliferation and apoptosis, and Müller glial cell overproduction in the developing retinas. Consequently, the retinas of these mice show localized rosette-like arrangements in the outer nuclear layer, and develop abnormal vascularization, broken down retinal pigment epithelium, photoreceptor loss and early onset of retinal degeneration. Although the retinal defect is associated with increased cyclin D1 and CDK4/6 expression, and RB phosphorylation and E2F-target gene upregulation, it is independent of the EOB phenotype and of JNK. The retinal developmental defect still occurs in knockout mice that have undergone tarsorrhaphy, but is absent in compound mutant Map3k1(+/ΔKD)Jnk1(-/-) and Map3k1(+/ΔKD)Jnk(+/-)Jnk2(+/-) mice that have EOB and reduced JNK signaling. Our results unveil a novel role for MAP3K1 in which it crosstalks with the cell cycle regulatory pathways in the prevention of retina malformation and degeneration.
Assuntos
Apoptose/genética , Proliferação de Células , MAP Quinase Quinase Quinase 1/genética , Retina/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Embrião de Mamíferos , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Inativação de Genes , MAP Quinase Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinase 1/fisiologia , Camundongos , Camundongos Knockout , Procedimentos Cirúrgicos Oftalmológicos , Retina/embriologia , Retina/metabolismo , Retina/cirurgia , SuturasRESUMO
Developmental eyelid closure is an evolutionarily conserved morphogenetic event requiring proliferation, differentiation, cytoskeleton reorganization, and migration of epithelial cells at the tip of the developing eyelid. Many signaling events take place during eyelid closure, but how the signals converge to regulate the morphogenetic process remains an open and intriguing question. Here we show that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) highly expressed in the developing eyelid epithelium, forms with c-Jun, a regulatory axis that orchestrates morphogenesis by integrating two different networks of eyelid closure signals. A TGF-α/EGFR-RhoA module initiates one of these networks by inducing c-Jun expression which, in a phosphorylation-independent manner, binds to the Map3k1 promoter and causes an increase in MAP3K1 expression. RhoA knockout in the ocular surface epithelium disturbs this network by decreasing MAP3K1 expression, and causes delayed eyelid closure in Map3k1 hemizygotes. The second network is initiated by the enzymatic activity of MAP3K1, which phosphorylates and activates a JNK-c-Jun module, leading to AP-1 transactivation and induction of its downstream genes, such as Pai-1. MAP3K1 inactivation reduces AP-1 activity and PAI-1 expression both in cells and developing eyelids. MAP3K1 is therefore the nexus of an intracrine regulatory loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c-Jun-AP-1 pathways in developmental eyelid closure.
Assuntos
Pálpebras/embriologia , MAP Quinase Quinase Quinase 1/metabolismo , Animais , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , MAP Quinase Quinase Quinase 1/deficiência , MAP Quinase Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Modelos Biológicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Mitogen-activated protein 3 kinase 1 (MAP3K1) has a plethora of cell type-specific functions not yet fully understood. Herein, we describe a role for MAP3K1 in female reproductive tract (FRT) development. MAP3K1 kinase domain-deficient female mice exhibited an imperforate vagina, labor failure and infertility. These defects corresponded with shunted Müllerian ducts (MDs), the embryonic precursors of FRT, that manifested as a contorted caudal vagina and abrogated vaginal-urogenital sinus fusion in neonates. The MAP3K1 kinase domain is required for optimal activation of the Jun-N-terminal kinase (JNK) and cell polarity in the MD epithelium, and for upregulation of WNT signaling in the mesenchyme surrounding the caudal MD. The MAP3K1-deficient epithelial cells and MD epithelium had reduced expression of WNT7B ligands. Correspondingly, conditioned media derived from MAP3K1-competent, but not -deficient, epithelial cells activated a TCF/Lef-luciferase reporter in fibroblasts. These observations indicate that MAP3K1 regulates MD caudal elongation and FRT development, in part through the induction of paracrine factors in the epithelium that trans-activate WNT signaling in the mesenchyme.
