RESUMO
Polycystic kidney diseases are characterized by numerous bilateral renal cysts that continuously enlarge and, through compression of intact nephrons, lead to a decline in kidney function over time. We previously showed that cyst enlargement is accompanied by regional hypoxia, which results in the stabilization of hypoxia-inducible transcription factor-1α (HIF-1α) in the cyst epithelium. Here we demonstrate a correlation between cyst size and the expression of the HIF-1α-target gene, glucose transporter 1, and report that HIF-1α promotes renal cyst growth in two in vitro cyst models-principal-like MDCK cells (plMDCKs) within a collagen matrix and cultured embryonic mouse kidneys stimulated with forskolin. In both models, augmenting HIF-1α levels with the prolyl hydroxylase inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate enhanced cyst growth. In addition, inhibition of HIF-1α degradation through tubule-specific knockdown of the von Hippel-Lindau tumor suppressor increased cyst size in the embryonic kidney cyst model. In contrast, inhibition of HIF-1α by chetomin and knockdown of HIF-1α both decreased cyst growth in these models. Consistent with previous reports, plMDCK cyst enlargement was driven largely by transepithelial chloride secretion, which consists, in part, of a calcium-activated chloride conductance. plMDCKs deficient for HIF-1α almost completely lacked calcium-activated chloride secretion. We conclude that regional hypoxia in renal cysts contributes to cyst growth, primarily due to HIF-1α-dependent calcium-activated chloride secretion. These findings identify the HIF system as a novel target for inhibition of cyst growth.
Assuntos
Cloretos/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Doenças Renais Policísticas/etiologia , Animais , Canais de Cloreto/metabolismo , Cães , Feminino , Regulação da Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Hipóxia/fisiopatologia , Células Madin Darby de Rim Canino , Masculino , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas/metabolismoRESUMO
The inhibitor of apoptosis protein survivin is a bifunctional molecule that regulates cellular division and survival. We have previously shown that survivin protein can be found at high concentrations in the adult kidney, particularly in the proximal tubules. Here, survivin is localized primarily at the apical membrane, a pattern that may indicate absorption of the protein. Several proteins in primary urine are internalized by megalin, an endocytosis receptor, which is in principle found in the same localization as survivin. Immunolabeling for survivin in different species confirmed survivin signal localizing to the apical membrane of the proximal tubule. Immunoelectron microscopy also showed apical localization of survivin in human kidneys. Furthermore, in polarized human primary tubular cells endogenous as well as external recombinant survivin is stored in the apical region of the cells. Costaining of survivin and megalin by immunohistochemistry and immunoelectron microscopy confirmed colocalization. Finally, by surface plasmon resonance we were able to demonstrate that survivin binds megalin and cubilin and that megalin knockout mice lose survivin through the urine. Survivin accumulates at the apical membrane of the renal tubule by reuptake, which is achieved by the endocytic receptor megalin, collaborating with cubilin. For this to occur, survivin will have to circulate in the blood and be filtered into the primary urine. It is not known at this stage what the functional role of tubular survivin is. However, a small number of experimental and clinical reports implicate that renal survivin is important for functional integrity of the kidney.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Túbulos Renais Proximais/fisiologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Proteínas Repressoras/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Knockout , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Receptores de Superfície Celular/metabolismo , SurvivinaRESUMO
Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.
Assuntos
Hipóxia Celular/fisiologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Animais , Western Blotting , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Células Endoteliais/efeitos dos fármacos , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Camundongos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure. Aberrant epithelial cell proliferation is a major cause of progressive cyst enlargement in ADPKD. Since activation of the Ras/Raf signaling system has been detected in cyst-lining epithelia, inhibition of Raf kinase has been proposed as an approach to retard the progression of ADPKD. Methods and results. PLX5568, a novel selective small molecule inhibitor of Raf kinases, attenuated proliferation of human ADPKD cyst epithelial cells. It reduced in vitro cyst growth of Madin-Darby Canine Kidney cells and of human ADPKD cells within a collagen gel. In male cy/+ rats with polycystic kidneys, PLX5568 inhibited renal cyst growth along with a significant reduction in the number of proliferating cell nuclear antigen- and phosphorylated extracellular signal-regulated kinase-positive cyst-lining epithelial cells. Furthermore, treated animals showed increased capacity to concentrate urine. However, PLX5568 did not lead to a consistent improvement of renal function. Moreover, although relative cyst volume was decreased, total kidney-to-body weight ratio was not significantly reduced by PLX5568. Further analyses revealed a 2-fold increase of renal and hepatic fibrosis in animals treated with PLX5568. CONCLUSIONS: PLX5568 attenuated cyst enlargement in vitro and in a rat model of ADPKD without improving kidney function, presumably due to increased renal fibrosis. These data suggest that effective therapies for the treatment of ADPKD will need to target fibrosis as well as the growth of cysts.
