RESUMEN
Previously, we showed that receptor for activated C kinase 1 (Rack1) regulates growth of colon cells in vitro, partly by suppressing Src kinase activity at key cell cycle checkpoints, in apoptotic and cell survival pathways and at cell-cell adhesions. Here, we generated mouse models of Rack1 deficiency to assess Rack1's function in intestinal epithelia in vivo. Intestinal Rack1 deficiency resulted in proliferation of crypt cells, diminished differentiation of crypt cells into enterocyte, goblet, and enteroendocrine cell lineages, and expansion of Paneth cell populations. Following radiation injury, the morphology of Rack1-deleted small bowel was strikingly abnormal with development of large polypoid structures that contained many partly formed villi, numerous back-to-back elongated and regenerating crypts, and high-grade dysplasia in surface epithelia. These abnormalities were not observed in Rack1-expressing areas of intestine or in control mice. Following irradiation, apoptosis of enterocytes was strikingly reduced in Rack1-deleted epithelia. These novel findings reveal key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. NEW & NOTEWORTHY Our findings reveal novel functions for receptor for activated C kinase 1 (Rack1) in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia.
Asunto(s)
Apoptosis , Diferenciación Celular , Proliferación Celular , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Receptores de Cinasa C Activada/metabolismo , Regeneración , Animales , Apoptosis/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Linaje de la Célula , Proliferación Celular/efectos de la radiación , Células Epiteliales/patología , Células Epiteliales/efectos de la radiación , Genotipo , Mucosa Intestinal/patología , Mucosa Intestinal/efectos de la radiación , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Neoplasias Intestinales/prevención & control , Intestino Delgado/patología , Intestino Delgado/efectos de la radiación , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Inducidas por Radiación/metabolismo , Neoplasias Inducidas por Radiación/patología , Neoplasias Inducidas por Radiación/prevención & control , Fenotipo , Receptores de Cinasa C Activada/deficiencia , Receptores de Cinasa C Activada/genética , Regeneración/efectos de la radiación , Transducción de SeñalRESUMEN
Previously, we generated mouse models of Rack1 deficiency to identify key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. However, other than low body weight, we did not detect an overt phenotype in mice constitutively deleted of Rack1 in intestinal epithelia ( vil-Cre: Rack1fl/fl mice), presumably because Rack1 was deleted in <10% of the total surface area of the epithelia. To assess the effect of Rack1 loss throughout the entire intestinal epithelia, we generated another mouse model of Rack1 deficiency, vil-Cre-ERT2: Rack1fl/fl. Within 5-10 days of the initial tamoxifen treatment, the mice lost over 20% of their body weight, developed severe diarrhea that for some was bloody, became critically ill, and died, if not euthanized. Necropsies revealed mildly distended, fluid-, gas-, and sometimes blood-filled loops of small and large bowel, inguinal lymphadenopathy, and thrombocytosis. Rack1 was deleted in nearly 100% of the epithelia in both the small intestine and colon when assessed by immunofluorescent or immunoblot analyses. Rack1 expression in other tissues and organs was not different than in control mice, indicating tissue specificity of the recombination. Histopathology revealed a patchy, erosive, hemorrhagic, inflammatory enterocolitis with denuded, sloughed off surface epithelium, and crypt hyperplasia. These results suggest a protective function for Rack1 in maintaining the integrity of intestinal epithelia and for survival. NEW & NOTEWORTHY Our findings reveal a novel function for Rack1 in maintaining intestinal homeostasis by protecting the epithelial barrier. Rack1 loss results in a patchy, erosive, hemorrhagic, inflammatory enterocolitis, which resembles that of inflammatory bowel diseases (IBD) in humans. Understanding mechanisms that protect barrier function in normal intestine and how loss of that protection contributes to the pathogenesis of IBD could lead to improved therapies for these and other erosive diseases of the gastrointestinal tract.