Assuntos
Células Epiteliais , MAP Quinase Quinase Quinase 1 , Vagina , Animais , Feminino , Camundongos , Células Epiteliais/metabolismo , Epitélio/metabolismo , Vagina/metabolismo , Via de Sinalização Wnt , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismoRESUMO
Mitogen-Activated Protein 3 Kinase 1 (MAP3K1) is a dynamic signaling molecule with a plethora of cell-type specific functions, most of which are yet to be understood. Here we describe a role for MAP3K1 in the development of female reproductive tract (FRT). MAP3K1 kinase domain-deficient ( Map3k1 ΔKD ) females exhibit imperforate vagina, labor failure, and infertility. These defects correspond to a shunted Müllerian duct (MD), the principle precursor of the FRT, in embryos, while they manifest as a contorted caudal vagina with abrogated vaginal-urogenital sinus fusion in neonates. In epithelial cells, MAP3K1 acts through JNK and ERK to activate WNT, yet in vivo MAP3K1 is crucial for WNT activity in mesenchyme associated with the caudal MD. Expression of Wnt7b is high in wild type, but low in Map3k1 knockout MD epithelium and MAP3K1-deficient keratinocytes. Correspondingly, conditioned media derived from MAP3K1-competent epithelial cells activate TCF/Lef-luciferase reporter in fibroblasts, suggesting that MAP3K1-induced factors released from epithelial cells trans-activate WNT signaling in fibroblasts. Our results reveal a temporal-spatial and paracrine MAP3K1-WNT crosstalk contributing to MD caudal elongation and FRT development. Highlights: MAP3K1 deficient female mice exhibit imperforate vagina and infertilityLoss of MAP3K1 kinase activity impedes Müllerian duct (MD) caudal elongation and fusion with urogenital sinus (UGS) in embryogenesisThe MAP3K1-MAPK pathway up-regulates WNT signaling in epithelial cellsMAP3K1 deficiency down-regulates Wnt7b expression in the MD epithelium and prevents WNT activity in mesenchyme of the caudal MD.
RESUMO
Epithelial development starts with stem cell commitment to ectoderm followed by differentiation to the basal keratinocytes. The basal keratinocytes, first committed in embryogenesis, constitute the basal layer of the epidermis. They have robust proliferation and differentiation potential and are responsible for epidermal expansion, maintenance and regeneration. We generated basal epithelial cells in vitro through differentiation of mouse embryonic stem cells (mESCs). Early on in differentiation, the expression of stem cell markers, Oct4 and Nanog, decreased sharply along with increased ectoderm marker keratin (Krt) 18. Later on, Krt 18 expression was subdued when cells displayed basal keratinocyte characteristics, including regular polygonal shape, adherent and tight junctions and Krt 14 expression. These cells additionally expressed abundant Sca-1, Krt15 and p63, suggesting epidermal progenitor characteristics. Using Map3k1 mutant mESCs and environmental dioxin, we examined the gene and environment effects on differentiation. Neither Map3k1 mutation nor dioxin altered mESC differentiation to ectoderm and basal keratinocytes, but they, individually and in combination, potentiated Krt 1 expression and basal to spinous differentiation. Similar gene-environment effects were observed in vivo where dioxin exposure increased Krt 1 more substantially in the epithelium of Map3k1+/- than wild type embryos. Thus, the in vitro model of epithelial differentiation can be used to investigate the effects of genetic and environmental factors on epidermal development.