Assuntos
Proliferação de Células/efeitos dos fármacos , Cistos/patologia , Rim/fisiopatologia , Cirrose Hepática/induzido quimicamente , Doenças Renais Policísticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Células Cultivadas , Cistos/tratamento farmacológico , Cães , Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Rim/efeitos dos fármacos , Masculino , Proteínas Quinases Ativadas por Mitógeno , Fosforilação/efeitos dos fármacos , Doenças Renais Policísticas/enzimologia , Doenças Renais Policísticas/patologia , Inibidores de Proteínas Quinases/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
Proximal tubular epithelial cells differ from other epithelial cells in the expression of N-cadherin as major adherens junction protein instead of E-cadherin. Migration of proximal epithelial cells (HKC-8) was analyzed by scratch wounding and by a barrier assay, which allowed determination of migration velocity on different extracellular matrices. Migration velocity was about threefold higher on fibronectin compared to collagen IV. The differential migration velocity was reflected by the orientation of F-actin stress fibers. TGF-beta activated secretion of fibronectin and thus increased migration on collagen IV, but did not further promote migration on fibronectin. Pharmacological inhibition of Rho kinases (ROCKs) by Y-27632, hydroxyfasudil and H-1152, or siRNA against ROCKs significantly increased migration velocity independently of the extracellular matrix. Cells at the migration front showed long filopodia, which could not be mimicked by overexpression of consitutively active Cdc42, indicative of a more complex regulation of F-actin structures. N-cadherin was reorganized from tight zipper-like structures into loosened cell-cell contacts upon incubation with Y-27632, but HKC-8 cells still migrated as cohort. Migration through single cell pores in a modified Boyden chamber assay was also stimulated by ROCK inhibitors. ROCK inhibitors enhanced migration of primary cultures of renal tubular cells which consisted of proximal and distal tubular cells expressing N-cadherin and E-cadherin, respectively. There was no indication of a switch in cadherin expression in these cells or a preferential migration of N-cadherin expressing cells. Pharmacologic inhibition of ROCKs may thus favor repair processes in renal tubules by increasing the migratory capacity of tubular epithelial cells.
Assuntos
Movimento Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/citologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Actinas/metabolismo , Amidas/farmacologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Células Epiteliais/enzimologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Piridinas/farmacologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Tubular epithelial cells secrete connective tissue growth factor (CTGF, CCN2), which contributes to tubulointerstitial fibrosis. However, the molecular regulation of CTGF in human primary tubular epithelial cells (hPTECs) is not well defined. Therefore, CTGF expression was characterized in hPTECs isolated from healthy parts of tumor nephrectomies, with special emphasis on the regulation by transforming growth factor-beta (TGF-beta) and hypoxia, essential factors in the development of fibrosis. CTGF synthesis was strongly dependent on cell density. High CTGF levels were detected in sparse cells, whereas CTGF expression was reduced in confluent cells. Concomitantly, stimulation of CTGF by TGF-beta or the histone deacetylase inhibitor trichostatin was prevented in dense cells. Exposure of hPTECs to low oxygen tension (1% O2) or the hypoxia mimetic dimethyl-oxalylglycine for 24 h reduced CTGF gene expression in most of the 17 preparations analyzed. Preincubation of the cells under hypoxic conditions significantly reduced TGF-beta-mediated upregulation of CTGF. In line with these data, CTGF mRNA was only induced in interstitial cells, but not in tubular cells in kidneys of mice exposed to hypoxia. Longer exposure to hypoxia or TGF-beta (up to 72 h) did not induce hPTECs to adopt a mesenchymal phenotype characterized by upregulation of alpha-smooth muscle actin, downregulation of E-cadherin, or increased sensitivity of the cells in terms of CTGF expression. Sensitivity was restored by inhibition of DNA methylation. Taken together, our data provide evidence that exposure to hypoxia decreased CTGF gene expression. Furthermore, hypoxia per se was not sufficient to induce a mesenchymal phenotype in primary tubular epithelial cells.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Técnicas de Cultura de Células , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Metilação de DNA , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Túbulos Renais/efeitos dos fármacos , Oxigênio/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBV's oncogenic potential by activating nuclear factor-kappaB (NF-kappaB). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-kappaB binding sites in the miR-155 promoter; both sites recruited NF-kappaB complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-kappaB sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-kappaB binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target.
