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1.
J Neurochem ; 139(2): 197-207, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27501468

RESUMO

MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The norepinephrine transporter (NET) and glucocorticoid receptors are closely related to the homeostatic integration and regulation after stress. Our previous studies demonstrated that NET mRNA and protein levels in rats are regulated by chronic stress and by administration of corticosterone, which is mediated through glucocorticoid receptors. Whether miRNAs are intermediaries in the regulation of these proteins remains to be elucidated. This study was undertaken to determine possible regulatory effects of miRNAs on the expression of NET and glucocorticoid receptors in the noradrenergic neuronal cell line. Using computational target prediction, we identified several candidate miRNAs potentially targeting NET and glucocorticoid receptors. Western blot results showed that over-expression of miR-181a and miR-29b significantly repressed protein levels of NET, which is accompanied by a reduced [3 H] norepinephrine uptake, and glucocorticoid receptors in PC12 cells. Luciferase reporter assays verified that both miR-181a and miR-29b bind the 3'UTR of mRNA of NET and glucocorticoid receptors. Furthermore, exposure of PC12 cells to corticosterone markedly reduced the endogenous levels of miR-29b, which was not reversed by the application of glucocorticoid receptor antagonist mifepristone. These observations indicate that miR-181a and miR-29b can function as the negative regulators of NET and glucocorticoid receptor translation in vitro. This regulatory effect may be related to stress-induced up-regulation of the noradrenergic phenotype, a phenomenon observed in stress models and depressive patients. This study demonstrated that miR-29b and miR-181a, two short non-coding RNAs that provide global regulation of gene expression, markedly repressed protein levels of norepinephrine (NE) transporter and glucocorticoid receptor (GR), as well as NE uptake by binding the 3'UTR of their mRNAs in PC12 cells. Also, exposure of cells to corticosterone significantly reduced miR-29b levels through a GR-independent way.


Assuntos
MicroRNAs/genética , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/biossíntese , Receptores de Glucocorticoides/biossíntese , Regiões 3' não Traduzidas , Animais , Simulação por Computador , Corticosterona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , MicroRNAs/biossíntese , Mifepristona/farmacologia , Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Células PC12 , Ratos , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/genética
2.
J Neurochem ; 135(1): 38-49, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26212818

RESUMO

Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [(3) H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status.


Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Estresse Fisiológico/fisiologia , Ativação Transcricional/fisiologia , Ansiedade/metabolismo , Linhagem Celular , Humanos , Norepinefrina/metabolismo , RNA Mensageiro/metabolismo , Regulação para Cima/fisiologia
3.
Am J Physiol Renal Physiol ; 305(10): F1455-65, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23986516

RESUMO

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Contração Muscular , Força Muscular , Músculo Liso/metabolismo , Mutação , Bexiga Urinária/metabolismo , Animais , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Galinhas , Estimulação Elétrica , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Muscular/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Miosinas/metabolismo , Fenótipo , Cloreto de Potássio/farmacologia , Estrutura Terciária de Proteína , Fatores de Tempo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/inervação , Urodinâmica
4.
Biocell ; 35(3): 71-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22423483

RESUMO

Apigenin, a nonmutagenic flavonoid, has been shown to possess free radical scavenging activities, anticarcinogenic properties, antioxidant and anti-inflammatory effects. Recently, apigenin was reported to cause gastric relaxation in murine. To assess possible effects of apigenin on migration of bladder smooth muscle (SM) cell, we isolated SM cells from peri-cancer tissue of human bladder and established a cell model that was capable to overexpress transiently MEKK1 (MEK kinase 1). Results showed that overexpression of active human MEKK1 by adenoviruses infection induced migration of human bladder smooth muscle (hBSM) cells and phosphorylation of MAPKs, ERK, JNK and p38, which are the downstream molecules of MEKK1. Then, hBSM cell overexpressing MEKK1 were exposed to apigenin (50 microM). Our data indicated that apigenin inhibited significantly activation/phosphorylation of MAPKs and migration of hBSM cells induced by MEKK1 overexpression. Besides, apigenin inhibited actin polymerization, which underlines muscle contraction and cell migration. The results suggest that apigenin inhibits activation of MAPKs and thereby the cell migration. The mechanism might be that apigenin blocks signal transmission from MEKK1 to MAPKs.