Assuntos
Dioxinas , Queratinócitos , MAP Quinase Quinase Quinase 1 , Células-Tronco Embrionárias Murinas , Animais , Diferenciação Celular , Dioxinas/farmacologia , Células Epidérmicas , Epiderme/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , MAP Quinase Quinase Quinase 1/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , MutaçãoRESUMO
Epidermis is the most outer layer of the skin and a physical barrier protecting the internal tissues from mechanical and environmental insults. The basal keratinocytes, which, through proliferation and differentiation, supply diverse cell types for epidermal homeostasis and injury repair. Sustainable culture of murine keratinocyte, however, is a major obstacle. Here we developed murine keratinocyte lines using low-Ca2+ (0.06 mM) keratinocyte serum-free medium (KSFM-Ca2+) without feeder cells. Cells derived in this condition could be subcultured for >70 passages. They displayed basal epithelial cell morphology and expressed keratin (Krt) 14, but lacked the epithelial-characteristic intercellular junctions. Moreover, these cells could be adapted to grow in the Defined-KSFM (DKSFM) media containing 0.15 mM Ca2+, and the adapted cells established tight- and adherens-junctions and exhibited increased Krt1/10 expression while retained subculture capacity. Global gene expression studies showed cells derived in KSFM-Ca2+ media had enriched stem/proliferation markers and cells adapted in DKSFM media had epithelial progenitor signatures. Correspondingly, KSFM-Ca2+-derived cells exhibited a remarkable capacity of clonal expansion, whereas DKSFM-adapted cells could differentiate to suprabasal epithelial cell types in 3-dimentional (3D) organoids. The generation of stem-like murine keratinocyte lines and the conversion of these cells to epithelial progenitors capable of terminal differentiation provide the critically needed resources for skin research.
Assuntos
Queratinócitos/citologia , Células-Tronco Embrionárias Murinas/citologia , Cultura Primária de Células/métodos , Animais , Células Cultivadas , Meios de Cultura/química , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The MAP3K1 is responsible for transmitting signals to activate specific MAP2K-MAPK cascades. Following the initial biochemical characterization, genetic mouse models have taken center stage to elucidate how MAP3K1 regulates biological functions. To that end, mice were generated with the ablation of the entire Map3k1 gene, the kinase domain coding sequences, or ubiquitin ligase domain mutations. Analyses of the mutants identify diverse roles that MAP3K1 plays in embryonic survival, maturation of T/B cells, and development of sensory organs, including eye and ear. Specifically in eye development, Map3k1 loss-of-function was found to be autosomal recessive for congenital eye abnormalities, but became autosomal dominant in combination with Jnk and RhoA mutations. Additionally, Map3k1 mutation increased eye defects with an exposure to environmental agents such as dioxin. Data from eye developmental models reveal the nexus role of MAP3K1 in integrating genetic and environmental signals to control developmental activities. Here, we focus the discussions on recent advances in understanding the signaling mechanisms of MAP3K1 in eye development in mice and in sex differentiation from human genomics findings. The research works featured here lead to a deeper understanding of the in vivo signaling network, the mechanisms of gene-environment interactions, and the relevance of this multifaceted protein kinase in disease etiology and pathogenesis.
Assuntos
MAP Quinase Quinase Quinase 1/genética , Diferenciação Sexual/genética , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Interação Gene-Ambiente , Humanos , MAP Quinase Quinase Quinase 1/metabolismo , CamundongosRESUMO
The transparent ocular lens plays a crucial role in vision by focusing light on to the retina with loss of lens transparency leading to impairment of vision. While maintenance of epithelial phenotype is recognized to be essential for lens development and function, knowledge of the identity of different molecular mechanisms regulating lens epithelial characteristics remains incomplete. This study reports that CNN-3, the acidic isoform of calponin, an actin binding contractile protein, is expressed preferentially and abundantly relative to the basic and neutral isoforms of calponin in the ocular lens, and distributes predominantly to the epithelium in both mouse and human lenses. Expression and MEKK1-mediated threonine 288 phosphorylation of CNN-3 is induced by extracellular cues including TGF-ß2 and lysophosphatidic acid. Importantly, siRNA-induced deficiency of CNN3 in lens epithelial cell cultures and explants results in actin stress fiber reorganization, stimulation of focal adhesion formation, Yap activation, increases in the levels of α-smooth muscle actin, connective tissue growth factor and fibronectin, and decreases in E-cadherin expression. These results reveal that CNN3 plays a crucial role in regulating lens epithelial contractile activity and provide supporting evidence that CNN-3 deficiency is associated with the induction of epithelial plasticity, fibrogenic activity and mechanosensitive Yap/Taz transcriptional activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/metabolismo , Cristalino/metabolismo , Mecanotransdução Celular , Proteínas dos Microfilamentos/deficiência , Transativadores/metabolismo , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Células Epiteliais/patologia , Feminino , Fibrose , Cristalino/patologia , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Transativadores/genética , Proteínas de Sinalização YAPRESUMO
The mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1) mediates activin B signals required for eyelid epithelium morphogenesis during mouse fetal development. The present study investigates the role of MEKK1 in epithelial wound healing, another activin-regulated biological process. In a skin wound model, injury markedly stimulates MEKK1 expression and activity, which are in turn required for the expression of genes involved in extracellular matrix (ECM) homeostasis. MEKK1 ablation or down-regulation by interfering RNA significantly delays skin wound closure and impairs activation of Jun NH2-terminal kinases, induction of plasminogen activator inhibitor (PAI)-1, and restoration of cell-cell junctions of the wounded epidermis. Conversely, expression of wild-type MEKK1 accelerates reepithelialization of full-thickness skin and corneal debridement wounds by mechanisms involving epithelial cell migration, a cell function that is partially abolished by neutralizing antibodies for PAI-1 and metalloproteinase III. Our data suggest that MEKK1 transmits wound signals, leading to the transcriptional activation of genes involved in ECM homeostasis, epithelial cell migration, and wound reepithelialization.