Assuntos
Linfócitos B/virologia , MicroRNAs/genética , NF-kappa B/metabolismo , Ativação Transcricional , Proteínas da Matriz Viral/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Células Cultivadas , Herpesvirus Humano 4/fisiologia , Humanos , Camundongos , MicroRNAs/biossíntese , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: Hypoxia plays an important role in kidney injury. By the stabilization of the transcription factor HIF-1, hypoxia affects gene expression also in tubular epithelial cells. Increased expression of connective tissue growth factor (CTGF) is observed in different kidney diseases and is associated with deteriorating renal function. Therefore, we hypothesized that the expression of CTGF might be modulated under hypoxic conditions. METHODS: The human proximal tubular epithelial cell lines HK-2 and HKC-8 were treated with reduced oxygen tension (1% O(2)) or the hypoxia mimetic dimethyloxalyl glycine (DMOG). CTGF was analysed by Western blotting, real-time RT-PCR and luciferase gene expression assays. RESULTS: Exposure of HK-2 or HKC-8 cells to hypoxia or treatment with DMOG for up to 24 h reduced cellular as well as secreted CTGF protein synthesis. Downregulation was also detectable at the mRNA level and was confirmed by reporter gene assays. Hypoxic repression of CTGF synthesis was dependent on HIF-1, as shown by HIF-1alpha knockdown by siRNA. Furthermore, exposure to hypoxia reduced CTGF synthesis in response to TGF-beta. A negative correlation between HIF-1alpha accumulation and CTGF synthesis was also observed in renal cell carcinoma cells (RCC4 and RCC10). Reexpression of von Hippel-Lindau protein reduced HIF-1alpha and increased CTGF synthesis. CONCLUSIONS: We provide evidence that hypoxia inhibits CTGF synthesis in human proximal tubular epithelial cells, involving HIF-1alpha. Under hypoxic conditions, induction of CTGF by TGF-beta was repressed. The reduced synthesis of the profibrotic factor CTGF may contribute to a potential protective effect of hypoxic preconditioning in acute renal injury.
Assuntos
Hipóxia Celular , Fator de Crescimento do Tecido Conjuntivo/genética , Túbulos Renais Proximais/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Carcinoma de Células Renais/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Renais/metabolismo , Túbulos Renais Proximais/citologia , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta1/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologiaRESUMO
BACKGROUND: Hepatocyte growth factor (HGF) is a pleiotropic protein with renoprotective functions, which have been attributed at least in part to its ability to counteract the profibrotic effects of transforming growth factor beta (TGF-beta). A major downstream mediator of TGF-beta is connective tissue growth factor (CTGF). However, the molecular mechanisms of CTGF regulation by HGF have not yet been investigated. METHODS: CTGF expression was analysed in human primary tubular epithelial cells (hPTECs) and the cell line HKC-8 by western blotting. Morphological alterations were analysed by immunocytochemistry. RESULTS: HGF induced a transient expression of CTGF, which was maximal after 6 h and returned to baseline after 24 h. Coincubation with TGF-beta increased CTGF protein at 6 h, whereas HGF significantly decreased CTGF induction by TGF-beta after 24 h. Furthermore, HGF induced cell scattering associated with reorganization of focal adhesions and formation of lamellipodia and filopodia. The early induction of CTGF was linked to the HGF-mediated alterations of cell morphology. The PP2 inhibitor of Src-family kinases, which regulate focal adhesion turnover, reduced HGF-mediated upregulation of CTGF. In addition, inhibition of the Rho-kinase, which modulates the actin cytoskeleton, impaired CTGF expression. Combination of both inhibitors further decreased CTGF expression. Comparable inhibitory effects were obtained, when CTGF was induced by the combination of HGF and TGF-beta. CONCLUSIONS: We provide evidence for a dual effect of HGF on CTGF regulation in human tubular epithelial cells: transient upregulation of CTGF in the absence of TGF-beta, which was related to alterations of cell morphology, and interference with TGF-beta-mediated CTGF induction after prolonged incubation.