Assuntos
Apigenina/farmacologia , Movimento Celular/efeitos dos fármacos , MAP Quinase Quinase Quinase 1/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Humanos , Immunoblotting , MAP Quinase Quinase Quinase 1/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/citologia
5.
Cytoskeleton (Hoboken) ; 68(3): 139-49, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20722044

RESUMO

Myosins are a superfamily of actin-based molecular motor proteins, which hydrolyze ATP and generate various forms of eukaryotic motility and muscle contraction. Myosin light chain 20 (MLC20) is small ring around the neck region of heavy chain of myosins. Phosphorylation of MLC20 is thought to play a key role in regulation of smooth muscle contraction. Calcium- and calmodulin-dependent myosin light chain kinase (MLCK) is considered the primary regulator of MLC20 phosphorylation. However, several observations in smooth muscle contraction cannot be explained by the mode of phosphorylation. By performing a series of experiments in vitro and in vivo, we report here MLCK-independent MLC20 phosphorylation. Gene expression study reveals that expression of MLCK in smooth muscles is inconsistent with MLC20 phosphorylation at Ser19. None of inactivating calmodulin/MLCK, depriving of calcium and silencing MLCK expression by siRNA blocks effectively the phosphorylation of MLC20 at Ser19. In addition, by overexpressing active human MAP (mitogen-activated protein)-ERK kinase kinase-1 (MEKK1) and blocking its downstream messengers, we have demonstrated a new regulatory system of MLC phosphorylation via MEKK1, which downregulates Ser19 phosphorylation of MLC20 through its downstream molecules, p38, JNK, and ERK in human bladder smooth muscle cells.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Bexiga Urinária/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Movimento Celular , Células Cultivadas , Humanos , Imunoprecipitação , Camundongos , Contração Muscular , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/genética , Fosforilação , RNA Interferente Pequeno/genética , Bexiga Urinária/citologia
6.
J Urol ; 182(5): 2497-503, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19765744

RESUMO

PURPOSE: Partial bladder outlet obstruction in male rabbits causes detrusor smooth muscle hypertrophy and voiding dysfunction similar to that observed in men with benign prostate hyperplasia. Using this model, we analyzed the protein expression and ultrastructure of caveolae and the intermediate size filament in detrusor smooth muscle following partial bladder outlet obstruction induced hypertrophy. MATERIALS AND METHODS: Detrusor smooth muscle sections from bladder body were processed for immunofluorescence and electron microscopy. Western analysis was performed to determine the expression of caveolin isoform-1, 2 and 3, and intermediate size filament proteins. RESULTS: Detrusor smooth muscle cells from both normal and hypertrophied bladders contain orderly arrays of thick and thin myofilaments, interspersed with dense bodies. In addition, there was an increase in intermediate size filaments in the hypertrophic detrusor smooth muscle cells. The dense plaques in the inner membrane of hypertrophied detrusor smooth muscle were longer than those of the control. Detrusor smooth muscle from hypertrophied bladder revealed a decreased number of caveolae and a lack of their orderly distribution at the plasma membrane. Western blotting showed decreased expression of caveolin-1, 2 and 3 in hypertrophied detrusor smooth muscle. CONCLUSIONS: Caveolae serve as platforms for proteins and receptors that have a role in signal transduction. The decreased number of caveolae and caveolin protein expression in hypertrophied detrusor smooth muscle might contribute to alterations in signal transduction pathways that regulate the downstream effects of agonist induced contraction, including calcium sensitization, observed in obstructed bladder. In addition, the increased number of intermediate size filaments in the hypertrophied detrusor smooth muscle is likely to alter the cytoskeletal structure and affect the cellular transmission of passive and/or active force.


Assuntos
Caveolinas/biossíntese , Músculo Liso/metabolismo , Músculo Liso/patologia , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Animais , Cavéolas , Hipertrofia , Técnicas In Vitro , Masculino , Microscopia Eletrônica , Músculo Liso/ultraestrutura , Coelhos , Bexiga Urinária/ultraestrutura
7.
Cell Motil Cytoskeleton ; 64(12): 951-65, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17868135

