RESUMO
The Wilms tumor suppressor gene wt1 encodes a zinc finger DNA binding protein, WT1, that functions as a transcriptional repressor. The fetal mitogen insulin-like growth factor II (IGF-II) is overexpressed in Wilms tumors and may have autocrine effects in tumor progression. The major fetal IGF-II promoter was defined in transient transfection assays as a region spanning from nucleotides -295 to +135, relative to the transcription start site. WT1 bound to multiple sites in this region and functioned as a potent repressor of IGF-II transcription in vivo. Maximal repression was dependent on the presence of WT1 binding sites on each side of the transcriptional initiation site. These findings provide a molecular basis for overexpression of IGF-II in Wilms tumors and suggest that WT1 negatively regulates blastemal cell proliferation by limiting the production of a fetal growth factor in the developing vertebrate kidney.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes do Tumor de Wilms/fisiologia , Fator de Crescimento Insulin-Like II/genética , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , DNA/química , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Rim/embriologia , Rim/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Homologia de Sequência do Ácido Nucleico , Transfecção , Proteínas WT1 , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Dedos de ZincoRESUMO
Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.
Assuntos
Cálcio/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico/biossíntese , Animais , Drosophila melanogaster/genética , Proteínas de Choque Térmico/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Glândulas Salivares/metabolismoRESUMO
The Wilms' tumor suppressor, WT1, is a zinc finger transcriptional regulator which exists as multiple forms owing to alternative mRNA splicing. The most abundant splicing variants contain a nine-nucleotide insertion encoding lysine, threonine, and serine (KTS) in the H-C link region between the third and fourth WT1 zinc fingers which disrupts binding to a previously defined WT1-EGR1 binding site. We have identified WT1[+KTS] binding sites in the insulin-like growth factor II gene and show that WT1[+KTS] represses transcription from the insulin-like growth factor II P3 promoter. The highest affinity WT1[+KTS] DNA binding sites included nucleotide contacts involving all four WT1 zinc fingers. We also found that different subsets of three WT1 zinc fingers could bind to distinct DNA recognition elements. A tumor-associated, WT1 finger 3 deletion mutant was shown to bind to juxtaposed nucleotide triplets for the remaining zinc fingers 1, 2, and 4. The characterization of novel WT1 DNA recognition elements adds a new level of complexity to the potential gene regulatory activity of WT1. The results also present the possibility that altered DNA recognition by the dominant WT1 zinc finger 3 deletion mutant may contribute to tumorigenesis.
Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/biossíntese , DNA/metabolismo , Variação Genética , RNA Mensageiro/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Genes do Tumor de Wilms , Vetores Genéticos , Humanos , Fator de Crescimento Insulin-Like II/biossíntese , Cinética , Metilação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , RNA Mensageiro/biossíntese , Transfecção , Células Tumorais Cultivadas , Proteínas WT1 , Dedos de Zinco/genéticaRESUMO
The kidney of the zebrafish shares many features with other vertebrate kidneys including the human kidney. Similar cell types and shared developmental and patterning mechanisms make the zebrafish pronephros a valuable model for kidney organogenesis. Here we review recent advances in studies of zebrafish pronephric development and provide experimental protocols to analyze kidney cell types and structures, measure nephron function, live image kidney cells in vivo, and probe mechanisms of kidney regeneration after injury.