Assuntos
Epitélio/enzimologia , Epitélio/fisiologia , MAP Quinase Quinase Quinase 1/metabolismo , Cicatrização/imunologia , Ativinas/metabolismo , Animais , Animais Recém-Nascidos , Movimento Celular , Córnea/citologia , Ativação Enzimática , Células Epidérmicas , Epiderme/patologia , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/citologia , Pele/patologiaRESUMO
Activins and other members of the transforming growth factor beta family play a critical role in morphological changes of the epidermis that require epithelial cell movement. We investigated the molecular pathways in the transmission of activin signals that lead to actin reorganization and epithelial cell migration. We found that activins cause the activation of RhoA but not of Rac and CDC42, leading to MEKK1-dependent phosphorylation of JNK and transcription factor c-Jun. Through a RhoA-independent mechanism, the activins also induce p38 activity in keratinocytes from wild-type but not from MEKK1-deficient mice. Although neither pathway is dependent on Smad activation, the MEKK1-mediated JNK and p38 activities are both essential for activin-stimulated and transcription-dependent keratinocyte migration. Only JNK is involved in transcription-independent actin stress fiber formation, which needs also the activity of ROCK. Because ROCK is required for JNK activation by RhoA and its overexpression leads to MEKK1 activation, we propose a RhoA-ROCK-MEKK1-JNK pathway and a MEKK1-p38 pathway as Smad-independent mechanisms in the transmission of activin signals. Together, these pathways lead to the control of actin cytoskeleton reorganization and epithelial cell migration, contributing to the physiologic and pathological effects of activins on epithelial morphogenesis.
Assuntos
Actinas/metabolismo , Ativinas/metabolismo , Queratinócitos/metabolismo , MAP Quinase Quinase Quinase 1/fisiologia , Transdução de Sinais , Animais , Western Blotting , Movimento Celular , Citoesqueleto/metabolismo , Ativação Enzimática , Células Epiteliais/citologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4 , MAP Quinase Quinase Quinase 1/metabolismo , Camundongos , Microscopia de Fluorescência , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Mutação , Fosforilação , Retroviridae/genética , Fatores de Tempo , Cicatrização , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND AND PURPOSE: Meibomian glands (MGs) play an important role in the maintenance of ocular surface health, but the mechanisms of their development are still poorly understood. The MGs arise from the epithelium at the junction of eyelid fusion, raising the possibility that defective eyelid fusion disturbs the formation of MGs. METHODS: We examined, histologically and functionally, the development of MGs in mice with either normal or defective eyelid fusion, displaying eye-closed at birth (ECB) or eye-open at birth (EOB) phenotypes, respectively. RESULTS: The Meibomian anlage was detected in the epithelium at the eyelid fusion junction immediately after birth at postnatal day 0 (PD0), and it extended into the eyelid stroma at PD1 and started to branch and produce meibum at PD7 in the ECB mice. In contrast, few if any MG structures were detectable in the EOB mice in the early postnatal periods. The Meibomian gland ductile system was seen aligned along the eyelid margin in the adult ECB mice, but was absent or scarce in that of the EOB mice. While MG abnormalities were found in all EOB mice, the severity varied and corresponded to the position and the size of eye opening but not the genetic defects of the mice. CONCLUSION: Proper Meibomian gland formation and development require eyelid closure and fusion.
