RESUMO
FgfrL1 is a novel growth factor receptor that is primarily expressed in musculoskeletal tissues and the kidney. FgfrL1-deficient mice have a malformed diaphragm and no kidneys. Such animals die immediately after birth because they are not able to inflate their lungs. The FgfrL1 molecule is composed of three extracellular Ig domains, a transmembrane helix and a short intracellular domain. To investigate the contribution of each of these domains to the function of the novel receptor, we generated mice with deletions of the individual domains. Mice lacking the intracellular domain are viable and phenotypically normal. Mice lacking the first (N-terminal) Ig domain are also viable and normal, but have a reduced life span. Mice lacking the Ig2 or the Ig3 domain are born alive, but die within 24 âh after birth. Ig2-deficient animals exhibit substantially smaller kidneys than wild-type littermates and contain a lower number of glomeruli. Ig3-deficient mice completely lack metanephric kidneys. Interestingly, both the Ig2 and the Ig3-deficient animals show only minor alterations in the diaphragm, which still enables them to inflate their lungs after birth. Our results demonstrate that the principal function of the FgfrL1 receptor is to control the growth of the metanephric kidneys by regulating nephrogenesis. It appears that this function is primarily accomplished by the Ig3 domain with some contribution of the Ig2 domain. It is conceivable that the two domains interact with an Fgf ligand and another molecule from the surface of neighboring cells to induce condensation of the metanephric mesenchyme to renal epithelia and glomeruli.
Assuntos
Diafragma/anormalidades , Rim/embriologia , Sistema Musculoesquelético/embriologia , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organogênese/genética , Organogênese/fisiologia , Domínios Proteicos/genéticaRESUMO
ATPase H+-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H+-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na+-K+-2Cl- cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity.
Assuntos
Rim/enzimologia , ATPases Translocadoras de Prótons/fisiologia , Receptores de Superfície Celular/fisiologia , Sistema Renina-Angiotensina/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Animais , Feminino , Masculino , CamundongosRESUMO
The mammalian kidney develops from the ureteric bud and the metanephric mesenchyme. In mice, the ureteric bud invades the metanephric mesenchyme at day E10.5 and begins to branch. The tips of the ureteric bud induce the metanephric mesenchyme to condense and form the cap mesenchyme. Some cells of this cap mesenchyme undergo a mesenchymal-to-epithelial transition and differentiate into renal vesicles, which further develop into nephrons. The developing kidney expresses Fibroblast growth factor (Fgf)1, 7, 8, 9, 10, 12 and 20 and Fgf receptors Fgfr1 and Fgfr2. Fgf7 and Fgf10, mainly secreted by the metanephric mesenchyme, bind to Fgfr2b of the ureteric bud and induce branching. Fgfr1 and Fgfr2c are required for formation of the metanephric mesenchyme, however the two receptors can substitute for one another. Fgf8, secreted by renal vesicles, binds to Fgfr1 and supports survival of cells in the nascent nephrons. Fgf9 and Fgf20, expressed in the metanephric mesenchyme, are necessary to maintain survival of progenitor cells in the cortical region of the kidney. FgfrL1 is a novel member of the Fgfr family that lacks the intracellular tyrosine kinase domain. It is expressed in the ureteric bud and all nephrogenic structures. Targeted deletion of FgfrL1 leads to severe kidney dysgenesis due to the lack of renal vesicles. FgfrL1 is known to interact mainly with Fgf8. It is therefore conceivable that FgfrL1 restricts signaling of Fgf8 to the precise location of the nascent nephrons. It might also promote tight adhesion of cells in the condensed metanephric mesenchyme as required for the mesenchymal-to-epithelial transition.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Rim/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Transição Epitelial-Mesenquimal , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica , Rim/crescimento & desenvolvimento , Néfrons/crescimento & desenvolvimento , Néfrons/metabolismo , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de SinaisRESUMO
Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.
Assuntos
Cobre/metabolismo , Cobre/toxicidade , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/metabolismo , Vitamina K 2/metabolismo , Membrana Celular/metabolismo , Elementos de DNA Transponíveis , Técnicas de Inativação de Genes , Mutagênese Insercional , Oxirredução , Succinato-CoA Ligases/genéticaRESUMO
The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.
Assuntos
Diferenciação Celular , Expressão Gênica , Células Gigantes/citologia , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Células CHO , Fusão Celular , Cricetinae , Cricetulus , Células Gigantes/metabolismo , Células HEK293 , Células HeLa , Humanos , Mioblastos/citologia , Mioblastos/metabolismo , Estrutura Terciária de Proteína , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/químicaRESUMO
Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.
Assuntos
Rim , Néfrons , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Apoptose/fisiologia , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Feminino , Expressão Gênica , Rim/anormalidades , Rim/embriologia , Rim/metabolismo , Mesoderma/fisiologia , Camundongos , Camundongos Transgênicos , Néfrons/anormalidades , Néfrons/embriologia , Néfrons/metabolismo , Gravidez , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Técnicas de Cultura de TecidosRESUMO
To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two-dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding site of CopY-type copper-responsive repressors. A genome-wide search for cop boxes revealed 28 such sequence motifs. They were tested by electrophoretic mobility shift assays for the interaction with purified CopR, the CopY-type repressor of L. lactis. Seven of the cop boxes interacted with CopR in a copper-sensitive manner. They were present in the promoter region of five genes, lctO, ytjD, copB, ydiD, and yahC; and two polycistronic operons, yahCD-yaiAB and copRZA. Induction of these genes by copper was confirmed by real-time quantitative PCR. The copRZA operon encodes the CopR repressor of the regulon; a copper chaperone, CopZ; and a putative copper ATPase, CopA. When expressed in Escherichia coli, the copRZA operon conferred copper resistance, suggesting that it functions in copper export from the cytoplasm. Other member genes of the CopR regulon may similarly be involved in copper metabolism.
Assuntos
Proteínas de Bactérias/fisiologia , Cobre/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Lactococcus lactis/fisiologia , Regulon , Transativadores/fisiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Sequência Consenso , Cobre/toxicidade , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Farmacorresistência Bacteriana , Eletroforese em Gel Bidimensional , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Perfilação da Expressão Gênica , Lactococcus lactis/genética , Lactoilglutationa Liase/biossíntese , Oxigenases de Função Mista/análise , Nitrorredutases/biossíntese , Regiões Promotoras Genéticas , Ligação Proteica , Proteoma/análise , Transativadores/genética , Transativadores/isolamento & purificaçãoRESUMO
Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.
RESUMO
Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.
Assuntos
Perfilação da Expressão Gênica , Rim/metabolismo , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Animais , Biomarcadores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Rim/embriologia , Camundongos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/genética , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
Lactococcus lactis IL1403 is a Gram-positive bacterium of great biotechnological interest for food grade applications. Its use is however hampered by the difficulty to efficiently transform this strain. We here describe a detailed, optimized electrotransformation protocol which yields a transformation efficiency of 10(6) cfu/microg of DNA with the two E. coli Gram-positive shuttle vectors pC3 and pVA838. The utility of the protocol was demonstrated by the generation of single- and double-knock-out mutants by homologous recombination.