Assuntos
Rim/crescimento & desenvolvimento , Organogênese/genética , Regeneração/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Néfrons/crescimento & desenvolvimento , Pronefro/crescimento & desenvolvimento , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The floor plate of the neural tube serves an important function as a source of signals that pattern cell fates in the nervous system as well as directing proper axon pathfinding. We have cloned a novel zebrafish wnt family member, wnt4b, which is expressed exclusively in the floor plate. To place wnt4b in the context of known regulators of midline development, its expression was analyzed in the zebrafish mutants cyclops (cyc), floating head (flh), you-too (yot), and sonic you (syu). wnt4b expression in the medial and lateral floor plate are shown to be regulated independently: medial floor plate expression occurs in the absence of a notochord, while lateral floor plate expression requires a functional notochord, sonic hedgehog and gli-2.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Oncogênicas/genética , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Transativadores , Fatores de Transcrição/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Proteínas Hedgehog , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , Proteínas Wnt , Proteína Wnt4 , Peixe-Zebra/embriologia , Proteína GLI1 em Dedos de ZincoRESUMO
Heat shock causes significant changes in intracellular free calcium ([Ca2+]i) which occur rapidly following temperature elevation. The resting level of free calcium in single Drosophila melanogaster larval salivary gland cells measured with the fluorescent indicator fura-2 is 198 +/- 31 nM (n = 4). It increases approximately 10-fold to 1870 +/- 770 nM (n = 4), during a heat shock. When salivary glands are incubated in calcium-free, EGTA-buffered medium the resting free calcium is reduced to 80 +/- 7 nM (n = 3) and heat shock results in a 4-fold increase in free calcium to 353 +/- 90 nM (n = 3). Drosophila Kc cells show a heat shock-induced increase in [Ca2+]i from 118.4 +/- 2 nM (n = 11) to 323 +/- 18 nM. Procedures were devised to block the effects of heat shock on the increase in intracellular calcium and assess its role in the induction of heat shock proteins and in the stress-induced rearrangement of the vimentin cytoskeleton. We report here the changes in [Ca2+]i are not required for a complete induction of the heat shock response or for the collapse of the vimentin cytoskeleton.
Assuntos
Cálcio/fisiologia , Citoesqueleto/ultraestrutura , Proteínas de Choque Térmico/biossíntese , Animais , Benzofuranos , Drosophila melanogaster , Fura-2 , Indicadores e ReagentesRESUMO
The Egr family of transcriptional regulators comprises a group of genes which encode members of the Cys2-His2 class of zinc-finger proteins. We have isolated a zebrafish egr1 homologue by screening a zebrafish genomic library with a mouse Egr1 zinc finger probe. Southern blotting indicated the existence of a single zebrafish egr1 gene and, as in higher vertebrates, the presence of related members of a larger gene family. Sequence analysis of the zebrafish egr1 coding region revealed a high level of homology to the mouse, rat, and human Egr1 genes with the notable exception of a polymorphic, triplet nucleotide repeat sequence in the region coding for the amino terminus of the Egr1 protein. The predicted DNA-binding, zinc-finger domain protein sequence was strictly conserved. The 5' region of the zebrafish egr1 gene contained a variety of transcription factor binding sites, also present in the mouse gene, for serum response factor, CREB and c-Ets. The zebrafish egr1 transcript was approximately 3.4 kb in size and was expressed in adult zebrafish brain and muscle RNA, a pattern of expression similar to that observed in mice. The potential for zebrafish egr1 to function as a transcriptional regulator was tested by constructing an expression vector containing zebrafish egr1 coding sequences under the control of a cytomegalovirus promoter. This construct was found to activate transcription of a reporter plasmid bearing multiple Egr1 binding sites when transiently cotransfected into mouse 3T3 cells. Our results indicate that the structure, regulation, and function of the Egr1 gene have been highly conserved during vertebrate evolution and suggest an important role for this gene in growth and development.
Assuntos
Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas Imediatamente Precoces , Fatores de Transcrição/genética , Transcrição Gênica , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína 1 de Resposta de Crescimento Precoce , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Peixe-ZebraRESUMO
The Egr family of transcriptional regulators comprises a group of genes that encode members of the Cys2-His2 class of zinc finger proteins. We have isolated a zebrafish egr1 homolog by screening a zebrafish genomic library with a mouse Egr1 zinc finger probe. Southern blotting indicated the existence of single zebrafish egr1 gene and, as in higher vertebrates, the presence of related members of a larger gene family. Sequence analysis of the zebrafish egr1 coding region revealed a high level of homology to the mouse, rat, and human egr1 genes with the notable exception of a polymorphic, triplet nucleotide repeat sequence in the region coding for the amino terminus of the Egr1 protein. The predicted DNA-binding, zinc finger domain protein sequence was strictly conserved. The 5' region of the zebrafish egr1 gene contained a variety of transcription factor binding sites, also present in the mouse gene, for serum response factor, CREB, and c-ets. The zebrafish egr1 transcript was approximately 3.4 kb in size and was expressed in adult zebrafish brain and muscle RNA, a pattern of expression similar to that observed in mice. The potential for zebrafish egr1 to function as a transcriptional regulator was tested by constructing an expression vector containing zebrafish egr1 coding sequences under the control of a cytomegalovirus promoter. This construct was found to activate transcription of a reporter plasmid bearing multiple Egr1 binding sites when transiently cotransfected into mouse 3T3 cells. Our results indicate that the structure, regulation, and function of the Egr1 gene have been highly conserved during vertebrate evolution and suggest an important role for this gene in growth and development.
Assuntos
Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas Imediatamente Precoces , Fatores de Transcrição/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteína 1 de Resposta de Crescimento Precoce , Regulação da Expressão Gênica , Humanos , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Transcrição Gênica , Peixe-ZebraRESUMO
The zebrafish, as a model system for vertebrate development, offers distinct experimental advantages for studies of organogenesis. The simplicity of the zebrafish pronephros, the feasibility of isolating large numbers of mutants, and the growth in infrastructure for genomics makes the zebrafish an attractive system for the analysis of kidney development. Mutants affecting several aspects of nephrogenesis, including differentiation of the intermediate mesoderm, nephron patterning, epithelial polarity, and angiogenesis, have been isolated. Analysis of mutant phenotypes and the cloning of mutant genes has revealed: (1) a role for bone morphogenetic proteins in patterning the ventral mesoderm, (2) an essential role for the pax2.1 gene in pronephric development, (3) multiple loci required for establishing epithelial polarity in the pronephric duct, (4) a central role for podocytes in directing glomerulogenesis, and (5) 15 loci associated with cystic maldevelopment in the pronephros. The striking similarities of pronephric cell types to those found in higher vertebrates, as well as the conservation of kidney-specific gene expression patterns, suggest that insights gained from studies in zebrafish will be broadly applicable to cell differentiation in the kidney.
Assuntos
Néfrons/embriologia , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/fisiologia , Técnicas Genéticas , Mesoderma/fisiologia , Mutação , Neovascularização Fisiológica , Néfrons/irrigação sanguínea , Peixe-Zebra/genéticaRESUMO
During differentiation of Dictyostelium discoideum, cAMP functions as a diffusible, extracellular signal to direct chemotaxis and regulate developmental gene expression. The availability of signal-transduction mutants of Dictyostelium now makes it feasible to pursue a genetic analysis of cAMP signaling. The synag 7 mutant is defective in receptor-mediated adenylate cyclase stimulation and cannot relay a cAMP signal. To further characterize this mutant, mRNA levels of several cAMP-regulated genes were measured during development. cAMP-regulated gene expression was found to be dramatically altered in synag 7:several different genes which require cAMP for expression in wild-type cells were induced in synag 7 in the absence of cAMP. In addition, the gene-encoding discoidin I, which is normally expressed in starved cells and repressed by cAMP, is expressed at very low levels in starved synag 7 cells, possibly due to precocious repression. These results suggest that a pleiotropic regulator of cAMP-regulated gene expression is uncoupled from its normal controls during development in synag 7.