Assuntos
Glândulas Tarsais , Animais , Doenças Palpebrais , Camundongos , MorfogêneseRESUMO
IkB kinase ß (IKKß) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKß in the cornea using Ikkß(ΔCS) mice in which the Ikkß gene was specifically deleted in the corneal stromal keratocytes. The Ikkß(ΔCS) corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the Ikkß(F/F) corneas that restored transparency in 2 weeks after injury, over 50% of the Ikkß(ΔCS) corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence ß-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the Ikkß(ΔCS) corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKß in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing.
Assuntos
Lesões da Córnea/fisiopatologia , Ceratócitos da Córnea/fisiologia , Quinase I-kappa B/fisiologia , Transdução de Sinais/fisiologia , Cicatrização/fisiologia , Animais , Apoptose/fisiologia , Córnea/metabolismo , Córnea/fisiopatologia , Lesões da Córnea/complicações , Ceratócitos da Córnea/metabolismo , Opacidade da Córnea/etiologia , Opacidade da Córnea/fisiopatologia , Camundongos , Camundongos Knockout , Miofibroblastos/fisiologia , Estresse Oxidativo/fisiologiaRESUMO
Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase ß (IKKß) and the transforming growth factor ß (TGFß) signaling pathways. We show that in vitro ablation of Ikkß in fibroblasts led to progressive ROS accumulation and TGFß activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKß activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKß-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfß2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFß loop IKKß plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Assuntos
Comunicação Autócrina/fisiologia , Senescência Celular , Quinase I-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular , Movimento Celular , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Miofibroblastos/citologia , Miofibroblastos/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/genética , Regulação para CimaRESUMO
PURPOSE: Mammalian eye development requires temporary fusion of the upper and lower eyelids in embryogenesis. Failure of lid closure in mice leads to an eye open at birth (EOB) phenotype. Many genetic mutant strains develop this phenotype and studies of the mutants lead to a better understanding of the signaling mechanisms of morphogenesis. The present study investigates the roles of lid closure in eye development. METHODS: Seven mutant mouse strains were generated by different gene ablation strategies that inactivated distinct signaling pathways. These mice, including systemic ablation of Map3k1 and Dkk2, ocular surface epithelium (OSE) knockout of c-Jun and Egfr, conditional knockout of Shp2 in stratified epithelium (SE), as well as the Map3k1/Jnk1 and Map3k1/Rhoa compound mutants, all exhibited defective eyelid closure. The embryonic and postnatal eyes in these mice were characterized by histology and immunohistochemistry. RESULTS: Some eye abnormalities, such as smaller lens in the Map3k1-null mice and Harderian gland hypoplasia in the Dkk2-null mice, appeared to be mutant strain-specific, whereas other abnormalities were seen in all mutants examined. The common defects included corneal erosion/ulceration, meibomian gland hypoplasia, truncation of the eyelid tarsal muscles, failure of levator palpebrae superioris (LPS) extension into the upper eyelid and misplacement of the inferior oblique (IO) muscle and inferior rectus (IR) muscle. The muscle defects were traced to the prenatal fetuses. CONCLUSIONS: In addition to providing a protective barrier for the ocular surface, eyelid closure in embryogenesis is required for the development of ocular adnexa, including eyelid and extraocular muscles.
Assuntos
DNA/genética , Desenvolvimento Embrionário/genética , Proteínas do Olho/genética , Pálpebras/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Glândulas Tarsais/embriologia , Prenhez , Animais , Movimento Celular , Proliferação de Células , Proteínas do Olho/biossíntese , Feminino , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Fenótipo , Gravidez , Transdução de SinaisRESUMO
Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury.