RESUMEN
The main laminin-binding integrins α3ß1, α6ß1 and α6ß4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.
Asunto(s)
Integrinas , Túbulos Renales Colectores , Animales , Células Epiteliales , Inflamación/genética , Integrina alfa3beta1 , Integrinas/genética , Laminina/genética , Ratones , FN-kappa B/genéticaRESUMEN
A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2-Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.
Asunto(s)
Actomiosina/metabolismo , Túbulos Renales Colectores/metabolismo , Neuropéptidos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , Polaridad Celular/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Miosina Tipo II/metabolismo , Transducción de Señal/fisiologíaAsunto(s)
Carcinoma de Células Renales , Eliminación de Gen , Neoplasias Renales , Riñón/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Animales , Carcinoma de Células Renales/enzimología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Humanos , Riñón/patología , Neoplasias Renales/enzimología , Neoplasias Renales/genética , Neoplasias Renales/patología , Ratones , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismoAsunto(s)
Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Adhesión Celular/fisiología , Línea Celular , Femenino , Adhesiones Focales/fisiología , Riñón/patología , Proteínas con Dominio LIM/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Simulación de Dinámica Molecular , Paxillin/metabolismo , Dominios Proteicos/fisiología , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
Polycystic kidney disease (PKD) is a common genetic disorder characterized by the growth of fluid-filled cysts in the kidneys. Several studies reported that the serine-threonine kinase Lkb1 is dysregulated in PKD. Here we show that genetic ablation of Lkb1 in the embryonic ureteric bud has no effects on tubule formation, maintenance, or growth. However, co-ablation of Lkb1 and Tsc1, an mTOR repressor, results in an early developing, aggressive form of PKD. We find that both loss of Lkb1 and loss of Pkd1 render cells dependent on glutamine for growth. Metabolomics analysis suggests that Lkb1 mutant kidneys require glutamine for non-essential amino acid and glutathione metabolism. Inhibition of glutamine metabolism in both Lkb1/Tsc1 and Pkd1 mutant mice significantly reduces cyst progression. Thus, we identify a role for Lkb1 in glutamine metabolism within the kidney epithelia and suggest that drugs targeting glutamine metabolism may help reduce cyst number and/or size in PKD.
Asunto(s)
Glutamina/metabolismo , Enfermedades Renales Poliquísticas/enzimología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Quinasas Activadas por AMP , Animales , Progresión de la Enfermedad , Femenino , Humanos , Riñón/embriología , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Noqueados , Enfermedades Renales Poliquísticas/embriología , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of α and ß subunits; crucial integrins in the kidney collecting system express the ß1 subunit. The ß1 cytoplasmic tail has two NPxY motifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the ß1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the ß1 integrin subunit NPxY motif.
Asunto(s)
Integrina beta1/metabolismo , Morfogénesis , Talina/metabolismo , Uréter/citología , Uréter/embriología , Uniones Adherentes/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Adhesión Celular , Membrana Celular/metabolismo , Polaridad Celular , Regulación del Desarrollo de la Expresión Génica , Integrina beta1/química , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/embriología , Ratones Endogámicos C57BL , Mutación/genética , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismo , Uréter/metabolismoRESUMEN
Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.
Asunto(s)
Membrana Basal/metabolismo , Integrina alfa6beta1/genética , Integrina alfa6beta4/genética , Túbulos Renales Colectores/metabolismo , Laminina/genética , Obstrucción Ureteral/metabolismo , Animales , Apoptosis , Membrana Basal/patología , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibrosis , Regulación de la Expresión Génica , Humanos , Integrina alfa6beta1/deficiencia , Integrina alfa6beta4/deficiencia , Túbulos Renales Colectores/patología , Laminina/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Transducción de Señal , Uréter/cirugía , Obstrucción Ureteral/patología , Obstrucción Ureteral/cirugíaRESUMEN
The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development and maintenance, its exact molecular function in kidney development is not well understood. In this study, we define the specific role of Cdc42 during murine kidney epithelial tubulogenesis by deleting it selectively at the initiation of ureteric bud or metanephric mesenchyme development. Deletion in either lineage results in abnormal tubulogenesis, with profound defects in polarity, lumen formation and the actin cytoskeleton. Ultimately, these defects lead to renal failure. Additionally, in vitro analysis of Cdc42-null collecting duct cells shows that Cdc42 controls these processes by regulating the polarity Par complex (Par3-Par6-aPKC-Cdc42) and the cytoskeletal proteins N-Wasp and ezrin. Thus, we conclude that the principal role of Cdc42 in ureteric bud and metanephric mesenchyme development is to regulate epithelial cell polarity and the actin cytoskeleton.