Assuntos
AMP Cíclico/fisiologia , Dictyostelium/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Lectinas , Proteínas de Protozoários , Actinas/biossíntese , Discoidinas , Proteínas Fúngicas/biossíntese , Receptores de AMP Cíclico , Transdução de SinaisRESUMO
The role of oxidative stress in the induction of heat-shock proteins (HSPs) was studied in Drosophila Kc cells by comparing the effects of two different inducers, temperature stress and reoxygenation following a period of anoxia, on cellular respiration, thiol status, and the accumulation of HSPs. A heat shock from 25 to 37 degrees C caused a 60% increase in the rate of O2 uptake but caused little oxidative stress as indicated by a constant level of reduced glutathione, a slight increase in oxidized glutathione, and no change in protein sulfhydryls. Heat shock resulted in a pronounced accumulation of HSPs which was not inhibited by anoxic conditions. A different HSP inducer, reoxygenation following anoxia, resulted in an overall inhibition of respiration, the appearance of CN -insensitive O2 uptake, a 50% decrease in the level of reduced glutathione and a fourfold increase in the ratio of oxidized to reduced glutathione. Despite these indicators of oxidative stress, HSP synthesis was less pronounced than observed during heat shock and was not affected by antioxidants. Oxidative stress may induce HSP synthesis in some cases but is not responsible for HSP synthesis during a heat shock.
Assuntos
Proteínas de Choque Térmico/biossíntese , Consumo de Oxigênio , Animais , Linhagem Celular , Drosophila , Glutationa/biossíntese , Glutationa Redutase/biossíntese , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Fatores de TempoRESUMO
We have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell-surface cAMP receptor in suspension-starved Dictyostelium discoideum cells by measuring ammonium sulfate-stabilized binding of [3-H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+ phosphate buffer, pH 6.5, plus 1 mM CaCl2 and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/10(7) cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine-treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30-40 mM was found to significantly inhibit the induction of cAMP receptors.
Assuntos
Adenosina/farmacologia , Cafeína/farmacologia , Dictyostelium/crescimento & desenvolvimento , Receptores de AMP Cíclico/metabolismo , Soluções Tampão , AMP Cíclico/metabolismo , Dictyostelium/efeitos dos fármacos , Dictyostelium/metabolismo , CinéticaRESUMO
In zebrafish, the pronephric glomerulus occupies a midline position underneath the notochord and is vascularized through angiogenic capillary ingrowth from the dorsal aorta. The midline mutants floating head (flh), sonic you (syu), and you-too (yot) provide the opportunity to study glomerular differentiation in the absence of the notochord and vascularization from the dorsal aorta. In flh, syu, and yot mutants, glomeruli differentiate at ectopic lateral positions within the embryo and contain morphologically identifiable podocyte and endothelial cell types. In the absence of the dorsal aorta, endothelia from an alternate source are recruited by podocytes during glomerular vascularization to make functional glomeruli. Our results suggest that midline signals are required for proper glomerular morphogenesis but not for the differentiation of podocytes. Podocytes appear to play an important role in directing cellular recruitment events leading to glomerular differentiation. Furthermore, we find defects in sclerotomal development that correlate with defects in glomerular morphogenesis suggesting a possible link between the formation of these embryonic structures.
Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Glomérulos Renais/citologia , Mutação , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra , Peixe-Zebra/genética , Animais , Embrião não Mamífero/citologia , Glomérulos Renais/ultraestrutura , Microscopia EletrônicaRESUMO
The physiological functions of the zebrafish pronephros are blood plasma filtration and osmoregulation. The pronephric glomerulus is vascularized through a capillary network sprouting from the dorsal aorta. Vascularization of the glomerulus, visualized by flk-1 expression and alkaline phosphatase reactivity, involves the intimate association between podocytes and endothelial cells and the formation of an intervening glomerular basement membrane (GBM). Cell-cell interactions between podocytes and endothelial cells are thought to play an important role in glomerular angiogenesis. In order to determine whether endothelial cell-derived signals were required for podocyte differentiation, we employed in situ hybridization and electron microscopy to investigate glomerulogenesis in the zebrafish mutant cloche (clo), where endothelial cell development is blocked at an early stage. In clo mutants, glomerular epithelial cells expressing the podocyte specific marker wt1 display well-formed foot processes and are able to form a GBM, suggesting podocytes are able to morphologically differentiate in the absence of endothelia or endothelial-derived signals. The presence of irregular aggregates in the clo GBM as well as the apparent effacement of podocyte foot processes implies a role for endothelial cells in the maintenance of the mature glomerular filtration barrier.