RESUMO

Caldesmon (CaD), a component of microfilaments in all cells and thin filaments in smooth muscle cells, is known to bind to actin, tropomyosin, calmodulin, and myosin and to inhibit actin-activated ATP hydrolysis by smooth muscle myosin. Thus, it is believed to regulate smooth muscle contraction, cell motility and the cytoskeletal structure. Using bladder smooth muscle cell cultures and RNA interference (RNAi) technique, we show that the organization of actin into microfilaments in the cytoskeleton is diminished by siRNA-mediated CaD silencing. CaD silencing significantly decreased the amount of polymerized actin (F-actin), but the expression of actin was not altered. Additionally, we find that CaD is associated with 10 nm intermediate-sized filaments (IF) and in vitro binding assay reveals that it binds to vimentin and desmin proteins. Assembly of vimentin and desmin into IF is also affected by CaD silencing, although their expression is not significantly altered when CaD is silenced. Electronmicroscopic analyses of the siRNA-treated cells showed the presence of myosin filaments and a few surrounding actin filaments, but the distribution of microfilament bundles was sparse. Interestingly, the decrease in CaD expression had no effect on tubulin expression and distribution of microtubules in these cells. These results demonstrate that CaD is necessary for the maintenance of actin microfilaments and intermediate-sized filaments in the cytoskeletal structure. This finding raises the possibility that the cytoskeletal structure in smooth muscle is affected when CaD expression is altered, as in smooth muscle de-differentiation and hypertrophy seen in certain pathological conditions.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Proteínas de Ligação a Calmodulina/fisiologia , Filamentos Intermediários/ultraestrutura , Músculo Liso/ultraestrutura , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/análise , Actinas/metabolismo , Animais , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular , Filamentos Intermediários/química , Filamentos Intermediários/metabolismo , Músculo Liso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Coelhos , Bexiga Urinária/citologia
8.
Mol Biol Cell ; 17(8): 3446-55, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16760432

RESUMO

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/patologia
9.
Wei Sheng Yan Jiu ; 34(6): 701-4, 2005 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-16535840

RESUMO

OBJECTIVE: To investigate the effects of exogenous nucleotides on apoptosis of a normal rat small intestinal epithelial cell line, IEC-6. METHODS: Cultured IEC-6 cells were treated by four kinds of monophosphate nucleotides and their mixture prepared according to their composition in human milk, then the cell apoptosis was determined by flow cytometry measurement, morphologic characterization, and electron-microscope observation. RESULTS: IEC-6 cells treated with AMP or GMP showed a apotosis peak in flow cytometry measurement, but only AMP produce typical apoptosis characteristics in electron-microscope observation. Pyrimidine nucleotides (UMP and CMP)and nucleotides mixture could not induce apoptosis. However, UMP could significantly eliminate the apoptosis-inducing effects of AMP or GMP. CONCLUSION: Purine nucleotides induce apoptosis of IEC-6, inducing effects of purine nucleotides. pyrimidine nucleotides UMP could abolish the apoptosis-inducing effects of purine nucleotides.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/citologia , Intestino Delgado/citologia , Nucleotídeos de Purina/farmacologia , Nucleotídeos de Pirimidina/farmacologia , Animais , Linhagem Celular , Ratos
10.
Mol Cell Biol ; 25(1): 60-5, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15601830

RESUMO

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/metabolismo
11.
Zhonghua Yu Fang Yi Xue Za Zhi ; 38(6): 383-7, 2004 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-15569509

RESUMO

OBJECTIVE: To explore the effects of Bisphenol A in adult rats and its possible mechanisms. METHODS: BPA (in corn oil) was administered orally to 9-week-old male Sprague-Dawley rats for 14 days (0, 1 and 5 g/kg bw), and incubated primary Sertoli cells from pubertal SD rats with 0, 10(-7), 10(-6), 10(-5), 10(-4) mol/L BPA. RESULTS: After oral administration, a significant decrease in right testis weight was observed in 5 g/kg dose group, but not in the 1 g/kg bw dose group. Germ cells were detached from basement membrane of seminiferous tubules and Sertoli cells in BPA-treated groups. Administration of BPA at 1 g/kg bw and 5 g/kg bw produced both nucleus pycnosis and vacuolized nucleus in germ cells and Sertoli cells. A marked loss in vimentin staining in Sertoli cells from testis of BPA-treated rats was detected. No change in levels of serum estradiol and testosterone was observed after two-week exposure to BPA. In Sertoli cell primary culture, BPA destroyed the cytoskeleton and cell-cell junctions, and elongated Sertoli cells. CONCLUSION: These results suggest that BPA may injure reproductive function of male rats by destroying the cytoskeleton and changing the form of Sertoli cells.