Asunto(s)
Polaridad Celular/fisiología , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales/embriología , Proteína de Unión al GTP cdc42/metabolismo , Animales , Citoesqueleto/genética , Células Epiteliales/citología , Ratones , Proteína de Unión al GTP cdc42/genéticaRESUMEN
The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin α3ß1 is crucial for this developmental program; however, the LM types and LM/integrin α3ß1-dependent signaling pathways are poorly defined. We show that α3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3ß1-dependent collecting duct cell functions. We demonstrate that integrin α3ß1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin ß1 subunit and regulator of integrin α3ß1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3ß1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways.
Asunto(s)
Integrina alfa3beta1/metabolismo , Túbulos Renales Colectores/embriología , Morfogénesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Túbulos Renales Colectores/metabolismo , Ratones , Ratones Noqueados , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/genética , UbiquitinaciónRESUMEN
Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-ß mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-ß receptor (TßRII), which in turn promotes a TßRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TßRII is considered the principal regulatory mechanism of TßRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TßRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TßRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TßRII tail tyrosine residues, resulting in inhibition of TßR-dependent fibrotic signaling. The collagen-binding receptor integrin α1ß1 was required for recruitment of TCPTP to the TßRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TßRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1ß1 and TßRII that is essential for TßRII-mediated SMAD activation and fibrotic signaling pathways.
Asunto(s)
Integrina alfa1beta1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Smad/metabolismo , Animales , Colágeno/biosíntesis , Transición Epitelial-Mesenquimal , Fibrosis , Integrina alfa1/genética , Integrina alfa1/metabolismo , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/química , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Tirosina/química , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin ß1 subunit in these cells converts them from a "loose" epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a "tight" epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin ß1 cytoplasmic tail and does not entail ß1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the ß1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin ß1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.
Asunto(s)
Eliminación de Gen , Integrina beta1/genética , Integrina beta1/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Permeabilidad de la Membrana Celular , Células Cultivadas , Claudina-2/genética , Claudina-2/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Integrina beta1/análisis , Ratones , Permeabilidad , Regulación hacia Arriba , Orina/químicaRESUMEN
Loss of ß1 integrin expression inhibits renal collecting-system development. Two highly conserved NPXY motifs in the distal ß1 tail regulate integrin function by associating with phosphtyrosine binding (PTB) proteins, such as talin and kindlin. Here, we define the roles of these two tyrosines in collecting-system development and delineate the structural determinants of the distal ß1 tail using nuclear magnetic resonance (NMR). Mice carrying alanine mutations have moderate renal collecting-system developmental abnormalities relative to ß1-null mice. Phenylalanine mutations did not affect renal collecting-system development but increased susceptibility to renal injury. NMR spectra in bicelles showed the distal ß1 tail is disordered and does not interact with the model membrane surface. Alanine or phenylalanine mutations did not alter ß1 structure or interactions between α and ß1 subunit transmembrane/cytoplasmic domains; however, they did decrease talin and kindlin binding. Thus, these studies highlight the fact that the functional roles of the NPXY motifs are organ dependent. Moreover, the ß1 cytoplasmic tail, in the context of the adjacent transmembrane domain in bicelles, is significantly different from the more ordered, membrane-associated ß3 integrin tail. Finally, tyrosine mutations of ß1 NPXY motifs induce phenotypes by disrupting their interactions with critical integrin binding proteins like talins and kindlins.