Assuntos
Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Membrana Basal/embriologia , Membrana Basal/ultraestrutura , Diferenciação Celular , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Endotélio Vascular/fisiologia , Glomérulos Renais/fisiologia , Microscopia Eletrônica , Mutação , Peixe-Zebra/fisiologiaRESUMO
AIMS: For studies of kidney organogenesis, clonal human fetal kidney (HFK) cell lines that represent various stages of kidney epithelial development would provide valuable tools for in vitro approaches to kidney cell differentiation. METHODS: Cell cultures from the cortices of 12- to 16-week HFKs were immortalized by retroviral expression of human papilloma virus (HPV) E6/E7 genes, and clonal isolates were characterized with regard to morphology, expression of molecular markers, growth on different matrices, and their ability to differentiate further in vitro. RESULTS: Three immortal, clonal cell lines displayed homogeneous but distinct morphologies in vitro, ranging from mesenchymal to epithelial. Northern blotting with stage-specific markers allowed us to assign particular cell lines to discrete stages in kidney cell differentiation. These results were confirmed by assessing cell growth on type-I collagen versus Matrigel matrices. We have also demonstrated that for one of the cell lines fetal calf serum contains a differentiation factor that induced morphological differentiation and enhanced expression of epithelial-specific genes. CONCLUSION: Immortalization by HPV-E6/E7 expression preserved important phenotypic characteristics of fetal kidney cells. The creation of these lines provides useful tools for in vitro approaches to kidney growth and development.
Assuntos
Embrião de Mamíferos , Rim/citologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas Repressoras , Retroviridae/genética , Northern Blotting , Caderinas/análise , Diferenciação Celular , Linhagem Celular Transformada , Colágeno/análise , Vetores Genéticos , Humanos , Queratinas/análise , Rim/química , Rim/virologia , Proteínas E7 de Papillomavirus , Vimentina/análiseRESUMO
Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, we have used the zebrafish pax2.1 mutant no isthmus to investigate the role for pax2.1 in development of the pronephros. We demonstrate a requirement for pax2.1 in multiple aspects of pronephric development including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi(- )larvae specifically lack pronephric tubules while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na(+)/K(+) ATPase (&agr;)1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Ligação a DNA/fisiologia , Túbulos Renais/embriologia , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Glomérulos Renais/embriologia , Mutagênese , Fator de Transcrição PAX2 , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-ZebraRESUMO
To identify growth factors which may play a role in kidney organogenesis, we have analyzed culture supernatants from the pediatric kidney tumor cell line G401. G401 cells were found to secrete fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells, OP-1/BMP7, an epithelial cell growth inhibitor, and midkine (MK). Northern blotting confirmed expression of FGF2, OP-1/BMP7 and MK mRNA, as well as Wnt5A mRNA in G401 cells. In situ hybridization and immunocytochemistry on human fetal kidney demonstrated FGF2 expression in epithelial cells of the branching ureteric bud epithelium, nephron precursors ("S-shaped bodies"), proximal tubule epithelium and the parietal epithelium of the glomerulus. FGF2 protein in condensed "caps" of induced mesenchymal cells was also detected by immunocytochemistry. FGF2 protein was found to be concentrated in nuclei, particularly in proximal tubule epithelial cells. Recombinant FGF2 was found to act as a mitogen on primary mouse fetal kidney cell cultures. The results demonstrate G401 cells secrete a variety of fetal kidney growth factors and that FGF2 may act as a mitogen for fetal kidney cells and thus could play a role in the morphogenesis of the kidney.