Assuntos
Fenóis/toxicidade , Células de Sertoli/citologia , Vimentina/metabolismo , Animais , Compostos Benzidrílicos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Células de Sertoli/efeitos dos fármacos , Testículo/anatomia & histologia , Testículo/citologia , Testículo/efeitos dos fármacos
12.
Mol Vis ; 9: 584-93, 2003 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-14627958

RESUMO

PURPOSE: To study the in vivo role of MEK kinase 1 (MEKK1) in corneal development. METHODS: Wild type and Mekk1DeltaKD/DeltaKD mice eye tissues were examined by staining with hematoxylin and eosin for morphogenesis and Masson's trichrome for extracellular matrix (ECM) deposition. The cells expressing ECM gene transcripts of Collagen I, Keratocan, and Lumican in corneal stroma were identified by in situ hybridization and the level of Collagen I mRNA in the developing cornea was quantified by real-time RT-PCR. Immunohistochemistry staining was employed to study the expression and N-terminal phosphorylation of c-Jun and the expression of epithelium differentiation markers and intercellular structural proteins of the corneal epithelium. RESULTS: Mekk1DeltaKD/DeltaKD mice exhibited the "eye open at birth" phenotype (EOB), and developed eye defects and severe pathology secondary to impaired eyelid formation. The corneal stroma of Mekk1DeltaKD/DeltaKD fetuses, although exhibiting normal morphology, thickness, and keratocyte proliferation, showed reduced extracellular matrix (ECM) accumulation, corresponding to a decrease in transcription of Lumican, Keratocan, and Collagen I. Immunohistochemistry studies demonstrated that MEKK1 ablation caused a remarkable reduction in the expression of occludin and zonula occluden protein-1 (ZO-1), components of tight junction, but had no effect on the expression of E-cadherin and beta-catenin for adherens junctions, desmoplakin and desmoglein for desmosomes and cytokeratins 12 and 14 for cornea-type epithelial differentiation in the developing cornea. c-Jun was abundantly expressed in the developing corneal epithelium and its phosphorylation was considerably reduced in Mekk1DeltaKD/DeltaKD fetuses. CONCLUSIONS: In addition to its role in eyelid morphogenesis, MEKK1 is crucial for corneal development such that its ablation caused a reduction of ECM deposition in corneal stroma and disturbance of tight junctions in corneal epithelium. c-Jun phosphorylation in corneal epithelium is a downstream event of the MEKK1 pathway, likely contributing to corneal development and function. Altogether, MEKK1 plays a major role in ocular surface morphogenesis and its ablation leads to damage and various eye manifestations at postnatal stages.


Assuntos
Córnea/embriologia , MAP Quinase Quinase Quinase 1 , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Apoptose , Diferenciação Celular , Divisão Celular , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Córnea/crescimento & desenvolvimento , Doenças da Córnea/genética , Doenças da Córnea/patologia , Substância Própria/metabolismo , Substância Própria/patologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Sulfato de Queratano/genética , Sulfato de Queratano/metabolismo , Lumicana , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Morfogênese , Ocludina , Fosfoproteínas/metabolismo , Fosforilação , Proteoglicanas/genética , Proteoglicanas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1
13.
Wei Sheng Yan Jiu ; 31(3): 168-71, 2002 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-12545752

RESUMO

The effect of exogenous nucleotides on proliferation and migration of a normal rat small intestinal epithelial cell line IEC-6 is studied. The concentration-effects as well as interaction of exogenous nucleotides on proliferation of IEC-6 are measured by MTT. Migration of IEC-6 after wounded is determined by an in vitro model of intestinal epithelial restitution of IEC-6 monolayer. Expression of TGF beta is detected by immunohistochemistry. The results show AMP and GMP remarkably inhibit proliferation of IEC-6 in concentration-dependent manner respectively at 30 mumol/L or 150 mumol/L and more. CMP, UMP and nucleotides mixture can not enhance or inhibit the growth with the exception of inhibition of CMP on proliferation at very high concentration (1440 mumol/L). In contrast, CMP, especially UMP, can remarkably abolish the proliferation-inhibiting effects of AMP or GMP on the cell, when AMP or GMP is supplemented. Nucleotides mixture significantly facilitate migration of IEC-6 after wounded but fail to promote the expression of TGF beta. It is concluded that purine nucleotides inhibits proliferation of IEC-6. Pyrimidine nucleotides can abolish the inhibitive effects of purine nucleotides, and Nucleotides mixture promotes migration of IEC-6 after wounded by a TGF beta-independented way.


Assuntos
Células Epiteliais/citologia , Intestino Delgado/citologia , Nucleotídeos/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Ratos
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