Asunto(s)
Integrina beta1/metabolismo , Túbulos Renales Colectores/crecimiento & desarrollo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Citosol/metabolismo , Humanos , Integrina beta1/genética , Integrina beta3/química , Integrina beta3/metabolismo , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias/química , Unión Proteica , Conformación Proteica , Talina/química , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMEN
In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of α6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and ß1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of γ1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.
Asunto(s)
Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/metabolismo , Laminina/metabolismo , Animales , Membrana Basal/embriología , Membrana Basal/metabolismo , Diabetes Insípida/embriología , Diabetes Insípida/genética , Diabetes Insípida/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/metabolismo , Heparitina Sulfato/metabolismo , Hidronefrosis/embriología , Hidronefrosis/genética , Hidronefrosis/metabolismo , Integrina beta1/metabolismo , Túbulos Renales Colectores/anomalías , Laminina/deficiencia , Laminina/genética , Masculino , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Embarazo , Transducción de SeñalRESUMEN
The integrin-linked kinase (ILK), pinch and parvin ternary complex connects the cytoplasmic tails of beta1 integrins to the actin cytoskeleton. We recently showed that constitutive expression of ILK and alpha parvin in both the ureteric bud and the metanephric mesenchyme of the kidney is required for kidney development. In this study, we define the selective role of ILK in the ureteric bud of the mouse kidney in renal development by deleting it in the ureteric cell lineage before the onset of branching morphogenesis (E10.5). Although deleting ILK resulted in only a moderate decrease in branching, the mice died at 8 weeks of age from obstruction due to the unprecedented finding of intraluminal collecting duct cellular proliferation. ILK deletion in the ureteric bud resulted in the inability of collecting duct cells to undergo contact inhibition and to activate p38 mitogen-activated protein kinase (MAPK) in vivo and in vitro. p38 MAPK activation was not dependent on the kinase activity of ILK. Thus, we conclude that ILK plays a crucial role in activating p38 MAPK, which regulates cell cycle arrest of epithelial cells in renal tubulogenesis.
Asunto(s)
Ciclo Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Uréter/embriología , Uréter/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Uréter/citologíaRESUMEN
Extracellular matrix (ECM) remodeling regulates multiple cellular functions required for normal development and tissue repair. Matrix metalloproteinases (MMPs) are key mediators of this process and membrane targeted MMPs (MT-MMPs) in particular have been shown to be important in normal development of specific organs. In this study we investigated the role of MT1-MMP in kidney development. We demonstrate that loss of MT1-MMP leads to a renal phenotype characterized by a moderate decrease in ureteric bud branching morphogenesis and a severe proliferation defect. The kidneys of MT1-MMP-null mice have increased deposition of collagen IV, laminins, perlecan, and nidogen and the phenotype is independent of the MT-1MMP target, MMP-2. Utilizing in vitro systems we demonstrated that MTI-MMP proteolytic activity is required for renal tubule cells to proliferate in three dimensional matrices and to migrate on collagen IV and laminins. Together these data suggest an important role for MT1-MMP in kidney development, which is mediated by its ability to regulate cell proliferation and migration by proteolytically cleaving kidney basement membrane components.
Asunto(s)
Membrana Basal/metabolismo , Riñón/crecimiento & desarrollo , Metaloproteinasa 14 de la Matriz/fisiología , Animales , Membrana Basal/citología , Movimiento Celular , Proliferación Celular , Matriz Extracelular , Riñón/citología , Metaloproteinasa 14 de la Matriz/deficiencia , Ratones , Ratones Noqueados , FenotipoRESUMEN
TGF-beta plays a key role in upregulating matrix production in injury-induced renal fibrosis, but how TGF-beta signaling in distinct compartments of the kidney, such as specific segments of the nephron, affects the response to injury is unknown. In this study, we determined the role of TGF-beta signaling both in development of the renal collecting system and in response to injury by selectively deleting the TGF-beta type II receptor in mice at the initiation of ureteric bud development. These mice developed normally but demonstrated a paradoxic increase in fibrosis associated with enhanced levels of active TGF-beta after unilateral ureteral obstruction. Consistent with this observation, TGF-beta type II receptor deletion in cultured collecting duct cells resulted in excessive integrin alphavbeta6-dependent TGF-beta activation that increased collagen synthesis in co-cultured renal interstitial fibroblasts. These results suggest that inhibiting TGF-beta receptor-mediated function in collecting ducts may exacerbate renal fibrosis by enhancing paracrine TGF-beta signaling between epithelial and interstitial cells.