Assuntos
Feto/metabolismo , Fator 2 de Crescimento de Fibroblastos/fisiologia , Substâncias de Crescimento/metabolismo , Neoplasias Renais/metabolismo , Rim/embriologia , Animais , Meios de Cultura/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Substâncias de Crescimento/genética , Humanos , Rim/metabolismo , Córtex Renal/citologia , Córtex Renal/efeitos dos fármacos , Córtex Renal/embriologia , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Mitógenos/farmacologia , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Animal cells respond to calcium ionophore (A23187) treatment with the coordinate induction of a set of genes encoding proteins identical to the glucose-regulated proteins (GRPs). By monitoring the intracellular free calcium with the fluorescent indicator fura-2 while employing both intracellular and extracellular calcium buffers, we demonstrated that A23187 can induce the GRP94 and GRP78 genes without an increase in cytoplasmic calcium ([Ca2+]i). Induction of GRP mRNA during glucose starvation was also independent of [Ca2+]i. Instead, gene induction by A23187 was closely correlated with the depletion of intracellular calcium stores. We conclude that perturbations of sequestered calcium ions by A23187 can serve as a stimulus for gene expression.
Assuntos
Calcimicina/farmacologia , Cálcio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Biossíntese de Proteínas , Animais , Linhagem Celular , Cricetinae , Cricetulus , Meios de Cultura/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Líquido Intracelular/metabolismo , Pulmão , Proteínas/genética , Ativação TranscricionalRESUMO
The Wilms' tumor suppressor gene WT1 encodes a zinc-finger DNA-binding protein that functions as a transcriptional repressor. WT1 is expressed in a dramatic spatial and temporal pattern during kidney development and is thought to be critical during mesenchymal-epithelial conversion. The WT1 protein bound multiple sites in the WT1 promoter and functioned as a powerful transcriptional repressor of its gene in vivo (> 50-fold). The WT1 protein carrying an NH2-terminal 17-amino acid insertion and a 3-amino acid insertion (KTS) between zinc fingers 3 and 4, arising from the most abundant of four alternatively spliced transcripts, was the most powerful repressor. Of importance, a subset of WT1-binding sites differs from the Egr-1 consensus sequence, which has been shown to bind one splice variant of the WT1 protein (WT1(-KTS)). We characterized two of these sites and show that they bind both -KTS and +KTS forms of the WT1 zinc-finger protein and can confer repression on a heterologous promoter construct. Our data demonstrate that WT1, in addition to its known effects on insulin-like growth factor II, platelet-derived growth factor A, and Pax-2 transcription, is a powerful repressor of its own gene. These observations emphasize its critical role as a transcriptional regulatory protein during normal kidney development.
Assuntos
Regulação da Expressão Gênica , Genes do Tumor de Wilms , Sequência de Bases , Sítios de Ligação , Células Cultivadas , DNA/metabolismo , Regulação para Baixo , Humanos , Dados de Sequência Molecular , Néfrons/metabolismo , Regiões Promotoras GenéticasRESUMO
The insulin-like growth factor-I receptor (IGF-I-R) has been implicated in the etiology and/or progression of Wilms' tumor, a pediatric malignancy of the kidney that is often associated with deletion or mutation of the WT1 tumor suppressor gene. The expression of the IGF-I-R gene is increased in Wilms' tumor as compared with normal kidney tissue. Furthermore, the levels of IGF-I-R mRNA in individual tumors have been shown to be inversely correlated to the levels of WT1 mRNA, suggesting that the expression of the IGF-I-R gene is under the negative control of WT1. The activity of an IGF-I-R promoter/luciferase construct in Chinese hamster ovary cells was reduced by cotransfection of a WT1 expression vector. An analysis of various reporter constructs containing different portions of the IGF-I-R 5'-flanking and 5'-untranslated regions suggested that the effect of WT1 depends on the number of WT1 binding sites present, with sites located both upstream and downstream of the IGF-I-R transcription start site involved in mediating this effect. Using the purified zinc finger domain of WT1 in gel retardation and DNase I footprinting assays, we mapped five sites in the 5'-flanking and six sites in the 5'-untranslated regions that were involved in WT1 binding. In addition, the initiator element of the IGF-I-R gene contains a sequence that binds WT1. Thus, the repression of IGF-I-R promoter activity by the WT1 tumor suppressor gene product involves multiple interactions of its zinc finger domain with WT1 binding sites located both 5' and 3' of the transcription initiation site.