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Epiteliais/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Túbulos Renais/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologia , Técnicas de Cultura de Células , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Staufen1, the mammalian homolog of Drosophila Staufen, assembles into ribonucleoprotein particles (RNPs), which are thought to transport and localize RNA into dendrites of mature hippocampal neurons. We therefore investigated whether additional components of the RNA localization complex besides Staufen are conserved. One candidate is the mammalian homolog of Drosophila Barentsz (Btz), which is essential for the localization of oskar mRNA to the posterior pole of the Drosophila oocyte and is a component of the oskar RNA localization complex along with Staufen. In this study, we report the characterization of mammalian Btz, which behaves like a nucleocytoplasmic shuttling protein. When expressed in the Drosophila egg chamber, mammalian Btz is still able to interact with Drosophila Staufen and reach the posterior pole in the wild-type oocyte, but does not rescue the btz mutant phenotype. Most interestingly, we show by immunoprecipitation assays that Btz interacts with mammalian Staufen in an RNA-dependent manner through a conserved domain, which encompasses the region of homology to the Drosophila Btz protein and contains a novel conserved motif. One candidate for an RNA that mediates this interaction is the dendritically localized brain cytoplasmic 1 transcript. In addition, Btz and Staufen1 colocalize within particles in the cell body and, to a more variable extent, in dendrites of mature hippocampal neurons. Together, our data suggest that the mRNA transport machinery is conserved during evolution, and that mammalian Btz is an additional component of the dendritic RNPs in hippocampal neurons.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Sequência Conservada , Dendritos/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias , Neurônios/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oócitos/metabolismo , Especificidade de Órgãos , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Renal tubular epithelial cells of proximal and distal origin differ markedly in their physiological functions. Therefore, we hypothesized that they also differ in their capacity to undergo epithelial to mesenchymal alterations. RESULTS: We used cultures of freshly isolated primary human tubular cells. To distinguish cells of different tubular origin we took advantage of the fact that human proximal epithelial cells uniquely express N-cadherin instead of E-cadherin as major cell-cell adhesion molecule. To provoke mesenchymal alteration we treated these cocultures with TGF-ß for up to 6 days. Within this time period, the morphology of distal tubular cells was barely altered. In contrast to tubular cell lines, E-cadherin was not down-regulated by TGF-ß, even though TGF-ß signal transduction was initiated as demonstrated by nuclear localization of Smad2/3. Analysis of transcription factors and miRNAs possibly involved in E-cadherin regulation revealed high levels of miRNAs of the miR200-family, which may contribute to the stability of E-cadherin expression in human distal tubular epithelial cells. By contrast, proximal tubular epithelial cells altered their phenotype when treated with TGF-ß. They became elongated and formed three-dimensional structures. Rho-kinases were identified as modulators of TGF-ß-induced morphological alterations. Non-specific inhibition of Rho-kinases resulted in stabilization of the epithelial phenotype, while partial effects were observed upon downregulation of Rho-kinase isoforms ROCK1 and ROCK2. The distinct reactivity of proximal and distal cells was retained when the cells were cultured as polarized cells. CONCLUSIONS: Interference with Rho-kinase signaling provides a target to counteract TGF-ß-mediated mesenchymal alterations of epithelial cells, particularly in proximal tubular epithelial cells. Furthermore, primary distal tubular cells differed from cell lines by their high phenotypic stability which included constant expression of E-cadherin. Our cell culture system of primary epithelial cells is thus suitable to understand and modulate cellular remodeling processes of distinct tubular cells relevant for human renal disease.
Assuntos
Caderinas/metabolismo , Células Epiteliais/metabolismo , Mesoderma/metabolismo , Western Blotting , Caderinas/genética , Adesão Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Túbulos Renais Distais/citologia , Túbulos Renais Proximais/citologia , Mesoderma/citologia , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Cell migration plays a pivotal role in many biological processes and is modulated by cytokines and growth factors. In vivo, cells are embedded in an extracellular matrix (ECM). ECM proteins are linked to the cellular cytoskeleton by integrin adhesion receptors, which transmit extracellular signals into the cell, thereby affecting cell adhesion and migration as well as gene expression. We describe a cell migration assay that uses a barrier device to separate the cells. The assay enables quantification of the migration of adherent cells on defined matrix proteins and the ability to evaluate migration-associated characteristics of individual cells. Thus, the barrier cell migration assay is a useful tool for exploring matrix-dependent migration of adherent cells.