Asunto(s)
Túbulos Renales Colectores , Riñón/patología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Animales , Células Cultivadas , Fibrosis/etiología , Ratones , Obstrucción UreteralRESUMEN
The kidney collecting system develops from branching morphogenesis of the ureteric bud (UB). This process requires signaling by growth factors such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factors (FGFs) as well as cell extracellular matrix interactions mediated by integrins. The importance of integrin signaling in UB development was investigated by deleting integrin beta1 at initiation (E10.5) and late (E18.5) stages of development. Deletion at E10.5 resulted in a severe branching morphogenesis phenotype. Deletion at E18.5 did not alter renal development but predisposed the collecting system to severe injury following ureteric obstruction. beta1 integrin was required for renal tubular epithelial cells to mediate GDNF- and FGF-dependent signaling despite normal receptor localization and activation in vitro. Aberrations in the same signaling molecules were present in the beta1-null UBs in vivo. Thus beta1 integrins can regulate organ branching morphogenesis during development by mediating growth-factor-dependent signaling in addition to their well-defined role as adhesion receptors.
Asunto(s)
Integrina beta1/metabolismo , Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/metabolismo , Uréter/embriología , Uréter/metabolismo , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Sustancias de Crecimiento/metabolismo , Integrina beta1/genética , Túbulos Renales Colectores/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Morfogénesis , Técnicas de Cultivo de Órganos , Transducción de SeñalRESUMEN
The collagen IV binding receptor integrin alpha1beta1 has been shown to regulate lung cancer due to its proangiogenic properties; however, it is unclear whether this receptor also plays a direct role in promoting primary lung tumors. To investigate this possibility, integrin alpha1-null mice were crossed with KrasLA2 mice that carry an oncogenic mutation of the Kras gene (G12D) and develop spontaneous primary tumors with features of non-small cell lung cancer. We provide evidence that KrasLA2/alpha1-null mice have a decreased incidence of primary lung tumors and longer survival compared with KrasLA2/alpha1 wild-type controls. Tumors from KrasLA2/alpha1-null mice were also smaller, less vascularized, and exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end staining, respectively. Moreover, tumors from the KrasLA2/alpha1-null mice showed diminished extracellular signal-regulated kinase (ERK) but enhanced p38 mitogen-activated protein kinase activation. Primary lung tumor epithelial cells isolated from KrasLA2/alpha1-null mice showed a significant decrease in anchorage-independent colony formation, collagen-mediated cell proliferation, ERK activation, and, most importantly, tumorigenicity when injected into nude mice compared with KrasLA2/alpha1 wild-type tumor cells. These results indicate that loss of the integrin alpha1 subunit decreases the incidence and growth of lung epithelial tumors initiated by oncogenic Kras, suggesting that both Kras and integrin alpha1beta1 cooperate to drive the growth of non-small cell lung cancer in vivo.
Asunto(s)
Genes ras , Integrina alfa1beta1/fisiología , Neoplasias Pulmonares/genética , Animales , Adhesión Celular , Proliferación Celular , Colágeno Tipo IV/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunohistoquímica , Integrina alfa1beta1/genética , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones Noqueados , Análisis de SupervivenciaRESUMEN
The collecting system of the kidney, derived from the ureteric bud (UB), undergoes repetitive bifid branching events during early development followed by a phase of tubular growth and elongation. Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. In this study, we demonstrate that members of the R-Ras family of proteins, R-Ras and TC21, are expressed in the murine collecting system at E13.5, whereas H-Ras is only detected at day E17.5. Using murine UB cells expressing activated H-Ras, R-Ras, and TC21, we demonstrate that R-Ras-expressing cells show increased branching morphogenesis and cell growth, TC21-expressing cells branch excessively but lose their ability to migrate, whereas H-Ras-expressing cells migrated the most and formed long unbranched tubules. These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules, events that occur later in development.