Asunto(s)
Enterocolitis/metabolismo , Células Epiteliales/metabolismo , Absorción Intestinal , Mucosa Intestinal/metabolismo , Receptores de Cinasa C Activada/metabolismo , Animales , Diarrea/genética , Diarrea/metabolismo , Diarrea/patología , Enterocolitis/genética , Enterocolitis/patología , Células Epiteliales/patología , Hemorragia Gastrointestinal/genética , Hemorragia Gastrointestinal/metabolismo , Hemorragia Gastrointestinal/patología , Predisposición Genética a la Enfermedad , Homeostasis , Mucosa Intestinal/patología , Ratones Noqueados , Permeabilidad , Fenotipo , Receptores de Cinasa C Activada/deficiencia , Receptores de Cinasa C Activada/genética , Transducción de Señal , Pérdida de PesoRESUMEN
Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Células Enteroendocrinas/metabolismo , Mucosa Intestinal/lesiones , Mucosa Intestinal/metabolismo , Yeyuno/lesiones , Yeyuno/metabolismo , Células Madre/metabolismo , Animales , Antígenos de Diferenciación/genética , Células Enteroendocrinas/patología , Regulación de la Expresión Génica , Mucosa Intestinal/patología , Yeyuno/patología , Ratones , Ratones Transgénicos , Células Madre/patologíaAsunto(s)
Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/diagnóstico por imagen , Enfermedades Pulmonares/complicaciones , Enfermedades Pulmonares/diagnóstico por imagen , Adulto , Antiinflamatorios/administración & dosificación , Enfermedad de Crohn/tratamiento farmacológico , Quimioterapia Combinada , Humanos , Enfermedades Pulmonares/tratamiento farmacológico , MasculinoAsunto(s)
Amebicidas/uso terapéutico , Disentería Amebiana/tratamiento farmacológico , Absceso Hepático Amebiano/tratamiento farmacológico , Antidiarreicos/uso terapéutico , Colonoscopía , Quimioterapia Combinada , Disentería Amebiana/diagnóstico , Humanos , Yodoquinol/uso terapéutico , Leucocitosis/tratamiento farmacológico , Absceso Hepático Amebiano/diagnóstico , Loperamida/uso terapéutico , Masculino , Metronidazol/uso terapéutico , Persona de Mediana Edad , Paromomicina/uso terapéutico , Recurrencia , Tinidazol/uso terapéutico , Resultado del TratamientoRESUMEN
BACKGROUND: Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported. METHODOLOGY/PRINCIPAL FINDINGS: In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the 'tail' domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively. CONCLUSIONS/SIGNIFICANCE: Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic.
Asunto(s)
Queratina-19/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Queratina-19/química , Queratina-19/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , FosforilaciónAsunto(s)
Neoplasias de los Conductos Biliares/diagnóstico , Conductos Biliares Extrahepáticos , Colangiocarcinoma/diagnóstico , Colangitis/diagnóstico , Neoplasias de los Conductos Biliares/diagnóstico por imagen , Neoplasias de los Conductos Biliares/cirugía , Biopsia con Aguja , Colangiocarcinoma/patología , Colangiocarcinoma/cirugía , Colangiopancreatografia Retrógrada Endoscópica/métodos , Diagnóstico Diferencial , Servicio de Urgencia en Hospital , Estudios de Seguimiento , Hematemesis/diagnóstico , Hematemesis/etiología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pancreaticoduodenectomía/métodos , Medición de Riesgo , Tomografía Computarizada por Rayos X/métodos , Resultado del TratamientoRESUMEN
Cascades of kinases and phosphatases are regulated by selective protein-protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the over- or underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here, we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G1, and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein-protein interactions.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Fase G1/fisiología , Neuropéptidos/metabolismo , Péptidos/administración & dosificación , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Fase S/fisiología , Animales , Fase G1/efectos de los fármacos , Ratones , Células 3T3 NIH , Receptores de Cinasa C Activada , Fase S/efectos de los fármacosRESUMEN
Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.
Asunto(s)
Fase G1 , Proteínas Musculares , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas , Fase S , Familia-src Quinasas/metabolismo , Animales , Quinasas CDC2-CDC28/metabolismo , Ciclo Celular , División Celular , Ciclina D1/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Citometría de Flujo , Proteínas de Unión al GTP , Genes Reporteros , Humanos , Immunoblotting , Ratones , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Células 3T3 NIH , Neoplasias/metabolismo , Proteínas Oncogénicas/metabolismo , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-vav , Receptores de Cinasa C Activada , Receptores de Superficie Celular , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.
Asunto(s)
Proteína Oncogénica pp60(v-src)/metabolismo , Péptidos/fisiología , Actinas/metabolismo , Actinas/ultraestructura , Animales , Adhesión Celular/fisiología , Recuento de Células , División Celular/fisiología , Línea Celular Transformada , Medios de Cultivo , Proteínas del Citoesqueleto/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Ratones , Células 3T3 NIH , Proteína Oncogénica pp60(v-src)/antagonistas & inhibidores , Proteína Oncogénica pp60(v-src)/genética , Paxillin , Péptidos/genética , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Cinasa C Activada , Suero , Transfección , Transformación Genética , Tirosina/metabolismoRESUMEN
RACK1 is the founding member of a family of receptors for activated C kinase collectively called RACKs. Upon activation of PKC, RACK1 co-localizes with the Src tyrosine kinase at the plasma membrane and functions as a substrate, binding partner and inhibitor of Src (as measured in vitro), and a growth inhibitor in NIH 3T3 cells. To further analyze the function of RACK1 in Src and PKC signaling, we utilized cell-permeable peptides that modulate the interaction of RACK1 and betaIIPKC, thereby affecting betaIIPKC translocation and function. We found that the association of betaIIPKC and RACK1 is necessary for Src phosphorylation of RACK1. Src activity is required for tyrosine phosphorylation of RACK1, and for RACK1 binding to Src, but not to betaIIPKC. Endogenous Src kinase activity, as measured by phosphorylation of Sam68 (a mitotic-specific Src substrate involved in cell cycle regulation and RNA splicing) or p190RhoGAP (a Src substrate and GTPase-activating protein involved in actin reorganization), increases with disruption of the Src-RACK1 complex, and decreases with enhanced complex formation. RACK1 inhibits Src-mediated p190RhoGAP signaling and actin cytoskeleton rearrangement. Thus, RACK1 functions as an endogenous inhibitor of the Src kinase in diverse signaling pathways that regulate distinct cellular functions. Our results demonstrate the potential for using peptide modulators of Src activity as a tool for uncovering the function of Src in cells.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Nucleares/metabolismo , Proteína Quinasa C/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Superficie Celular/metabolismo , Familia-src Quinasas/metabolismo , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al ADN , Proteínas Activadoras de GTPasa , Ratones , Fosfoproteínas , Receptores de Cinasa C Activada , Proteínas Represoras , Transducción de SeñalRESUMEN
Cytomegalovirus (CMV) infection is reported to be a cause of steroid-refractory ulcerative colitis (UC), but the strength of this association has not been tested in a case control study. Controlled studies have also not been performed to determine the sensitivity of available immunohistochemical techniques to detect CMV in this setting. The pathology database at Stanford Hospital was searched for UC patients with a diagnosis of "severe colitis" between the years 1992 and 2002 and medical records were reviewed. Forty patients were identified with refractory UC, defined as poor response to highdose systemic steroids for >2 weeks. Another group of 40 patients with severe, but nonrefractory, UC was case-matched for age and year of biopsy. A series of 40 patients who underwent colectomy for reasons other than inflammatory bowel disease with representative sections of "normal" colon were selected as noncolitis controls. CMV inclusions were detected on hematoxylin and eosin (H&E) in 2 of 40 patients with refractory UC, but not in other patients. Immunohistochemistry (IHC) detected CMV in 10 of 40 (25%) patients with refractory UC and 1 of 40 (2.5%) patients with nonrefractory UC (P = 0.007). The CMV-positive cases initially identified on IHC but not on H&E were re-reviewed for viral inclusions on H&E: 3 had rare, but typical, inclusions; 3 had atypical inclusions; and 3 had no inclusions. CMV was not detected by H&E or IHC in 40 noncolitis controls. Of 10 steroid-refractory UC patients with CMV detected, 7 were refractory to cyclosporin or 6-mercaptopurine/azathioprine (70%) and 6 had undergone proctocolectomy (60%) prior to detection of the CMV. Two patients with recognized CMV infection were treated with gancyclovir, improved, and were able to taper off steroids and avoid proctocolectomy. This study provides evidence that unrecognized and therefore untreated CMV infection is significantly associated with steroid-refractory UC. Moreover, IHC is more sensitive than H&E for detection of CMV and should be considered as part of the routine evaluation of steroid-refractory UC patients, before proceeding with other medical or surgical therapy that may be unnecessary once the CMV is treated.
Asunto(s)
Colitis Ulcerosa/virología , Infecciones por Citomegalovirus/complicaciones , Citomegalovirus/aislamiento & purificación , Esteroides , Adolescente , Adulto , Antígenos Virales/análisis , California/epidemiología , Estudios de Casos y Controles , Niño , Colitis Ulcerosa/tratamiento farmacológico , Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/epidemiología , Resistencia a Medicamentos , Femenino , Humanos , Huésped Inmunocomprometido , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Esteroides/uso terapéuticoAsunto(s)
Proteínas Quinasas/metabolismo , Técnicas del Sistema de Dos Híbridos , Animales , Proteínas de Unión al GTP , Biblioteca de Genes , Genes Reporteros , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteínas Quinasas/genética , Receptores de Cinasa C Activada , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
RACK1 is one of a group of PKC-interacting proteins collectively called RACKs (Receptors for Activated C-Kinases). Previously, we showed that RACK1 also interacts with the Src tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various tyrosine kinases in vitro, and by kinase-active and inactive mutants of Src in vivo. We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor tyrosine kinases and is involved in the regulation of Src function and cell growth.